Majority Of Amyotrophic Lateral Sclerosis Patients Research Articles

Non-coding genome contribution to ALS.

The majority of amyotrophic lateral sclerosis (ALS) is caused by a complex gene-environment interaction. Despite high estimates of heritability, the genetic basis of disease in the majority of ALS patients are unknown. This limits the development of targeted genetic therapies which require an understanding of patient-specific genetic drivers. There is good evidence that the majority of these missing genetic risk factors are likely to be found within the non-coding genome. However, a major challenge in the discovery of non-coding risk variants is determining which variants are functional in which specific CNS cell type. We summarise current discoveries of ALS-associated genetic drivers within the non-coding genome and we make the case that improved cell-specific annotation of genomic function is required to advance this field, particularly via single-cell epigenetic profiling and spatial transcriptomics. We highlight the example of TBK1 where an apparent paradox exists between pathogenic coding variants which cause loss of protein function, and protective non-coding variants which cause reduced gene expression; the paradox is resolved when it is understood that the non-coding variants are acting primarily via change in gene expression within microglia, and the effect of coding variants is most prominent in neurons. We propose that cell-specific functional annotation of ALS-associated genetic variants will accelerate discovery of the genetic architecture underpinning disease in the vast majority of patients.

Optineurin Deficiency and Insufficiency Lead to Higher Microglial TDP-43 Protein Levels.

Mutations in optineurin, a ubiquitin-binding adaptor protein, cause amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease of motor neurons linked to chronic inflammation and protein aggregation. The majority of ALS patients, including those carrying the optineurin mutations, exhibit cytoplasmic mislocalization, ubiquitination, and aggregation of nuclear TAR DNA-binding protein 43 kDa (TDP-43). To address the crosstalk between optineurin and TDP-43, we generated optineurin knockout (KO) neuronal and microglial cell lines using the CRISPR/Cas9 approach. Interestingly, we observed that loss of optineurin resulted in elevated TDP-43 protein expression in microglial BV2 but not neuronal Neuro 2a and NSC-34 cell lines. No changes were observed at the mRNA level, suggesting that this increase was post-translationally regulated. To confirm this observation in primary cells, we then used microglia and macrophages from an optineurin loss-of-function mouse model that lacks the C-terminal ubiquitin-binding region (Optn470T), mimicking optineurin truncations in ALS patients. As observed in the BV2 cells, we also found elevated basal levels of TDP-43 protein in Optn470T microglia and bone marrow-derived macrophages. To test if inflammation could further enhance TDP-43 accumulation in cells lacking functional optineurin, we stimulated them with lipopolysaccharide (LPS), and we observed a significant increase in TDP-43 expression following LPS treatment of WT cells. However, this was absent in both BV2 Optn KO and primary Optn470T microglia, which exhibited the same elevated TDP-43 levels as in basal conditions. Furthermore, we did not observe nuclear TDP-43 depletion or cytoplasmic aggregate formation in either Optn470T microglia or LPS-treated WT or Optn470T microglia. Taken together, our results show that optineurin deficiency and insufficiency post-translationally upregulate microglial TDP-43 protein levels and that elevated TDP-43 levels in cells lacking functional optineurin could not be further increased by an inflammatory stimulus, suggesting the presence of a plateau.

Transcriptome analysis using patient iPSC-derived skeletal myocytes: Bet1L as a new molecule possibly linked to neuromuscular junction degeneration in ALS

Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular disease in which patients gradually become paralyzed due to loss of motor function. Many genetically inheritable mutations have been linked to ALS; however, the majority of ALS patients are considered sporadic. Therefore, there is a need for a common therapy that is effective for all ALS patients. Although there is evidence of the disease beginning in the periphery at the neuromuscular junction (NMJ), the specific processes involved in skeletal muscle and at the NMJ are still largely unknown. To study common disease mechanisms in ALS skeletal muscle, we performed RNA sequencing of skeletal myocytes differentiated from induced pluripotent stem cells (iPSCs) derived from familial ALS (with C9ORF72, SOD1, or TARDBP mutations) and sporadic ALS patients. Compared to healthy control lines, the myocytes from all ALS lines showed downregulation of four genes: BET1L, DCX, GPC3, and HNRNPK. We next measured the expression levels of these four genes in hind limb muscle samples from a rat model of familial ALS (SOD1G93A transgenic) and found that only the Bet1L gene, which encodes Bet1 Golgi Vesicular Membrane Trafficking Protein Like, was commonly downregulated. Bet1L protein appeared to be localized to the basal lamina of the NMJ, with decreased expression over time in SOD1G93A transgenic rats. Importantly, the expression levels began to decrease early in the disease process. Our results indicate that loss of Bet1L at the NMJ could be of interest for better understanding ALS disease progression.

