Sodium formononetin-3'-sulphonate alleviates cerebral ischemia-reperfusion injury in rats via suppressing endoplasmic reticulum stress-mediated apoptosis.

BACKGROUND: Sodium formononetin-3'-sulphonate (Sul-F) may alleviate I/R injury in vivo with uncertain mechanism. Endoplasmic reticulum (ER) stress-mediated apoptosis participates in the process of cerebral ischemia-reperfusion (I/R) injury. Our aim is to figure out the effect of Sul-F on cerebral I/R injury and to verify whether it works through suppressing ER stress-mediated apoptosis. RESULTS: The cerebral lesions of middle cerebral artery occlusion (MCAO) model in SD rats were aggravated after 24 h of reperfusion, including impaired neurological function, increased infarct volume, intensified inflammatory response and poor cell morphology. After intervention, the edaravone (EDA, 3 mg/kg) group and Sul-F high-dose (Sul-F-H, 80 mg/kg) group significantly alleviated I/R injury via decreasing neurological score, infarct volume and the serum levels of inflammatory factors (TNF-α, IL-1ß and IL-6), as well as alleviating pathological injury. Furthermore, the ER stress level and apoptosis rate were elevated in the ischemic penumbra of MCAO group, and were significantly blocked by EDA and Sul-F-H. In addition, EDA and Sul-F-H significantly down-regulated the ER stress related PERK/eIF2α/ATF4 and IRE1 signal pathways, which led to reduced cell apoptosis rate compared with the MCAO group. Furthermore, there was no difference between the EDA and Sul-F-H group in terms of therapeutic effect on cerebral I/R injury, indicating a therapeutic potential of Sul-F for ischemic stroke. CONCLUSIONS: Sul-F-H can significantly protects against cerebral I/R injury through inhibiting ER stress-mediated apoptosis in the ischemic penumbra, which might be a novel therapeutic target for ischemic stroke.

The deletion of mutant SOD1 via CRISPR/Cas9/sgRNA prolongs survival in an amyotrophic lateral sclerosis mouse model.

The superoxide dismutase 1 (SOD1) mutation is one of the most notable causes of amyotrophic lateral sclerosis (ALS), and modifying the mutant SOD1 gene is the best approach for the treatment of patients with ALS linked to the mutations in this gene. Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas9)/sgRNA delivered by the adeno-associated virus (AAV) system is a powerful tool for genome editing in the central nervous system (CNS). Here, we tested the capacity of the AAV-SaCas9-sgRNA system to modify mutant SOD1 in SOD1G93A transgenic mice and found that AAV9-SaCas9-sgRNA5 deleted the SOD1 gene, improved the lifespan of SOD1G93A mice by 54.6%, and notably ameliorated the performance of ALS transgenic mice. An immunochemical analysis showed that the expression of mutant SOD1 was very weak in motor neurons expressing SaCas9-sgRNA5. Consequently, the area showing muscle atrophy was more notably restored in the group treated with SaCas9-sgRNA5 compared with the group treated with SaCas9-sgLacZ. In addition, deep sequencing did not show the indel mutation in the gene highly matched to sgRNA5. Hence, AAV9-SaCas9-sgRNA-based gene editing is a feasible potential treatment for patients with ALS linked to SOD1 mutations.

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.

FGF9 modulates Schwann cell myelination in developing nerves and induces a pro-inflammatory environment during injury.

Myelin sheath is critical for the proper functioning of the peripheral nervous system (PNS), which allows the effective conduction of nerve impulses. Fibroblast growth factor 9 (FGF9) is an autocrine and paracrine protein in the fibroblast growth factor family that regulates cell differentiation and proliferation. Fgf9 Schwann cell (SC) conditional knockout mice were developed to detect the role of FGF9 in the PNS. In our study, the absence of Fgf9 led to delayed myelination in early development. The expression of mature SC-related genes decreased, and the expression of genes associated with immature SCs increased in the Fgf9 knockout mice. These data were consistent with the morphology and praxeology we observed during the development of the peripheral nerves. Extracellular-regulated kinases 1/2 (ERK1/2) are key signals for myelination, and our results showed that Fgf9 ablation led to the inactivation of ERK1/2. Further research was performed to detect the role of FGF9 in peripheral nerve injury. In superoxide dismutase 1-G93A mice with Fgf9 SC knockout, we found that Fgf9 ablation inhibited the expressions of Cd68, Il-1ß, and Cd86, which contributed to the degeneration of the axon and myelin sheath.

