Michael Joseph Strong

As we have entered the endemic phase of COVID-19, there is a need to assess the long-term effects of SARS-CoV-2 infection on the trajectory of neurodegenerative disorders. Herein, we investigate whether the SARS-CoV-2 RNA-binding nucleocapsid protein (NCP; N-protein) may influence molecular processes relevant to motor neuron degeneration in amyotrophic lateral sclerosis (ALS). We detected N-protein immunoreactivity in ALS spinal cord motor neurons, including cytoplasmic N-protein-positive puncta that colocalized with cytoplasmic TAR DNA-binding protein 43 (TDP-43). In vitro, optogenetically induced N-protein biomolecular condensates (BMCs) showed reversible, liquid-like behavior and recovered after photobleaching. Using this model, we found that these condensates colocalized with TDP-43 and the RNA-binding proteins G3BP1/2, PABPC1 and TIA1. Condensate formation was associated with TDP-43 cytoplasmic mislocalization, altered TARDBP expression, and altered exon 3 splicing of POLDIP3 . The N-terminal portion of N-protein influenced BMC formation, whereas truncated C-terminal proteoforms did not form detectable BMCs or show detectable TDP-43 colocalization in this assay. Extending these observations to a human cell model, we show that optogenetically induced N-protein BMCs colocalize with TDP-43 in human iPSC-derived motor neurons. Collectively, these findings support further investigation into potential molecular links between SARS-CoV-2 exposure and the trajectory of neurodegenerative disease.

Currently, pathological inclusions of ubiquitinated TDP-43 are considered central to the pathogenesis of amyotrophic lateral sclerosis (ALS). However, this view has yielded sparse attention to covert alternative pathways of aggregation in the disease. Here, we identified a pathological axis independent of TDP-43 led by ARPP21, a SUZ domain-containing RNA-binding protein encoded by a gene recently described in strong association with ALS. ARPP21 showed large fibrillar non-ubiquitinated aggregates in all the non-SOD1 ALS cases studied, consistently segregating from TDP-43 pathology. Biophysically, ARPP21 undergoes spontaneous phase separation, with a higher propensity for condensate formation than TDP-43 but without co-aggregating with it. ARPP21 also exhibited very slow soluble behavior and lower condensate dynamics compared to TDP-43, an effect that was more pronounced for the ARPP21-P529L disease-related variant. In human iPSC-derived motor neurons, expression of ARPP21 variants was sufficient to drive condensate formation and reduce cell viability. Mechanistically, ARPP21 promoted intercellular propagation by inducing tunnelling nanotube formation, enabling efficient cell-to-cell spreading of aggregates. Genetic analyses further identified multiple ARPP21 variants in ALS, supporting its clinical relevance. Together, our findings uncover ARPP21 as a previously unrecognized TDP-43-independent aggregation pathway in ALS, with implications for disease heterogeneity and therapeutic targeting.

Cognitive and behavioural dysfunction in frontotemporal dementia (FTD) evolves along a clinical continuum from a clinically silent stage of disease, through a prodromal period, often referred to as mild cognitive impairment (MCI) and/or mild behavioural impairment (MBI), and into a clinically manifest stage recognized by the syndromes of behavioural variant FTD and primary progressive aphasia (PPA). Similar cognitive and behavioural manifestations of varying severity are also encountered in individuals with amyotrophic lateral sclerosis (ALS). Notwithstanding this phenotypic overlap as well as a degree of shared genetic risk and underlying pathology, the approach to phenotypic characterization and description meaningfully differs between the ALS and FTD communities. The problem is particularly evident within the ALS community when describing impairment in the prodromal versus clinically manifest disease stages. Pertinent considerations include the nature of cognitive and behavioural assessments, the nosology used to describe symptoms identified, including those encompassed by the terms ‘behavioural’ and ‘neuropsychiatric’, the operationalized criteria that define prodromal syndromes and clinically manifest disease, and the extent to which these criteria rely on longitudinal (versus cross-sectional) data. A multi-stakeholder workshop, held in London, Ontario (12–14 May 2025), brought together neurologists, psychiatrists, neuropsychologists, neuropathologists, neuroscientists and neurogeneticists studying ALS and FTD, to promote interdisciplinary dialogue and to define a research agenda that might be jointly tackled to address these critical issues. A new nosology for classifying frontotemporal-related behavioural and neuropsychiatric dysfunction, which could be used to support empirical evidence collection, is proposed alongside a list of cognitive, language, behavioural and neuropsychiatric symptoms to be considered in the evaluation of individuals with ALS. Research priorities for each of the ALS and FTD communities, as well as initiatives that should be undertaken jointly, are highlighted. The long-term goal is to harmonize approaches to phenotypic characterization, the nosology used to describe symptoms and the criteria used to define landmark stages along the clinical continua of the motor neuron and frontotemporal axes, irrespective of clinical stage.