Dysregulation of AMPA receptor subunit expression in sporadic ALS post-mortem brain.

Amyotrophic lateral sclerosis (ALS) is characterised by progressive motor neuron degeneration. Although there are over 40 genes associated with causal monogenetic mutations, the majority of ALS patients are not genetically determined. Causal ALS mutations are being increasingly mechanistically studied, though how these mechanisms converge and diverge between the multiple known familial causes of ALS (fALS) and sporadic forms of ALS (sALS) and furthermore between different neuron types, is poorly understood. One common pathway that is implicated in selective motor neuron death is enhanced α‐amino‐3‐hydroxyl‐5‐methyl‐4‐isoxazole‐propionate (AMPAR)‐mediated excitoxicity. Specifically, human in vitro and pathological evidence has linked the C9orf72 repeat expansion mutation to a relative increase in the Ca2+‐permeable AMPAR population due to AMPAR subunit dysregulation. Here, we provide the first comparative quantitative assessment of the expression profile of AMPAR subunit transcripts, using BaseScope, in post‐mortem lower motor neurons (spinal cord, anterior horn), upper motor neurons (motor cortex) and neurons of the pre‐frontal cortex in sALS and fALS due to mutations in SOD1 and C9orf72. Our data indicated that AMPAR dysregulation is prominent in lower motor neurons in all ALS cases. However, sALS and mutant C9orf72 cases exhibited GluA1 upregulation whereas mutant SOD1 cases displayed GluA2 down regulation. We also showed that sALS cases exhibited widespread AMPAR dysregulation in the motor and pre‐frontal cortex, though the exact identity of the AMPAR subunit being dysregulated was dependent on brain region. In contrast, AMPAR dysregulation in mutant SOD1 and C9orf72 cases was restricted to lower motor neurons only. Our data highlight the complex dysregulation of AMPAR subunit expression that reflects both converging and diverging mechanisms at play between different brain regions and between ALS cohorts. © 2019 Authors. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Modeling sporadic ALS in iPSC-derived motor neurons identifies a potential therapeutic agent

Amyotrophic lateral sclerosis (ALS) is a heterogeneous motor neuron disease for which no effective treatment is available, despite decades of research into SOD1-mutant familial ALS (FALS). The majority of ALS patients have no familial history, making the modeling of sporadic ALS (SALS) essential to the development of ALS therapeutics. However, as mutations underlying ALS pathogenesis have not yet been identified, it remains difficult to establish useful models of SALS. Using induced pluripotent stem cell (iPSC) technology to generate stem and differentiated cells retaining the patients' full genetic information, we have established a large number of in vitro cellular models of SALS. These models showed phenotypic differences in their pattern of neuronal degeneration, types of abnormal protein aggregates, cell death mechanisms, and onset and progression of these phenotypes in vitro among cases. We therefore developed a system for case clustering capable of subdividing these heterogeneous SALS models by their in vitro characteristics. We further evaluated multiple-phenotype rescue of these subclassified SALS models using agents selected from non-SOD1 FALS models, and identified ropinirole as a potential therapeutic candidate. Integration of the datasets acquired in this study permitted the visualization of molecular pathologies shared across a wide range of SALS models.

