APOE4 expression in iPSC-derived neurons increases AlphaBeta production and tau phosphorylation causing GABA neuron degeneration.

Gain of toxic Apolipoprotein E4 effects in Human iPSC-Derived Neurons Is Ameliorated by a Small-Molecule Structure Corrector

Efforts to develop drugs for Alzheimer's disease (AD) have shown promise in animal studies, only to fail in human trials, suggesting a pressing need to study AD in human model systems. Using human neurons derived from induced pluripotent stem cells that expressed apolipoprotein E4 (ApoE4), a variant of the APOE gene product and the major genetic risk factor for AD, we demonstrated that ApoE4-expressing neurons had higher levels of tau phosphorylation, unrelated to their increased production of amyloid-β (Aβ) peptides, and that they displayed GABAergic neuron degeneration. ApoE4 increased Aβ production in human, but not in mouse, neurons. Converting ApoE4 to ApoE3 by gene editing rescued these phenotypes, indicating the specific effects of ApoE4. Neurons that lacked APOE behaved similarly to those expressing ApoE3, and the introduction of ApoE4 expression recapitulated the pathological phenotypes, suggesting a gain of toxic effects from ApoE4. Treatment of ApoE4-expressing neurons with a small-molecule structure corrector ameliorated the detrimental effects, thus showing that correcting the pathogenic conformation of ApoE4 is a viable therapeutic approach for ApoE4-related AD.

ErbB4-neuregulin signaling modulates synapse development and dendritic arborization through distinct mechanisms.

Perturbations in neuregulin-1 (NRG1)/ErbB4 function have been associated with schizophrenia. Affected patients exhibit altered levels of these proteins and display hypofunction of glutamatergic synapses as well as altered neuronal circuitry. However, the role of NRG1/ErbB4 in regulating synapse maturation and neuronal process formation has not been extensively examined. Here we demonstrate that ErbB4 is expressed in inhibitory interneurons at both excitatory and inhibitory postsynaptic sites. Overexpression of ErbB4 postsynaptically enhances size but not number of presynaptic inputs. Conversely, knockdown of ErbB4 using shRNA decreases the size of presynaptic inputs, demonstrating a specific role for endogenous ErbB4 in synapse maturation. Using ErbB4 mutant constructs, we demonstrate that ErbB4-mediated synapse maturation requires its extracellular domain, whereas its tyrosine kinase activity is dispensable for this process. We also demonstrate that depletion of ErbB4 decreases the number of primary neurites and that stimulation of ErbB4 using a soluble form of NRG1 results in exuberant dendritic arborization through activation of the tyrosine kinase domain of ErbB4 and the phosphoinositide 3-kinase pathway. These findings demonstrate that NRG1/ErbB4 signaling differentially regulates synapse maturation and dendritic morphology via two distinct mechanisms involving trans-synaptic signaling and tyrosine kinase activity, respectively.

Reprogramming factor stoichiometry influences the epigenetic state and biological properties of induced pluripotent stem cells.

We compared two genetically highly defined transgenic systems to identify parameters affecting reprogramming of somatic cells to a pluripotent state. Our results demonstrate that the level and stoichiometry of reprogramming factors during the reprogramming process strongly influence the resulting pluripotency of iPS cells. High expression of Oct4 and Klf4 combined with lower expression of c-Myc and Sox2 produced iPS cells that efficiently generated "all-iPSC mice" by tetraploid (4n) complementation, maintained normal imprinting at the Dlk1-Dio3 locus, and did not create mice with tumors. Loss of imprinting (LOI) at the Dlk1-Dio3 locus did not strictly correlate with reduced pluripotency though the efficiency of generating "all-iPSC mice" was diminished. Our data indicate that stoichiometry of reprogramming factors can influence epigenetic and biological properties of iPS cells. This concept complicates efforts to define a "generic" epigenetic state of iPSCs and ESCs and should be considered when comparing different iPS and ES cell lines.

PP2A regulates tau phosphorylation directly and also indirectly via activating GSK-3beta.

Abnormal hyperphosphorylation of tau appears to be crucial in neurofibrillary degeneration in Alzheimer's disease (AD). Previous studies suggest that a down-regulation of protein phosphatase 2A (PP2A), the major tau phosphatase in human brain, contributes to tau hyperphosphorylation in AD. However, the effects of PP2A down-regulation on site-specific tau hyperphosphorylation is not well understood. In the present study, we showed that PP2A dephosphorylated tau at several phosphorylation sites with different efficiencies. Among the sites studied, Thr205, Thr212, Ser214, and Ser262 were the most favorable sites, and Ser199 and Ser404 were the least favorable sites for PP2A in vitro. Inhibition of PP2A with okadaic acid in metabolically active rat brain slices caused inhibition of glycogen synthase kinase-3beta (GSK-3beta) via an increase in its phosphorylation at Ser9. GSK-3beta phosphorylated tau at many sites, with Ser199, Thr205, and Ser396 being the most favorable sites in cells. The overall alterations in tau phosphorylation induced by PP2A inhibition were the result of the combined effects of both reduced tau dephosphorylation due to PP2A inhibition directly and reduced phosphorylation by GSK-3beta due to its inhibition. Because the impacts of tau phosphorylation on its biological activity and on neurofibrillary degeneration are site-specific, this study provides a new insight into the role of PP2A down-regulation in neurofibrillary degeneration in AD.

Pituitary adenylate cyclase-activating polypeptide (PACAP(1-38)) enhances N-methyl-D-aspartate receptor function and brain-derived neurotrophic factor expression via RACK1.

We recently identified a novel mechanism for modulation of the phosphorylation state and function of the N-methyl-d-aspartate (NMDA) receptor via the scaffolding protein RACK1. We found that RACK1 binds both the NR2B subunit of the NMDA receptor and the nonreceptor protein-tyrosine kinase, Fyn. RACK1 inhibits Fyn phosphorylation of NR2B and decreases NMDA receptor-mediated currents in CA1 hippocampal slices (Yaka, R., Thornton, C., Vagts, A. J., Phamluong, K., Bonci, A., and Ron, D. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 5710-5715). Here, we identified the signaling cascade by which RACK1 is released from the NMDA receptor complex and identified the consequences of the dissociation. We found that activation of the cAMP/protein kinase A pathway in hippocampal slices induced the release of RACK1 from NR2B and Fyn. This resulted in the induction of NR2B phosphorylation and the enhancement of NMDA receptor-mediated activity via Fyn. We identified the neuropeptide, pituitary adenylate cyclase activating polypeptide (PACAP(1-38)), as a ligand that induced phosphorylation of NR2B and enhanced NMDA receptor potentials. Finally, we found that activation of the cAMP/protein kinase A pathway induced the movement of RACK1 to the nuclear compartment in dissociated hippocampal neurons. Nuclear RACK1 in turn was found to regulate the expression of brain-derived neurotrophic factor induced by PACAP(1-38). Taken together our results suggest that activation of adenylate cyclase by PACAP(1-38) results in the release of RACK1 from the NMDA receptor and Fyn. This in turn leads to NMDA receptor phosphorylation, enhanced activity mediated by Fyn, and to the induction of brain-derived neurotrophic factor expression by RACK1.

Apolipoprotein E: high-avidity binding to beta-amyloid and increased frequency of type 4 allele in late-onset familial Alzheimer disease.

Apolipoprotein E is immunochemically localized to the senile plaques, vascular amyloid, and neurofibrillary tangles of Alzheimer disease. In vitro, apolipoprotein E in cerebrospinal fluid binds to synthetic beta A4 peptide (the primary constituent of the senile plaque) with high avidity. Amino acids 12-28 of the beta A4 peptide are required. The gene for apolipoprotein E is located on chromosome 19q13.2, within the region previously associated with linkage of late-onset familial Alzheimer disease. Analysis of apolipoprotein E alleles in Alzheimer disease and controls demonstrated that there was a highly significant association of apolipoprotein E type 4 allele (APOE-epsilon 4) and late-onset familial Alzheimer disease. The allele frequency of the APOE-epsilon 4 in 30 random affected patients, each from a different Alzheimer disease family, was 0.50 +/- 0.06; the allele frequency of APOE-epsilon 4 in 91 age-matched unrelated controls was 0.16 +/- 0.03 (Z = 2.44, P = 0.014). A functional role of the apolipoprotein E-E4 isoform in the pathogenesis of late-onset familial Alzheimer disease is suggested.

MicroRNA-22 (miR-22) Overexpression Is Neuroprotective via General Anti-Apoptotic Effects and May also Target Specific Huntington’s Disease-Related Mechanisms

BACKGROUND Whereas many causes and mechanisms of neurodegenerative diseases have been identified, very few therapeutic strategies have emerged in parallel. One possible explanation is that successful treatment strategy may require simultaneous targeting of more than one molecule of pathway. A new therapeutic approach to have emerged recently is the engagement of microRNAs (miRNAs), which affords the opportunity to target multiple cellular pathways simultaneously using a single sequence. METHODOLOGY/PRINCIPAL FINDINGS We identified miR-22 as a potentially neuroprotective miRNA based on its predicted regulation of several targets implicated in Huntington's disease (histone deacetylase 4 (HDAC4), REST corepresor 1 (Rcor1) and regulator of G-protein signaling 2 (Rgs2)) and its diminished expression in Huntington's and Alzheimer's disease brains. We then tested the hypothesis that increasing cellular levels of miRNA-22 would achieve neuroprotection in in vitro models of neurodegeneration. As predicted, overexpression of miR-22 inhibited neurodegeneration in primary striatal and cortical cultures exposed to a mutated human huntingtin fragment (Htt171-82Q). Overexpression of miR-22 also decreased neurodegeneration in primary neuronal cultures exposed to 3-nitropropionic acid (3-NP), a mitochondrial complex II/III inhibitor. In addition, miR-22 improved neuronal viability in an in vitro model of brain aging. The mechanisms underlying the effects of miR-22 included a reduction in caspase activation, consistent with miR-22's targeting the pro-apoptotic activities of mitogen-activated protein kinase 14/p38 (MAPK14/p38) and tumor protein p53-inducible nuclear protein 1 (Tp53inp1). Moreover, HD-specific effects comprised not only targeting HDAC4, Rcor1 and Rgs2 mRNAs, but also decreasing focal accumulation of mutant Htt-positive foci, which occurred via an unknown mechanism. CONCLUSIONS These data show that miR-22 has multipartite anti-neurodegenerative activities including the inhibition of apoptosis and the targeting of mRNAs implicated in the etiology of HD. These results motivate additional studies to evaluate the feasibility and therapeutic efficacy of manipulating miR-22 in vivo.

Fasting Activation of AgRP Neurons Requires NMDA Receptors and Involves Spinogenesis and Increased Excitatory Tone

AgRP neuron activity drives feeding and weight gain whereas that of nearby POMC neurons does the opposite. However, the role of excitatory glutamatergic input in controlling these neurons is unknown. To address this question, we generated mice lacking NMDA receptors (NMDARs) on either AgRP or POMC neurons. Deletion of NMDARs from AgRP neurons markedly reduced weight, body fat and food intake whereas deletion from POMC neurons had no effect. Activation of AgRP neurons by fasting, as assessed by c-Fos, Agrp and Npy mRNA expression, AMPA receptor-mediated EPSCs, depolarization and firing rates, required NMDARs. Furthermore, AgRP but not POMC neurons have dendritic spines and increased glutamatergic input onto AgRP neurons caused by fasting was paralleled by an increase in spines, suggesting fasting induced synaptogenesis and spinogenesis. Thus glutamatergic synaptic transmission and its modulation by NMDARs play key roles in controlling AgRP neurons and determining the cellular and behavioral response to fasting.

Nuclear accumulation of HDAC4 in ATM deficiency promotes neurodegeneration in ataxia-telangiectasia

Ataxia telangiectasia is a neurodegenerative disease caused by mutation of the Atm gene. Here we report that ataxia telangiectasia mutated (ATM) deficiency causes nuclear accumulation of histone deacetylase 4 (HDAC4) in neurons and promotes neurodegeneration. Nuclear HDAC4 binds to chromatin, as well as to myocyte enhancer factor 2A (MEF2A) and cAMP-responsive element binding protein (CREB), leading to histone deacetylation and altered neuronal gene expression. Blocking either HDAC4 activity or its nuclear accumulation blunts these neurodegenerative changes and rescues several behavioral abnormalities of ATM-deficient mice. Full rescue of the neurodegeneration, however, also requires the presence of HDAC4 in the cytoplasm, suggesting that the ataxia telangiectasia phenotype results both from a loss of cytoplasmic HDAC4 as well as its nuclear accumulation. To remain cytoplasmic, HDAC4 must be phosphorylated. The activity of the HDAC4 phosphatase, protein phosphatase 2A (PP2A), is downregulated by ATM-mediated phosphorylation. In ATM deficiency, enhanced PP2A activity leads to HDAC4 dephosphorylation and the nuclear accumulation of HDAC4. Our results define a crucial role of the cellular localization of HDAC4 in the events leading to ataxia telangiectasia neurodegeneration.

Control of cortical GABA circuitry development by Nrg1 and ErbB4 signalling

Schizophrenia is a complex disorder that interferes with the function of several brain systems required for cognition and normal social behaviour. Although the most notable clinical aspects of the disease only become apparent during late adolescence or early adulthood, many lines of evidence suggest that schizophrenia is a neurodevelopmental disorder with a strong genetic component. Several independent studies have identified neuregulin 1 (NRG1) and its receptor ERBB4 as important risk genes for schizophrenia, although their precise role in the disease process remains unknown. Here we show that Nrg1 and ErbB4 signalling controls the development of inhibitory circuitries in the mammalian cerebral cortex by cell-autonomously regulating the connectivity of specific GABA (γ-aminobutyric acid)-containing interneurons. In contrast to the prevalent view, which supports a role for these genes in the formation and function of excitatory synapses between pyramidal cells, we found that ErbB4 expression in the mouse neocortex and hippocampus is largely confined to certain classes of interneurons. In particular, ErbB4 is expressed by many parvalbumin-expressing chandelier and basket cells, where it localizes to axon terminals and postsynaptic densities receiving glutamatergic input. Gain- and loss-of-function experiments, both in vitro and in vivo, demonstrate that ErbB4 cell-autonomously promotes the formation of axo-axonic inhibitory synapses over pyramidal cells, and that this function is probably mediated by Nrg1. In addition, ErbB4 expression in GABA-containing interneurons regulates the formation of excitatory synapses onto the dendrites of these cells. By contrast, ErbB4 is dispensable for excitatory transmission between pyramidal neurons. Altogether, our results indicate that Nrg1 and ErbB4 signalling is required for the wiring of GABA-mediated circuits in the postnatal cortex, providing a new perspective to the involvement of these genes in the aetiology of schizophrenia.

NuA4 lysine acetyltransferase Esa1 is targeted to coding regions and stimulates transcription elongation with Gcn5.

NuA4, the major H4 lysine acetyltransferase (KAT) complex in Saccharomyces cerevisiae, is recruited to promoters and stimulates transcription initiation. NuA4 subunits contain domains that bind methylated histones, suggesting that histone methylation should target NuA4 to coding sequences during transcription elongation. We show that NuA4 is cotranscriptionally recruited, dependent on its physical association with elongating polymerase II (Pol II) phosphorylated on the C-terminal domain by cyclin-dependent kinase 7/Kin28, but independently of subunits (Eaf1 and Tra1) required for NuA4 recruitment to promoters. Whereas histone methylation by Set1 and Set2 is dispensable for NuA4's interaction with Pol II and targeting to some coding regions, it stimulates NuA4-histone interaction and H4 acetylation in vivo. The NuA4 KAT, Esa1, mediates increased H4 acetylation and enhanced RSC occupancy and histone eviction in coding sequences and stimulates the rate of transcription elongation. Esa1 cooperates with the H3 KAT in SAGA, Gcn5, to enhance these functions. Our findings delineate a pathway for acetylation-mediated nucleosome remodeling and eviction in coding sequences that stimulates transcription elongation by Pol II in vivo.

