Distinct roles for motor neuron autophagy early and late in the SOD1G93A mouse model of ALS

Mutations in autophagy genes can cause familial and sporadic amyotrophic lateral sclerosis (ALS). However, the role of autophagy in ALS pathogenesis is poorly understood, in part due to the lack of cell type-specific manipulations of this pathway in animal models. Using a mouse model of ALS expressing mutant superoxide dismutase 1 (SOD1G93A), we show that motor neurons form large autophagosomes containing ubiquitinated aggregates early in disease progression. To investigate whether this response is protective or detrimental, we generated mice in which the critical autophagy gene Atg7 was specifically disrupted in motor neurons (Atg7 cKO). Atg7 cKO mice were viable but exhibited structural and functional defects at a subset of vulnerable neuromuscular junctions. By crossing Atg7 cKO mice to the SOD1G93A mouse model, we found that autophagy inhibition accelerated early neuromuscular denervation of the tibialis anterior muscle and the onset of hindlimb tremor. Surprisingly, however, lifespan was extended in Atg7 cKO; SOD1G93A double-mutant mice. Autophagy inhibition did not prevent motor neuron cell death, but it reduced glial inflammation and blocked activation of the stress-related transcription factor c-Jun in spinal interneurons. We conclude that motor neuron autophagy is required to maintain neuromuscular innervation early in disease but eventually acts in a non–cell-autonomous manner to promote disease progression.

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Myomirnas Role in Als Suggest a Crosstalk between Muscle and Motor Neuron

10.20944/preprints201810.0709.v1 ◽

2018 ◽

Author(s):

Valentina Pegoraro ◽

Antonio Merico ◽

Corrado Angelini

Keyword(s):

Skeletal Muscle ◽

Motor Neuron ◽

Aerobic Exercise ◽

Muscle Atrophy ◽

Motor Neurons ◽

Neuromuscular Junctions ◽

Neurodegenerative Disorder ◽

Disease Process ◽

Muscle Fibres ◽

Wide Range

Amyotrophic lateral sclerosis (ALS) is a rare, progressive, neurodegenerative disorder caused by degeneration of upper and lower motor neurons. The disease process leads from lower motor neuron involvement to progressive muscle atrophy, weakness, fasciculations for the upper motor neuron involvement to spasticity. Muscle atrophy in ALS is caused by a dysregulation in the molecular network controlling fast and slow muscle fibres. Denervation and reinnervation processes in skeletal muscle occur in the course of ALS and are modulated by rehabilitation. MicroRNAs (miRNAs) are small non-coding RNAs that modulate a wide range of biological functions under various pathophysiological conditions. MiRNAs can be secreted by various cell types and they are markedly stable in body fluids. MiR-1, miR-133 a, miR-133b, and miR-206 are called “myomiRs” and are considered markers of myogenesis during muscle regeneration and neuromuscular junction stabilization or sprouting. We observed a positive effect of a standard aerobic exercise rehabilitative protocol conducted for six weeks in 18 ALS patients during hospitalization in our center. We correlated clinical scales with molecular data on myomiRs. After six weeks of moderate aerobic exercise, myomiRNAs were down-regulated, suggesting an active proliferation of satellite cells in muscle and increased neuromuscular junctions. Our data suggest that circulating miRNAs modulate during skeletal muscle recovery in response to physical rehabilitation in ALS.

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Multiple Presynaptic and Postsynaptic Sites of Inhibitory Modulation by Myomodulin at ARC Neuromuscular Junctions ofAplysia

Journal of Neurophysiology ◽

10.1152/jn.00140.2002 ◽

2003 ◽

Vol 89 (3) ◽

pp. 1488-1502 ◽

Cited By ~ 9

Author(s):

Irina V. Orekhova ◽

Vera Alexeeva ◽

Paul J. Church ◽

Klaudiusz R. Weiss ◽

Vladimir Brezina

Keyword(s):

