Histochemical Identification of Motor Fascicles Using Cholinesterase Staining in Rats Using a Mixed Nerve Model.

BACKGROUND: Inappropriate matching of motor and sensory fibers after nerve repair or grafting can lead to nerve recovery failure. Identifying the motor and sensory fascicles enables surgeons to match them accurately and correctly align nerve stumps, which is crucial for neural regeneration. Very few methods have been reported to differentiate between the sensory and motor nerve fascicles, and the replicability of these techniques remains unestablished. In this study, we aimed to assess the accuracy of axonal cholinesterase (CE) histochemical staining in distinguishing motor and sensory nerve fibers. METHODS: The femoral and sciatic nerves were harvested from rats. The specimens were immediately cut, frozen in isopentane, and cooled with liquid nitrogen. Nerve serial cross-sections were processed for hematoxylin and eosin staining, followed by CE histochemistry. The staining protocol solutions included acetylthiocholine iodide, phosphate buffer, cobalt sulfate hydrate, potassium phosphate monobasic, sulfuric acid, sodium bicarbonate, glutaraldehyde, and ammonium sulfide. RESULTS: Cross-sections of nerves containing efferent and afferent nerve fibers in segregated fascicles showed that CE activity was confined to motor neurons. A histochemical study revealed that motor fibers with high cholinesterase activity can be differentiated from sensory fibers. The motor branches of the femoral and sciatic nerves showed specific axonal staining, whereas the sensory branch did not show any specific staining. CONCLUSION: CE histochemical staining is a useful technique for distinguishing between motor and sensory nerve fibers. It can be potentially useful in improving the outcomes of nerve grafts or extremity allotransplantation surgery.

Activation of Muscle-Specific Kinase (MuSK) Reduces Neuromuscular Defects in the Delta7 Mouse Model of Spinal Muscular Atrophy (SMA).

Spinal muscular atrophy (SMA) is a motor neuron disease caused by insufficient levels of the survival motor neuron (SMN) protein. One of the most prominent pathological characteristics of SMA involves defects of the neuromuscular junction (NMJ), such as denervation and reduced clustering of acetylcholine receptors (AChRs). Recent studies suggest that upregulation of agrin, a crucial NMJ organizer promoting AChR clustering, can improve NMJ innervation and reduce muscle atrophy in the delta7 mouse model of SMA. To test whether the muscle-specific kinase (MuSK), part of the agrin receptor complex, also plays a beneficial role in SMA, we treated the delta7 SMA mice with an agonist antibody to MuSK. MuSK agonist antibody #13, which binds to the NMJ, significantly improved innervation and synaptic efficacy in denervation-vulnerable muscles. MuSK agonist antibody #13 also significantly increased the muscle cross-sectional area and myofiber numbers in these denervation-vulnerable muscles but not in denervation-resistant muscles. Although MuSK agonist antibody #13 did not affect the body weight, our study suggests that preservation of NMJ innervation by the activation of MuSK may serve as a complementary therapy to SMN-enhancing drugs to maximize the therapeutic effectiveness for all types of SMA patients.

Oral Treatment with RD2RD2 Impedes Development of Motoric Phenotype and Delays Symptom Onset in SOD1G93A Transgenic Mice.

Neuroinflammation is a pathological hallmark of several neurodegenerative disorders and plays a key role in the pathogenesis of amyotrophic lateral sclerosis (ALS). It has been implicated as driver of disease progression and is observed in ALS patients, as well as in the transgenic SOD1G93A mouse model. Here, we explore and validate the therapeutic potential of the d-enantiomeric peptide RD2RD2 upon oral administration in SOD1G93A mice. Transgenic mice were treated daily with RD2RD2 or placebo for 10 weeks and phenotype progression was followed with several behavioural tests. At the end of the study, plasma cytokine levels and glia cell markers in brain and spinal cord were analysed. Treatment resulted in a significantly increased performance in behavioural and motor coordination tests and a decelerated neurodegenerative phenotype in RD2RD2-treated SOD1G93A mice. Additionally, we observed retardation of the average disease onset. Treatment of SOD1G93A mice led to significant reduction in glial cell activation and a rescue of neurons. Analysis of plasma revealed normalisation of several cytokines in samples of RD2RD2-treated SOD1G93A mice towards the levels of non-transgenic mice. In conclusion, these findings qualify RD2RD2 to be considered for further development and testing towards a disease modifying ALS treatment.

