Motor Neuron Cell Research Articles

Nanoparticles Encapsulating Phosphatidylinositol Derivatives Promote Neuroprotection and Functional Improvement via a long-lasting activation of TRPML1 lysosomal channel in Preclinical Models of ALS

Background and purposeAmyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease currently incurable, in which motor neuron degeneration leads to voluntary skeletal muscle atrophy. Molecularly, ALS is characterized by protein aggregation, synaptic and organellar dysfunction, and Ca2+ dyshomeostasis. Of interest, autophagy dysfunction is emerging as one of the main putative targets of ALS therapy. A tune regulation of this cleansing process is affordable by a proper stimulation of TRPML1, one of the main lysosomal channels. However, TRPML1 activation by PI(3,5)P2 has low open probability to remain in an active conformation. To overcome this drawback we developed a lipid-based formulation of PI(3,5)P2 whose putative therapeutic potential has been tested in in vitro and in vivo ALS models. Experimental ApproachPharmacodynamic properties of PI(3,5)P2 lipid-based formulations (F1 and F2) on TRPML1 activity have been characterized by means of patch-clamp electrophysiology and Fura-2AM video-imaging in motor neuronal cells. Once selected for the ability to stabilize TRPML1 activity, the most effective preparation F1 was studied in vivo to measure neuromuscular function and survival of SOD1G93A ALS mice, thereby establishing its therapeutic profile.Key ResultsF1, but not PI(3,5)P2 alone, stabilized the open state of the lysosomal channel TRPML1 and increased the persistence of intracellular calcium concentration ([Ca2+]i). Then, F1 was effective in delaying motor neuron loss, improving innervated endplants and muscle performance in SOD1G93A mice, extending overall lifespan by an average of 10 days. Of note F1 prevented gliosis and autophagy dysfunction in ALS mice by restoring PI(3,5)P2 level. Conclusion and implicationsOur novel self-assembling lipidic formulation for PI(3,5)P2 delivery exerts a neuroprotective effect in preclinical models of ALS mainly regulating dysfunctional autophagy through TRPML1 activity stabilization.

Spinal V1 inhibitory interneuron clades differ in birthdate, projections to motoneurons, and heterogeneity.

The complexity of spinal interneuron diversity and circuit organization represents a challenge to understand neural control of movement in normal adults as well as during motor development and in disease. Inhibitory interneurons are a core element of these spinal circuits. V1 interneurons comprise the largest group of inhibitory interneurons in the ventral horn, and their organization remains unclear. Here we present a comprehensive examination of V1 subtypes according to neurogenesis, placement in spinal motor circuits, and motoneuron synaptic targeting. V1 diversity increases during evolution from axial-swimming fishes to limb-based mammalian terrestrial locomotion. This increased diversity is reflected in the size and heterogeneity of the Foxp2-V1 clade, a group closely associated with limb motor pools. We show that Foxp2-V1 interneurons establish the densest direct inhibitory input to motoneurons, especially on cell bodies. These findings are particularly significant because recent studies have shown that motor neurodegenerative diseases like amyotrophic lateral sclerosis (ALS) affect inhibitory V1 synapses on motoneuron cell bodies and Foxp2-V1 interneurons themselves in the earliest stages of pathology.

The effect of repetitive magnetic stimulation on neuronal apoptosis and PI3K/Akt protein expression in rats with incomplete spinal cord injury.

