dynamic neural stem - cell proliferation zones apparent in the developing central nervous system are also present in the adult brain , primarily restricted to the dentate gyrus subgranular zone ( sgz ; altman and das , 1965 ; cameron et al . , 1993 ; reviewed in liu and brinton , 2010 ) of the hippocampus and the subventricular zone ( svz ) of the lateral ventricle ( altman , 1969 ; luskin , 1993 ; reviewed in liu and brinton , 2010 ) .
these regions retain regenerative potential throughout the life span with marked declines in aged brain ( kuhn et al . , 1996 ;
adult mammalian neurogenesis has been confirmed in humans ( eriksson et al . , 1998 ) .
quantitatively a remarkable degree of neurogenesis occurs in the rat dentate gyrus on a daily basis with 9,400 cells proliferating with a cell cycle time of 25  h ( cameron and mckay , 2001 ) . by this estimate ,
9,000 new cells would be generated in the dentate gyrus each day or more than 250,000 per month . within 12  weeks of 5-bromo-2-deoxyuridine ( brdu ) injection ,
5060% of all brdu - labeled ( brdu+ ) cells in the dentate gyrus express neuron - specific markers .
the number of new granule neurons generated each month is 6% of the total size of the granule cell population and 3060% of the size of the afferent and efferent populations .
although most newborn granule cells do not survive , 3% of these cells are replaced every month which corresponds to 0.1% per day in young adult mice and rats ( kempermann et al .
, 1997 ; cameron and mckay , 1999 ; liu and brinton , 2010 ) . in the rat dentate gyrus
, 85% of the granule cells are formed postnatally ( bayer and altman , 1974 ) .
immature granule cells reach their highest level within the second postnatal week , while mature granule cells reach asymptotic levels at 2  months of age ( bayer and altman , 1974 ) .
proliferation of granule cell precursors is age - associated and proliferation of these cells declines with increased age ( kuhn et al . , 1996 ) .
a decline in granule cell proliferation correlated with high levels of corticosteroids that decreased neurogenic potential ( cameron and mckay , 2001 ) .
the rat svz is estimated to produce 30,000 neuroblasts per day ( alvarez - buylla et al . , 2000 ) . together with the 9,000 new granule cells , roughly 40,000 new cells are available to regenerate the adult rat brain every day .
relative to juvenile rats , year - old rats displayed a 94% reduction in neurogenesis due to a 92% drop in cell production ( mcdonald and wojtowicz , 2005 ) .
aging alone does not overtly weaken the intrinsic neuronal properties of newborn granule cells . in the aged rodent , the relatively few new neurons that survive in the dentate gyrus
develop typical granule cell morphology and spine density ( morgenstern et al . , 2008 ) .
surviving neurons possess a synaptic density of afferent glutamatergic connections comparable to that of neurons born in young adults ( morgenstern et al . , 2008 ) .
decreased neurogenesis has been proposed as a factor in the age - related decline of cognitive ability ( bizon and gallagher , 2003 ; drapeau et al .
newborn granule cells arise from the hippocampal sgz within the dentate gyrus and functionally integrate into the preexisting neural circuitry ( van praag et al . ,
2002 ) to influence hippocampal - dependent processes including spatial pattern recognition ( clelland et al . , 2009 ) .
experimental methods that influence neurogenesis include exercise ( van praag et al . , 1999 , 2002 ; pereira et al . , 2007 ; rodriguez et al . , 2011
) , environmental enrichment ( thuret et al . , 2009 ; rodriguez et al . , 2011 ) , deep brain stimulation ( toda et al . , 2008 ; encinas et al . , 2011 ; stone et al . ,
2009 ) , and pharmacologic intervention by peptide growth factor delivery ( massa et al . , 2010 ) or small molecules ( malberg et al . ,
2000 ; mayo et al . , 2005 ; wang et al . , 2005 ; taupin , 2009 ; pieper et al . ,
glutamatergic and cholinergic synaptic input from the surrounding hippocampal circuitry penetrate the sgz of the adult dentate gyrus .
however , the local microenvironment of the adult dentate gyrus sgz retains an embryonic - like state wherein nearly exclusive gamma - aminobutyric acid type a receptor ( gabaar ) chloride channel depolarizing inputs surround progenitors in the neurogenic niche ( tozuka et al . , 2005 ) . while gaba is typically an inhibitory neurotransmitter , gaba is excitatory to neural progenitor cells and triggers an efflux of chloride to modulate a cascade of molecular events that regulate cell proliferation ( figure 1 ) .
adult - generated granule cells initially receive gabaergic input from local interneurons , isolated from extrinsic excitatory input ( overstreet wadiche et al . , 2005 ) .
adult - generated granule cells mature at a slow rate in comparison to the perinatal granule cells which are exposed to the hyperproliferative environment required for brain development .
nevertheless , gabaar excitation initiates a cascade of events leading to calcium influx in adult neuroprogenitor cells subsequently inducing the accumulation of a neurogenic transcription factor , neurod ( tozuka et al . , 2005 ) .
work by hisatsune s group found significant inward currents in type-2 cells but not type-1 cells following gaba exposure ( tozuka et al . , 2005 ) .
in response to depolarization events , transcription factor - activated processes promote mitosis , and subsequent granule cell maturation ( overstreet - wadiche et al . , 2006 )
( 1 ) ap activates the gaba type a receptor to initiate the efflux of chloride ions ( cl ) from neural progenitor and neural stem cells .
( 2 ) extrusion of cl from the intracellular compartment leads to membrane depolarization and activation of the voltage dependent l - type calcium channel .
( 3 ) the intracellular calcium ( ca ) rise activates cadependent kinases that ultimately lead to regulation of gene expression and protein synthesis of cell cycle proteins . ( 4 ) involving the transcription factor cyclic amp response element binding protein ( creb ) signaling pathway , ap up - regulates the expression of cell cycle genes that promote neural progenitor mitosis while simultaneously down regulating genes that repress cell division .