Feedback Circuit among INK4 Tumor Suppressors Constrains Human Glioblastoma Development

We have developed a nonheuristic genome topography scan (GTS) algorithm to characterize the patterns of genomic alterations in human glioblastoma (GBM), identifying frequent p18(INK4C) and p16(INK4A) codeletion. Functional reconstitution of p18(INK4C) in GBM cells null for both p16(INK4A) and p18(INK4C) resulted in impaired cell-cycle progression and tumorigenic potential. Conversely, RNAi-mediated depletion of p18(INK4C) in p16(INK4A)-deficient primary astrocytes or established GBM cells enhanced tumorigenicity in vitro and in vivo. Furthermore, acute suppression of p16(INK4A) in primary astrocytes induced a concomitant increase in p18(INK4C). Together, these findings uncover a feedback regulatory circuit in the astrocytic lineage and demonstrate a bona fide tumor suppressor role for p18(INK4C) in human GBM wherein it functions cooperatively with other INK4 family members to constrain inappropriate proliferation.

Neuregulin 1 promotes excitatory synapse development and function in GABAergic interneurons.

Neuregulin 1 (NRG1) and its receptor ErbB4 are both susceptibility genes of schizophrenia. However, little is known about the underlying mechanisms of their malfunction. Although ErbB4 is enriched in GABAergic interneurons, the role of NRG1 in excitatory synapse formation in these neurons remains poorly understood. We showed that NRG1 increased both the number and size of PSD-95 puncta and the frequency and amplitude of miniature EPSCs (mEPSCs) in GABAergic interneurons, indicating that NRG1 stimulates the formation of new synapses and strengthens existing synapses. In contrast, NRG1 treatment had no effect on either the number or size of excitatory synapses in glutamatergic neurons, suggesting its synaptogenic effect is specific to GABAergic interneurons. Ecto-ErbB4 treatment diminished both the number and size of excitatory synapses, suggesting that endogenous NRG1 may be critical for basal synapse formation. NRG1 could stimulate the stability of PSD-95 in the manner that requires tyrosine kinase activity of ErbB4. Finally, deletion of ErbB4 in parvalbumin-positive interneurons led to reduced frequency and amplitude of mEPSCs, providing in vivo evidence that ErbB4 is important in excitatory synaptogenesis in interneurons. Together, our findings suggested a novel synaptogenic role of NRG1 in excitatory synapse development, possibly via stabilizing PSD-95, and this effect is specific to GABAergic interneurons. In light of the association of the genes of both NRG1 and ErbB4 with schizophrenia and dysfunction of GABAergic system in this disorder, these results provide insight into its potential pathological mechanism.

Brain region-specific up-regulation of mouse apolipoprotein E by pharmacological estrogen treatments.

Cerebral apolipoprotein E (apoE) has been implicated in neuronal protection and repair. Due to the variable levels and types of estrogen receptors within different brain regions, the effect of estrogen on apoE and the mechanism of this effect may vary within different regions. Ovariectomized female C57BL/6 mice were treated with pharmacological levels of 17 beta-estradiol or placebo for 5 days, resulting in supraphysiological plasma levels of estradiol in the treated mice. ApoE and glial fibrillary acidic protein (GFAP) levels were measured in the cortex, hippocampus and diencephalon. 17 beta-Estradiol up-regulated apoE but not GFAP in the cortex and diencephalon, whereas in the hippocampus, GFAP and apoE were equally up-regulated. Treatment of estrogen receptor (ER) alpha knockout mice with 17 beta-estradiol or treatment of C57BL/6 mice with 17 alpha-estradiol, a poor estrogen receptor agonist, specifically induced apoE in the cortex, but not in the diencephalon. These results indicate that 17 beta-estradiol effects on apoE are either directly or indirectly mediated by ER alpha in the diencephalon, while the effects in the cortex may be mediated by a non-classical mechanism or by ER beta. Measurement of mRNA levels in estrogen versus placebo-treated wild-type mice indicated that the effect of 17 beta-estradiol on apoE was not associated with changes in apoE mRNA levels.

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP), a Neuron-Derived Peptide Regulating Glial Glutamate Transport and Metabolism

In the brain, glutamatergic neurotransmission is terminated predominantly by the rapid uptake of synaptically released glutamate into astrocytes through the Na(+)-dependent glutamate transporters GLT-1 and GLAST and its subsequent conversion into glutamine by the enzyme glutamine synthetase (GS). To date, several factors have been identified that rapidly alter glial glutamate uptake by post-translational modification of glutamate transporters. The only condition known to affect the expression of glial glutamate transporters and GS is the coculturing of glia with neurons. We now demonstrate that neurons regulate glial glutamate turnover via pituitary adenylate cyclase-activating polypeptide (PACAP). In the cerebral cortex PACAP is synthesized by neurons and acts on the subpopulation of astroglia involved in glutamate turnover. Exposure of astroglia to PACAP increased the maximal velocity of [(3)H]glutamate uptake by promoting the expression of GLT-1, GLAST, and GS. Moreover, the stimulatory effects of neuron-conditioned medium on glial glutamate transporter expression were attenuated in the presence of PACAP-inactivating antibodies or the PACAP receptor antagonist PACAP 6-38. In contrast to PACAP, vasoactive intestinal peptide promoted glutamate transporter expression only at distinctly higher concentrations, suggesting that PACAP exerts its effects on glial glutamate turnover via PAC1 receptors. Although PAC1 receptor-dependent activation of protein kinase A (PKA) was sufficient to promote the expression of GLAST, the activation of both PKA and protein kinase C (PKC) was required to promote GLT-1 expression optimally. Given the existence of various PAC1 receptor isoforms that activate PKA and PKC to different levels, these findings point to a complex mechanism by which PACAP regulates glial glutamate transport and metabolism. Disturbances of these regulatory mechanisms could represent a major cause for glutamate-associated neurological and psychiatric disorders.

Pioglitazone prevents tau oligomerization.

Tau aggregation and amyloid β protein (Aβ) deposition are the main causes of Alzheimer's disease (AD). Peroxisome proliferator-activated receptor γ (PPARγ) activation modulates Aβ production. To test whether the PPARγ agonist pioglitazone (PIO) is also effective in preventing tau aggregation in AD, we used a cellular model in which wild-type tau protein (4R0N) is overexpressed (M1C cells) (Hamano et al., 2012) as well as primary neuronal cultures. PIO reduced both phosphorylated and total tau levels, and inactivated glycogen synthase kinase 3β, a major tau kinase, associated with activation of Akt. In addition, PIO decreased cleaved caspase3 and C-terminal truncated tau species by caspase, which is expected to decrease tau aggregation. A fractionation study showed that PIO reduced high molecular-weight (120 kDa), oligomeric tau species in Tris Insoluble, sarkosyl-soluble fractions. Tau decrease was reversed by adding GW9662, a PPARγ antagonist. Together, our current results support the idea that PPARγ agonists may be useful therapeutic agents for AD.

Aberrant Expression of RCAN1 in Alzheimer’s Pathogenesis: A New Molecular Mechanism and a Novel Drug Target

AD, a devastating neurodegenerative disorder, is the most common cause of dementia in the elderly. Patients with AD are characterized by three hallmarks of neuropathology including neuritic plaque deposition, neurofibrillary tangle formation, and neuronal loss. Growing evidences indicate that dysregulation of regulator of calcineurin 1 (RCAN1) plays an important role in the pathogenesis of AD. Aberrant RCAN1 expression facilitates neuronal apoptosis and Tau hyperphosphorylation, leading to neuronal loss and neurofibrillary tangle formation. This review aims to describe the recent advances of the regulation of RCAN1 expression and its physiological functions. Moreover, the AD risk factors-induced RCAN1 dysregulation and its role in promoting neuronal loss, synaptic impairments and neurofibrillary tangle formation are summarized. Furthermore, we provide an outlook into the effects of RCAN1 dysregulation on APP processing, Aβ generation and neuritic plaque formation, and the possible underlying mechanisms, as well as the potential of targeting RCAN1 as a new therapeutic approach.

Amyloid accumulation is a late event in sporadic Alzheimer's disease-like pathology in nontransgenic rats

The amyloid cascade hypothesis posits that deposition of the amyloid β (Aβ) peptide in the brain is a key event in the initiation of Alzheimer's disease (AD). Nonetheless, it now seems increasingly unlikely that amyloid toxicity is the cause of sporadic AD, which leads to cognitive decline. Here, using accelerated-senescence nontransgenic OXYS rats, we confirmed that aggregation of Aβ is a later event in AD-like pathology. We showed that an age-dependent increase in the levels of Aβ₁₋₄₂ and extracellular Aβ deposits in the brain of OXYS rats occur later than do synaptic losses, neuronal cell death, mitochondrial structural abnormalities, and hyperphosphorylation of the tau protein. We identified the variants of the genes that are strongly associated with the risk of either late-onset or early-onset AD, including App, Apoe4, Bace1, Psen1, Psen2, and Picalm. We found that in OXYS rats nonsynonymous SNPs were located only in the genes Casp3 and Sorl1. Thus, we present proof that OXYS rats may be a model of sporadic AD. It is possible that multiple age-associated pathological processes may precede the toxic amyloid accumulation, which in turn triggers the final stage of the sporadic form of AD and becomes a hallmark event of the disease.

Neurons derived from reprogrammed fibroblasts functionally integrate into the fetal brain and improve symptoms of rats with Parkinson's disease.

The long-term goal of nuclear transfer or alternative reprogramming approaches is to create patient-specific donor cells for transplantation therapy, avoiding immunorejection, a major complication in current transplantation medicine. It was recently shown that the four transcription factors Oct4, Sox2, Klf4, and c-Myc induce pluripotency in mouse fibroblasts. However, the therapeutic potential of induced pluripotent stem (iPS) cells for neural cell replacement strategies remained unexplored. Here, we show that iPS cells can be efficiently differentiated into neural precursor cells, giving rise to neuronal and glial cell types in culture. Upon transplantation into the fetal mouse brain, the cells migrate into various brain regions and differentiate into glia and neurons, including glutamatergic, GABAergic, and catecholaminergic subtypes. Electrophysiological recordings and morphological analysis demonstrated that the grafted neurons had mature neuronal activity and were functionally integrated in the host brain. Furthermore, iPS cells were induced to differentiate into dopamine neurons of midbrain character and were able to improve behavior in a rat model of Parkinson's disease upon transplantation into the adult brain. We minimized the risk of tumor formation from the grafted cells by separating contaminating pluripotent cells and committed neural cells using fluorescence-activated cell sorting. Our results demonstrate the therapeutic potential of directly reprogrammed fibroblasts for neuronal cell replacement in the animal model.

Early loss of interneurons and delayed subunit-specific changes in GABA(A)-receptor expression in a mouse model of mesial temporal lobe epilepsy.

Unilateral injection of kainic acid (KA) into the dorsal hippocampus of adult mice induces spontaneous recurrent partial seizures and replicates histopathological changes observed in human mesial temporal lobe epilepsy (MTLE) (Bouilleret V et al., Neuroscience 1999; 89:717-729). Alterations in pre- and postsynaptic components of GABAergic neurotransmission were investigated immunohistochemically at different time points (1-120 days) in this mouse model of MTLE. Markers of GABAergic interneurons (parvalbumin, calbindin-D28k, and calretinin), the type-1 GABA transporter (GAT1), and major GABA(A)-receptor subunits expressed in the hippocampal formation were analyzed. Acutely, KA injection produced a profound loss of hilar cells but only limited damage to CA1 and CA3 pyramidal cells. In addition, parvalbumin and calbindin-D28k staining of interneurons disappeared irreversibly in CA1 and dentate gyrus (DG), whereas calretinin staining was spared. The prominent GABA(A)-receptor alpha1 subunit staining of interneurons also disappeared after KA treatment, suggesting acute degeneration of these cells. Likewise, GAT1 immunoreactivity revealed degenerating terminals at 24 h post-KA in CA1 and DC and subsided almost completely thereafter. Loss of CA1 and, to a lesser extent, CA3 neurons became evident at 7-15 days post-KA. It was more accentuated after 1 month, accompanied by a corresponding reduction of GABA(A)-receptor staining. In contrast, DC granule cells were markedly enlarged and dispersed in the molecular layer and exhibited a prominent increase in GABA(A)-receptor subunit staining. After 4 months, the dorsal CA1 area was lost almost entirely, CA3 was reduced, and the DG represented most of the remaining dorsal hippocampal formation. No significant morphological alterations were detected contralaterally. These results suggest that loss of hilar cells and GABAergic neurons contributes to epileptogenesis in this model of MTLE. In contrast, long-term degeneration of pyramidal cells and granule cell dispersion may reflect distinct responses to recurrent seizures. Finally, GABA(A)-receptor upregulation in the DG may represent a compensatory response persisting for several months in epileptic mice.

Atg5-independent autophagy regulates mitochondrial clearance and is essential for iPSC reprogramming

Successful generation of induced pluripotent stem cells entails a major metabolic switch from mitochondrial oxidative phosphorylation to glycolysis during the reprogramming process. The mechanism of this metabolic reprogramming, however, remains elusive. Here, our results suggest that an Atg5-independent autophagic process mediates mitochondrial clearance, a characteristic event involved in the metabolic switch. We found that blocking such autophagy, but not canonical autophagy, inhibits mitochondrial clearance, in turn, preventing iPSC induction. Furthermore, AMPK seems to be upstream of this autophagic pathway and can be targeted by small molecules to modulate mitochondrial clearance during metabolic reprogramming. Our work not only reveals that the Atg5-independent autophagy is crucial for establishing pluripotency, but it also suggests that iPSC generation and tumorigenesis share a similar metabolic switch.

The developmental potential of iPSCs is greatly influenced by reprogramming factor selection.

Induced pluripotent stem cells (iPSCs) are commonly generated by transduction of Oct4, Sox2, Klf4, and Myc (OSKM) into cells. Although iPSCs are pluripotent, they frequently exhibit high variation in terms of quality, as measured in mice by chimera contribution and tetraploid complementation. Reliably high-quality iPSCs will be needed for future therapeutic applications. Here, we show that one major determinant of iPSC quality is the combination of reprogramming factors used. Based on tetraploid complementation, we found that ectopic expression of Sall4, Nanog, Esrrb, and Lin28 (SNEL) in mouse embryonic fibroblasts (MEFs) generated high-quality iPSCs more efficiently than other combinations of factors including OSKM. Although differentially methylated regions, transcript number of master regulators, establishment of specific superenhancers, and global aneuploidy were comparable between high- and low-quality lines, aberrant gene expression, trisomy of chromosome 8, and abnormal H2A.X deposition were distinguishing features that could potentially also be applicable to human.

Rapamycin treatment augments motor neuron degeneration in SOD1(G93A) mouse model of amyotrophic lateral sclerosis.

Aberrant protein misfolding may contribute to the pathogenesis of amyotrophic lateral sclerosis (ALS) but the detailed mechanisms are largely unknown. Our previous study has shown that autophagy is altered in the mouse model of ALS. In the present study, we systematically investigated the correlation of the autophagic alteration with the motor neurons (MNs) degeneration in the ALS mice. We have demonstrated that the autophagic protein marker LC3-II is markedly and specifically increased in the spinal cord MNs of the ALS mice. Electron microscopy and immunochemistry studies have shown that autophagic vacuoles are significantly accumulated in the dystrophic axons of spinal cord MNs of the ALS mice. All these changes in the ALS mice appear at the age of 90 d when the ALS mice display modest clinical symptoms; and they become prominent at the age of 120 d. The clinical symptoms are correlated with the progression of MNs degeneration. Moreover, we have found that p62/SQSTM1 is accumulated progressively in the spinal cord, indicating that the possibility of impaired autophagic flux in the SOD1(G93A) mice. Furthermore, to our surprise, we have found that treatment with autophagy enhancer rapamycin accelerates the MNs degeneration, shortens the life span of the ALS mice, and has no obvious effects on the accumulation of SOD1 aggregates. In addition, we have demonstrated that rapamycin treatment in the ALS mice causes more severe mitochondrial impairment, higher Bax levels and greater caspase-3 activation. These findings suggest that selective degeneration of MNs is associated with the impairment of the autophagy pathway and that rapamycin treatment may exacerbate the pathological processing through apoptosis and other mechanisms in the ALS mice.

The death receptor CD95 activates adult neural stem cells for working memory formation and brain repair.