The pathological and physiological process of spinal cord injury is complex, and there is currently no effective treatment method. Magnetic stimulation is an emerging electromagnetic therapy method in recent years, and studies have shown its potential to reduce cell apoptosis. This study used an improved Allen's method to replicate an incomplete spinal cord injury rat model, and repetitive magnetic stimulation (rMS) intervention was performed on the rats for 21 days. The research plan consists of two parts. The first part aims to observe the effects of rMS on motor function and neuronal cell apoptosis in rats. The Basso-Beattie-Bresnahan (BBB) score results indicate that rMS promotes the recovery of motor function in rats; H&E staining showed that rMS improved spinal cord structural damage and inflammatory infiltration; TUNEL and NeuN staining suggest that rMS can reduce cell apoptosis and promote neuronal cell survival. The second part aims to explore the mechanism of action of rMS. Immunofluorescence staining showed that after rMS intervention, the positive counts of PI3K and Akt increased, whereas the positive counts of caspase-3 decreased. Western blot showed that after rMS intervention, the expression of phospho-phosphatidylinositol-3 kinase (p-PI3K)/PI3K, phospho (p)-Akt/Akt, and Bcl-2 increased, whereas the expression of Bcl-2-associated X protein(Bax) and caspase-3 decreased. In summary, rMS can significantly reduce cell apoptosis in the damaged spinal cord and promote neuronal cell survival. Its mechanism of action may be related to promoting the expression of PI3K/Akt pathway proteins, upregulating the antiapoptotic protein Bcl-2, downregulating the proapoptotic protein Bax, and thereby inhibiting the expression of apoptotic protein caspase-3. NEW & NOTEWORTHY Spinal cord injury is a serious disabling central nervous system disease. Recently, research on magnetic stimulation therapy for spinal cord injury has been increasing, and its potential has gradually attracted the attention of experts. This study found that repetitive magnetic stimulation (rMS) can improve motor function and reduce neuronal apoptosis in spinal cord injury rats. The mechanism may be related to increasing the expression of phosphatidylinositol-3 kinase (PI3K)/Akt protein, thereby inhibiting cell apoptosis and promoting neuronal survival.

Hepatocyte Growth Factor Delivery to Injured Cervical Spinal Cord Using an Engineered Biomaterial Protects Respiratory Neural Circuitry and Preserves Functional Diaphragm Innervation.

A major portion of spinal cord injury (SCI) cases occur in the cervical region, where essential components of the respiratory neural circuitry are located. Phrenic motor neurons (PhMNs) housed at cervical spinal cord level C3-C5 directly innervate the diaphragm, and SCI-induced damage to these cells severely impairs respiratory function. In this study, we tested a biomaterial-based approach aimed at preserving this critical phrenic motor circuitry after cervical SCI by locally delivering hepatocyte growth factor (HGF). HGF is a potent mitogen that promotes survival, proliferation, migration, repair, and regeneration of a number of different cell and tissue types in response to injury. We developed a hydrogel-based HGF delivery system that can be injected into the intrathecal space for local delivery of high levels of HGF without damaging the spinal cord. Implantation of HGF hydrogel after unilateral C5 contusion-type SCI in rats preserved diaphragm function, as assessed by invivo recordings of both compound muscle action potentials and inspiratory electromyography amplitudes. HGF hydrogel also preserved PhMN innervation of the diaphragm, as assessed by both retrograde PhMN tracing and detailed neuromuscular junction morphological analysis. Furthermore, HGF hydrogel significantly decreased lesion size and degeneration of cervical motor neuron cell bodies, as well as reduced levels surrounding the injury site of scar-associated chondroitin sulfate proteoglycan molecules that limit axon growth capacity. Our findings demonstrate that local biomaterial-based delivery of HGF hydrogel to injured cervical spinal cord is an effective strategy for preserving respiratory circuitry and diaphragm function.

CAMP/PKA signaling regulates TDP-43 aggregation and mislocalization

Cytoplasmic mislocalization and aggregation of TDP-43 protein are hallmarks of amyotrophic lateral sclerosis (ALS) and are observed in the vast majority of both familial and sporadic cases. How these two interconnected processes are regulated on a molecular level, however, remains enigmatic. Genome-wide screens for modifiers of the ALS-associated genes TDP-43 and FUS have identified the phospholipase D (Pld) pathway as a key regulator of ALS-related phenotypes in the fruit fly Drosophila melanogaster [M. W. Kankel et al., Genetics 215, 747-766 (2020)]. Here, we report the results of our search for downstream targets of the enzymatic product of Pld, phosphatidic acid. We identify two conserved negative regulators of the cAMP/PKA signaling pathway, the phosphodiesterase dunce and the inhibitory subunit PKA-R2, as modifiers of pathogenic phenotypes resulting from overexpression of the Drosophila TDP-43 ortholog TBPH. We show that knockdown of either of these genes results in a mitigation of both TBPH aggregation and mislocalization in larval motor neuron cell bodies, as well as an amelioration of adult-onset motor defects and shortened lifespan induced by TBPH. We determine that PKA kinase activity is downstream of both TBPH and Pld and that overexpression of the PKA target CrebA can rescue TBPH mislocalization. These findings suggest a model whereby increasing cAMP/PKA signaling can ameliorate the molecular and functional effects of pathological TDP-43.

