Integrating hydrogels manipulate ECM deposition after spinal cord injury for specific neural reconnections via neuronal relays.

A bioinspired hydrogel composed of hyaluronic acid-graft-dopamine (HADA) and a designer peptide HGF-(RADA)4-DGDRGDS (HRR) was presented to enhance tissue integration following spinal cord injury (SCI). The HADA/HRR hydrogel manipulated the infiltration of PDGFRß+ cells in a parallel pattern, transforming dense scars into an aligned fibrous substrate that guided axonal regrowth. Further incorporation of NT3 and curcumin promoted axonal regrowth and survival of interneurons at lesion borders, which served as relays for establishing heterogeneous axon connections in a target-specific manner. Notable improvements in motor, sensory, and bladder functions resulted in rats with complete spinal cord transection. The HADA/HRR + NT3/Cur hydrogel promoted V2a neuron accumulation in ventral spinal cord, facilitating the recovery of locomotor function. Meanwhile, the establishment of heterogeneous neural connections across the hemisected lesion of canines was documented in a target-specific manner via neuronal relays, significantly improving motor functions. Therefore, biomaterials can inspire beneficial biological activities for SCI repair.

Sex differences in PD-L1-induced analgesia in paclitaxel-induced peripheral neuropathy mice depend on TRPV1-based inhibition of CGRP.

AIMS: Paclitaxel (PTX) is extensively utilized in the management of diverse solid tumors, frequently resulting in paclitaxel-induced peripheral neuropathy (PIPN). The present study aimed to investigate sex differences in the behavioral manifestations and underlying pathogenesis of PIPN and search for clinically efficacious interventions. METHODS: Male and female C57BL/6 mice (5-6 weeks and 12 months, weighing 18-30 g) were intraperitoneally (i.p.) administered paclitaxel diluted in saline (NaCl 0.9%) at a dose of 2 mg/kg every other day for a total of 4 injections. Von Frey and hot plate tests were performed before and after administration to confirm the successful establishment of the PIPN model and also to evaluate the pain of PIPN and the analgesic effect of PD-L1. On day 14 after PTX administration, PD-L1 protein (10 ng/pc) was injected into the PIPN via the intrathecal (i.t.) route. To knock down TRPV1 in the spinal cord, adeno-associated virus 9 (AAV9)-Trpv1-RNAi (5 µL, 1 × 1013 vg/mL) was slowly injected via the i.t. route. Four weeks after AAV9 delivery, the downregulation of TRPV1 expression was verified by immunofluorescence staining and Western blotting. The levels of PD-L1, TRPV1 and CGRP were measured via Western blotting, RT-PCR, and immunofluorescence staining. The levels of TNF-α and IL-1ß were measured via RT-PCR. RESULTS: TRPV1 and CGRP protein and mRNA levels were higher in the spinal cords of control female mice than in those of control male mice. PTX-induced nociceptive behaviors in female PIPN mice were greater than those in male PIPN mice, as indicated by increased expression of TRPV1 and CGRP. The analgesic effects of PD-L1 on mechanical hyperalgesia and thermal sensitivity were significantly greater in female mice than in male mice, with calculated relative therapeutic levels increasing by approximately 2.717-fold and 2.303-fold, respectively. PD-L1 and CGRP were partly co-localized with TRPV1 in the dorsal horn of the mouse spinal cord. The analgesic effect of PD-L1 in PIPN mice was observed to be mediated through the downregulation of TRPV1 and CGRP expression following AAV9-mediated spinal cord specific decreased TRPV1 expression. CONCLUSIONS: PTX-induced nociceptive behaviors and the analgesic effect of PD-L1 in PIPN mice were sexually dimorphic, highlighting the significance of incorporating sex as a crucial biological factor in forthcoming mechanistic studies of PIPN and providing insights for potential sex-specific therapeutic approaches.

Asymptomatic herpes simplex virus brain infection elicits cellular senescence phenotypes in the central nervous system of mice suffering multiple sclerosis-like disease.