Motor Neuron ◽

Motor Neurons ◽

Neuromuscular Junctions ◽

Definitive Evidence ◽

Multiple Parameters ◽

Intact Muscle ◽

Single Arc ◽

Excitatory Junction Potentials ◽

K Current ◽

Neuron Terminals

The functional activity of even simple cellular ensembles is often controlled by surprisingly complex networks of neuromodulators. One such network has been extensively studied in the accessory radula closer (ARC) neuromuscular system of Aplysia. The ARC muscle is innervated by two motor neurons, B15 and B16, which release modulatory peptide cotransmitters to shape ACh-mediated contractions of the muscle. Previous analysis has shown that key to the combinatorial ability of B15 and B16 to control multiple parameters of the contraction is an asymmetry in their peptide modulatory actions. B16, but not B15, releases myomodulin, which, among other actions, inhibits the contraction. Work in single ARC muscle fibers has identified a distinctive myomodulin-activated K current as a candidate postsynaptic mechanism of the inhibition. However, definitive evidence for this mechanism has been lacking. Here, working with the single fibers and then motor neuron-elicited excitatory junction potentials (EJPs) and contractions of the intact ARC muscle, we have confirmed two central predictions of the K-current hypothesis: the myomodulin inhibition of contraction is associated with a correspondingly large inhibition of the underlying depolarization, and the inhibition of both contraction and depolarization is blocked by 4-aminopyridine (4-AP), a potent and selective blocker of the myomodulin-activated K current. However, in the intact muscle, the experiments revealed a second, 4-AP-resistant component of myomodulin inhibition of both B15- and B16-elicited EJPs. This component resembles, and mutually occludes with, inhibition of the EJPs by another peptide modulator released from both B15 and B16, buccalin, which acts by a presynaptic mechanism, inhibition of ACh release from the motor neuron terminals. Direct measurements of peptide release showed that myomodulin also inhibits buccalin release from B15 terminals. At the level of contractions, nevertheless, the postsynaptic K-current mechanism is responsible for much of the myomodulin inhibition of peak contraction amplitude. The presynaptic mechanism, which is most evident during the initial build-up of the EJP waveform, underlies instead an increase of contraction latency.

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Comparison of the efficacy of MOE and PMO modifications of systemic antisense oligonucleotides in a severe SMA mouse model

Nucleic Acids Research ◽

10.1093/nar/gkaa126 ◽

2020 ◽

Vol 48 (6) ◽

pp. 2853-2865 ◽

Cited By ~ 3

Author(s):

Lei Sheng ◽

Frank Rigo ◽

C Frank Bennett ◽

Adrian R Krainer ◽

Yimin Hua

Keyword(s):

Mouse Model ◽

Motor Neurons ◽

Time Course ◽

Neuromuscular Junctions ◽

Muscular Atrophy ◽

Body Weight Gain ◽

Target Sequence ◽

Advantages And Disadvantages ◽

Extended Target ◽

The Central Nervous System

Abstract Spinal muscular atrophy (SMA) is a motor neuron disease. Nusinersen, a splice-switching antisense oligonucleotide (ASO), was the first approved drug to treat SMA. Based on prior preclinical studies, both 2′-O-methoxyethyl (MOE) with a phosphorothioate backbone and morpholino with a phosphorodiamidate backbone—with the same or extended target sequence as nusinersen—displayed efficient rescue of SMA mouse models. Here, we compared the therapeutic efficacy of these two modification chemistries in rescue of a severe mouse model using ASO10-29—a 2-nt longer version of nusinersen—via subcutaneous injection. Although both chemistries efficiently corrected SMN2 splicing in various tissues, restored motor function and improved the integrity of neuromuscular junctions, MOE-modified ASO10-29 (MOE10-29) was more efficacious than morpholino-modified ASO10-29 (PMO10-29) at the same molar dose, as seen by longer survival, greater body-weight gain and better preservation of motor neurons. Time-course analysis revealed that MOE10-29 had more persistent effects than PMO10-29. On the other hand, PMO10-29 appears to more readily cross an immature blood-brain barrier following systemic administration, showing more robust initial effects on SMN2 exon 7 inclusion, but less persistence in the central nervous system. We conclude that both modifications can be effective as splice-switching ASOs in the context of SMA and potentially other diseases, and discuss the advantages and disadvantages of each.

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Downregulating carnitine palmitoyl transferase 1 affects disease progression in the SOD1 G93A mouse model of ALS

Communications Biology ◽

10.1038/s42003-021-02034-z ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Michael Sloth Trabjerg ◽

Dennis Christian Andersen ◽

Pam Huntjens ◽

Kirsten Egelund Oklinski ◽

Luise Bolther ◽

...

