The sense of antisense therapies in ALS.

Treatment of patients with amyotrophic lateral sclerosis (ALS) has entered a new era now that encouraging results about antisense oligonucleotides (ASOs) are becoming available and a first ASO therapy for ALS has been approved by the FDA. Moreover, there is hope not only that ALS can be stopped but also that symptoms can be reversed. Until now, degrading ASOs seemed to be successful mostly for rarer forms of familial ALS. However, the first attempts to correct mis-splicing events in sporadic ALS are underway, as well as a clinical trial examining interference with a genetic modifier. In this review, we discuss the current status of using ASOs in ALS and the possibilities and pitfalls of this therapeutic strategy.

Differences in Cerebral Glucose Metabolism in ALS Patients with and without C9orf72 and SOD1 Mutations.

Amyotrophic lateral sclerosis (ALS) is characterized by progressive loss of upper and lower motor neurons. In 10% of patients, the disorder runs in the family. Our aim was to study the impact of ALS-causing gene mutations on cerebral glucose metabolism. Between October 2010 and October 2022, 538 patients underwent genetic testing for mutations with strong evidence of causality for ALS and 18F-2-fluoro-2-deoxy-D-glucose-PET (FDG PET), at University Hospitals Leuven. We identified 48 C9orf72-ALS and 22 SOD1-ALS patients. After propensity score matching, two cohorts of 48 and 21 matched sporadic ALS patients, as well as 20 healthy controls were included. FDG PET images were assessed using a voxel-based and volume-of-interest approach. We observed widespread frontotemporal involvement in all ALS groups, in comparison to healthy controls. The degree of relative glucose metabolism in SOD1-ALS in motor and extra-motor regions did not differ significantly from matched sporadic ALS patients. In C9orf72-ALS, we found more pronounced hypometabolism in the peri-rolandic region and thalamus, and hypermetabolism in the medulla extending to the pons, in comparison to matched sporadic ALS patients. Our study revealed C9orf72-dependent differences in glucose metabolism in the peri-rolandic region, thalamus, and brainstem (i.e., medulla, extending to the pons) in relation to matched sporadic ALS patients.

Multiomics and machine-learning identify novel transcriptional and mutational signatures in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis is a fatal and incurable neurodegenerative disease that mainly affects the neurons of the motor system. Despite the increasing understanding of its genetic components, their biological meanings are still poorly understood. Indeed, it is still not clear to which extent the pathological features associated with amyotrophic lateral sclerosis are commonly shared by the different genes causally linked to this disorder. To address this point, we combined multiomics analysis covering the transcriptional, epigenetic and mutational aspects of heterogenous human induced pluripotent stem cell-derived C9orf72-, TARDBP-, SOD1- and FUS-mutant motor neurons as well as datasets from patients' biopsies. We identified a common signature, converging towards increased stress and synaptic abnormalities, which reflects a unifying transcriptional program in amyotrophic lateral sclerosis despite the specific profiles due to the underlying pathogenic gene. In addition, whole genome bisulphite sequencing linked the altered gene expression observed in mutant cells to their methylation profile, highlighting deep epigenetic alterations as part of the abnormal transcriptional signatures linked to amyotrophic lateral sclerosis. We then applied multi-layer deep machine-learning to integrate publicly available blood and spinal cord transcriptomes and found a statistically significant correlation between their top predictor gene sets, which were significantly enriched in toll-like receptor signalling. Notably, the overrepresentation of this biological term also correlated with the transcriptional signature identified in mutant human induced pluripotent stem cell-derived motor neurons, highlighting novel insights into amyotrophic lateral sclerosis marker genes in a tissue-independent manner. Finally, using whole genome sequencing in combination with deep learning, we generated the first mutational signature for amyotrophic lateral sclerosis and defined a specific genomic profile for this disease, which is significantly correlated to ageing signatures, hinting at age as a major player in amyotrophic lateral sclerosis. This work describes innovative methodological approaches for the identification of disease signatures through the combination of multiomics analysis and provides novel knowledge on the pathological convergencies defining amyotrophic lateral sclerosis.

