Neuronal Cell Death Research Articles

Chemical composition and studying the possible neuroprotective effect of iridoids-rich fraction from Pentas lanceolata leaves using rotenone model of Parkinson's disease in mice.

Parkinsonism is an age-related neurodegenerative illness that affects motor coordination leading to loss of dopaminergic neurons. Many medications are used for the treatment of Parkinson's disease but are only symptomatic and have a limited effect on the progression of this ailment. Therefore, bioactive compounds which derived from plants have been examined for their ability to improve the neuronal damage and cell death happened in parkinsonian patients. In this study the iridoids-rich fraction isolated from Pentas lanceolata (PIRF) leaves was investigated for its phytoconstituents. Seven iridoids (1-7) and one flavonol diglycoside (8) were isolated, and their chemical structures were achieved by 1H and 13C nuclear magnetic resonance and ESI-MS spectral data.Compound 1 (6β,7β-epoxy-8-epi-splendoside) and 5 (gaertneroside) were isolated for the first time from Pentas genus as well as compound 8 (kaempferol-3-O-robinobioside). The current study aims to investigate the possible anti-parkinsonian effect of PIRF using a rotenone model of Parkinsonism in mice. Behavioural tests (wirehanging, stair and wooden-walking tests) were done to examine the motor coordination in mice after treatment. Biochemical and histopathological examinations for brain striatum in different groups were also evaluated. Results revealed that rotenone-treated mice had poor motor functions described by depletion of dopamine and Ach levels, a significant increase in proinflammatory cytokines, IL-1B, TNF-α and Mcp-1 and oxidative biomarkers with subsequent reduction in antioxidant mediators. Disorganization of striatum, degenerated neurocytes, slight vacuolation, shrunken neurons with pyknotic nuclei and apoptotic cells are displayed by histopathological examinations. Treatment with PIRF ameliorates the neurodegeneration-induced by rotenone in the brain of mice. The anti-parkinsonian effect of PIRF could be attributed to their bioactive constituents of iridoids.

The role of P-selectin/PSGL-1 in regulating NETs as a novel mechanism in cerebral ischemic injury.

In recent years, substantial advancements have been made in understanding the pathophysiology of ischemic stroke. Despite these developments, therapeutic options for cerebral ischemia remain limited due to stringent time windows and various contraindications. Consequently, there has been a concentrated effort to elucidate the underlying mechanisms of cerebral ischemic injury. Emerging research indicates that neutrophil extracellular traps (NETs) exacerbate inflammation and damage in ischemic brain tissue, contributing to neuronal cell death. The inhibition of NETs has shown potential in preventing thrombosis and the infiltration of immune cells. Central to the formation of NETs are P-selectin and its ligand, P-selectin glycoprotein ligand-1 (PSGL-1), which represent promising therapeutic targets. This review explores the detrimental impact of P-selectin, PSGL-1, and NETs on cerebral ischemia. Additionally, it delineates the processes by which P-selectin and PSGL-1 stimulate NETs production and provides evidence that blocking these molecules reduces NETs formation. This novel insight highlights a potential therapeutic avenue that warrants further investigation by researchers in the field.

Step-down infusion of barbiturate improves neurofunction in a new rat model of rebleeding subarachnoid hemorrhage

Rebleeding from a ruptured aneurysm is a risk factor for fatal prognosis in subarachnoid hemorrhage (SAH). A novel barbiturate treatment, step-down infusion of barbiturate (sd-B), was previously developed; it showed beneficial effects on patients with severe traumatic brain injuries. The present study established a rebleeding SAH model in rats and evaluated the effect of sd-B. Fifty male Sprague-Dawley rats were divided into sham-operation with distilled water, sham-operation with barbiturate, SAH-rebleeding with distilled water, and SAH-rebleeding with barbiturate groups. For posttreatment with sd-B, thiamylal was intraperitoneally administered at 3 mg/kg/h on days 0–1, 2 mg/kg/h on days 1–2, and 1 mg/kg/h on days 2–3 after SAH using osmotic minipumps. We monitored neurofunction and case fatality as the primary endpoints and evaluated brain injuries, including brain edema and cortical neuronal cell death, as the secondary endpoints. Posttreatment with sd-B improved the modified Garcia test, reduced the brain water content, and inhibited the loss of neuronal cells and microglial expressions in the rat model. Our results revealed that sd-B ameliorated neurofunction and brain injuries on the rebleeding SAH model, suggesting that the novel treatment is a good candidate drug for patients with SAH with rebleeding.