Determining the optimal timing of disease-modifying therapies for neurodegenerative disorders will necessitate identification of when the underlying pathobiological process becomes active, well in advance of the point at which clinical manifestations appear. Phenoconversion, the emergence of clinically manifest syndromes, may be preceded by years to decades of silent pathobiological activity that can only be mapped by an array of biomarkers. Amyotrophic lateral sclerosis (ALS) and frontotemporal dementias (FTD), traditionally identified as distinct clinical syndromes, are increasingly recognized to exist along a spectrum of clinical syndromes with shared genetic risk and underlying pathology. This clinicopathological spectrum is underpinned by cytoplasmic aggregation of TAR DNA-binding protein 43 (TDP-43) as the common neuropathological hallmark. In contrast, the majority of neuropathologically-defined frontotemporal lobar degeneration (FTLD) is associated with alterations in either TDP-43 metabolism (FTLD-TDP) or of the microtubule associated protein tau (FTLD-tau), with a smaller percentage associated with either autosomal dominant genetic mutations or impairments in the ubiquitin proteasome system. As the field of neurodegenerative disorders increasingly shifts towards the frameworks of a pathobiological definition of disease, there is a growing imperative to develop biomarkers that reflect the varied pathobiologies that underly these disorders, and to determine the sensitivity of such biomarkers to detect the presence of these pathobiologies before phenoconversion. To that end, an international workshop was convened in London, Canada in 2025 to review the evidence for existing or evolving biomarkers suitable for (i) the detection of either ALS or FTD pathobiology prior to phenoconversion and/or (ii) predict phenoconversion in at-risk individuals. Such biomarkers might be conceptualized as ‘biotypic biomarkers’, capturing their ability to describe an underlying pathophysiology whilst being agnostic to the emergent clinical manifestations. While currently no single biotypic marker can predict the emergence of ALS, FTD or their intersection, a multimodal approach to developing a biotypic biomarker profile holds promise for the detection of relevant pathobiological processes. The strength of such an approach would be augmented by also addressing issues of resiliency/susceptibility both in terms of genetic risk susceptibility profiles and developing sensitive biomarkers of genomic and cellular ageing. By including such non-traditional markers of disease, a more robust picture of not only the degenerative process but also of those factors that might potentially mitigate or drive a heightened probability of disease can be derived.

Background: This study aimed at identifying neuropsychological sub‐phenotypes in amyotrophic lateral sclerosis (ALS) within the mild cognitive impairment (MCI) and mild behavioral impairment (MBI) frameworks. Methods: We used individual task−/item‐level data from the cognitive and behavioral sections of the Edinburgh Cognitive and Behavioral ALS Screen (ECAS) from 901 non‐demented ALS to derive neuropsychological sub‐phenotypes pursuant to classical MCI and MBI frameworks and in accordance with an expanded version of Strong's criteria, which also addressed memory and visuo‐spatial measures. Results: The prevalence of MCI and MBI was 39% and 37%, respectively in this retrospective review. The following MCI sub‐phenotypes were identified: dysexecutive MCI—single‐ and multiple‐domain (dMCI‐sd: 63%; dMCI‐md: 24%, respectively); non‐dysexecutive MCI—single‐ and multiple‐domain (ndMCI‐sd: 12%; ndMCI‐md: 1%, respectively). MBI was classified as follows: apathetic MBI—single‐ and multiple‐domain (aMBI‐sd: 40%; aMBI‐md: 20%, respectively); apathetic‐disinihibited/perseverative MBI—multiple domain (ad/pMBI‐md: 21%); disinihibited/perseverative MBI—multiple domain (d/pMBI‐md: 7%); psychotic MBI—single‐ and multiple‐domain (psyMBI‐sd: 2%; psyMBI‐md: 3%, respectively); unclassifiable MBI—multiple domain (uMBI‐md: 1%). 143 (16%) of patients exhibited mild cognitive and behavioral impairment (MCBI). Conclusions: This study delivers a provisional, ECAS‐based classification for the neuropsychological sub‐phenotyping of non‐demented ALS patients, which, with further validation, might be useful for both research and clinical purposes.