Experimental autoimmune encephalomyelitis (EAE) is a demyelinating disease affecting the central nervous system (CNS) in animals that parallels several clinical and molecular traits of multiple sclerosis in humans. Herpes simplex virus type 1 (HSV-1) infection mainly causes cold sores and eye diseases, yet eventually, it can also reach the CNS, leading to acute encephalitis. Notably, a significant proportion of healthy individuals are likely to have asymptomatic HSV-1 brain infection with chronic brain inflammation due to persistent latent infection in neurons. Because cellular senescence is suggested as a potential factor contributing to the development of various neurodegenerative disorders, including multiple sclerosis, and viral infections may induce a premature senescence state in the CNS, potentially increasing susceptibility to such disorders, here we examine the presence of senescence-related markers in the brains and spinal cords of mice with asymptomatic HSV-1 brain infection, EAE, and both conditions. Across all scenarios, we find a significant increases of senescence biomarkers in the CNS with some differences depending on the analyzed group. Notably, some senescence biomarkers are exclusively observed in mice with the combined conditions. These results indicate that asymptomatic HSV-1 brain infection and EAE associate with a significant expression of senescence biomarkers in the CNS.

IL-6 Inhibition as a Therapeutic Target in Aged Experimental Autoimmune Encephalomyelitis.

Multiple sclerosis (MS) onset at an advanced age is associated with a higher risk of developing progressive forms and a greater accumulation of disability for which there are currently no effective disease-modifying treatments. Immunosenescence is associated with the production of the senescence-associated secretory phenotype (SASP), with IL-6 being one of the most prominent cytokines. IL-6 is a determinant for the development of autoimmunity and neuroinflammation and is involved in the pathogenesis of MS. Herein, we aimed to preclinically test the therapeutic inhibition of IL-6 signaling in experimental autoimmune encephalomyelitis (EAE) as a potential age-specific treatment for elderly MS patients. Young and aged mice were immunized with myelin oligodendrocyte protein (MOG)35-55 and examined daily for neurological signs. Mice were randomized and treated with anti-IL-6 antibody. Inflammatory infiltration was evaluated in the spinal cord and the peripheral immune response was studied. The blockade of IL-6 signaling did not improve the clinical course of EAE in an aging context. However, IL-6 inhibition was associated with an increase in the peripheral immunosuppressive response as follows: a higher frequency of CD4 T cells producing IL-10, and increased frequency of inhibitory immune check points PD-1 and Tim-3 on CD4+ T cells and Lag-3 and Tim-3 on CD8+ T cells. Our results open the window to further studies aimed to adjust the anti-IL-6 treatment conditions to tailor an effective age-specific therapy for elderly MS patients.

CBP Expression Contributes to Neuropathic Pain via CREB and MeCP2 Regulation in the Spared Nerve Injury Rat Model.

Background and Objectives: This study aimed to investigate the relationship between neuropathic pain and CREB-binding protein (CBP) and methyl-CpG-binding protein 2 (MeCP2) expression levels in a rat model with spared nerve injury (SNI). Materials and Methods: Rat (male Sprague-Dawley white rats) models with surgical SNI (n = 6) were prepared, and naive rats (n = 5) were used as controls. The expression levels of CBP and MeCP2 in the spinal cord and dorsal root ganglion (DRG) were compared through immunohistochemistry at 7 and 14 days after surgery. The relationship between neuropathic pain and CBP/MeCP2 was also analyzed through intrathecal siRNA administration. Results: SNI induced a significant increase in the number of CBPs in L4 compared with contralateral DRG as well as with naive rats. The number of MeCP2 cells in the dorsal horn on the ipsilateral side decreased significantly compared with the contralateral dorsal horn and the control group. SNI induced a significant decrease in the number of MeCP2 neurons in the L4 ipsilateral DRG compared with the contralateral DRG and naive rats. The intrathecal injection of CBP siRNA significantly inhibited mechanical allodynia induced by SNI compared with non-targeting siRNA treatment. MeCP2 siRNA injection showed no significant effect on mechanical allodynia. Conclusions: The results suggest that CBP and MeCP2 may play an important role in the generation of neuropathic pain following peripheral nerve injury.

Inhibiting spinal cord-specific hsp90 isoforms reveals a novel strategy to improve the therapeutic index of opioid treatment.

