ALS-like Pathology Research Articles

Severe dynein dysfunction in cholinergic neurons exacerbates ALS-like phenotypes in a new mouse model

Cytoplasmic dynein 1, a motor protein essential for retrograde axonal transport, is increasingly implicated in the pathogenesis of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). In this study, we developed a novel mouse model that combines the Legs at odd angles (Loa, F580Y) point mutation in the dynein heavy chain with a cholinergic neuron-specific knockout of the dynein heavy chain. This model, for the first time, allows us to investigate the impact of Loa allele exclusivity in these neurons into adulthood. Our findings reveal that this selective increase in dynein dysfunction exacerbated the phenotypes observed in heterozygous Loa mice including pre-wean survival, reduced body weight and grip strength. Additionally, it induced ALS-like pathology in neuromuscular junctions (NMJs) not seen in heterozygous Loa mice. Notably, we also found a previously unobserved significant increase in neurons displaying TDP-43 puncta in both Loa mutants, suggesting early TDP-43 mislocalisation – a hallmark of ALS. The novel model also exhibited a concurrent rise in p62 puncta that did not co-localise with TDP-43, indicating broader impairments in autophagic clearance mechanisms. Overall, this new model underscores the fact that dynein impairment alone can induce ALS-like pathology and provides a valuable platform to further explore the role of dynein in ALS.

Desloratadine alleviates ALS-like pathology in hSOD1G93A mice via targeting 5HTR2A on activated spinal astrocytes.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with progressive loss of motor neurons in the spinal cord, cerebral cortex and brain stem. ALS is characterized by gradual muscle atrophy and dyskinesia. The limited knowledge on the pathology of ALS has impeded the development of therapeutics for the disease. Previous studies have shown that autophagy and astrocyte-mediated neuroinflammation are involved in the pathogenesis of ALS, while 5HTR2A participates in the early stage of astrocyte activation, and 5HTR2A antagonism may suppress astrocyte activation. In this study, we evaluated the therapeutic effects of desloratadine (DLT), a selective 5HTR2A antagonist, in human SOD1G93A (hSOD1G93A) ALS model mice, and elucidated the underlying mechanisms. HSOD1G93A mice were administered DLT (20 mg·kg-1·d-1, i.g.) from the age of 8 weeks for 10 weeks or until death. ALS onset time and lifespan were determined using rotarod and righting reflex tests, respectively. We found that astrocyte activation accompanying with serotonin receptor 2 A (5HTR2A) upregulation in the spinal cord was tightly associated with ALS-like pathology, which was effectively attenuated by DLT administration. We showed that DLT administration significantly delayed ALS symptom onset time, prolonged lifespan and ameliorated movement disorders, gastrocnemius injury and spinal motor neuronal loss in hSOD1G93A mice. Spinal cord-specific knockdown of 5HTR2A by intrathecal injection of adeno-associated virus9 (AAV9)-si-5Htr2a also ameliorated ALS pathology in hSOD1G93A mice, and occluded the therapeutic effects of DLT administration. Furthermore, we demonstrated that DLT administration promoted autophagy to reduce mutant hSOD1 levels through 5HTR2A/cAMP/AMPK pathway, suppressed oxidative stress through 5HTR2A/cAMP/AMPK/Nrf2-HO-1/NQO-1 pathway, and inhibited astrocyte neuroinflammation through 5HTR2A/cAMP/AMPK/NF-κB/NLRP3 pathway in the spinal cord of hSOD1G93A mice. In summary, 5HTR2A antagonism shows promise as a therapeutic strategy for ALS, highlighting the potential of DLT in the treatment of the disease. DLT as a 5HTR2A antagonist effectively promoted autophagy to reduce mutant hSOD1 level through 5HTR2A/cAMP/AMPK pathway, suppressed oxidative stress through 5HTR2A/cAMP/AMPK/Nrf2-HO-1/NQO-1 pathway, and inhibited astrocytic neuroinflammation through 5HTR2A/cAMP/AMPK/NF-κB/NLRP3 pathway in the spinal cord of hSOD1G93A mice.