Systemic inflammation suppresses spinal respiratory motor plasticity via mechanisms that require serine/threonine protein phosphatase activity.

BACKGROUND: Inflammation undermines multiple forms of neuroplasticity. Although inflammation and its influence on plasticity in multiple neural systems has been extensively studied, its effects on plasticity of neural networks controlling vital life functions, such as breathing, are less understood. In this study, we investigated the signaling mechanisms whereby lipopolysaccharide (LPS)-induced systemic inflammation impairs plasticity within the phrenic motor system-a major spinal respiratory motor pool that drives contractions of the diaphragm muscle. Here, we tested the hypotheses that lipopolysaccharide-induced systemic inflammation (1) blocks phrenic motor plasticity by a mechanism that requires cervical spinal okadaic acid-sensitive serine/threonine protein phosphatase (PP) 1/2A activity and (2) prevents phosphorylation/activation of extracellular signal-regulated kinase 1/2 mitogen activated protein kinase (ERK1/2 MAPK)-a key enzyme necessary for the expression of phrenic motor plasticity. METHODS: To study phrenic motor plasticity, we utilized a well-characterized model for spinal respiratory plasticity called phrenic long-term facilitation (pLTF). pLTF is characterized by a long-lasting, progressive enhancement of inspiratory phrenic nerve motor drive following exposures to moderate acute intermittent hypoxia (mAIH). In anesthetized, vagotomized and mechanically ventilated adult Sprague Dawley rats, we examined the effect of inhibiting cervical spinal serine/threonine PP 1/2A activity on pLTF expression in sham-vehicle and LPS-treated rats. Using immunofluorescence optical density analysis, we compared mAIH-induced phosphorylation/activation of ERK 1/2 MAPK with and without LPS-induced inflammation in identified phrenic motor neurons. RESULTS: We confirmed that mAIH-induced pLTF is abolished 24 h following low-dose systemic LPS (100 µg/kg, i.p.). Cervical spinal delivery of the PP 1/2A inhibitor, okadaic acid, restored pLTF in LPS-treated rats. LPS also prevented mAIH-induced enhancement in phrenic motor neuron ERK1/2 MAPK phosphorylation. Thus, a likely target for the relevant okadaic acid-sensitive protein phosphatases is ERK1/2 MAPK or its upstream activators. CONCLUSIONS: This study increases our understanding of fundamental mechanisms whereby inflammation disrupts neuroplasticity in a critical population of motor neurons necessary for breathing, and highlights key roles for serine/threonine protein phosphatases and ERK1/2 MAPK kinase in the plasticity of mammalian spinal respiratory motor circuits.

Inhibition of Cytosolic Phospholipase A2 Has Neuroprotective Effects on Motoneuron and Muscle Atrophy after Spinal Cord Injury.

Surviving motoneurons undergo dendritic atrophy after spinal cord injury (SCI), suggesting an important therapeutic target for neuroprotective strategies to improve recovery of function after SCI. Our previous studies showed that cytosolic phospholipase A2 (PLA2) may play an important role in the pathogenesis of SCI. In the present study, we investigated whether blocking cytosolic PLA2 (cPLA2) pharmacologically with arachidonyl trifluoromethyl ketone (ATK) or genetically using cPLA2 knockout (KO) mice attenuates motoneuron atrophy after SCI. C57BL/6 mice received either sham or contusive SCI at the T10 level. At 30 min after SCI, mice were treated with ATK or vehicle. Four weeks later, motoneurons innervating the vastus lateralis muscle of the quadriceps were labeled with cholera toxin-conjugated horseradish peroxidase, and dendritic arbors were reconstructed in three dimensions. Soma volume, motoneuron number, lesion volume, and tissue sparing were also assessed, as were muscle weight, fiber cross-sectional area, and motor endplate size and density. ATK administration reduced percent lesion volume and increased percent volume of spared white matter, compared to the vehicle-treated control animals. SCI with or without ATK treatment had no effect on the number or soma volume of quadriceps motoneurons. However, SCI resulted in a decrease in dendritic length of quadriceps motoneurons in untreated animals, and this decrease was completely prevented by treatment with ATK. Similarly, vastus lateralis muscle weights of untreated SCI animals were smaller than those of sham surgery controls, and these reductions were prevented by ATK treatment. No effects on fiber cross-sectional areas, motor endplate area, or density were observed across treatment groups. Remarkably, genetically deleting cPLA2 in cPLA2 KO mice attenuated dendritic atrophy after SCI. These findings suggest that, after SCI, cord tissue damage and regressive changes in motoneuron and muscle morphology can be reduced by inhibition of cPLA2, further supporting a role for cPLA2 as a neurotherapeutic target for SCI treatment.