Opioids are the gold standard for the treatment of chronic pain but are limited by adverse side effects. In our earlier work, we showed that Heat shock protein 90 (Hsp90) has a crucial role in regulating opioid signaling in spinal cord; Hsp90 inhibition in spinal cord enhances opioid anti-nociception. Building on these findings, we injected the non-selective Hsp90 inhibitor KU-32 by the intrathecal route into male and female CD-1 mice, showing that morphine anti-nociceptive potency was boosted by 1.9-3.5-fold in acute and chronic pain models. At the same time, tolerance was reduced from 21-fold to 2.9 fold and established tolerance was rescued, while the potency of constipation and reward was unchanged. These results demonstrate that spinal Hsp90 inhibition can improve the therapeutic index of morphine. However, we also found that systemic non-selective Hsp90 inhibition blocked opioid pain relief. To avoid this effect, we used selective small molecule inhibitors and CRISPR gene editing to identify 3 Hsp90 isoforms active in spinal cord (Hsp90α, Hsp90ß, and Grp94) while only Hsp90α was active in brain. We thus hypothesized that a systemically delivered selective inhibitor to Hsp90ß or Grp94 could selectively inhibit spinal cord Hsp90 activity, resulting in enhanced opioid therapy. We tested this hypothesis using intravenous delivery of KUNB106 (Hsp90ß) and KUNG65 (Grp94), showing that both drugs enhanced morphine anti-nociceptive potency while rescuing tolerance. Together, these results suggest that selective inhibition of spinal cord Hsp90 isoforms is a novel, translationally feasible strategy to improve the therapeutic index of opioids.

Hydrogen Sulfide can Scavenge Free Radicals to Improve Spinal Cord Injury by Inhibiting the p38MAPK/mTOR/NF-κB Signaling Pathway.

Spinal cord injury (SCI) causes irreversible cell loss and neurological dysfunctions. Presently, there is no an effective clinical treatment for SCI. It can be the only intervention measure by relieving the symptoms of patients such as pain and fever. Free radical-induced damage is one of the validated mechanisms in the complex secondary injury following primary SCI. Hydrogen sulfide (H2S) as an antioxidant can effectively scavenge free radicals, protect neurons, and improve SCI by inhibiting the p38MAPK/mTOR/NF-κB signaling pathway. In this report, we analyze the pathological mechanism of SCI, the role of free radical-mediated the p38MAPK/mTOR/NF-κB signaling pathway in SCI, and the role of H2S in scavenging free radicals and improving SCI.

Astrocytic stress response is induced by exposure to astrocyte-binding antibodies expressed by plasmablasts from pediatric patients with acute transverse myelitis.

BACKGROUND: Pediatric acute transverse myelitis (ATM) accounts for 20-30% of children presenting with a first acquired demyelinating syndrome (ADS) and may be the first clinical presentation of a relapsing ADS such as multiple sclerosis (MS). B cells have been strongly implicated in the pathogenesis of adult MS. However, little is known about B cells in pediatric MS, and even less so in pediatric ATM. Our lab previously showed that plasmablasts (PB), the earliest B cell subtype producing antibody, are expanded in adult ATM, and that these PBs produce self-reactive antibodies that target neurons. The goal of this study was to examine PB frequency and phenotype, immunoglobulin selection, and B cell receptor reactivity in pediatric patients presenting with ATM to gain insight to B cell involvement in disease. METHODS: We compared the PB frequency and phenotype of 5 pediatric ATM patients and 10 pediatric healthy controls (HC) and compared them to previously reported adult ATM patients using cytometric data. We purified bulk IgG from the plasma samples and cloned 20 recombinant human antibodies (rhAbs) from individual PBs isolated from the blood. Plasma-derived IgG and rhAb autoreactivity was measured by mean fluorescence intensity (MFI) in neurons and astrocytes of murine brain or spinal cord and primary human astrocytes. We determined the potential impact of these rhAbs on astrocyte health by measuring stress and apoptotic response. RESULTS: We found that pediatric ATM patients had a reduced frequency of peripheral blood PB. Serum IgG autoreactivity to neurons in EAE spinal cord was similar in the pediatric ATM patients and HC. However, serum IgG autoreactivity to astrocytes in EAE spinal cord was reduced in pediatric ATM patients compared to pediatric HC. Astrocyte-binding strength of rhAbs cloned from PBs was dependent on somatic hypermutation accumulation in the pediatric ATM cohort, but not HC. A similar observation in predilection for astrocyte binding over neuron binding of individual antibodies cloned from PBs was made in EAE brain tissue. Finally, exposure of human primary astrocytes to these astrocyte-binding antibodies increased astrocytic stress but did not lead to apoptosis. CONCLUSIONS: Discordance in humoral immune responses to astrocytes may distinguish pediatric ATM from HC.