ALS-like pathology diminishes swelling of spinal astrocytes in the SOD1 animal model.

Astrocytes are crucial for the functioning of the nervous system as they maintain the ion homeostasis via volume regulation. Pathological states, such as amyotrophic lateral sclerosis (ALS), affect astrocytes and might even cause a loss of such functions. In this study, we examined astrocytic swelling/volume recovery in both the brain and spinal cord of the SOD1 animal model to determine the level of their impairment caused by the ALS-like pathology. Astrocyte volume changes were measured in acute brain or spinal cord slices during and after exposure to hyperkalemia. We then compared the results with alterations of extracellular space (ECS) diffusion parameters, morphological changes, expression of the Kir4.1 channel and the potassium concentration measured in the cerebrospinal fluid, to further disclose the link between potassium and astrocytes in the ALS-like pathology. Morphological analysis revealed astrogliosis in both the motor cortex and the ventral horns of the SOD1 spinal cord. The activated morphology of SOD1 spinal astrocytes was associated with the results from volume measurements, which showed decreased swelling of these cells during hyperkalemia. Furthermore, we observed lower shrinkage of ECS in the SOD1 spinal ventral horns. Immunohistochemical analysis then confirmed decreased expression of the Kir4.1 channel in the SOD1 spinal cord, which corresponded with the diminished volume regulation. Despite astrogliosis, cortical astrocytes in SOD1 mice did not show alterations in swelling nor changes in Kir4.1 expression, and we did not identify significant changes in ECS parameters. Moreover, the potassium level in the cerebrospinal fluid did not deviate from the physiological concentration. The results we obtained thus suggest that ALS-like pathology causes impaired potassium uptake associated with Kir4.1 downregulation in the spinal astrocytes, but based on our data from the cortex, the functional impairment seems to be independent of the morphological state.

UndERACting ion channels in neurodegeneration

In a recent study, Guo and colleagues characterised the function of an elusive endoplasmic reticulum (ER) anion channel protein, Chloride Channel CLiC Like 1 (CLCC1), and identified rare CLCC1 variants in people with amyotrophic lateral sclerosis (ALS). CLCC1 mutants disrupted ER function in vitro and promoted ALS-like pathology and neurodegeneration in mice. This work reveals a previously uncharacterised pathway involved in ER calcium release and highlights new pathogenic mechanisms underlying neurodegeneration.

Cortical glia in SOD1(G93A) mice are subtly affected by ALS-like pathology

The role of glia in amyotrophic lateral sclerosis (ALS) is undeniable. Their disease-related activity has been extensively studied in the spinal cord, but only partly in the brain. We present herein a comprehensive study of glia in the cortex of SOD1(G93A) mice—a widely used model of ALS. Using single-cell RNA sequencing (scRNA-seq) and immunohistochemistry, we inspected astrocytes, microglia, and oligodendrocytes, in four stages of the disease, respecting the factor of sex. We report minimal changes of glia throughout the disease progression and regardless of sex. Pseudobulk and single-cell analyses revealed subtle disease-related transcriptional alterations at the end-stage in microglia and oligodendrocytes, which were supported by immunohistochemistry. Therefore, our data support the hypothesis that the SOD1(G93A) mouse cortex does not recapitulate the disease in patients, and we recommend the use of a different model for future studies of the cortical ALS pathology.