Upregulation of JHDM1D-AS1 alleviates neuroinflammation and neuronal injury via targeting miR-101-3p-DUSP1 in spinal cord after brachial plexus injury.

BACKGROUND: Neuroinflammation in the spinal cord following acute brachial plexus injury (BPI) remains a vital cause that leads to motor dysfunction and neuropathic pain. In this study, we aim to explore the role of long non-coding RNA JHDM1D antisense 1 (JHDM1D-AS1) in mediating BPI-induced neuroinflammation and neuronal injury. METHODS: A total brachial plexus root avulsion (tBPRA) model in adult rats and IL-1ß-treated motor neuron-like NSC-34 cells and LPS-treated microglia cell line BV2 were conducted for in vivo and in vitro experiments, respectively. The expressions of JHDM1D-AS1, miR-101-3p and DUSP1, p38, NF-κB, TNF-α, IL-1ß, and IL-6 were detected by RT-PCR and western blot seven days after tBPI. Immunohistochemistry (IHC) was used to detect neuronal apoptosis. CCK8 assay, Tunel assay and LDH kit were used for the detection of neuronal injury. The targeted relationships between JHDM1D-AS1 and miR-101-3p, miR-101-3p and DUSP1 were verified by RNA immunoprecipitation (RIP) and dual-luciferase reporter gene assay. RESULTS: We found significant downregulated expression of JHDM1D-AS1 and DUSP1 but upregulated expression of miR-101-3p in the spinal cord after tBPI. Overexpression of JHDM1D-AS1 had a prominent neuroprotective effect by suppressing neuronal apoptosis and microglial inflammation through reactivation of DUSP1. Further exploration revealed that JHDM1D-AS1 may act as a competitive endogenous RNA targeting miR-101-3p, which bound on the 3'UTR of DUSP1 mRNA. In addition, overexpression of miR-101-3p could reverse the neuroprotective effects of JHDM1D-AS1 upregulation by blocking DUSP1. CONCLUSIONS: JHDM1D-AS1 exerted neuroprotective and anti-inflammatory effects in a rat model of tBPI by regulating miR-101-3p/DUSP1 axis.

Neuron-specific deletion of CuZnSOD leads to an advanced sarcopenic phenotype in older mice.

Age-associated loss of muscle mass and function (sarcopenia) has a profound effect on the quality of life in the elderly. Our previous studies show that CuZnSOD deletion in mice (Sod1-/- mice) recapitulates sarcopenia phenotypes, including elevated oxidative stress and accelerated muscle atrophy, weakness, and disruption of neuromuscular junctions (NMJs). To determine whether deletion of Sod1 initiated in neurons in adult mice is sufficient to induce muscle atrophy, we treated young (2- to 4-month-old) Sod1flox/SlickHCre mice with tamoxifen to generate i-mn-Sod1KO mice. CuZnSOD protein was 40-50% lower in neuronal tissue in i-mn-Sod1KO mice. Motor neuron number in ventral spinal cord was reduced 28% at 10 months and more than 50% in 18- to 22-month-old i-mn-Sod1KO mice. By 24 months, 22% of NMJs in i-mn-Sod1KO mice displayed a complete lack of innervation and deficits in specific force that are partially reversed by direct muscle stimulation, supporting the loss of NMJ structure and function. Muscle mass was significantly reduced by 16 months of age and further decreased at 24 months of age. Overall, our findings show that neuronal-specific deletion of CuZnSOD is sufficient to cause motor neuron loss in young mice, but that NMJ disruption, muscle atrophy, and weakness are not evident until past middle age. These results suggest that loss of innervation is critical but may not be sufficient until the muscle reaches a threshold beyond which it cannot compensate for neuronal loss or rescue additional fibers past the maximum size of the motor unit.

