The Role of RNA-Binding Proteins and RNA Metabolism in Neurological Development and Disease*

Date/Time: Sunday, September 10, 2023 - 9:30 AM – 11:30 AM
Track: Plenary
Room: Salons E-F (5th Floor)
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Chair: Sheng-Han Kuo, MD

Co-Chair: Vikram Khurana, MD, PhD

Cells modify their protein repertoire in response to a specific stimulus, much of which is controlled at the post-transcriptional level by the coordinated activity of RNA-binding proteins (RBPs). RBPs regulate their targets' transport, localization, translation, metabolism, and decay. These functions seem to be particularly critical in the brain, where the precise amount of mRNA transcripts and thus the resultant protein must be available at the right amount. Increasing evidence points to abnormal RNA metabolism as a common pathogenic mechanism in a number of neurodevelopmental and neurodegenerative diseases.
However, while little is known about the functions of RBPs and RNA regulation in brain development or disease, recent studies have been able to pinpoint the root cause and mechanisms of disease, as well as therapeutic options. We will discuss how alterations in RNA and their related RBPs can lead to neurodevelopmental and/or neurodegenerative disorders, as well as potential therapeutic approaches.

Learning Objectives:

  • Learn the basic RNA biology and RNA binding protein homeostasis.
  • Understand the new development of anti-sense oligonucleotide and CRISPR technologies.
  • Learn the implications for neurodegenerative and neurodevelopmental disorders.

Same gene, different variants, different phenotypes: the puzzling case of the RNA-binding protein Pumilio1 (SCA47)

Speaker: Vincenzo A. Gennarino, PhD

Several years ago, we discovered that the RNA-binding protein Pumilio1 (Pum1) is essential for cerebellar function, which prompted us to search for human patients with mutations in PUM1. Unexpectedly, we found patients with two distinct phenotypes: some patients had a mild, late-onset cerebellar ataxia, while others had infantile-onset developmental delay, ataxia, and seizures. The more severe phenotype was reminiscent of what we observed in Pum1 knockout mice, rather than the heterozygous mice. What could account for such a severe phenotype? Our data suggest that the mild phenotype results from the deregulation of PUM1 targets, while the severe syndrome stems from disruption of PUM1's interactions with its native partners. We therefore generated a Pum1 interactome from the mouse brain. We have also generated mouse models that recapitulate PRCA and PADDAS. As we have continued recruiting patients, we have identified 37 different PUM1 variants that cause additional phenotypes, including seizures-only, an early-onset ataxia without any other symptoms, and a severe form of PADDAS that involves skeletal/cardiac defects. Our data suggest that understanding phenotypic heterogeneity in other non-expansion ataxias will require examining specific variants in their physiological context. Developing tissue-specific interactomes should provide a more realistic picture of how RNA-binding proteins cooperate and compete in neurons.

Disruption of RNA metabolism in neurodegeneration and emerging therapeutic strategies  

Speaker: Clotilde Lagieer-Tourenne, MD, PhD, FANA

Alteration of RNA metabolism has emerged as a central theme in neurodegenerative diseases with mutations and/or mislocalization of RNA binding proteins, including TDP-43 and FUS, in amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD) and Alzheimer’s disease. Following the recognition of their crucial role in neurodegeneration, we have used genome wide approaches to define their role in regulating expression and splicing of their RNA targets. We recently demonstrated that the human RNA most affected by loss of nuclear TDP-43 is encoding the neuronal growth-associated factor called stathmin-2. Reduced levels in stathmin-2 is a hallmark in sporadic and familial ALS/FTD, and restoration of stathmin-2 expression emerges as an attractive therapeutic strategy in TDP-43 proteinopathies. Using newly generated cellular and animal models, we have determined stathmin-2’s essential role for neuronal regeneration and axonal maintenance and have established antisense oligonucleotides (ASOs) as a therapeutically viable approach to rescue stathmin-2 in TDP-43 proteinopathies. Other potential therapeutic approaches aim at directly targeting the cellular mislocalization and abnormal phase transition of TDP-43 and FUS. We use small molecules and genetic screens, including high content optical screens, to identify modifiers of disease-associated phenotypes that represent new therapeutic targets.

