OPTN gene therapy increases autophagy and protects mitochondria in SOD1-G93A-expressing transgenic mice and cells.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by progressive motor neuron (MN) death. Mutation of the superoxide dismutase 1 (SOD1) gene, which results in abnormal protein aggregation, is one of the causes of familial ALS. Autophagic dysfunction occurs in SOD1-G93A mutant mice as the disease progresses, but the etiology of this disease is still unclear. Optineurin (OPTN) is an adaptor that is involved in autophagy and participates in aggrephagy and mitophagy. Previous studies have established that OPTN mutations contribute to diseases such as glaucoma and ALS. However, the function of OPTN in autophagy and mitophagy has not been intensively investigated in models of ALS. In this study, we assessed the beneficial effect of OPTN on autophagy and mitochondrial function by intrathecally injecting adeno-associated virus 9 (AAV9)-OPTN into SOD1-G93A transgenic mice and by administering lentivirus (LV)-OPTN to cells expressing the SOD1-G93A mutant protein. The expression of voltage-dependent anion channel 1 (VDAC1) was increased and autophagy was elevated after OPTN gene therapy, as shown by a lower level of p62 and a higher level of microtubule-associated protein 1A/1B-light chain 3 (LC3)-II. Moreover, using electron microscopy, we observed a hyperpolarized mitochondrial transmembrane potential and reversal of mitochondrial morphological abnormalities. Furthermore, the protein level of TANK-binding kinase 1 (TBK1) was increased, suggesting that mitophagy was increased. Our findings from both animal and cell line studies strongly suggest that OPTN gene therapy is a powerful strategy to increase autophagy and protect mitochondria to prevent the progression of ALS and could be effective in the treatment of ALS.

High-throughput Sequencing and Bioinformatics Analysis Reveals the Neurogenesis Key Targets of Curcumin Action in Mouse Brain with MCAO.

BACKGROUND: Experimental studies have shown that curcumin exerts neuroprotective effects in animal models with middle cerebral artery occlusion (MCAO). However, the mechanisms of protective effects of curcumin in MCAO are not fully understood. OBJECTIVE: This study aims to investigate the key neurogenesis targets of curcumin action in mouse brain with MCAO. METHODS: The MCAO models were established in mice. High-throughput sequencing was used to identify differentially expressed mRNA, lncRNA, and circRNA. The reverse expressed mRNAs, lncRNA, and circRNA in sham vs. MCAO and MCAO vs. curcumin were identified. Biological functions were determined by gene ontology (GO) analyses. The protein-protein interaction (PPI) network of neurogenesis-related genes was constructed. Next, neurogenesis-related lncRNA/ circRNA-miRNA-mRNA ceRNA networks were constructed. RESULTS: The total of reverse expressed 1215 mRNAs, 32 lncRNAs, and 43 circRNAs were filtered based on the 2 series (sham vs. MCAO and MCAO vs. Curcumin). The functional enrichment analysis of 1215 reverse expressed mRNAs found that they were involved in neurogenesis, neuron generation, neurogenesis regulation, and others. The PPI network of neurogenesis-related genes consisted of 115 nodes, including 27 down-regulated genes and 36 up-regulated genes. Furthermore, the neurogenesis-related lncRNA/circRNA-miRNA-mRNA ceRNAs networks were constructed, and 5 lncRNA ceRNA networks and 3 circRNA ceRNA networks were explored. CONCLUSION: Our study revealed that curcumin exerts neuroprotective effects by regulating neurogenesis. The neurogenesis-related lncRNA/circRNA-miRNA-mRNA ceRNA networks are potential therapeutic targets of curcumin in MCAO. This study provided a theoretical basis for curcumin exerting neuroprotective effects in MCAO.

Ligustrazine exerts neuroprotective effects via circ_0008146/miR-709/Cx3cr1 axis to inhibit cell apoptosis and inflammation after cerebral ischemia/reperfusion injury.