Dose-Dependent Alterations of Lysosomal Activity and Alpha-Synuclein in Peripheral Blood Monocyte-Derived Macrophages and SH-SY5Y Neuroblastoma Cell Line by upon Inhibition of MTOR Protein Kinase – Assessment of the Prospects of Parkinson’s Disease Therapy

To date, the molecular mechanisms of the common neurodegenerative disorder Parkinson’s disease (PD) are unknown and, as a result, there is no neuroprotective therapy that may stop or slow down the process of neuronal cell death. The aim of the current study was to evaluate the prospects of using the mTOR molecule as a potential target for PD therapy due to the dose-dependent effect of mTOR kinase activity inhibition on cellular parameters associated with, PD pathogenesis. The study used peripheral blood monocyte-derived macrophages and SH-SY5Y neuroblastoma cell line. As a result, we have for the first time showed that inhibition of mTOR by Torin1 only at a concentration of 100 nM affects the level of the lysosomal enzyme glucocerebrosidase (GCase), encoded by the GBA1 gene. Mutations in GBA1 are considered a high-risk factor for PD development. This concentration led a decrease in pathological phosphorylated alpha-synuclein (Ser129), an increase in its stable tetrameric form with no changes in the lysosomal enzyme activities and concentrations of lysosphingolipids. Our findings suggest that inhibition of the mTOR protein kinase could be a promising approach for developing therapies for PD, particularly for GBA1-associated PD.

Hypomyelinated vps16 Mutant Zebrafish Exhibit Systemic and Neurodevelopmental Pathologies.

Homotypic Fusion and Protein Sorting (HOPS) and Class C-core Vacuole/Endosome Tethering (CORVET) complexes regulate the correct fusion of endolysosomal bodies. Mutations in core proteins (VPS11, VPS16, VPS18, and VPS33) have been linked with multiple neurological disorders, including mucopolysaccharidosis (MPS), genetic leukoencephalopathy (gLE), and dystonia. Mutations in human Vacuolar Protein Sorting 16 (VPS16) have been associated with MPS and dystonia. In this study, we generated and characterized a zebrafish vps16(-/-) mutant line using immunohistochemical and behavioral approaches. The loss of Vps16 function caused multiple systemic defects, hypomyelination, and increased neuronal cell death. Behavioral analysis showed a progressive loss of visuomotor response and reduced motor response and habituation to acoustic/tap stimuli in mutants. Finally, using a novel multiple-round acoustic/tap stimuli test, mutants showed intermediate memory deficits. Together, these data demonstrate that zebrafish vps16(-/-) mutants show systemic defects, neurological and motor system pathologies, and cognitive impairment. This is the first study to report behavior abnormalities and memory deficiencies in a zebrafish vps16(-/-) mutant line. Finally, we conclude that the deficits observed in vps16(-/-) zebrafish mutants do not mimic pathologies associated with dystonia, but more align to abnormalities associated with MPS and gLE.

Current View on PPAR-α and Its Relation to Neurosteroids in Alzheimer's Disease and Other Neuropsychiatric Disorders: Promising Targets in a Therapeutic Strategy.

