Identification and characterization of novel genetic variants in the first Chinese family of mucopolysaccharidosis IIIC (Sanfilippo C syndrome).

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 is HGSNAT, 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 the HGSNAT gene 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.

Rapid and high-purity differentiation of human medium spiny neurons reveals LMNB1 hypofunction and subtype necessity in modeling Huntington's disease.

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.

Characterization of the circRNA Landscape in Interleukin-4 Induced Anti-Inflammatory Microglia.

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.

Transcription Factor-Mediated Differentiation of Motor Neurons from Human Pluripotent Stem Cells.

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.

The efficient generation of knockout microglia cells using a dual-sgRNA strategy by CRISPR/Cas9.

Gene deletion in microglia has become an important and exciting approach for studying neuroinflammation, especially after the development of the CRISPR/Cas9 system for genome editing during the last decade. In this study, we described a protocol for the highly efficient generation of knockout microglia cells using a dual-short guide RNA (sgRNA) strategy by CRISPR/Cas9. Leucine-rich repeat kinase 2 (LRRK2), a pathogenic gene of Parkinson's disease (PD), has played versatile roles during the disease development. Despite many key insights into LRRK2 studies, the normal and disease-related functions of LRRK2 in microglia and neuroinflammation remain to be fully investigated. Given the importance of LRRK2 in PD pathogenesis, we designed and applied the protocol to target LRRK2. Specifically, we designed two sgRNAs targeting the N terminus of LRRK2, spanning the 5' untranslated region (UTR) and exon 1, and screened knockout cells by single-cell expansion. In practice, the dual-sgRNA system can facilitate in obtaining knockout cells in a more convenient, rapid, and accurate way. Candidate knockout cells can be easily distinguished by genomic PCR and running on agarose gels, based on the different band sizes. Successful knockouts were further verified by Sanger sequencing and Western blot. Using this protocol, we obtained an LRRK2-deficient microglia cell line, which was characterized by longer cellular processes, enhanced adhesion, and weakened migration capacity. The knockout microglia may further serve as an important cellular tool to reveal conserved and novel aspects of LRRK2 functions in the development and progression of PD. Our protocol using dual-sgRNA targeting guarantees > 60% targeting efficiency and could also be applied to targeting other genes/loci, especially non-coding RNAs and regulatory elements.

The heterogeneity of microglial activation and its epigenetic and non-coding RNA regulations in the immunopathogenesis of neurodegenerative diseases.

Microglia are resident immune cells in the brain and play a central role in the development and surveillance of the nervous system. Extensive gliosis is a common pathological feature of several neurodegenerative diseases, such as Alzheimer's disease (AD), the most common cause of dementia. Microglia can respond to multiple inflammatory insults and later transform into different phenotypes, such as pro- and anti-inflammatory phenotypes, thereby exerting different functions. In recent years, an increasing number of studies based on both traditional bulk sequencing and novel single-cell/nuclear sequencing and multi-omics analysis, have shown that microglial phenotypes are highly heterogeneous and dynamic, depending on the severity and stage of the disease as well as the particular inflammatory milieu. Thus, redirecting microglial activation to beneficial and neuroprotective phenotypes promises to halt the progression of neurodegenerative diseases. To this end, an increasing number of studies have focused on unraveling heterogeneous microglial phenotypes and their underlying molecular mechanisms, including those due to epigenetic and non-coding RNA modulations. In this review, we summarize the epigenetic mechanisms in the form of DNA and histone modifications, as well as the general non-coding RNA regulations that modulate microglial activation during immunopathogenesis of neurodegenerative diseases and discuss promising research approaches in the microglial era.

The Double-Faceted Role of Leucine-Rich Repeat Kinase 2 in the Immunopathogenesis of Parkinson's Disease.

Leucine-rich repeat kinase 2 (LRRK2) is one of the most common causative genes in Parkinson's disease (PD). The complex structure of this multiple domains' protein determines its versatile functions in multiple physiological processes, including migration, autophagy, phagocytosis, and mitochondrial function, among others. Mounting studies have also demonstrated the role of LRRK2 in mediating neuroinflammation, the prominent hallmark of PD, and intricate functions in immune cells, such as microglia, macrophages, and astrocytes. Of those, microglia were extensively studied in PD, which serves as the resident immune cell of the central nervous system that is rapidly activated upon neuronal injury and pathogenic insult. Moreover, the activation and function of immune cells can be achieved by modulating their intracellular metabolic profiles, in which LRRK2 plays an emerging role. Here, we provide an updated review focusing on the double-faceted role of LRRK2 in regulating various cellular physiology and immune functions especially in microglia. Moreover, we will summarize the latest discovery of the three-dimensional structure of LRRK2, as well as the function and dysfunction of LRRK2 in immune cell-related pathways.

