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

Author Correction: Blocking Zika virus vertical transmission.

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Uncovering novel roles for glia in neurodegenerative diseases

The complexity of the central nervous system poses a tremendous challenge for understanding the intricate interplay between the myriad cell types that comprise the brain and spinal cord. Understanding these interactions becomes especially challenging when examining dynamic processes, such as aging or disease. In genetic disorders, an additional layer of complexity emerges when multiple cell types express the disease-causing or disease-associated proteins, as this expression can contribute to pathophysiology through primary, cell autonomous effects and secondary, non-cell autonomous mechanisms. Identifying these cell type-specific changes and interpreting them in the overall context of the disease have remained difficult, especially in the case of chronic neurodegenerative disorders. As a result, although extensive effort has been invested into studying these processes, effective therapeutic interventions for the majority of neurodegenerative diseases are lacking. The work presented here describes my graduate studies in Dr. Janghoo Lim’s lab, collectively aimed at improving approaches to neurodegenerative diseases through (1) elucidating the molecular mechanisms underlying degeneration of selectively vulnerable neuronal populations, (2) uncovering previously undescribed roles for non-neuronal cells in dysfunction and degeneration of affected tissues, and (3) developing and testing novel therapeutic strategies in pre-clinical animal models of disease. In the first chapter, I comprehensively review the previous literature describing the pathogenic mechanisms underlying spinocerebellar ataxia type 1 (SCA1), which has been focused on Purkinje cells (PCs), a rare cerebellar cell type that degenerates at late stages in the disease. I then introduce a novel framework that extends the analysis of cellular and molecular disease mechanisms beyond a single cell type by dissecting how many cell types within a heterogenous tissue simultaneously contribute to the pathogenesis and progression of a disease. By performing single-nucleus RNA-sequencing of the mouse and human SCA1 cerebellum and constructing continuous dynamic trajectories of each population, we define the unique temporal dynamics of different subsets of cells throughout all stages of disease. Furthermore, we specifically report the precise transcriptional changes that precede loss of PCs and identified early oligodendroglial impairments that can profoundly impact cerebellar function. This work uncovers new roles for diverse cerebellar cell types in SCA1 and provides a generalizable analysis framework for studying the mechanisms underlying the process of neurodegeneration. In the second and third chapters, I introduce two novel functions of Nemo-like kinase (Nlk), a protein previously implicated in several neurodegenerative disorders for its role in phosphorylating the disease-causing proteins. In Chapter 2, I report that Nlk is a negative regulator of lysosome-associated gene transcription, and that its reduction promotes functional lysosome biogenesis in motor neurons of the brain and spinal cord. Furthermore, genetic or pharmacological reduction of Nlk enhanced clearance of aggregated TDP-43 and ameliorated pathological, behavioral, and lifespan deficits in two distinct mouse models of TDP-43 proteinopathy. In Chapter 3, I report that Nlk is also a regulator of receptor-mediated endocytosis and that modulation of endocytosis via reduction of Nlk in microglia, but not neurons, can alter total brain levels of Progranulin (Pgrn), a protein for which haploinsufficiency is causal for frontotemporal lobar degeneration. We demonstrate that Nlk reduction promotes Pgrn degradation by enhancing its trafficking through endocytosis-lysosomal pathway, revealing a new mechanism for Pgrn level regulation in the brain through the active catabolism by microglia. Taken together, this work reveals new roles for different glial cell types in several neurodegenerative disorders and provides insights into the mechanisms regulating protein trafficking and lysosomal function, with broad implications for a variety of protein aggregation disorders

Using induced pluripotent stem cells and CRISPR/Cas9- mediated targeted mutagenesis to create a model to study Aicardi-Goutières Syndrome /

Aicardi-Goutières Syndrome is a neurological disease resulting in a variable array of symptoms, of which include microcephaly, calcification of the basal ganglia, leukodystrophy, and other neurological defects, all of which are accompanied by a mild to severe mental handicap seen in patients with the disease. In this disease, there is a mishandling of nucleic acids due to mutations in one of several nucleases and this mishandling is hypothesized to be involved in an autoimmune response triggered by the anomalous DNA or RNA. In order to better study the effects of these mutations, in particular mutations in three-prime repair exonuclease 1 (TREX1), I have described a method to generate neural progenitor cells, neurons, and astrocytes from induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) to obviate the need to isolate brain tissue from patients or utilize post-mortem brain tissue. By reprogramming AGS1 patient fibroblasts with a V201D mutation into iPSCs and by generating H9 embryonic stem cell wild-type/mutant TREX1 isogenic pairs, along with being able to successful differentiate these pluripotent stem cells into cells of the neural lineage, I have begun to establish an in vitro platform that allows for direct observation of the effects of TREX1 mutations on the development and function of various cell types that make up the brai

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

A Novel Missense Mutation in <i>ERCC8</i> 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

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

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