Progressive degeneration of human neural stem cells caused by pathogenic LRRK2

Nuclear architecture defects have been shown to correlate with the manifestation of a number of human diseases as well as aging1-4. It is then plausible that diseases whose manifestations correlate with aging might be connected to the appearance of nuclear aberrations over time. We decided to evaluate nuclear organization in the context of aging-associated disorders by focusing on a Leucine Rich Repeat Kinase 2 (LRRK2) dominant mutation (G2019S) shown to associate with familial and sporadic Parkinson’s Disease (PD), as well as impairment of adult neurogenesis in mice5. Here, we report on the generation of PD patient-derived induced pluripotent stem cells (iPSCs) and the implications of LRRK2(G2019S) in human neural stem cell (NSC) populations. Mutant NSCs showed increased susceptibility to proteasomal stress as well as passage-dependent deficiencies in clonal expansion and neuronal differentiation. Disease phenotypes were rescued by targeted correction of the LRRK2(G2019S) mutation with its wild-type counterpart in PD-iPSCs and recapitulated upon targeted knock-in of LRRK2(G2019S) in human embryonic stem cells (hESCs). Analysis of human brain tissue showed nuclear envelope impairment in clinically diagnosed Parkinson’s patients. Altogether, our results identify the nucleus as a previously unknown cellular organelle in Parkinson’s pathology and may help open new avenues for PD diagnoses as well as potential development of therapeutics targeting this fundamental cell structure.

