Development and application of platforms for functional assessment of oligogenic factors in amyotrophic lateral sclerosis

Motor neuron disease (MND) refers to a group of incurable neurodegenerative diseases where motor neurons progressively degenerate and ultimately culminates in death. Amyotrophic lateral sclerosis (ALS) is the most common form of MND, and is typically synonymous with “MND” in Australia and the United Kingdom. Approximately 10% of ALS cases have a family history and are classified as familial, whilst the remaining 90% are considered to be sporadic. At present, the only proven aetiology are genetic mutations, with over 30 genes accounting for over two-thirds of familial cases.

In Australia, 70% of familial ALS cases are monogenic, where a single causative mutation leads to disease. The remaining unsolved 30% display reduced disease penetrance or non-Mendelian inheritance, which is challenging to solve. Recently, evidence has demonstrated that some ALS cases are oligogenic, where two or more risk or pathogenic variants are responsible for disease. In the United States and United Kingdom, 1–4% of ALS patients harbour more than one known ALS risk or causative variant. In our Australian sporadic ALS patient cohort, this frequency is higher at 6.81%. As such, we updated our novel discovery strategy to investigate oligogenic factors.

The novel discovery strategy, comprising genetic, in silico, in vitro and in vivo pipelines, aims to prioritize candidate variants in families and rapidly assess their potential role in disease in a high-throughput manner. When applied to family MQ1, an Australian ALS family that screened negative for known ALS genes, the genetics and in silico pipelines prioritized two novel candidate variants in acetyl-CoA carboxylase beta (ACACB) and vacuolar protein sorting-associated protein 29 (VPS29) respectively, as well as the presence of an intermediate expansion in ataxin-2 (ATXN2). All three of these genetic factors lie in close proximity to each other at a locus on chromosome 12q24.

To understand the importance and role of ATXN2 in the context of ALS, a comprehensive review was undertaken (Manuscript I). Intermediate ATXN2 CAG expansions are known to confer ALS risk in most ethnicities, and this thesis was the first to demonstrate that these expansions are associated with Australian ALS (p=0.039, Manuscript II). Further analysis revealed that patients who carried an intermediate expansion had a shorter disease duration (median reduction of 10.6 months, p=0.023), compared to the rest of the cohort. Methods for molecular manipulation of the ATXN2 CAG expansion were also optimized, which revealed that the family MQ1 proband intermediate expansion allele harboured three CAA interruptions, which have previously been reported to influence disease onset (Yu et al., 2011).

Expanded ATXN2 was characterized in vitro and in vivo as per the relevant functional prioritization arms of the novel discovery strategy. In HEK293T cells, both expanded ATXN2 and normal length ATXN2 recombinant proteins displayed diffuse cytosolic morphology and localization, and occasionally formed cytoplasmic inclusions. No change in ATXN2 solubility was detected. Transient expression of expanded ATXN2 mRNA in zebrafish did not lead to increased cell death or aberrant primary motor neuron morphology. As in vivo models of intermediate ATXN2 expansions are lacking, Tol2 constructs were generated to establish transgenic zebrafish lines that constitutively express normal length or expanded GFP-tagged family MQ1 proband ATXN2. These provide a suitable in vivo platform for further screening of novel oligogenic ALS candidate variants via the in vivo arm of the novel discovery strategy and potential to facilitate high-throughput drug screening.

To further investigate oligogenic disease effects, this thesis also presents novel platforms developed for functional validation of oligogenic candidate genes. To overcome current limitations in co-expression studies, a novel modular molecular platform was generated by leveraging two 2A peptide motifs in tandem. The fluorescently tagged candidate genes flanked this motif, which was cleaved during translation to produce the candidate proteins ACACB and VPS29 separately, resulting in their co-expression. To validate this platform, potential positional effects of the candidate genes within the expression vector were examined. This revealed that recombinant protein expression levels and cytotoxicity were affected by gene position in the construct. However, these effects did not alter the ALS-related phenotypic outcomes assessed via the in vitro functional pipeline using HEK293T cells.

This novel molecular platform facilitated the preliminary in vitro screen of ACACB and VPS29 in combination with ATXN2. Immunocytochemistry using HEK293T cells revealed that when co-expressed, candidate variants ACACB p.I619T and VPS29 p.P70L colocalized and formed cytosolic aggregates. ATXN2 remained diffuse in the cytoplasm regardless of expansion status or mutations status of ACACB and VPS29. Colocalization analysis suggested that ACACB p.I619T may recruit normal length ATXN2 to aggregates, whereas expanded ATXN2 may be inherently prone to aggregation. No effect on cytotoxicity was observed in Neuro-2A cells, however further investigation is required in more relevant cell types, including iPSC differentiated motor neurons.

The analysis of family MQ1 candidates has begun to unravel the web of pathogenic mechanisms responsible for ALS. The new innovative strategies presented in this thesis will facilitate screening of all oligogenic candidate genes and bring a fresh approach to the difficult task of functional validation of candidates disease genes.