MS785-MS27 Reactive Misfolded/Non-Native Zn-Deficient SOD1 Species Exhibit Cytotoxicity and Adopt Heterozygous Conformations in Motor Neurons.

Misfolding of superoxide dismutase-1 (SOD1) is a pathological hallmark of amyotrophic lateral sclerosis (ALS) with SOD1 mutations. The development of antibodies specific for misfolded SOD1 deepens our understanding of how the protein participates in ALS pathogenesis. Since the term "misfolding" refers to various disordered conformers other than the natively folded one, which misfolded species are recognized by specific antibodies should be determined. Here, we molecularly characterized the recognition by MS785-MS27, an antibody cocktail experimentally confirmed to recognize over 100 ALS-linked SOD1 mutants. Indirect ELISA revealed that the antibody cocktail recognized Zn-deficient wild-type and mutated SOD1 species. It also recognized conformation-disordered wild-type and mutated SOD1 species, such as unfolded and oligomeric forms, but had less affinity for the aggregated form. Antibody-reactive SOD1 exhibited cytotoxicity to a motor neuron cell model, which was blocked by Zn treatment with Zn-deficient SOD1. Immunohistochemistry revealed antibody-reactive SOD1 mainly in spinal motor neurons of SOD1G93A mice throughout the disease course, and the distribution after symptomatic stages differed from that of other misfolded SOD1 species. This suggests that misfolded/non-native SOD1 species exist as heterogeneous populations. In conclusion, MS785-MS27 recognizes various conformation-disordered SOD1 species lacking the Zn ion.

Intrinsic motoneuron properties in typical human development.

Motoneuron properties and their firing patterns undergo significant changes throughout development and in response to neuromodulators such as serotonin. Here, we examined the age-related development of self-sustained firing and general excitability of tibialis anterior motoneurons in a young development (7-17years), young adult (18-28years) and adult (32-53years) group, as well as in a separate group of participants taking selective serotonin reuptake inhibitors (SSRIs, aged 11-28years). Self-sustained firing, as measured by ΔF, was larger in the young development (∼5.8Hz, n=20) compared to the young adult (∼4.9Hz, n=13) and adult (∼4.8Hz, n=8) groups, consistent with a developmental decrease in self-sustained firing mediated by persistent inward currents (PIC). ΔF was also larger in participants taking SSRIs (∼6.5Hz, n=9) compared to their age-matched controls (∼5.3Hz, n=26), consistent with increased levels of spinal serotonin facilitating the motoneuron PIC. Participants in the young development and SSRI groups also had higher firing rates and a steeper acceleration in initial firing rates (secondary ranges), consistent with the PIC producing a steeper acceleration in membrane depolarization at the onset of motoneuron firing. In summary, both the young development and SSRI groups exhibited increased intrinsic motoneuron excitability compared to the adults, which, in the young development group, was also associated with a larger unsteadiness in the dorsiflexion torque profiles. We propose several intrinsic and extrinsic factors that affect both motoneuron PICs and cell discharge which vary during development, with a time course similar to the changes in motoneuron firing behaviour observed in the present study. KEY POINTS: Neurons in the spinal cord that activate muscles in the limbs (motoneurons) undergo increases in excitability shortly after birth to help animals stand and walk. We examined whether the excitability of human ankle flexor motoneurons also continues to change from child to adulthood by recording the activity of the muscle fibres they innervate. Motoneurons in children and adolescents aged 7-17years (young development group) had higher signatures of excitability that included faster firing rates and more self-sustained activity compared to adults aged ≥18years. Participants aged 11-28years of age taking serotonin reuptake inhibitors had the highest measures of motoneuron excitability compared to their age-matched controls. The young development group also had more unstable contractions, which might partly be related to the high excitability of the motoneurons.

