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.

This study aimed to compare different algorithms based on the Edinburgh Cognitive and Behavioural ALS Screen (ECAS) to classify patients with amyotrophic lateral sclerosis (ALS) according to their neuropsychological phenotype to identify possible discrepancies among these systems. ECAS-Cognitive and -Carer Interview (ECAS-C/-CI) scores of N = 901 patients with ALS without a formal diagnosis of dementia were retrospectively retrieved. Patients were classified, pursuant to Strong et al.’s criteria, as cognitively and behaviourally normal (ALScbn), cognitively and/or behaviourally impaired (ALSci/bi/cbi), or Possible ALS-FTD, according the following ECAS-based algorithms: (1) Abrahams’, solely addressing ECAS-C total and ALS-Specific subtotals; (2) Poletti et al.’s, addressing single task-level ECAS-C scores; (3) “Subscale”, addressing ECAS-C subscales (i.e., Language, Executive, Fluency, Memory and Visuospatial). All algorithms relied on single-item-level ECAS-CI scores for behavioural classifications. Whilst agreement rates among these classifications were moderate to high (84–86

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 manifestions appear. Phenoconversion, the emergence of clinically manifest syndomes, may be preceded by years to decades of silent pathobiological activity that can only be mapped by an array of biomarkers. ALS and FTD, traditionally identified as distinct clinical syndromes, are increasingly recognized to exist along a spectrum of clinical syndromes with shared genetic risk and shared 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 (1) the detection of either ALS or FTD pathobiology prior to phenoconversion and/or (2) 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. Whereas no single biotypic marker is yet able to 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 aging. By including such nontraditional 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.

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.

As the use of genetic testing for neurological diseases increases exponentially, interpreting variants of uncertain significance (VUS) has become a challenging problem. Nowhere has this become more evident than in amyotrophic lateral sclerosis (ALS) and the frontotemporal dementias (FTDs), both of which are complex heterogeneous disorders in which an increasing array of disease-causative and modifying genetic variants are observed. Moreover, while traditionally identified as distinct clinical syndromes, ALS and FTD are increasingly recognized to exist along a spectrum of clinical syndromes (termed the frontotemporal spectrum disorders of ALS; ALS-FTSD) with shared genetic risk. While VUS are generally not used in clinic for decision-making or counseling given their obvious limitations in being medically actionable, their correct interpretation is dynamic and rapidly evolving. VUS are increasingly the subject of intensive study as potential determinants of biological outcomes. It is timely therefore to review the current state of VUS interpretation and to critically evaluate how this can be applied to enhance both patient care and treatment decisions in the broader context of neurological disorders and more specifically in the context of ALS and ALS-FTSD. In doing so, we also explore the evolving challenge of defining pathogenicity of oligogenic inheritance in the context of multiple VUS detection in a single individual using an illustrative case example.

BACKGROUND AND OBJECTIVES:Plasma biomarkers of Alzheimer disease (AD), neuroinflammation, and neurodegeneration are increasingly being used in clinical trials for diagnosis and monitoring of dementia. However, their association with longitudinal structural brain MRI changes, an important outcome measure across neurodegenerative and cerebrovascular diseases, is less known. We investigated how baseline plasma biomarkers reflect MRI markers of progression over time in patients with neurodegenerative and cerebrovascular diseases. METHODS:This longitudinal cohort study included patients from the Ontario Neurodegenerative Disease Research Initiative diagnosed with AD or mild cognitive impairment (AD/MCI), Parkinson disease (PD), frontotemporal dementia spectrum disorders (FTD), or cerebrovascular disease (CVD), followed annually for 2 years. Recruitment took place at specialized university-based dementia, movement disorders, and/or stroke clinics in the province of ON, Canada. MRI outcomes included markers of cerebral atrophy (ventricular CSF and regional gray matter volumes) and of small vessel disease pathology (white matter hyperintensity [WMH], perivascular spaces, and lacunar volumes). Hemorrhagic markers at baseline were also included. Plasma levels of glial fibrillary acidic protein (GFAP), neurofilament light chain (NfL), phosphorylated tau181 and tau217 (p-tau181, p-tau217), and β-amyloid (Aβ42/40) were quantified from blood samples collected at baseline using Simoa and used as predictors in linear mixed models adjusted for time (months), age, sex, apolipoprotein E (APOE)-ε4 carrier status, kidney function, vascular risk factors, microtubule-associated protein tau (MAPT) diplotypes, waist-hip circumference ratio, and disease duration. RESULTS:We analyzed 1,240 MRIs from 473 patients (age: 69.2 ± 7.4 [range: 49-87]; 32.8% women). Elevated baseline levels of GFAP, NfL, p-tau181, and p-tau217, and to a lesser extent decreased levels of Aβ42/40, were significantly associated with more cerebral atrophy and WMH burden at baseline (|B| = 0.02 to 1.69, p = 0.044 to <0.001) and with progression over time (|B| = 0.001 to 0.028, p = 0.049 to <0.001) in the pooled disease-agnostic group. Within disease-specific cohorts, GFAP and NfL were associated with cerebral atrophy and/or small vessel disease copathology in AD/MCI, PD, FTD, or CVD. P-tau181 and p-tau217 were associated with cerebral atrophy and/or small vessel disease copathology in AD/MCI, CVD, PD-MCI, or PD-dementia. DISCUSSION:Selected plasma biomarkers seem useful as prognosis and monitoring tools of longitudinal imaging changes within real-world populations of neurodegenerative and/or cerebrovascular diseases, and provide insight into overlap across diseases in shared pathologic burden.

