Elucidating the Genetic Landscape of the Frontotemporal Dementias using Next-Generation Sequencing and In-Silico Analyses

Frontotemporal Dementia and Amyotrophic Lateral Sclerosis comprise a spectrum of heterogenous disorders that lie on the "FTD/ALS" spectrum, characterized by similar pathology and genetics but highly variable clinical symptoms that can im- pact behaviour, cognition or/and motor skills. These diseases have a late age at onset, rapid progression and debilitating symptoms that have devastated tens of thousands of families over the last few decades. To-date, treatment includes only symptom management. Rapid advances in genomic technologies over the last decade have enabled scientists to investigate the complexities that underlie disease progression and identify some at-risk populations, but much of the genetic variability is still undiscovered. In this dissertation, we apply an in-depth and systematic approach to study the genetic landscape of FTD/ALS in protein coding genes as well as in non-coding genetic el- ements called long non-coding RNAs (lncRNAs). In chapters 2 and 3, we apply a step-wise genetic screen of FTD/ALS patients to study the frequencies of both pathogenic and potentially pathogenic mutations in known neurodegenerative disease (NDD) genes. We discover an overlap of path- ways previously thought to be associated with other NDDs such as Alzheimer’s Dis- ease and type B Kufs disease. As a result of our findings, we propose the inclusion of two genes, CTSF and SERPINA1, in future genetic screens for FTD/ALS. Through rare-variant association tests, we also find an excessive burden of rare, damaging variants in human autophagy associated genes in FTD/ALS cases versus controls. In chapters 4 and 5, we perform a two-phase study to investigate the role of lncR- NAs in NDDs and healthy ageing. In phase 1, we perform antisense oligonucleotide based knockdowns in highly expressed lncRNAs in the brain. Additionally, we test these lncRNAs for evidence of cis-regulation of proximal genes. In phase 2, we de- sign a genomewide CRISPRi experiment, including a novel sgRNA library targeting ∼4000 lncRNAs and 360 negative controls. This, to the best of our knowledge, is the first sgRNA library targeting a genomewide set of lncRNAs expressed in neu- ronal cell lines. Finally, we perform a series of in-silico analyses using both in-house and public data to gather functional evidence of lncRNAs inageing, cognitive im- pairment, antisense regulation of NDD genes, eQTL associatedgene regulation as well as those that were differentially expressed in FTD cases versus controls. As a result, we curate a list of 119 lncRNAs with evidence of function in human NDDs and healthy ageing. This is one of the first ever large-scale studies investigating the role of lncRNAs in neurodegeneration

Repeat expansions in NOP56 are a cause of spinocerebellar ataxia Type 36 in the British population

Spinocerebellar ataxias form a clinically and genetically heterogeneous group of neurodegenerative disorders characterized by progressive cerebellar ataxia. Their prevalence varies among populations and ethnicities. Spinocerebellar ataxia 36 is caused by a GGCCTG repeat expansion in the first intron of the NOP56 gene and is characterized by late-onset ataxia, sensorineural hearing loss and upper and lower motor neuron signs, including tongue fasciculations. Spinocerebellar ataxia 36 has been described mainly in East Asian and Western European patients and was thought to be absent in the British population. Leveraging novel bioinformatic tools to detect repeat expansions from whole-genome sequencing, we analyse the NOP56 repeat in 1257 British patients with hereditary ataxia and in 7506 unrelated controls. We identify pathogenic repeat expansions in five families (seven patients), representing the first cohort of White British descent patients with spinocerebellar ataxia 36. Employing in silico approaches using whole-genome sequencing data, we found an 87 kb shared haplotype in among the affected individuals from five families around the NOP56 repeat region, although this block was also shared between several controls, suggesting that the repeat arises on a permissive haplotype. Clinically, the patients presented with slowly progressive cerebellar ataxia with a low rate of hearing loss and variable rates of motor neuron impairment. Our findings show that the NOP56 expansion causes ataxia in the British population and that spinocerebellar ataxia 36 can be suspected in patients with a late-onset, slowly progressive ataxia, even without the findings of hearing loss and tongue fasciculation

Drug screen in iPSC-Neurons identifies nucleoside analogs as inhibitors of (G(4)C(2))(n) expression in C9orf72 ALS/FTD

An intronic (G(4)C(2))(n) expansion in C9orf72 causes amyotrophic lateral sclerosis and frontotemporal dementia primarily through gain-of-function mechanisms: the accumulation of sense and antisense repeat RNA foci and dipeptide repeat (DPR) proteins (poly-GA/GP/GR/PA/PR) translated from repeat RNA. To therapeutically block this pathway, we screen a library of 1,430 approved drugs and known bioactive compounds in patient-derived induced pluripotent stem cell-derived neurons (iPSC-Neurons) for inhibitors of DPR expression. The clinically used guanosine/cytidine analogs decitabine, entecavir, and nelarabine reduce poly-GA/GP expression, with decitabine being the most potent. Hit compounds nearly abolish sense and antisense RNA foci and reduce expression of the repeat-containing nascent C9orf72 RNA transcript and its mature mRNA with minimal effects on global gene expression, suggesting that they specifically act on repeat transcription. Importantly, decitabine treatment reduces (G(4)C(2))(n) foci and DPRs in C9orf72 BAC transgenic mice. Our findings suggest that nucleoside analogs are a promising compound class for therapeutic development in C9orf72 repeat-expansion-associated disorders

Medin co-aggregates with vascular amyloid-β in Alzheimer's disease.

Aggregates of medin amyloid (a fragment of the protein MFG-E8, also known as lactadherin) are found in the vasculature of almost all humans over 50 years of age1,2, making it the most common amyloid currently known. We recently reported that medin also aggregates in blood vessels of ageing wild-type mice, causing cerebrovascular dysfunction3. Here we demonstrate in amyloid-β precursor protein (APP) transgenic mice and in patients with Alzheimer's disease that medin co-localizes with vascular amyloid-β deposits, and that in mice, medin deficiency reduces vascular amyloid-β deposition by half. Moreover, in both the mouse and human brain, MFG-E8 is highly enriched in the vasculature and both MFG-E8 and medin levels increase with the severity of vascular amyloid-β burden. Additionally, analysing data from 566 individuals in the ROSMAP cohort, we find that patients with Alzheimer's disease have higher MFGE8 expression levels, which are attributable to vascular cells and are associated with increased measures of cognitive decline, independent of plaque and tau pathology. Mechanistically, we demonstrate that medin interacts directly with amyloid-β to promote its aggregation, as medin forms heterologous fibrils with amyloid-β, affects amyloid-β fibril structure, and cross-seeds amyloid-β aggregation both in vitro and in vivo. Thus, medin could be a therapeutic target for prevention of vascular damage and cognitive decline resulting from amyloid-β deposition in the blood vessels of the brain