Progressive Degeneration and Inhibition of Peripheral Nerve Regeneration in the SOD1-G93A Mouse Model of Amyotrophic Lateral Sclerosis.

BACKGROUND: Myelination, degeneration and regeneration are implicated in crucial responses to injury in the peripheral nervous system. Considering the progression of amyotrophic lateral sclerosis (ALS), we used the superoxide dismutase 1 (SOD1)-G93A transgenic mouse model of ALS to investigate the effects of mutant SOD1 on the peripheral nerves. METHODS: Changes in peripheral nerve morphology were analyzed in SOD1 mutant mice at various stages of the disease by toluidine blue staining and electron microscopy (EM). Schwann cell proliferation and recruitment of inflammatory factors were detected by immunofluorescence staining and quantitative reverse transcription PCR and were compared between SOD1 mutant mice and control mice. Furthermore, western blotting (WB) and TUNEL staining were used to investigate axonal damage and Schwann cell survival in the sciatic nerves of mice in both groups. RESULTS: An analysis of the peripheral nervous system in SOD1-G93A mice revealed the following novel features: (i) Schwann cells and axons in mutant mice underwent changes that were similar to those seen in the control mice during the early development of peripheral nerves. (ii) The peripheral nerves of SOD1-G93A mice developed progressive neuropathy, which presented as defects in axons and myelin, leading to difficulty in walking and reduced locomotor capacity at a late stage of the disease. (iii) Macrophages were recruited and accumulated, and nerve injury and a deficit in the blood-nerve barrier were observed. (iv) Proliferation and the inflammatory micro-environment were inhibited, which impaired the regeneration and remyelination of axons after crush injury in the SOD1-G93A mice. CONCLUSIONS: The mutant human SOD1 protein induced axonal and myelin degeneration during the progression of ALS and participated in axon remyelination and regeneration in response to injury.

Diallyl Trisulfide Protects Motor Neurons from the Neurotoxic Protein TDP-43 via Activating Lysosomal Degradation and the Antioxidant Response.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive motor neuron disease for which only limited effective therapeutics are available. Currently, TAR DNA-binding protein 43 (TDP-43) is recognized as a pathological and biochemical marker for ALS. Increases in the levels of aggregated or mislocalized forms of TDP-43 might result in ALS pathology. Therefore, clearance pathways for intracellular protein aggregates have been suggested as potential therapeutic targets for the treatment of ALS. Here we report that treatment of motor neuron-like NSC34 cells overexpressing TDP-43 with diallyl trisulfide (DATS) induced neuronal autophagy and lysosomal clearance of TDP-43 and C-terminal TDP-43 fragments. We also observed that the antioxidant transcription factor NF-E2-related factor 2 (Nrf2) was accumulated in the nucleus and the expression of the antioxidant enzymes heme oxygenase1 (HO-1) and NAD(P)H:quinone oxidoreductase (NQO1) was increased. Consequently, DATS suppressed the increase in the levels of reactive oxygen species induced by TDP-43 expression. This study extends the findings of prior reports indicating that lower doses of DATS mediate cell survival in part by inducing autophagy and activating the Nrf2/antioxidant response element pathway.

Intrathecal Delivery of ssAAV9-DAO Extends Survival in SOD1G93A ALS Mice.