Keyword(s):

Lipid Metabolism ◽

Mouse Model ◽

Disease Progression ◽

Motor Neurons ◽

High Fat Diet ◽

High Fat ◽

Carnitine Palmitoyl Transferase ◽

Disease Symptoms ◽

Lateral Sclerosis ◽

Sod1 G93a Mouse

AbstractAmyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease characterized by death of motor neurons. The etiology and pathogenesis remains elusive despite decades of intensive research. Herein, we report that dysregulated metabolism plays a central role in the SOD1 G93A mouse model mimicking ALS. Specifically, we report that the activity of carnitine palmitoyl transferase 1 (CPT1) lipid metabolism is associated with disease progression. Downregulation of CPT1 activity by pharmacological and genetic methods results in amelioration of disease symptoms, inflammation, oxidative stress and mitochondrial function, whereas upregulation by high-fat diet or corticosterone results in a more aggressive disease progression. Finally, we show that downregulating CPT1 shifts the gut microbiota communities towards a protective phenotype in SOD1 G93A mice. These findings reveal that metabolism, and specifically CPT1 lipid metabolism plays a central role in the SOD1 G93A mouse model and shows that CPT1 might be a therapeutic target in ALS.

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Ultrastructural characterization of peripheral denervation in a mouse model of Type III spinal muscular atrophy

Journal of Neural Transmission ◽

10.1007/s00702-021-02353-9 ◽

2021 ◽

Author(s):

Federica Fulceri ◽

Francesca Biagioni ◽

Fiona Limanaqi ◽

Carla L. Busceti ◽

Larisa Ryskalin ◽

...

Keyword(s):

Mouse Model ◽

Spinal Muscular Atrophy ◽

Motor Neuron ◽

Motor Neurons ◽

Muscular Atrophy ◽

Disease Onset ◽

Muscle Spindles ◽

Neuromuscular Disorder ◽

Type Iii ◽

Smn Protein

AbstractSpinal muscular atrophy (SMA) is a heritable, autosomal recessive neuromuscular disorder characterized by a loss of the survival of motor neurons (SMN) protein, which leads to degeneration of lower motor neurons, and muscle atrophy. Despite SMA being nosographically classified as a motor neuron disease, recent advances indicate that peripheral alterations at the level of the neuromuscular junction (NMJ), involving the muscle, and axons of the sensory-motor system, occur early, and may even precede motor neuron loss. In the present study, we used a mouse model of slow progressive (type III) SMA, whereby the absence of the mouse SMN protein is compensated by the expression of two human genes (heterozygous SMN1A2G, and SMN2). This leads to late disease onset and prolonged survival, which allows for dissecting slow degenerative steps operating early in SMA pathogenesis. In this purely morphological study carried out at transmission electron microscopy, we extend the examination of motor neurons and proximal axons towards peripheral components, including distal axons, muscle fibers, and also muscle spindles. We document remarkable ultrastructural alterations being consistent with early peripheral denervation in SMA, which may shift the ultimate anatomical target in neuromuscular disease from the spinal cord towards the muscle. This concerns mostly mitochondrial alterations within distal axons and muscle, which are quantified here through ultrastructural morphometry. The present study is expected to provide a deeper knowledge of early pathogenic mechanisms in SMA.

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Dissociation of disease onset, progression and sex differences from androgen receptor levels in a mouse model of amyotrophic lateral sclerosis

Scientific Reports ◽

10.1038/s41598-021-88415-0 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Doris Tomas ◽

Victoria M. McLeod ◽

Mathew D. F. Chiam ◽

Nayomi Wanniarachchillage ◽

Wah C. Boon ◽

...

Keyword(s):