Arctigenin derivative A-1 ameliorates motor dysfunction and pathological manifestations in SOD1G93A transgenic mice via the AMPK/SIRT1/PGC-1α and AMPK/SIRT1/IL-1ß/NF-κB pathways.

AIM: Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative disease characterized by progressive death of upper and lower motor neurons, leading to generalized muscle atrophy, paralysis, and even death. Mitochondrial damage and neuroinflammation play key roles in the pathogenesis of ALS. In the present study, the efficacy of A-1, a derivative of arctigenin with AMP-activated protein kinase (AMPK) and silent information regulator 1 (SIRT1) activation for ALS, was investigated. METHODS: A-1 at 33.3 mg/kg was administrated in SOD1G93A transgenic mice orally from the 13th week for a 6-week treatment period. Motor ability was assessed before terminal anesthesia. Muscle atrophy and fibrosis, motor neurons, astrocytes, and microglia in the spinal cord were evaluated by H&E, Masson, Sirius Red, Nissl, and immunohistochemistry staining. Protein expression was detected with proteomics analysis, Western blotting, and ELISA. Mitochondrial adenosine triphosphate (ATP) and malondialdehyde (MDA) levels were measured using an assay kit. RESULTS: A-1 administration in SOD1G93A mice enhanced mobility, decreased skeletal muscle atrophy and fibrosis, mitigated loss of spinal motor neurons, and reduced glial activation. Additionally, A-1 treatment improved mitochondrial function, evidenced by elevated ATP levels and increased expression of key mitochondrial-related proteins. The A-1 treatment group showed decreased levels of IL-1ß, pIκBα/IκBα, and pNF-κB/NF-κB. CONCLUSIONS: A-1 treatment reduced motor neuron loss, improved gastrocnemius atrophy, and delayed ALS progression through the AMPK/SIRT1/PGC-1α pathway, which promotes mitochondrial biogenesis. Furthermore, the AMPK/SIRT1/IL-1ß/NF-κB pathway exerted neuroprotective effects by reducing neuroinflammation. These findings suggest A-1 as a promising therapeutic approach for ALS.

Topography of the GLP-1/GLP-1 receptor system in the spinal cord of male mice.

Glucagon-like peptide-1 receptor (GLP-1R) agonists are now commonly used to treat type 2 diabetes and obesity. GLP-1R signaling in the spinal cord has been suggested to account for the mild tachycardia caused by GLP-1R agonists, and may also be involved in the therapeutic effects of these drugs. However, the neuroanatomy of the GLP-1/GLP-1R system in the spinal cord is still poorly understood. Here we applied in situ hybridization and immunohistochemistry to characterize this system, and its relation to cholinergic neurons. GLP-1R transcript and protein were expressed in neuronal cell bodies across the gray matter, in matching distribution patterns. GLP-1R-immunolabeling was also robust in dendrites and axons, especially in laminae II-III in the dorsal horn. Cerebrospinal fluid-contacting neurons expressed GLP-1R protein at exceedingly high levels. Only small subpopulations of cholinergic neurons expressed GLP-1R, including a subset of sympathetic preganglionic neurons at the rostral tip of the intermediolateral nucleus. GLP-1 axons innervated all regions where GLP-1R neurons were distributed, except laminae II-III. Scattered preproglucagon (Gcg) mRNA-expressing neurons were identified in the cervical and lumbar enlargements. The results will facilitate further studies on how GLP-1 regulates the sympathetic system and other autonomic and somatic functions via the spinal cord.

Astaxanthin alleviates fibromyalgia pain and depression via NLRP3 inflammasome inhibition.