BACKGROUND: Ligustrazine is a traditional Chinese herbal medicine that has long been used to treat cerebral ischemic disorders. However, the molecular mechanisms of ligustrazine in cerebral ischemia/reperfusion (I/R) damage have not been clear elucidated. The aim of this study was to examine the neuroprotective mechanisms of ligustrazine in cerebral I/R. METHODS: 9 C57BL/6 mice were randomly divided to three groups: Sham group (n = 3), Middle cerebral artery occlusion (MCAO) group (n = 3), and MCAO + Ligustrazine group (n = 3). The neurological deficit score was evaluated, the cerebral infarct volume was measured by triphenylterazolium chloride (TTC) staining. Differentially expressed (DE) messenger RNAs (mRNAs), long noncoding RNAs (lncRNAs) and circular RNAs (circRNAs) were analyzed using the R package DEseq2 based on P-value < 0.05 and Log2 |fold change (FC)| ≥ 2 in sham group vs MCAO group and MCAO group vs ligustrazine group by high-throughput sequencing. Function enrichment analysis, the protein-protein interaction (PPI) of neurogenesis related genes were performed. The neurogenesis related competitive endogenous RNA (ceRNA) network was constructed. RESULTS: The expression of circ_0008146 was considerably higher in the MCAO group than the Sham group, and ligustrazine treatment markedly decreased the expression of circ_0008146 in MCAO. Next, the circ_0008146 ceRNA network was established, including circ_0008146-miR-709-Cx3cr1 ceRNA network. Besides, real time quantitative polymerase chain reaction (RT-qPCR) assay identified that miR-709 expression was considerably lower and Cx3cr1 expression was higher in the MCAO group than Sham group, and ligustrazine treatment markedly increased the miR-709 expression and reduced Cx3cr1 expression in MCAO. Further, silencing of circ_0008146 inhibited the concentration of Interleukin 6 (IL-6), Tumor Necrosis Factor alpha (TNF-α) and reduced neuron cell death and up-regulated miR-709 expression and down-regulated Cx3cr1 expression in Lipopolysaccharide (LPS) induced BV-2 cells. Dual-Luciferase reporter gene assay verified that circ_0008146 targeted miR-709. CONCLUSION: Ligustrazine targets circ_0008146/miR-709/Cx3cr1 axis to inhibit cell apoptosis and inflammation after cerebral ischemia/reperfusion injury.

The construction of neurogenesis-related ceRNA network of ischemic stroke treated by oxymatrine.

BACKGROUND: Known as a disease associated with high mortality, disability and a significant financial burden, ischemic stroke ranks as one of the three diseases threatening human health. Recent advances in omics technology created opportunities to uncover the mechanism in ischemic stroke occurrence and treatment. In this study, we aimed to construct the competitive endogenous RNA (ceRNA) networks of ischemic stroke treated by oxymatrine intervention. METHOD: The middle cerebral artery occlusion (MCAO) mouse model of ischemic stroke was constructed, and oxymatrine was administered. Then RNA-Sequencing was performed and integrated analysis of mRNAs, lncRNAs and circRNAs was conducted to reveal the pharmacology of oxymatrine. Functional enrichment analysis was performed to explore the underlying mechanism of differentially expressed (DE) mRNAs. The protein-protein interaction (PPI) network of neurogenesis-related genes and long noncoding RNAs (lncRNAs)/circular RNAs (circRNAs) based ceRNA networks were constructed. RESULTS: First, this study revealed the DE-mRNAs, DE-lncRNAs and DE-circRNAs between Oxymatrine treated group and the MCAO group. Then, the common 1231 DE-mRNAs, 32 DE-lncRNAs and 31 DE-circRNAs with opposite trends were identified. The Kyoto Encyclopedia of Genes and Genomes to identify the functional enrichment of 1231 DE-mRNAs were enriched in neurogenesis-related biological processes. Based on neurogenesis-related DE-mRNAs, the PPI network was constructed, and hub genes were identified based on centrality. Finally, both the lncRNA-based and circRNAs-based ceRNA networks were constructed. CONCLUSION: In summary, this study identified novel coding and noncoding ischemic stroke targets of oxymatrine-treated MCAO. Most importantly, we identified lncRNAs and circRNAs candidates as potential oxymatrine targets and constructed the neurogenesis-related ceRNA networks.

Neurosyphilis with a rare magnetic resonance imaging pattern confirmed by metagenomic next-generation sequencing: a case report.