The phosphorylation of tau is a critical determinant of both its physiological function and the induction of pathological misfolding and aggregation. We have previously provided evidence that tau phosphorylation at Thr175 results in the exposure of the N-terminal phosphatase-activating domain (PAD) leading to the subsequent phosphorylation of Thr231, and formation of tau oligomers. A number of tauopathies, including chronic traumatic encephalopathy (CTE), amyotrophic lateral sclerosis with cognitive impairment (ALSci), and experimental traumatic brain injury (TBI) have been proposed to be associated with this cascade of events. However, the cellular mechanism by which Thr175 tau is phosphorylated remains unclear. In this study we identified ERK2, JNK1, and p38 as candidate kinases through molecular and histological analyses in a rodent model of TBI, where increased kinase activity and protein interaction were associated with pThr175 tau. We confirmed that both ERK2 and JNK1 are capable of phosphorylating Thr175 tau in vitro, but only ERK2-mediated phosphorylation of Thr175 tau induced the pathological cascade characterized by PAD exposure and the generation of oligomeric, truncated and neurofibrillary tau. Thr175 phosphorylation was also associated with an altered interaction between tau and the molecular chaperone protein DnaJC7, which regulates tau misfolding. Additionally, we observed that pThr175 and pThr231 tau were increased by oxidative stress, which was associated with the activation of the MAPK signaling pathways. These findings further clarify the mechanisms leading to Thr175 tau phosphorylation and its role in pathological tau formation by identifying ERK1 and JNK2 as important cellular mediators.

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 tauT175D) co-expressing an ALS-associated TAR DNA-binding protein 43 (TDP-43) mutant (TDP-43M337V) 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-wtTDP-43 or EGFP-tau constructs were expressed in HEK293 or SH-SY5Y cells. The secretome and EV fractions contained wtTDP-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.

Spinal metastases can be contained in the bone or have epidural spread. Whether the extent of epidural involvement changes tumor response to therapy is unknown. The decision of when to treat disease progression with focal radiation therapy with or without surgery vs. systemic therapy is debated. The present study compared outcomes and local tumor control after stereotactic body radiation therapy (SBRT) between patients with spine metastases localized to the bone (Bilsky 0) vs. patients with mild epidural spread (Bilsky 1). A retrospective analysis of a prospectively maintained database of adult oncological patients who underwent SBRT to the spine at a single, large, tertiary care facility from August 2010 to January 2021 was performed. Patients with Bilsky grades 1a, 1b and 1c were grouped and compared. Approximately half (53.7%) of the 255 patients identified had Bilsky grade 1 epidural disease. Of the 311 spine treatment sites, 86 (27.7%) had a radiosensitive histology, 116 (37.3%) had intermediate radiosensitivity and 109 (35.0%) had a radioresistant histology. Patients with Bilsky grade 1 were more predisposed to receive surgery followed by SBRT compared with those with Bilsky grade 0 (21.0% vs. 6.3%; P=0.0002). Patients with Bilsky grade 0 compression had 92.0% local control at 12 months and 85.8% local control at 24 months; patients with Bilsky grade 1 compression had 85.6% local control at 12 months and 77.6% local control at 24 months. Biologically effective dose and infield progression between patients presenting with Bilsky grade 0 and 1 compression were not statistically different. Local control rates did not differ significantly between Bilsky grade 0 and grade 1 patients following treatment with spinal SBRT. However, patients with grade 1 disease were more likely to receive surgery before SBRT. Overall, evidence indicates that patients may benefit from treatment with SBRT before epidural disease progresses to requiring separation surgery.