Amyotrophic lateral sclerosis (ALS) is an adult-onset, irreversible neurodegenerative disease that leads to progressive paralysis and inevitable death 3-5 years after diagnosis. The mechanisms underlying this process remain unknown, but new evidence indicates that accumulating levels of D-serine result from the downregulation of D-amino acid oxidase (DAO) and that this is a novel mechanism that leads to motoneuronal death in ALS via N-methyl-D-aspartate receptor-mediated cell toxicity. Here, we explored a new therapeutic approach to ALS by overexpressing DAO in the lumbar region of the mouse spinal cord using a single stranded adeno-associated virus serotype 9 (ssAAV9) vector. A single intrathecal injection of ssAAV9-DAO was made in SOD1G93A mice, a well-established mouse model of ALS. Treatment resulted in moderate expression of exogenous DAO in motorneurons in the lumbar spinal cord, reduced immunoreactivity of D-serine, alleviated motoneuronal loss and glial activation, and extended survival. The potential mechanisms underlying these effects were associated with the down-regulation of NF-κB and the restoration of the phosphorylation of Akt. In conclusion, administering ssAAV9-DAO may be an effective complementary approach to gene therapy to extend lifespans in symptomatic ALS.

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.

Inhibiting NF-κB inducing kinase improved the motor performance of ALS animal model.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a typical neurodegenerative disorder typically characterized by inflammation activation. However, the relationship between non-canonical NF-κB (ncNF-κB) pathway activation and ALS progression is not clear. METHODS: We tested the ncNF-κB pathway in the ALS animal model including hSOD1-G93A transgenic mice and TBK1 deletion mice.We treated age-matched SOD1-G93A mice with B022 (a NIK inhibitor) to investigate the role of NIK in the ALS animal model. We also established a new mice model by crossing SOD1-G93A mice with NIK+/- mice to further evaluate the interrelationship between the NIK and the disease progression in ALS animal model. RESULTS: In this study, we found the ncNF-κB pathway was activated in SOD1-G93A animal model and TBK1 deletion model. Inhibition of NIK activity by small molecule B022 significantly improved the motor performance of the ALS animal model. However, NIK deletion enhanced the mutant SOD1 toxicity by inflammatory infiltration. CONCLUSION: TBK1 deletion and mutant SOD1 shared the common pathological feature possibly via effects on NIK activation and inhibitor of NIK could be a novel strategy for treating ALS.

The modest impact of transcription factor Nrf2 on the course of disease in an ALS animal model.

Oxidative stress is associated with the pathogenesis of amyotrophic lateral sclerosis (ALS). Nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway is one of the major cellular defense mechanisms against oxidative stress. However, the role of Nrf2-mediated neuroprotection (antioxidant defense) in the disease development of ALS remains unclear. To further investigate the role of Nrf2 in ALS, we genetically eliminate the Nrf2 gene from SOD1-G93A mice, a commonly used ALS mouse model, by generating a double mutant (Nrf2-/- SOD1-G93A mice). We found that it only had a modest impact on the course of disease by knocking out Nrf2 gene in these mice. Further studies demonstrated that, among previously known Nrf2-regulated phase II enzymes, only NAD(P)H: quinone oxidoreductase 1 induction was significantly affected by the elimination of Nrf2 gene in SOD1-G93A mice. Taken together, our data suggested that Nrf2 is not the sole mediator for the induction of antioxidant genes in SOD1-G93A mice, and Nrf2-mediated neuroprotection is not the key protective mechanism against neurodegeneration in those mice.

IRF5 knockdown reverses TDP-related phenotypes partially by increasing TBK1 expression.

Interferon-regulatory factor 5 (IRF5) participates in the regulation of apoptosis, affects the phenotype of inflammatory macrophages and plays an essential role in the inflammatory response. However, the role of IRF5 in the progression of amyotrophic lateral sclerosis (ALS) remains largely unknown. Here, we show that IRF5 mainly accumulated in the nucleus in cells expressing the truncated 25 kD C-terminal fragments of TDP-43 (TDP-25, named TDP-25 cells hereafter). IRF5 knockdown using a lentivirus carrying an shRNA in TDP-25 cells exerted a protective effect and reduced the level of the apoptosis-related protein cleaved caspase-9 and the cell cycle arrest protein p21, while increasing the expression of the antioxidant transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) and its target molecule glutamate-cysteine ligase modulatory subunit (GCLM). Furthermore, IRF5-knockdown cells showed improved mitochondrial swelling and cristae dilation. In addition, we found that IRF5 mediated neuronal injury partly through the negative regulation of TANK-binding kinase 1 (TBK1). These data indicate that the loss of IRF5 in TDP-25 cells exerts a protective effect mainly by inhibiting apoptosis, regulating cell cycle arrest and alleviating oxidative stress.

