Investigating the neuropathological correlates of clinical heterogeneity in amyotrophic lateral sclerosis

Abstract

BACKGROUND: Research efforts over the last three decades have advanced our understanding of amyotrophic lateral sclerosis (ALS) as a highly heterogeneous, progressive multi-system neurodegenerative disease. ALS exhibits significant phenotypic and genetic heterogeneity, and a diverse range of mechanisms are currently implicated in the disease pathogenesis. Around only 10% of cases harbour an identifiable ALS causative mutation; the aetiology of the majority of cases is thought to result from complex interactions between latent genetic variants and environmental factors. Dysproteostasis features across all cases at post-mortem, however, an intricate understanding of the disease pathogenesis is still lacking. In the absence of identifiable therapeutic targets, currently licenced disease modifying therapies are of limited efficacy and are reported to extend survival by an order of only months.

Disease duration is one of the most variable features of ALS, the average disease duration is less than 2 years. However, disease trajectories are unpredictable and a minority of patients survive beyond ten years. Understanding the pathophysiology underlying disease progression is critical to developing urgently needed targeted therapies.

The studies presented in this thesis were undertaken to investigate the molecular profiles associated with phenotypic variation; to identify candidate genes and pathways associated with differential disease duration and impaired cognition.

METHODS: A tiered approach was used consisting of an initial systematic review and meta-analysis to summarise the existing ALS neuropathological literature, informing the subsequent experimental study design. In order to investigate clinicopathological correlates of clinical variation in ALS a phenotypically defined neuropathological cohort comprised of 17 ALS cases stratified by disease duration was first curated using the CARE-MND national research database. The cohort included 11 age and sex matched non-neurological control samples sourced from the MRC sudden death brain bank. In this study sporadic ALS cases were defined as both; C9orf72 repeat negative and without affected first or second-degree relatives. Using the ENCALS median observed survival times from symptom onset, cohort cases were stratified into long (≥44.6m) and short disease (<44.6m) duration groups. Clinicopathological correlates of pTDP-43 pathology were assessed across an extended range of 14 motor and extra-motor cohort cerebral regions.

Highly sensitive transcriptional profiling techniques, optimised for use on post-mortem tissue were used in the subsequent experimental discovery stages. Using NanoString™ molecular profiling a comparative transcriptomic analysis of the 17 ALS motor cortex case tissues and non-neurological control samples was performed. Differential gene expression analyses comparing ALS versus control samples and long versus short ALS samples were performed using DESeq2 followed by Gene Ontology (GO) enrichment and cell-type specific association analyses.

Spatially resolved validation studies of identified candidate genes were then performed on a subset of motor cortex and spinal cord sections using an RNA in-situ hybridisation technology, BaseScope™. Slides were analysed using a bespoke semi-automated quantitative Fiji image analysis workflow. Western blotting was also performed on cohort case motor cortex and spinal cord tissue to assess the protein expression levels of candidate genes. Finally, to further investigate the pathological transcriptomic correlates of cognitive neuroresilience, NanoString™ sequencing using the same neuropathology gene panel was applied to a separate cohort of ALS case cerebral extra-motor regions stratified by cognition.

RESULTS: The systematic review and meta-analysis of neuropathological studies identified extensive dysregulation spanning the transcriptome to the epigenome; extending beyond the CNS to muscle tissue. The mechanisms of: dysproteostasis, neuroinflammation, immune response, neuronal development, oxidative stress and neurotransmission were identified as dominant reported themes irrespective of mutation status. Important methodological limitations were consistently identified across studies, highlighting the need to improve future experimental design and omics reporting practices. Identification of concordantly dysregulated miRNAs and mRNAs characterise an immune-inflammatory neuropathological microenvironment, several of the identified candidate genes have also been identified in recent neuro-oncology studies as potential prognostic biomarkers. Profiling of pTDP-43 immunostaining across 14 cortical regions identified extensive deposition of pTDP-43 in both neuronal and glial cells. BA4 was the most commonly affected region in 94% of all cases, followed by BA6 and regions associated with executive and language function. In contrast BA11 and BA19 were the least commonly affected regions. The overall pTDP-43 burden or regional BA4 pTDP-43 severity score were not found to be associated with the clinical features of; site of onset, age or disease duration. The total regional burden of glial pTDP-43 in cognitive regions was found to be associated with a detectable cognitive deficit on ECAS testing.

Comparative transcriptional profiling of motor cortex samples of longer and short disease duration cases identified differential neuroimmune microenvironments characterised by upregulation of microglial and endothelial associated genes. Significantly increased AQP4 mRNA perinuclear localisation and clustering was identified in the motor cortex white matter regions of short disease duration cases (p. adj <0.05). Differential perinuclear AQP4 mRNA clustering was not observed in motor cortex grey matter, spinal cord anterior or posterior horn regions.

Application of the same Nanostring™ neuropathology gene panel to a separate cohort of ALS cases stratified by cognition identified differences in the immune-inflammatory profile between cognitively affected and unaffected cortical regions, including upregulation of NLRP3 inflammasome in cognitively affected cases. The expression profile of cognitively resilient cases compared to cognitively affected cases identified upregulation of candidate neuroprotective genes critically involved in autophagy and excitotoxicity.

SUMMARY: Overall, these experimental results are consistent with the existing literature evidencing a neuroimmune microenvironment in ALS. In addition, these findings suggest that differential neuroimmune and neurovascular responses may modify the disease pathogenesis of ALS.

The significance of the observed increased AQP4 mRNA perinuclear localisation in motor cortex white matter regions of short disease duration ALS cases requires further investigation. Perinuclear AQP4 mRNA mis-localisation may result from nucleocytoplasmic trafficking deficits, reduced expression of astrocytic AQP4 protein and glymphatic dysfunction is one possible consequence. The role of the blood brain barrier and brain spinal cord barrier in the context of the ALS neurodegenerative microenvironment is an understudied area. While the results of these neuropathological studies may reflect response mechanisms to a primary pathogenic mechanism, importantly in-vivo human and animal studies have identified features of immune-inflammatory dysregulation at earlier time points during the disease course and even prior to symptom onset. The results presented in this thesis support further functional studies to investigate neuroimmune dysregulation and glymphatic dysfunction as potential disease modifiers in ALS.