Tau protein plays a critical role in the physiological functioning of the central nervous system by providing structural integrity to the cytoskeletal architecture of neurons and glia through microtubule assembly and stabilization. Under certain pathological conditions, tau is aberrantly phosphorylated and aggregates into neurotoxic fibrillary tangles. The aggregation and cell-to-cell propagation of pathological tau leads to the progressive deterioration of the nervous system. The clinical entity of traumatic brain injury (TBI) ranges from mild to severe and can promote tau aggregation by inducing cellular mechanisms and signalling pathways that increase tau phosphorylation and aggregation. Chronic traumatic encephalopathy (CTE), which is a consequence of repetitive TBI, is a unique tauopathy characterized by pathological tau aggregates located at the depths of the sulci and surrounding blood vessels. The mechanisms leading to increased tau phosphorylation and aggregation in CTE remain to be fully defined but are likely the result of the primary and secondary injury sequelae associated with TBI. The primary injury includes physical and mechanical damage resulting from the head impact and accompanying forces that cause blood–brain barrier disruption and axonal shearing, which primes the central nervous system to be more vulnerable to the subsequent secondary injury mechanisms. A complex interplay of neuroinflammation, oxidative stress, excitotoxicity, and mitochondrial dysfunction activate kinase and cell death pathways, increasing tau phosphorylation, aggregation and neurodegeneration. In this review, we explore the most recent insights into the mechanisms of tau phosphorylation associated with TBI and propose how multiple cellular pathways converge on tau phosphorylation, which may contribute to CTE progression.

Lumbar stenosis with grade 1 spondylolisthesis can be treated with decompression with or without fusion, while fusion providing better quality-of-life metrics and reduced re-operation rates. Patients with larger amounts of anterolisthesis are thought to be at higher risk of progression with decompression alone rather than decompression and fusion. Endoscopic interlaminar laminectomy is an ultra-minimally invasive approach that preserves the posterior tension band and has less muscle disruption than traditional minimally invasive tubular decompressions. All patients who underwent endoscopic interlaminar lumbar stenosis with spondylolisthesis at a large tertiary academic center were reviewed. To exclude trivial spondylolistheses, inclusion criteria were a minimal of 5mm of slip and existing pre-operative and post-operative flexion/extension lumbar x-rays for review of stability. Clinical and radiographic measures were assessed. Twenty-four patients underwent endoscopic interlaminar laminectomy for spondylolisthesis during the study period. Of these, 4 patients met inclusion criteria. Three patients were elective for neurogenic claudication and discharged home from PACU, and 1 was emergent for worsening cauda equina symptoms who was discharged home after 3 days. All had clinical improvement. Average age was 77.3, BMI was 27.6, EBL of 8.7mL, and surgical length of 167min. The degree of anterolistheis did not change in extension (8.00 ± 0.82 pre-op vs 8.03 ± 0.92 post-op, p = 0.89) nor in flexion (8.55 pre-op ± 0.66 vs 8.9 ± 0.91, p = 0.26). All patients were treated at L4-5 and no patients underwent re-operation with an average follow up time of 8.1 ± 0.7 months. Endoscopic interlaminar laminectomy is an effective treatment for lumbar stenosis with grade 1 spondylolisthesis >5mm without notable risk of progression of radiographic instability.