High expression of NF-κB inducing kinase in the bulge region of hair follicle induces tumor.

The bulge region, a reservoir of multipotent stem cells, is possibly responsible for tumorigenesis. NF-κB-inducing kinase (NIK) is a kinase involved in the activation of the noncanonical NF-κB pathway and exhibits positive staining in tumor cells. However, whether high expression of NIK can result in tumorigenesis has not been reported in published papers. By establishing Nik-coe (Nik-stopF/F crossed with Chat-cre) and Nik-soe (Nik-stopF/F crossed with Sox9-cre) mice, we found that overexpression of Nik in the bulge region of hair follicles induced hair follicle loss and tumorigenesis. Furthermore, RNA sequencing, proteomic and phosphopeptide analyses revealed that multiple cancer pathways are involved in tumor formation. Taken together, these findings indicate that constitutive activation of Nik in the bulge region induces tumorigenesis.

Herbal medicine for amyotrophic lateral sclerosis: A systematic review and meta-analysis.

Background: The effect of herbal medicine (HM) on amyotrophic lateral sclerosis (ALS) is controversial. Clinical trials investigating HMs continue; however, the use of HM is still questioned. We aimed to systematically review the literature pertaining to the effects and safety of HM in ALS. Methods: Randomised controlled trials (RCTs) that investigated the efficacy of HMs in ALS patients compared to any types of controls were identified. Nine databases and six registers were searched from their inception dates to 25 March 2022. Per the PRISMA guidelines, trials were identified and extracted. The risk of bias was evaluated using the Cochrane's tool. Certainty of evidence was assessed as per the GRADE criteria. Forest plots were constructed to assess the effect size and corresponding 95% CIs using fixed-effect models, and random-effect models were employed when required. The primary outcome was the activity limitation measured by validated tools, such as the revised ALS Functional Rating Scale. Results: Twenty studies (N = 1,218) were eligible. Of these, only five studies were double-blinded, and two were placebo-controlled. Fourteen HMs (fifty-one single botanicals) were involved; Astragalus mongholicus Bunge, Atractylodes macrocephala Koidz., and Glycyrrhiza glabra L. were commonly used in nine, eight, and six trials, respectively. For delaying activity limitation, Jiweiling injection (MD, 2.84; 95% CI, 1.21 to 4.46; p = 0.0006) and Shenmai injection (SMD, 1.07; 0.69 to 1.45; p < 0.00001) were significantly more efficacious than Riluzole, but the evidence was low quality. For ameliorating motor neuron loss, Jiweiling injection [right abductor pollicis brevis (APB): MD, 32.42; 7.91 to 56.93; p = 0.01 and left APB: MD, 34.44; 12.85 to 56.03; p = 0.002] was favoured, but the evidence was very low quality. Nine studies reported one hundred and twenty-three adverse events, twenty-six of which occurred in the treatment groups and ninety-seven in the control groups. Conclusion: Very low to low quality of evidence suggests that HMs seem to produce superior treatment responses for ALS without increased risk of adverse events. Additional studies with homogeneous participants, reduced methodological issues, and more efficient outcome measures are required to provide confirmatory evidence. Systematic Review Registration: https://www.crd.york.ac.uk/PROSPERO/, identifier CRD42021277443.

Activation of nuclear factor erythroid 2-related factor 2 cytoprotective signaling by curcumin protect primary spinal cord astrocytes against oxidative toxicity.