diagnosed Parkinson's patients. Altogether, our results identify the nucleus as a previously unknown cellular organelle in Parkinson's pathology and may help open new avenues for PD diagnoses as well as potential development of therapeutics targeting this fundamental cell structure.
To study the role that the nuclear envelope might have during aging-related neurodegenerative processes we chose to focus our attention on Parkinson's disease (PD), the second most prevalent neurodegenerative disease among aging individuals. LRRK2 is a large multidomain protein bearing kinase activity 10 whose mutations correlate with inherited and sporadic PD 10,11 . Specifically, G2019S mutation leads to an increase in LRRK2 kinase activity correlating with the manifestation of disease 3,10-13 . However, the molecular and cellular implications of LRRK2(G2019S) during PD manifestation and progression remain elusive.
To this end, we first decide to evaluate potential novel interactions. The fact that LRRK2(G2019S) leads to impaired adult neurogenesis in mice 5 , led us to take advantage of iPSC technologies and their potential for directed differentiation into specific human cell lineages ( Supplementary Figs. 3 and 4). Differentiation of iPSCs into neural progenitors followed by serial passaging resembling cellular "aging" 14 showed progressive deterioration of nuclear architecture in mutant but not in wild-type NSCs (ipsNSCs-LRRK2(G2019S) and ipsNSCs-wt respectively). Nuclear aberrations started to manifest by passage 14 and progressively resulted in compartmentalized pedal-like nuclei ( Fig. 1a- 19), demonstrated a markedly enlarged nuclear area, accompanied by a significant decrease in nuclear circularity, in ipsNSCs-LRRK2(G2019S) (Fig.1c-e, Supplementary Fig. 5c and Supplementary Fig. 8a). Noticeably, late passage ipsNSCs-LRRK2(G2019S) exhibited local loss of Lamin B1 and B2 at specific folds of the nuclear envelope (Fig. 1c,f and g, and Supplementary Figs 6e and 7) whereas all other examined nuclear components remained correctly localized ( Fig. 1f and Supplementary Figs. 6e and 7). Co-immunoprecipitation experiments revealed that ectopically expressed LRRK2(G2019S) associated in protein complexes with both, Lamin B1 and Lamin B2 (Fig. 1h). Furthermore, increased phosphorylation of Lamin B1 and Lamin B2 was observed in late passage ipsNSC-LRRK2(G2019S) (Supplementary Fig. 8b), suggesting that excessive LRRK2 kinase activation may directly or indirectly stimulate phosphorylation of B-type lamins in NSCs. Lastly, analysis of mitochondrial structure, an organelle frequently implicated in PD pathogenesis 10 , demonstrated the normal expression of the outer mitochondrial membrane receptor TOM20 as well as the mitochondrial heat shock protein HSP60 in both NSCs-wt and NSCs-LRRK2(G2019S) (Supplementary Fig. 9). We next wondered whether LRRK2(G2019S)-elicited nuclear defects contributed to epigenetic alterations such as those observed during cellular aging 1,2,15 . Fluorescence in situ hybridization revealed a dramatic increase of centromeric signals accompanied by reorganization of centromeric, but not telomeric, heterochromatin in passage 15 ipsNSCs-LRRK2(G2019S) (Supplementary Fig. 10a). Genome-wide epigenetic analysis demonstrated passage-dependent differences in global H3K4me3 modifications, an epigenetic mark associated with human neuronal aging 16 , at the analyzed promoter regions ( Fig. 2a and Supplementary Fig. 10b). At passage 15, 437 genes in ipsNSCs-LRRK2(G2019S) displayed ≥5-fold enrichment for the epigenetic mark H3K4me3 as compared to ipsNSCs-wt whereas no significant differences were observed when comparing undifferentiated iPSCs. Interestingly, most of these genes were associated with neurogenesis and neural function (Supplementary Table 2).
These results led us to hypothesize that LRRK2(G2019S) might progressively affect other in vitro NSC functions such as clonal expansion and neural differentiation. Newly generated ipsNSCs-wt and ipsNSCs-LRRK2(G2019S) were similarly capable of both clonal expansion and neural differentiation, whereas only ipsNSCs-wt retained clonogenic and differentiation capacity upon extensive passaging (≥14) ( To further validate that the observed cellular phenotypes were related to NSCs carrying the LRRK2(G2019S) mutation, we evaluated different somatic cell types as well as generated two different unbiased cellular systems, an isogenic control iPSC line by targeted correction of the LRRK2(G2019S) mutation as well as the reversal by specific knock-in of LRRK2(G2019S) into human Embryonic Stem Cells (hESCs) 18 Supplementary Figs. 13 and 14). Altogether, our results suggested a specific role for LRRK2(G2019S) during the manifestation of passage-dependent NSC abnormal phenotypes and highlighted the nucleus as a potential novel organelle affected in PD.
Chemical inhibition has been previously shown to protect against LRRK2 associated toxicity in vitro and in vivo 13,22 . Accordingly, treating late-passage ipsNSCs-LRRK2(G2019S) with LRRK2-In-1 12, 22 resulted in reduced phosphorylation of LRRK2 downstream targets and significantly rescued the aberrant cellular parameters observed in late-passage ipsNSCs-LRRK2(G2019S), (Fig. 4b-f, Supplementary Fig. 6a-c, and Supplementary Movie 1). Microarray analysis demonstrated that 5-day incubation of passage 14 esNSCs-LRRK2(G2019S) with LRRK2-In-1 was sufficient to restore a gene expression signature similar to that of wild-type esNSCs and significantly different from untreated controls (Fig. 4g; Supplementary Table 3). Further studies on cross-talking signaling pathways suggested that LRRK2-In-1 was capable of restoring both nuclear morphology and colony-like growth in late passage ipsNSCs-LRRK2(G2019S), whereas, LY294002, a PI3K inhibitor, partially rescued nuclear morphology (Supplementary Fig. 16c  and 17).
Lastly, we investigated nuclear morphology in post-mortem human brain samples. Lamin B1 immunostaining demonstrated a high proportion of cells displaying altered nuclear morphologies within the hippocampal dentate gyrus, but not in non-neurogenic areas such as cortex regions, in all the analyzed LRRK2(G2019S)-PD patients' samples as compared to their respective age-matched controls (78.82%±6.28 and 16.49%±4.26 respectively) (Fig.  4h, Supplementary Table 5 and Supplementary Fig. 18). Interestingly, nuclear aberrations were not only present in LRRK2(G2019S) but also in several idiopathic PD human brain samples (47.28%±4.217). Of note, control samples also displayed nuclear disruption to a certain extent, which might be potentially due to the described association of physiological aging and the nuclear envelope 27 (Fig. 4h and Supplementary Fig. 18). Whereas due to the reduced number of samples (n=5) we cannot conclude that the above mentioned phenotypes have a 100% penetrance in all PD patients, these results indicate the possibility that dysfunctional NSC pools, and/or their respective neuronal derivatives at early stages of maturation, might contribute to the hippocampal and subventricular zone-related agedependent non-motor symptoms, including depression, anxiety and hyposmia, associated with LRRK2(G2019S)-bearing individuals and PD 5,29 .
Altogether, LRRK2(G2019S)-elicited aberrant phenotypic manifestations seemed particularly prominent in human NSC populations and were more noticeable at late passages. Of most relevance, our studies further indicate the suitability of iPSC-based models of disease and aging not only for the in vitro recapitulation of known disease phenotypes, as extensively described, but also as a platform for discovery and study of otherwise novel and elusive cell parameters and populations 30 (Supplementary Fig. 1). Furthermore, through the analysis of human brain samples, our study demonstrates that these cellular aberrations can also be found in vivo. The fact that this validation was only possible in human samples once a reliable iPSC in vitro system was employed further highlights the potential of patient-specific iPSCs, not only for disease modeling but, most importantly, for the advancement of disease pathology knowledge.

Method summary iPSC generation
Parkinson's disease fibroblasts were obtained from Telethon Genetic Biobank Network and brain patient samples from the Biobank of hospital clinic (IDIBAPS) in Barcelona. Normal fibroblasts were purchased from Coriell Cell Repository. Fibroblasts were reprogrammed with retroviruses expressing OCT4, SOX2, KLF4. The generated iPSC lines were maintained in hESC medium on iMEF feeder cells or in mTeSR on Matrigel 1, 18 .

Statistical analysis
Results are presented as mean±s.d. or mean±s.e.m. for at least three independent biological replicates. Comparisons were performed with student's t-test. Distributions of nuclear area and nuclear envelope (NE) circularity were analyzed with the Kolmogorov-Smirnov test.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.