FUS-ALS hiPSC-derived astrocytes impair human motor units through both gain-of-toxicity and loss-of-support mechanisms.

BACKGROUND: Astrocytes play a crucial, yet not fully elucidated role in the selective motor neuron pathology in amyotrophic lateral sclerosis (ALS). Among other responsibilities, astrocytes provide important neuronal homeostatic support, however this function is highly compromised in ALS. The establishment of fully human coculture systems can be used to further study the underlying mechanisms of the dysfunctional intercellular interplay, and has the potential to provide a platform for revealing novel therapeutic entry points. METHODS: In this study, we characterised human induced pluripotent stem cell (hiPSC)-derived astrocytes from FUS-ALS patients, and incorporated these cells into a human motor unit microfluidics model to investigate the astrocytic effect on hiPSC-derived motor neuron network and functional neuromuscular junctions (NMJs) using immunocytochemistry and live-cell recordings. FUS-ALS cocultures were systematically compared to their CRISPR-Cas9 gene-edited isogenic control systems. RESULTS: We observed a dysregulation of astrocyte homeostasis, which resulted in a FUS-ALS-mediated increase in reactivity and secretion of inflammatory cytokines. Upon coculture with motor neurons and myotubes, we detected a cytotoxic effect on motor neuron-neurite outgrowth, NMJ formation and functionality, which was improved or fully rescued by isogenic control astrocytes. We demonstrate that ALS astrocytes have both a gain-of-toxicity and loss-of-support function involving the WNT/ß-catenin pathway, ultimately contributing to the disruption of motor neuron homeostasis, intercellular networks and NMJs. CONCLUSIONS: Our findings shine light on a complex, yet highly important role of astrocytes in ALS, and provides further insight in to their pathological mechanisms.

CRISPR/Cas9 screen in human iPSC-derived cortical neurons identifies NEK6 as a novel disease modifier of C9orf72 poly(PR) toxicity.

INTRODUCTION: The most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are hexanucleotide repeats in chromosome 9 open reading frame 72 (C9orf72). These repeats produce dipeptide repeat proteins with poly(PR) being the most toxic one. METHODS: We performed a kinome-wide CRISPR/Cas9 knock-out screen in human induced pluripotent stem cell (iPSC) -derived cortical neurons to identify modifiers of poly(PR) toxicity, and validated the role of candidate modifiers using in vitro, in vivo, and ex-vivo studies. RESULTS: Knock-down of NIMA-related kinase 6 (NEK6) prevented neuronal toxicity caused by poly(PR). Knock-down of nek6 also ameliorated the poly(PR)-induced axonopathy in zebrafish and NEK6 was aberrantly expressed in C9orf72 patients. Suppression of NEK6 expression and NEK6 activity inhibition rescued axonal transport defects in cortical neurons from C9orf72 patient iPSCs, at least partially by reversing p53-related DNA damage. DISCUSSION: We identified NEK6, which regulates poly(PR)-mediated p53-related DNA damage, as a novel therapeutic target for C9orf72 FTD/ALS.

Effect of neurofilament analysis on the diagnostic delay in amyotrophic lateral sclerosis.

AIMS: The aim of this study was to investigate whether neurofilament light (NfL) and phosphorylated neurofilament heavy (pNfH) in cerebrospinal fluid (CSF), sampled prior to referral to a neuromuscular reference center (NMRC), shorten the diagnostic delay in patients with amyotrophic lateral sclerosis. METHODS: In this retrospective study, patients with ALS were included with (i) determination of neurofilaments (Nfs) before referral to the NMRC (preC-Nfs ALS, n = 58), (ii) determination of Nfs at the NMRC (C-Nfs, n = 54) or (iii) with no determination of Nfs (C-No Nfs, n = 180). Fifty-six disease controls were included. RESULTS: The preC-Nfs cohort had CSF sampled 2.2 months (range: 0.6-12.0 months) before referral to the NMRC. In this cohort, the diagnostic delay was significantly shorter [median (range): 8.24 (2.37-49.7) months] than in the C-Nfs cases [median (range): 11.4 (2.93-86.5) months; p < 0.05], but not in the C-No Nfs cases. When including the disease progression rate and the presence of a genetic mutation as covariates, the difference ceased to exist (p = 0.14). pNfH and NfL levels in the preC-Nfs cohort were significantly higher than in disease controls (p < 0.0001). Both Nfs showed a similar discriminating performance. CONCLUSIONS: CSF Nfs assessed before the diagnosis of ALS at a NMRC decreased the diagnostic delay in specific cases by 3 months and only when other covariates were not taken into account.