Oxidative damage plays a critical role in many neurodegenerative diseases. Astrocytes are involved in supporting the survival and protection of neurons against oxidative damage. The dysfunction of antioxidant in astrocytes has been implicated in a variety of neurodegenerative disorders, such as amyotrophic lateral sclerosis (ALS), spinalmuscularatrophy (SMA). The loss of motor neuron in spinal cord has been attributed to deterioration of astrocytes. The activation of antioxidantive function in astrocytes may serve as a therapeutic strategy for neurodegenerative diseases. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a master transcriptional regulator of phase II antioxidantive genes. We report herein that curcumin significantly activates Nrf2 target genes in primary spinal cord astrocytes, decreases the level of intracellular reactive oxygen species (ROS), and attenuates oxidative damage and mitochondrial dysfunction.

Myeloid TBK1 Deficiency Induces Motor Deficits and Axon Degeneration Through Inflammatory Cell Infiltration.

BACKGROUND: TANK-binding kinase1 (TBK1) haploinsufficiency has been shown to cause both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD); however, the mechanism is unclear. METHODS: Myeloid Tbk1 knockout mice (Tbk1-LKO mice) were established and motor function and pathological analyses were also performed. The level of p-TBK1 was analyzed in the ALS animal model and in patient samples using flow cytometry. The expression of inflammatory proteins and mRNAs was analyzed via western blotting and RT-PCR, respectively. RESULTS: The latency to fall in seven-month-old Tbk1-LKO mice was significantly reduced in evaluations conducted on two consecutive days. Overall, 25.6% of Tbk1-LKO mice presented paralysis symptoms and signs, along with a loosened myelin sheath and axon degeneration at 14-16 months of age. Furthermore, the Tbk1 deficiency in myeloid cells induced inflammatory cell infiltration and dysbacteriosis in the digestive tract. Additionally, p-TBK1 levels were reduced by 29.5% and 14.8% in monocytes of patients with definite ALS and probable ALS and decreased by 27.6% and 45.5% in monocytes and microglia of ALS animals, respectively. The use of PEI-mannose-TBK1 or PEI-mannose-SaCas9-sgRNA to delete mutant SOD1 in macrophages significantly delayed disease onset and prolonged survival in the mouse model of ALS. CONCLUSIONS: Based on these data, inflammatory monocyte and macrophage infiltration and impaired innate immune defenses contribute to ALS and FTD.

Deletion of FGF9 in GABAergic neurons causes epilepsy.

Fibroblast growth factor 9 (FGF9) has long been assumed to modulate multiple biological processes, yet very little is known about the impact of FGF9 on neurodevelopment. Herein, we found that loss of Fgf9 in olig1 progenitor cells induced epilepsy in mice, with pathological changes in the cortex. Then depleting Fgf9 in different neural populations revealed that epilepsy was associated with GABAergic neurons. Fgf9 CKO in GABAergic neuron (CKOVGAT) mice exhibited not only the most severe seizures, but also the most severe growth retardation and highest mortality. Fgf9 deletion in CKOVGAT mice caused neuronal apoptosis and decreased GABA expression, leading to a GABA/Glu imbalance and epilepsy. The adenylate cyclase/cyclic AMP and ERK signaling pathways were activated in this process. Recombinant FGF9 proteoliposomes could significantly decrease the number of seizures. Furthermore, the decrease of FGF9 was commonly observed in serum of epileptic patients, especially those with focal seizures. Thus, FGF9 plays essential roles in GABAergic neuron survival and epilepsy pathology, which could serve as a new target for the treatment of epilepsy.

Protective effect of combination of sulforaphane and riluzole on glutamate-mediated excitotoxicity.

Threohydroxyaspartate (THA) causes glutamate excitotoxicity in motor neurons in organotypic culture of rat spinal cord. Some drugs, including sulforaphane (SF) and riluzole, can protect motor neuron against excitotoxicity. It has been demonstrated that SF is a potent inducer of Phase II enzymes, while riluzole is a classic anti-glutamate agent. The objective of the current study is to investigate whether the combination of SF and riluzole is superior to either one used alone. In our study, the combination of SF with riluzole not only stimulates the expression of nuclear factor erythroid 2-related factor 2 (Nrf2), reduced nicotinamide adenine dinucleotide phosphate (NADPH): quinone oxidoreductase 1 (NQO1) and heme oxygenase 1 (HO-1), but also reduces the extracellular accumulation of glutamate. When used at optimal doses, SF (10 microM) and riluzole (5 microM), either alone or in combination, all exert significant and similar neuroprotection, as measured by the number of motor neuron, medium malondialdehyde (MDA) level and lactate dehydrogenase (LDH) level. When used at low doses, the combination is better than each agent used alone. In conclusion, these results suggest the potential utility of combination use of SF and riluzole for protection of motor neuron against excitotoxicity.

