Maize C. Cao

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.

TDP-43 dysfunction is a molecular hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). A major hypothesis of TDP-43 dysfunction in disease is the loss of normal nuclear function, resulting in impaired RNA regulation and the emergence of cryptic exons. Cryptic exons and exon changes are emerging as promising markers of lost TDP-43 function in addition to revealing biological pathways involved in neurodegeneration in ALS/FTD. In this brief report, we identified markers of TDP-43 loss of function by depleting TARDBP from post-mortem human brain pericytes, a manipulable in vitro primary human brain cell model, and identifying differential exon usage events with bulk RNA-sequencing analysis. We present these data in an interactive database ( https://www.scotterlab.auckland.ac.nz/research-themes/tdp43-lof-db-v2/ ) together with seven other TDP-43-depletion datasets we meta-analysed previously, for user analysis of differential expression and splicing signatures. Differential exon usage events that were validated by qPCR were then compiled into a ‘differential exon usage panel’ with other well-established TDP-43 loss-of-function exon markers. This differential exon usage panel was investigated in ALS and control motor cortex tissue to verify whether, and to what extent, TDP-43 loss of function occurs in ALS. We find that profiles of TDP-43-regulated cryptic exons and changed exon usage discriminate ALS brain tissue from controls, verifying TDP-43 loss of function as a pathomechanism in ALS. We propose that TDP-43-regulated splicing markers with most predictive value for therapeutic intervention will be those based on splicing events that occur both in tissues/biofluids amenable to sampling, and in brain tissue susceptible to disease.