Characterising ALS disease progression according to El Escorial and Gold Coast criteria.

BACKGROUND: The Gold Coast criteria (GCC) have been proposed as a means of selecting patients for amyotrophic lateral sclerosis (ALS) clinical trials. We aimed to characterise disease progression according to the GCC. METHODS: Data from population-based ALS registries from the Netherlands and Belgium were analysed. The GCC additionally define ALS as lower motor neuron (LMN) dysfunction in ≥2 body regions without upper motor neuron dysfunction. Therefore, the revised El Escorial criteria (rEEC) were supplemented with a 'Gold Coast ALS' category for patients with only LMN dysfunction in ≥2 body regions. We assessed survival time, ALS Functional Rating Scale (ALSFRS-R) progression rates and between-patient variability per diagnostic category. RESULTS: We included 5957 ALS patients, of whom 600 (10.1%) fulfilled the GCC but not the rEEC, and 95 (1.6%) fulfilled only the rEEC. ALSFRS-R progression rates were similar for the rEEC (0.84 points/month) and GCC (0.81 points/month) with similar variability (standard deviation of 0.59 vs. 0.60) and median survival time (17.8 vs.18.7 months). Survival time and average progression rates varied (p<0.001) between categories. Per category, however, there was considerable between-patient variability with progression rates ranging from: -2.10 to -0.14 (definite), -1.94 to -0.06 (probable), -2.10 to -0.02 (probable laboratory supported), -1.79 to -0.02 (possible) and -1.31 to 0.08 (Gold Coast). CONCLUSIONS: The GCC broaden the definition of ALS, allowing more patients to participate in trials, while minimally impacting population heterogeneity. Given the large variability per diagnostic category, selecting only specific categories for trials may not result in a more homogeneous study population.

Respiratory onset of amyotrophic lateral sclerosis in a pregnant woman with a novel SOD1 mutation.

BACKGROUND AND PURPOSE: With the advent of gene therapies for amyotrophic lateral sclerosis (ALS), the importance of gene testing in ALS is increasing. This will likely lead to the identification of new variants for which the pathogenicity is not established. We aimed to study the pathogenicity of a newly identified variant in superoxide dismutase 1 (SOD1). METHODS: Gene testing was performed using Sanger sequencing. SOD1 activity in erythrocytes was measured using spectrophotometry. Postmortem brain and spinal cord sections were stained with antibodies against phospho-TDP-43 and SOD1. RESULTS: We identified a novel c.416G>T (p.Gly139Val) mutation in SOD1, which caused a rapidly progressive respiratory onset form of ALS. The mutation resulted in a 50% drop of SOD1 activity. Postmortem examination confirmed the absence of TDP-43 pathology and displayed typical SOD1 inclusions in remaining motor neurons, confirming the pathogenic nature of the mutation. CONCLUSIONS: Novel variants of unknown pathogenicity will be identified as a result of a surge in gene testing in people with ALS. An in-depth study of a newly identified p.Gly139Val mutation in SOD1 confirmed the pathogenicity of this mutation. Future patients with this particular mutation should qualify for SOD1 silencing or editing therapies.

Insights into the identification of a molecular signature for amyotrophic lateral sclerosis exploiting integrated microRNA profiling of iPSC-derived motor neurons and exosomes.