MATR3 P154S knock-in mice do not exhibit motor, muscle or neuropathologic features of ALS

Matrin 3 is a nuclear matrix protein that has many roles in RNA processing including splicing and transport of mRNA. Many missense mutations in the Matrin 3 gene (MATR3) have been linked to familial forms of amyotrophic lateral sclerosis (ALS) and distal myopathy. However, the exact role of MATR3 mutations in ALS and myopathy pathogenesis is not understood. To demonstrate a role of MATR3 mutations in vivo, we generated a novel CRISPR/Cas9 mediated knock-in mouse model harboring the MATR3 P154S mutation expressed under the control of the endogenous promoter. The P154S variant of the MATR3 gene has been linked to familial forms of ALS. Heterozygous and homozygous MATR3 P154S knock-in mice did not develop progressive motor deficits compared to wild-type mice. In addition, ALS-like pathology did not develop in nervous or muscle tissue in either heterozygous or homozygous mice. Our results suggest that the MATR3 P154S variant is not sufficient to produce ALS-like pathology in vivo.

Slow motor neurons resist pathological TDP-43 and mediate motor recovery in the rNLS8 model of amyotrophic lateral sclerosis

In the intermediate stages of amyotrophic lateral sclerosis (ALS), surviving motor neurons (MNs) that show intrinsic resistance to TDP-43 proteinopathy can partially compensate for the loss of their more disease-susceptible counterparts. Elucidating the mechanisms of this compensation may reveal approaches for attenuating motor impairment in ALS patients. In the rNLS8 mouse model of ALS-like pathology driven by doxycycline-regulated neuronal expression of human TDP-43 lacking a nuclear localization signal (hTDP-43ΔNLS), slow MNs are more resistant to disease than fast-fatigable (FF) MNs and can mediate recovery following transgene suppression. In the present study, we used a viral tracing strategy to show that these disease-resistant slow MNs sprout to reinnervate motor endplates of adjacent muscle fibers vacated by degenerated FF MNs. Moreover, we found that neuromuscular junctions within fast-twitch skeletal muscle (tibialis anterior, TA) reinnervated by SK3-positive slow MNs acquire resistance to axonal dieback when challenged with a second course of hTDP-43ΔNLS pathology. The selective resistance of reinnervated neuromuscular junctions was specifically induced by the unique pattern of reinnervation following TDP-43-induced neurodegeneration, as recovery from unilateral sciatic nerve crush did not produce motor units resistant to subsequent hTDP-43ΔNLS. Using cross-reinnervation and self-reinnervation surgery in which motor axons are disconnected from their target muscle and reconnected to a new muscle, we show that FF MNs remain hTDP-43ΔNLS-susceptible and slow MNs remain resistant, regardless of which muscle fibers they control. Collectively, these findings demonstrate that MN identity dictates the susceptibility of neuromuscular junctions to TDP-43 pathology and slow MNs can drive recovery of motor systems due to their remarkable resilience to TDP-43-driven degeneration. This study highlights a potential pathway for regaining motor function with ALS pathology in the advent of therapies that halt the underlying neurodegenerative process.

Spreading of TDP-43 pathology via pyramidal tract induces ALS-like phenotypes in TDP-43 transgenic mice

Transactive response DNA-binding protein 43 kDa (TDP-43) has been identified as the major component of ubiquitinated inclusions found in patients with sporadic amyotrophic lateral sclerosis (ALS). Increasing evidence suggests prion-like transmission of TDP-43 aggregates via neuroanatomic connection in vitro and pyramidal tract in vivo. However, it is still unknown whether the spreading of pathological TDP-43 sequentially via pyramidal tract can initiate ALS-like pathology and phenotypes. In this study, we reported that injection of TDP-43 preformed fibrils (PFFs) into the primary motor cortex (M1) of Thy1-e (IRES-TARDBP) 1 mice induced the spreading of pathological TDP-43 along pyramidal tract axons anterogradely. Moreover, TDP-43 PFFs-injected Thy1-e (IRES-TARDBP) 1 mice displayed ALS-like neuropathological features and symptoms, including motor dysfunctions and electrophysiological abnormalities. These findings provide direct evidence that transmission of pathological TDP-43 along pyramidal tract induces ALS-like phenotypes, which further suggest the potential mechanism for TDP-43 proteinopathy.

