Anthony Wing Sang Chan

Trinucleotide repeats (TNRs) are dispersed throughout the human genome. About 20 loci are related to human diseases, such as Huntington’s disease (HD). A larger TNR instability is predominantly observed in the paternal germ cells in some TNR disorders. Suppressing the expansion during spermatogenesis can provide a unique opportunity to end the vicious cycle of genetic anticipation. Here, using an in vitro differentiation method to derive advanced spermatogenic cells, we investigated the efficacy of two therapeutic agents, araC (cytarabine) and aspirin, on stabilizing TNRs in spermatogenic cells. Two WT patient-derived induced pluripotent stem cell (iPSC) lines and two HD hiPSC lines, with 44 Q and 180 Q, were differentiated into spermatogonial stem cell-like cells (SSCLCs). Both HD cell lines showed CAG tract expansion in SSCLC. When treated with araC and aspirin, HD1 showed moderate but not statistically significant stabilization of TNR. In HD2, 10 nM of aspirin and araC showed significant stabilization of TNR. All cell lines showed increased DNA damage response (DDR) gene expression in SSCLCs while more genes were significantly induced in HD SSCLC. In HD1, araC and aspirin treatment showed general suppression of DNA damage response genes. In HD2, onlyFAN1,OGG1, andPCNAshowed significant suppression. When the methylation profile of HD cells was analyzed,FAN1andOGG1showed significant hypermethylation after the aspirin and araC treatment in SSCLC compared to the control. This study underscores the utility of our in vitro spermatogenesis model to study and develop therapies for TNR disorders such as HD.

Huntington’s Disease (HD) is an autosomal dominant disease that results in severe neurodegeneration with no cure. HD is caused by the expanded CAG trinucleotide repeat (TNR) on the Huntingtin gene (HTT). Although the somatic and germline expansion of the CAG repeats has been well-documented, the underlying mechanisms had not been fully delineated. Increased CAG repeat length is associated with a more severe phenotype, greater TNR instability, and earlier age of onset. The direct relationship between CAG repeat length and molecular pathogenesis makes TNR instability a useful measure of symptom severity and tissue susceptibility. Thus, we examined the tissue-specific TNR instability of transgenic nonhuman primate models of Huntington’s disease. Our data show a similar profile of CAG repeat expansion in both rHD1 and rHD7, where high instability was observed in testis, liver, caudate, and putamen. CAG repeat expansion was observed in all tissue samples, and tissue- and CAG repeat size-dependent expansion was observed. Correlation analysis of CAG repeat expansion and the gene expression profile of four genes in different tissues, clusterin (CLU), transferrin (TF), ribosomal protein lateral stalk subunit P1 (RPLP1), and ribosomal protein L13a (RPL13A), showed a strong correlation with CAG repeat instability. Overall, our data, along with previously published studies, can be used for studying the biology of CAG repeat instability and identifying new therapeutic targets.

Gene regulatory divergence is thought to play a central role in determining human-specific traits. However, our ability to link divergent regulation to divergent phenotypes is limited. Here, we utilized human–chimpanzee hybrid induced pluripotent stem cells to study gene expression separating these species. The tetraploid hybrid cells allowed us to separate cis- from trans-regulatory effects, and to control for nongenetic confounding factors. We differentiated these cells into cranial neural crest cells, the primary cell type giving rise to the face. We discovered evidence of lineage-specific selection on the hedgehog signaling pathway, including a human-specific sixfold down-regulation of EVC2 (LIMBIN), a key hedgehog gene. Inducing a similar down-regulation of EVC2 substantially reduced hedgehog signaling output. Mice and humans lacking functional EVC2 show striking phenotypic parallels to human–chimpanzee craniofacial differences, suggesting that the regulatory divergence of hedgehog signaling may have contributed to the unique craniofacial morphology of humans.

Purpose The expansion of CAG (glutamine; Q) trinucleotide repeats (TNRs) predominantly occurs through male lineage in Huntington’s disease (HD). As a result, offspring will have larger CAG repeats compared to their fathers, which causes an earlier onset of the disease called genetic anticipation. This study aims to develop a novel in vitro model to replicate CAG repeat instability in early spermatogenesis and demonstrate the biological process of genetic anticipation by using the HD stem cell model for the first time. Methods HD rhesus monkey embryonic stem cells (rESCs) were cultured in vitro for an extended period. Male rESCs were used to derive spermatogenic cells in vitro with a 10-day differentiation. The assessment of CAG repeat instability was performed by GeneScan and curve fit analysis. Results Spermatogenic cells derived from rESCs exhibit progressive expansion of CAG repeats with high daily expansion rates compared to the extended culture of rESCs. The expansion of CAG repeats is cell type–specific and size-dependent. Conclusions Here, we report a novel stem cell model that replicates genome instability and CAG repeat expansion in in vitro derived HD monkey spermatogenic cells. The in vitro spermatogenic cell model opens a new opportunity for studying TNR instability and the underlying mechanism of genetic anticipation, not only in HD but also in other TNR diseases.

Amitriptyline is a tricyclic antidepressant commonly prescribed in humans for pain and sleep disorders and in non-human primates for self-injurious behaviors. Here, we report a clinical case on the teratogenic effect of maternal-fetal amitriptyline exposure.