Amyotrophic lateral sclerosis (ALS) is a rare neurodegenerative disorder characterized by progressive degeneration of motor neurons (MNs). Most cases are sporadic, whereas 10% are familial. The pathological mechanisms underlying the disease are partially understood, but it is increasingly being recognized that alterations in RNA metabolism and deregulation of microRNA (miRNA) expression occur in ALS. In this study, we performed miRNA expression profile analysis of iPSC-derived MNs and related exosomes from familial patients and healthy subjects. We identified dysregulation of miR-34a, miR-335 and miR-625-3p expression in both MNs and exosomes. These miRNAs regulate genes and pathways which correlate with disease pathogenesis, suggesting that studying miRNAs deregulation can contribute to deeply investigate the molecular mechanisms underlying the disease. We also assayed the expression profile of these miRNAs in the cerebrospinal fluid (CSF) of familial (fALS) and sporadic patients (sALS) and we identified a significant dysregulation of miR-34a-3p and miR-625-3p levels in ALS compared to controls. Taken together, all these findings suggest that miRNA analysis simultaneously performed in different human biological samples could represent a promising molecular tool to understand the etiopathogenesis of ALS and to develop new potential miRNA-based strategies in this new propitious therapeutic era.

The role of inflammation in neurodegeneration: novel insights into the role of the immune system in C9orf72 HRE-mediated ALS/FTD.

Neuroinflammation is an important hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). An inflammatory reaction to neuronal injury is deemed vital for neuronal health and homeostasis. However, a continued activation of the inflammatory response can be detrimental to remaining neurons and aggravate the disease process. Apart from a disease modifying role, some evidence suggests that neuroinflammation may also contribute to the upstream cause of the disease. In this review, we will first focus on the role of neuroinflammation in the pathogenesis of chromosome 9 open reading frame 72 gene (C9orf72) hexanucleotide repeat expansions (HRE)-mediated ALS/FTD (C9-ALS/FTD). Additionally, we will discuss evidence from ex vivo and in vivo studies and finally, we briefly summarize the trials and progress of anti-inflammatory therapies.

The Brain-Heart Link: A Case Report of a Critically Located Multiple Sclerosis Lesion in the Brainstem Leading to Recurrent Takotsubo Syndrome.

Various central nervous system (CNS) diseases, including neurovascular and neuroinflammatory diseases, can lead to stress cardiomyopathy, also known as Takotsubo syndrome (TTS). We present a case of a 69-year-old woman with cardiovascular comorbidities, suffering from repeated episodes of TTS and respiratory failure due to a critical lesion in the brainstem, leading to a diagnosis of multiple sclerosis (MS). Despite aggressive treatment, intractable and recurrent symptoms in our patient occurred. Repeated bouts of autonomic dysfunction and respiratory failure ultimately led to installment of palliative care and the patient passing away. TTS should raise suspicion for underlying neurological diseases. Thorough questioning of previous neurological symptoms and extensive neurological workup is warranted. MS should be considered as a trigger of TTS also in elderly patients with cardiovascular risk factors.

Distinguishing Primary Lateral Sclerosis from Parkinsonian Syndromes with the Help of Advanced Imaging.

We describe a unique case of a patient presenting with unilateral mild paresis, slowing of the upper limb, and parkinsonism, who underwent a full imaging work-up including MRI, 123I-FP-CIT PET, 18F-FE-PE2I PET, and 18F-FDG PET. This case demonstrates that imaging may aid substantially in the diagnostic work-up of complex neurologic disorders.

Caspr2 autoantibody-associated Morvan syndrome predating thymoma relapse by 30 months.

Morvan's syndrome (MoS) is a rare autoimmune disorder characterized by central nervous system involvement, autonomic dysfunction and peripheral nerve hyperexcitability. MoS is believed to be caused by autoantibodies targeting contactin-associated protein 2 (Caspr2), a subunit of the neuronal voltage-gated potassium channel (VGKC) complex, usually in association with thymoma, less commonly with other malignancies. This case highlights an exceptional case of severe sleep disturbances and behavioural changes due to MoS, in a patient who would present with and be treated successfully for a second relapse of thymoma 30 months later. Originally he suffered from ocular myasthenia, another autoimmune disorder, which led to diagnosis of his original thymoma and first relapse.

Serum neurofilament heavy chains as early marker of motor neuron degeneration.

