Identification of adrenergic presynaptic and postsynaptic protein locations at neuromuscular junctions, their decrease during aging, and recovery by nicotinamide mononucleotide administration.

Neuromuscular junctions are innervated by motor and sympathetic nerves. The sympathetic modulation of motor innervation shows functional decline during aging, but the cellular and molecular mechanism of this change is not fully known. This study aimed to evaluate the effect of aging on sympathetic nerves and synaptic proteins at mouse neuromuscular junctions. Sympathetic nerves, presynaptic, and postsynaptic proteins of sympathetic nerves at neuromuscular junctions were visualized using immunohistochemistry, and aging-related changes were compared between adult-, aged-, and nicotinamide mononucleotide (NMN) administered aged mice. Sympathetic nerves were detected by anti-tyrosine hydroxylase antibody, and presynaptic protein vesicular monoamine transporter 2 colocalized with the sympathetic nerves. These two signals surrounded motor nerve terminals and acetylcholine receptor clusters. Postsynaptic neurotransmitter receptor ß2-adrenergic receptors colocalized with motor nerve terminals and resided in reduced density at extrasynaptic sarcolemma. The signal intensity of the sympathetic nerve marker did not show a significant difference at neuromuscular junctions between 8.5-month-old adult mice and 25-month-old aged mice. However, the signal intensity of vesicular monoamine transporter 2 and ß2-adrenergic receptors showed age-related decline at neuromuscular junctions. Interestingly, both age-related declines reverted to the adult level after 1 month of oral administration of NMN by drinking water. In contrast, NMN administration did not alter the expression level of sympathetic marker tyrosine hydroxylase at neuromuscular junctions. The results suggest a functional decline of sympathetic nerves at aged neuromuscular junctions due to decreases in presynaptic and postsynaptic proteins, which can be reverted to the adult level by NMN administration.

Neuronal Plasticity and Age-Related Functional Decline in the Motor Cortex.

Physiological aging causes a decline of motor function due to impairment of motor cortex function, losses of motor neurons and neuromuscular junctions, sarcopenia, and frailty. There is increasing evidence suggesting that the changes in motor function start earlier in the middle-aged stage. The mechanism underlining the middle-aged decline in motor function seems to relate to the central nervous system rather than the peripheral neuromuscular system. The motor cortex is one of the responsible central nervous systems for coordinating and learning motor functions. The neuronal circuits in the motor cortex show plasticity in response to motor learning, including LTP. This motor cortex plasticity seems important for the intervention method mechanisms that revert the age-related decline of motor function. This review will focus on recent findings on the role of plasticity in the motor cortex for motor function and age-related changes. The review will also introduce our recent identification of an age-related decline of neuronal activity in the primary motor cortex of middle-aged mice using electrophysiological recordings of brain slices.

Coenzyme Q10 supplementation improves the motor function of middle-aged mice by restoring the neuronal activity of the motor cortex.

Physiological aging causes motor function decline and anatomical and biochemical changes in the motor cortex. We confirmed that middle-aged mice at 15-18 months old show motor function decline, which can be restored to the young adult level by supplementing with mitochondrial electron transporter coenzyme Q10 (CoQ10) as a water-soluble nanoformula by drinking water for 1 week. CoQ10 supplementation concurrently improved brain mitochondrial respiration but not muscle strength. Notably, we identified an age-related decline in field excitatory postsynaptic potential (fEPSP) amplitude in the pathway from layers II/III to V of the primary motor area of middle-aged mice, which was restored to the young adult level by supplementing with CoQ10 for 1 week but not by administering CoQ10 acutely to brain slices. Interestingly, CoQ10 with high-frequency stimulation induced NMDA receptor-dependent long-term potentiation (LTP) in layer V of the primary motor cortex of middle-aged mice. Importantly, the fEPSP amplitude showed a larger input‒output relationship after CoQ10-dependent LTP expression. These data suggest that CoQ10 restores the motor function of middle-aged mice by improving brain mitochondrial function and the basal fEPSP level of the motor cortex, potentially by enhancing synaptic plasticity efficacy. Thus, CoQ10 supplementation may ameliorate the age-related decline in motor function in humans.

Forelimb Resistance Exercise Protects Against Neuromuscular Junction Denervation in the SOD1-G93A Rat Model of ALS.