Social cognition is impacted early in the disease progression of many neurodegenerative diseases (ND). The Salience network (SN) is an intrinsically connected brain network responsible for social cognitive function. Keys hubs of this brain network, the anterior insula (AI) and anterior cingulate cortex (ACC), are reported to incorporate ‘bottom-up’ signals from subcortical regions such as the amygdala and periaqueductal gray (PAG), but this mechanism and the subcortical contribution to SN connectivity is poorly understood. Our aim was to investigate the contribution of cortical and subcortical structures to SN functional connectivity and to social cognition across NDs. 76 participants (21 Alzheimer’s disease, 13 behavioural variant frontotemporal dementia, and 42 Parkinson’s disease) from the Ontario Neurodegenerative Research Initiative (ONDRI) baseline or one-year follow up visits with resting state fMRI, Montreal Cognitive Assessment (MoCA) total scores, and informant-reported socioemotional sensitivity scores using the Revised Self-Monitoring Scale (RSMS) were included (higher score, indicating higher function). All groups were age- and sex-matched. Fisher-transformed correlation coefficients of functional connectivity from an ROI-to-ROI analysis between cortical and subcortical SN ROIs were used to create a mean cortical SN value and mean subcortical SN value to use in linear regression modelling with behavioural scores. Mean cortical and subcortical SN connectivity were significantly associated with RSMS total score (b = 2.94, p = 0.041; (b = 3.60, p = 0.014, respectively), independent of cognitive function, with higher connectivity predicting higher score. The interaction between cortical and subcortical connectivity was not significantly associated with RSMS total score. Mean cortical and subcortical connectivity was significantly associated with RSMS-EX (expressive behaviour of others) and RSMS-SP (self-presentation) subscores (b = 1.36, p = 0.049; b = 1.44, p = 0.040; b coef = 1.58, p-value = 0.033; b coef = 2.15, p-value = 0.005, respectively). Our results indicate a stronger contribution of subcortical structures to social cognition-related functional connectivity across various neurodegenerative diseases. Despite previous associations with cortical regions, our evidence suggests that alterations in subcortical structures mediate changes in social cognition. Further exploration in larger cohorts is necessary, as impaired social cognition in patients with ND is associated with increased caregiver distress.

Paraspeckles are nuclear condensates formed by NEAT1_2 lncRNA and different RNA-binding proteins. In general, these membraneless organelles function in the regulation of gene expression and translation and in miRNA processing, and in doing this, they regulate cellular homeostasis and mediate pro-survival in the cell. Despite evidence showing the importance of paraspeckles in the stress response, the dynamics of paraspeckles and their components under conditions of osmotic stress remain unknown. We exposed HEK293T cells to sorbitol and examined NEAT1_2 expression using real-time PCR. Localization and quantification of the main paraspeckle components, NEAT1_2, PSPC1, NONO, and SFPQ, in different cellular compartments was performed using smFISH and immunofluorescence. Our findings showed a significant decrease in total NEAT1_2 expression in cells after osmotic stress. Sorbitol shifted the subcellular localization of NEAT1_2, PSPC1, NONO, and SFPQ from the nucleus to the cytoplasm and decreased the number and size of NEAT1_2 foci in the nucleus. PSPC1 formed immunoreactive cytoplasmic fibrils under conditions of osmotic stress, which slowly disassembled under recovery. Our study deepens the paraspeckle dynamics in response to stress, suggesting a novel role for NEAT1_2 in the cytoplasm in osmotic stress and physiological conditions.

Using bio-based metal-organic frameworks (bio-MOFs) holds promise for innovative approaches in cancer treatment by leveraging the unique properties of these materials for targeted therapeutic applications. In this study, a novel bio-Schiff base ligand derived from vanillin was successfully synthesized, and it was used to create a bio-based copper-based MOF through a one -pot solvothermal method. The bio-MOF was then comprehensively characterized and utilized to encapsulate the anticancer drug curcumin (CUR). The BET analysis revealed an average pore diameter of approximately 2.25 nm for the bio-MOF, highlighting its suitability for CUR loading due to its smaller size relative to the pore diameter. The entrapment efficiency and loading capacity of CUR were established at approximately 84.35% and 28.11%, respectively, resulting in CUR@bio-MOF. Furthermore, the CUR@bio-MOF was coated with the carboxymethyl cellulose (CMC) biopolymer, resulting in the CMC/ CUR@bio-MOF biocomposite hydrogel designed for pH-responsive delivery under physiological conditions at pH 7.4 and the acidic environment of tumor cells at a pH of 5.0. The in vitro CUR release study indicated that the CMC/bio-MOF exhibited more attractive performances than the pristine bio-MOF. Analysis of CUR release from bio-MOF and CMC/bio-MOF aligned most closely with the Higuchi model at both pH levels of 5.0 and 7.4. In vitro cytotoxicity screening displayed the high efficacy of CUR@bio-MOF against cancer cells. Furthermore, using confocal laser microscopy, it was observed that CUR@bio-MOF exhibited the ability to efficiently deliver drugs into cells. The findings presented here indicate that the developed bio-based carrier is an effective delivery system for the controlled and pH-sensitive release of curcumin.