From Vesicle Trafficking to mRNA Metabolism: a Double-Life for the Parkinson’s Protein Alpha-Synuclein

Speaker: Vikram Khurana, MD, PhD

Alpha-synuclein (αSyn) is a conformationally plastic protein that reversibly binds to cellular membranes. It aggregates and is genetically linked to Parkinson’s disease (PD). Our laboratory has identified molecular perturbations at proteome-scale that result from misfolding and mistrafficking of αSyn. These investigations have strongly suggested that the resultant toxicity is closely related to its native function and molecular interactions. While αSyn has been closely tied to vesicle trafficking at the presynaptic terminal, we have surprisingly found that αSyn to be in close proximity to numerous RNA-binding proteins (RBPs). Moreover, αSyn toxicity was both impacted by genetic modulation of RBPs and associated with striking alterations of the RBP proteome. Recently, we showed that αSyn is directly involved in gene regulation through RBP interactions, and that this is likely important in PD. More specifically, αSyn directly modulates Processing-bodies (P-bodies), membraneless organelles that function in mRNA turnover and storage. The N-terminus of αSyn, but not other synucleins, dictates mutually exclusive binding either to cellular membranes or to P-bodies in the cytosol. αSyn associates with multiple decapping proteins in close proximity on the Edc4 scaffold. As αSyn pathologically accumulates, aberrant interaction with Edc4 occurs at the expense of physiologic decapping-module interactions. mRNA-decay kinetics within PD-relevant pathways are correspondingly disrupted in PD patient neurons and brain. Genetic modulation of P-body components alters αSyn toxicity, and human genetic analysis lends support to the disease-relevance of these interactions. Beyond revealing an unexpected aspect of αSyn function and pathology, our data highlight the versatility of conformationally plastic proteins with high intrinsic disorder and their role in intracellular signaling.

Soriano Lectureship Award:
Repeating Themes in Human Neurologic Disease

Speaker / Award Recipient: Peter Todd, MD, PhD, FANA

Over the past 30 years, nucleotide repeat expansions have emerged as common causes of many neurologic conditions, including ALS, FTD, Ataxia and Autism. Repeats create dynamic elements as DNA, RNA and translated proteins to drive disease pathogenesis. In this lecture I will describe how the structures of repetitive RNA elements directly influence their metabolism, binding partners, localization, translation, and degradation. I will then discuss how these same properties of repeats create unique molecular targets for modular therapeutic development through use of emerging technologies.

Improved Survival, Strength, And Neuroinflammation In A Mouse Model Of Sporadic ALS After Novel AAV-mediated Delivery Of RNAi Targeting Atxn2