Introduction: The symptoms of Amyotrophic Lateral Sclerosis (ALS) include muscle weakness and eventual paralysis. These symptoms result from denervation of the neuromuscular junction (NMJ) and motor neuron cell death in the brain and spinal cord. Due to the "dying back" pattern of motor neuron degeneration, protecting NMJs should be a therapeutic priority. Although exercise has the potential to protect against NMJ denervation, its use in ALS has been controversial. Most preclinical studies have focused on aerobic exercise, which report that exercise can be beneficial at moderate intensities. The effects of resistance exercise on NMJ preservation in limb muscles have not been explored. Methods: We trained male SOD1-G93A rats, which model ALS, to perform a unilateral isometric forelimb resistance exercise task. This task allows within-animal comparisons of trained and untrained forelimbs. We then determined the effects of isometric resistance exercise on NMJ denervation and AMP kinase (AMPK) activation in forelimb muscles. Results: Our results revealed that SOD1-G93A rats were able to learn and perform the task similarly to wildtype rats, even after loss of body weight. SOD1-G93A rats exhibited significantly greater NMJ innervation in their trained vs their untrained forelimb biceps muscles. Measures of activated (phosphorylated) AMPK (pAMPK) were also greater in the trained vs untrained forelimb triceps muscles. Discussion: These results demonstrate that isometric resistance exercise may protect against NMJ denervation in ALS. Future studies are required to determine the extent to which our findings generalize to female SOD1-G93A rats and to other subtypes of ALS.

Similarity and Diversity of Presynaptic Molecules at Neuromuscular Junctions and Central Synapses.

Synaptic transmission is essential for controlling motor functions and maintaining brain functions such as walking, breathing, cognition, learning, and memory. Neurotransmitter release is regulated by presynaptic molecules assembled in active zones of presynaptic terminals. The size of presynaptic terminals varies, but the size of a single active zone and the types of presynaptic molecules are highly conserved among neuromuscular junctions (NMJs) and central synapses. Three parameters play an important role in the determination of neurotransmitter release properties at NMJs and central excitatory/inhibitory synapses: the number of presynaptic molecular clusters, the protein families of the presynaptic molecules, and the distance between presynaptic molecules and voltage-gated calcium channels. In addition, dysfunction of presynaptic molecules causes clinical symptoms such as motor and cognitive decline in patients with various neurological disorders and during aging. This review focuses on the molecular mechanisms responsible for the functional similarities and differences between excitatory and inhibitory synapses in the peripheral and central nervous systems, and summarizes recent findings regarding presynaptic molecules assembled in the active zone. Furthermore, we discuss the relationship between functional alterations of presynaptic molecules and dysfunction of NMJs or central synapses in diseases and during aging.

Oxaloacetate treatment preserves motor function in SOD1G93A mice and normalizes select neuroinflammation-related parameters in the spinal cord.

Amyotrophic lateral sclerosis (ALS) remains a devastating motor neuron disease with limited treatment options. Oxaloacetate treatment has a neuroprotective effect in rodent models of seizure and neurodegeneration. Therefore, we treated the ALS model superoxide dismutase 1 (SOD1) G93A mice with oxaloacetate and evaluated their neuromuscular function and lifespan. Treatment with oxaloacetate beginning in the presymptomatic stage significantly improved neuromuscular strength measured during the symptomatic stage in the injected mice compared to the non-treated group. Oxaloacetate treatment starting in the symptomatic stage significantly delayed limb paralysis compared with the non-treated group. For lifespan analysis, oxaloacetate treatment did not show a statistically significant positive effect, but the treatment did not shorten the lifespan. Mechanistically, SOD1G93A mice showed increased levels of tumor necrosis factor-α (TNFα) and peroxisome proliferative activated receptor gamma coactivator 1α (PGC-1α) mRNAs in the spinal cord. However, oxaloacetate treatment reverted these abnormal levels to that of wild-type mice. Similarly, the altered expression level of total NF-κB protein returned to that of wild-type mice with oxaloacetate treatment. These results suggest that the beneficial effects of oxaloacetate treatment in SOD1G93A mice may reflect the effects on neuroinflammation or bioenergetic stress.

Impairment Mechanisms and Intervention Approaches for Aged Human Neuromuscular Junctions.