Identification and characterization of two novel noncoding tyrosinase (TYR) gene variants leading to oculocutaneous albinism type 1.

Oculocutaneous albinism type 1 (OCA1), resulting from pathogenic variants in the tyrosinase (TYR) gene, refers to a group of phenotypically heterogeneous autosomal recessive disorders characterized by a partial or a complete absence of pigment in the skin/hair and is also associated with common developmental eye defects. In this study, we identified two novel compound heterozygous TYR variants from a Chinese hypopigmentary patient by whole-exome sequencing. Specifically, the two variants were c.-89T>G, located at the core of the initiator E-box (Inr E-box) of the TYR promoter, and p.S16Y (c.47C>A), located within the signal sequence. We performed both in silico analysis and experimental validation and verified these mutations as OCA1 variants that caused either impaired or complete loss of function of TYR. Mechanistically, the Inr E-box variant dampened TYR binding to microphthalmia-associated transcription factor, a master transcriptional regulator of the melanocyte development, whereas the S16Y variant contributed to endoplasmic reticulum retention, a common and principal cause of impaired TYR activity. Interestingly, we found that the Inr E-box variant creates novel protospacer adjacent motif sites, recognized by nucleases SpCas9 and SaCas9-KKH, respectively, without compromising the functional TYR coding sequence. We further used allele-specific genomic editing by CRISPR activation to specifically target the variant promoter and successfully activated its downstream gene expression, which could lead to potential therapeutic benefits. In conclusion, this study expands the spectrum of TYR variants, especially those within the promoter and noncoding regions, which can facilitate genetic counseling and clinical diagnosis of OCA1.

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OBJECTIVES: To investigate the risk factors for serious infections among hospitalized systemic lupus erythematosus (SLE) patients, and to provide the advice for preventing serious infections in SLE patients. METHODS: Information of SLE patients hospitalized from March 2017 to February 2019 at the Department of Rheumatology and Immunology, Xiangya Hospital, Central South University was obtained. The patients were assigned into a serious infection group and a non-serious infection group. The risk factors for serious infections among SLE inpatients were identified by comparison between the 2 groups and multivariate logistic regression analysis. RESULTS: There were 463 SLE inpatients in total, and 144 were in the serious infection group and 319 in the non-serious infection group. Multivariate logistic regression analysis showed that age ≥54.50 years old (OR=4.958, P<0.001), cardiovascular involvement (OR=6.287, P<0.001), hematologic involvement (OR=2.643, P=0.003), serum albumin <20 g/L (OR=2.340, P=0.036), C-reaction protein (CRP)/erythrocyte sedimentation rate (ESR)≥0.12 (OR=2.430, P=0.002), glucocorticoid dose ≥8.75 mg/d prednisone-equivalent (OR=2.465, P=0.002), and the combined use of immunosuppressive agents (OR=2.847, P=0.037) were the risk factors for serious infections in SLE inpatients. CONCLUSIONS: SLE patients with older age, cardiovascular involvement, hematologic involvement, low serum albumin are prone to suffering serious infections. Increased CRP/ESR ratio indicates serious infections in SLE inpatients. High-dose glucocorticoid and the combined use of immunosuppressive agents can increase the risk of serious infections in SLE inpatients.

An induced pluripotent stem cell line (CSUi004-A) from skin fibroblasts of a healthy individual.

We established an induced pluripotent stem cell (iPSC) reprogrammed from dermal fibroblasts of a 53-year-old healthy man. Retroviruses expressing Yamanaka factors (OCT4, SOX2, KLF4 and c-MYC) were employed to initiate the reprogramming and were silenced in iPSCs. The generated iPSCs were pluripotent as determined by immunostaining and expression of pluripotency markers. They were further induced as embryoid bodies in vitro and teratomas in vivo, reminiscent of their full capacity of differentiating into three germ layers. They are with a normal karyotype and genetically identical to donor fibroblasts. This iPSC line provides excellent cell sources for studying and modeling human-specific physiology.