Adult neurogenesis persists in the subventricular zone and the dentate gyrus and can be induced upon central nervous system injury. However, the final contribution of newborn neurons to neuronal networks is limited. Here we show that in neural stem cells, stimulation of the "death receptor" CD95 does not trigger apoptosis but unexpectedly leads to increased stem cell survival and neuronal specification. These effects are mediated via activation of the Src/PI3K/AKT/mTOR signaling pathway, ultimately leading to a global increase in protein translation. Induction of neurogenesis by CD95 was further confirmed in the ischemic CA1 region, in the naive dentate gyrus, and after forced expression of CD95L in the adult subventricular zone. Lack of hippocampal CD95 resulted in a reduction in neurogenesis and working memory deficits. Following global ischemia, CD95-mediated brain repair rescued behavioral impairment. Thus, we identify the CD95/CD95L system as an instructive signal for ongoing and injury-induced neurogenesis.

C9orf72 repeat expansions cause neurodegeneration in Drosophila through arginine-rich proteins

An expanded GGGGCC repeat in C9orf72 is the most common genetic cause of frontotemporal dementia and amyotrophic lateral sclerosis. A fundamental question is whether toxicity is driven by the repeat RNA itself and/or by dipeptide repeat proteins generated by repeat-associated, non-ATG translation. To address this question, we developed in vitro and in vivo models to dissect repeat RNA and dipeptide repeat protein toxicity. Expression of pure repeats, but not stop codon–interrupted “RNA-only” repeats in Drosophila caused adult-onset neurodegeneration. Thus, expanded repeats promoted neurodegeneration through dipeptide repeat proteins. Expression of individual dipeptide repeat proteins with a non-GGGGCC RNA sequence revealed that both poly-(glycine-arginine) and poly-(proline-arginine) proteins caused neurodegeneration. These findings are consistent with a dual toxicity mechanism, whereby both arginine-rich proteins and repeat RNA contribute to C9orf72-mediated neurodegeneration.

Modeling neurogenesis impairment in Down syndrome with induced pluripotent stem cells from Trisomy 21 amniotic fluid cells.

Down syndrome (DS), or Trisomy 21 (T21) syndrome, one of the most common chromosomal abnormalities, is caused by an extra duplication of chromosome 21. In studies of neuron development, experimental models based on human cells are considered to be the most desired and accurate for basic research. The generation of diseased induced pluripotetn stem (iPS) cell is a critical step in understanding the developmental stages of complex neuronal diseases. Here, we generated human DS iPS cell lines from second trimester amniotic fluid (AF) cells with T21 by co-expressing Yamanaka factors through lentiviral delivery and subsequently differentiated them into neuronal progenitor cells (NPCs) for further analyses. T21 AF-iPS cells were characterized for the expression of pluripotent markers and for their ability to differentiate into all three germ layers by forming embryoid bodies in vitro and teratomas in vivo. The T21 AF-iPS cells maintained their unique pattern of chromosomal karyotypes: three pairs of chromosome 21. The level of amyloid precursor protein was significantly increased in NPCs derived from T21 AF-iPS cells compared with NPCs from normal AF-iPS cells. The expression levels of miR-155 and miR-802 in T21 AF-iPS-NPCs were highly elevated in the presence of low expression of MeCP2. We observed that T21 iPS-NPCs generated fewer neurons compared with controls. T21 iPS-NPCs exhibit developmental defects during neurogenesis. Our findings suggest that T21 AF-iPS cells serve as a good source to further elucidate the impairment neurogenesis of DS and the onset of Alzheimer's disease.

Obesity occurring in apolipoprotein E-knockout mice has mild effects on fertility.

The Apolipoprotein (Apo) family is implicated in lipid metabolism. There are five types of Apo: Apoa, Apob, Apoc, Apod, and Apoe. Apoe has been demonstrated to play a central role in lipoprotein metabolism and to be essential for efficient receptor-mediated plasma clearance of chylomicron remnants and VLDL remnant particles by the liver. Apoe-deficient (Apoe(-/-)) mice develop atherosclerotic plaques spontaneously, followed by obesity. In this study, we investigated whether lipid deposition caused by Apoe knockout affects reproduction in female mice. The results demonstrated that Apoe(-/-) mice were severely hypercholesterolemic, with their cholesterol metabolism disordered, and lipid accumulating in the ovaries causing the ovaries to be heavier compared with the WT counterparts. In addition, estrogen and progesterone decreased significantly at D 100. Quantitative PCR analysis demonstrated that at D 100 the expression of cytochromeP450 aromatase (Cyp19a1), 3β-hydroxysteroid dehydrogenase (Hsd3b), mechanistic target of rapamycin (Mtor), and nuclear factor-κB (Nfkb) decreased significantly, while that of BCL2-associated agonist of cell death (Bad) and tuberous sclerosis complex 2 (Tsc2) increased significantly in the Apoe(-/-) mice. However, there was no difference in the fertility rates of the Apoe(-/-) and WT mice; that is, obesity induced by Apoe knockout has no significant effect on reproduction. However, the deletion of Apoe increased the number of ovarian follicles and the ratio of ovarian follicle atresia and apoptosis. We believe that this work will augment our understanding of the role of Apoe in reproduction.

The mammalian target of rapamycin signaling pathway mediates epileptogenesis in a model of temporal lobe epilepsy.

Understanding molecular mechanisms mediating epileptogenesis is critical for developing more effective therapies for epilepsy. We recently found that the mammalian target of rapamycin (mTOR) signaling pathway is involved in epileptogenesis, and mTOR inhibitors prevent epilepsy in a mouse model of tuberous sclerosis complex. Here, we investigated the potential role of mTOR in a rat model of temporal lobe epilepsy initiated by status epilepticus. Acute kainate-induced seizures resulted in biphasic activation of the mTOR pathway, as evident by an increase in phospho-S6 (P-S6) expression. An initial rise in P-S6 expression started approximately 1 h after seizure onset, peaked at 3-6 h, and returned to baseline by 24 h in both hippocampus and neocortex, reflecting widespread stimulation of mTOR signaling by acute seizure activity. After resolution of status epilepticus, a second increase in P-S6 was observed in hippocampus only, which started at 3 d, peaked 5-10 d, and persisted for several weeks after kainate injection, correlating with the development of chronic epileptogenesis within hippocampus. The mTOR inhibitor rapamycin, administered before kainate, blocked both the acute and chronic phases of seizure-induced mTOR activation and decreased kainate-induced neuronal cell death, neurogenesis, mossy fiber sprouting, and the development of spontaneous epilepsy. Late rapamycin treatment, after termination of status epilepticus, blocked the chronic phase of mTOR activation and reduced mossy fiber sprouting and epilepsy but not neurogenesis or neuronal death. These findings indicate that mTOR signaling mediates mechanisms of epileptogenesis in the kainate rat model and that mTOR inhibitors have potential antiepileptogenic effects in this model.

Human iPSC-based modeling of late-onset disease via progerin-induced aging.

Reprogramming somatic cells to induced pluripotent stem cells (iPSCs) resets their identity back to an embryonic age and, thus, presents a significant hurdle for modeling late-onset disorders. In this study, we describe a strategy for inducing aging-related features in human iPSC-derived lineages and apply it to the modeling of Parkinson's disease (PD). Our approach involves expression of progerin, a truncated form of lamin A associated with premature aging. We found that expression of progerin in iPSC-derived fibroblasts and neurons induces multiple aging-related markers and characteristics, including dopamine-specific phenotypes such as neuromelanin accumulation. Induced aging in PD iPSC-derived dopamine neurons revealed disease phenotypes that require both aging and genetic susceptibility, such as pronounced dendrite degeneration, progressive loss of tyrosine hydroxylase (TH) expression, and enlarged mitochondria or Lewy-body-precursor inclusions. Thus, our study suggests that progerin-induced aging can be used to reveal late-onset age-related disease features in hiPSC-based disease models.

Disclosure of APOE genotype for risk of Alzheimer's disease.

BACKGROUND The apolipoprotein E (APOE) genotype provides information on the risk of Alzheimer's disease, but the genotyping of patients and their family members has been discouraged. We examined the effect of genotype disclosure in a prospective, randomized, controlled trial. METHODS We randomly assigned 162 asymptomatic adults who had a parent with Alzheimer's disease to receive the results of their own APOE genotyping (disclosure group) or not to receive such results (nondisclosure group). We measured symptoms of anxiety, depression, and test-related distress 6 weeks, 6 months, and 1 year after disclosure or nondisclosure. RESULTS There were no significant differences between the two groups in changes in time-averaged measures of anxiety (4.5 in the disclosure group and 4.4 in the nondisclosure group, P=0.84), depression (8.8 and 8.7, respectively; P=0.98), or test-related distress (6.9 and 7.5, respectively; P=0.61). Secondary comparisons between the nondisclosure group and a disclosure subgroup of subjects carrying the APOE epsilon4 allele (which is associated with increased risk) also revealed no significant differences. However, the epsilon4-negative subgroup had a significantly lower level of test-related distress than did the epsilon4-positive subgroup (P=0.01). Subjects with clinically meaningful changes in psychological outcomes were distributed evenly among the nondisclosure group and the epsilon4-positive and epsilon4-negative subgroups. Baseline scores for anxiety and depression were strongly associated with post-disclosure scores of these measures (P<0.001 for both comparisons). CONCLUSIONS The disclosure of APOE genotyping results to adult children of patients with Alzheimer's disease did not result in significant short-term psychological risks. Test-related distress was reduced among those who learned that they were APOE epsilon4-negative. Persons with high levels of emotional distress before undergoing genetic testing were more likely to have emotional difficulties after disclosure. (ClinicalTrials.gov number, NCT00571025.)

Phosphorylation of centromeric histone H3 variant regulates chromosome segregation in Saccharomyces cerevisiae

The centromeric histone H3 variant (CenH3) is essential for chromosome segregation in eukaryotes. We identify posttranslational modifications of Saccharomyces cerevisiae CenH3, Cse4. Functional characterization of cse4 phosphorylation mutants shows growth and chromosome segregation defects when combined with kinetochore mutants okp1 and ame1. Using a phosphoserine-specific antibody, we show that the association of phosphorylated Cse4 with centromeres increases in response to defective microtubule attachment or reduced cohesion. We determine that evolutionarily conserved Ipl1/Aurora B contributes to phosphorylation of Cse4, as levels of phosphorylated Cse4 are reduced at centromeres in ipl1 strains in vivo, and in vitro assays show phosphorylation of Cse4 by Ipl1. Consistent with these results, we observe that a phosphomimetic cse4-4SD mutant suppresses the temperature-sensitive growth of ipl1-2 and Ipl1 substrate mutants dam1 spc34 and ndc80, which are defective for chromosome biorientation. Furthermore, cell biology approaches using a green fluorescent protein-labeled chromosome show that cse4-4SD suppresses chromosome segregation defects in dam1 spc34 strains. On the basis of these results, we propose that phosphorylation of Cse4 destabilizes defective kinetochores to promote biorientation and ensure faithful chromosome segregation. Taken together, our results provide a detailed analysis, in vivo and in vitro, of Cse4 phosphorylation and its role in promoting faithful chromosome segregation.

Generation of induced pluripotent stem cells from Asian patients with chronic neurodegenerative diseases.

Induced pluripotent stem (iPS) cells derived from disease patients are an invaluable resource for biomedical research and may provide a source for replacement therapies. In this study, we have generated iPS cells from Asian patients with chronic degenerative diseases of the nervous system, including spinal muscular atrophy (SMA), Parkinson disease (PD) and amyotrophic lateral sclerosis (ALS) by transduction with four factors (KLF4, SOX2, OCT4 and c-MYC). All of the iPS cells showed pluripotency similar to that of human embryonic stem cells (hESCs) and were able to differentiate into various somatic cell types in vitro and in vivo. Furthermore, the iPS cells also can be committed to differentiate into neural cells, the cell type that is affected in chronic degenerative diseases. Therefore, the patient-specific iPS cells we generated offer a cellular model in which to investigate disease mechanisms, discover and test novel drugs and develop new therapies for chronic neurodegenerative diseases.

Integration-free induced pluripotent stem cells model genetic and neural developmental features of down syndrome etiology.

Down syndrome (DS) is the most frequent cause of human congenital mental retardation. Cognitive deficits in DS result from perturbations of normal cellular processes both during development and in adult tissues, but the mechanisms underlying DS etiology remain poorly understood. To assess the ability of induced pluripotent stem cells (iPSCs) to model DS phenotypes, as a prototypical complex human disease, we generated bona fide DS and wild-type (WT) nonviral iPSCs by episomal reprogramming. DS iPSCs selectively overexpressed chromosome 21 genes, consistent with gene dosage, which was associated with deregulation of thousands of genes throughout the genome. DS and WT iPSCs were neurally converted at >95% efficiency and had remarkably similar lineage potency, differentiation kinetics, proliferation, and axon extension at early time points. However, at later time points DS cultures showed a twofold bias toward glial lineages. Moreover, DS neural cultures were up to two times more sensitive to oxidative stress-induced apoptosis, and this could be prevented by the antioxidant N-acetylcysteine. Our results reveal a striking complexity in the genetic alterations caused by trisomy 21 that are likely to underlie DS developmental phenotypes, and indicate a central role for defective early glial development in establishing developmental defects in DS brains. Furthermore, oxidative stress sensitivity is likely to contribute to the accelerated neurodegeneration seen in DS, and we provide proof of concept for screening corrective therapeutics using DS iPSCs and their derivatives. Nonviral DS iPSCs can therefore model features of complex human disease in vitro and provide a renewable and ethically unencumbered discovery platform.

Knockdown of the co-chaperone Hop promotes extranuclear accumulation of Stat3 in mouse embryonic stem cells.

A key event in the mechanism of mouse embryonic stem cell (mESC) pluripotency is phosphorylation, dimerisation and translocation to the nucleus of the signal transducer and activator of transcription3, Stat3. We used RNAi to suppress the levels of the co-chaperone Hsp70/Hsp90 organising protein (Hop) in an mESC line. Hop knockdown caused 68% depletion in Stat3 mRNA levels, decreased soluble pYStat3 levels, and led to an extranuclear accumulation of Stat3. The major binding partner of Hop, Hsp90, co-localised with a small non-nuclear fraction of Stat3 in mESCs, and both Stat3 and Hop co-precipitated with Hsp90. Hop knockdown did not affect Nanog and Oct4 protein levels; however, Nanog mRNA levels were decreased. We found that in the absence of Hop, mESCs lost their pluripotent ability to form embryoid bodies with a basement membrane. These data suggest that Hop facilitates the phosphorylation and nuclear translocation of Stat3, implying a role for the Hsp70/Hsp90 chaperone heterocomplex machinery in pluripotency signalling.

Schizophrenia susceptibility pathway neuregulin 1–ErbB4 suppresses Src upregulation of NMDA receptors

Hypofunction of the N-methyl D-aspartate subtype of glutamate receptor (NMDAR) is hypothesized to be a mechanism underlying cognitive dysfunction in individuals with schizophrenia. For the schizophrenia-linked genes NRG1 and ERBB4, NMDAR hypofunction is thus considered a key detrimental consequence of the excessive NRG1-ErbB4 signaling found in people with schizophrenia. However, we show here that neuregulin 1β–ErbB4 (NRG1β-ErbB4) signaling does not cause general hypofunction of NMDARs. Rather, we find that, in the hippocampus and prefrontal cortex, NRG1β-ErbB4 signaling suppresses the enhancement of synaptic NMDAR currents by the nonreceptor tyrosine kinase Src. NRG1β-ErbB4 signaling prevented induction of long-term potentiation at hippocampal Schaffer collateral–CA1 synapses and suppressed Src-dependent enhancement of NMDAR responses during theta-burst stimulation. Moreover, NRG1β-ErbB4 signaling prevented theta burst–induced phosphorylation of GluN2B by inhibiting Src kinase activity. We propose that NRG1-ErbB4 signaling participates in cognitive dysfunction in schizophrenia by aberrantly suppressing Src-mediated enhancement of synaptic NMDAR function.

Apolipoprotein E and atherosclerosis: beyond lipid effect.