Trimetazidine Improves Mitochondrial Dysfunction in SOD1G93A Cellular Models of Amyotrophic Lateral Sclerosis through Autophagy Activation.

Amyotrophic Lateral Sclerosis (ALS) is considered the prototype of motor neuron disease, characterized by motor neuron loss and muscle waste. A well-established pathogenic hallmark of ALS is mitochondrial failure, leading to bioenergetic deficits. So far, pharmacological interventions for the disease have proven ineffective. Trimetazidine (TMZ) is described as a metabolic modulator acting on different cellular pathways. Its efficacy in enhancing muscular and cardiovascular performance has been widely described, although its molecular target remains elusive. We addressed the molecular mechanisms underlying TMZ action on neuronal experimental paradigms. To this aim, we treated murine SOD1G93A-model-derived primary cultures of cortical and spinal enriched motor neurons, as well as a murine motor-neuron-like cell line overexpressing SOD1G93A, with TMZ. We first characterized the bioenergetic profile of the cell cultures, demonstrating significant mitochondrial dysfunction that is reversed by acute TMZ treatments. We then investigated the effect of TMZ in promoting autophagy processes and its impact on mitochondrial morphology. Finally, we demonstrated the effectiveness of TMZ in terms of the mitochondrial functionality of ALS-rpatient-derived peripheral blood mononuclear cells (PBMCs). In summary, our results emphasize the concept that targeting mitochondrial dysfunction may represent an effective therapeutic strategy for ALS. The findings demonstrate that TMZ enhances mitochondrial performance in motor neuron cells by activating autophagy processes, particularly mitophagy. Although further investigations are needed to elucidate the precise molecular pathways involved, these results hold critical implications for the development of more effective and specific derivatives of TMZ for ALS treatment.

HERV-K (HML-2) Envelope Protein Induces Mitochondrial Depolarization and Neurotoxicity via Endolysosome Iron Dyshomeostasis.

Human endogenous retroviruses (HERVs) are associated with the pathogenesis of amyotrophic lateral sclerosis (ALS); a disease characterized by motor neuron degeneration and cell death. The HERV-K subtype HML-2 envelope protein (HERV-K Env) is expressed in the brain, spinal cord, and cerebrospinal fluid of people living with ALS and through CD98 receptor-linked interactions causes neurodegeneration. HERV-K Env-induced increases in oxidative stress are implicated in the pathogenesis of ALS, and ferrous iron (Fe2+) generates reactive oxygen species (ROS). Endolysosome stores of Fe2+ are central to iron trafficking and endolysosome deacidification releases Fe2+ into the cytoplasm. Because HERV-K Env is an arginine-rich protein that is likely endocytosed and arginine is a pH-elevating amino acid, it is important to determine HERV-K Env effects on endolysosome pH and whether HERV-K Env-induced neurotoxicity is downstream of Fe2+ released from endolysosomes. Here, we showed using SH-SY5Y human neuroblastoma cells and primary cultures of human cortical neurons (HCNs, information on age and sex was not available) that HERV-K Env (1) is endocytosed via CD98 receptors, (2) concentration dependently deacidified endolysosomes, (3) decreased endolysosome Fe2+ concentrations, (4) increased cytosolic and mitochondrial Fe2+ and ROS levels, (5) depolarized mitochondrial membrane potential, and (6) induced cell death, effects blocked by an antibody against the CD98 receptor and by the endolysosome iron chelator deferoxamine. Thus, HERV-K Env-induced increases in cytosolic and mitochondrial Fe2+ and ROS as well as cell death appear to be mechanistically caused by HERV-K Env endocytosis, endolysosome deacidification, and endolysosome Fe2+ efflux into the cytoplasm.

Restoration of injured motoneurons reduces microglial proliferation in the adult rat facial nucleus.

