Longitudinal single-cell transcriptional dynamics throughout neurodegeneration in SCA1.

Neurodegeneration is a protracted process involving progressive changes in myriad cell types that ultimately results in the death of vulnerable neuronal populations. To dissect how individual cell types within a heterogeneous tissue contribute to the pathogenesis and progression of a neurodegenerative disorder, we performed longitudinal single-nucleus RNA sequencing of mouse and human spinocerebellar ataxia type 1 (SCA1) cerebellar tissue, establishing continuous dynamic trajectories of each cell population. Importantly, we defined the precise transcriptional changes that precede loss of Purkinje cells and, for the first time, identified robust early transcriptional dysregulation in unipolar brush cells and oligodendroglia. Finally, we applied a deep learning method to predict disease state accurately and identified specific features that enable accurate distinction of wild-type and SCA1 cells. Together, this work reveals new roles for diverse cerebellar cell types in SCA1 and provides a generalizable analysis framework for studying neurodegeneration.

Reduction of nemo-like kinase increases lysosome biogenesis and ameliorates TDP-43-related neurodegeneration.

Protein aggregation is a hallmark of many neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). Although mutations in TARDBP, encoding transactive response DNA-binding protein 43 kDa (TDP-43), account for less than 1% of all ALS cases, TDP-43-positive aggregates are present in nearly all ALS patients, including patients with sporadic ALS (sALS) or carrying other familial ALS-causing (fALS-causing) mutations. Interestingly, TDP-43 inclusions are also present in subsets of patients with frontotemporal dementia, Alzheimer's disease, and Parkinson's disease; therefore, methods of activating intracellular protein quality control machinery capable of clearing toxic cytoplasmic TDP-43 species may alleviate disease-related phenotypes. Here, we identify a function of nemo-like kinase (Nlk) as a negative regulator of lysosome biogenesis. Genetic or pharmacological reduction of Nlk increased lysosome formation and improved clearance of aggregated TDP-43. Furthermore, Nlk reduction ameliorated pathological, behavioral, and life span deficits in 2 distinct mouse models of TDP-43 proteinopathy. Because many toxic proteins can be cleared through the autophagy/lysosome pathway, targeted reduction of Nlk represents a potential approach to therapy development for multiple neurodegenerative disorders.

A Novel Missense Mutation in ERCC8 Co-Segregates with Cerebellar Ataxia in a Consanguineous Pakistani Family.

Autosomal-recessive cerebellar ataxias (ARCAs) are heterogeneous rare disorders mainly affecting the cerebellum and manifest as movement disorders in children and young adults. To date, ARCA causing mutations have been identified in nearly 100 genes; however, they account for less than 50% of all cases. We studied a multiplex, consanguineous Pakistani family presenting with a slowly progressive gait ataxia, body imbalance, and dysarthria. Cerebellar atrophy was identified by magnetic resonance imaging of brain. Using whole exome sequencing, a novel homozygous missense mutation ERCC8:c.176T>C (p.M59T) was identified that co-segregated with the disease. Previous studies have identified homozygous mutations in ERCC8 as causal for Cockayne Syndrome type A (CSA), a UV light-sensitive syndrome, and several ARCAs. ERCC8 plays critical roles in the nucleotide excision repair complex. The p.M59T, a substitution mutation, is located in a highly conserved WD1 beta-transducin repeat motif. In silico modeling showed that the structure of this protein is significantly affected by the p.M59T mutation, likely impairing complex formation and protein-protein interactions. In cultured cells, the p.M59T mutation significantly lowered protein stability compared to wildtype ERCC8 protein. These findings expand the role of ERCC8 mutations in ARCAs and indicate that ERCC8-related mutations should be considered in the differential diagnosis of ARCAs.

Differential effects of Wnt-ß-catenin signaling in Purkinje cells and Bergmann glia in spinocerebellar ataxia type 1.

