Progressive degeneration of human neural stem cells caused by pathogenic LRRK2

Abstract

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.

Large-scale generation of human iPSC-derived neural stem cells/early neural progenitor cells and their neuronal differentiation

“…Compelling evidence now indicates that iPSC-based models can be used to model selected aspects of neurological and neurodegenerative disorders. [5][6][7] Besides their potential to provide important molecular insights into pathogenic mechanisms, iPSC-based cellular platforms can also be used for drug discovery in specific differentiated cell types. 8 Such platforms require replicable, efficient, and cost effective protocols to generate uniform cultures of neurons in sufficient numbers to enable screening of potentially thousands of different compounds.…”

Global DNA methylation and transcriptional analyses of human ESC-derived cardiomyocytes

“…Differentiation of H9 hESC into hNSC was followed by the protocol described previously (Liu et al, 2012).…”

“…hESC-derived hNSCs ( Liu et al, 2012) were used as a control population. All the cells shared the same genetic background (H9), allowing for an unbiased side-by-side comparison of their gene expression profile.…”

Use of the CRISPR/Cas9 system as an intracellular defense against HIV-1 infection in human cells

“…For HdAdV production, Venus complementary DNA and Venus-expression HdAdV (pHdAdVenus-geo-TK) were kindly provided by Drs A. Miyawaki and K. Mitani 42,43 . The HdAdV production, purification and titration were shown in a previous paper 44 HIV-1 reporter virus preparation and infection. HIV-1 NL43-GFP was generated from pNL4-3 Nef þ IRES eGFP vector 45 .…”