Establishment of an induced pluripotent stem cell line (CSUi003-A) from fibroblasts of a healthy elderly individual.

Skin fibroblasts derived from a 71-year-old healthy woman were reprogrammed into induced pluripotent stem cells (iPSCs) by the transduction of retroviruses expressing OCT4, SOX2, KLF4 and c-MYC, respectively. The generated iPSCs maintained a normal karyotype and expressed various pluripotency markers. Moreover, they could be induced to form embryoid bodies in vitro and teratomas in vivo, indicating the full capacity of differentiating into three germ layers. This iPSC line could be differentiated into multiple cell subtypes for cellular modeling and drug discovery.

Generation of patient-specific induced pluripotent stem cell line (CSUi002-A) from a patient with isolated dystonia carrying TOR1A mutation.

Early onset isolated dystonia (DYT1) is a hereditary neurological movement disease caused by a single amino-acid deletion in torsin A (TOR1A), a gene encoding a membrane-embedded ATPase. In this study, we generated an induced pluripotent stem cell (iPSC) line from fibroblasts of a DYT1 patient by the retroviral transduction of Yamanaka factors. The iPSCs retained the heterozygous TOR1A mutation (p.Glu303del), showed a normal karyotype, expressed pluripotency markers and exhibited the potential to differentiate into three germ layers both in vitro and in vivo. This DYT1 patient-specific iPSC will be used for modeling the dystonia pathophysiology and probably drug screening.

Revisiting the Immune Balance Theory: A Neurological Insight Into the Epidemic of COVID-19 and Its Alike.

As the pandemic of COVID-19 is raging around the world, the mysteriousness of severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) coronavirus is being revealed by the concerted endeavors of scientists. Although fever and pneumonia are typical symptoms, COVID-19 patients exhibit multiple neurological complications. In this interim review, we will summarize the neurological manifestations and their potential causes in COVID-19. Similar to the other two fatal respiratory coronaviruses, SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV), SARS-CoV-2 also shows to be neuroinvasive that may spread from the periphery to brain, probably by the retrograde axonal transport. The invaded viruses may directly disrupt the complex neural circuits, and raise a chronic activation of immune responses. In another hand, multiple organ failure in severe COVID-19 is caused by the systemic acute immune responses, and unsurprisingly caused the brain inflammation and led to encephalitis. However, in the central nervous system (CNS), the activation of resident immune cells including microglia and astrocytes may lead to chronic immune imbalance, which underlies the potential long-term effects in synaptic changes and neuropsychiatric impairments. The neuroinvasive biology also provides a possible link with the Braak staging of neurodegenerative diseases such as Parkinson's disease (PD). Although with considerable advances, the neurotropic potential and chronic neurological effects caused by SARS-CoV-2 infections merit further investigations.

Expression and role of ERAP1 in placental tissues of preeclampsia.

Objective: This study aimed to investigate the expression and role of ERAP1 in preeclampsia pathogenesis.Methods: ERAP1 expression of placental tissues was analyzed by Real-Time PCR and western blot. The location of ERAP1 was detected by immunohistochemistry. Cellular preeclampsia model was established by hypoxia treatment of HTR-8/SVneo cells (2% oxygen).Results: The present study revealed that ERAP1 expression were significantly elevated in placental tissues of preeclampsia, and hypoxiaincreased ERAP1 expression in trophoblast, suggesting that ERAP-1 may be involved in the development of preeclampsia.Conclusion: These finds highlight the role of ERAP1 in the pathogenesis of preeclampsia.

Allele-specific genome targeting in the development of precision medicine.

The CRISPR-based genome editing holds immense potential to fix disease-causing mutations, however, must also handle substantial natural genetic variations between individuals. Previous studies have shown that mismatches between the single guide RNA (sgRNA) and genomic DNA may negatively impact sgRNA efficiencies and lead to imprecise specificity prediction. Hence, the genetic variations bring about a great challenge for designing platinum sgRNAs in large human populations. However, they also provide a promising entry for designing allele-specific sgRNAs for the treatment of each individual. The CRISPR system is rather specific, with the potential ability to discriminate between similar alleles, even based on a single nucleotide difference. Genetic variants contribute to the discrimination capabilities, once they generate a novel protospacer adjacent motif (PAM) site or locate in the seed region near an available PAM. Therefore, it can be leveraged to establish allele-specific targeting in numerous dominant human disorders, by selectively ablating the deleterious alleles. So far, allele-specific CRISPR has been increasingly implemented not only in treating dominantly inherited diseases, but also in research areas such as genome imprinting, haploinsufficiency, spatiotemporal loci imaging and immunocompatible manipulations. In this review, we will describe the working principles of allele-specific genome manipulations by virtue of expanding engineering tools of CRISPR. And then we will review new advances in the versatile applications of allele-specific CRISPR targeting in treating human genetic diseases, as well as in a series of other interesting research areas. Lastly, we will discuss their potential therapeutic utilities and considerations in the era of precision medicine.