Does wild-type Cu/Zn-superoxide dismutase have pathogenic roles in amyotrophic lateral sclerosis?

Amyotrophic lateral sclerosis (ALS) is characterized by adult-onset progressive degeneration of upper and lower motor neurons. Increasing numbers of genes are found to be associated with ALS; among those, the first identified gene, SOD1 coding a Cu/Zn-superoxide dismutase protein (SOD1), has been regarded as the gold standard in the research on a pathomechanism of ALS. Abnormal accumulation of misfolded SOD1 in affected spinal motor neurons has been established as a pathological hallmark of ALS caused by mutations in SOD1 (SOD1-ALS). Nonetheless, involvement of wild-type SOD1 remains quite controversial in the pathology of ALS with no SOD1 mutations (non-SOD1 ALS), which occupies more than 90% of total ALS cases. In vitro studies have revealed post-translationally controlled misfolding and aggregation of wild-type as well as of mutant SOD1 proteins; therefore, SOD1 proteins could be a therapeutic target not only in SOD1-ALS but also in more prevailing cases, non-SOD1 ALS. In order to search for evidence on misfolding and aggregation of wild-type SOD1 in vivo, we reviewed pathological studies using mouse models and patients and then summarized arguments for and against possible involvement of wild-type SOD1 in non-SOD1 ALS as well as in SOD1-ALS.

N-Acylethanolamine Acid Amidase contributes to disease progression in a mouse model of multiple sclerosis.

N-Acylethanolamine acid amidase (NAAA) deactivates the endogenous peroxisome proliferator-activated receptor-α (PPAR-α) agonist palmitoylethanolamide (PEA). NAAA-regulated PEA signaling participates in the control of peripheral inflammation, but evidence suggests also a role in the modulation of neuroinflammatory pathologies such as multiple sclerosis (MS). Here we show that disease progression in the mouse experimental autoimmune encephalomyelitis (EAE) model of MS is accompanied by induction of NAAA expression in spinal cord, which in presymptomatic animals is confined to motor neurons and oligodendrocytes but, as EAE progresses, extends to microglia/macrophages and other cell types. As previously reported for NAAA inhibition, genetic NAAA deletion delayed disease onset and attenuated symptom intensity in female EAE mice, suggesting that accrued NAAA expression may contribute to pathology. To further delineate the role of NAAA in EAE, we generated a mouse line that selectively overexpresses the enzyme in macrophages, microglia and other monocyte-derived cells. Non-stimulated alveolar macrophages from these NaaaCD11b+ mice contain higher-than-normal levels of inducible nitric oxide synthase and display an activated morphology. Furthermore, intranasal lipopolysaccharide injections cause greater alveolar leukocyte accumulation in NaaaCD11b+ than in control mice. NaaaCD11b+ mice also display a more aggressive clinical response to EAE induction, compared to their wild-type littermates. The results identify NAAA as a critical control step in EAE pathogenesis, and point to this enzyme as a possible target for the treatment of MS.

Calpain system is altered in survival motor neuron-reduced cells from in vitro and in vivo spinal muscular atrophy models.