Spinocerebellar ataxia type 1 (SCA1) is a dominantly inherited neurodegenerative disease characterized by progressive ataxia and degeneration of specific neuronal populations, including Purkinje cells (PCs) in the cerebellum. Previous studies have demonstrated a critical role for various evolutionarily conserved signaling pathways in cerebellar patterning, such as the Wnt-ß-catenin pathway; however, the roles of these pathways in adult cerebellar function and cerebellar neurodegeneration are largely unknown. In this study, we found that Wnt-ß-catenin signaling activity was progressively enhanced in multiple cell types in the adult SCA1 mouse cerebellum, and that activation of this signaling occurs in an ataxin-1 polyglutamine (polyQ) expansion-dependent manner. Genetic manipulation of the Wnt-ß-catenin signaling pathway in specific cerebellar cell populations revealed that activation of Wnt-ß-catenin signaling in PCs alone was not sufficient to induce SCA1-like phenotypes, while its activation in astrocytes, including Bergmann glia (BG), resulted in gliosis and disrupted BG localization, which was replicated in SCA1 mouse models. Our studies identify a mechanism in which polyQ-expanded ataxin-1 positively regulates Wnt-ß-catenin signaling and demonstrate that different cell types have distinct responses to the enhanced Wnt-ß-catenin signaling in the SCA1 cerebellum, underscoring an important role of BG in SCA1 pathogenesis.

Microglia regulate brain progranulin levels through the endocytosis/lysosomal pathway.

Genetic variants in Granulin (GRN), which encodes the secreted glycoprotein progranulin (PGRN), are associated with several neurodegenerative diseases, including frontotemporal lobar degeneration, neuronal ceroid lipofuscinosis, and Alzheimer's disease. These genetic alterations manifest in pathological changes due to a reduction of PGRN expression; therefore, identifying factors that can modulate PGRN levels in vivo would enhance our understanding of PGRN in neurodegeneration and could reveal novel potential therapeutic targets. Here, we report that modulation of the endocytosis/lysosomal pathway via reduction of Nemo-like kinase (Nlk) in microglia, but not in neurons, can alter total brain Pgrn levels in mice. We demonstrate that Nlk reduction promotes Pgrn degradation by enhancing its trafficking through the endocytosis/lysosomal pathway, specifically in microglia. Furthermore, genetic interaction studies in mice showed that Nlk heterozygosity in Grn haploinsufficient mice further reduces Pgrn levels and induces neuropathological phenotypes associated with PGRN deficiency. Our results reveal a mechanism for Pgrn level regulation in the brain through the active catabolism by microglia and provide insights into the pathophysiology of PGRN-associated diseases.

Cortical organoids model early brain development disrupted by 16p11.2 copy number variants in autism.

Reciprocal deletion and duplication of the 16p11.2 region is the most common copy number variation (CNV) associated with autism spectrum disorders. We generated cortical organoids from skin fibroblasts of patients with 16p11.2 CNV to investigate impacted neurodevelopmental processes. We show that organoid size recapitulates macrocephaly and microcephaly phenotypes observed in the patients with 16p11.2 deletions and duplications. The CNV dosage affects neuronal maturation, proliferation, and synapse number, in addition to its effect on organoid size. We demonstrate that 16p11.2 CNV alters the ratio of neurons to neural progenitors in organoids during early neurogenesis, with a significant excess of neurons and depletion of neural progenitors observed in deletions. Transcriptomic and proteomic profiling revealed multiple pathways dysregulated by the 16p11.2 CNV, including neuron migration, actin cytoskeleton, ion channel activity, synaptic-related functions, and Wnt signaling. The level of the active form of small GTPase RhoA was increased in both, deletions and duplications. Inhibition of RhoA activity rescued migration deficits, but not neurite outgrowth. This study provides insights into potential neurobiological mechanisms behind the 16p11.2 CNV during neocortical development.

Pharmacological reversal of synaptic and network pathology in human MECP2-KO neurons and cortical organoids.

Duplication or deficiency of the X-linked MECP2 gene reliably produces profound neurodevelopmental impairment. MECP2 mutations are almost universally responsible for Rett syndrome (RTT), and particular mutations and cellular mosaicism of MECP2 may underlie the spectrum of RTT symptomatic severity. No clinically approved treatments for RTT are currently available, but human pluripotent stem cell technology offers a platform to identify neuropathology and test candidate therapeutics. Using a strategic series of increasingly complex human stem cell-derived technologies, including human neurons, MECP2-mosaic neurospheres to model RTT female brain mosaicism, and cortical organoids, we identified synaptic dysregulation downstream from knockout of MECP2 and screened select pharmacological compounds for their ability to treat this dysfunction. Two lead compounds, Nefiracetam and PHA 543613, specifically reversed MECP2-knockout cytologic neuropathology. The capacity of these compounds to reverse neuropathologic phenotypes and networks in human models supports clinical studies for neurodevelopmental disorders in which MeCP2 deficiency is the predominant etiology.