( 5 ) the mechanism of ap-induced neurogenesis takes advantage of the developmentally regulated direction of cl flux to induce neurogenesis in those cells that are phenotypically competent to divide while not activating those mechanisms in mature neurons ( wang et al . , 2005 ; brinton and wang , 2006 ) .
adult hippocampal neural progenitor cells ( brdu+ cells immunolabeled green ; neun+ cells are colored red ; coronal section of mouse hippocampal dentate gyrus ; scale bar  =  50  m ) in the image above are shown as an illustrative example of neurogenesis within the mouse dentate gyrus subgranular zone ( sgz ) .
newly born granule cells proliferate and develop into neurons and glia along the border between the hilus and the granule cell layer .
migration ( through the vertical space of the granule cell layer ( gcl ) ; arrows indicate sequence of temporal development ) and integration of these cells occurs within the days and weeks following proliferation .
newly born neurons that survive will continue to mature and send axonal projections to form mossy fiber synapses in the ca3 subfield and dendrites to extend into the molecular cell layer ( mcl ) to receive glutamatergic afferents from the perforant pathway of the entorhinal cortex .
neurosteroids bind to gabaar at sites that differ from gaba , benzodiazepines , ethanol , and barbiturate binding sites and can act as positive or negative modulators of gabaar function ( gee et al .
allopregnanolone ( ap ; 3-hydroxy-5-pregnan-20-one ; also known as ap , allo , or thp ) is a potent positive allosteric activator of the gabaar channels which at nanomolar concentrations enhances the action of gaba at gabaar and at higher concentrations directly activates gabaar .
ap binds to two transmembrane sites of the heteropentameric gabaar ( hosie et al . , 2006 ) .
the binding sites are conserved throughout all gabaar subtypes with the general subunit stoichiometry 2:2:1 with the  subunit replaced by the neurosteroid - sensitive  subunit in some extrasynaptic channel complexes .
gaba plays a key role in generation of the spontaneous network activity of immature dentate granule cells ( owens and kriegstein , 2002 ; sipila et al . , 2004 ) . in neuroprogenitor cells , gabaergic depolarization of the uniquely reversed membrane potential
adult dentate granule cells , devoid of the 1 subunit , are also subjected to tonic gabaergic signaling via  subunit containing gabaars mediated by surrounding synaptic boutons of local interneurons ( overstreet wadiche et al . , 2005 ) .
gabaars have been observed by electron microscopy at membrane sites on brdu co - labeled adult hippocampal sgz progenitor cells ( mayo et al . , 2005 ) .
ap-potentiated gabaergic stimulation of neural progenitor cells elicits an efflux of chloride and a concomitant influx of calcium that contributes to the induction of cell signaling events leading to gene transcription of mitotic genes and downregulation of anti - mitotic genes ( figure 1 ) .
gabaergic signaling in the svz also controls proliferation of adult progenitor cells within the neurogenic niche ( liu et al . , 2005 ) .
the survival and maturation potential of newborn neural cells as evidenced through enhanced learning and memory ( figure 2 ; wang et al .
, 2010 ; singh et al . , 2011 ) is achieved through modulation of gabaergic signaling with optimal pulses of ap ( figure 3 ; chen et al . , 2011 ) .
triple - transgenic alzheimer s disease ( 3xtgad ) mice were exposed to a single dose of ap. data were plotted as percent change relative to age - matched vehicle control to assess the age - related differences in response to ap administered at 3- , 6- , 9- , or 12-months of age .
mice were treated with either ap ( subcutaneous , 10  mg / kg ) or vehicle and 1  h later with bromodeoxyuridine ( brdu ) ( intraperitoneal , 100  mg / kg ) .
learning and memory performances were measured by trace eyeblink conditioning , a hippocampal - dependent task .
mice were trained by pairing delivery of a tone ( conditioned stimulus ; cs , 250  ms , 2  khz , 85  db ) as the conditioned stimulus followed by a 250-ms period of no stimuli , followed by a mild periorbital shock ( unconditioned stimulus ; us , 100  ms ) to elicit an eyeblink response .
mice received two blocks of 30 trials per day ( 3060  s intertrial intervals , 34  h interblock intervals ) .
this behavioral paradigm is subthreshold for inducing neurogenesis ( wang et al . , 2010 ; singh et al . , 2011 ) .
one week following a single dose of ap , mice were subjected to trace eyeblink conditioning , with 1  day of habituation and 5  days of paired training . following paired training ,
mice were left undisturbed in their home cages for 9  days and subsequently were tested for memory . following the final learning trial ,
brdu+ cell survival / hippocampus was measured at the end of the study , 3-weeks following a single dose of ap and the thymidine analog dna - synthesis marker brdu .
bars represent percent change    sem in response to a single exposure to ap compared to age - matched vehicle at 3- , 6- , 9- , 12-months of age in 3xtgad mice ( n    7 ; wang et al . , 2010 ;
within 3  weeks following a single exposure to ap , neurogenesis , maximal learning , and memory indicators were increased 100% relative to age - matched vehicle control in adult male 3xtgad mice when administered at ages prior to overt ad pathology .
the 3xtgad mouse model displays age - associated decrements in endogenous neurogenic cell survival in the subgranular zone ( sgz ) as compared with the non - transgenic mice in addition to age - associated increments in a pathology burden ( depicted supra to bar graph ) . at 12-months of age , intra- and extraneuronal a 6e10
antibody staining is apparent and plaque structures are developed in subiculum ( wang et al . , 2010 ;
the therapeutic response to ap was specific to the transgenic ad phenotype , as the age - matched non - transgenic mice did not benefit from a single exposure to ap. remarkably , a single exposure to ap increased neurogenesis and subsequent cell survival in aged non - transgenic mice when administered at 15-months of age ( non - tg data not in figure ; singh et al . , 2011 ) . at 12-months of age , the point when extraneuronal plaques are known to be present in this ad mouse model , a single exposure to ap was ineffective . at ages
prior to extraneuronal a plaques , ap significantly ( p  <  0.05 ) increased brdu+ cell survival , maximal learning , and memory function relative to age - matched vehicle control .