Spinal muscular atrophy (SMA) is a severe neuromuscular disorder caused by loss of the survival motor neuron 1 (SMN1) gene. SMA is characterized by the degeneration of spinal cord motoneurons (MNs), progressive skeletal muscle atrophy, and weakness. The cellular and molecular mechanisms causing MN loss of function are only partially known. Recent advances in SMA research postulate the role of calpain protease regulating survival motor neuron (SMN) protein and the positive effect on SMA phenotype of treatment with calpain inhibitors. We analyzed the level of calpain pathway members in mice and human cellular SMA models. Results indicate an increase of calpain activity in SMN-reduced MNs. Spinal cord analysis of SMA mice treated with calpeptin, a calpain inhibitor, showed an increase of SMN, calpain, and its endogenous inhibitor calpastatin in MNs. Finally, in vitro calpeptin treatment prevented microtubule-associated protein 1A/1B-light chain 3 (LC3) increase in MNs neurites, indicating that calpain inhibition may reduce autophagosome accumulation in neuron prolongations, but not in soma. Thus, our results show that calpain activity is increased in SMA MNs and its inhibition may have a beneficial effect on SMA phenotype through the increase of SMN in spinal cord MNs.

Artemisinin protects motoneurons against axotomy-induced apoptosis through activation of the PKA-Akt signaling pathway and promotes neural stem/progenitor cells differentiation into NeuN+ neurons.

Brachial plexus axotomy is a common peripheral nerve trauma. Artemisinin, an FDA-approved antimalarial drug, has been described to possess neuroprotective properties. However, the specific mechanisms by which artemisinin protects neurons from axotomy-induced neurotoxicity remain to be elucidated. In this study, we assessed the neuroprotective effects of artemisinin on an experimental animal model of brachial plexus injury and explored the possible mechanisms involved. Artemisinin treatment restored both athletic ability and sensation of the affected upper limb, rescued motoneurons and attenuated the inflammatory response in the ventral horn of the spinal cord. Additionally, artemisinin inhibited the molecular signals of apoptosis, activated signaling pathways related to cell survival and induced NSCPs differentiation into NeuN-positive neurons. Further validation of the involved key signaling molecules, using an in vitro model of hydrogen peroxide-induced neurotoxicity, revealed that both the inhibition of PKA signaling pathway or the silencing of Akt reversed the neuroprotective action of artemisinin on motoneurons. Our results indicate that artemisinin provides neuroprotection against axotomy and hydrogen peroxide-induced neurotoxicity, an effect that might be mediated by the PKA-Akt signaling pathway.

Synaptic restoration by cAMP/PKA drives activity-dependent neuroprotection to motoneurons in ALS.

Excessive excitation is hypothesized to cause motoneuron (MN) degeneration in amyotrophic lateral sclerosis (ALS), but actual proof of hyperexcitation in vivo is missing, and trials based on this concept have failed. We demonstrate, by in vivo single-MN electrophysiology, that, contrary to expectations, excitatory responses evoked by sensory and brainstem inputs are reduced in MNs of presymptomatic mutSOD1 mice. This impairment correlates with disrupted postsynaptic clustering of Homer1b, Shank, and AMPAR subunits. Synaptic restoration can be achieved by activation of the cAMP/PKA pathway, by either intracellular injection of cAMP or DREADD-Gs stimulation. Furthermore, we reveal, through independent control of signaling and excitability allowed by multiplexed DREADD/PSAM chemogenetics, that PKA-induced restoration of synapses triggers an excitation-dependent decrease in misfolded SOD1 burden and autophagy overload. In turn, increased MN excitability contributes to restoring synaptic structures. Thus, the decrease of excitation to MN is an early but reversible event in ALS. Failure of the postsynaptic site, rather than hyperexcitation, drives disease pathobiochemistry.

Shorter axon initial segments do not cause repetitive firing impairments in the adult presymptomatic G127X SOD-1 Amyotrophic Lateral Sclerosis mouse.

Increases in axonal sodium currents in peripheral nerves are some of the earliest excitability changes observed in Amyotrophic Lateral Sclerosis (ALS) patients. Nothing is known, however, about axonal sodium channels more proximally, particularly at the action potential initiating region - the axon initial segment (AIS). Immunohistochemistry for Nav1.6 sodium channels was used to investigate parameters of AISs of spinal motoneurones in the G127X SOD1 mouse model of ALS in adult mice at presymptomatic time points (~190 days old). In vivo intracellular recordings from lumbar spinal motoneurones were used to determine the consequences of any AIS changes. AISs of both alpha and gamma motoneurones were found to be significantly shorter (by 6.6% and 11.8% respectively) in G127X mice as well as being wider by 9.8% (alpha motoneurones). Measurements from 20-23 day old mice confirmed that this represented a change during adulthood. Intracellular recordings from motoneurones in presymptomatic adult mice, however, revealed no differences in individual action potentials or the cells ability to initiate repetitive action potentials. To conclude, despite changes in AIS geometry, no evidence was found for reduced excitability within the functional working range of firing frequencies of motoneurones in this model of ALS.

