Rita Sattler - US grants

DESCRIPTION (provided by applicant): The overall aim of this proposal is to study the pathology of the expanded hexanucleotide repeats in the C9orf72 gene using human iPS differentiated neurons and glia cells. The expanded GGGGCC hexanucleotide repeat in the non-coding region of the C9orf72 gene on chromosome 9p21 has been discovered as the cause of approximately 30-50% of familial and up to 10% of sporadic ALS cases as well as 12% of familial FTD cases, making this the most common known genetic cause of ALS/FTD to date. Expanded repeats are highly unstable and potentially yield toxic RNAs that accumulate in the nucleus and are hypothesized to cause cellular dysfunction via aberrant DNA and protein binding. Preliminary data from our laboratory confirm (GGGGCC)n nuclear RNA foci, aberrant gene expression and nuclear retention of RNA binding proteins (RBPs) in C9orf72 patient derived fibroblasts and iPS differentiated neurons. We therefore propose to elaborate on these early findings by generating an extensive genetic profile of ALS/FTD human iPS neurons and glia cells through the use of microarray and validate whether the iPS changes are relevant by comparing to human autopsy C9orf72 brain tissues. Furthermore, we will use C9orf72 iPS cell lines to investigate the RNA toxicity/pathology thru the identification of aberrant accumulation and binding of RNA binding proteins. Finally we will determine if we can abrogate C9orf72 genomic toxicity and pathology with antisense oligonucleotides already designed and validated in our laboratory. The likelihood of success will be greatly enhanced through a collaborative working relationship, the availability and experience of using iPS cells and human tissues. The extensive use of iPS cells to model disease, to cross correlate with human tissues and their use to validate ameliorative antisense therapy provides an important and possibly new direction for understanding disease pathophysiology and therapeutics development. !

Project Summary This application seeks support for the Gordon Research Conference (GRC) on ?ALS and Related Motor Neuron Diseases,? which will take place from July 21-26th, 2019 at Mount Snow, West Dover, VT. This second biennial meeting brings together both senior and junior scientists and clinicians who investigate, clinically examine and therapeutically treat Amyotrophic Lateral Sclerosis (ALS) and related motor neurons diseases including spinal-bulbar muscular atrophy (SBMA), as well as spinal muscular atrophy (SMA), progressive muscular atrophy (PMA) and multisystem proteinopathy (MSP). The GRC will be accompanied by a Gordon Research Seminar (GRS), which takes place July 20-21st, 2019 at the same location, and is specifically targeted at and organized by students and postdoctoral fellows of the aforementioned research community. The GRC will be divided into nine scientific sessions and daily poster presentations. National and international experts of the field will be invited as speakers to present unpublished research. In addition, we will give selected poster abstract authors, which are generally young investigators or trainees, an opportunity to present among these experts. This GRC provides a unique opportunity for scientists of interdisciplinary expertise to exchange ideas and hypotheses that will lead to new collaborative efforts towards finding new therapeutic targets and treatments for patients affected by these diseases.