The neuromuscular junction (NMJ) is a chemical synapse formed between a presynaptic motor neuron and a postsynaptic muscle cell. NMJs in most vertebrate species share many essential features; however, some differences distinguish human NMJs from others. This review will describe the pre- and postsynaptic structures of human NMJs and compare them to NMJs of laboratory animals. We will focus on age-dependent declines in function and changes in the structure of human NMJs. Furthermore, we will describe insights into the aging process revealed from mouse models of accelerated aging. In addition, we will compare aging phenotypes to other human pathologies that cause impairments of pre- and postsynaptic structures at NMJs. Finally, we will discuss potential intervention approaches for attenuating age-related NMJ dysfunction and sarcopenia in humans.

A deletion in the N-terminal polymerizing domain of laminin ß2 is a new mouse model of chronic nephrotic syndrome.

The importance of the glomerular basement membrane (GBM) in glomerular filtration is underscored by the manifestations of Alport and Pierson syndromes, caused by defects in type IV collagen α3α4α5 and the laminin ß2 chain, respectively. Lamb2 null mice, which model the most severe form of Pierson syndrome, exhibit proteinuria prior to podocyte foot process effacement and are therefore useful for studying GBM permselectivity. We hypothesize that some LAMB2 missense mutations that cause mild forms of Pierson syndrome induce GBM destabilization with delayed effects on podocytes. While generating a CRISPR/Cas9-mediated analogue of a human LAMB2 missense mutation in mice, we identified a 44-amino acid deletion (LAMB2-Del44) within the laminin N-terminal domain, a domain mediating laminin polymerization. Laminin heterotrimers containing LAMB2-Del44 exhibited a 90% reduction in polymerization in vitro that was partially rescued by type IV collagen and nidogen. Del44 mice showed albuminuria at 1.8-6.0 g/g creatinine (ACR) at one to two months, plateauing at an average 200 g/g ACR at 3.7 months, when GBM thickening and hallmarks of nephrotic syndrome were first observed. Despite the massive albuminuria, some Del44 mice survived for up to 15 months. Blood urea nitrogen was modestly elevated at seven-nine months. Eight to nine-month-old Del44 mice exhibited glomerulosclerosis and interstitial fibrosis. Similar to Lamb2-/- mice, proteinuria preceded foot process effacement. Foot processes were widened but not effaced at one-two months despite the high ACRs. At three months some individual foot processes were still observed amid widespread effacement. Thus, our chronic model of nephrotic syndrome may prove useful to study filtration mechanisms, long-term proteinuria with preserved kidney function, and to test therapeutics.

Super-resolution microscopy for analyzing neuromuscular junctions and synapses.

Super-resolution microscopy techniques offer subdiffraction limited resolution that is two- to ten-fold improved compared to that offered by conventional confocal microscopy. This breakthrough in resolution for light microscopy has contributed to new findings in neuroscience and synapse biology. This review will focus on the Structured Illumination Microscopy (SIM), Stimulated emission depletion (STED) microscopy, and Stochastic optical reconstruction microscopy (STORM) / Single molecule localization microscopy (SMLM) techniques and compare them for the better understanding of their differences and their suitability for the analysis of synapse biology. In addition, we will discuss a few practical aspects of these microscopic techniques, including resolution, image acquisition speed, multicolor capability, and other advantages and disadvantages. Tips for the improvement of microscopy will be introduced; for example, information resources for recommended dyes, the limitations of multicolor analysis, and capabilities for live imaging. In addition, we will summarize how super-resolution microscopy has been used for analyses of neuromuscular junctions and synapses.

Practical Anatomy of the Neuromuscular Junction in Health and Disease.

Neuromuscular junctions (NMJs) form between nerve terminals of spinal cord motor neurons and skeletal muscles, and perisynaptic Schwann cells and kranocytes cap NMJs. One muscle fiber has one NMJ, which is innervated by one motor nerve terminal. NMJs are excitatory synapses that use P/Q-type voltage-gated calcium channels to release the neurotransmitter acetylcholine. Acetylcholine receptors accumulate at the postsynaptic specialization called the end plate on the muscle fiber membrane, the sarcolemma. Proteins essential for the organization of end plates include agrin secreted from nerve terminals, Lrp4 and MuSK receptors for agrin, and Dok-7 and rapsyn cytosolic proteins in the muscle.