FGF9 knockout in GABAergic neurons induces apoptosis and inflammation via the Fas/caspase-3 pathway in the cerebellum of mice.

Fibroblast growth factor 9 (FGF9) is a member of the fibroblast growth factor family and is widely expressed in the central nervous system (CNS). However, it is not clear how the working mechanism of FGF9 is involved in cerebellar development. To address this question, we deleted the Fgf9 gene specifically in GABAergic neurons or glutamatergic neurons, and demonstrated that Fgf9 ablation in GABAergic neurons rather than the glutamatergic neurons caused severe ataxia. We showed that FGF9 played a key role in the survival and development of Purkinje cells. GABAergic neuron-specific knockout of FGF9 (Fgf9VGAT) significantly affected the survival and development of Purkinje cells, disrupting Bergmann fiber scaffold formation and granule neuron migration in mice. RNA sequencing revealed that 976 differentially expressed genes (DEGs) were identified between Fgf9VGAT and control mice. The DEGs with significantly upregulated expression were found to be involved in apoptotic and inflammatory signaling. Selected genes including Fas, Bid, Mapk11, Cxcl10, CCl2, Bik and Fos, were validated by qRT-PCR and exhibited increases in expression in Fgf9VGAT mice compared to control mice similar to those seen in the RNA-sequencing data. The expression levels of apoptosis- and inflammation-related proteins were also increased, especially those of Fas and caspase-3 pathway related proteins. Interestingly, activated ERK signaling has been observed in apoptosis and inflammatory responses induced by deleting Fgf9 in GABAergic neurons.

The role of insulin-like growth factor 1 in ALS cell and mouse models: A mitochondrial protector.

Amyotrophic lateral sclerosis (ALS) is a common neurodegenerative disorder, but little is known about the exact causes and pathophysiology of this disease. In transgenic mouse models of ALS, mitochondrial abnormalities develop during the disease and might contribute to the progression of ALS. Gene therapy was recently shown to induce beneficial effects. For example, the delivery of human insulin-like growth factor-1 (hIGF-1) by self-complementary adeno-associated virus (AAV) vectors has been shown to prolong the lifespan of ALS transgenic mice. However, the function of IGF-1 in mitochondria has not been systematically studied in ALS models. In this study, scAAV9-hIGF-1 was intramuscularly injected into transgenic SOD1G93A mice and administered to cell lines expressing the ∼25-kDa C-terminal fragment of transactive response DNA-binding protein (TDP-25). The mitochondrial electrical transmembrane potential was hyperpolarized, and electron microscopy findings revealed that the abnormal mitochondria were transformed. Moreover, the intrinsic mitochondrial apoptotic process was modified through the upregulation of anti-apoptotic proteins (B-cell lymphoma-extra large (Bcl-xl) and B-cell lymphoma-2 (Bcl-2)), the downregulation of pro-apoptotic proteins (Bcl-2-associated x protein (Bax) and Bcl-2 homologous antagonist killer (Bak)) and a reduction in mitochondrial cytochrome c release. Mitophagy was also increased after scAAV9-hIGF-1 treatment, as evidenced by a decrease in the p62 level and an increase in the LC3-II level. Furthermore, the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) system was used to delete the IGF-1 gene in SOD1G93A model mice via an intrathecal injection of scAAV9-sgRNA-IGF1-Cas9 to confirm these findings. The protective effect of IGF-1 on the mitochondria decreased after genetic deletion. These novel findings demonstrate that IGF-1 strongly protects mitochondria from apoptosis and upregulates mitophagy in mouse and cell models of ALS. Therefore, therapies that specifically protect mitochondrial function might be promising strategies for treating ALS.

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.