Overriding FUS autoregulation in mice triggers gain-of-toxic dysfunctions in RNA metabolism and autophagy-lysosome axis.

Mutations in coding and non-coding regions of FUS cause amyotrophic lateral sclerosis (ALS). The latter mutations may exert toxicity by increasing FUS accumulation. We show here that broad expression within the nervous system of wild-type or either of two ALS-linked mutants of human FUS in mice produces progressive motor phenotypes accompanied by characteristic ALS-like pathology. FUS levels are autoregulated by a mechanism in which human FUS downregulates endogenous FUS at mRNA and protein levels. Increasing wild-type human FUS expression achieved by saturating this autoregulatory mechanism produces a rapidly progressive phenotype and dose-dependent lethality. Transcriptome analysis reveals mis-regulation of genes that are largely not observed upon FUS reduction. Likely mechanisms for FUS neurotoxicity include autophagy inhibition and defective RNA metabolism. Thus, our results reveal that overriding FUS autoregulation will trigger gain-of-function toxicity via altered autophagy-lysosome pathway and RNA metabolism function, highlighting a role for protein and RNA dyshomeostasis in FUS-mediated toxicity.

Metabolic therapy with Deanna Protocol supplementation delays disease progression and extends survival in amyotrophic lateral sclerosis (ALS) mouse model.

Amyotrophic Lateral Sclerosis (ALS), also known as Lou Gehrig’s disease, is a neurodegenerative disorder of motor neurons causing progressive muscle weakness, paralysis, and eventual death from respiratory failure. There is currently no cure or effective treatment for ALS. Besides motor neuron degeneration, ALS is associated with impaired energy metabolism, which is pathophysiologically linked to mitochondrial dysfunction and glutamate excitotoxicity. The Deanna Protocol (DP) is a metabolic therapy that has been reported to alleviate symptoms in patients with ALS. In this study we hypothesized that alternative fuels in the form of TCA cycle intermediates, specifically arginine-alpha-ketoglutarate (AAKG), the main ingredient of the DP, and the ketogenic diet (KD), would increase motor function and survival in a mouse model of ALS (SOD1-G93A). ALS mice were fed standard rodent diet (SD), KD, or either diets containing a metabolic therapy of the primary ingredients of the DP consisting of AAKG, gamma-aminobutyric acid, Coenzyme Q10, and medium chain triglyceride high in caprylic triglyceride. Assessment of ALS-like pathology was performed using a pre-defined criteria for neurological score, accelerated rotarod test, paw grip endurance test, and grip strength test. Blood glucose, blood beta-hydroxybutyrate, and body weight were also monitored. SD+DP-fed mice exhibited improved neurological score from age 116 to 136 days compared to control mice. KD-fed mice exhibited better motor performance on all motor function tests at 15 and 16 weeks of age compared to controls. SD+DP and KD+DP therapies significantly extended survival time of SOD1-G93A mice by 7.5% (p = 0.001) and 4.2% (p = 0.006), respectively. Sixty-three percent of mice in the KD+DP and 72.7% of the SD+DP group lived past 125 days, while only 9% of the control animals survived past that point. Targeting energy metabolism with metabolic therapy produces a therapeutic effect in ALS mice which may prolong survival and quality of life in ALS patients.

Brain and Spinal Cord Trauma as a Risk Factor for Amyotrophic Lateral Sclerosis: A Mini-Review

Trauma to the central nervous system (CNS) has been investigated as a risk factor for amyotrophic lateral sclerosis (ALS) despite conflicting epidemiological reports. Studies have suggested a link between ALS and traumatic axonal injury which complements the “dying back” theory of ALS. The theory suggests that neuronal dysfunction first occurs at the neuromuscular junction, and subsequent axonal impairment leads to dysfunction of the cell body. A pathological link has been shown between CNS trauma and ALS, further supporting this relationship. Another proposed hypothesis is that differences in “molecular thresholds” based on individual genetic backgrounds could explain some individuals developing ALS or ALS-like pathology subsequent to trauma, as well as elucidate the seemingly increased risk for ALS associated with multiple traumas. However, it is still unclear how trauma to the CNS might directly or indirectly trigger ALS. The current minireview re-examines the relationship between CNS trauma and risk of developing ALS or an ALS-like pathology, and explores potential explanations for discrepant study results.