BACKGROUND: Neurosyphilis refers to infection of the central nervous system by Treponema pallidum. The clinical presentation is variable and nonspecific. Neuroimaging findings are complex and that the diagnosis is based on clinical presentation, cerebrospinal fluid (CSF) parameters, and serologic and CSF evidence of syphilis. To date, there is no case report describing Treponema pallidum detected by metagenomic next-generation sequencing (mNGS) in CSF. CASE PRESENTATION: In this report, we describe a case of neurosyphilis in a HIV-negative, 29-year-old man, who was admitted to our hospital with an epileptic seizure and progressive cognitive impairment. Brain magnetic resonance imaging (MRI) revealed fluid-attenuated inversion recovery (FLAIR) high signal intensities in bilateral medial and anterior temporal lobes, insula, right pulvinar of the thalami, precuneus, frontal and temporo-occipital lobes. Laboratory examination showed positive results by means of nontreponemal or specific treponemal test in serum and CSF. mNGS of the CSF was also performed to identify Treponema pallidum for the first time. CONCLUSIONS: This case underscores the importance of considering neurosyphilis as a potential cause of mesiotemporal abnormality. In addition, the rapid improvement and wide usability of mNGS technology will bring new breakthroughs in the clinical diagnosis of neurosyphilis.

FGF9 alters the Wallerian degeneration process by inhibiting Schwann cell transformation and accelerating macrophage infiltration.

In vitro experiments have proved that Fibroblast Growth Factor 9 (FGF9) was decreased in Schwann cells (SCs) in which Wallerian degeneration (WD) occurred after nerve injury. We hypothesize that FGF9 downregulation in WD has some biological influence on Schwann cells (SCs) and macrophages - the two most important cell components involved in WD. In this study, we employed strategies to regulate FGF9 in sciatic nerve crush by generating a mouse model, wherein Fgf9 was specifically knocked-out in SCs, and an intraneural injection of human FGF9 protein administered to overexpress FGF9 independently. Furthermore, an inhibitor of extracellular-regulated kinases 1/2 (ERK1/2), PD0325901, was used to clarify the underlying downstream mechanism of ERK1/2 activated by FGF9. Analysis of WD revealed the novel features of FGF9: (i) FGF9 was widely expressed in axons and SCs, and was decreased during WD process. (ii) Fgf9 knockout in SCs impaired the debris clearance and eventually impeded the regeneration of nerve fibers after damage. (iii) Fgf9 knockout in SCs promoted the dedifferentiation of SCs and delayed the infiltration of macrophages by decreasing Mcp1, Tnfα, Il1ß levels and leaky blood-nerve-barrier (BNB) in WD. (iv) FGF9 injection preserved the nerve fibers, inhibited SCs dedifferentiation and accelerated macrophages infiltration. (v) ERK1/2 phosphorylation was increased by exogenous FGF9 injection. P75, Cyclin D1, Mcp1, Tnfα, Il1ß, c-Jun changes by FGF9 intraneural injection were partially reversed by the ERK1/2 inhibitor. Conclusion was that FGF9 inhibited the dedifferentiation of SCs and accelerated the accumulation of macrophages in WD, and exogenous FGF9 took effects partially by ERK1/2.

The Protective Effect of Aromatase on NSC-34 Cells with Stably Expressed hSOD1-G93A.

As an adult-onset neurodegenerative disease, amyotrophic lateral sclerosis (ALS) results in progressive muscular atrophy and paralysis. However, the mechanism of ALS has not yet been elucidated, and no cure has been found. Research has revealed that a mutation of the Cu/Zn superoxide dismutase (SOD1) gene is linked to familial ALS and that potential sex discrepancies exist in ALS incidence. Here, NSC-34 cells stably expressing hSOD1-G93A (hSOD1-G93A cells) were transiently transfected with Cyp19a1 mouse open reading frame (ORF) cDNA or a short hairpin RNA (ShRNA) plasmid. Overexpression of aromatase resulting from Cyp19a1 mouse ORF cDNA plasmid transfection enhanced cell proliferation and reduced cell damage in hSOD1-G93A cells. This protective effect occurred through anti-apoptotic pathways related to estrogen receptor-alpha (ER-α) activation. Meanwhile, knockdown of aromatase with Cyp19a1 ShRNA plasmid transfection reduced cell proliferation, increased cell damage, promoted apoptosis, and decreased ER-α expression in hSOD1-G93A cells, and the induced apoptotic effects could be reversed by estradiol (E2). In brief, the results of our study suggest that aromatase plays a neuroprotective role against apoptosis in hSOD1-G93A cells by activating ER-α and may become a new intervention target for ALS treatment.

Evidence for a protective role of the CX3CL1/CX3CR1 axis in a model of amyotrophic lateral sclerosis.