triple - transgenic alzheimer s disease ( 3xtgad ) mice were treated subcutaneously with allopregnanolone ( ap ; 10  mg / kg ) once per month ( 1/month ) , once per week ( 4/month ) , or every other day ( 12/month ) to determine extent of neurogenesis ( depicted by red colored arrow ) and pathology reduction capacity ( depicted by black colored arrow ) on double y - axes ( chen et al . , 2011 ) .
all three ap treatments were initiated when 3xtgad mice reached 3  months of age . upon completion of each treatment paradigm ,
both the 1/month ap treatment and the 4/month ap treatment induced a significant increase in neurogenesis , with the latter regimen yielding the greater increase in neurogenesis .
however , the 3/week/3  months ( 12/month ) treatment induced a significant decrease in neurogenesis . brain sections from 3xtgad mice treated with ap or vehicle were immunostained .
a immunoreactivity was detected and indicated that the 1/week/6  months ( 4/month ) ap treatment significantly decreased a immunoreactivity .
efficacy of a reduction in 4/month was comparable to the 12/month ap treatment whereas the 1/month ap treatment was ineffective at reducing a immunoreactivity ( chen et al . , 2011 ) .
from these dosing frequency studies , we conclude that the optimal treatment regimen for ad is to intervene as early as possible with once per week administration of ap to simultaneously promote neurogenesis and subsequent cell survival .
neurosteroids are synthesized in the central and peripheral nervous system , particularly in myelinating glial cells , astrocytes , and several neuronal cell types .
a region - specific expression pattern of progesterone converting enzymes is evident in both hippocampus and cortex . in steroidogenic cells ,
biosynthesis of neuroactive steroids is controlled by the 18-kda outer mitochondrial membrane translocator protein ( tspo ; rupprecht et al . , 2010 ) and tspo - associated proteins including the steroidogenic acute regulatory protein ( star ) to form a macromolecular signaling complex which transports cholesterol through the mitochondrial inner membrane to the mitochondrial matrix to be converted into pregnenolone by the cytochrome p450 side - chain cleavage ( cyp450scc ) enzyme ( liu et al . , 2006 ) .
pregnenolone subsequently diffuses back into the cytoplasm whereby its conversion to progesterone is facilitated by the 3-hydroxysteroid dehydrogenase ( 3-hsd ) enzyme .
ap is synthesized in the brain from progesterone by the sequential action of 5-reductase ( 5-r ) type - i and 3-hydroxysteroid dehydrogenase ( 3-hsd ; mellon et al . , 2001
the rate - limiting step is the unidirectional reduction of progesterone to 5-dihydroprogesterone by 5-r .
subsequently , 3-hsd catalyzes conversion of 5-dihydroprogesterone into ap. interestingly , 5-r and 3-hsd are functionally expressed in pluripotent progenitor cells ( melcangi et al . , 1996 ) .
ap and other trophic factors are diminished in the brains of alzheimer s disease ( ad ) patients compared to age - matched controls ( weill - engerer et al . , 2002 ; marx et al . , 2006 ;
is the loss of episodic and semantic memory ( perry et al . , 2000 ) .
ad diagnostic imaging studies using volumetric mri reveal a decreased hippocampal volume due to atrophy of gray matter , i.e. , neurodegeneration in amnestic subtype mild cognitive impairment in the progression to ad ( whitwell et al . , 2007 ) .
neuropathological hallmarks of ad include extracellular and intracellular deposition of -amyloid ( a ) protein , neurofibrillary tangles and neurodegeneration .
ad causes extensive neurodegeneration to the cholinergic neurons of the nucleus basalis and to a greater extent to the noradrenergic neurons of the locus coeruleus ( zarow et al . , 2003 ) .
in addition to the usual culprits that lead to neurodegeneration , it was discovered that a subpopulation of dysfunctional neurons in ad display aberrant entry into the cell cycle and replicate their genomes but fail to divide leading to a fatal apoptotic mechanism ( busser et al . , 1998 ;
there have been no neurogenic therapies developed to prevent neurodegenerative mechanisms of aberrant cell cycle entry .
several studies have reported decreased neurogenesis in ad mouse models ( lazarov and marr , 2010 ) . however , there are few human studies to confirm or reject the importance of neurogenesis in ad .
( 2004 ) showed increased expression of doublecortin ( dcx ) , a marker for new neurons , in a cohort of senile patients and suggested that neurogenesis is increased as a compensatory mechanism of neurodegeneration .
( 2006 ) challenged the findings reported by jin et al . and demonstrated that in human presenile ad brain , most of the proliferation could be accounted for by glial and vasculature - associated changes and found no evidence for altered neurogenesis in the dentate gyrus .
thus , in ad brain , the extent of neural proliferation and its potential for regenerative therapeutic responsiveness remains an active area of investigation .
our discovery investigations of ap effects in immature hippocampal cells began nearly two decades ago beginning in vitro then advancing to in vivo studies , all reviewed herein ( brinton , 1994 ) .
it was known that seizure activity was associated with aberrant hippocampal nerve cell growth and that ap protected against seizure activity ( brinton , 1994 ) .
what was not known at the time was whether ap prevented against aberrant circuit development .
our initial attempts to address this question were in vitro analyses using videomicroscopy of hippocampal neurons in culture to determine the impact of ap on neurite outgrowth in real time ( brinton , 1994 ) .
results of these analyses revealed that ap induced a significant decrease in the area and length of neurites within 40  min of exposure .
a concomitant decrement in the number and length of filopodia decorating neuritic extensions also occurred within the same time frame .
