Yu Tang

DYT1 is an autosomal dominant form of isolated dystonia, which is basically caused by an in-frame 3-bp GAG deletion in the TOR1A gene, leading to loss of a single glutamic acid residue (ΔE) at the C-terminus. TOR1A has been strongly implicated in various biological processes, such as protein quality control and regulation of ER stress. Many of these functions involve as protein multimers between TOR1A and its partners, whereas the ΔE mutant leads to destabilization of their binding, thereby reducing ATPase activation. Despite controversy over its functional model, the dominant-negative nature of TOR1A ^ΔE has been demonstrated in a number of ways. Therefore, it is promising to develop an allele-specific intervention strategy that specifically silences the pathogenic TOR1A allele while preserving the wild-type allele to perform its normal function. In this study, we systematically evaluated the allele-specific targeting of TOR1A ^ΔE using over 20 Cas endonucleases. We found that NmCas9, one of the compact Cas endonucleases yet with high-fidelity, selectively targeted the TOR1A ^ΔE allele, with a 3-nt deletion located in the spacer region of sgRNAs. The discriminatory Nm-sgRNAs were verified both exogenously and endogenously that showed high specificity in disrupting the TOR1A ^ΔE allele but not the wild-type one. Functionally, this strategy efficiently ameliorated the ubiquitin accumulation in DYT1 fibroblasts. Overall, our study demonstrates that the allele-specific targeting of mutant TOR1A with NmCas9 is a promising alternative approach for the treatment of DYT1.

Mucopolysaccharidosis type IIIC (MPS IIIC) is one of inherited lysosomal storage disorders, caused by deficiencies in lysosomal hydrolases degrading acidic mucopolysaccharides. The gene responsible for MPS IIIC isHGSNAT, which encodes an enzyme that catalyses the acetylation of the terminal glucosamine residues of heparan sulfate. So far, few studies have focused on the genetic landscape of MPS IIIC in China, where IIIA and IIIB were the major subtypes. In this study, we utilized whole‐exome sequencing (WES) to identify novel compound heterozygous variants in theHGSNATgene from a Chinese patient with typical MPS IIIC symptoms: c.743G>A; p.Gly248Glu and c.1030C>T; p.Arg344Cys. We performed in silico analysis and experimental validation, which confirmed the deleterious pathogenic nature of both variants, as evidenced by the loss of HGSNAT activity and failure of lysosomal localization. To the best of our knowledge, the MPS IIIC is first confirmed by clinical, biochemical and molecular genetic findings in China. Our study thus expands the spectrum of MPS IIIC pathogenic variants, which is of importance to dissect the pathogenesis and to carry out clinical diagnosis of MPS IIIC. Moreover, this study helps to depict the natural history of Chinese MPS IIIC populations.

Background: Different neural subtypes are selectively lost in diverse neurodegenerative diseases. Huntington’s disease (HD) is an inherited neurodegenerative disease characterized by motor abnormalities that primarily affect the striatum. The Huntingtin (HTT) mutation involves an expanded CAG repeat, leading to insoluble polyQ, which renders GABA⁺ medium spiny neurons (MSN) more venerable to cell death. Human pluripotent stem cells (hPSCs) technology allows for the construction of disease-specific models, providing valuable cellular models for studying pathogenesis, drug screening, and high-throughput analysis. Methods: In this study, we established a method that allows for rapid and efficient generation of MSNs (> 90%) within 21 days from hPSC-derived neural progenitor cells, by introducing a specific combination of transcription factors. Results: We efficiently induced several neural subtypes, in parallel, based on the same cell source, and revealed that, compared to other neural subtypes, MSNs exhibited higher polyQ aggregation propensity and overexpression toxicity, more severe dysfunction in BDNF/TrkB signaling, greater susceptibility to BDNF withdrawal, and more severe disturbances in nucleocytoplasmic transport (NCT). We further found that the nuclear lamina protein LMNB1 was greatly reduced in HD neurons and mislocalized to the cytoplasm and axons. Knockdown of HTT or treatment with KPT335, an orally selective inhibitor of nuclear export (SINE), effectively attenuated the pathological phenotypes and alleviated neuronal death caused by BDNF withdrawal. Conclusions: This study thus establishes an effective method for obtaining MSNs and underscores the necessity of using high-purity MSNs to study HD pathogenesis, especially the MSN-selective vulnerability.