Selective vulnerability of motor neurons is a defining feature of amyotrophic lateral sclerosis (ALS) and provides a valuable framework for uncovering mechanisms that distinguish resilient from vulnerable neuronal populations. We investigated whether dysregulation of neuroprotective microRNAs (miRNAs), miR-9-5p and miR-124-3p, contributes to the differential susceptibility of motor neuron subtypes. We focused on cervical spinal motor neurons (SMNs), which undergo drastic degeneration in ALS, and oculomotor neurons (OMNs), which remain functionally intact and rarely degenerate, allowing preservation of eye movement in ALS patients. Using a modified multiplexed fluorescent in situ hybridization protocol combined with immunofluorescence, we quantified the expression of miR-9-5p and miR-124-3p in cervical SMNs and OMNs from ALS and control cases. We observed significant downregulation of both miRNAs in ALS SMNs, while their expression was maintained in ALS OMNs. Stratification of ALS SMNs by TDP-43 pathological status revealed similarly reduced miRNA expression in neurons with and without cytoplasmic inclusions, suggesting that miRNA downregulation occurs independently of visible TDP-43 pathology. We assessed the localization of the Dicer cofactor TRBP and found that it colocalized with TDP-43 inclusions in ALS SMNs, suggesting that TRBP sequestration could prevent proper miRNA processing. However, TRBP remained normally localized in neurons without cytoplasmic inclusions, indicating that sequestration cannot fully account for miRNA reduction across all ALS motor neurons. These findings support a model in which early or subtle disruptions, preceding visible pathology, may also contribute to miRNA downregulation in ALS. By identifying preserved miRNA networks as correlates of oculomotor neuron resilience in ALS, this work also exposes new therapeutic targets potentially capable of reinstating miRNA expression and reprogramming vulnerable SMNs.

Background: The blood-brain barrier (BBB) is an intricate structure of the vascular endothelium that regulates trafficking required for cerebral homeostasis via tightly regulated influx and efflux transport mechanisms. It also performs a critical function in mediating communication between the periphery and the central nervous system (CNS). The BBB is designed to maintain a precisely regulated microenvironment conducive to normal neuronal activity. The development of in vitro BBB models has been driven by the need for a fast, reliable, and cost-effective tool to study the complexities of the BBB and to screen potential CNS drugs. Aim: This review provides readers with a concise yet comprehensive introduction to the fundamental histology of the BBB. This background is essential for interpreting model design constraints and limitations in BBB simulation. Following that, we thoroughly examine in vitro models, providing a comprehensive analysis of their applications and the equations used to assess specific BBB parameters. Relevance for patients: From a translational and clinical perspective, in vitro models of the BBB have the potential to provide novel insights into brain pathophysiology under adverse conditions, including brain tumors, traumatic injuries, strokes, and neurodegenerative disorders, and ultimately aid in the discovery of effective treatment strategies.

While most human genes express mRNA 3′untranslated region (3′UTR) variants of different lengths, their impact on cell physiology and disease remains largely unknown. Here, we studied 3′UTR length heterogeneity in amyotrophic lateral sclerosis (ALS) and determined that three ALS‐linked transcripts exhibit lengthening of their 3′UTRs in patient samples. We investigated phenotypical effects in a neuronal cell line expressing these 3′UTRs and observed that expression of these unique 3′UTRs induces morphological changes at different levels. Among the most expressed 3′UTR variants in ALS, NEFH 3′UTR‐Long induces the formation of nuclear RNA clusters, and Superoxide Dismutase 1 3′UTR‐Long diminishes filopodia in the plasma membrane. Sequestosome 1 3′UTR‐Long did not show major changes in nuclear RNA clusters or filopodia. Our findings provide the first evidence that 3′UTRs can modulate cellular phenotype independent of the coding region, further expanding the impact of alterations in mRNA biogenesis in ALS.

Perturbations in the metabolism of microtubule‐associated protein tau (tau) underlie the pathology of a broad array of dementias, including chronic traumatic encephalopathy, amyotrophic lateral sclerosis (ALS) with cognitive impairment (ALSci) and approximately half of the dementias associated with frontotemporal lobar degeneration. We recently observed significantly increased hippocampal tau pathology in rats injected with pseudophosphorylated human tau (2N4R tau ^T175D ) co‐expressing an ALS‐associated TAR DNA‐binding protein 43 (TDP‐43) mutant (TDP‐43 ^M337V ) when compared to wild‐type rats. To understand this mechanism, we examined whether the extracellular vesicles (EVs) derived from wild‐type TDP‐43 (wtTDP‐43) or tau‐expressing cells could transfer expression of these proteins to recipient cells, and whether co‐localization of these proteins occurs. mCherry‐ wt TDP‐43 or EGFP‐tau constructs were expressed in HEK293 or SH‐SY5Y cells. The secretome and EV fractions contained wt TDP‐43 or 2N4R tau protein and RNA, and could transfer proteins into nontransfected cells. Co‐localization was also detected in the cytosol of recipient cells. In silico modeling of tau and TDP‐43 interactions suggests hydrogen bonding underlies this interaction. These studies further our understanding of the interaction between tau and TDP‐43 by demonstrating their ability to co‐aggregate and in providing a mechanism by which cell–cell transfer of either protein via extracellular vesicles can lead to these synergistic interactions.