Genetic Risk of Autism Spectrum Disorder in a Pakistani Population.

Autism spectrum disorder (ASD) is a group of complex multifactorial neurodevelopmental and neuropsychiatric disorders in children characterized by impairment of communication and social interaction. Several genes with associated single nucleotide polymorphisms (SNPs) have been identified for ASD in different genetic association studies, meta-analyses, and genome-wide association studies (GWAS). However, associations between different SNPs and ASD vary from population to population. Four SNPs in genes CNTNAP2, EIF4E, ATP2B2, CACNA1C, and SNP rs4307059 (which is found between CDH9 and CDH10 genes) have been identified and reported as candidate risk factors for ASD. The aim of the present study was, for the first time, to assess the association of SNPs in these genes with ASD in the Pakistani population. PCR-based genotyping was performed using allele-specific primers in 93 ASD and 93 control Pakistani individuals. All genetic associations, genotype frequencies, and allele frequencies were computed as odds' ratios (ORs) using logistic regression with a threshold of p ≤ 0.01 to determine statistical significance. We found that the homozygous genotypes of mutant T alleles of CNTNAP2 and ATP2B2 were significantly associated with Pakistani ASD patients in unadjusted ORs (p < 0.01), but their significance score was lost in the adjusted model. Other SNPs such as rs4307059, rs17850950 of EIF4E, and rs1006737 of CACNA1C were not statistically significant. Based on this, we conclude that SNPs are not associated with, or are not the main cause of, autism in the Pakistani population, indicating the involvement of additional players, which need to be investigated in future studies in a large population size. One of the limitations of present study is its small sample size. However, this study, being the first on Pakistani ASD patients, may lay the foundations for future studies in larger samples.

Pathogenic mechanisms underlying spinocerebellar ataxia type 1.

The family of hereditary cerebellar ataxias is a large group of disorders with heterogenous clinical manifestations and genetic etiologies. Among these, over 30 autosomal dominantly inherited subtypes have been identified, collectively referred to as the spinocerebellar ataxias (SCAs). Generally, the SCAs are characterized by a progressive gait impairment with classical cerebellar features, and in a subset of SCAs, accompanied by extra-cerebellar features. Beyond the common gait impairment and cerebellar atrophy, the wide range of additional clinical features observed across the SCAs is likely explained by the diverse set of mutated genes that encode proteins with seemingly disparate functional roles in nervous system biology. By synthesizing knowledge obtained from studies of the various SCAs over the past several decades, convergence onto a few key cellular changes, namely ion channel dysfunction and transcriptional dysregulation, has become apparent and may represent central mechanisms of cerebellar disease pathogenesis. This review will detail our current understanding of the molecular pathogenesis of the SCAs, focusing primarily on the first described autosomal dominant spinocerebellar ataxia, SCA1, as well as the emerging common core mechanisms across the various SCAs.

Author Correction: Blocking Zika virus vertical transmission.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Association of CACNA1C with bipolar disorder among the Pakistani population.

Many single nucleotide polymorphisms (SNPs) have been identified for Bipolar disorder (BD), but association between SNPs and BD can vary depending on the population tested. SNPs rs10994336 and rs9804190 in ANK3 and rs1006737 in CACNA1C have emerged as the most highly replicated SNPs significantly associated with BD. The aim of the present study was to assess the association of these SNPs with BD in the Pakistani population, which has never before been examined. A total of 120 BD and 120 control individuals from Pakistan were examined in this analysis. Genotyping results indicated that rs1006737 in CACNA1C was significantly associated with BD, while rs10994336 or rs9804190 in ANK3 was not significant when examined individually. However, risk score assessment found that the presence of two or more risk alleles was significantly associated with disease, indicating that risk alleles from ANK3 and CACNA1C may additively contribute to BD. A protein-protein interaction network was generated using STRING to probe the relationship between ANK3 and CACNA1C interactors and their associations with BD. While none of the interactors are directly linked to BD, they play a role in pathways linked to BD, including oxytocin and dopamine signaling pathways. Collectively, these results reveal a significant association of CACNA1C with BD among the Pakistani population, extending results from other ethnic groups to the Pakistani population for the first time.