Fibromyalgia is characterised by widespread chronic pain and is often accompanied by comorbidities such as sleep disorders, anxiety, and depression. Because it is often accompanied by many adverse symptoms and lack of effective treatment, it is important to search for the pathogenesis and treatment of fibromyalgia. Astaxanthin, a carotenoid pigment known for its anti-inflammatory and antioxidant properties, has demonstrated effective analgesic effects in neuropathic pain. However, its impact on fibromyalgia remains unclear. Therefore, in this study, we constructed a mouse model of fibromyalgia and investigated the effect of astaxanthin on chronic pain and associated symptoms through multiple intragastrical injections. We conducted behavioural assessments to detect pain and depression-like states in mice, recorded electroencephalograms to monitor sleep stages, examined c-Fos activation in the anterior cingulate cortex, measured activation of spinal glial cells, and assessed levels of inflammatory factors in the brain and spinal cord, including interleukin (IL)-1ß, IL-6, and tumour necrosis factor- α(TNF-α).Additionally, we analysed the expression levels of IL-6, IL-10, NOD-like receptor thermal protein domain associated protein 3 (NLRP3), Apoptosis-associated speck-like protein containing CARD, and Caspase-1 proteins. The findings revealed that astaxanthin significantly ameliorated mechanical and thermal pain in mice with fibromyalgia and mitigated sleep disorders and depressive-like symptoms induced by pain. A potential mechanism underlying these effects is the anti-inflammatory action of astaxanthin, likely mediated through the inhibition of the NLRP3 inflammasome, which could be one of the pathways through which astaxanthin alleviates fibromyalgia. In conclusion, our study suggests that astaxanthin holds promise as a potential analgesic medication for managing fibromyalgia and its associated symptoms.

Longitudinal Neuropathological Consequences of Extracranial Radiation Therapy in Mice.

Cancer-related cognitive impairment (CRCI) is a consequence of chemotherapy and extracranial radiation therapy (ECRT). Our prior work demonstrated gliosis in the brain following ECRT in SKH1 mice. The signals that induce gliosis were unclear. Right hindlimb skin from SKH1 mice was treated with 20 Gy or 30 Gy to induce subclinical or clinical dermatitis, respectively. Mice were euthanized at 6 h, 24 h, 5 days, 12 days, and 25 days post irradiation, and the brain, thoracic spinal cord, and skin were collected. The brains were harvested for spatial proteomics, immunohistochemistry, Nanostring nCounter® glial profiling, and neuroinflammation gene panels. The thoracic spinal cords were evaluated by immunohistochemistry. Radiation injury to the skin was evaluated by histology. The genes associated with neurotransmission, glial cell activation, innate immune signaling, cell signal transduction, and cancer were differentially expressed in the brains from mice treated with ECRT compared to the controls. Dose-dependent increases in neuroinflammatory-associated and neurodegenerative-disease-associated proteins were measured in the brains from ECRT-treated mice. Histologic changes in the ECRT-treated mice included acute dermatitis within the irradiated skin of the hindlimb and astrocyte activation within the thoracic spinal cord. Collectively, these findings highlight indirect neuronal transmission and glial cell activation in the pathogenesis of ECRT-related CRCI, providing possible signaling pathways for mitigation strategies.

Seeding activity of human superoxide dismutase 1 aggregates in familial and sporadic amyotrophic lateral sclerosis postmortem neural tissues by real-time quaking-induced conversion.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive neurodegenerative disease with average lifespan of 2-5 years after diagnosis. The identification of novel prognostic and pharmacodynamic biomarkers are needed to facilitate therapeutic development. Metalloprotein human superoxide dismutase 1 (SOD1) is known to accumulate and form aggregates in patient neural tissue with familial ALS linked to mutations in their SOD1 gene. Aggregates of SOD1 have also been detected in other forms of ALS, including the sporadic form and the most common familial form linked to abnormal hexanucleotide repeat expansions in the Chromosome 9 open reading frame 72 (C9ORF72) gene. Here, we report the development of a real-time quaking-induced conversion (RT-QuIC) seed amplification assay using a recombinant human SOD1 substrate to measure SOD1 seeding activity in postmortem spinal cord and motor cortex tissue from persons with different ALS etiologies. Our SOD1 RT-QuIC assay detected SOD1 seeds in motor cortex and spinal cord dilutions down to 10-5. Importantly, we detected SOD1 seeding activity in specimens from both sporadic and familial ALS cases, with the latter having mutations in either their SOD1 or C9ORF72 genes. Analyses of RT-QuIC parameters indicated similar lag phases in spinal cords of sporadic and familial ALS patients, but higher ThT fluorescence maxima by SOD1 familial ALS specimens and sporadic ALS thoracic cord specimens. For a subset of sporadic ALS patients, motor cortex and spinal cords were examined, with seeding activity in both anatomical regions. Our results suggest SOD1 seeds are in ALS patient neural tissues not linked to SOD1 mutation, suggesting that SOD1 seeding activity may be a promising biomarker, particularly in sporadic ALS cases for whom genetic testing is uninformative.