Presynaptic active zones of mammalian neuromuscular junctions: Nanoarchitecture and selective impairments in aging.

Neurotransmitter release occurs at active zones, which are specialized regions of the presynaptic membrane. A dense collection of proteins at the active zone provides a platform for molecular interactions that promote recruitment, docking, and priming of synaptic vesicles. At mammalian neuromuscular junctions (NMJs), muscle-derived laminin ß2 interacts with presynaptic voltage-gated calcium channels to organize active zones. The molecular architecture of presynaptic active zones has been revealed using super-resolution microscopy techniques that combine nanoscale resolution and multiple molecular identification. Interestingly, the active zones of adult NMJs are not stable structures and thus become impaired during aging due to the selective degeneration of specific active zone proteins. This review will discuss recent progress in the understanding of active zone nanoarchitecture and the mechanisms underlying active zone organization in mammalian NMJs. Furthermore, we will summarize the age-related degeneration of active zones at NMJs, and the role of exercise in maintaining active zones.

A potential role for neuronal connexin 36 in the pathogenesis of amyotrophic lateral sclerosis.

Neuronal gap junctional protein connexin 36 (Cx36) contributes to neuronal death following a range of acute brain insults such as ischemia, traumatic brain injury and epilepsy. Whether Cx36 contributes to neuronal death and pathological outcomes in chronic neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), is not known. We show here that the expression of Cx36 is significantly decreased in lumbar segments of the spinal cord of both human ALS subjects and SOD1G93A mice as compared to healthy human and wild-type mouse controls, respectively. In purified neuronal cultures prepared from the spinal cord of wild-type mice, knockdown of Cx36 reduces neuronal death caused by overexpression of the mutant human SOD1-G93A protein. Taken together, these data suggest a possible contribution of Cx36 to ALS pathogenesis. A perspective for the use of blockers of Cx36 gap junction channels for ALS therapy is discussed.

Human Parvovirus B19 Utilizes Cellular DNA Replication Machinery for Viral DNA Replication.

Human parvovirus B19 (B19V) infection of human erythroid progenitor cells (EPCs) induces a DNA damage response and cell cycle arrest at late S phase, which facilitates viral DNA replication. However, it is not clear exactly which cellular factors are employed by this single-stranded DNA virus. Here, we used microarrays to systematically analyze the dynamic transcriptome of EPCs infected with B19V. We found that DNA metabolism, DNA replication, DNA repair, DNA damage response, cell cycle, and cell cycle arrest pathways were significantly regulated after B19V infection. Confocal microscopy analyses revealed that most cellular DNA replication proteins were recruited to the centers of viral DNA replication, but not the DNA repair DNA polymerases. Our results suggest that DNA replication polymerase δ and polymerase α are responsible for B19V DNA replication by knocking down its expression in EPCs. We further showed that although RPA32 is essential for B19V DNA replication and the phosphorylated forms of RPA32 colocalized with the replicating viral genomes, RPA32 phosphorylation was not necessary for B19V DNA replication. Thus, this report provides evidence that B19V uses the cellular DNA replication machinery for viral DNA replication.IMPORTANCE Human parvovirus B19 (B19V) infection can cause transient aplastic crisis, persistent viremia, and pure red cell aplasia. In fetuses, B19V infection can result in nonimmune hydrops fetalis and fetal death. These clinical manifestations of B19V infection are a direct outcome of the death of human erythroid progenitors that host B19V replication. B19V infection induces a DNA damage response that is important for cell cycle arrest at late S phase. Here, we analyzed dynamic changes in cellular gene expression and found that DNA metabolic processes are tightly regulated during B19V infection. Although genes involved in cellular DNA replication were downregulated overall, the cellular DNA replication machinery was tightly associated with the replicating single-stranded DNA viral genome and played a critical role in viral DNA replication. In contrast, the DNA damage response-induced phosphorylated forms of RPA32 were dispensable for viral DNA replication.

Mouse Behavior Tracker: An economical method for tracking behavior in home cages.