In the axotomized facial nucleus (axotFN), the levels of choline acetyltransferase, vesicular acetylcholine transporter, and gamma amino butyric acid A receptor α1 are decreased, after which the microglia begin to proliferate around injured motoneuron cell bodies. We conjectured that an injury signal released from the injured motoneurons triggers the microglial proliferation in the axotFN. However, it is unclear whether the level of microglial proliferation is dependent on the degree of motoneuronal insult. In this study, we investigated the relationship between the extents of motoneuronal injury and microglial proliferation in a rat axotFN model. Administration of glial cell line-derived neurotrophic factor, N-acetyl L-cysteine, or salubrinal at the transection site ameliorated the increase in c-Jun and the reductions in levels of phosphorylated cAMP response element binding protein (p-CREB) and functional molecules in the injured motoneurons. Concurrently, the levels of the microglial marker ionized calcium-binding adapter molecule 1 and of macrophage colony-stimulating factor (cFms), proliferating cell nuclear antigen, and p-p38/p38 were significantly downregulated in microglia. These results demonstrate that the recovery of motoneuron function resulted in the reduction in microglial proliferation. We conclude that the degree of neuronal injury regulates the levels of microglial proliferation in the axotFN.

Functional consequences of familial ALS-associated SOD1L84F in neuronal and muscle cells.

Amyotrophic lateral sclerosis is a fatal neurodegenerative disorder characterized by progressive skeletal muscle denervation and loss of motor neurons that results in muscle atrophy and eventual death due to respiratory failure. Previously, we identified a novel SOD1L84F variation in a familial ALS case. In this study, we examined the functional consequences of SOD1L84F overexpression in the mouse motor neuron cell line (NSC-34). The cells expressing SOD1L84F showed increased oxidative stress and increased cell death. Interestingly, SOD1L84F destabilized the native dimer and formed high molecular weight SDS-resistant protein aggregates. Furthermore, SOD1L84F also decreased the percentage of differentiated cells and significantly reduced neurite length. A plethora of evidence suggested active involvement of skeletal muscle in disease initiation and progression. We observed differential processing of the mutant SOD1 and perturbations of cellular machinery in NSC-34 and muscle cell line C2C12. Unlike neuronal cells, mutant protein failed to accumulate in muscle cells probably due to the activated autophagy, as evidenced by increased LC3-II and reduced p62. Further, SOD1L84F altered mitochondrial dynamics only in NSC-34. In addition, microarray analysis also revealed huge variations in differentially expressed genes between NSC-34 and C2C12. Interestingly, SOD1L84F hampered the endogenous FUS autoregulatory mechanism in NSC-34 by downregulating retention of introns 6 and 7 resulting in a two-fold upregulation of FUS. No such changes were observed in C2C12. Our findings strongly suggest the differential processing and response towards the mutant SOD1 in neuronal and muscle cell lines.

Coffee and amyotrophic lateral sclerosis (ALS).

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder characterized by progressive loss of motor neurons. The effective treatments for ALS remain elusive, necessitating exploration into novel preventive strategies. ALS pathogenesis is triggered by oxidative stress which results in neuroinflammation, exicitotoxicity and neuronal cell death. Nutritional mechanism for halting progression of neurodegeneration is through dietary compounds with antioxidants, anti-inflammatory or neuromodulating activity. Coffee is a widely consumed beverage made up of polyphenols, caffeine and other compounds with possible antioxidants and neuro-protective roles. It is important to say that various epidemiological studies have documented association between coffee intake and ALS. This chapter is aimed to present a comprehensive review of existing literature on coffee consumption and ALS, involving epidemiological studies, preclinical research, and its mechanism of actions in animal model of ALS. It highlights key findings regarding the potential neuroprotective properties of coffee constituents such as caffeine, polyphenols, and other bioactive compounds. Furthermore, it discusses possible pathways through which coffee may modulate ALS pathogenesis, including suppressing oxidative stress and neuroinflammation while boosting adenosine function via the adenosine receptor two on the motor neuron cells membrane in the spinal cord to enhance motor function via the corticospinal tract. Overall, this chapter underscores the significance of further research to unravel the specific mechanisms by which coffee exerts its neuroprotective effects in ALS, with the ultimate goal of identifying dietary strategies for ALS prevention and management.