Myelin degeneration induced by mutant superoxide dismutase 1 accumulation promotes amyotrophic lateral sclerosis.

Myelin is a specialized membrane that wraps around nerve fibers and is essential for normal axonal conduction in neurons. In the central nervous system, oligodendrocytes are responsible for myelin formation. Recent studies have reported pathological abnormalities in oligodendrocytes in human patients with amyotrophic lateral sclerosis (ALS) and a mouse model of ALS expressing the G93A mutation of the human superoxide dismutase 1 (mtSOD1). However, it is unclear whether oligodendrocyte pathology in ALS represents the primary dysfunction induced by mtSOD1 and how mtSOD1 contributes to oligodendrocyte degeneration and ALS pathogenesis. We analyzed GAL4-VP16-UAS transgenic zebrafish selectively expressing mtSOD1 in mature oligodendrocytes. We observed that mtSOD1 directly induced oligodendrocyte degeneration by disrupting the myelin sheath and downregulating monocarboxylate transporter 1 (MCT1), thereby causing spinal motor neuron degeneration. Pathological changes observed in this transgenic zebrafish were similar to the pathology observed in the SOD1G93A mouse model of ALS, which is characterized by expression of mtSOD1 in all cells. In addition, oligodendrocyte dysfunction induced by mtSOD1 was associated with anxiety-related behavioral abnormalities, learning impairments, and motor defects in the early symptomatic stage. We also found that treatment with potassium channel inhibitors rescued behavioral abnormalities without rescuing MCT1 expression, suggesting that myelin disruption induces behavioral abnormalities independently of MCT1. These results indicate that mtSOD1-induced dysfunction of mature oligodendrocytes is sufficient to induce motor neuron degeneration, thus informing future therapeutic strategies targeted at oligodendrocytes in ALS.

Age- and gender-dependent D-amino acid oxidase activity in mouse brain and peripheral tissues: implication for aging and neurodegeneration.

D-amino acid oxidase (DAO) is a flavoenzyme, catalysing oxidative deamination of D-amino acids to produce corresponding α-keto acids, ammonia and hydrogen peroxide. In our search for DAO activity among various tissues, we developed a sensitive assay based on hydrogen peroxide production involving enzyme-coupled colorimetric assay with peroxidase. We first optimized buffer components to extract DAO protein from mouse tissues. Here we show that DAO activity was detected in kidney, cerebellum, medulla oblongata, midbrain and spinal cord, but not in liver. In addition, we observed that DAO activity and expression were decreased in thoracic and lumbar regions of spinal cord in aged mice when compared with young mice, indicating that decreased DAO is involved in motoneuron degeneration during senescence. We also found gender difference in DAO activity in the kidney, suggesting that DAO activity is influenced by sexual dimorphism. We newly detected DAO activity in the epididymis, although undetected in testis. Furthermore, DAO activity was significantly higher in the caput region than corpus and cauda regions of epididymis, indicating that D-amino acids present in the testis are eliminated in epididymis. Taken together, age- and gender-dependent DAO activity in each organ may underlie the human pathophysiology regulated by D-amino acid metabolism.

DNA methylation inhibitor attenuates polyglutamine-induced neurodegeneration by regulating Hes5.