OBJECTIVE: To determine whether serum phosphorylated neurofilament heavy chain (pNfH) levels are elevated before patients were diagnosed with sporadic or familial ALS, and what the prognostic value of these prediagnostic pNfH levels is. METHODS: pNfH was measured via ELISA in leftovers of serum drawn for routine purposes before the time of diagnosis. These prediagnostic samples were retrieved from the biobank of the University Hospitals Leuven for 95 patients who in follow-up received a diagnosis of ALS. Additionally, 35 patients with mild cognitive impairment (MCI) and 85 healthy controls (HC) were included in this retrospective study. RESULTS: The median disease duration (range) from onset to prediagnostic sampling and from onset to diagnosis was 6.5 (-71.9-36.1) and 9.9 (2.0-40.7) months, respectively. Fifty-eight percent of the prediagnostic samples had serum pNfH levels above the 95th percentile of pNfH levels measured in HC. Serum pNfH levels (median (range)) were elevated up to 18 months before the diagnosis of ALS (91 pg/mL (6-342 pg/mL)) in comparison with HC (30 pg/mL (6-146 pg/mL); P = 0.05), and increased during the prediagnostic stage, which was not observed in patients with MCI. Furthermore, prediagnostic pNfH levels were a univariate predictor of survival in ALS (hazard ratio (95% CI): 2.16 (1.20-3.87); P = 0.01). INTERPRETATION: Our findings demonstrate that serum pNfH is elevated well before the time of diagnosis in mainly sporadic ALS patients. These results encourage to prospectively explore if pNfH has an added value to shorten the diagnostic delay in ALS.

Loss of paraplegin drives spasticity rather than ataxia in a cohort of 241 patients with SPG7.

OBJECTIVE: We took advantage of a large multinational recruitment to delineate genotype-phenotype correlations in a large, trans-European multicenter cohort of patients with spastic paraplegia gene 7 (SPG7). METHODS: We analyzed clinical and genetic data from 241 patients with SPG7, integrating neurologic follow-up data. One case was examined neuropathologically. RESULTS: Patients with SPG7 had a mean age of 35.5 ± 14.3 years (n = 233) at onset and presented with spasticity (n = 89), ataxia (n = 74), or both (n = 45). At the first visit, patients with a longer disease duration (>20 years, n = 62) showed more cerebellar dysarthria (p < 0.05), deep sensory loss (p < 0.01), muscle wasting (p < 0.01), ophthalmoplegia (p < 0.05), and sphincter dysfunction (p < 0.05) than those with a shorter duration (<10 years, n = 93). Progression, measured by Scale for the Assessment and Rating of Ataxia evaluations, showed a mean annual increase of 1.0 ± 1.4 points in a subgroup of 30 patients. Patients homozygous for loss of function (LOF) variants (n = 65) presented significantly more often with pyramidal signs (p < 0.05), diminished visual acuity due to optic atrophy (p < 0.0001), and deep sensory loss (p < 0.0001) than those with at least 1 missense variant (n = 176). Patients with at least 1 Ala510Val variant (58%) were older (age 37.6 ± 13.7 vs 32.8 ± 14.6 years, p < 0.05) and showed ataxia at onset (p < 0.05). Neuropathologic examination revealed reduction of the pyramidal tract in the medulla oblongata and moderate loss of Purkinje cells and substantia nigra neurons. CONCLUSIONS: This is the largest SPG7 cohort study to date and shows a spasticity-predominant phenotype of LOF variants and more frequent cerebellar ataxia and later onset in patients carrying at least 1 Ala510Val variant.

Recurrent dysphasia due to nivolumab-induced encephalopathy with presence of Hu autoantibody.

A 58-year-old man was being treated for squamous non-small-cell lung cancer with nivolumab. At the 17th of biweekly administrations he presented with global dysphasia, dysarthria and myoclonus in the right upper extremity. MRI showed multiple T2/FLAIR hyperintense lesions in the left hemisphere; lumbar puncture showed lymphocytic pleiocytosis in the CSF without identifiable pathogens. Hu antibodies were present in serum and CSF. Nivolumab was discontinued and corticosteroids were administered. The neurological symptoms gradually improved; MRI showed complete remission of cerebral lesions. After rechallenge with nivolumab his symptoms and cerebral lesions recurred, proving the causal relationship with nivolumab. After tapering of corticosteroids, a second relapse occurred.