an unforeseen outcome was the observation that within ap-treated neurons , retrograde transport of intracellular organelles occurred .
ap-induced regression of neuronal morphology and retrograde transport of organelles was only observed in hippocampal cells that had not yet established contact with other neuronal or glial cells in culture .
the cellular selectivity for the morphological effect of ap was an early indicator of a more significant effect of ap which we later discovered .
neither the inactive stereoisomer 3-hydroxy-5-pregnan-20-one nor progesterone had a significant effect upon any of the morphological parameters assessed ( brinton , 1994 ) . in more mature hippocampal cultures in which structural synaptic connections had been established , ap protected neurons against picrotoxin - induced cell death . ap activated chloride ion channels , proved to be an initial step in the biochemical mechanism underlying both the retraction and later neurogenesis .
interestingly , 17-estradiol a known neurotrophic factor ( brinton , 2009 ) rapidly reversed the filopodial regression induced by ap indicating that neurosteroids acting singly and in combination play complex roles in generation and differentiation of developing neural cells .
subsequent to the observation of ap-induced neurite regression of hippocampal neurons in culture , we made the serendipitous discovery that ap was increasing mitosis of hippocampal neurons in culture .
we then understood that the ap-induced regression of neurites was a prelude to ap-induced mitosis . at the time
this discovery was made , understanding of neurogenesis in brain was limited to embryonic development in the mammalian brain and regeneration of neurons required for song in the bird brain . from the vantage point of our current understanding ,
the hippocampal neurons undergoing mitosis in the primary hippocampal cell cultures were likely neural progenitor cells .
this early discovery of ap-induced mitosis was supported by biochemical analyses demonstrating that ap induced a significant increase in h - thymidine uptake indicating increased dna synthesis during the s phase of the cell cycle ( wang et al . , 2005 ) .
these early foundational studies were followed up with the advent of an understanding of the neural progenitor pool within the adult mammalian brain .
we determined ap proliferative efficacy in rodent embryonic and adult neural progenitor cells and embryonic derived human neural progenitor cells . in each of these cell types , ap induced a significant increase in markers of dna synthesis , brdu and h - thymidine incorporation , mulv - gfp - labeled mitotic neural progenitor cells by facs analysis and unbiased quantitative stereology of brdu - positive cells in both the subgranular and subventricular proliferative zones ( wang et al . , 2005 ) . in vitro ,
the magnitude of ap-induced neurogenesis ranged from 20 to 30% in the rat hippocampal neural progenitor cells to 3749% in human cortical neural progenitors ( wang et al . , 2005 ) .
the efficacy of ap as a neurogenic factor is comparable to that induced by bfgf  +  heparin .
as was the case for cultured embryonic hippocampal neurons , ap-induced progenitor cell proliferation was stereoisomer specific , as 3-hydroxy-5-pregnan-20-one did not increase h - thymidine uptake ( wang et al . , 2005 ) .
npc markers nestin and tuj1 were identified by immunofluorescence and indicated that the newly formed cells were indeed neuronal . to determine the mechanism of action induced by ap in progenitor cells , microarray analysis of cell cycle genes demonstrated that ap increased the expression of mitotic genes and inhibited the expression of anti - mitotic genes .
ap-induced proliferation of neural progenitors was antagonized by the both voltage - gated l - type calcium channel blocker nifedipine indicating a calcium - dependent mechanism for neuroproliferation ( figure 1 ; wang et al . , 2005 ) .
consistent with a calcium - dependent mechanism for neuroproliferation , ap induced a rapid increase in intracellular calcium in hippocampal neurons via a gabaar - activated l - type calcium channel ( wang and brinton , 2008 ) .
following ap treatment , regulation of intracellular calcium concentration was measured in e18 rat hippocampal neurons using ratiometric fura2-am imaging .
results indicated that ap rapidly increased the intracellular calcium concentration in a dose - dependent and developmentally regulated manner , with an ec50 of 110    15  nm and a maximal response occurring at 3  days in vitro .
the stereoisomers 3-hydroxy-5-hydroxy - pregnan-20-one and 3-hydroxy-5-hydroxy - pregnan-20-one , as well as progesterone , were without significant effect .
ap-induced intracellular calcium concentration increase was not observed in calcium depleted medium and was blocked in the presence of the broad spectrum calcium channel blocker lanthanum ion , or the l - type calcium channel blocker nifedipine .
furthermore , the gabaar blockers bicuculline and picrotoxin abolished the ap-induced intracellular calcium concentration rise ( wang and brinton , 2008 ) . the in vitro neurogenic properties of ap
coupled with a low molecular weight , easy penetration of the blood brain barrier and lack of toxicity , were key elements that guided our efforts toward in vivo studies of ap as a neurogenic regenerative therapeutic with the ambitious aim of restoring synaptic connections in victims of ad .
to evaluate ap as a potential therapy for ad it is essential to obtain preclinical in vivo evidence of ap safety and efficacy . the triple - transgenic mouse model of alzheimer s ( 3xtgad ) , an ad model is characterized by overexpression of swedish mutant app , mutant p301l tau in the homozygous mutant of presenilin 1 ( m146v ) knock - in mouse ( oddo et al .
these mice have been used extensively as an ad research model as they age - dependently develop hallmarks that include hippocampal tangle - like pathology , neurological deficits , and intraneuronal and extraneuronal a deposition .
several completed in vivo studies have demonstrated the pro - neurogenic and promnesic effects of ap. we review here three studies from our laboratory in which a correlation was made between ap-induced neural progenitor cell survival and improved memory function in 3xtgad mice ( wang et al . , 2010 ; chen et al . , 2011 ; singh et al . , 2011 ) .