In humans, apolipoprotein E (apoE) is a polymorphic multifunctional protein.1 It is coded by three alleles (e2, e3, e4) of a modulator gene (level, variability, and susceptibility gene) at the apoE locus on chromosome 19, determining six apoE genotypes and plasma phenotypes. Its pleiotropic effects are exerted on plasma lipoprotein metabolism, coagulation, oxidative processes, macrophage, glial cell and neuronal cell homeostasis, adrenal function, central nervous system physiology, inflammation, and cell proliferation.2,3 ApoE polymorphism modulates susceptibility to many diseases. It is, however, particularly notorious for its role in neurodegenerative disorders4 and atherosclerotic arterial disease.5,6 The e4 allele (phenotypes E4/4 and E4/3) that is associated with higher low density lipoprotein cholesterol (LDL-C) is considered proatherogenic, whereas the presence of the e2 allele (E3/2, E2/2), being associated with lower LDL-C levels, is deemed to have the opposite effect (although it may be associated with increased plasma triglycerides and lipoprotein remnants). This simple equation, however, is an oversimplification because these properties are subject to many environmental and genetic influences. ApoE has allele- and gender-dependent effects on reverse cholesterol transport, platelet aggregation, and oxidative processes that are likely to affect the overall atherogenic potential ascribed to modulation of lipoprotein metabolism.2,3,6 Notwithstanding the context dependency, a recent meta-analysis fully supports the presence of the e4 allele as a significant risk factor for coronary artery disease.7 Several mechanisms have been evoked to link apoE with atherosclerosis, but the relationship is not fully unraveled in humans. Nevertheless, some apoE mimetic peptides that promote LDL clearance are currently tested in animals for potential clinical applications.8,9  See page 436   The situation is relatively simpler in animals. The mouse model has been prominently useful to test mechanisms …

Ataxia, dementia, and hypogonadotropism caused by disordered ubiquitination.

BACKGROUND The combination of ataxia and hypogonadism was first described more than a century ago, but its genetic basis has remained elusive. METHODS We performed whole-exome sequencing in a patient with ataxia and hypogonadotropic hypogonadism, followed by targeted sequencing of candidate genes in similarly affected patients. Neurologic and reproductive endocrine phenotypes were characterized in detail. The effects of sequence variants and the presence of an epistatic interaction were tested in a zebrafish model. RESULTS Digenic homozygous mutations in RNF216 and OTUD4, which encode a ubiquitin E3 ligase and a deubiquitinase, respectively, were found in three affected siblings in a consanguineous family. Additional screening identified compound heterozygous truncating mutations in RNF216 in an unrelated patient and single heterozygous deleterious mutations in four other patients. Knockdown of rnf216 or otud4 in zebrafish embryos induced defects in the eye, optic tectum, and cerebellum; combinatorial suppression of both genes exacerbated these phenotypes, which were rescued by nonmutant, but not mutant, human RNF216 or OTUD4 messenger RNA. All patients had progressive ataxia and dementia. Neuronal loss was observed in cerebellar pathways and the hippocampus; surviving hippocampal neurons contained ubiquitin-immunoreactive intranuclear inclusions. Defects were detected at the hypothalamic and pituitary levels of the reproductive endocrine axis. CONCLUSIONS The syndrome of hypogonadotropic hypogonadism, ataxia, and dementia can be caused by inactivating mutations in RNF216 or by the combination of mutations in RNF216 and OTUD4. These findings link disordered ubiquitination to neurodegeneration and reproductive dysfunction and highlight the power of whole-exome sequencing in combination with functional studies to unveil genetic interactions that cause disease. (Funded by the National Institutes of Health and others.).

Exercise-linked FNDC5/irisin rescues synaptic plasticity and memory defects in Alzheimer’s models

Defective brain hormonal signaling has been associated with Alzheimer's disease (AD), a disorder characterized by synapse and memory failure. Irisin is an exercise-induced myokine released on cleavage of the membrane-bound precursor protein fibronectin type III domain-containing protein 5 (FNDC5), also expressed in the hippocampus. Here we show that FNDC5/irisin levels are reduced in AD hippocampi and cerebrospinal fluid, and in experimental AD models. Knockdown of brain FNDC5/irisin impairs long-term potentiation and novel object recognition memory in mice. Conversely, boosting brain levels of FNDC5/irisin rescues synaptic plasticity and memory in AD mouse models. Peripheral overexpression of FNDC5/irisin rescues memory impairment, whereas blockade of either peripheral or brain FNDC5/irisin attenuates the neuroprotective actions of physical exercise on synaptic plasticity and memory in AD mice. By showing that FNDC5/irisin is an important mediator of the beneficial effects of exercise in AD models, our findings place FNDC5/irisin as a novel agent capable of opposing synapse failure and memory impairment in AD.

Neuron-specific expression of CuZnSOD prevents the loss of muscle mass and function that occurs in homozygous CuZnSOD-knockout mice.

Deletion of copper-zinc superoxide dismutase (CuZnSOD) in Sod1(-/-) mice leads to accelerated loss of muscle mass and force during aging, but the losses do not occur with muscle-specific deletion of CuZnSOD. To determine the role of motor neurons in the muscle decline, we generated transgenic Sod1(-/-) mice in which CuZnSOD was expressed under control of the synapsin 1 promoter (SynTgSod1(-/-) mice). SynTgSod1(-/-) mice expressed CuZnSOD in brain, spinal cord, and peripheral nerve, but not in other tissues. Sciatic nerve CuZnSOD content in SynTgSod1(-/-) mice was ~20% that of control mice, but no reduction in muscle mass or isometric force was observed in SynTgSod1(-/-) mice compared with control animals, whereas muscles of age-matched Sod1(-/-) mice displayed 30-40% reductions in mass and force. In addition, increased oxidative damage and adaptations in stress responses observed in muscles of Sod1(-/-) mice were absent in SynTgSod1(-/-) mice, and degeneration of neuromuscular junction (NMJ) structure and function occurred in Sod1(-/-) mice but not in SynTgSod1(-/-) mice. Our data demonstrate that specific CuZnSOD expression in neurons is sufficient to preserve NMJ and skeletal muscle structure and function in Sod1(-/-) mice and suggest that redox homeostasis in motor neurons plays a key role in initiating sarcopenia during aging.

Transcriptional Signature and Memory Retention of Human-Induced Pluripotent Stem Cells

Genetic reprogramming of somatic cells to a pluripotent state (induced pluripotent stem cells or iPSCs) by over-expression of specific genes has been accomplished using mouse and human cells. However, it is still unclear how similar human iPSCs are to human Embryonic Stem Cells (hESCs). Here, we describe the transcriptional profile of human iPSCs generated without viral vectors or genomic insertions, revealing that these cells are in general similar to hESCs but with significant differences. For the generation of human iPSCs without viral vectors or genomic insertions, pluripotent factors Oct4 and Nanog were cloned in episomal vectors and transfected into human fetal neural progenitor cells. The transient expression of these two factors, or from Oct4 alone, resulted in efficient generation of human iPSCs. The reprogramming strategy described here revealed a potential transcriptional signature for human iPSCs yet retaining the gene expression of donor cells in human reprogrammed cells free of viral and transgene interference. Moreover, the episomal reprogramming strategy represents a safe way to generate human iPSCs for clinical purposes and basic research.

FOXO4 is necessary for neural differentiation of human embryonic stem cells.

Proteostasis is critical for maintaining cell function and proteome stability may play an important role in human embryonic stem cell (hESC) immortality. Notably, hESC populations exhibit a high assembly of active proteasomes, a key node of the proteostasis network. FOXO4, an insulin/IGF-1 responsive transcription factor, regulates proteasome activity in hESCs. We find that loss of FOXO4 reduces the potential of hESCs to differentiate into neural lineages. Therefore, FOXO4 crosses evolutionary boundaries and links hESC function to invertebrate longevity modulation.

Disrupted-in-schizophrenia 1 and neuregulin 1 are required for the specification of oligodendrocytes and neurones in the zebrafish brain.

Schizophrenia may arise from subtle abnormalities in brain development due to alterations in the functions of candidate susceptibility genes such as Disrupted-in-schizophrenia 1 (DISC1) and Neuregulin 1 (NRG1). To provide novel insights into the functions of DISC1 in brain development, we mapped the expression of zebrafish disc1 and set out to characterize its role in early embryonic development using morpholino antisense methods. These studies revealed a critical requirement for disc1 in oligodendrocyte development by promoting specification of olig2-positive cells in the hindbrain and other brain regions. Since NRG1 has well-documented roles in myelination, we also analyzed the roles of nrg1 and ErbB signalling in zebrafish brain development and we observed strikingly similar defects to those seen in disc1 morphant embryos. In addition to their effects on oligodendrocyte development, knock-down of disc1 or nrg1 caused near total loss of olig2-positive cerebellar neurones, but caused no apparent loss of spinal motor neurones. These findings suggest that disc1 and nrg1 function in common or related pathways controlling development of oligodendrocytes and neurones from olig2-expressing precursor cells. Like DISC1 and NRG1, OLIG2 and ERBB4 are promising candidate susceptibility genes for schizophrenia. Hence our findings in the zebrafish embryo suggest that hitherto unappreciated neurodevelopmental connections may exist between key human schizophrenia susceptibility genes. These connections could be investigated in Disc1 and Nrg1 mouse models and in genetically defined groups of patients in order to determine whether they are relevant to the pathobiology of schizophrenia. GenBank accession number for Danio rerio disc1: EU273350.

Mild overexpression of MeCP2 causes a progressive neurological disorder in mice.

Mutations in the X-linked methyl-CpG-binding protein 2 (MECP2), encoding a transcriptional repressor, cause Rett syndrome and a variety of related neurodevelopmental disorders. The vast majority of mutations associated with human disease are loss-of-function mutations, but precisely what aspect of MeCP2 function is responsible for these phenotypes remains unknown. We overexpressed wild-type human protein in transgenic mice using a large genomic clone containing the entire human MECP2 locus. Detailed neurobehavioral and electrophysiological studies in transgenic line MeCP2(Tg1), which expresses MeCP2 at approximately 2-fold wild-type levels, demonstrated onset of phenotypes around 10 weeks of age. Surprisingly, these mice displayed enhanced motor and contextual learning and enhanced synaptic plasticity in the hippocampus. After 20 weeks of age, however, these mice developed seizures, became hypoactive and approximately 30% of them died by 1 year of age. These data demonstrate that MeCP2 levels must be tightly regulated in vivo, and that even mild overexpression of this protein is detrimental. Furthermore, these results support the possibility that duplications or gain-of-function mutations in MECP2 might underlie some cases of X-linked delayed-onset neurobehavioral disorders.

Phosphorylation of ATM by Cdk5 mediates DNA damage signaling and regulates neuronal death

The phosphatidylinositol-3-kinase-like kinase ATM (ataxia-telangiectasia mutated) has a central role in coordinating DNA damage responses, including cell-cycle checkpoint control, DNA repair and apoptosis. Mutations of ATM cause a spectrum of defects ranging from neurodegeneration to cancer predisposition. However, the mechanism by which DNA damage activates ATM is poorly understood. Here we show that Cdk5 (cyclin-dependent kinase 5), activated by DNA damage, directly phosphorylates ATM at Ser 794 in post-mitotic neurons. Phosphorylation at Ser 794 precedes, and is required for, ATM autophosphorylation at Ser 1981, and activates ATM kinase activity. The Cdk5-ATM signal regulates phosphorylation and function of the ATM targets p53 and H2AX. Interruption of the Cdk5-ATM pathway attenuates DNA-damage-induced neuronal cell cycle re-entry and expression of the p53 targets PUMA and Bax, protecting neurons from death. Thus, activation of Cdk5 by DNA damage serves as a critical signal to initiate the ATM response and regulate ATM-dependent cellular processes.

Increased dendritic complexity and axonal length in cultured mouse cortical neurons overexpressing methyl-CpG-binding protein MeCP2

Rett syndrome is caused by loss-of-function mutations in the gene encoding the methyl DNA-binding factor MeCP2. As brain mass and neuronal complexity tend to be diminished in Rett patients, we tested whether MeCP2 directly influences the morphological complexity of developing neurons. Our results show that cultured mouse neurons overexpressing MeCP2beta (MECP2A) develop more complex morphologies, having longer axonal and dendritic processes, and an increased number of axonal and dendritic terminal endings. We then tested whether overexpressing a mutant form of MeCP2beta lacking its carboxyl terminus would elicit the same effects. Interestingly, while neurons overexpressing this mutant failed to enhance axonal and dendritic process elongation, the complexity of their axonal and dendritic processes remained significantly elevated. Taken together, these data support the hypothesis that MeCP2 directly regulates neuronal maturation and/or synaptogenesis, and provides evidence that MeCP2 may influence neuritic elongation and process branching through different mechanisms.

A Null Mutation in Inositol Polyphosphate 4-Phosphatase Type I Causes Selective Neuronal Loss in Weeble Mutant Mice

Weeble mutant mice have severe locomotor instability and significant neuronal loss in the cerebellum and in the hippocampal CA1 field. Genetic mapping was used to localize the mutation to the gene encoding inositol polyphosphate 4-phosphatase type I (Inpp4a), where a single nucleotide deletion results in a likely null allele. The substrates of INPP4A are intermediates in a pathway affecting intracellular Ca(2+) release but are also involved in cell cycle regulation through binding the Akt protooncogene; dysfunction in either may account for the neuronal loss of weeble mice. Although other mutations in phosphoinositide enzymes are associated with synaptic defects without neuronal loss, weeble shows that Inpp4a is critical for the survival of a subset of neurons during postnatal development in mice.

Lithium Therapy Improves Neurological Function and Hippocampal Dendritic Arborization in a Spinocerebellar Ataxia Type 1 Mouse Model

Background  Spinocerebellar ataxia type 1 (SCA1) is a dominantly inherited neurodegenerative disorder characterized by progressive motor and cognitive dysfunction. Caused by an expanded polyglutamine tract in ataxin 1 (ATXN1), SCA1 pathogenesis involves a multifactorial process that likely begins with misfolding of ATXN1, which has functional consequences on its interactions, leading to transcriptional dysregulation. Because lithium has been shown to exert neuroprotective effects in a variety of conditions, possibly by affecting gene expression, we tested the efficacy of lithium treatment in a knock-in mouse model of SCA1 (Sca1154Q/2Q mice) that replicates many features of the human disease. Methods and Findings  Sca1154Q/2Q mice and their wild-type littermates were fed either regular chow or chow that contained 0.2% lithium carbonate. Dietary lithium carbonate supplementation resulted in improvement of motor coordination, learning, and memory in Sca1154Q/2Q mice. Importantly, motor improvement was seen when treatment was initiated both presymptomatically and after symptom onset. Neuropathologically, lithium treatment attenuated the reduction of dendritic branching in mutant hippocampal pyramidal neurons. We also report that lithium treatment restored the levels of isoprenylcysteine carboxyl methyltransferase (Icmt; alternatively, Pccmt), down-regulation of which is an early marker of mutant ATXN1 toxicity. Conclusions  The effect of lithium on a marker altered early in the course of SCA1 pathogenesis, coupled with its positive effect on multiple behavioral measures and hippocampal neuropathology in an authentic disease model, make it an excellent candidate treatment for human SCA1 patients.

Butyrate greatly enhances derivation of human induced pluripotent stem cells by promoting epigenetic remodeling and the expression of pluripotency-associated genes.

We report here that butyrate, a naturally occurring fatty acid commonly used as a nutritional supplement and differentiation agent, greatly enhances the efficiency of induced pluripotent stem (iPS) cell derivation from human adult or fetal fibroblasts. After transient butyrate treatment, the iPS cell derivation efficiency is enhanced by 15- to 51-fold using either retroviral or piggyBac transposon vectors expressing 4 to 5 reprogramming genes. Butyrate stimulation is more remarkable (>100- to 200-fold) on reprogramming in the absence of either KLF4 or MYC transgene. Butyrate treatment did not negatively affect properties of iPS cell lines established by either 3 or 4 retroviral vectors or a single piggyBac DNA transposon vector. These characterized iPS cell lines, including those derived from an adult patient with sickle cell disease by either the piggyBac or retroviral vectors, show normal karyotypes and pluripotency. To gain insights into the underlying mechanisms of butyrate stimulation, we conducted genome-wide gene expression and promoter DNA methylation microarrays and other epigenetic analyses on established iPS cells and cells from intermediate stages of the reprogramming process. By days 6 to 12 during reprogramming, butyrate treatment enhanced histone H3 acetylation, promoter DNA demethylation, and the expression of endogenous pluripotency-associated genes, including DPPA2, whose overexpression partially substitutes for butyrate stimulation. Thus, butyrate as a cell permeable small molecule provides a simple tool to further investigate molecular mechanisms of cellular reprogramming. Moreover, butyrate stimulation provides an efficient method for reprogramming various human adult somatic cells, including cells from patients that are more refractory to reprogramming.