Analysis of mouse behavior often requires expensive equipment and transfer of the mice to new test environments, which could trigger confounding behavior alterations. Here, we describe a system for tracking mouse behavior in home cages using a low-cost USB webcam and free software (Fiji and wrMTrck). We demonstrate the effectiveness of this method by tracking differences in distance traveled, speed, and movement tracks between wild-type mice and amyotrophic lateral sclerosis (ALS) model mice (SOD1G93A).

Post-transcriptional Inhibition of Hsc70-4/HSPA8 Expression Leads to Synaptic Vesicle Cycling Defects in Multiple Models of ALS.

Amyotrophic lateral sclerosis (ALS) is a synaptopathy accompanied by the presence of cytoplasmic aggregates containing TDP-43, an RNA-binding protein linked to ∼97% of ALS cases. Using a Drosophila model of ALS, we show that TDP-43 overexpression (OE) in motor neurons results in decreased expression of the Hsc70-4 chaperone at the neuromuscular junction (NMJ). Mechanistically, mutant TDP-43 sequesters hsc70-4 mRNA and impairs its translation. Expression of the Hsc70-4 ortholog, HSPA8, is also reduced in primary motor neurons and NMJs of mice expressing mutant TDP-43. Electrophysiology, imaging, and genetic interaction experiments reveal TDP-43-dependent defects in synaptic vesicle endocytosis. These deficits can be partially restored by OE of Hsc70-4, cysteine-string protein (Csp), or dynamin. This suggests that TDP-43 toxicity results in part from impaired activity of the synaptic CSP/Hsc70 chaperone complex impacting dynamin function. Finally, Hsc70-4/HSPA8 expression is also post-transcriptionally reduced in fly and human induced pluripotent stem cell (iPSC) C9orf72 models, suggesting a common disease pathomechanism.

Impaired Mitophagy Plays a Role in Denervation of Neuromuscular Junctions in ALS Mice.

Motor neurons in amyotrophic lateral sclerosis (ALS) patients and animal models show degeneration from the nerve terminal, known as dying-back neuropathy. To investigate the mechanism underlying this neuropathy, we analyzed the neuromuscular junctions (NMJs) and motor neuron cell bodies in SOD1G93A mice using electron microscopy. NMJs of SOD1G93A mice exhibited significantly higher numbers of autophagosomes and degenerated mitochondria compared to wild-type controls. Mitophagosomes were identified in the NMJ presynaptic terminals of wild-type mice and SOD1G93A mice. However, the number of mitophagosomes did not increase significantly in SOD1G93A NMJs indicating a defect in mitophagy, the autophagic process to degrade mitochondria. Consistent with this, proteins essential for mitophagy, p62/SQSTM1, Bnip3, Pink1, and Parkin were down-regulated in motor neurons in SOD1G93A mice. Importantly, SQSTM1 is one of the genes mutated in familial ALS patients. We evaluated the effect of impaired mitophagy on motor neurons by analyzing the double knockout mice of Pink1 and Parkin, two genes responsible for sensing depolarized mitochondria and delivering degenerated mitochondria to mitophagosomes. The double knockout mice exhibited NMJ degeneration, including axon swelling and NMJ fragmentation at 4 months of age. These phenotypes were rarely observed in wild-type control mice of the same age. The protein level of ATP synthase ß subunit increased in the NMJ presynaptic terminals, suggesting the accumulation of mitochondria at NMJs of the double knockout mice. Importantly, NMJ denervation was observed in the double knockout mice. These data suggest that the reduced mitophagy function in motor neurons of SOD1G93A mice is one of the mechanisms causing degeneration of ALS NMJs.

Immunization of mice with LRP4 induces myasthenia similar to MuSK-associated myasthenia gravis.

Since the first report of experimental animal models of myasthenia gravis (MG) with autoantibodies against low-density lipoprotein receptor-related protein 4 (LRP4), there have not been any major reports replicating the pathogenicity of anti-LRP4 antibodies (Abs). Recent clinical studies have cast doubt on the specificity and pathogenicity of anti-LRP4 antibodies for MG, highlighting the need for further research. In this study, we purified antigens corresponding to the extracellular region of human LRP4 stably expressed with chaperones in 293 cells and used these antigens to immunize female A/J mice. Immunization with LRP4 protein caused mice to develop myasthenia having similar electrophysiological and histological features as are observed in MG patients with circulating Abs against muscle-specific kinase (MuSK). Our results clearly demonstrate that active immunization of mice with LRP4 proteins causes myasthenia similar to the MG induced by anti-MuSK Abs. Further experimental and clinical studies are required to prove the pathogenicity of anti-LRP4 Abs in MG patients.