A Switch from Glial to Neuronal Gene Expression Alterations in the Spinal Cord of SIV-infected Macaques on Antiretroviral Therapy.

Despite antiretroviral therapy (ART), HIV-associated peripheral neuropathy remains one of the most prevalent neurologic manifestations of HIV infection. The spinal cord is an essential component of sensory pathways, but spinal cord sampling and evaluation in people with HIV has been very limited, especially in those on ART. The SIV/macaque model allows for assessment of the spinal cord at key time points throughout infection with and without ART. In this study, RNA was isolated from the spinal cord of uninfected, SIV+, and SIV + ART animals to track alterations in gene expression using global RNA-seq. Next, the SeqSeek platform was used to map changes in gene expression to specific cell types. Pathway analysis of differentially expressed genes demonstrated that highly upregulated genes in SIV-infected spinal cord aligned with interferon and viral response pathways. Additionally, this upregulated gene set significantly overlapped with those expressed in myeloid-derived cells including microglia. Downregulated genes were involved in cholesterol and collagen biosynthesis, and TGF-b regulation of extracellular matrix. In contrast, enriched pathways identified in SIV + ART animals included neurotransmitter receptors and post synaptic signaling regulators, and transmission across chemical synapses. SeqSeek analysis showed that upregulated genes were primarily expressed by neurons rather than glia. These findings indicate that pathways activated in the spinal cord of SIV + ART macaques are predominantly involved in neuronal signaling rather than proinflammatory pathways. This study provides the basis for further evaluation of mechanisms of SIV infection + ART within the spinal cord with a focus on therapeutic interventions to maintain synaptodendritic homeostasis.

Probiotics and microbial metabolites maintain barrier and neuromuscular functions and clean protein aggregation to delay disease progression in TDP43 mutation mice.

Amyotrophic lateral sclerosis (ALS) is a neuromuscular disease. The ALS mice expressing human mutant of transactive response DNA binding protein of 43 kDa (hmTDP43) showed intestinal dysfunction before neuromuscular symptoms. We hypothesize that restoring the intestinal and microbial homeostasis with a bacterial metabolite or probiotics delays the ALS disease onset. We investigate the pathophysiological changes in the intestine and neurons, intestinal and blood-brain barriers, and inflammation during the ALS progression. We then cultured enteric glial cells (EGCs) isolated from TDP43 mice for mechanistic studies. TDP43 mice had significantly decreased intestinal mobility, increased permeability, and weakened muscle, compared with the age-matched wild-type mice. We observed increased hmTDP43 and Glial fibrillary acidic protein (GFAP), and decreased expression of α-smooth muscle actin (α-SMA), tight junction proteins (ZO-1 and Claudin-5) in the colon, spinal cord, and brain in TDP43 mice. TDP43 mice had reduced Butyryl-coenzyme A CoA transferase, decreased butyrate-producing bacteria Butyrivibrio fibrisolvens, and increased Bacteroides fragilis, compared to the WT mice. Serum inflammation cytokines (IL-6, IL-17, and IFN-γ) and LPS were elevated in TDP43 mice. EGCs from TDP43 mice showed aggregation of hmTDP43 associated with increased GFAP and ionized calcium-binding adaptor molecule (IBA1, a microglia marker). TDP43 mice treated with butyrate or probiotic VSL#3 had significantly increased rotarod time, increased intestinal mobility and decreased permeability, compared to the untreated group. Butyrate or probiotics treatment decreased the expression of GFAP, TDP43, and increased α-SMA, ZO-1, and Claudin-5 in the colon, spinal cord, and brain. Also, butyrate or probiotics treatment enhanced the Butyryl-coenzyme A CoA transferase, Butyrivibrio fibrisolvens, and reduced inflammatory cytokines in TDP43 mice. The TDP43 EGCs treated with butyrate or probiotics showed reduced GFAP, IBA1, and TDP43 aggregation. Restoring the intestinal and microbial homeostasis by beneficial bacteria and metabolites provide a potential therapeutic strategy to treat ALS.