Rho guanine nucleotide exchange factor (RGNEF) is a guanine nucleotide exchange factor (GEF) mainly involved in regulating the activity of Rho-family GTPases. It is a bi-functional protein, acting both as a guanine exchange factor and as an RNA-binding protein. RGNEF is known to act as a destabilizing factor of neurofilament light chain RNA (NEFL) and it could potentially contribute to their sequestration in nuclear cytoplasmic inclusions. Most importantly, RGNEF inclusions in the spinal motor neurons of ALS patients have been shown to co-localize with inclusions of TDP-43, the major well-known RNA-binding protein aggregating in the brain and spinal cord of human patients. Therefore, it can be hypothesized that loss-of-function of both proteins following aggregation may contribute to motor neuron death/survival in ALS patients. To further characterize their relationship, we have compared the transcriptomic profiles of neuronal cells depleted of TDP-43 and RGNEF and show that these two factors predominantly act in an antagonistic manner when regulating the expression of axon guidance genes. From a mechanistic point of view, our experiments show that the effect of these genes on the processivity of long introns can explain their mode of action. Taken together, our results show that loss-of-function of factors co-aggregating with TDP-43 can potentially affect the expression of commonly regulated neuronal genes in a very significant manner, potentially acting as disease modifiers. This finding further highlights that neurodegenerative processes at the RNA level are the result of combinatorial interactions between different RNA-binding factors that can be co-aggregated in neuronal cells. A deeper understanding of these complex scenarios may lead to a better understanding of pathogenic mechanisms occurring in patients, where more than one specific protein may be aggregating in their neurons.

The progressive degeneration of motor neurons in amyotrophic lateral sclerosis (ALS) is accompanied by the formation of a broad array of cytoplasmic and nuclear neuronal inclusions (protein aggregates) largely containing RNA-binding proteins such as TAR DNA-binding protein 43 (TDP-43) or fused in sarcoma/translocated in liposarcoma (FUS/TLS). This process is driven by a liquid-to-solid phase separation generally from proteins in membrane-less organelles giving rise to pathological biomolecular condensates. The formation of these protein aggregates suggests a fundamental alteration in the mRNA expression or the levels of the proteins involved. Considering the role of the epigenome in gene expression, alterations in DNA methylation, histone modifications, chromatin remodeling, non-coding RNAs, and RNA modifications become highly relevant to understanding how this pathological process takes effect. In this review, we explore the evidence that links epigenetic mechanisms with the formation of protein aggregates in ALS. We propose that a greater understanding of the role of the epigenome and how this inter-relates with the formation of pathological LLPS in ALS will provide an attractive therapeutic target.

The SARS-CoV-2 nucleocapsid protein (N protein) is critical in viral replication by undergoing liquid-liquid phase separation to seed the formation of a ribonucleoprotein (RNP) complex to drive viral genomic RNA (gRNA) translation and in suppressing both stress granules and processing bodies, which is postulated to increase uncoated gRNA availability. The N protein can also form biomolecular condensates with a broad range of host endogenous proteins including RNA binding proteins (RBPs). Amongst these RBPs are proteins that are associated with pathological, neuronal, and glial cytoplasmic inclusions across several adult-onset neurodegenerative disorders, including TAR DNA binding protein 43 kDa (TDP-43) which forms pathological inclusions in over 95% of amyotrophic lateral sclerosis cases. In this study, we demonstrate that the N protein can form biomolecular condensates with TDP-43 and that this is dependent on the N protein C-terminus domain (N-CTD) and the intrinsically disordered C-terminus domain of TDP-43. This process is markedly accelerated in the presence of RNA. In silico modeling suggests that the biomolecular condensate that forms in the presence of RNA is composed of an N protein quadriplex in which the intrinsically disordered TDP-43 C terminus domain is incorporated.

Ethical animal use follows the 3R’s: Replacement, Reduction and Refinement. Here, we present the use of simultaneous jugular vein and cisterna magna catheterization via a port system in rats for repeated fluid sampling for 14 consecutive days without loss of catheter patency. This technique allows repeated intra-animal sampling without anesthesia and, if used with pooling samples from a cohort of animals, replaces the need for terminal collections for sufficient sample volumes.