Effects of Tongue Force Training on Bulbar Motor Function in the Female SOD1-G93A Rat Model of Amyotrophic Lateral Sclerosis.

BACKGROUND: The use of exercise in amyotrophic lateral sclerosis (ALS) is controversial. Although moderate exercise appears to be beneficial for limb muscles in ALS, the effects of exercise on bulbar muscles such as the tongue have not been studied. OBJECTIVE: To determine the effects of tongue force training on bulbar motor function in the SOD1-G93A rat model of ALS. METHODS: We compared the effects of tongue force training on bulbar motor function and neuromuscular junction innervation in female SOD1-G93A rats and age-matched female wild-type controls. Half of each group underwent afternoon tongue force training sessions, and all rats were tested under minimal force conditions in the mornings. RESULTS: Tongue force did not differ between the SOD1-G93A rats and healthy controls during the morning testing sessions, nor was it affected by training. Surprisingly, decreases in tongue motility, the number of licks per session, and body weight were greater in the tongue force-trained SOD1-G93A rats. Forelimb grip force, survival, and denervation of the genioglossus (GG) muscle did not differ between the trained and untrained SOD1-G93A rats. GG innervation was correlated with changes in tongue force but not tongue motility in SOD1-G93A rats at end stage. CONCLUSIONS: The results indicate a potential deleterious effect of tongue force training on tongue motility in female SOD1-G93A rats. The lack of a relationship between GG innervation and tongue motility suggests that factors other than lower-motor neuron integrity likely accounted for this effect.

The role of laminins in the organization and function of neuromuscular junctions.

The synapse between motor neurons and skeletal muscle is known as the neuromuscular junction (NMJ). Proper alignment of presynaptic and post-synaptic structures of motor neurons and muscle fibers, respectively, is essential for efficient motor control of skeletal muscles. The synaptic cleft between these two cells is filled with basal lamina. Laminins are heterotrimer extracellular matrix molecules that are key members of the basal lamina. Laminin α4, α5, and ß2 chains specifically localize to NMJs, and these laminin isoforms play a critical role in maintenance of NMJs and organization of synaptic vesicle release sites known as active zones. These individual laminin chains exert their role in organizing NMJs by binding to their receptors including integrins, dystroglycan, and voltage-gated calcium channels (VGCCs). Disruption of these laminins or the laminin-receptor interaction occurs in neuromuscular diseases including Pierson syndrome and Lambert-Eaton myasthenic syndrome (LEMS). Interventions to maintain proper level of laminins and their receptor interactions may be insightful in treating neuromuscular diseases and aging related degeneration of NMJs.

Dual-color STED microscopy reveals a sandwich structure of Bassoon and Piccolo in active zones of adult and aged mice.

Presynaptic active zones play a pivotal role as synaptic vesicle release sites for synaptic transmission, but the molecular architecture of active zones in mammalian neuromuscular junctions (NMJs) at sub-diffraction limited resolution remains unknown. Bassoon and Piccolo are active zone specific cytosolic proteins essential for active zone assembly in NMJs, ribbon synapses, and brain synapses. These proteins are thought to colocalize and share some functions at active zones. Here, we report an unexpected finding of non-overlapping localization of these two proteins in mouse NMJs revealed using dual-color stimulated emission depletion (STED) super resolution microscopy. Piccolo puncta sandwiched Bassoon puncta and aligned in a Piccolo-Bassoon-Piccolo structure in adult NMJs. P/Q-type voltage-gated calcium channel (VGCC) puncta colocalized with Bassoon puncta. The P/Q-type VGCC and Bassoon protein levels decreased significantly in NMJs from aged mouse. In contrast, the Piccolo levels in NMJs from aged mice were comparable to levels in adult mice. This study revealed the molecular architecture of active zones in mouse NMJs at sub-diffraction limited resolution, and described the selective degeneration mechanism of active zone proteins in NMJs from aged mice. Interestingly, the localization pattern of active zone proteins described herein is similar to active zone structures described using electron microscope tomography.