Spinal and bulbar muscular atrophy (SBMA) is a polyglutamine-mediated neuromuscular disease caused by a CAG repeat expansion in the androgen receptor (AR) gene. While transcriptional dysregulation is known to play a critical role in the pathogenesis of SBMA, the underlying molecular pathomechanisms remain unclear. DNA methylation is a fundamental epigenetic modification that silences the transcription of various genes that have a CpG-rich promoter. Here, we showed that DNA methyltransferase 1 (Dnmt1) is highly expressed in the spinal motor neurons of an SBMA mouse model and in patients with SBMA. Both genetic Dnmt1 depletion and treatment with RG108, a DNA methylation inhibitor, ameliorated the viability of SBMA model cells. Furthermore, a continuous intracerebroventricular injection of RG108 mitigated the phenotype of SBMA mice. DNA methylation array analysis identified hairy and enhancer of split 5 (Hes5) as having a CpG island with hyper-methylation in the promoter region, and the Hes5 expression was strongly silenced in SBMA. Moreover, Hes5 over-expression rescued the SBMA cells possibly by inducing Smad2 phosphorylation. Our findings suggest DNA hyper-methylation underlies the neurodegeneration in SBMA.

MAP4K4 Activation Mediates Motor Neuron Degeneration in Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting both upper and lower motor neurons (MNs). To date, its underlying mechanisms have yet to be clarified completely, and there are no truly effective treatments. Here, we show that MAP4K4, a MAP kinase family member, regulates MN death, with its suppression not only promoting survival but preventing neurite degeneration and decreasing mutant SOD1 levels through autophagy activation. Moreover, we report that MAP4K4 signaling specifically modulates MN viability via phosphorylated JNK3 and activation of the canonical c-Jun apoptotic pathway. Finally, we show the feasibility of MAP4K4 as a drug target by using an available MAP4K4-specific inhibitor, which improves survival of ESC and/or iPSC-derived MNs and MNs cultured from mouse spinal cords. In summary, our studies highlight a MAP4K4-initiated signaling cascade that induces MN degeneration, shedding light on the mechanism underlying MN degeneration and providing a druggable target for ALS therapeutics.

Trehalose induces autophagy via lysosomal-mediated TFEB activation in models of motoneuron degeneration.

Macroautophagy/autophagy, a defense mechanism against aberrant stresses, in neurons counteracts aggregate-prone misfolded protein toxicity. Autophagy induction might be beneficial in neurodegenerative diseases (NDs). The natural compound trehalose promotes autophagy via TFEB (transcription factor EB), ameliorating disease phenotype in multiple ND models, but its mechanism is still obscure. We demonstrated that trehalose regulates autophagy by inducing rapid and transient lysosomal enlargement and membrane permeabilization (LMP). This effect correlated with the calcium-dependent phosphatase PPP3/calcineurin activation, TFEB dephosphorylation and nuclear translocation. Trehalose upregulated genes for the TFEB target and regulator Ppargc1a, lysosomal hydrolases and membrane proteins (Ctsb, Gla, Lamp2a, Mcoln1, Tpp1) and several autophagy-related components (Becn1, Atg10, Atg12, Sqstm1/p62, Map1lc3b, Hspb8 and Bag3) mostly in a PPP3- and TFEB-dependent manner. TFEB silencing counteracted the trehalose pro-degradative activity on misfolded protein causative of motoneuron diseases. Similar effects were exerted by trehalase-resistant trehalose analogs, melibiose and lactulose. Thus, limited lysosomal damage might induce autophagy, perhaps as a compensatory mechanism, a process that is beneficial to counteract neurodegeneration. Abbreviations: ALS: amyotrophic lateral sclerosis; AR: androgen receptor; ATG: autophagy related; AV: autophagic vacuole; BAG3: BCL2-associated athanogene 3; BECN1: beclin 1, autophagy related; CASA: chaperone-assisted selective autophagy; CTSB: cathepsin b; DAPI: 4',6-diamidino-2-phenylindole; DMEM: Dulbecco's modified Eagle's medium; EGFP: enhanced green fluorescent protein; fALS, familial amyotrophic lateral sclerosis; FRA: filter retardation assay; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GLA: galactosidase, alpha; HD: Huntington disease; hIPSCs: human induced pluripotent stem cells; HSPA8: heat shock protein A8; HSPB8: heat shock protein B8; IF: immunofluorescence analysis; LAMP1: lysosomal-associated membrane protein 1; LAMP2A: lysosomal-associated membrane protein 2A; LGALS3: lectin, galactose binding, soluble 3; LLOMe: L-leucyl-L-leucine methyl ester; LMP: lysosomal membrane permeabilization; Lys: lysosomes; MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; MCOLN1: mucolipin 1; mRNA: messenger RNA; MTOR: mechanistic target of rapamycin kinase; NDs: neurodegenerative diseases; NSC34: neuroblastoma x spinal cord 34; PBS: phosphate-buffered saline; PD: Parkinson disease; polyQ: polyglutamine; PPARGC1A: peroxisome proliferative activated receptor, gamma, coactivator 1 alpha; PPP3CB: protein phosphatase 3, catalytic subunit, beta isoform; RT-qPCR: real-time quantitative polymerase chain reaction; SBMA: spinal and bulbar muscular atrophy; SCAs: spinocerebellar ataxias; siRNA: small interfering RNA; SLC2A8: solute carrier family 2, (facilitated glucose transporter), member 8; smNPCs: small molecules neural progenitors cells; SOD1: superoxide dismutase 1; SQSTM1/p62: sequestosome 1; STED: stimulated emission depletion; STUB1: STIP1 homology and U-box containing protein 1; TARDBP/TDP-43: TAR DNA binding protein; TFEB: transcription factor EB; TPP1: tripeptidyl peptidase I; TREH: trehalase (brush-border membrane glycoprotein); WB: western blotting; ZKSCAN3: zinc finger with KRAB and SCAN domains 3.