Targeting N-Terminal Huntingtin with a Dual-sgRNA Strategy by CRISPR/Cas9.

Huntington's disease (HD) is an autosomal dominant progressive neurodegenerative disorder, caused by a CAG/polyglutamine (polyQ) repeat expansion in the Huntingtin (HTT) gene. The polyQ tract is located in and transcribed from N-terminal HTT of exon 1. HTT is a large multifaceted protein, which participates in a range of cellular functions. Previous studies have shown that truncated HTT, which lacks N-terminus, retains specific functions that can produce neuroprotective benefits. It gives an insight that it is possible to repair HD by removing deleterious N-terminal HTT with CRISPR/Cas9, without compromising functions of remaining HTT peptides. To successfully generate functional truncated HTT proteins, an alternative downstream ATG start codon that is capable of initiating truncated HTT expression is required. In this study, we searched all possible in-frame ATGs before exon 7 and demonstrated that one of them can indeed initiate the downstream GFP expression in plasmids. We then tried to remove endogenous N-terminal HTT with an optimized dual-sgRNA strategy by CRISPR/Cas9; however, we cannot detect obvious traits of truncated HTT expression. Our results suggest that noncanonical ATGs of N-terminal HTT may not be effective in the genomic context, as in the construct context. Nevertheless, our study examined the therapeutic efficacy of downstream noncanonical ATGs for protein translation and also provided an optimized dual-sgRNA strategy for further genome manipulation of the HTT gene.

Protective Microglia and Their Regulation in Parkinson's Disease.

Microglia-mediated neuroinflammation is a hallmark of Parkinson's disease (PD). In the brains of patients with PD, microglia have both neurotoxic and neuroprotective effects, depending on their activation state. In this review, we focus on recent research demonstrating the neuroprotective role of microglia in PD. Accumulating evidence indicates that the protective mechanisms of microglia may result from their regulation of transrepression pathways via nuclear receptors, anti-inflammatory responses, neuron-microglia crosstalk, histone modification, and microRNA regulation. All of these mechanisms work together to suppress the production of neurotoxic inflammatory components. However, during the progression of PD, the detrimental effects of inflammation overpower the protective actions of microglia. Therefore, an in-depth exploration of the mechanisms underlying microglial neuroprotection, and a means of promoting the transformation of microglia to the protective phenotype, are urgently needed for the treatment of PD.

Insufficient screening knowledge in Chinese interns: a survey in ten leading medical schools.

OBJECTIVE: This study aimed to investigate Chinese medical interns' cancer knowledge and associated factors, focusing on cancer screening. METHODS: A questionnaire survey was conducted in ten leading Chinese medical schools from June to July in 2011. Medical interns were invited to fill the questionnaire. RESULTS: Out of the 1350 copies sent, 1135 eligible responses were returned. Around 50% of interns had positive attitude toward oncology, but the knowledge score was low, particularly in screening. The percentages of scores were 44.8% (8.95/20) for overall and only 29.6% (2.07/7) for screening. The majority of internship length in oncology department was eight to fourteen days. Screening and prevention was ranked as third most taught, following diagnosis and treatment. Multivariate analysis showed that positive attitude to oncology correlated with positive self-evaluated overall (OR = 1.76, 95% CI (1.45, 2.12)) and screening (OR = 1.62, 95% CI (1.35, 1.95)) competence, but unexpectedly predicted lower screening score (OR = 0.77, 95% CI (0.61, 0.97)). Interns with positive self-evaluated screening competence were not found to possess higher cancer screening knowledge. CONCLUSION: Current medical education in Chinese medical schools fails to equip interns with optimal cancer knowledge, particularly in screening, even in interns who hold positive view to oncology. Interns' self-evaluated competence is not proportional to their knowledge scores.