PROJECT ABSTRACT The GGGGCC (G4C2) hexanucleotide repeat expansion (HRE) in the first intron of the gene C9orf72, is the most common genetic abnormality associated with frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). The disease pathogenesis ultimately leads to the concurrent degeneration of cortical forebrain and spinal motor neurons, and result in the clinical deficits of motor function and dementia. While the C9orf72- FTD/ALS disease pathogenesis has been well characterized in spinal motor neurons and a contribution of the observed neurodegeneration has been attributed to spinal cord astrocytes, there is little known about the pathobiology in cortical astrocytes and their role in cortical neurodegeneration, which is proposed to contribute to the dementia symptoms in this patient population. Here, we hypothesize that cortical astrocytes play an integral role in the non-cell autonomous disease pathology contributing to the degeneration of cortical neurons in C9orf72-FTD/ALS. To test this hypothesis, we will investigate hiPSC-derived C9orf72-FTD/ALS cortical astrocytes in monoculture and in co-culture with cortical neurons (Aim 1). We will characterize C9orf72- FTD/ALS hiPSC-cortical astrocytes by assessing astrocyte function and determine C9orf72 HRE-specific pathobiology. Furthermore, we will establish the relationship between cortical astrocytes and cortical neurons using co-cultures of control and C9orf72-FTD/ALS lines. The co-cultures will be assessed for changes in astrocyte function, neuronal function and viability, and C9orf72-disease pathobiology. In addition, we will examine transcriptomic alterations in the diseased hiPSC-derived cortical astrocytes (Aim 2). Transcriptomic profiles of the diseased and control cortical astrocytes in both monoculture and co-culture conditions will be generated using RNA-sequencing. In addition, we will analyze existing single nuclei RNA seq data already generated in the lab and identify overlapping candidate genes that are specifically dysregulated in cortical astrocytes in C9orf72-FTD/ALS patients and hiPSC lines. We will validate these candidate genes on the RNA and protein level in postmortem patient tissue samples via RNAscope and immunohistochemistry, respectively. Select validated top hits will undergo preliminary mechanistic validation through genetic manipulation of these candidate genes in the hiPSC in vitro model. These studies will for the first time elucidate the contributing role of cortical astrocytes in the neurodegeneration of cortical neurons in C9orf72-FTD/ALS, addressing the disease mechanisms of dementia in this spectrum disorder. Additionally, this work will provide novel opportunities for drug target identification with the hope of identifying novel therapeutics for the affected patient populations.

Project Abstract The role of microglia in the C9orf72 (C9) amyotrophic lateral sclerosis (ALS)/ frontotemporal dementia (FTD) disease spectrum remains poorly understood. Early investigations found that microglia activation was significantly higher in ALS with dementia and impaired executive function, suggesting that microglia activation correlates with FTD-like symptoms in ALS. More recent neuropathologic examinations of microglia in FTLD patient autopsy brains with mutations in progranulin versus C9orf72 concluded that the observed microglia dysfunction was different between the two genetically different patient subgroups suggesting specificity of microglia dysfunction depending on the etiology of the patient population. One interesting aspect of microglia- neuron communication is the role of microglia in the maintenance and refinement of synaptic networks through the selective pruning of synapses, which occurs predominantly during development but has been shown to also be triggered in Alzheimer's disease (AD) and related dementias, including FTD. The degree of synapse loss in AD strongly correlates with cognitive decline, even more than the amount of plaque, tangles or neuronal loss, and a recent study of ALS postmortem tissue confirmed increased synapse loss in the prefrontal cortex of patients with reported cognitive impairments. Our laboratory has preliminary data supporting the hypothesis that there is an altered neural-immune interaction in the cortical forebrain regions of C9orf72 patients with confirmed FTD in which microglia and neurons modify each other's function. Using patient-derived hiPSC microglia and cortical neurons, we are able to show that C9 patient-derived hiPSC microglia mono-cultures do have intrinsic phenotypes, including altered gene profiles, phagocytic activities and lysosomal function. Most interestingly, preliminary data suggests that C9 microglia do regulate neuronal excitability and survival of C9 iPSC neurons. To further investigate the role and contribution of microglia in C9 cortical degeneration, we propose to thoroughly investigate the intrinsic properties of C9 hiPSC-microglia (from all patient subgroups: FTD, FTD/ALS, ALS; Aim1). For the first time, we will then co-culture these microglia with C9 and healthy control hiPSC cortical neurons to better understand the co-regulation between these two cell types (Aim 2). Finally, in the third aim, we will study microglia activation and pathology in C9 patient postmortem autopsy tissue. This will include cell-type specific genetic profiling from existing snRNA seq data sets, immunohistochemistry of microgliosis and multi-label immunostaining for microglial-specific candidate genes/proteins in conjunction with C9 neuronal disease pathology markers (TDP-43 and C9 DPRs) to gain novel knowledge on whether microglia are preferentially altered in close vicinity to neuronal pathologies.