Control of glutamate clearance and synaptic efficacy by glial coverage of neurons.

Analysis of excitatory synaptic transmission in the rat hypothalamic supraoptic nucleus revealed that glutamate clearance and, as a consequence, glutamate concentration and diffusion in the extracellular space, is associated with the degree of astrocytic coverage of its neurons. Reduction in glutamate clearance, whether induced pharmacologically or associated with a relative decrease of glial coverage in the vicinity of synapses, affected transmitter release through modulation of presynaptic metabotropic glutamate receptors. Astrocytic wrapping of neurons, therefore, contributes to the regulation of synaptic efficacy in the central nervous system.

Effects of Hypoxia and Oxidative Stress on Expression of Neprilysin in Human Neuroblastoma Cells and Rat Cortical Neurones and Astrocytes

Pathogenesis of Alzheimer’s disease (AD), which is characterised by accumulation of extracellular deposits of β-amyloid peptide (Aβ) in the brain, has recently been linked to vascular disorders such as ischemia and stroke. Aβ is constantly produced in the brain from amyloid precursor protein (APP) through its cleavage by β- and γ-secretases and certain Aβ species are toxic for neurones. The brain has an endogenous mechanism of Aβ removal via proteolytic degradation and the zinc metalloproteinase neprilysin (NEP) is a critical regulator of Aβ concentration. Down-regulation of NEP could predispose to AD. By comparing the effects of hypoxia and oxidative stress on expression and activity of the Aβ-degrading enzyme NEP in human neuroblastoma NB7 cells and rat primary cortical neurones we have demonstrated that hypoxia reduced NEP expression at the protein and mRNA levels as well as its activity. On contrary in astrocytes hypoxia increased NEP mRNA expression.

Lentiviral vector design and imaging approaches to visualize the early stages of cellular reprogramming.

Induced pluripotent stem cells (iPSCs) can be derived from somatic cells by gene transfer of reprogramming transcription factors. Expression levels of these factors strongly influence the overall efficacy to form iPSC colonies, but additional contribution of stochastic cell-intrinsic factors has been proposed. Here, we present engineered color-coded lentiviral vectors in which codon-optimized reprogramming factors are co-expressed by a strong retroviral promoter that is rapidly silenced in iPSC, and imaged the conversion of fibroblasts to iPSC. We combined fluorescence microscopy with long-term single cell tracking, and used live-cell imaging to analyze the emergence and composition of early iPSC clusters. Applying our engineered lentiviral vectors, we demonstrate that vector silencing typically occurs prior to or simultaneously with the induction of an Oct4-EGFP pluripotency marker. Around 7 days post-transduction (pt), a subfraction of cells in clonal colonies expressed Oct4-EGFP and rapidly expanded. Cell tracking of single cell-derived iPSC colonies supported the concept that stochastic epigenetic changes are necessary for reprogramming. We also found that iPSC colonies may emerge as a genetic mosaic originating from different clusters. Improved vector design with continuous cell tracking thus creates a powerful system to explore the subtle dynamics of biological processes such as early reprogramming events.

Transcriptional regulation of neuronal genes and its effect on neural functions: expression and function of forkhead transcription factors in neurons.

Forkhead box transcription factor, class O (FOXO) is a mammalian homologue of DAF-16, which is known to regulate the lifespan of Caenorhabditis elegans and includes subfamilies of forkhead transcription factors such as AFX, FKHRL1, and FKHR. FKHR is phosphorylated on three sites (Thr-24, Ser-256, and Ser-319) in a phosphatidylinositol 3-kinase (PI3K)/Akt-dependent manner, thereby inhibiting death signals. We here documented dephosphorylation of FKHR following transient forebrain ischemia with its concomitant translocation into the nucleus in neurons in gerbil and mouse brains. The activation of FKHR preceded delayed neuronal death in the vulnerable hippocampal regions following ischemic brain injury. The FKHR activation was accompanied by an increase in DNA binding activity for FKHR-responsive element on the Fas ligand promoter. We also defined FKHR-induced downstream targets such as Fas ligand and Bim in brain ischemia. Therefore, we propose a new strategy to rescue neurons from delayed neuronal death by promoting the survival signaling. Sodium orthovanadate, a protein tyrosine phosphatase inhibitor, up-regulated Akt activity in the brain and in turn rescue neurons from delayed neuronal death by inhibiting FKHR-dependent or -independent death signals in neurons.

Modulation of mitochondrial function and morphology by interaction of Omi/HtrA2 with the mitochondrial fusion factor OPA1.

Loss of Omi/HtrA2 function leads to nerve cell loss in mouse models and has been linked to neurodegeneration in Parkinson's and Huntington's disease. Omi/HtrA2 is a serine protease released as a pro-apoptotic factor from the mitochondrial intermembrane space into the cytosol. Under physiological conditions, Omi/HtrA2 is thought to be involved in protection against cellular stress, but the cytological and molecular mechanisms are not clear. Omi/HtrA2 deficiency caused an accumulation of reactive oxygen species and reduced mitochondrial membrane potential. In Omi/HtrA2 knockout mouse embryonic fibroblasts, as well as in Omi/HtrA2 silenced human HeLa cells and Drosophila S2R+ cells, we found elongated mitochondria by live cell imaging. Electron microscopy confirmed the mitochondrial morphology alterations and showed abnormal cristae structure. Examining the levels of proteins involved in mitochondrial fusion, we found a selective up-regulation of more soluble OPA1 protein. Complementation of knockout cells with wild-type Omi/HtrA2 but not with the protease mutant [S306A]Omi/HtrA2 reversed the mitochondrial elongation phenotype and OPA1 alterations. Finally, co-immunoprecipitation showed direct interaction of Omi/HtrA2 with endogenous OPA1. Thus, we show for the first time a direct effect of loss of Omi/HtrA2 on mitochondrial morphology and demonstrate a novel role of this mitochondrial serine protease in the modulation of OPA1. Our results underscore a critical role of impaired mitochondrial dynamics in neurodegenerative disorders.

Acetylation of apurinic/apyrimidinic endonuclease-1 regulates Helicobacter pylori-mediated gastric epithelial cell apoptosis.

BACKGROUND & AIMS Helicobacter pylori-induced gastric epithelial cell (GEC) apoptosis is a complex process that includes activation of the tumor suppressor p53. p53-mediated apoptosis involves p53 activation, bax transcription, and cytochrome c release from mitochondria. Apurinic/apyrimidinic endonuclease-1 (APE-1) regulates transcriptional activity of p53, and H pylori induce APE-1 expression in human GECs. H pylori infection increases intracellular calcium ion concentration [Ca2+]i of GECs, which induces APE-1 acetylation. We investigated the effects of H pylori infection and APE-1 acetylation on GEC apoptosis. METHODS AGS cells (wild-type or with suppressed APE-1), KATO III cells, and cells isolated from gastric biopsy specimens were infected with H pylori. Effects were examined by immunoblotting, real-time reverse-transcription polymerase chain reaction, immunoprecipitation, immunofluorescence microscopy, chromatin immunoprecipitation, mobility shift, DNA binding, and luciferase assays. RESULTS H pylori infection increased [Ca2+]i and acetylation of APE-1 in GECs, but the acetylation status of APE-1 did not affect the transcriptional activity of p53. In GECs, expression of a form of APE-1 that could not be acetylated increased total and mitochondrial levels of Bax and induced release of cytochrome c and fragmentation of DNA; expression of wild-type APE-1 reduced these apoptotic events. We identified a negative calcium response element in the human bax promoter and found that poly (adenosine diphosphate-ribose) polymerase 1 recruited the acetylated APE-1/histone deacetylase-1 repressor complex to bax nCaRE. CONCLUSIONS H pylori-mediated acetylation of APE-1 suppresses Bax expression; this prevents p53-mediated apoptosis when H pylori infect GECs.

Glutamate mediates the function of melanocortin receptor 4 on Sim1 neurons in body weight regulation.

The melanocortin receptor 4 (MC4R) is a well-established mediator of body weight homeostasis. However, the neurotransmitter(s) that mediate MC4R function remain largely unknown; as a result, little is known about the second-order neurons of the MC4R neural pathway. Single-minded 1 (Sim1)-expressing brain regions, which include the paraventricular nucleus of hypothalamus (PVH), represent key brain sites that mediate melanocortin action. We conditionally restored MC4R expression in Sim1 neurons in the background of Mc4r-null mice. The restoration dramatically reduced obesity in Mc4r-null mice. The anti-obesity effect was completely reversed by selective disruption of glutamate release from those same Sim1 neurons. The reversal was caused by lower energy expenditure and hyperphagia. Corroboratively, selective disruption of glutamate release from adult PVH neurons led to rapid obesity development via reduced energy expenditure and hyperphagia. Thus, this study establishes glutamate as the primary neurotransmitter that mediates MC4Rs on Sim1 neurons in body weight regulation.

Depletion of glutathione induces 4-hydroxynonenal protein adducts and hydroxyurea teratogenicity in the organogenesis stage mouse embryo.

Glutathione (GSH) homeostasis is important during organogenesis. To elucidate the impact of GSH depletion in organogenesis stage embryos on oxidative stress and drug teratogenicity, l-buthionine-S,R-sulfoximine (BSO) was given to timed pregnant CD-1 mice 4 h before exposure to a model teratogen, hydroxyurea (HU) [400 mg/kg (HU-400) or 600 mg/kg (HU-600)]. Treatment with BSO or HU alone or with BSO plus HU-400 did not alter the ratios of glutathione disulfide/GSH in the embryo; in contrast, the combination of BSO plus HU-600 did increase this ratio at both 0.5 and 3 h post-HU, indicating the induction of oxidative stress in the embryos. Immunoreactivity to a product of lipid peroxidation, 4-hydroxynonenal (4-HNE) protein adducts, was detected in saline-treated embryos; the intensity and nuclear localization of 4-HNE protein adduct immunoreactivity in specific regions in the embryo was significantly increased by exposure to BSO alone or BSO and either dose of HU. BSO pretreatment increased the spectrum and incidence of external and skeletal malformations (curly tail, hind limb malformations, hydrocephaly, exencephaly, open eye, spina bifida, and gastroschisis) induced by HU-400 and HU-600; BSO exposure did not alter the effects of HU on fetal mortality or fetal weights or HU induction of c-Fos heterodimer-dependent activator protein 1 DNA binding activity. The formation of 4-HNE protein adducts in teratogen-exposed embryos was localized to regions of the embryo that were highly susceptible to insult, namely the somites and caudal neural tube, correlating the presence of 4-HNE adducts with the disruption of pattern formation during organogenesis.

Chromatin modifying enzymes as modulators of reprogramming

Generation of induced pluripotent stem cells (iPSCs) by somatic cell reprogramming involves global epigenetic remodelling. Whereas several proteins are known to regulate chromatin marks associated with the distinct epigenetic states of cells before and after reprogramming, the role of specific chromatin-modifying enzymes in reprogramming remains to be determined. To address how chromatin-modifying proteins influence reprogramming, we used short hairpin RNAs (shRNAs) to target genes in DNA and histone methylation pathways, and identified positive and negative modulators of iPSC generation. Whereas inhibition of the core components of the polycomb repressive complex 1 and 2, including the histone 3 lysine 27 methyltransferase EZH2, reduced reprogramming efficiency, suppression of SUV39H1, YY1 and DOT1L enhanced reprogramming. Specifically, inhibition of the H3K79 histone methyltransferase DOT1L by shRNA or a small molecule accelerated reprogramming, significantly increased the yield of iPSC colonies, and substituted for KLF4 and c-Myc (also known as MYC). Inhibition of DOT1L early in the reprogramming process is associated with a marked increase in two alternative factors, NANOG and LIN28, which play essential functional roles in the enhancement of reprogramming. Genome-wide analysis of H3K79me2 distribution revealed that fibroblast-specific genes associated with the epithelial to mesenchymal transition lose H3K79me2 in the initial phases of reprogramming. DOT1L inhibition facilitates the loss of this mark from genes that are fated to be repressed in the pluripotent state. These findings implicate specific chromatin-modifying enzymes as barriers to or facilitators of reprogramming, and demonstrate how modulation of chromatin-modifying enzymes can be exploited to more efficiently generate iPSCs with fewer exogenous transcription factors.

Pathways disrupted in human ALS motor neurons identified through genetic correction of mutant SOD1.

Although many distinct mutations in a variety of genes are known to cause Amyotrophic Lateral Sclerosis (ALS), it remains poorly understood how they selectively impact motor neuron biology and whether they converge on common pathways to cause neuronal degeneration. Here, we have combined reprogramming and stem cell differentiation approaches with genome engineering and RNA sequencing to define the transcriptional and functional changes that are induced in human motor neurons by mutant SOD1. Mutant SOD1 protein induced a transcriptional signature indicative of increased oxidative stress, reduced mitochondrial function, altered subcellular transport, and activation of the ER stress and unfolded protein response pathways. Functional studies demonstrated that these pathways were perturbed in a manner dependent on the SOD1 mutation. Finally, interrogation of stem-cell-derived motor neurons produced from ALS patients harboring a repeat expansion in C9orf72 indicates that at least a subset of these changes are more broadly conserved in ALS.

Tyrosine phosphorylation and regulation of the AMPA receptor by SRC family tyrosine kinases.

Phosphorylation of AMPA receptors is a major mechanism for the regulation of receptor function and underlies several forms of synaptic plasticity in the CNS. Although serine and threonine phosphorylation of AMPA receptors has been well studied, the potential role of tyrosine phosphorylation of AMPA receptors has not been investigated. Here, we show that the GluR2 subunit of AMPA receptors is tyrosine phosphorylated in vitro and in vivo by Src family tyrosine kinases on tyrosine 876 near its C terminus. In addition, GluR agonist treatment of cultured cortical neurons increased phosphorylation of tyrosine 876. The association with GluR2-interacting molecules GRIP1/2 was decreased by tyrosine phosphorylation of GluR2, whereas PICK1 interaction was not influenced. Moreover, mutation of tyrosine 876 eliminated AMPA- and NMDA-induced internalization of the GluR2 subunit. These data indicate that tyrosine phosphorylation of tyrosine 876 on the GluR2 C terminus by Src family tyrosine kinases is important for the regulation of AMPA receptor function and may be important for synaptic plasticity.

Effect of cyclic AMP-dependent protein phosphorylating conditions on the pH-dependent activity of tyrosine hydroxylase from beef and rat striata.

Tyrosine hydroxylase (TH, EC 1.14.16.2) from beef brain striata was purified 23-fold from an extract of an acetone powder. If this enzyme preparation is treated with a cyclic AMP[-dependent protein phosphorylation system, there is a change in the pH dependence of the enzyme activity. The pH optimum at saturating tetrahydrobiopterin (BH4) concentration is shifted from below pH 6 to about pH 6.7. At pH 7, activation is expressed mainly as an increase in Vmax, whereas at pH 6, activation is expressed mainly as a decrease in Km for the pterin cofactor. Further, even with the control enzyme the Km for pterin cofactor declines precipitously as the pH is increased from 6 toward neutrality. Similar data were obtained with G-25 Sephadex-treated rat striatal TH. Experiments in which rat striatal synaptosomes were used demonstrated that the in situ activation of TH by phosphorylating conditions is expressed primarily as an increase in the maximum rate of dopamine synthesis. These results indicate that changes in TH activity caused by cyclic AMP-dependent protein phosphorylation will depend to a large extent on the pH of the TH environment.

Synaptic Incorporation of AMPA Receptors during LTP Is Controlled by a PKC Phosphorylation Site on GluR1

Incorporation of GluR1-containing AMPA receptors into synapses is essential to several forms of neural plasticity, including long-term potentiation (LTP). Numerous signaling pathways that trigger this process have been identified, but the direct modifications of GluR1 that control its incorporation into synapses are unclear. Here, we show that phosphorylation of GluR1 by PKC at a highly conserved serine 818 residue is increased during LTP and critical for LTP expression. GluR1 is phosphorylated by PKC at this site in vitro and in vivo. In addition, acute phosphorylation at GluR1 S818 by PKC, as well as a phosphomimetic mutation, promotes GluR1 synaptic incorporation. Conversely, preventing GluR1 S818 phosphorylation reduces LTP and blocks PKC-driven synaptic incorporation of GluR1. We conclude that the phosphorylation of GluR1 S818 by PKC is a critical event in the plasticity-driven synaptic incorporation of AMPA receptors.