Characterizing the multiple roles of FGF-2 in SOD1G93A ALS mice in vivo and in vitro.

We have previously shown that knockout of fibroblast growth factor-2 (FGF-2) and potential compensatory effects of other growth factors result in amelioration of disease symptoms in a transgenic mouse model of amyotrophic lateral sclerosis (ALS). ALS is a rapidly progressive neurological disorder leading to degeneration of cortical, brain stem, and spinal motor neurons followed by subsequent denervation and muscle wasting. Mutations in the superoxide dismutase 1 (SOD1) gene are responsible for approximately 20% of familial ALS cases and SOD1 mutant mice still are among the models best mimicking clinical and neuropathological characteristics of ALS. The aim of the present study was a thorough characterization of FGF-2 and other growth factors and signaling effectors in vivo in the SOD1G93A mouse model. We observed tissue-specific opposing gene regulation of FGF-2 and overall dysregulation of other growth factors, which in the gastrocnemius muscle was associated with reduced downstream extracellular-signal-regulated kinases (ERK) and protein kinase B (AKT) activation. To further investigate whether the effects of FGF-2 on motor neuron death are mediated by glial cells, astrocytes lacking FGF-2 were cocultured together with mutant SOD1 G93A motor neurons. FGF-2 had an impact on motor neuron maturation indicating that astrocytic FGF-2 affects motor neurons at a developmental stage. Moreover, neuronal gene expression patterns showed FGF-2- and SOD1 G93A -dependent changes in ciliary neurotrophic factor, glial-cell-line-derived neurotrophic factor, and ERK2, implying a potential involvement in ALS pathogenesis before the onset of clinical symptoms.

Loss of tubulin deglutamylase CCP1 causes infantile-onset neurodegeneration.

A set of glutamylases and deglutamylases controls levels of tubulin polyglutamylation, a prominent post-translational modification of neuronal microtubules. Defective tubulin polyglutamylation was first linked to neurodegeneration in the Purkinje cell degeneration (pcd) mouse, which lacks deglutamylase CCP1, displays massive cerebellar atrophy, and accumulates abnormally glutamylated tubulin in degenerating neurons. We found biallelic rare and damaging variants in the gene encoding CCP1 in 13 individuals with infantile-onset neurodegeneration and confirmed the absence of functional CCP1 along with dysregulated tubulin polyglutamylation. The human disease mainly affected the cerebellum, spinal motor neurons, and peripheral nerves. We also demonstrate previously unrecognized peripheral nerve and spinal motor neuron degeneration in pcd mice, which thus recapitulated key features of the human disease. Our findings link human neurodegeneration to tubulin polyglutamylation, entailing this post-translational modification as a potential target for drug development for neurodegenerative disorders.