NAP (davunetide) modifies disease progression in a mouse model of severe neurodegeneration: Protection against impairments in axonal transport

NAP (davunetide) is a novel neuroprotective compound with mechanism of action that appears to involve microtubule (MT) stabilization and repair. To evaluate, for the first time, the impact of NAP on axonal transport in vivo and to translate it to neuroprotection in a severe neurodegeneration, the SOD1-G93A mouse model for amyotrophic lateral sclerosis (ALS) was used. Manganese-enhanced magnetic resonance imaging (MRI), estimating axonal transport rates, revealed a significant reduction of the anterograde axonal transport in the ALS mice compared to healthy control mice. Acute NAP treatment normalized axonal transport rates in these ALS mice. Tau hyperphosphorylation, associated with MT dysfunction and defective axonal transport, was discovered in the brains of the ALS mice and was significantly reduced by chronic NAP treatment. Furthermore, in healthy wild type (WT) mice, NAP reversed axonal transport disruption by colchicine, suggesting drug-dependent protection against axonal transport impairment through stabilization of the neuronal MT network. Histochemical analysis showed that chronic NAP treatment significantly protected spinal cord motor neurons against ALS-like pathology. Sequential MRI measurements, correlating brain structure with ALS disease progression, revealed a significant damage to the ventral tegmental area (VTA), indicative of impairments to the dopaminergic pathways relative to healthy controls. Chronic daily NAP treatment of the SOD1-G93A mice, initiated close to disease onset, delayed degeneration of the trigeminal, facial and hypoglossal motor nuclei as was significantly apparent at days 90–100 and further protected the VTA throughout life. Importantly, protection of the VTA was significantly correlated with longevity and overall, NAP treatment significantly prolonged life span in the ALS mice.

Clinical and neuropathologic heterogeneity of c9FTD/ALS associated with hexanucleotide repeat expansion in C9ORF72

Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are part of a disease spectrum associated with TDP-43 pathology. Strong evidence supporting this is the existence of kindreds with family members affected by FTD, ALS or mixed features of FTD and ALS, referred to as FTD-MND. Some of these families have linkage to chromosome 9, with hexanucleotide expansion mutation in a noncoding region of C9ORF72. Discovery of the mutation defines c9FTD/ALS. Prior to discovery of mutations in C9ORF72, it was assumed that TDP-43 pathology in c9FTD/ALS was uniform. In this study, we examined the neuropathology and clinical features of 20 cases of c9FTD/ALS from a brain bank for neurodegenerative disorders. Included are six patients clinically diagnosed with ALS, eight FTD, one FTD-MND and four Alzheimer-type dementia. Clinical information was unavailable for one patient. Pathologically, the cases all had TDP-43 pathology, but there were three major pathologic groups: ALS, FTLD-MND and FTLD-TDP. The ALS cases were morphologically similar to typical sporadic ALS with almost no extramotor TDP-43 pathology; all had oligodendroglial cytoplasmic inclusions. The FTLD-MND showed predominantly Mackenzie Type 3 TDP-43 pathology, and all had ALS-like pathology in motor neurons, but more extensive extramotor pathology, with oligodendroglial cytoplasmic inclusions and infrequent hippocampal sclerosis. The FTLD-TDP cases had several features similar to FTLD-TDP due to mutations in the gene for progranulin, including Mackenzie Type 1 TDP-43 pathology with neuronal intranuclear inclusions and hippocampal sclerosis. FTLD-TDP patients were older and some were thought to have Alzheimer-type dementia. In addition to the FTD and ALS clinical presentations, the present study shows that c9FTD/ALS can have other presentations, possibly related to age of onset and the presence of hippocampal sclerosis. Moreover, there is pathologic heterogeneity not only between ALS and FTLD, but also within the FTLD group. Further studies are needed to address the molecular mechanism of clinical and pathological heterogeneity of c9FTD/ALS due to mutations in C9ORF72.

