Heledd Brown-Wright

Extra-motor symptoms are increasingly recognised in amyotrophic lateral sclerosis (ALS), encompassing cognitive, social, and behavioural deficits that can substantially impact quality of life. TAR DNA binding protein 43 (TDP-43) pathology is the central disease marker of almost all cases of ALS and approximately half of all frontotemporal dementia (FTD). However, the mechanisms linking TDP-43 pathology with extra-motor symptoms in TDP-43-associated neurodegenerative diseases remain unresolved. In this study, we used the rNLS8 mouse model, which expresses human TDP-43 with an ablated nuclear localisation sequence (hTDP-43 ^ΔNLS ) in a doxycycline-regulatable manner causing progressive motor decline reminiscent of ALS, to delineate the molecular changes associated with disease-relevant phenotypes. We found that in addition to previously reported dramatic motor decline, rNLS8 mice also develop extra-motor phenotypes consistent with FTD, including disinhibition-like and anxiety-like behaviours, and social interaction impairments. These changes began in the earliest disease stages and remained readily detectable even when rNLS8 mice became severely motor impaired. Notably, extra-motor deficits persisted in rNLS8 mice that had recovered motor function upon hTDP-43 ^ΔNLS transgene suppression, regardless of whether recovery was initiated at timepoints prior to or after overt neurodegeneration begins. Transcriptomic analysis of rNLS8 mouse cortex tissues revealed early alterations in expression of 321 genes, most notably involving neuroinflammatory-related pathway activation, and all but 2 of these genes returned to control levels upon suppression of hTDP-43 ^ΔNLS expression. Further, 814 genes showed differential exon usage, indicating changes in alternative splicing, in rNLS8 mouse cortex. Of these, differential exon usage of 10 neuronal genes persisted after hTDP-43 ^ΔNLS transgene suppression, including synapse component genes Nrxn1 , Unc13a , and Gls . Similarly, proteomics analysis of the cortex of rNLS8 mice revealed depletion of synaptic proteins, particularly those involved in glutamatergic signalling pathways, which also persisted following hTDP-43 ^ΔNLS transgene suppression. Similar glutamatergic pathway changes were detected in human ALS and FTD post-mortem cortex tissues. Our findings indicate that extra-motor phenotypes emerge early in disease in rNLS8 mice and remain evident despite progressive motor impairments. Further, extra-motor phenotypes persist even upon motor recovery, correlating with specific synaptic gene expression and splicing changes. Overall, this study suggests the potential utility of an expanded suite of behavioural paradigms in preclinical testing using rNLS8 mice, with enhanced relevance to the diversity of TDP-43 proteinopathies including FTD. Our findings further suggest that targeting glutamatergic synaptic components may be an avenue to correct extra-motor deficits associated with TDP-43 pathology.

Amyotrophic lateral sclerosis (ALS) is characterised by motor neuron (MN) death; however, astrocytes play a key role in disease pathogenesis. Developments in the field of artificial intelligence (AI) have the potential to impact drug discovery in multiple ways, including the rapid identification of drug repurposing candidates. A combination of natural language processing and deep learning algorithms was used to generate a knowledge graph based on scientific literature, omics and chemical databases, and other public sources with the aim to identify drug repurposing candidates for ALS. The aim of the study was to determine the effect of a cancer compound identified by AI, gefitinib, on MN survival, and to decipher its mode of action in in vitro and in vivo models of ALS. We used co-cultures of healthy motor neurons with ALS patient-derived astrocytes (iAstrocytes), obtained through a semi-direct conversion protocol, to assess the neuroprotective properties of gefitinib. Compound treatment led to a significant rescue of MNs cultured with ALS iAstrocytes and a significant reduction in the levels of cleaved TDP-43 fragments in ALS iAstrocytes. Our data suggest that gefitinib-mediated activation of autophagy decreased the 35 kDa fragments of TDP-43. In a proof-of-concept in vivo study in SOD1 ^G93A mice, gefitinib treatment significantly delayed the onset of neurological symptoms, thus showing the potential of AI-augmented drug discovery for neurodegenerative disorders. Significance Statement: This study presents an AI-augmented method of identifying potential repurposing candidates for disease with an unprecedented speed. The AI’s results were validated in vitro using iAstrocytes differentiated from induced neuronal progenitor cells (iNPCs), which are pathophysiologically relevant models suitable for studying neurodegeneration. iNPCs recapitulate many pathological hallmarks of the disease and they retain the ageing phenotype of the patient that they are obtained from. TDP-43 proteinopathy is one of the disease hallmarks observed in patients and is present in 97% of ALS patients. Here, we show gefitinib, a repurposing candidate identified by AI, improves survival of MNs in a co-culture with patient-derived astrocytes and can modulate TDP-43 proteinopathy.

Understanding the mechanisms that drive TDP-43 pathology is integral to combating amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD) and other neurodegenerative diseases. Here we generated a longitudinal quantitative proteomic map of the cortex from the cytoplasmic TDP-43 rNLS8 mouse model of ALS and FTLD, and developed a complementary open-access webtool, TDP-map (https://shiny.rcc.uq.edu.au/TDP-map/). We identified distinct protein subsets enriched for diverse biological pathways with temporal alterations in protein abundance, including increases in protein folding factors prior to disease onset. This included increased levels of DnaJ homolog subfamily B member 5, DNAJB5, which also co-localized with TDP-43 pathology in diseased human motor cortex. DNAJB5 over-expression decreased TDP-43 aggregation in cell and cortical neuron cultures, and knockout of Dnajb5 exacerbated motor impairments caused by AAV-mediated cytoplasmic TDP-43 expression in mice. Together, these findings reveal molecular mechanisms at distinct stages of ALS and FTLD progression and suggest that protein folding factors could be protective in neurodegenerative diseases.