Peroxisome proliferator-activated receptors (PPARs) may play an important role in the pathomechanism/pathogenesis of Alzheimer's disease (AD) and several other neurological/neuropsychiatric disorders. AD leads to progressive alterations in the redox state, ion homeostasis, lipids, and protein metabolism. Significant alterations in molecular processes and the functioning of several signaling pathways result in the degeneration and death of synapses and neuronal cells, leading to the most severe dementia. Peroxisome proliferator-activated receptor alpha (PPAR-α) is among the processes affected by AD; it regulates the transcription of genes related to the metabolism of cholesterol, fatty acids, other lipids and neurotransmission, mitochondria biogenesis, and function. PPAR-α is involved in the cholesterol transport to mitochondria, the substrate for neurosteroid biosynthesis. PPAR-α-coding enzymes, such as sulfotransferases, which are responsible for neurosteroid sulfation. The relation between PPAR-α and cholesterol/neurosteroids may have a significant impact on the course and progression of neurodegeneration/neuroprotection processes. Unfortunately, despite many years of intensive studies, the pathogenesis of AD is unknown and therapy for AD and other neurodegenerative diseases is symptomatic, presenting a significant goal and challenge today. This review presents recent achievements in therapeutic approaches for AD, which are targeting PPAR-α and its relation to cholesterol and neurosteroids in AD and neuropsychiatric disorders.

Incomplete autophagy and increased cholesterol synthesis during neuronal cell death caused by a synthetic cannabinoid, CP-55,940

There is a propensity for synthetic cannabinoid abuse to spread worldwide. CP-55,940, a synthetic cannabinoid having the ability to activate both CB1 and CB2 receptors, has been shown to induce cell death in neurons as well as other cells. Here we investigate molecular events underling the adverse effects of CP-55,940 on neuronal cells. Exposure of mouse neuroblastoma Neuro2a cells to 10–50 µM CP-55,940 results in concentration-dependent cell death that is not accompanied by an induction of apoptosis. CP-55,940 also stimulates autophagy, but the stimulation is not followed by an increase in autophagic degradation. Transcriptome analysis using DNA microarray revealed the increased expression of genes for the cholesterol biosynthesis pathway that is associated with the activation of SREBP-2, the master transcriptional regulator of cholesterol biosynthesis. However, free cholesterol is localized mainly to cytoplasmic structures, although it is localized to the plasma membrane in healthy cells. Thus, cellular trafficking of cholesterol seems to be somewhat disrupted in CP-55,940 stimulated cells. These results show for the first time that CP-55,940 stimulates autophagy as well as cholesterol biosynthesis, although not all the processes involved in the cellular response to CP-55,940 seem to be complete in these cells.

Bifunctional halloysite nanotube-based Ripk1 siRNA delivery system rescues cognitive impairment by targeting amyloid β plaques for clearance and reducing necrotic neurons in Alzheimer’s disease mice

Alzheimer’s disease (AD) is exacerbated by abnormal accumulation of toxic β-amyloid (Aβ) aggregates and Aβ-mediated neuronal cell death. Currently, no effective drugs for AD treatment are mainly due to the blood–brain barrier hindrance, which limits the most drugs for application in AD treatment. Accumulating evidence suggests that increased levels of Ripk1 and enhancement of Ripk1-mediated neuronal necroptosis are known to play an important role in neuronal death in AD. Targeting Ripk1 by pharmacological or genetic inhibition of Ripk1 has been demonstrated to prevent neuronal necroptosis and improve memory deficits in AD mice models. Here, we showed that halloysite (HNTs), one of the nanoclays, absorbed Aβ and reduced toxic Aβ aggregate production by inhibiting fibril formation and promoting the clearance of Aβ plaques. We also presented evidence that RVG29, a brain-penetrating peptide, bound directly to Aβ species and inhibited Aβ aggregation. In addition, Ripk1 is more dramatically expressed around Aβ plaques than those far from plaques in the brain of AD mice. Therefore, we designed a novel Aβ plaque-targeted nanomedicine (SH-RVG/siRipk1) via functionalizing with RVG29 onto short HNTs (SH) and loading Ripk1 siRNA (siRipk1) into the lumen of SH to prevent siRipk1 degradation. As we expected, SH-RVG/siRipk1 not only reduced plaques and improved memory impairment but also rescued necrotic neuronal loss around plaques by reducing Ripk1 expression around plaques in AD mice. These findings indicate that SH-RVG/siRipk1 may be an effective strategy for the treatment of AD.