Oxidative stress sensitivity in ALS muscle cells

Introduction. Amyotrophic lateral sclerosis (ALS) is a rapidly progressive and invariably lethal neurodegenerative condition affecting one to three new individuals out of 100,000 per year, which ranks ALS as the most common neurodegenerative disease after Alzheimer's and Parkinson's diseases. ALS leads to death within 3 to 5 years after onset through the selective degeneration of both upper (cortico-spinal) and lower (bulbar and spinal) motor neurons, muscle atrophy and paralysis. While the majority of ALS cases are not linked to a known family history and are, hence, termed sporadic, the remainder (20%) are of genetic origin, generally transmitted with an autosomal dominant inheritance. In recent years, several genes including angiogenin, vapb, dynactin, tdp43, fus/tls and fig4 have been linked to familial forms of ALS, and how these specific mutations trigger ALS is currently the subject of intense research (Boillee et al., 2006a,b, Gonzalez de Aguilar et al., 2007). On the contrary, the mechanisms underlying ALS linked to mutations in the sod1 gene have been extensively studied. The sod1 gene was the first gene linked to familial ALS in 1993 and encodes for the cytosolic copper-zinc superoxide dismutase (SOD1), an enzyme involved in oxidative stress resistance. More than 150 mutations in the sod1 gene have been identified, all leading to ALS. The identification of sod1 as a causative gene for ALS allowed the generation of multiple lines of transgenic mice expressing SOD1 mutations of human or murine origin (Boillee et al., 2006a,b, Bruijn et al., 1997; Ripps et al., 1995; Wong et al., 1995) (mSOD1 mice). These mice exhibit a transgene dose-dependent ALS-like pathology, featuring a progressive paralysis, typical histopathological ALS-like lesions in the spinal cord and culminating to premature death. mSOD1 mice currently represent the only reliable animal model for ALS either familial or sporadic. Pathophysiological mechanisms of ALS are still elusive. These are probably multiple and converge to trigger motor neuron death. Among the mechanisms at work in ALS, intrinsinc neuronal defects,

Activation of the Nrf2–ARE pathway in muscle and spinal cord during ALS-like pathology in mice expressing mutant SOD1

Oxidative stress plays a key role in the neuronal loss exhibited in amyotrophic lateral sclerosis (ALS), an event precipitating irreversible muscle atrophy. By crossing ALS mouse models (SOD G93A and SOD H46RH48Q) with an antioxidant response element (ARE) reporter mouse, we identified activation characteristics of the ARE system throughout the timecourse of motor neuron disease. Surprisingly, the earliest and most significant activation of this genetic sensor of oxidative stress occurred in the distal muscles of mutant SOD mice. The resultant data supports existing hypotheses that the muscle is somehow implicated during the initial pathology of these mice. Subsequently, Nrf2–ARE activation appears to progress in a retrograde fashion along the motor pathway. These data provide timely information concerning the contributions of the Nrf2–ARE pathway in ALS disease progression.

The metabolic hypothesis in amyotrophic lateral sclerosis: insights from mutant Cu/Zn-superoxide dismutase mice

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by selective loss of motor neurons and progressive muscle atrophy. A subset of patients harbors point mutations in the gene encoding Cu/Zn-superoxide dismutase (SOD1), which allowed the generation of transgenic mice that express different SOD1 mutations and develop an ALS-like pathology. Recently, we reported in these mice the occurrence of a characteristic defect in energy homeostasis and the beneficial effect on the course of the disease of a high-energy fat-enriched diet. In this review, we discuss the implication of these findings in the light of classical clinical observations concerning metabolic alterations in human ALS.