Characterization of spinal cord tissue-derived extracellular vesicles in neuroinflammation.

Extracellular vesicles (EVs) are released by all cells, can cross the blood-brain barrier, and have been shown to play an important role in cellular communication, substance shuttling, and immune modulation. In recent years EVs have shifted into focus in multiple sclerosis (MS) research as potential plasma biomarkers and therapeutic vehicles. Yet little is known about the disease-associated changes in EVs in the central nervous system (CNS). To address this gap, we characterized the physical and proteomic changes of mouse spinal cord-derived EVs before and at 16 and 25 days after the induction of experimental autoimmune encephalomyelitis (EAE), a neuroinflammatory model of MS. Using various bioinformatic tools, we found changes in inflammatory, glial, and synaptic proteins and pathways, as well as a shift in the predicted contribution of immune and glial cell types over time. These results show that EVs provide snapshots of crucial disease processes such as CNS-compartmentalized inflammation, re/de-myelination, and synaptic pathology, and might also mediate these processes. Additionally, inflammatory plasma EV biomarkers previously identified in people with MS were also altered in EAE spinal cord EVs, suggesting commonalities of EV-related pathological processes during EAE and MS and overlap of EV proteomic changes between CNS and circulating EVs.

SIRT1 mediates the excitability of spinal CaMKIIα-positive neurons and participates in neuropathic pain by controlling Nav1.3.

AIMS: Neuropathic pain is a common chronic pain disorder, which is largely attributed to spinal central sensitization. Calcium/calmodulin-dependent protein kinase II alpha (CaMKIIα) activation in the spinal dorsal horn (SDH) is a major contributor to spinal sensitization. However, the exact way that CaMKIIα-positive (CaMKIIα+) neurons in the SDH induce neuropathic pain is still unclear. This study aimed to explore the role of spinal CaMKIIα+ neurons in neuropathic pain caused by chronic constriction injury (CCI) and investigate the potential epigenetic mechanisms involved in CaMKIIα+ neuron activation. METHODS: CCI-induced neuropathic pain mice model, Sirt1loxP/loxP mice, and chemogenetic virus were used to investigate whether the activation of spinal CaMKIIα+ neurons is involved in neuropathic pain and its involved mechanism. Transcriptome sequence, western blotting, qRT-PCR, and immunofluorescence analysis were performed to assay the expression of related molecules and activation of neurons. Co-immunoprecipitation was used to observe the binding relationship of protein. Chromatin immunoprecipitation (ChIP)-PCR was applied to analyze the acetylation of histone H3 in the Scn3a promoter region. RESULTS: The expression of sodium channel Nav1.3 was increased and the expression of SIRT1 was decreased in the spinal CaMKIIα+ neurons of CCI mice. CaMKIIα neurons became overactive after CCI, and inhibiting their activation relieved CCI-induced pain. Overexpression of SIRT1 reversed the increase of Nav1.3 and alleviated pain, while knockdown of SIRT1 or overexpression of Nav1.3 promoted CaMKIIα+ neuron activation and induced pain. By knocking down spinal SIRT1, the acetylation of histone H3 in the Scn3a (encoding Nav1.3) promoter region was increased, leading to an increased expression of Nav1.3. CONCLUSION: The findings suggest that an aberrant reduction of spinal SIRT1 after nerve injury epigenetically increases Nav1.3, subsequently activating CaMKIIα+ neurons and causing neuropathic pain.