Introduction: Cholesterol gallstone (CGS) is a biliary tract disorder requiring treatment in approximately 20% of patients. The efficacy of Chaihu Shugan in preventing CGS recurrence after successful treatment remains uncertain.Methods: We examined the in vivo preventive efficacy of Chaihu Shugan using a CGS mouse model and used multi-omics to study the interplay between gut microbiota, metabolism, and gene expression.Results: The intestinal microbiota was severely dysregulated during the formation of CGS, showing a marked decrease in the abundance of beneficial microbiota, especially Lactobacillus and Akkermansia. Chaihu Shugan prevented CGS formation by restoring the composition of the gut microbiota and reversing the metabolic disturbances caused by dysbiosis. This preventive effect of Chaihu Shugan was paralleled by changes in the expression of metabolism-related genes in the liver. A network pharmacology analysis of Chaihu Shugan revealed that obacunone may be the key active metabolite in regulating bile acid metabolism. Multi-omics and correlation analyses elucidated the interplay between gut microbiota, metabolism, and gene alterations in the dose-dependent effect of Chaihu Shugan.Conclusion: Our data show that Chaihu Shugan can prevent CGS and indicate its mechanisms of action.

Microglia are resident innate immune cells that play an essential role in the development and surveillance of the central nervous system as well as the shared pathogenesis of neurodegenerative diseases. Microglia rapidly respond to multiple inflammatory stimuli and activate towards different phenotypes, such as pro-inflammatory and anti-inflammatory phenotypes. Cytokines, epigenetic and long non-coding RNA modulations have been shown to regulate microglial activation; however, the role of circRNAs in microglia-mediated neuroinflammation remains elusive. Here, we performed circRNA sequencing in IL-4-treated anti-inflammatory microglia and discovered 120 differentially expressed circRNAs. We systemically verified the identities of circRNAs by assays of PCR, RNase R treatment and fluorescent in situ hybridization (FISH), among others. We found that circAdgre1 promoted IL-4-induced anti-inflammatory responses and further conferred neuroprotective effects upon lipopolysaccharide (LPS) stimuli. Taken together, our results show that circRNAs might be possible therapeutic targets for microglia-mediated neuroinflammation and neurodegenerative diseases.

Studying the pathogenesis of neurological diseases with animal models might not always truly recapitulate their pathophysiology, due to species differences. Fortunately, human pluripotent stem cells (hPSCs) including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), especially derived from patients, have been widely employed to induce neural progenitor cells (NPCs) and further multiple neural subtypes. Particularly in the past decade, hPSC-based cell sources have been applied in studying neural development, cell therapy, disease modeling, and drug screening, among others. The generation of unlimited amount of neurons also facilitates a variety of biochemical assays, mass spectrometry, omic analysis, and next-generation sequencing, which thus provides an excellent tool in modeling neurodegenerative and neurodevelopmental diseases. Dysfunction or death of motor neurons (MNs) in the spinal cord and motor cortex is implicated in various motor neuron diseases (MNDs). Yet, producing high-purity and high-yield MNs remains a major challenge due to the complexity of MN specification during development. In this chapter, we describe a method of generating functional MNs via lentiviral delivery of transcription factors, based on the preservable NPC platform derived from hPSCs. Specifically, we transduce NPCs with a single lentivirus co-expressing three transcription factors including NGN2, ISL1, and LHX3, which is necessary and sufficient to induce mature MNs with high efficiencies (~90%) within 3 weeks. This chapter thus provides a robust method to generate high-purity hPSC-MNs at very high yields, enabling the acquisition of rich patient-specific MNs to be used for modeling the molecular underpinnings of MNDs. Key words Pluripotent stem cells Neural differentiation Neural progenitor cells Motor neuron Transcription factor

GGC repeat expansions within NOTCH2NLC have been identified as the genetic cause of neuronal intranuclear inclusion disease (NIID). To understand the molecular pathogenesis of NIID, here, we established both a transgenic mouse model and a human neural progenitor cells (hNPCs) model. Expression of the NOTCH2NLC with expanded GGC repeats produced widespread intranuclear and perinuclear polyglycine (polyG), polyalanine (polyA), and polyarginine (polyR) inclusions, leading to behavioral deficits and severe neurodegeneration, which faithfully mimicked the clinical and pathological features associated with NIID. Furthermore, conserved alternative splicing events were identified between the NIID mouse and hNPC models, among which was the enrichment of the binding motifs of hnRNPM, an RNA binding protein known as alternative splicing regulator. Expanded NOTCH2NLC-polyG and NOTCH2NLC-polyA could interact with and sequester hnRNPM, while overexpression of hnRNPM could ameliorate the cellular toxicity. These results together suggested that dysfunction of hnRNPM could play an important role in the molecular pathogenesis of NIID.