the basal concentration of ap in blood plasma ( 0.47  ng / ml    0.88 ) was significantly lower ( p  <  0.05 ) than in cortex ( 10.36  ng / g    1.43 ) in young adult male non - transgenic ( non - tg ) mice , indicating a higher brain accumulation , consistent with locally synthesized ap in hippocampus and cortex . in contrast , 3xtgad mice exhibited a lower basal level of ap in the cerebral cortex ( 3xtgad , 6.49  ng / g    2.02 versus non - tg , 10.36  ng / g    1.43 ) , suggesting either impairment of ap synthesis or accelerated ap metabolism in 3xtgad mice brain ( wang et al . , 2010 ) . a decline in neurogenesis occurs within the sgz and svz in male and female 3xtgad mice in correlation with ad pathology ( brinton and wang , 2006 ; rodriguez et al . , 2008 , 2009 ;
neural progenitor cell proliferation and subsequent cell survival was determined by analysis of brdu incorporation .
we verified the phenotype of in vivo newly formed brdu+ cells by triple - immunolabeling coronal sections of mouse hippocampi adjacent to those that were stereologically analyzed and derived from 3-month - old 3xtgad mice treated with a single subcutaneous injection of 10  mg / kg ap ( wang et al . , 2010 ) .
the phenotypic markers assessed were doublecortin dcx , to label young immature neurons ; neun , to label mature neurons ; and glial fibrillary acidic protein ( gfap ) , to label astrocytes .
confocal microscopy identified brdu - positive cells colocalized with dcx alone or together with neun .
newly born cell survival was further confirmed by immunolabeling coronal sections derived from ap ( subcutaneous , 10  mg / kg)-treated 3xtgad mouse dentate gyrus 22  days post - ap treatment and post - behavioral analyses .
brdu+ cells were located deep within the granular cell layer , indicating the migration of newly formed cells from the sgz to the granule cell layer ( wang et al . , 2010 ) .
collectively , these data indicated that newly formed cells , generated following ap treatment , express a neuronal phenotype .
learning and memory function was assessed using the hippocampal - dependent trace eyeblink conditioning paradigm . at 3  months of age ,
the basal level of brdu+ cells in the sgz of 3xtgad mice was significantly lower relative to non - transgenic ( non - tg ) mice , despite the lack of evident ad pathology ( wang et al . , 2010 ) .
ap significantly increased , in a dose - dependent manner , brdu+ cells in the sgz of 3xtgad mice and restored sgz proliferation to normal magnitude of non - tg mice ( wang et al .
coinciding with deficits in adult neural progenitor proliferation , 3xtgad mice exhibited deficits in learning and memory .
ap reversed the cognitive deficits to restore learning and memory performance to the level of normal non - tg mice ( wang et al .
ap increased proliferation and promoted the survival of newly born hippocampal dentate granule cells ( wang et al . , 2010 ) .
interestingly , the early deficits in neurogenesis detected in this genetic model of ad were evident prior to immunodetectable -amyloid ( a ) .
to broaden this preclinical discovery work , studies were extended to 3xtgad mice aged 6- , 9- , 12-months , therapeutically relevant to early and mid - stage ad .
comparable to the 3-month mice , ap increased proliferative activity , promoted the survival of newly born hippocampal dentate granule cells , and restored cognition affected by ad pathology in 6- , 9-month - old 3xtgad mice while having no significant impact on their age - matched normal non - tg counterparts ( singh et al . , 2011 ) .
brdu+ neural progenitor cell survival was assessed after 3  weeks , following a single exposure to ap ( 10  mg / kg ) . during the 3-week period ,
a hippocampal - dependent associative learning and memory task was performed which evidently allowed granule cells sufficient time to proliferate and migrate at the border between the hilus and the granule cell layer subsequent to their incorporation into the existing neural network .
ap significantly increased survival of brdu+ cells and recovered hippocampal - dependent cognition in 6- , and 9-month - old 3xtgad mice , in the presence of intraneuronal a , whereas ap was ineffective subsequent to development of extraneuronal a plaques in 12-month - old mice .
surprisingly , cognition was restored to maximum by the first day of trace eyeblink conditioning , only 1  week following a single exposure to ap. hippocampal - dependent associative learning accomplished by repeated trials of an auditory tone followed by a mildly aversive shock stimulus , was sustained throughout behavioral training .
learning and memory function in ap-treated 3xtgad mice was 100% greater in magnitude compared to the age - matched vehicle - treated group ( figure 2 ) and was remarkably comparable to the maximal normal non - tg mouse performance .
furthermore , we observed an upward trend albeit not statistically significant , toward efficacy in non - tg at 12  months of age .
the behavioral experiment was then extended to include 15-month - old non - tg mice to determine whether or not ap presented a therapeutic benefit to older non - ad mice that may have a greater degree of age - related diminution of neurogenic growth factors .
our results indicated that hippocampal brdu+ cell survival was significantly increased by a single exposure to ap and cognitive performance was significantly enhanced in 15-month - old male non - tg mice ( singh et al . , 2011 ) .
in normal non - ad men as early as age 40 , a decline in ap has been reported ( genazzani et al . , 1998 ) .
the therapeutic benefit found in aged non - tg mice suggests that at least in males , ap therapy could supplement an age- and gender - related decline in this neurogenic factor .
these findings provided preclinical evidence of ap-promoted survival of newly generated cells and paralleled restoration of cognitive performance in the pre - plaque phase of ad pathology and in late - stage normal aging .
to further advance efforts to assess the preclinical efficacy of ap for ad , our group designed long - term studies .
the studies were designed to test ap using the same age of enrollment ( 3xtgad male 3  months of age ; prior to overt intraneuronal a ) , neurogenically efficacious dose of 10  mg / kg via subcutaneous route of administration , matching our previous studies .
in addition to neurogenic efficacy , the long - term studies were extended to determine the disease modifying effects afforded by the therapeutic regimen
2011 ) tested three treatment regimens  once per month , once per week , and every other day ( figure 3 ) .
based on measured endpoints of a oligomers by immunostain and immunoblot approaches , the every other day treatment regimen was maximally efficacious but did not increase neurogenesis .