Blocking Zika virus vertical transmission.

The outbreak of the Zika virus (ZIKV) has been associated with increased incidence of congenital malformations. Although recent efforts have focused on vaccine development, treatments for infected individuals are needed urgently. Sofosbuvir (SOF), an FDA-approved nucleotide analog inhibitor of the Hepatitis C (HCV) RNA-dependent RNA polymerase (RdRp) was recently shown to be protective against ZIKV both in vitro and in vivo. Here, we show that SOF protected human neural progenitor cells (NPC) and 3D neurospheres from ZIKV infection-mediated cell death and importantly restored the antiviral immune response in NPCs. In vivo, SOF treatment post-infection (p.i.) decreased viral burden in an immunodeficient mouse model. Finally, we show for the first time that acute SOF treatment of pregnant dams p.i. was well-tolerated and prevented vertical transmission of the virus to the fetus. Taken together, our data confirmed SOF-mediated sparing of human neural cell types from ZIKV-mediated cell death in vitro and reduced viral burden in vivo in animal models of chronic infection and vertical transmission, strengthening the growing body of evidence for SOF anti-ZIKV activity.

Modeling neuro-immune interactions during Zika virus infection.

Although Zika virus (ZIKV) infection is often asymptomatic, in some cases, it can lead to birth defects in newborns or serious neurologic complications in adults. However, little is known about the interplay between immune and neural cells that could contribute to the ZIKV pathology. To understand the mechanisms at play during infection and the antiviral immune response, we focused on neural precursor cells (NPCs)-microglia interactions. Our data indicate that human microglia infected with the current circulating Brazilian ZIKV induces a similar pro-inflammatory response found in ZIKV-infected human tissues. Importantly, using our model, we show that microglia interact with ZIKV-infected NPCs and further spread the virus. Finally, we show that Sofosbuvir, an FDA-approved drug for Hepatitis C, blocked viral infection in NPCs and therefore the transmission of the virus from microglia to NPCs. Thus, our model provides a new tool for studying neuro-immune interactions and a platform to test new therapeutic drugs.

Hunting for the mutant without the MAP(K).

In a paper recently published in Cell Research, Yu et al. identify two MAPK-related kinases, MAPK11 and HIPK3, as positive regulators of levels of mutant huntingtin protein, a toxic species highly involved in Huntington's disease (HD) pathology. The identification and validation of these kinases as therapeutic targets for knockdown in multiple relevant experimental model systems reveal novel potential approaches for treatment of HD.

Modeling of TREX1-Dependent Autoimmune Disease using Human Stem Cells Highlights L1 Accumulation as a Source of Neuroinflammation.

Three-prime repair exonuclease 1 (TREX1) is an anti-viral enzyme that cleaves nucleic acids in the cytosol, preventing accumulation and a subsequent type I interferon-associated inflammatory response. Autoimmune diseases, including Aicardi-Goutières syndrome (AGS) and systemic lupus erythematosus, can arise when TREX1 function is compromised. AGS is a neuroinflammatory disorder with severe and persistent intellectual and physical problems. Here we generated a human AGS model that recapitulates disease-relevant phenotypes using pluripotent stem cells lacking TREX1. We observed abundant extrachromosomal DNA in TREX1-deficient neural cells, of which endogenous Long Interspersed Element-1 retrotransposons were a major source. TREX1-deficient neurons also exhibited increased apoptosis and formed three-dimensional cortical organoids of reduced size. TREX1-deficient astrocytes further contributed to the observed neurotoxicity through increased type I interferon secretion. In this model, reverse-transcriptase inhibitors rescued the neurotoxicity of AGS neurons and organoids, highlighting their potential utility in therapeutic regimens for AGS and related disorders.