Emerging Scholar Speaker: Defne Amado, MD, PhD

Amyotrophic lateral sclerosis (ALS) is a fatal disease characterized by death of motor neurons, which at autopsy show cytoplasmic aggregates of Tar-DNA binding protein of 43kDa (TDP-43). TDP-43 associates with cytoplasmic stress granules (SGs) and leads to toxicity through both cytoplasmic gain- and nuclear loss-of-function. In 2017, a seminal study showed that inhibiting SG formation through downregulation of the SG-associated protein Ataxin-2 (Atxn2) using antisense oligonucleotides (ASOs) prolongs survival by 35% in a mouse model of sporadic ALS (Becker et al., Nature 2017), a strategy that is now in human clinical trials. However, frequent CNS administrations are required for sustained knockdown, and the intrathecal approach may have limited efficacy in reaching the brain, limiting safety and efficacy. Our group therefore developed an approach using AAV-mediated RNAi delivery to achieve lasting and targeted knockdown, a strategy that could be used to treat sporadic ALS. We designed and tested miRNAs targeting Atxn2 in cultured cells, packaging the top candidate into a novel AAV9 variant, AAV1999, that we engineered for superior CNS targeting in mice and nonhuman primates. Mouse dosing studies demonstrated 55% Atxn2 knockdown in frontal cortex and 25% knockdown throughout brainstem and cervical and lumbar spinal cord after intracerebroventricular injection. We then conducted an efficacy study in the same ALS mouse model used in the ASO study, in which wildtype human TDP-43 is overexpressed in neurons and mice exhibit a rapid decline in strength and survival. After treatment, mean and median survival were increased by 54% and 45% respectively (p<0.002). Mice showed a 47% recovery of weight and marked improvement across strength-related measures, including rotarod (2.6X duration, p<0.001); composite gait (40% improvement, p<0.00005); clasping (24% improvement, p<0.05); kyphosis (75% improvement, p<0.00001); tremor (39% improvement, p<0.001); foot angling (57% improvement, p<0.005); and limping (29% improvement, p<0.05). Interestingly, treated mice showed an increase in vertical activity above that seen in wildtypes, perhaps suggesting an unmasking of an FTD phenotype with improved strength. Histologically, there was an increase in lumbar motor neurons, and a marked reduction in inflammatory markers in the brain and to a lesser degree, the spinal cord. Spinal cord transcriptomics revealed markedly dysregulated transcriptomes in mutant mice that were partly alleviated by treatment. AAV-mediated RNAi against Atxn2 is therefore a promising treatment strategy for the 97% of ALS characterized by TDP-43 pathology.

Experimental Confirmation of PI3KR1 Gene Mutation as a Cause of ALS-Like Syndrome Associated with Primary Immunodeficiency

Emerging Scholar Speaker: Farinaz Safavi, MD, PhD

Inborn Errors of Immunity typically present with recurrent infections and inflammatory diseases, but neurological complications are very common in these patients. How immune-related gene defects manifest in the nervous system is very unclear. A 42-year-old man with recurrent infections and hypogammaglobulinemia presenting in childhood was found to have a pathogenic heterozygous mutation (c.1710dup) in the PIK3R1 gene. He developed spastic paraparesis at the age of 30, but an extensive neuroinfectious and neuroinflammatory workup was unrevealing. Significant weakness in all limbs and bulbar symptoms progressed despite treatment with IVIg, cyclophosphamide, and rituximab targeting of potential neuroinflammatory processes. Further neurological evaluation several years later showed both upper and lower motor neuron disease clinically consistent with the diagnosis of ALS. We used our newly developed pipeline to evaluate the potential contribution of mutated PIK3R1 to his CNS disease. CSF cell immunophenotyping indicated active antigen presentation in myeloid cells and T cell activation in the CNS compartment, suggesting immune activation possibly due to neuronal destruction. To investigate the role of PIK3R1 in neurons, we generated an iPSC line from patient CD34+ cells and repaired the mutation with CRISPR-Cas9 technology. We then differentiated patient-derived native and gene-edited cells into motor neurons (MNs), as well as iPSCs from a healthy donor. iPSCs from all three lines differentiated normally into neuroepithelial cells, motor neuron precursors, and terminally differentiated MNs. Electrophysiology showed significantly lower activity in patient-derived neurons compared to the healthy donor. NMDA treatment increased firing in motor neurons from the healthy but not the patient-derived cells. Finally, patient-derived neurons were more prone to apoptosis than control, as shown by higher expression of Caspase 3. The electrophysiology, NMDA activation and apoptosis abnormalities normalized in edited differentiated patient-derived neurons, indicating that this specific mutation in PI3KR1 critically degraded neuronal function. These findings demonstrate a significant role for PIK3R1 signaling in neuronal function and survival, identifying new and targetable pathways for neurodegeneration.