the once per month ap treatment was efficacious for proliferation of neural progenitor cells in the sgz ( and svz , unpublished ) but not for decreased a pathology .
the once per month treatment regimen ( chen et al . , 2011 ) was similar to the single exposure treatment paradigm that improved learning and memory performance after a single exposure ( singh et al . , 2011 ) .
analyses to determine the optimal treatment paradigm indicated that ap administered once per week for 6  months was maximally efficacious for both neurogenic and anti - amyloidogenic endpoints ( figure 3 ) . in parallel to the 3-month - old mice administered once per week ap , we simultaneously began treatment of a 6-month - old male 3xtgad group . when ap was administered beginning at 6  months of age , the age at which intraneuronal plaques are apparent in this mouse model , the appearance of a pathology paralleled cessation of ap efficacy . once intraneuronal a is extracellularly localized , the efficacy of ap is largely diminished .
this suggested to us that intraneuronal a accumulation is a determining factor that focuses the window of ap therapeutic efficacy on the early stages of ad .
increasing evidence indicates that altered cholesterol metabolism is linked to ad pathology ( schumacher et al . , 2004 ;
in addition to the mechanism of action whereby ap induces neurogenesis through excitation of gabaar chloride channels in neural progenitor cells ( figure 1 ; wang et al . , 2005 , 2008 , 2010 ; brinton and wang , 2006 ; wang and brinton , 2008 ) , ap regulates cholesterol homeostasis via the liver - x - receptor ( lxr ) and pregnane - x - receptor ( pxr ) system ( chen et al . , 2011 ) .
lxr , a nuclear hormone receptor abundant in the brain ( whitney et al . , 2002 )
, acts as a molecular sensor of cholesterol levels and initiates cholesterol clearance ( whitney et al . , 2002 ) .
lxr activation increases cholesterol efflux through up - regulating abca1 and apoe expression , and prevents the hyper - activation of -secretase and over - production of a ( whitney et al . , 2002
lxr activation has been demonstrated to improve cognitive function in multiple mouse models of amyloidogenesis ( schultz et al . , 2000 ;
, 2006 ; xiong et al . , 2008 ; donkin et al . , 2010 ;
importantly , lxr ligands have been shown to activate pregnane - x - receptor ( pxr ; riddell et al . , 2007 ) .
results from our analyses indicated that in parallel with an ap-induced increase in lxr expression in the pre - pathology condition , ap also increased pxr expression in the pre - pathology 3xtgad mouse brain ( chen et al . , 2011 ) .
pxr activation induces cytochrome p450 3a ( cyp3a ) enzymes including cyp3a4 and cyp3a13 and leads to cholesterol hydroxylation and activation of organic anion transporters for cholesterol extrusion ( sun et al . ,
in addition to increased lxr and pxr expression , ap treatment initiated in pre - a pathology 3-month - old 3xtgad mice treated once per week for 6  months displayed increased expression of 3-hydroxy-3-methyl - glutaryl - coa - reductase ( hmg - coa - r ; chen et al . , 2011 ) . the increase in hmg - coa - r
is at first paradoxical as it is the rate - limiting enzyme in cholesterol synthesis .
hmg - coa reductase is also required for oxysterol generation which activate lxr and pxr - mediated gene transcription for cholesterol and lipid transport proteins ( leduc et al . , 2010 ) .
thus , the ap-induced increase in brain lxr and pxr leads to increased cholesterol efflux , thereby reducing -secretase activation by cholesterol - laden lipid rafts .
increased cholesterol efflux provides a plausible mechanism to explain how ap decreased the generation of both 27 and 56  kda intraneuronal a oligomers after 6  months of once per week treatment ( chen et al .
our findings suggest that in vivo , brain cholesterol homeostasis and intraneuronal a are tightly coupled with ap efficacy ( chen et al . , 2011 ) .
deposition of a in the extracellular compartment disconnected this coupled pathway and led to a loss of ap efficacy in advanced stages of ad - like pathology in the 3xtgad model .
ap significantly reduced a generation in hippocampus , cortex , and amygdala , which was paralleled by decreased mitochondrial a-binding alcohol dehydrogenase ( abad ) and reduced microglia activation assessed as reduced expression of ox42 ( chen et al . , 2011 ) .
ap has also been shown to increase myelin basic protein in organotypic slice cultures of rat cerebellum ( schumacher et al . , 2004 ) and
delay demyelination in niemann  pick c mice ( mellon et al . , 2008 ) .
ap may stimulate oligodendrocyte progenitor cells in addition to neural progenitor cells . in oligodendrocytes ,
the myelin marker cnpase was increased by once per week ap , indicating pro - myelinating capabilities in this mouse model ( chen et al . , 2011 ) .
collectively , ap is a multifaceted neurosteroid that promotes neurogenesis while simultaneously reducing ad pathology in the 3xtgad mouse model .
currently there are no effective treatments to delay progression of alzheimer s , halt the degenerative process or effectively treat the disease . in fact , since 2003 , there have been no new drug approvals for ad by the united states food and drug administration ( fda ) .
of the five fda - approved ad drugs , four are cholinesterase inhibitors and memantine is an n - methyl - d - aspartate ( nmda ) receptor antagonist . as part of the recent ad neuroimaging initiative or  adni  , mild cognitive impairment patients who received cholinesterase inhibitors with or without memantine were more functionally impaired , showed greater decline in scores , and progressed to dementia sooner than patients who did not receive treatment ( schneider et al . , 2011 ) .
phase 3 clinical trials have failed to therapeutically modify ad via the inhibition of amyloid cascade targets including a gamma - secretase modulator ( tarenflurbil ) , gamma - secretase inhibitor ( semagacestat ) , and beta - secretase inhibitor ( tramiprosate ; karran et al . , 2011 ) .