A comparison of confocal and epifluorescence microscopy for quantification of RNAScope and immunohistochemistry fluorescent images

BackgroundQuantification of RNA expression and protein production in fluorescent stainings provides critical information concerning neurodevelopment. A trustable independent quantification technique requires acquisition of reliable images prior to image processing. There is uncertainty in existing literature regarding the use of confocal microscopy compared to standard epifluorescence microscopy, especially in the context of RNA in situ hybridization protocols. New methodThe hindbrains of developing rat embryos from embryologic day 14 (E14) to E20 were sectioned and stained for expression of Hoxb1, Hoxb2, and Phox2b using both RNAScope and immunohistochemistry. Islet1 was used for identification of hindbrain motoneuron cell bodies. Slides were imaged using both confocal and epifluorescence microscopy. ResultsExpression patterns of both mRNA and protein were similar in both imaging modalities. Analyses of Hoxb1 and Hoxb2 mRNA expression were particularly concordant between-scopes, with similar p-values and posthoc differences between timepoints. Confocal imaging of Hoxb2 protein yielded a significant peak at E18, but this level of significance was not reached using epifluorescence microscopy. Although similar trends were observed, only Phox2b RNAScope results were statistically significant when analyzed with confocal microscopy. In contrast, Phox2b immunostaining analyses showed significant differences using both microscopes. Comparison with existing methodsResearchers may save time and financial resources if epifluorescence microscopy provides comparable or equal results as confocal. ConclusionsEpifluorescence microscopy appears sufficient for quantification of RNAScope experiments with relatively low puncta per cell, while confocal microscopy gives clearer definition to immunohistochemical protein relationships and may be preferable especially in targets with low protein production.

LRP4-related signalling pathways and their regulatory role in neurological diseases

The mechanism of action of low-density lipoprotein receptor related protein 4 (LRP4) is mediated largely via the Agrin-LRP4-MuSK signalling pathway in the nervous system. LRP4 contributes to the development of synapses in the peripheral nervous system (PNS). It interacts with signalling molecules such as the amyloid beta-protein precursor (APP) and the wingless type protein (Wnt). Its mechanisms of action are complex and mediated via interaction between the pre-synaptic motor neuron and post-synaptic muscle cell in the PNS, which enhances the development of the neuromuscular junction (NMJ). LRP4 may function differently in the central nervous system (CNS) than in the PNS, where it regulates ATP and glutamate release via astrocytes. It mayaffect the growth and development of the CNS by controlling the energy metabolism. LRP4 interacts with Agrin to maintain dendrite growth and density in the CNS. The goal of this article is to review the current studies involving relevant LRP4 signaling pathways in the nervous system. The review also discusses the clinical and etiological roles of LRP4 in neurological illnesses, such as myasthenia gravis, Alzheimer's disease and epilepsy. In this review, we provide a theoretical foundation for the pathogenesis and therapeutic application of LRP4 in neurologic diseases.

Human motor neurons derived from induced pluripotent stem cells are susceptible to SARS-CoV-2 infection.

COVID-19 typically causes Q7 respiratory disorders, but a high proportion of patients also reports neurological and neuromuscular symptoms during and after SARSCoV-2 infection. Despite a number of studies documenting SARS-CoV-2 infection of various neuronal cell populations, the impact of SARS-CoV-2 exposure on motor neuronal cells specifically has not been investigated so far. Thus, by using human iPSC-derived motor neurons (iPSC-MNs) we assessed: (i) the expression of SARS-CoV-2 main receptors; (ii) iPSC-MN infectability by SARS-CoV-2; and (iii) the effect of SARS-CoV-2 exposure on iPSC-MN transcriptome. Gene expression profiling and immunofluorescence (IF) analysis of the main host cell receptors recognized by SARS-CoV-2 revealed that all of them are expressed in iPSC-MNs, with CD147 and NRP1 being the most represented ones. By analyzing SARS-CoV-2 N1 and N2 gene expression over time, we observed that human iPSC-MNs were productively infected by SARS-CoV-2 in the absence of cytopathic effect. Supernatants collected from SARS-CoV-2-infected iPSC-MNs were able to re-infect VeroE6 cells. Image analyses of SARS-CoV-2 nucleocapsid proteins by IF confirmed iPSC-MN infectability. Furthermore, SARS-CoV-2 infection in iPSCMNs significantly altered the expression of genes (IL-6, ANG, S1PR1, BCL2, BAX, Casp8, HLA-A, ERAP1, CD147, MX1) associated with cell survival and metabolism, as well as antiviral and inflammatory response. These results suggest for the very first time that SARS-CoV-2 can productively infect human iPSC-derived MNs probably by binding CD147 and NRP1 receptors. Such information will be important to unveil the biological bases of neuromuscular disorders characterizing SARS-CoV-2 infection and the so called long-COVID symptoms.