Single-Cell Expression Analyses during Cellular Reprogramming Reveal an Early Stochastic and a Late Hierarchic Phase

During cellular reprogramming, only a small fraction of cells become induced pluripotent stem cells (iPSCs). Previous analyses of gene expression during reprogramming were based on populations of cells, impeding single-cell level identification of reprogramming events. We utilized two gene expression technologies to profile 48 genes in single cells at various stages during the reprogramming process. Analysis of early stages revealed considerable variation in gene expression between cells in contrast to late stages. Expression of Esrrb, Utf1, Lin28, and Dppa2 is a better predictor for cells to progress into iPSCs than expression of the previously suggested reprogramming markers Fbxo15, Fgf4, and Oct4. Stochastic gene expression early in reprogramming is followed by a late hierarchical phase with Sox2 being the upstream factor in a gene expression hierarchy. Finally, downstream factors derived from the late phase, which do not include Oct4, Sox2, Klf4, c-Myc, and Nanog, can activate the pluripotency circuitry.

Neuronal activity regulates remyelination via glutamate signalling to oligodendrocyte progenitors

Myelin regeneration can occur spontaneously in demyelinating diseases such as multiple sclerosis (MS). However, the underlying mechanisms and causes of its frequent failure remain incompletely understood. Here we show, using an in-vivo remyelination model, that demyelinated axons are electrically active and generate de novo synapses with recruited oligodendrocyte progenitor cells (OPCs), which, early after lesion induction, sense neuronal activity by expressing AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid)/kainate receptors. Blocking neuronal activity, axonal vesicular release or AMPA receptors in demyelinated lesions results in reduced remyelination. In the absence of neuronal activity there is a ∼6-fold increase in OPC number within the lesions and a reduced proportion of differentiated oligodendrocytes. These findings reveal that neuronal activity and release of glutamate instruct OPCs to differentiate into new myelinating oligodendrocytes that recover lost function. Co-localization of OPCs with the presynaptic protein VGluT2 in MS lesions implies that this mechanism may provide novel targets to therapeutically enhance remyelination.

The dystonia-associated protein torsinA modulates synaptic vesicle recycling.

The loss of a glutamic acid residue in the AAA-ATPase (ATPases associated with diverse cellular activities) torsinA is responsible for most cases of early onset autosomal dominant primary dystonia. In this study, we found that snapin, which binds SNAP-25 (synaptosome-associated protein of 25,000 Da) and enhances the association of the SNARE complex with synaptotagmin, is an interacting partner for both wild type and mutant torsinA. Snapin co-localized with endogenous torsinA on dense core granules in PC12 cells and was recruited to perinuclear inclusions containing mutant DeltaE-torsinA in neuroblastoma SH-SY5Y cells. In view of these observations, synaptic vesicle recycling was analyzed using the lipophilic dye FM1-43 and an antibody directed against an intravesicular epitope of synaptotagmin I. We found that overexpression of wild type torsinA negatively affects synaptic vesicle endocytosis. Conversely, overexpression of DeltaE-torsinA in neuroblastoma cells increases FM1-43 uptake. Knockdown of snapin and/or torsinA using small interfering RNAs had a similar inhibitory effect on the exo-endocytic process. In addition, down-regulation of torsinA causes the persistence of synaptotagmin I on the plasma membrane, which closely resembles the effect observed by the overexpression of the DeltaE-torsinA mutant. Altogether, these findings suggest that torsinA plays a role together with snapin in regulated exocytosis and that DeltaE-torsinA exerts its pathological effects through a loss of function mechanism. This may affect neuronal uptake of neurotransmitters, such as dopamine, playing a role in the development of dystonic movements.

Neuronal origin of a cerebral amyloid: neurofibrillary tangles of Alzheimer's disease contain the same protein as the amyloid of plaque cores and blood vessels.

The protein component of Alzheimer's disease amyloid [neurofibrillary tangles (NFT), amyloid plaque core and congophilic angiopathy] is an aggregated polypeptide with a subunit mass of 4 kd (the A4 monomer). Based on the degree of N-terminal heterogeneity, the amyloid is first deposited in the neuron, and later in the extracellular space. Using antisera raised against synthetic peptides, we show that the N terminus of A4 (residues 1-11) contains an epitope for neurofibrillary tangles, and the inner region of the molecule (residues 11-23) contains an epitope for plaque cores and vascular amyloid. The non-protein component of the amyloid (aluminum silicate) may form the basis for the deposition or amplification (possible self-replication) of the aggregated amyloid protein. The amyloid of Alzheimer's disease is similar in subunit size, composition but not sequence to the scrapie-associated fibril and its constituent polypeptides. The sequence and composition of NFT are not homologous to those of any of the known components of normal neurofilaments.

Directly reprogrammed fibroblasts show global epigenetic remodeling and widespread tissue contribution.

Ectopic expression of the four transcription factors Oct4, Sox2, c-Myc, and Klf4 is sufficient to confer a pluripotent state upon the fibroblast genome, generating induced pluripotent stem (iPS) cells. It remains unknown if nuclear reprogramming induced by these four factors globally resets epigenetic differences between differentiated and pluripotent cells. Here, using novel selection approaches, we have generated iPS cells from fibroblasts to characterize their epigenetic state. Female iPS cells showed reactivation of a somatically silenced X chromosome and underwent random X inactivation upon differentiation. Genome-wide analysis of two key histone modifications indicated that iPS cells are highly similar to ES cells. Consistent with these observations, iPS cells gave rise to viable high-degree chimeras with contribution to the germline. These data show that transcription factor-induced reprogramming leads to the global reversion of the somatic epigenome into an ES-like state. Our results provide a paradigm for studying the epigenetic modifications that accompany nuclear reprogramming and suggest that abnormal epigenetic reprogramming does not pose a problem for the potential therapeutic applications of iPS cells.

Differential effects of charybdotoxin on the activity of retinal ganglion cells in the dark- and light-adapted mouse retina

Patch-clamp recordings were made from retinal ganglion cells in the mouse retina. Under dark adaptation, blockage of BK(Ca) channels increases the spontaneous excitatory postsynaptic currents (EPSCs) and light-evoked On-EPSCs, while it decreases the light-evoked Off inhibitory postsynaptic currents (IPSCs). However, under light adaptation it decreases the light-evoked On-EPSCs, the spontaneous IPSCs and the light-evoked On- and Off-IPSCs. Blockage of BK(Ca) channels significantly altered the outputs of RGCs by changing their light-evoked responses into a bursting pattern and increasing the light-evoked depolarization of the membrane potentials, while it did not significantly change the peak firing rates of light-evoked responses.

The effects of genetic and pharmacological blockade of the CB1 cannabinoid receptor on anxiety.

The aim of this study was to compare the effects of the genetic and pharmacological disruption of CB1 cannabinoid receptors on the elevated plus-maze test of anxiety. In the first experiment, the behaviour of CB1-knockout mice and wild-type mice was compared. In the second experiment, the cannabinoid antagonist SR141716A (0, 1, and 3 mg/kg) was administered to both CB1-knockout and wild type mice. Untreated CB1-knockout mice showed a reduced exploration of the open arms of the plus-maze apparatus, thus appearing more anxious than the wild-type animals, however no changes in locomotion were noticed. The vehicle-injected CB1-knockout mice from the second experiment also showed increased anxiety as compared with wild types. Surprisingly, the cannabinoid antagonist SR141716A reduced anxiety in both wild type and CB1 knockout mice. Locomotor behaviour was only marginally affected. Recent evidence suggests the existence of a novel cannabinoid receptor in the brain. It has also been shown that SR141716A binds to both the CB1 and the putative novel receptor. The data presented here supports these findings, as the cannabinoid receptor antagonist affected anxiety in both wild type and CB1-knockout mice. Tentatively, it may be suggested that the discrepancy between the effects of the genetic and pharmacological blockade of the CB1 receptor suggests that the novel receptor plays a role in anxiety.

Enhancement of proteasome function by PA28α overexpression protects against oxidative stress.

The principal function of the proteasome is targeted degradation of intracellular proteins. Proteasome dysfunction has been observed in experimental cardiomyopathies and implicated in human congestive heart failure. Measures to enhance proteasome proteolytic function are currently lacking but would be beneficial in testing the pathogenic role of proteasome dysfunction and could have significant therapeutic potential. The association of proteasome activator 28 (PA28) with the 20S proteasome may play a role in antigen processing. It is unclear, however, whether the PA28 plays any important role outside of antigen presentation, although up-regulation of PA28 has been observed in certain types of cardiomyopathy. Here, we show that PA28α overexpression (PA28αOE) stabilized PA28β, increased 11S proteasomes, and enhanced the degradation of a previously validated proteasome surrogate substrate (GFPu) in cultured neonatal rat cardiomyocytes. PA28αOE significantly attenuated H(2)O(2)-induced increases in the protein carbonyls and markedly suppressed apoptosis in cultured cardiomyocytes under basal conditions or when stressed by H(2)O(2). We conclude that PA28αOE is sufficient to up-regulate 11S proteasomes, enhance proteasome-mediated removal of misfolded and oxidized proteins, and protect against oxidative stress in cardiomyocytes, providing a highly sought means to increase proteasomal degradation of abnormal cellular proteins.

Chk1 inhibits replication factory activation but allows dormant origin firing in existing factories

Replication origins are licensed by loading MCM2-7 hexamers before entry into S phase. However, only ∼10% of licensed origins are normally used in S phase, with the others remaining dormant. When fork progression is inhibited, dormant origins initiate nearby to ensure that all of the DNA is eventually replicated. In apparent contrast, replicative stress activates ataxia telangiectasia and rad-3-related (ATR) and Chk1 checkpoint kinases that inhibit origin firing. In this study, we show that at low levels of replication stress, ATR/Chk1 predominantly suppresses origin initiation by inhibiting the activation of new replication factories, thereby reducing the number of active factories. At the same time, inhibition of replication fork progression allows dormant origins to initiate within existing replication factories. The inhibition of new factory activation by ATR/Chk1 therefore redirects replication toward active factories where forks are inhibited and away from regions that have yet to start replication. This minimizes the deleterious consequences of fork stalling and prevents similar problems from arising in unreplicated regions of the genome.

Autophagy and mTORC1 regulate the stochastic phase of somatic cell reprogramming

We describe robust induction of autophagy during the reprogramming of mouse fibroblasts to induced pluripotent stem cells by four reprogramming factors (Sox2, Oct4, Klf4 and c-Myc), henceforth 4F. This process occurs independently of p53 activation, and is mediated by the synergistic downregulation of mechanistic target of rapamycin complex 1 (mTORC1) and the induction of autophagy-related genes. The 4F coordinately repress mTORC1, but bifurcate in their regulation of autophagy-related genes, with Klf4 and c-Myc inducing them but Sox2 and Oct4 inhibiting them. On one hand, inhibition of mTORC1 facilitates reprogramming by promoting cell reshaping (mitochondrial remodelling and cell size reduction). On the other hand, mTORC1 paradoxically impairs reprogramming by triggering autophagy. Autophagy does not participate in cell reshaping in reprogramming but instead degrades p62, whose accumulation in autophagy-deficient cells facilitates reprogramming. Our results thus reveal a complex signalling network involving mTORC1 inhibition and autophagy induction in the early phase of reprogramming, whose delicate balance ultimately determines reprogramming efficiency.

Tyrosine phosphorylation regulates the endocytosis and surface expression of GluN3A-containing NMDA receptors.

Selective control of receptor trafficking provides a mechanism for remodeling the receptor composition of excitatory synapses, and thus supports synaptic transmission, plasticity, and development. GluN3A (formerly NR3A) is a nonconventional member of the NMDA receptor (NMDAR) subunit family, which endows NMDAR channels with low calcium permeability and reduced magnesium sensitivity compared with NMDARs comprising only GluN1 and GluN2 subunits. Because of these special properties, GluN3A subunits act as a molecular brake to limit the plasticity and maturation of excitatory synapses, pointing toward GluN3A removal as a critical step in the development of neuronal circuitry. However, the molecular signals mediating GluN3A endocytic removal remain unclear. Here we define a novel endocytic motif (YWL), which is located within the cytoplasmic C-terminal tail of GluN3A and mediates its binding to the clathrin adaptor AP2. Alanine mutations within the GluN3A endocytic motif inhibited clathrin-dependent internalization and led to accumulation of GluN3A-containing NMDARs at the cell surface, whereas mimicking phosphorylation of the tyrosine residue promoted internalization and reduced cell-surface expression as shown by immunocytochemical and electrophysiological approaches in recombinant systems and rat neurons in primary culture. We further demonstrate that the tyrosine residue is phosphorylated by Src family kinases, and that Src-activation limits surface GluN3A expression in neurons. Together, our results identify a new molecular signal for GluN3A internalization that couples the functional surface expression of GluN3A-containing receptors to the phosphorylation state of GluN3A subunits, and provides a molecular framework for the regulation of NMDAR subunit composition with implications for synaptic plasticity and neurodevelopment.

Reprogramming factor expression initiates widespread targeted chromatin remodeling.

Despite rapid progress in characterizing transcription factor-driven reprogramming of somatic cells to an induced pluripotent stem cell (iPSC) state, many mechanistic questions still remain. To gain insight into the earliest events in the reprogramming process, we systematically analyzed the transcriptional and epigenetic changes that occur during early factor induction after discrete numbers of divisions. We observed rapid, genome-wide changes in the euchromatic histone modification, H3K4me2, at more than a thousand loci including large subsets of pluripotency-related or developmentally regulated gene promoters and enhancers. In contrast, patterns of the repressive H3K27me3 modification remained largely unchanged except for focused depletion specifically at positions where H3K4 methylation is gained. These chromatin regulatory events precede transcriptional changes within the corresponding loci. Our data provide evidence for an early, organized, and population-wide epigenetic response to ectopic reprogramming factors that clarify the temporal order through which somatic identity is reset during reprogramming.

Modelling and rescuing neurodevelopmental defect of Down syndrome using induced pluripotent stem cells from monozygotic twins discordant for trisomy 21

Down syndrome (trisomy 21) is the most common viable chromosomal disorder with intellectual impairment and several other developmental abnormalities. Here, we report the generation and characterization of induced pluripotent stem cells (iPSCs) derived from monozygotic twins discordant for trisomy 21 in order to eliminate the effects of the variability of genomic background. The alterations observed by genetic analysis at the iPSC level and at first approximation in early development illustrate the developmental disease transcriptional signature of Down syndrome. Moreover, we observed an abnormal neural differentiation of Down syndrome iPSCs in vivo when formed teratoma in NOD-SCID mice, and in vitro when differentiated into neuroprogenitors and neurons. These defects were associated with changes in the architecture and density of neurons, astroglial and oligodendroglial cells together with misexpression of genes involved in neurogenesis, lineage specification and differentiation. Furthermore, we provide novel evidence that dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A (DYRK1A) on chromosome 21 likely contributes to these defects. Importantly, we found that targeting DYRK1A pharmacologically or by shRNA results in a considerable correction of these defects.

Direct conversion of patient fibroblasts demonstrates non-cell autonomous toxicity of astrocytes to motor neurons in familial and sporadic ALS.

Amyotrophic lateral sclerosis (ALS) causes motor neuron degeneration, paralysis, and death. Accurate disease modeling, identifying disease mechanisms, and developing therapeutics is urgently needed. We previously reported motor neuron toxicity through postmortem ALS spinal cord-derived astrocytes. However, these cells can only be harvested after death, and their expansion is limited. We now report a rapid, highly reproducible method to convert adult human fibroblasts from living ALS patients to induced neuronal progenitor cells and subsequent differentiation into astrocytes (i-astrocytes). Non-cell autonomous toxicity to motor neurons is found following coculture of i-astrocytes from familial ALS patients with mutation in superoxide dismutase or hexanucleotide expansion in C9orf72 (ORF 72 on chromosome 9) the two most frequent causes of ALS. Remarkably, i-astrocytes from sporadic ALS patients are as toxic as those with causative mutations, suggesting a common mechanism. Easy production and expansion of i-astrocytes now enables rapid disease modeling and high-throughput drug screening to alleviate astrocyte-derived toxicity.