most ad researchers agree that it is ineffective to evaluate therapeutic efficacy in people that have reached the later stages of ad , when irreversible damage has occurred . to modify disease progression , therapies need to be instituted early to target specific physiological stages of ad progression .
one strategy to detect dysfunction of the neuroendocrine system would be to access blood levels of ap in persons at risk for ad .
preclinical biomarkers to predict clinical therapeutic potential and clinical biomarkers that confirm target engagement and dose selection are needed as prognostic and diagnostic tools for ad disease progression to select and track treatment strategies relevant to each pathological stage of dementia ( buckholtz , 2011 ) .
currently , large - scale multicenter controlled studies are being conducted in us , europe , japan , and australia ( adni ) to systematically develop and validate candidate biomarkers such as cerebrospinal fluid proteins and structural and functional imaging signatures ( hampel et al . , 2008 ) .
ideally , complementary brain imaging and fluid biomarkers will be developed for clinical evaluation of ad . as an endogenous metabolite of progesterone ,
men and women are exposed to ap throughout their lifetime . during reproductive years , women are chronically exposed to ap concentrations ranging from less than 1  nmol / l ( 0.32  ng / ml ) to over 4  nmol / l ( 1.27  ng / ml ) during the luteal phase ( genazzani et al . , 1998 ) . during pregnancy , blood production rate of ap can reach 100  mg/24  h ( dombroski et al . , 1997 ) and remain high throughout the third trimester of pregnancy at levels up to 157  nmol / l ( 50  ng / ml ) , which while associated with drowsiness , is not associated with adverse effects for either mother or fetus ( luisi et al . , 2000 ) . in response to acute stress ,
the fetal brain can increase synthesis of ap de novo independent of maternal supply and of the hypothalamic  pituitary  adrenal axis ( nguyen et al . , 2003 ) .
women receiving progesterone therapy can generate between 0.9  nmol / l ( 0.3  ng / ml ) and 4.2  nmol / l ( 1.3  ng / ml ) of ap , consistent with levels generated during the menstrual cycle .
analyses following a single intramuscular injection of 200  mg progesterone ( ap precursor ) indicated similar profiles in men and women with peak levels of ap of 35  nmol / l ( 11  ng / ml ) for men and 41  nmol / l ( 13  ng / ml ) for women in the first hours after administration ( soderpalm et al . , 2004 ) .
post - mortem human brain tissue from women revealed ap levels in the range of 1421  ng / g ( bixo et al . , 1997 ) .
oral delivery of ap would be convenient and readily tolerated however this route presents a challenge because of the low solubility properties of ap and first - pass metabolism in the digestive tract and liver .
results of our preclinical analyses predict that the optimal therapeutic ap regimen will be a once per week transdermal or subcutaneous administration ( figure 3 ) .
initial pharmacokinetic analyses of a topically applied formulation of ap in rabbit , indicated that ap is absorbed transdermally to reach the blood circulation with accumulation in the brain that is relatively slowly eliminated when compared to intravenous dosing ( table 1 ) .
several clinical studies by bckstrm s group at ume university , sweden have demonstrated that ap is safe with short - lived , mild self - reported sedation ( drowsiness ) .
following dosing of ap 0.09  mg / kg ( three cumulative doses of 0.015 , 0.03 , 0.045  mg / kg within 1  h ) fully bioavailable intravenous administration , van broekhoven et al . ( 2007 ) measured maximum blood levels of 150  nmol / l ( 48  ng / ml ) in men and 100  nmol / l ( 32  ng / ml ) in women ( table 1 ) .
interestingly , mean levels of ap were found to be higher in men compared to women at baseline ( men , 2.4  nmol / l or 0.76  ng / ml versus women , 0.4  nmol / l or 0.13  ng / ml ) and after each of three doses of intravenous ap. volume of distribution , elimination half - life , and the area under the curve ( auc ) adjusted for body weight did not differ between men and women in the clinical study ( van broekhoven et al . , 2007 ) . to date
pharmacokinetic properties are best studied by an intravenous ( iv ) dose as this route of administration has the greatest quantitative potential , assumed to be 100% bioavailable , and is useful for comparison to alternative routes of administration .
preclinical ap dosing studies with subcutaneous ( sc ) , transdermal ( td ) , intranasal ( in ) , intracerebroventricular ( icv ) , and iv routes of administration were tabulated with human iv dosing .
although the differences in the tabulated doses appear large , when bioavailability and species - specific allometric conversions are taken into account the preclinical studies that demonstrate neuroregenerative efficacy in an ad mouse model are within the range of the safe dosage for humans . in the rabbit pharmacokinetic study , the bioavailability of td ap was 1.5% ( ap td area under curve of 17.7  h*ng / ml versus iv 732.1  h*ng / ml plasma ) , brinton , sri , intl .
the conversion factor for ap is 3.1398 ( ap formula weight 318.49  g / mol ) to simplify mathematical conversion from nanograms per milliliter to nanomoles per liter .
ap 50  ng / ml    3.1398  =  ap 157  nmol / l corresponding to the upper level of plasma ap measured during the third trimester of human pregnancy ( luisi et al .
