Clarkson, Ben

Abstract Description RNA- and DNA-binding proteins are crucial for regulating gene expression by affecting DNA accessibility, transcription, and repair, as well as RNA splicing, transport, and stability. Dysfunction, mislocalization, aggregation, and liquid-liquid phase separation of these proteins are common in neurodegenerative and neuroinflammatory diseases. TDP43 mislocalization is notably implicated in neurodegenerative diseases like amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. In 97% of ALS cases, the loss of nuclear TDP43 and the formation of cytoplasmic aggregates (TDP43 proteinopathy) in motor neurons are observed. This loss or mislocalization of TDP43 disrupts nucleic acid sequestration and may activate cytosolic DNA and RNA sensors such as RIG1 and cGas-STING pathways, leading to the expression of interferon-stimulated genes (ISGs). Evidence suggests that the ubiquitin-like protein ISG15 contributes to neuron loss. Using primary murine cortical neurons, human IPSC-derived motor neurons, and mixed neural cultures, we report the impact of TDP43 knockdown and mislocalization on neuronal interferon signaling and ISG15 expression. We also use ISG15-deficient neurons to measure ISG15-dependent impacts on neuron viability. Our findings show ISG15-dependent effects on cGas-STING signaling, astrogliosis, and neuron loss in an AAV-C9orf72 mouse model of ALS using global and conditional ISG15 KO mice. Funding Sources Supported by Dominium Foundation Career Development Award (Clarkson). Topic Categories Neuroimmunology (NEUR)

Abstract Description Neurons upregulate MHC Class I during infection and neuroinflammation associated with demyelinating diseases, allowing them to present both self and foreign antigens to CD8+ T cells. We hypothesized that ablation of neuronal MHC I expression reduces neural antigen-specific CD8+ T cell recruitment to demyelinated CNS and reduces neuron loss. We have previously shown that demyelination induces expression of MHC Class I on neurons and axons, allowing neuron-specific neoantigens to be presented to CD8+ T cells, leading to neuroinflammatory damage. Elevated MHC-I and β2-microglobulin expression in white and grey matter tissue from MS patients further supported the role of CD8+ T cells in neuronal injury. Here we demonstrate that neuron specific β2-microglobulin ablation in the B6 MOG35-55 EAE model of multiple sclerosis reduces disease progression and severity via clinical scoring. Our preliminary findings suggest that MHC-I ablation may reduce peripheral activation and CNS recruitment of neural antigen-specific CD8+ T cells as well as CD8+ T cell-mediated neurodegeneration in in the context of demyelinating disease. Future studies are aimed at generating a humanized mouse model reconstituted with patient immune cells to understand the role of MHC I expression in neurons, astrocytes, and oligodendrocytes. Specifically, using this model, we will characterize brain infiltrating T cells from patients with CNS autoimmune disease and suspected T cell involvement. Funding Sources Generous donation from Don and Fran Herdrich. Topic Categories Neuroimmunology (NEUR)

Abstract MR imaging suggests an anatomic correspondence between WM and GM lesions in progressing MS patients, suggesting an underlying neuronal-signaling or neurodegenerative element to the progressive disease state. While retrograde signals evolving from stressed axons that alter soma gene expression have been previously described in models of axonal transection or crush injury, those resulting from inflammation and demyelination are unknown. Using microfluidic chambers that segregate neuronal soma and axons, we have previously demonstrated increased axonal MHC I following treatment with IFNg, a cytokine prominent in demyelinating MS lesions. Furthermore, expression of antigen-loaded MHC I predisposed axons to injury by antigen-specific CD8+ T cells in vitro. Here we report that neurons also upregulate expression of MHC class Ib mRNAs in response to axonal stimulation with IFNg. Genes most highly upregulated following cytokine treatment included H2-M3, H2-Q5, H2-Q6, H2-Q7, H2-Q8, H2-T10 , and H2-T23. We further report the development of an in vivo model system for measuring axonal vulnerability to antigen-specific immune attack following demyelination using an AAV vector to induce neuron-specific expression of axonally-targeted neoantigen (ovalbumin) tagged with cleavable eGFP. We also present optimized methods using AAV vectors in mice for infecting retinal, motor cortex, and hippocampal neurons to induce target gene expression in axons projecting to optic nerve and corpus collosum.