Amyotrophic Lateral Sclerosis ◽

Sex Differences ◽

Androgen Receptor ◽

Mouse Model ◽

Motor Neuron ◽

Motor Neurons ◽

Disease Onset ◽

Disease Course ◽

Male Mice ◽

Lateral Sclerosis

AbstractAmyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disorder caused by loss of motor neurons. ALS incidence is skewed towards males with typically earlier age of onset and limb site of onset. The androgen receptor (AR) is the major mediator of androgen effects in the body and is present extensively throughout the central nervous system, including motor neurons. Mutations in the AR gene lead to selective lower motor neuron degeneration in male spinal bulbar muscular atrophy (SBMA) patients, emphasising the importance of AR in maintaining motor neuron health and survival. To evaluate a potential role of AR in onset and progression of ALS, we generated SOD1G93A mice with either neural AR deletion or global human AR overexpression. Using a Cre-LoxP conditional gene knockout strategy, we report that neural deletion of AR has minimal impact on the disease course in SOD1G93A male mice. This outcome was potentially confounded by the metabolically disrupted Nestin-Cre phenotype, which likely conferred the profound lifespan extension observed in the SOD1G93A double transgenic male mice. In addition, overexpression of human AR produced no benefit to disease onset and progression in SOD1G93A mice. In conclusion, the disease course of SOD1G93A mice is independent of AR expression levels, implicating other mechanisms involved in mediating the sex differences in ALS. Our findings using Nestin-Cre mice, which show an inherent metabolic phenotype, led us to hypothesise that targeting hypermetabolism associated with ALS may be a more potent modulator of disease, than AR in this mouse model.

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Dynamic neuromuscular remodeling precedes motor-unit loss in a mouse model of ALS

eLife ◽

10.7554/elife.41973 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 29

Author(s):

Éric Martineau ◽

Adriana Di Polo ◽

Christine Vande Velde ◽

Richard Robitaille

Keyword(s):

Motor Neuron ◽

Motor Neurons ◽

Axonal Degeneration ◽

Synapse Formation ◽

Neuromuscular Junctions ◽

Motor Units ◽

Early Event ◽

Motor Neuron Degeneration ◽

Single Motor Units

Despite being an early event in ALS, it remains unclear whether the denervation of neuromuscular junctions (NMJ) is simply the first manifestation of a globally degenerating motor neuron. Using in vivo imaging of single axons and their NMJs over a three-month period, we identify that single motor-units are dismantled asynchronously in SOD1G37R mice. We reveal that weeks prior to complete axonal degeneration, the dismantling of axonal branches is accompanied by contemporaneous new axonal sprouting resulting in synapse formation onto nearby NMJs. Denervation events tend to propagate from the first lost NMJ, consistent with a contribution of neuromuscular factors extrinsic to motor neurons, with distal branches being more susceptible. These results show that NMJ denervation in ALS is a complex and dynamic process of continuous denervation and new innervation rather than a manifestation of sudden global motor neuron degeneration.

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Human motor units in microfluidic devices are impaired by FUS mutations and improved by HDAC6 inhibition

10.1101/2020.10.21.346874 ◽

2020 ◽

Author(s):

Katarina Stoklund Dittlau ◽

Emily N. Krasnow ◽

Laura Fumagalli ◽

Tijs Vandoorne ◽

Pieter Baatsen ◽

...

Keyword(s):

Motor Neuron ◽

Neurite Outgrowth ◽

Motor Neurons ◽

Neuromuscular Junctions ◽

Induced Pluripotent Stem Cell ◽

Microfluidic Devices ◽

Motor Units ◽

Human Motor ◽

Induced Pluripotent

AbstractNeuromuscular junctions (NMJs) ensure proper communication between motor neurons and muscle through the release of neurotransmitters. In motor neuron disorders, such as amyotrophic lateral sclerosis (ALS), NMJs degenerate resulting in muscle atrophy, paralysis and respiratory failure. The aim of this study was to establish a versatile and reproducible in vitro model of a human motor unit to study the effect of ALS-causing mutations. Therefore, we generated a co-culture of human induced pluripotent stem cell-derived motor neurons and human primary mesoangioblast-derived myotubes in microfluidic devices. A chemotactic and volumetric gradient facilitated the growth of motor neuron neurites through microgrooves resulting in the interaction with myotubes and the formation of NMJs. We observed that ALS-causing FUS mutations resulted in a reduced neurite outgrowth and in a decreased NMJ number. Interestingly, the selective HDAC6 inhibitor, Tubastatin A, improved the neurite outgrowth and the NMJ morphology of FUS-ALS co-cultures, further prompting HDAC6 inhibition as a potential therapeutic strategy for ALS.

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Assessment of motor neuron pathology and potential compensatory mechanisms in phenotypically normal mice with reduced Survival Motor Neuron levels.