Proteomic and genomic approaches reveal critical functions of H3K9 methylation and Heterochromatin Protein-1γ in reprogramming to pluripotency

Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) involves a marked reorganization of chromatin. To identify post-translational histone modifications that change in global abundance during this process, we have applied a quantitative mass-spectrometry-based approach. We found that iPSCs, compared with both the starting fibroblasts and a late reprogramming intermediate (pre-iPSCs), are enriched for histone modifications associated with active chromatin, and depleted for marks of transcriptional elongation and a subset of repressive modifications including H3K9me2/me3. Dissecting the contribution of H3K9 methylation to reprogramming, we show that the H3K9 methyltransferases Ehmt1, Ehmt2 and Setdb1 regulate global H3K9me2/me3 levels and that their depletion increases iPSC formation from both fibroblasts and pre-iPSCs. Similarly, we find that inhibition of heterochromatin protein-1γ (Cbx3), a protein known to recognize H3K9 methylation, enhances reprogramming. Genome-wide location analysis revealed that Cbx3 predominantly binds active genes in both pre-iPSCs and pluripotent cells but with a strikingly different distribution: in pre-iPSCs, but not in embryonic stem cells, Cbx3 associates with active transcriptional start sites, suggesting a developmentally regulated role for Cbx3 in transcriptional activation. Despite largely non-overlapping functions and the predominant association of Cbx3 with active transcription, the H3K9 methyltransferases and Cbx3 both inhibit reprogramming by repressing the pluripotency factor Nanog. Together, our findings demonstrate that Cbx3 and H3K9 methylation restrict late reprogramming events, and suggest that a marked change in global chromatin character constitutes an epigenetic roadblock for reprogramming.

A rare mutation in UNC5C predisposes to late-onset Alzheimer's disease and increases neuronal cell death

We have identified a rare coding mutation, T835M (rs137875858), in the UNC5C netrin receptor gene that segregated with disease in an autosomal dominant pattern in two families enriched for late-onset Alzheimer's disease and that was associated with disease across four large case-control cohorts (odds ratio = 2.15, Pmeta = 0.0095). T835M alters a conserved residue in the hinge region of UNC5C, and in vitro studies demonstrate that this mutation leads to increased cell death in human HEK293T cells and in rodent neurons. Furthermore, neurons expressing T835M UNC5C are more susceptible to cell death from multiple neurotoxic stimuli, including β-amyloid (Aβ), glutamate and staurosporine. On the basis of these data and the enriched hippocampal expression of UNC5C in the adult nervous system, we propose that one possible mechanism in which T835M UNC5C contributes to the risk of Alzheimer's disease is by increasing susceptibility to neuronal cell death, particularly in vulnerable regions of the Alzheimer's disease brain.

OPA1 gene therapy prevents retinal ganglion cell loss in a Dominant Optic Atrophy mouse model

Dominant optic atrophy (DOA) is a rare progressive and irreversible blinding disease which is one of the most frequent forms of hereditary optic neuropathy. DOA is mainly caused by dominant mutation in the OPA1 gene encoding a large mitochondrial GTPase with crucial roles in membrane dynamics and cell survival. Hereditary optic neuropathies are commonly characterized by the degeneration of retinal ganglion cells, leading to the optic nerve atrophy and the progressive loss of visual acuity. Up to now, despite increasing advances in the understanding of the pathological mechanisms, DOA remains intractable. Here, we tested the efficiency of gene therapy on a genetically-modified mouse model reproducing DOA vision loss. We performed intravitreal injections of an Adeno-Associated Virus carrying the human OPA1 cDNA under the control of the cytomegalovirus promotor. Our results provide the first evidence that gene therapy is efficient on a mouse model of DOA as the wild-type OPA1 expression is able to alleviate the OPA1-induced retinal ganglion cell degeneration, the hallmark of the disease. These results displayed encouraging effects of gene therapy for Dominant Optic Atrophy, fostering future investigations aiming at clinical trials in patients.

In vivo effect of chronic hypoxia on the neurochemical profile of the developing rat hippocampus.

The cognitive deficits observed in children with cyanotic congenital heart disease suggest involvement of the developing hippocampus. Chronic postnatal hypoxia present during infancy in these children may play a role in these impairments. To understand the biochemical mechanisms of hippocampal injury in chronic hypoxia, a neurochemical profile consisting of 15 metabolite concentrations and 2 metabolite ratios in the hippocampus was evaluated in a rat model of chronic postnatal hypoxia using in vivo 1H NMR spectroscopy at 9.4 T. Chronic hypoxia was induced by continuously exposing rats (n = 23) to 10% O2 from postnatal day (P) 3 to P28. Fifteen metabolites were quantified from a volume of 9-11 microl centered on the left hippocampus on P14, P21, and P28 and were compared with normoxic controls (n = 14). The developmental trajectory of neurochemicals in chronic hypoxia was similar to that seen in normoxia. However, chronic hypoxia had an effect on the concentrations of the following neurochemicals: aspartate, creatine, phosphocreatine, GABA, glutamate, glutamine, glutathione, myoinositol, N-acetylaspartate (NAA), phosphorylethanolamine, and phosphocreatine/creatine (PCr/Cr) and glutamate/glutamine (Glu/Gln) ratios (P < 0.001 each, except glutamate, P = 0.04). The increased PCr/Cr ratio is consistent with decreased brain energy consumption. Given the well-established link between excitatory neurotransmission and brain energy metabolism, we postulate that elevated glutamate, Glu/Gln ratio, and GABA indicate suppressed excitatory neurotransmission in an energy-limited environment. Decreased NAA and phosphorylethanolamine suggest reduced neuronal integrity and phospholipid metabolism. The altered hippocampal neurochemistry during its development may underlie some of the cognitive deficits present in human infants at risk of chronic hypoxia.

p63 Regulates adult neural precursor and newly born neuron survival to control hippocampal-dependent Behavior.

The molecular mechanisms that regulate adult neural precursor cell (NPC) survival, and thus maintain adult neurogenesis, are not well defined. Here, we investigate the role of p63, a p53 family member, in adult NPC function in mice. Conditional ablation of p63 in adult NPCs or p63 haploinsufficiency led to reduced numbers of NPCs and newborn neurons in the neurogenic zones of the hippocampus and lateral ventricles and in the olfactory bulb. These reductions were attributable to enhanced apoptosis of NPCs and newborn neurons and were rescued by inhibition of caspase activity, p53, or the p53 apoptotic effector PUMA (p53-upregulated modulator of apoptosis). Moreover, these cellular deficits were functionally important because they led to perturbations in hippocampus-dependent memory formation. These results indicate that p63 regulates the numbers of adult NPCs and adult-born neurons as well as neural stem cell-dependent cognitive functions, and that it does so, at least in part, by inhibiting p53-dependent cell death.

Antagonistic Controls of Autophagy and Glycogen Accumulation by Snf1p, the Yeast Homolog of AMP-Activated Protein Kinase, and the Cyclin-Dependent

In the yeast Saccharomyces cerevisiae, glycogen is accumulated as a carbohydrate reserve when cells are deprived of nutrients. Yeast mutated in SNF1, a gene encoding a protein kinase required for glucose derepression, has diminished glycogen accumulation and concomitant inactivation of glycogen synthase. Restoration of synthesis in an snf1 strain results only in transient glycogen accumulation, implying the existence of other SNF1-dependent controls of glycogen storage. A genetic screen revealed that two genes involved in autophagy, APG1 and APG13, may be regulated by SNF1. Increased autophagic activity was observed in wild-type cells entering the stationary phase, but this induction was impaired in an snf1 strain. Mutants defective for autophagy were able to synthesize glycogen upon approaching the stationary phase, but were unable to maintain their glycogen stores, because subsequent synthesis was impaired and degradation by phosphorylase, Gph1p, was enhanced. Thus, deletion of GPH1 partially reversed the loss of glycogen accumulation in autophagy mutants. Loss of the vacuolar glucosidase, SGA1, also protected glycogen stores, but only very late in the stationary phase. Gph1p and Sga1p may therefore degrade physically distinct pools of glycogen. Pho85p is a cyclin-dependent protein kinase that antagonizes SNF1 control of glycogen synthesis. Induction of autophagy in pho85 mutants entering the stationary phase was exaggerated compared to the level in wild-type cells, but was blocked in apg1 pho85 mutants. We propose that Snf1p and Pho85p are, respectively, positive and negative regulators of autophagy, probably via Apg1 and/or Apg13. Defective glycogen storage in snf1 cells can be attributed to both defective synthesis upon entry into stationary phase and impaired maintenance of glycogen levels caused by the lack of autophagy.

Histone deacetylases (HDACs) in frontotemporal lobar degeneration.

AIMS Frontotemporal lobar degeneration (FTLD) is clinically and pathologically heterogeneous. Although associated with variations in MAPT, GRN and C9ORF72, the pathogenesis of these, and of other nongenetic, forms of FTLD, remains unknown. Epigenetic factors such as histone regulation by histone deacetylases (HDAC) may play a role in the dysregulation of transcriptional activity, thought to underpin the neurodegenerative process. METHODS The distribution and intensity of HDACs 4, 5 and 6 was assessed semi-quantitatively in immunostained sections of temporal cortex with hippocampus, and cerebellum, from 33 pathologically confirmed cases of FTLD and 27 controls. RESULTS We found a significantly greater intensity of cytoplasmic immunostaining for HDAC4 and HDAC6 in granule cells of the dentate gyrus in cases of FTLD overall compared with controls, and specifically in cases of FTLD tau-Picks compared with FTLD tau-MAPT and controls. No differences were noted between FTLD-TDP subtypes, or between the different genetic and nongenetic forms of FTLD. No changes were seen in HDAC5 in any FTLD or control cases. CONCLUSIONS Dysregulation of HDAC4 and/or HDAC6 could play a role in the pathogenesis of FTLD-tau associated with Pick bodies, although their lack of immunostaining implies that such changes do not contribute directly to the formation of Pick bodies.

Increased atherosclerosis in mice reconstituted with apolipoprotein E null macrophages.

Macrophage-derived foam cells express apolipoprotein E (apoE) abundantly in atherosclerotic lesions. To examine the physiologic role of apoE secretion by the macrophage in atherogenesis, bone marrow transplantation was used to reconstitute C57BL/6 mice with macrophages that were either null or wild type for the apoE gene. After 13 weeks on an atherogenic diet, C57BL/6 mice reconstituted with apoE null marrow developed 10-fold more atherosclerosis than controls in the absence of significant differences in serum cholesterol levels or lipoprotein profiles. ApoE expression was absent in the macrophage-derived foam cells of C57BL/6 mice reconstituted with apoE null marrow. Thus, lack of apoE expression by the macrophage promotes foam cell formation. These data support a protective role for apoE expression by the macrophage in early atherogenesis.

Optogenetics reveal delayed afferent synaptogenesis on grafted human-induced pluripotent stem cell-derived neural progenitors.

Reprogramming of somatic cells into pluripotency stem cell state has opened new opportunities in cell replacement therapy and disease modeling in a number of neurological disorders. It still remains unknown, however, to what degree the grafted human-induced pluripotent stem cells (hiPSCs) differentiate into a functional neuronal phenotype and if they integrate into the host circuitry. Here, we present a detailed characterization of the functional properties and synaptic integration of hiPSC-derived neurons grafted in an in vitro model of hyperexcitable epileptic tissue, namely organotypic hippocampal slice cultures (OHSCs), and in adult rats in vivo. The hiPSCs were first differentiated into long-term self-renewing neuroepithelial stem (lt-NES) cells, which are known to form primarily GABAergic neurons. When differentiated in OHSCs for 6 weeks, lt-NES cell-derived neurons displayed neuronal properties such as tetrodotoxin-sensitive sodium currents and action potentials (APs), as well as both spontaneous and evoked postsynaptic currents, indicating functional afferent synaptic inputs. The grafted cells had a distinct electrophysiological profile compared to host cells in the OHSCs with higher input resistance, lower resting membrane potential, and APs with lower amplitude and longer duration. To investigate the origin of synaptic afferents to the grafted lt-NES cell-derived neurons, the host neurons were transduced with Channelrhodopsin-2 (ChR2) and optogenetically activated by blue light. Simultaneous recordings of synaptic currents in grafted lt-NES cell-derived neurons using whole-cell patch-clamp technique at 6 weeks after grafting revealed limited synaptic connections from host neurons. Longer differentiation times, up to 24 weeks after grafting in vivo, revealed more mature intrinsic properties and extensive synaptic afferents from host neurons to the lt-NES cell-derived neurons, suggesting that these cells require extended time for differentiation/maturation and synaptogenesis. However, even at this later time point, the grafted cells maintained a higher input resistance. These data indicate that grafted lt-NES cell-derived neurons receive ample afferent input from the host brain. Since the lt-NES cells used in this study show a strong propensity for GABAergic differentiation, the host-to-graft synaptic afferents may facilitate inhibitory neurotransmitter release, and normalize hyperexcitable neuronal networks in brain diseases, for example, such as epilepsy.

Disruption of tumor necrosis factor-alpha gene diminishes the development of atherosclerosis in ApoE-deficient mice.

Inflammatory cytokines, including tumor necrosis factor-alpha (TNF-alpha) have been implicated in atherogenesis. However, the precise role of TNF-alpha in atherogenesis is still unclear. To examine the effect of TNF-alpha on atherogenesis, we generated compound-deficient mice in apolipoprotein E (apoE) and TNF-alpha (apoE-/-/TNF-alpha-/-) and compared them with apoE-/- mice. Although serum total cholesterol levels were markedly elevated in both apoE-/-/TNF-alpha-/- and apoE-/- mice compared to wild-type mice, no differences were observed between apoE-/-/TNF-alpha-/- and apoE-/- mice. The atherosclerotic plaque area in the aortic luminal surface of apoE-/-/TNF-alpha-/- mice (n=8, 3.1+/-0.4%) was significantly smaller than that of apoE-/- mice (n=7, 4.7+/-0.4%, p<0.001) despite the lack of difference in serum cholesterol levels. The atherosclerotic lesion size in the aortic sinus of apoE-/-/TNF-alpha-/- mice (n=10, 5.1+/-0.3 x 10(5)microm(2)) was also significantly smaller than that of apoE-/- mice (n=11, 7.0+/-0.3 x 10(5)microm(2), p<0.0001). RT-PCR analysis indicated that the expression levels of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and monocyte chemoattractant protein-1 (MCP-1) were significantly higher in apoE-/- than apoE-/-/TNF-alpha-/- mice. Macrophages from apoE(-/-) mice showed higher uptake level of oxidized LDL and increased expression level of scavenger receptor class A (SRA) compared to those from apoE-/-/TNF-alpha-/- mice. These results indicate that TNF-alpha plays an atherogenic role by upregulating the expressions of ICAM-1, VCAM-1 and MCP-1 in the vascular wall, and by inducing SRA expression and oxidized LDL uptake in macrophages.

An Expanded Oct4 Interaction Network: Implications for Stem Cell Biology, Development, and Disease

The transcription factor Oct4 is key in embryonic stem cell identity and reprogramming. Insight into its partners should illuminate how the pluripotent state is established and regulated. Here, we identify a considerably expanded set of Oct4-binding proteins in mouse embryonic stem cells. We find that Oct4 associates with a varied set of proteins including regulators of gene expression and modulators of Oct4 function. Half of its partners are transcriptionally regulated by Oct4 itself or other stem cell transcription factors, whereas one-third display a significant change in expression upon cell differentiation. The majority of Oct4-associated proteins studied to date show an early lethal phenotype when mutated. A fraction of the human orthologs is associated with inherited developmental disorders or causative of cancer. The Oct4 interactome provides a resource for dissecting mechanisms of Oct4 function, enlightening the basis of pluripotency and development, and identifying potential additional reprogramming factors.

Chronic variable stress activates hematopoietic stem cells

Exposure to psychosocial stress is a risk factor for many diseases, including atherosclerosis. Although incompletely understood, interaction between the psyche and the immune system provides one potential mechanism linking stress and disease inception and progression. Known cross-talk between the brain and immune system includes the hypothalamic-pituitary-adrenal axis, which centrally drives glucocorticoid production in the adrenal cortex, and the sympathetic-adrenal-medullary axis, which controls stress-induced catecholamine release in support of the fight-or-flight reflex. It remains unknown, however, whether chronic stress changes hematopoietic stem cell activity. Here we show that stress increases proliferation of these most primitive hematopoietic progenitors, giving rise to higher levels of disease-promoting inflammatory leukocytes. We found that chronic stress induced monocytosis and neutrophilia in humans. While investigating the source of leukocytosis in mice, we discovered that stress activates upstream hematopoietic stem cells. Under conditions of chronic variable stress in mice, sympathetic nerve fibers released surplus noradrenaline, which signaled bone marrow niche cells to decrease CXCL12 levels through the β3-adrenergic receptor. Consequently, hematopoietic stem cell proliferation was elevated, leading to an increased output of neutrophils and inflammatory monocytes. When atherosclerosis-prone Apoe(-/-) mice were subjected to chronic stress, accelerated hematopoiesis promoted plaque features associated with vulnerable lesions that cause myocardial infarction and stroke in humans.