Background: Huntington’s Disease (HD) is a fatal neurodegenerative disorder caused by a CAG repeat expansion, resulting in a mutant huntingtin protein. While it is now clear that astrocytes are affected by HD and significantly contribute to neuronal dysfunction and pathogenesis, the alterations in the transcriptional and epigenetic profiles in HD astrocytes have yet to be characterized. Here, we examine global transcription and chromatin accessibility dynamics during in vitro astrocyte differentiation in a transgenic non-human primate model of HD.Results: We found global changes in accessibility and transcription across different stages of HD pluripotent stem cell differentiation, with distinct trends first observed in neural progenitor cells (NPCs), once cells have committed to a neural lineage. Transcription of p53 signaling and cell cycle pathway genes was highly impacted during differentiation, with depletion in HD NPCs and upregulation in HD astrocytes. E2F target genes also displayed this inverse expression pattern, and strong associations between E2F target gene expression and accessibility at nearby putative enhancers were observed.Conclusions: The results suggest that chromatin accessibility and transcription are altered throughout in vitro HD astrocyte differentiation and provide evidence that E2F dysregulation contributes to aberrant cell-cycle re-entry and apoptosis throughout the progression from NPCs to astrocytes.

The expanded CAG repeat results in somatic mosaicism and genetic anticipation in Huntington’s disease (HD). Here we report a longitudinal study examining CAG repeat instability in lymphocytes and sperm of transgenic throughout the whole life-span of

Huntington’s disease (HD) is a dominantly inherited monogenetic disorder characterized by motor and cognitive dysfunction due to neurodegeneration. The disease is caused by the polyglutamine (polyQ) expansion at the 5′ terminal of the exon 1 of the huntingtin (HTT) gene, IT15, which results in the accumulation of mutant HTT (mHTT) aggregates in neurons and cell death. The monogenetic cause and the loss of specific neural cell population make HD a suitable candidate for stem cell and gene therapy. In this study, we demonstrate the efficacy of the combination of stem cell and gene therapy in a transgenic HD mouse model (N171-82Q; HD mice) using rhesus monkey (Macaca mulatta) neural progenitor cells (NPCs). We have established monkey NPC cell lines from induced pluripotent stem cells (iPSCs) that can differentiate into GABAergic neurons in vitro as well as in mouse brains without tumor formation. Wild-type monkey NPCs (WT-NPCs), NPCs derived from a transgenic HD monkey (HD-NPCs), and genetically modified HD-NPCs with reduced mHTT levels by stable expression of small-hairpin RNA (HD-shHD-NPCs), were grafted into the striatum of WT and HD mice. Mice that received HD-shHD-NPC grafts showed a significant increase in lifespan compared to the sham injection group and HD mice. Both WT-NPC and HD-shHD-NPC grafts in HD mice showed significant improvement in motor functions assessed by rotarod and grip strength. Also, immunohistochemistry demonstrated the integration and differentiation. Our results suggest the combination of stem cell and gene therapy as a viable therapeutic option for HD treatment.

Huntington’s disease (HD) is a devastating monogenic, dominant, hereditary, neurodegenerative disease. HD is caused by the expansion of CAG repeats in exon 1 of the huntingtin (HTT) gene, IT15, resulting in an expanded polyglutamine (polyQ) residue in the N-terminus of the HTT protein. HD is characterized by the accumulation of mutant HTT (mHTT) in neural and somatic cells. Progressive brain atrophy occurs initially in the striatum and extends to different brain regions with progressive decline in cognitive, behavioral and motor functions. Astrocytes are the most abundant cell type in the brain and play an essential role in neural development and maintaining homeostasis in the central nervous system (CNS). There is increasing evidence supporting the involvement of astrocytes in the development of neurodegenerative diseases such as Parkinson’s disease (PD), Huntington’s disease (HD), Alzheimer’s disease (AD), and amyotrophic lateral sclerosis (ALS). We have generated neural progenitor cells (NPCs) from induced pluripotent stem cells (iPSCs) of transgenic HD monkeys as a model for studying HD pathogenesis. We have reported that NPCs can be differentiated in vitro into mature neural cells, such as neurons and glial cells, and are an excellent tool to study the pathogenesis of HD. To better understand the role of astrocytes in HD pathogenesis and discover new therapies to treat HD, we have developed an astrocyte differentiation protocol and evaluated the efficacy of RNAi to ameliorate HD phenotypes in astrocytes. The resultant astrocytes expressed canonical astrocyte-specific markers examined by immunostaining and real-time PCR. Flow cytometry (FACS) analysis showed that the majority of the differentiated NPCs (95.7%) were positive for an astrocyte specific marker, glial fibrillary acidic protein (GFAP). Functionalities of astrocytes were evaluated by glutamate uptake assay and electrophysiology. Expression of mHTT in differentiated astrocytes induced cytosolic mHTT aggregates and nuclear inclusions, suppressed the expression of SOD2 and PGC1, reduced ability to uptake glutamate, decreased 4-aminopyridine (4-AP) response, and shifted I/V plot measured by electrophysiology, which are consistent with previous reports on HD astrocytes and patient brain samples. However, expression of small-hairpin RNA against HTT (shHD) ameliorated and reversed aforementioned HD phenotypes in astrocytes. This represents a demonstration of a novel non-human primate (NHP) astrocyte model for studying HD pathogenesis and a platform for discovering novel HD treatments.