Elevated NLRP3 Inflammasome Activation Is Associated with Motor Neuron Degeneration in ALS.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by motor neuron degeneration in the central nervous system. Recent research has increasingly linked the activation of nucleotide oligomerization domain-like receptor protein 3 (NLRP3) inflammasome to ALS pathogenesis. NLRP3 activation triggers Caspase 1 (CASP 1) auto-activation, leading to the cleavage of Gasdermin D (GSDMD) and pore formation on the cellular membrane. This process facilitates cytokine secretion and ultimately results in pyroptotic cell death, highlighting the complex interplay of inflammation and neurodegeneration in ALS. This study aimed to characterize the NLRP3 inflammasome components and their colocalization with cellular markers using the wobbler mouse as an ALS animal model. Firstly, we checked the levels of miR-223-3p because of its association with NLRP3 inflammasome activity. The wobbler mice showed an increased expression of miR-223-3p in the ventral horn, spinal cord, and cerebellum tissues. Next, increased levels of NLRP3, pro-CASP 1, cleaved CASP 1 (c-CASP 1), full-length GSDMD, and cleaved GDSMD revealed NLRP3 inflammasome activation in wobbler spinal cords, but not in the cerebellum. Furthermore, we investigated the colocalization of the aforementioned proteins with neurons, microglia, and astrocyte markers in the spinal cord tissue. Evidently, the wobbler mice displayed microgliosis, astrogliosis, and motor neuron degeneration in this tissue. Additionally, we showed the upregulation of protein levels and the colocalization of NLRP3, c-CASP1, and GSDMD in neurons, as well as in microglia and astrocytes. Overall, this study demonstrated the involvement of NLRP3 inflammasome activation and pyroptotic cell death in the spinal cord tissue of wobbler mice, which could further exacerbate the motor neuron degeneration and neuroinflammation in this ALS mouse model.

Analysis and experimental validation of genes and their transcription factor prediction in contused rat spinal cord at the intermediate phase.

The intermediate phase of spinal cord injury (SCI) serves as an important target site for therapeutic mediation of SCI. However, there is a lack of insight into the mechanism of the intermediate phase of SCI. The present study aimed to investigate the molecular mechanism and the feasible treatment targets in the intermediate phase of SCI. We downloaded GSE2599 from GEO and identified 416 significant differentially expressed genes (DEGs), including 206 downregulated and 210 upregulated DEGs. Further enrichment analysis of DEGs revealed that many important biological processes and signal pathways were triggered in the injured spinal cord. Furthermore, a protein-protein interaction (PPI) network was constructed and the top 10 high-degree hub nodes were identified. Furthermore, 27 predicted transcription factors (TFs) and 136 predicted motifs were identified. We then selected insulin-like growth factor 1 (IGF1) and its predicted transcription factor, transcription factor A, mitochondrial (TFAM) for further investigation. We speculated and preliminarily confirmed that TFAM may regulate gene transcription of IGF1 and effected alterations in the function recovery of rats after SCI. These findings together provide novel information that may improve our understanding of the pathophysiological processes during the intermediate phase of SCI.

Il34 rescues metronidazole-induced impairment of spinal cord regeneration in zebrafish central nervous system.

Metronidazole (MTZ), a commonly used anti-infective drug in clinical practice, has also been employed as a prodrug in cell-targeted ablation systems in scientific research, exhibiting significant application value. However, it has been demonstrated that MTZ can induce neurotoxic symptoms to some extent during its use, and there is currently a lack of effective means to circumvent its toxicity in both clinical and research settings, which limits its application. Therefore, exploring the specific mechanisms underlying MTZ-induced neurotoxic symptoms and elucidating countermeasures will enhance the practical value of MTZ. In this study, using a zebrafish spinal cord injury regeneration model, we confirmed that MTZ neurotoxicity leads to impaired axon regeneration in the central nervous system. By overexpressing il34 in the central nervous system of zebrafish, we eliminated the inhibitory effect of MTZ on axonal regeneration and demonstrated that the pro-regenerative effect against MTZ neurotoxicity is not caused by excessive macrophages/microglia chemoattracted by interleukin 34(Il34). Transcriptome sequencing analysis and GO enrichment analysis of differentially expressed genes between groups revealed that Il34 may counteract MTZ neurotoxicity and promote spinal cord injury repair through biological processes that enhance cellular adhesion and cell location. In summary, our work uncovers a possible cause of MTZ neurotoxicity and provides a new perspective for eliminating MTZ toxicity.