Neuronal replacement from endogenous precursors in the adult brain after stroke

In the adult brain, new neurons are continuously generated in the subventricular zone and dentate gyrus, but it is unknown whether these neurons can replace those lost following damage or disease. Here we show that stroke, caused by transient middle cerebral artery occlusion in adult rats, leads to a marked increase of cell proliferation in the subventricular zone. Stroke-generated new neurons, as well as neuroblasts probably already formed before the insult, migrate into the severely damaged area of the striatum, where they express markers of developing and mature, striatal medium-sized spiny neurons. Thus, stroke induces differentiation of new neurons into the phenotype of most of the neurons destroyed by the ischemic lesion. Here we show that the adult brain has the capacity for self-repair after insults causing extensive neuronal death. If the new neurons are functional and their formation can be stimulated, a novel therapeutic strategy might be developed for stroke in humans.

Apolipoprotein E4 as a predictor of outcomes in pediatric mild traumatic brain injury.

The epsilon4 allele of the apolipoprotein E (APOE) gene has been linked to negative outcomes among adults with traumatic brain injury (TBI) across the spectrum of severity, with preliminary evidence suggesting a similar pattern among children. This study investigated the relationship of the APOE epsilon4 allele to outcomes in children with mild TBI. Participants in this prospective, longitudinal study included 99 children with mild TBI between the ages of 8 and 15 recruited from consecutive admissions to Emergency Departments at two large children's hospitals. Outcomes were assessed acutely in the Emergency Department and at follow-ups at 2 weeks, 3 months, and 12 months post-injury. Among the 99 participants, 28 had at least one epsilon4 allele. Children with and without an epsilon4 allele did not differ demographically. Children with an epsilon4 allele were significantly more likely than those without an epsilon4 allele to have a Glasgow Coma Scale score of less than 15, but the groups did not differ on any other measures of injury severity. Those with an epsilon4 allele exhibited better performance than children without an epsilon4 allele on a test of constructional skill, but the groups did not differ on any other neuropsychological tests. Children with and without an epsilon4 allele also did not differ on measures of post-concussive symptoms. Overall, the findings suggest that the APOE epsilon4 allele is not consistently related to the outcomes of mild TBI in children.

Kynurenic Acid Is a Nutritional Cue that Enables Behavioral Plasticity

The kynurenine pathway of tryptophan metabolism is involved in the pathogenesis of several brain diseases, but its physiological functions remain unclear. We report that kynurenic acid, a metabolite in this pathway, functions as a regulator of food-dependent behavioral plasticity in C. elegans. The experience of fasting in C. elegans alters a variety of behaviors, including feeding rate, when food is encountered post-fast. Levels of neurally produced kynurenic acid are depleted by fasting, leading to activation of NMDA-receptor-expressing interneurons and initiation of a neuropeptide-y-like signaling axis that promotes elevated feeding through enhanced serotonin release when animals re-encounter food. Upon refeeding, kynurenic acid levels are eventually replenished, ending the elevated feeding period. Because tryptophan is an essential amino acid, these findings suggest that a physiological role of kynurenic acid is in directly linking metabolism to activity of NMDA and serotonergic circuits, which regulate a broad range of behaviors and physiologies.

Molecular interplay of the noncoding RNA ANRIL and methylated histone H3 lysine 27 by polycomb CBX7 in transcriptional silencing of INK4a.

Expression of the INK4b/ARF/INK4a tumor suppressor locus in normal and cancerous cell growth is controlled by methylation of histone H3 at lysine 27 (H3K27me) as directed by the Polycomb group proteins. The antisense noncoding RNA ANRIL of the INK4b/ARF/INK4a locus is also important for expression of the protein-coding genes in cis, but its mechanism has remained elusive. Here we report that chromobox 7 (CBX7) within the polycomb repressive complex 1 binds to ANRIL, and both CBX7 and ANRIL are found at elevated levels in prostate cancer tissues. In concert with H3K27me recognition, binding to RNA contributes to CBX7 function, and disruption of either interaction impacts the ability of CBX7 to repress the INK4b/ARF/INK4a locus and control senescence. Structure-guided analysis reveals the molecular interplay between noncoding RNA and H3K27me as mediated by the conserved chromodomain. Our study suggests a mechanism by which noncoding RNA participates directly in epigenetic transcriptional repression.

Induction of pluripotent stem cells from mouse embryonic fibroblasts by Oct4 and Klf4 with small-molecule compounds.

Somatic cells can be induced into pluripotent stem cells (iPSCs) with a combination of four transcription factors, Oct4/Sox2/Klf4/c-Myc or Oct4/Sox2/Nanog/LIN28. This provides an enabling platform to obtain patient-specific cells for various therapeutic and research applications. However, several problems remain for this approach to be therapeutically relevant due to drawbacks associated with efficiency and viral genome integration. Recently, it was shown that neural progenitor cells (NPCs) transduced with Oct4/Klf4 can be reprogrammed into iPSCs. However, NPCs express Sox2 endogenously, possibly facilitating reprogramming in the absence of exogenous Sox2. In this study, we identified a small-molecule combination, BIX-01294 and BayK8644, that enables reprogramming of Oct4/Klf4-transduced mouse embryonic fibroblasts, which do not endogenously express the factors essential for reprogramming. This study demonstrates that small molecules identified through a phenotypic screen can compensate for viral transduction of critical factors, such as Sox2, and improve reprogramming efficiency.

Neurological outcomes of animal models of uterine artery ligation and relevance to human intrauterine growth restriction: a systematic review

AIM This review explores the molecular, neurological, and behavioural outcomes in animal models of uterine artery ligation. We analyse the relevance of this type of model to the pathological and functional phenotypes that are consistent with cerebral palsy and its developmental comorbidities in humans. METHOD A literature search of the PubMed database was conducted for research using the uterine artery ligation model published between 1990 and 2013. From the studies included, any relevant neuroanatomical and behavioural deficits were then summarized from each document and used for further analysis. RESULTS There were 25 papers that met the criteria included for review, and several outcomes were summarized from the results of these papers. Fetuses with growth restriction demonstrated a gradient of reduced body weight with a relative sparing of brain mass. There was a significant reduction in the size of the somatosensory cortex, hippocampus, and corpus callosum. The motor cortex appeared to be spared of identifiable deficits. Apoptotic proteins were upregulated, while those important to neuronal survival, growth, and differentiation were downregulated. Neuronal apoptosis and astrogliosis occurred diffusely throughout the brain regions. White matter injury involved oligodendrocyte precursor maturation arrest, hypomyelination, and an aberrant organization of existing myelin. Animals with growth restriction demonstrated deficits in gait, memory, object recognition, and spatial processing. INTERPRETATION This review concludes that neuronal death, white matter injury, motor abnormalities, and cognitive deficits are important outcomes of uterine artery ligation in animal models. Therefore, this is a clinically relevant type of model, as these findings resemble deficits in human cerebral palsy.

The essential Schizosaccharomyces pombe Pfh1 DNA helicase promotes fork movement past G-quadruplex motifs to prevent DNA damage

BACKGROUND G-quadruplexes (G4s) are stable non-canonical DNA secondary structures consisting of stacked arrays of four guanines, each held together by Hoogsteen hydrogen bonds. Sequences with the ability to form these structures in vitro, G4 motifs, are found throughout bacterial and eukaryotic genomes. The budding yeast Pif1 DNA helicase, as well as several bacterial Pif1 family helicases, unwind G4 structures robustly in vitro and suppress G4-induced DNA damage in S. cerevisiae in vivo. RESULTS We determined the genomic distribution and evolutionary conservation of G4 motifs in four fission yeast species and investigated the relationship between G4 motifs and Pfh1, the sole S. pombe Pif1 family helicase. Using chromatin immunoprecipitation combined with deep sequencing, we found that many G4 motifs in the S. pombe genome were associated with Pfh1. Cells depleted of Pfh1 had increased fork pausing and DNA damage near G4 motifs, as indicated by high DNA polymerase occupancy and phosphorylated histone H2A, respectively. In general, G4 motifs were underrepresented in genes. However, Pfh1-associated G4 motifs were located on the transcribed strand of highly transcribed genes significantly more often than expected, suggesting that Pfh1 has a function in replication or transcription at these sites. CONCLUSIONS In the absence of functional Pfh1, unresolved G4 structures cause fork pausing and DNA damage of the sort associated with human tumors.

AP-1/sigma1B-adaptin mediates endosomal synaptic vesicle recycling, learning and memory.

Synaptic vesicle recycling involves AP-2/clathrin-mediated endocytosis, but it is not known whether the endosomal pathway is also required. Mice deficient in the tissue-specific AP-1-sigma1B complex have impaired synaptic vesicle recycling in hippocampal synapses. The ubiquitously expressed AP-1-sigma1A complex mediates protein sorting between the trans-Golgi network and early endosomes. Vertebrates express three sigma1 subunit isoforms: A, B and C. The expressions of sigma1A and sigma1B are highest in the brain. Synaptic vesicle reformation in cultured neurons from sigma1B-deficient mice is reduced upon stimulation, and large endosomal intermediates accumulate. The sigma1B-deficient mice have reduced motor coordination and severely impaired long-term spatial memory. These data reveal a molecular mechanism for a severe human X-chromosome-linked mental retardation.

Wnt7a signaling promotes dendritic spine growth and synaptic strength through Ca²⁺/Calmodulin-dependent protein kinase II.

The balance between excitatory and inhibitory synapses is crucial for normal brain function. Wnt proteins stimulate synapse formation by increasing synaptic assembly. However, it is unclear whether Wnt signaling differentially regulates the formation of excitatory and inhibitory synapses. Here, we demonstrate that Wnt7a preferentially stimulates excitatory synapse formation and function. In hippocampal neurons, Wnt7a increases the number of excitatory synapses, whereas inhibitory synapses are unaffected. Wnt7a or postsynaptic expression of Dishevelled-1 (Dvl1), a core Wnt signaling component, increases the frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs), but not miniature inhibitory postsynaptic currents (mIPSCs). Wnt7a increases the density and maturity of dendritic spines, whereas Wnt7a-Dvl1-deficient mice exhibit defects in spine morphogenesis and mossy fiber-CA3 synaptic transmission in the hippocampus. Using a postsynaptic reporter for Ca(2+)/Calmodulin-dependent protein kinase II (CaMKII) activity, we demonstrate that Wnt7a rapidly activates CaMKII in spines. Importantly, CaMKII inhibition abolishes the effects of Wnt7a on spine growth and excitatory synaptic strength. These data indicate that Wnt7a signaling is critical to regulate spine growth and synaptic strength through the local activation of CaMKII at dendritic spines. Therefore, aberrant Wnt7a signaling may contribute to neurological disorders in which excitatory signaling is disrupted.

Interneuronal DISC1 regulates NRG1-ErbB4 signalling and excitatory-inhibitory synapse formation in the mature cortex.

Neuregulin-1 (NRG1) and its receptor ErbB4 influence several processes of neurodevelopment, but the mechanisms regulating this signalling in the mature brain are not well known. DISC1 is a multifunctional scaffold protein that mediates many cellular processes. Here we present a functional relationship between DISC1 and NRG1-ErbB4 signalling in mature cortical interneurons. By cell type-specific gene modulation in vitro and in vivo including in a mutant DISC1 mouse model, we demonstrate that DISC1 inhibits NRG1-induced ErbB4 activation and signalling. This effect is likely mediated by competitive inhibition of binding of ErbB4 to PSD95. Finally, we show that interneuronal DISC1 affects NRG1-ErbB4-mediated phenotypes in the fast spiking interneuron-pyramidal neuron circuit. Post-mortem brain analyses and some genetic studies have reported interneuronal deficits and involvement of the DISC1, NRG1 and ErbB4 genes in schizophrenia, respectively. Our results suggest a mechanism by which cross-talk between DISC1 and NRG1-ErbB4 signalling may contribute to these deficits.

Nuclear HuR accumulation through phosphorylation by Cdk1.

A predominantly nuclear RNA-binding protein, HuR translocates to the cytoplasm in response to stress and proliferative signals, where it stabilizes or modulates the translation of target mRNAs. Here, we present evidence that HuR phosphorylation at S202 by the G2-phase kinase Cdk1 influences its subcellular distribution. HuR was specifically phosphorylated in synchronous G2-phase cultures; its cytoplasmic levels increased by Cdk1-inhibitory interventions and declined in response to Cdk1-activating interventions. In keeping with the prominently cytoplasmic location of the nonphosphorylatable point mutant HuR(S202A), phospho-HuR(S202) was shown to be predominantly nuclear using a novel anti-phospho-HuR(S202) antibody. The enhanced cytoplasmic presence of unphosphorylated HuR was linked to its decreased association with 14-3-3 and to its heightened binding to target mRNAs. Our findings suggest that Cdk1 phosphorylates HuR during G2, thereby helping to retain it in the nucleus in association with 14-3-3 and hindering its post-transcriptional function and anti-apoptotic influence.

Excitotoxic brain injury in adult zebrafish stimulates neurogenesis and long-distance neuronal integration.

Zebrafish maintain a greater capacity than mammals for central nervous system repair after injury. Understanding differences in regenerative responses between different vertebrate species may shed light on mechanisms to improve repair in humans. Quinolinic acid is an excitotoxin that has been used to induce brain injury in rodents for modeling Huntington's disease and stroke. When injected into the adult rodent striatum, this toxin stimulates subventricular zone neurogenesis and neuroblast migration to injury. However, most new neurons fail to survive and lesion repair is minimal. We used quinolinic acid to lesion the adult zebrafish telencephalon to study reparative processes. We also used conditional transgenic lineage mapping of adult radial glial stem cells to explore survival and integration of neurons generated after injury. Telencephalic lesioning with quinolinic acid, and to a lesser extent vehicle injection, produced cell death, microglial infiltration, increased cell proliferation, and enhanced neurogenesis in the injured hemisphere. Lesion repair was more complete with quinolinic acid injection than after vehicle injection. Fate mapping of her4-expressing radial glia showed injury-induced expansion of radial glial stem cells that gave rise to neurons which migrated to injury, survived at least 8 weeks and formed long-distance projections that crossed the anterior commissure and synapsed in the contralateral hemisphere. These findings suggest that quinolinic acid lesioning of the zebrafish brain stimulates adult neural stem cells to produce robust regeneration with long-distance integration of new neurons. This model should prove useful for elucidating reparative mechanisms that can be applied to restorative therapies for mammalian brain injury.

Neuroprotective Effects of Four Phenylethanoid Glycosides on H2O2-Induced Apoptosis on PC12 Cells via the Nrf2/ARE Pathway

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key transcription factor against oxidative stress and neurodegenerative disorders. Phenylethanoid glycosides (PhGs; salidroside, acteoside, isoacteoside, and echinacoside) exhibit antioxidant and neuroprotective bioactivities. This study was performed to investigate the neuroprotective effect and molecular mechanism of PhGs. PhGs pretreatment significantly suppressed H₂O₂-induced cytotoxicity in PC12 cells by triggering the nuclear translocation of Nrf2 and reversing the downregulated protein expression of heme oxygenase 1 (HO-1), NAD(P)H quinone oxidoreductase 1 (NQO1), glutamate cysteine ligase-catalytic subunit (GCLC), and glutamate-cysteine ligase modifier subunit (GCLM). Nrf2 siRNA or HO-1 inhibitor zinc protoporphyrin (ZnPP) reduced the neuroprotective effect. PhGs showed potential interaction with the Nrf2 binding site in Kelch-like ECH-association protein 1 (Keap1). This result may support the hypothesis that PhGs are activators of Nrf2. We demonstrated the potential binding between PhGs and the Keap1-activated Nrf2/ARE pathway, and that PhGs with more glycosides had enhanced effects.