Aberrant microglial activation and neuroinflammation is a pathological hallmark of amyotrophic lateral sclerosis (ALS). Fractalkine (CX3CL1) is mostly expressed on neuronal cells. The fractalkine receptor (CX3CR1) is predominantly expressed on microglia. Many progressive neuroinflammatory disorders show disruption of the CX3CL1/CX3CR1 communication system. But the exact role of the CX3CL1/CX3CR1 in ALS pathology remains unknown. F1 nontransgenic/CX3CR1+/- females were bred with SOD1G93A/CX3CR1+/- males to produce F2 SOD1G93A/CX3CR1-/-, SOD1G93A/CX3CR1+/+. We analyzed end-stage (ES) SOD1G93A/CX3CR1-/- mice and progression-matched SOD1G93A/CX3CR1+/+ mice. Our study showed that the male SOD1G93A/CX3CR1-/- mice died sooner than male SOD1G93A/CX3CR1+/+ mice. In SOD1G93A/CX3CR1-/- mice demonstrated more neuronal cell loss, more microglial activation and exacerbated SOD1 aggregation at the end-stage of ALS. The NF-κB pathway was activated; the autophagy-lysosome degradation pathway and the autophagosome maturation were impaired. Our results indicated that the absence of CX3CR1/CX3CL1 signaling in the central nervous system (CNS) may worsen neurodegeneration. The CX3CL1/CX3CR1 communication system has anti-inflammatory and neuroprotective effects and plays an important role in maintaining autophagy activity. This effort may lead to new therapeutic strategies for neuroprotection and provide a therapeutic target for ALS patients.

IGF1 affects macrophage invasion and activation and TNF-α production in the sciatic nerves of female SOD1G93A mice.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease leading to paralysis and death within 3-5 years of its diagnosis. The SOD1G93A mouse is used extensively as an ALS animal model. Increasing evidence shows that non-neuronal cellscontribute to the pathogenesis and progression of ALS. Among them, many studies focus on microgliosis in the spinal cord (SC); while few on macrophage activation in the sciatic nerves. Substantial evidence shows that insulin-like growth factor 1 (IGF1) delays disease progression and increases the lifespan of SOD1G93A mice, and some studies indicate that IGF1 reduces inflammation in the SC of ALS mice. However, no studies have focused on the effect of IGF on sciatic nerve inflammation. Here, we find that ALS progression is characterized by increasing macrophage invasion and activation accompanied by significant TNF-α production in the sciatic nerve. Furthermore, IGF1 treatment and knockdown alleviate and aggravate these responses, respectively. Collectively, our findings show the first time that IGF1 has an anti-inflammatory effect in the sciatic nerves of SOD1G93A mice.

Characterization of aromatase expression in the spinal cord of an animal model of familial ALS.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease involving motor neurons in the motor cortex, brainstem and spinal cord. ALS leads to progressive, aggravated muscle weakness and paralysis. Although the precise pathogenesis remains unknown, several studies have shown that estrogens exert neuroprotective effects during the course of the disease. Aromatase is the key enzyme in estrogen synthesis. In the present study, we used immunohistochemistry, immunofluorescence and western blotting to observe the characteristics of aromatase expression in the spinal cords of copper-zinc superoxide dismutase-1 (SOD1)-G93A transgenic mice. Under normal and nearly normal (pre-symptomatic stage) conditions, the motor neurons in the spinal anterior horn expressed aromatase. After disease onset, astrocytes began to express aromatase. The total level of aromatase expression decreased with disease progression. These findings may provide the basis for the pathogenesis of ALS through glial aromatization during the progression of this disease.

Expression of aromatase and estrogen receptors in lumbar motoneurons of mice.

Estrogen exerts protective roles in amyotrophic lateral sclerosis (ALS). However, the expression of aromatase (ARO) and estrogen receptors (ERs) in the motoneurons of spinal cord, has not yet been elucidated. By immunohistochemistry, we found that ARO and ERs were present in the ventral horn of adult mice lumbar spinal cord, and colocalized with SMI-32, a motoneuron specific marker. Within motoneurons, we observed that ARO is detected primarily in the cytoplasm, with fewer ARO in the nucleus; ERα and ERß mainly localized in the nucleus with less in the cytoplasm; while GPR30 is located in soma and processes. In conclusion, we found that ERs and ARO are expressed in the motoneurons of lumbar spinal cord in adult mice. These findings suggest that estrogen may be useful as a promising therapeutic agent for prevention of damage and improvement of locomotor function in ALS.