, 2000 ) and safely achieved with human iv dosing ( van broekhoven et al . , 2007 ; kask et al . , 2008 ) .
neurosteroid levels from post - mortem brain tissue were measured in nanograms per gram and for comparison purposes are estimated to equal nanograms per milliliter and then converted to nanomoles per liter .
human , as well as rodent , brains exhibit a significant and profound reduction in the proliferative pool and regenerative potential of neural stem cells as they age , a phenomenon that may be exacerbated in prodromal and mild ad brains .
both the pool of neural stem cells and their proliferative potential are compromised in the course of ad ( lazarov et al . , 2010 ; winner et al . , 2011 ) .
in parallel , ap content , along with a host of other factors , is diminished in the brains of ad patients compared to age - matched controls ( weill - engerer et al . , 2002 ; marx et al . , 2006
while de novo synthesis of ap in brain is diminished , peripheral delivery could restore levels to normal .
a deficient milieu of endogenous neurosteroids and depleted neurogenesis demonstrate the therapeutic need for ap in the human brain .
decrements in learning and memory have been reported in animals and humans although these studies are either acute measures of memory performance within minutes of a sedative dose ( kask et al . , 2008 ) or chronic treatment paradigms that mimic stress conditions ( turkmen et al . , 2006 ) .
when high doses or chronic treatment regimens are employed , ap acts similar to benzodiazepines to reduce learning and impair memory .
ap administered twice daily at high doses to male rats for several consecutive days decreased performance on the morris water maze , escape latency , path length , and thigmotaxis and it was determined that pretreatment induced a partial tolerance against acute ap effects ( turkmen et al . , 2006 ) .
it is important to consider that gabaergic synapses are not universally influenced by ap and that many factors including gabaar subunit composition and receptor density influence the local cellular response .
thus for therapeutic use of ap , it is imperative to determine the proper dosing regimen which may be specific to each indication . aside from ad
, the proper ap dosing regimen has shown therapeutic potential in research models of several brain disorders or injuries including catamenial epilepsy ( reddy and rogawski , 2001 ; rogawski , 2003 ) , spontaneous seizures ( concas et al . , 1996 ) , diabetic neuropathy ( leonelli et al . , 2007 ) , niemann  pick type c neurodegenerative disorder ( griffin et al . , 2004 ;
ap is absorbed by multiple routes of administration , penetrates the blood brain barrier well , exhibits a wide margin of safety particularly when given transdermally or subcutaneously and for the 3xtgad mouse model is most efficacious when administered only once per week which also contributes to its margin of safety ( table 1 ) .
importantly , ap is a small molecular weight , blood brain barrier penetrant molecule with safety data in animals and humans ( timby et al . , 2006 ; van broekhoven et al . , 2007 ;
, ap is the only small molecule agent that both promotes regenerative function in the brain and simultaneously reduces ad pathology burden .
, 2005 , 2010 ) , recovery of learning and memory function ( wang et al .
, 2010 ; singh et al . , 2011 ) , and reduction of ad pathology burden ( chen et al . , 2011 ) in the 3xtgad mouse model
2005 ) and key regulators of cholesterol homeostasis ( chen et al . , 2011 )
provide mechanistic plausibility for its therapeutic efficacy to promote neurogenesis and cognitive function while reducing ad pathology .
our data show that regeneration is achieved with either once per month or once per week regimen of ap. reduction of ad pathology can be achieved with once per week or every other day regimens .
the combination of regeneration and reduction of pathology was achievable with the once per week ap treatment regimen .
together with the dosing frequency , the magnitude of pathology at the start of treatment intervention is critical to therapeutic efficacy .
administration of ap prior to and during the early stages of ad pathology significantly increased the regenerative response in brain while also reducing burden of pathology in an ad mouse model .
ap treatment initiated at the point of a plaque generation was not efficacious indicating that ap targets regenerative and pathology - reducing mechanisms present during the early to mid - stages of the disease .
based on the therapeutic efficacy of ap in a preclinical ad mouse model and in normal aged mice , we predict that ap has potential therapeutic benefit in humans to delay progression in persons with familial early onset ad and to prevent and delay disease in late onset ad . in these populations
, ap could be an effective therapy to promote the regenerative potential and myelination capacity of the brain to prevent or delay progression of mild cognitive impairment to clinically diagnosed ad . in summary
, targeting a unique mechanism of action , ap promotes the innate regenerative capability of the brain by increasing the number and survival of newly generated neurons .
patents pending on allopregnanolone as a therapeutic for mild cognitive impairment and alzheimer s disease .
 the proliferative pool and regenerative potential of neural stem cells diminishes with age , a phenomenon that may be exacerbated in prodromal and mild alzheimer s disease ( ad ) brains . in parallel 
 , the neuroactive progesterone metabolite , allopregnanolone ( ap ) , along with a host of other factors , is decreased in the ad brain . 
 results of preclinical analyses demonstrate that ap is a potent inducer of neural progenitor proliferation of both rodent and human derived neural progenitor cells in vitro . in vivo , 
 ap significantly increased neurogenesis within the subgranular zone of the dentate gyrus and subventricular zone of the 3xtgad mouse model . 
 functionally , ap reversed the learning and memory deficits of 3xtgad mice prior to and following the onset of ad pathology and was comparably efficacious in aged normal mice . 
 in addition to inducing regenerative responses in mouse models of ad , ap significantly reduced beta - amyloid burden , beta - amyloid binding alcohol dehydrogenase load , and microglial activation . in parallel , ap increased markers of white matter generation and cholesterol homeostasis . 
 analyses to determine the optimal treatment regimen in the 3xtgad mouse brain indicated that a treatment regimen of ap once per week was optimal for both inducing neurogenesis and reducing ad pathology . 
 pharmacokinetic analyses indicated that ap is rapidly increased in both plasma and brain following a single dose . 
 ap is most efficacious when administered once per week which will contribute to its margin of safety . 
 further , analyses in both animals and humans have provided parameters for safe ap dosage exposure in humans . from a translational perspective 
 , ap is a small molecular weight , blood brain barrier penetrant molecule with substantial preclinical efficacy data as a potential alzheimer s therapeutic with existing safety data in animals and humans . to our knowledge 
 , ap is the only small molecule that both promotes neural progenitor regeneration in brain and simultaneously reduces ad pathology burden . 
Introduction
Preclinical Discovery of Allopregnanolone as a Pro-Neurogenic Agent 
Allopregnanolone as a Multifaceted Pro-Neurogenic Agent in the Triple-Transgenic AD Mouse Model
Translation of Preclinical Allopregnanolone Studies to Clinical Alzheimers Disease Therapy
Conclusion
Conflict of Interest Statement