Pectolinarigenin Improves Oxidative Stress and Apoptosis in Mouse NSC-34 Motor Neuron Cell Lines Induced by C9-ALS-Associated Proline-Arginine Dipeptide Repeat Proteins by Enhancing Mitochondrial Fusion Mediated via the SIRT3/OPA1 Axis.

Amyotrophic lateral sclerosis (ALS) is considered a fatal progressive degeneration of motor neurons (MN) caused by oxidative stress and mitochondrial dysfunction. There are currently no treatments available. The most common inherited form of ALS is the C9orf72 mutation (C9-ALS). The proline-arginine dipeptide repeat protein (PR-DPR) produced by C9-ALS has been confirmed to be a functionally acquired pathogenic factor that can cause increased ROS, mitochondrial defects, and apoptosis in motor neurons. Pectolinarigenin (PLG) from the traditional medicinal herb Linaria vulgaris has antioxidant and anti-apoptotic properties. I established a mouse NSC-34 motor neuron cell line model expressing PR-DPR and confirmed the neuroprotective effect of PLG. The results showed that ROS production and apoptosis caused by PR-DPR could be improved by PLG treatment. In terms of mechanism research, PR-DPR inhibited the activity of the mitochondrial fusion proteins OPA1 and mitofusin 2. Conversely, the expression of fission protein fission 1 and dynamin-related protein 1 (DRP1) increased. However, PLG treatment reversed these effects. Furthermore, I found that PLG increased the expression and deacetylation of OPA1. Deacetylation of OPA1 enhances mitochondrial fusion and resistance to apoptosis. Finally, transfection with Sirt3 small interfering RNA abolished the neuroprotective effects of PLG. In summary, the mechanism by which PLG alleviates PR-DPR toxicity is mainly achieved by activating the SIRT3/OPA1 axis to regulate the balance of mitochondrial dynamics. Taken together, the potential of PLG in preclinical studies for C9-ALS drug development deserves further evaluation.

Spinal Cord Injury Model Mitochondria Connect Altered Function with Defects of Mitochondrion Morphology: an Ultrastructural Study.

The key role of mitochondria in neurodegenerative disease patients is well documented. Recent studies claimed that mitochondrial regulatory dysfunction might play a role in ongoing cell death and dysfunction. In the present study, we characterized ultrastructural morphometry of mitochondrial alterations occurring at the level of motor neuron cell bodies in SCI-induced rats. We applied 17β-estradiol (E2) to determine whether it can improve mitochondria structural integrity of motor neurons. We used a rat model of acute SCI generated by spinal cord contusion at the T9-T10 level, followed by tissue processing 21days post-SCI. Samples were dividedintofivegroups: laminectomy, SCI, vehicle, SCI + 25µg/kg E2, and SCI + 10µg/kg E2. Assessments included analysis of hind limb motor recovery, quantifying tissue repair, and evaluation of morphological changes in the ultrastructure of mitochondria in motor neurons by transmission electron microscopy. In the E2-treated groups, especially the group receiving 25µg/kg E2, less irregular mitochondria were observed, as there was a significant reduction in swelling or vacuolization, or fragmentation compared to the SCI group. Furthermore, E2 significantly reduced membrane rupture in the SCI group. E2 could be a proper therapeutic agent to relieve mitochondrial deleterious effects on neurons in neurodegenerative diseases.