An analysis of characteristics of nerve conduction in 154 cases of amyotrophic lateral sclerosis

Objective: To analyze the features of nerve conduction in patients with amyotrophic lateral sclerosis (ALS), and explore the correlation between compound muscle action potential (CMAP) amplitude and disease duration and revised amyotrophic lateral sclerosis functional rating scale (ALSFRS-R). Methods: Standard motor and sensory nerve conduction studies were performed in 154 patients with ALS. The following parameters were collected including CMAP amplitude, distal motor latency (DML), motor conduction velocity, sensory conduction velocity and sensory nerve action potential amplitude. Regression study was done to explore the correlation between CMAP amplitude and disease duration and ALSFRS-R. Results: Motor nerve conduction abnormalities were presented in a majority of the patients with prolonged DML in the tibial nerve, median nerve and ulnar nerve as the most common form (61.06%-81.42%), followed by decreased CMAP amplitude (30.12%-53.98%), decreased MCV (12.05%-16.81%) and absence of CMAP (2.65%-9.73%). Sensory nerve conduction abnormalities were detected in a small proportion of patients and the decreased SCV, decreased SNAP amplitude and absence of SNAP in the sural nerve, median nerve and ulnar nerve were found in 1.22%-2.73%, 0-1.82% and 0-1.22% patients respectively. No correlation was found between CMAP of the common peroneal nerve, tibial nerve, median nerve and ulnar nerve and the disease duration (P>0.05), while significant positive correlation was established between CMAP amplitude of the median nerve and ulnar nerve and ALSFRS-R (r=0.273, P=0.016; r=0.357, P=0.001). Conclusions: Motor nerve conduction is abnormal in a majority of ALS patients with prolonged DML as the most common form, while abnormal sensory nerve conduction is only found in a few of ALS patients. CMAP amplitude of the median nerve and ulnar nerve might be of certain clinical value in evaluating the severity of ALS.

Reduced Cognitive Function in Chinese Patients With Early Amyotrophic Lateral Sclerosis and Associated Factors

ABSTRACTThe aims of this study were to investigate the cognitive function of Chinese amyotrophic lateral sclerosis (ALS) patients. Twenty-two patients with ALS were enrolled and a group of 60 healthy individuals were enrolled as a control group. The Extended Scale for Dementia (ESD) was used to evaluate cognitive function and the Hamilton Depression Scale was used for the assessment of depression. In addition, the Modified Norris Scale was used to assess medulla oblongata and limb function. The overall ESD scores were significantly lower in the ALS patients; however, no correlation was found between individual scores of cognitive function and the overall Modified Norris Scale scores. A majority of ALS patients had significantly reduced cognitive function, suggesting the pathological involvement of areas in the brain outside the motor area. This study shows that through the assessment of cognitive function, abnormalities in cognitive function in ALS patients can be identified.

Predictors of noninvasive ventilation tolerance in patients with amyotrophic lateral sclerosis

Noninvasive ventilation (NIV) appears to improve survival and quality of life in patients with amyotrophic lateral sclerosis (ALS), but little is known about predictors of NIV tolerance. NIV use was assessed and clinical predictors of tolerance were investigated, using predictive modeling, in ALS patients diagnosed and followed in our clinic until death over a 4-year time period. Patients were prescribed NIV based on current practice parameters when respiratory symptoms were present or forced vital capacity was less than 50%. We prescribed NIV in 52% (72) of patients. For those prescribed NIV, information regarding tolerance was available for 50 patients, with 72% (36) tolerant to its use. Tolerance was six times more likely in limb-onset than bulbar-onset ALS patients, with a trend toward reduced tolerance in those with lower forced vital capacity at NIV initiation. Age, gender, and duration of disease were not predictors of NIV tolerance. We conclude that a majority of ALS patients who are prescribed NIV can successfully become tolerant to its use.

Oculomotor nuclear pathology in amyotrophic lateral sclerosis

We examined the oculomotor and/or trochlear nuclei of 27 amyotrophic lateral sclerosis (ALS) patients and 10 controls by histological and immunohistological methods. Their neurons were relatively well preserved. In 7 of 22 sporadic ALS patients (including 3/3 ALS with ophthalmoplegia) and in 4 of 5 ALS patients with dementia, some morphological changes similar to those in anterior horns (Bunina bodies, ubiquitin-positive skein-like inclusions, Lewy body-like inclusions, conglomerate inclusions and spheroids) were rarely, but clearly seen. These changes were not observed in controls. Our results suggest that the oculomotor and trochlear nuclei in ALS patients are slightly affected in a manner similar to that in the anterior horns, but the degree is less than that necessary for development of ophthalmoplegia in the majority of ALS patients.