Journal of Student Science and Technology ◽

10.13034/jsst.v8i1.42 ◽

2015 ◽

Vol 8 (1) ◽

Author(s):

Rebecca Xu Xu ◽

Lyndsay M. Murray M. Murray Murray ◽

Yves De Repentigny De Repentigny ◽

Rashmi Kothary Kothary

Keyword(s):

Motor Neuron ◽

Mouse Models ◽

Motor Neurons ◽

Neuromuscular Junctions ◽

Muscular Atrophy ◽

Neuromuscular Disorder ◽

Survival Motor Neuron ◽

Compensatory Mechanisms ◽

Protein Levels ◽

Smn Protein

Spinal muscular atrophy (SMA) is a destructive pediatric neuromuscular disorder caused by low survival motor neuron (Smn) protein levels due to mutations and deletions within the survival motor neuron 1 (SMN1) gene. Motor neurons are the main pathological targets, and along with neuromuscular junctions (NMJs), they play an early significant role in the pathogenesis of SMA. Previous studies demonstrate that a pathological reduction in Smn levels can lead to significant remodeling defects in both the outgrowth of axonal sprouts and in the nerve-directed clustering of AChRs in mouse models. However, whether this pathological reduction in Smn leads to ubclinical features has not been investigated. Here, we have employed the Smn2B/2B and Smn+/- mouse models to study whether similar SMA pathology is present sub-clinically, and if so whether there is any compensation present. We show a decrease in the motor neuron number in the mouse models, no change in myelin thickness and modest NMJ pathology in both mouse models. Additionally, compensation through the expansion of the motor unit size is suggested.L’amyotrophie spinale (AMS) est un trouble neuromusculaire pédiatrique destructif causé par le niveau bas de protéine du neurone de moteur de survie (NMS) en raison des mutations et des effacements dans le neurone de moteur de survie 1 gène (NMS1). Des neurones du moteur sont les cibles pathologiques principales, et ce, avec des jonctions neuromusculaires (JNMs), ils jouent, en avance, un rôle significatif dans la pathogénie de AMS. Des études précédentes démontrent qu’une réduction pathologique de niveaux de NMS peut mener aux défauts importants de réorganisation tant dans l’excroissance axonale que dans l’agrégation du récepteur de l’acétylcholine (AChR) sous la terminaison nerveuse dans des modèles de souris. Cependant, si cette reduction pathologique de NMS mène aux caractéristiques infracliniques n’a pas été à l’étude. Ici, nous avons employé le NMS2B/2B et NMS +/- des modèles de souris afin de déterminer si une pathologie semblable à l’AMS est présente infracliniquement, ainsi s’il y a présence de quelconque compensation. Nous montrons une diminution dans le nombre des neurones du moteur dans les modèles de souris, aucun changement de l’épaisseur du myelin et une pathologie modeste de JNM dans les deux modèles de souris. De plus, une compensation par l’expansion de la taille d’unité du moteur est suggérée.

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Motor organization in pharynx of Helix pomatia

Journal of Neurophysiology ◽

10.1152/jn.1984.52.3.389 ◽

1984 ◽

Vol 52 (3) ◽

pp. 389-409 ◽

Cited By ~ 29

Author(s):

M. Peters ◽

U. Altrup

Keyword(s):

Motor Neuron ◽

Motor Neurons ◽

Neuromuscular Junctions ◽

Muscle Tension ◽

Helix Pomatia ◽

Single Motor ◽

Cobalt Staining ◽

Excitatory Junction Potentials ◽

Individual Motor ◽

Phase Relationships

Identified motor neurons in the buccal ganglia of Helix pomatia and pharynx muscles innervated by them were studied with intracellular recording and cobalt staining. Retrograde cobalt staining via the buccal nerves indicated that neurons occupy relatively constant positions within the ganglia. With intracellular cobalt staining it was shown that the shape of a representative motor neuron (B4) is similar in different preparations. In some cases, however, deviations from the normal pattern of axon distribution were found. Presumed motor endings of neuron B4 in the muscle were also visualized with intracellular staining. Recordings from individual motor neurons show typical phase relationships of spontaneous spike activity. Most motor neurons are active in the retraction phase of the radula. Only excitatory motor neurons were found. Most neurons directly supply more than one muscle. Amplitude of excitatory junction potentials (EJP) and plasticity at neuromuscular junctions from one neuron are similar in different muscles. Single muscle fibers receive polyneuronal innervation. Activity of single motor neurons already leads to muscle contraction even without spiking of the muscle cells. Muscle tension depends on integrated EJP size. Most motor neurons supply typical combinations of a set of muscles. Thus, several muscles can be activated synchronously by activity of a single motor neuron. In this way muscle combinations are predetermined morphologically by the peripheral branching patterns of the respective neurons.

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