The Neuroprotective Role of TERT Influences the Expression of SOD1 in Motor Neurons and Mouse Brain: Implications for fALS

Amyotrophic lateral sclerosis (ALS) disease is characterized by degeneration of motor neurons and elevation of brain oxidative stress. Previous studies demonstrated the neuroprotective effects of Telomerase reverse transcriptase (TERT) from oxidative stress. We showed that increasing TERT expression in the brain of the Tg hSOD1G93A mouse ALS model attenuated the disease pathology and increased the survival of motor neurons exposed to oxidative stress. How TERT increased the survival of motor neurons exposed to oxidative stress is not yet clear. Here we investigated the consequence of TERT depletion in motor neuron cells under normal and oxidative stress conditions and in mouse brains of TERT knockout mice, on the expression and activity of SOD1 and catalase enzymes. Depletion of mouse TERT caused mitochondrial dysfunction and impaired catalase and SOD1 activity. Compensation with hTERT restored the activity of SOD1. SOD1 expression increased in the brain of TERT KO and in ALS mice and decreased in the brain of WT mice treated with telomerase-increasing compounds. We suggest that the ability of TERT to protect neurons from oxidative stress affects the expression and activity of SOD1, in a TERT-dependent manner, and supports the notion of TERT as a therapeutic target for neurodegenerative diseases like ALS.

Glycosphingolipids are linked to elevated neurotransmission and neurodegeneration in a Drosophila model of Niemann Pick type C.

The lipid storage disease Niemann Pick type C (NPC) causes neurodegeneration owing primarily to loss of NPC1. Here, we employed a Drosophila model to test links between glycosphingolipids, neurotransmission and neurodegeneration. We found that Npc1a nulls had elevated neurotransmission at the glutamatergic neuromuscular junction (NMJ), which was phenocopied in brainiac (brn) mutants, impairing mannosyl glucosylceramide (MacCer) glycosylation. Npc1a; brn double mutants had the same elevated synaptic transmission, suggesting that Npc1a and brn function within the same pathway. Glucosylceramide (GlcCer) synthase inhibition with miglustat prevented elevated neurotransmission in Npc1a and brn mutants, further suggesting epistasis. Synaptic MacCer did not accumulate in the NPC model, but GlcCer levels were increased, suggesting that GlcCer is responsible for the elevated synaptic transmission. Null Npc1a mutants had heightened neurodegeneration, but no significant motor neuron or glial cell death, indicating that dying cells are interneurons and that elevated neurotransmission precedes neurodegeneration. Glycosphingolipid synthesis mutants also had greatly heightened neurodegeneration, with similar neurodegeneration in Npc1a; brn double mutants, again suggesting that Npc1a and brn function in the same pathway. These findings indicate causal links between glycosphingolipid-dependent neurotransmission and neurodegeneration in this NPC disease model.

Transcriptomic Profiling after In Vitro Δ8-THC Exposure Shows Cytoskeletal Remodeling in Trauma-Injured NSC-34 Cell Line.

Neuronal cell death is a physiological process that, when uncontrollable, leads to neurodegenerative disorders like spinal cord injury (SCI). SCI represents one of the major causes of trauma and disabilities worldwide for which no effective pharmacological intervention exists. Herein, we observed the beneficial effects of Δ8-Tetrahydrocannabinol (Δ8-THC) during neuronal cell death recovery. We cultured NSC-34 motoneuron cell line performing three different experiments. A traumatic scratch injury was caused in two experiments. One of the scratched was pretreated with Δ8-THC to observe the role of the cannabinoid following the trauma. An experimental control group was neither scratched nor pretreated. All the experiments underwent RNA-seq analysis. The effects of traumatic injury were observed in scratch against control comparison. Comparison of scratch models with or without pretreatment highlighted how Δ8-THC counteracts the traumatic event. Our results shown that Δ8-THC triggers the cytoskeletal remodeling probably due to the activation of the Janus Kinase Signal Transducer and Activator of Transcription (JAK/STAT) signaling pathway and the signaling cascade operated by the Mitogen-Activated Protein (MAP) Kinase signaling pathway. In light of this evidence, Δ8-THC could be a valid pharmacological approach in the treatment of abnormal neuronal cell death occurring in motoneuron cells.