Neuroprotective effects of chlorogenic acid: Modulation of Akt/Erk1/2 signaling to prevent neuronal apoptosis in Parkinson's disease

As a prevalent neurodegenerative disorder, Parkinson's disease is associated with oxidative stress. Our recent investigations revealed that reactive oxygen species (ROS) and PD-toxins like 6-hydroxydopamine (6-OHDA) can induce neuronal apoptosis through over-activation of Akt signaling. Chlorogenic acid (CGA), a natural acid phenol abundant in the human diet, is well-documented for its ability to mitigate intracellular ROS. In this study, we utilized CGA to treat experimental models of PD both in vitro and in vivo. Our study results demonstrated that SH-SY5Y and primary neurons exhibited cell apoptosis in response to 6-OHDA. Pretreatment with CGA significantly attenuated PD toxins-induced large amount of ROS, inhibiting Erk1/2 activation, preventing Akt inhibition, and hindering neuronal cell death. Combining the Erk1/2 inhibitor U0126 with CGA could reverse 6-OHDA-induced Akt inhibition, ROS, and apoptosis in the cells. Crucially, the Akt activator SC79 and ROS scavenger NAC both could eliminate excessive ROS via Akt and Erk1/2 signaling pathways, and CGA further potentiated these effects in PD models. Behavioral experiments revealed that CGA could alleviate gait abnormalities in PD model mice. The neuroprotective effects have been demonstrated in several endocrine regions and in the substantia nigra tissue, which shows the positive tyrosine hydroxylase (TH). Overall, our results suggest that CGA prevents the activation of Erk1/2 and inactivation of Akt by removing excess ROS in PD models. These findings propose a potential strategy for mitigating neuronal degeneration in Parkinson's disease by modulating the Akt/Erk1/2 signaling pathway through the administration of CGA and/or the use of antioxidants to alleviate oxidative stress.

PACAP glycosides promote cell outgrowth in vitro and reduce infarct size after stroke in a preclinical model

Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) is a pleiotropic peptide known to promote many beneficial processes following neural damage and cell death after stroke. Despite PACAP’s known neurotrophic and anti-inflammatory properties, it has not realized its translational potential due to a poor pharmacokinetic profile (non-linear PK/PD), and limited Blood-Brain Barrier Penetration (BBB) permeability. We have previously shown that glycosylation of PACAP increases stability and enhances BBB penetration. In addition, our prior studies showed reduced neuronal cell death and neuroinflammation in models of Parkinson’s disease and Traumatic Brain Injury (TBI). In this study we show that a PACAP(1−27) glucoside retains the known neurotrophic activity of native PACAP(1−27)in vitro and a 5-day daily treatment regimen (100 nM) leads to neurite-like extensions in PC12 cells. In addition, we show that intraperitoneal injection of a PACAP(1−27) lactoside (10 mg/kg) with improved BBB-penetration, given 1-hour after reperfusion in a Transient Middle Cerebral Artery Occlusion (tMCAO) mouse model, reduces the infarct size after the ischemic injury in males significantly by ∼ 36 %, and the data suggest a dose-dependency. In conclusion, our data support further development of PACAP glycopeptides as promising novel drug candidates for the treatment of stroke, an area with an urgent clinical need.

New rat model of spinal cord infarction with long-lasting functional disabilities generated by intraspinal injection of endothelin-1

BackgroundThe current method for generating an animal model of spinal cord (SC) infarction is highly invasive and permits only short-term observation, typically limited to 28 days.ObjectiveWe aimed to establish a...

MDB-39. HOW DO TUMOUR DERIVED EXTRACELLULAR VESICLES INTERACT WITH THE DEVELOPING NERVOUS SYSTEM AND LEAD TO ALTERED PAIN PROCESSING IN CANCER SURVIVORS?

Abstract BACKGROUND Childhood cancer-related pain and its treatment lack sufficient clinical attention due to limited understanding of the underlying mechanisms. While chemotherapy-induced peripheral neuropathic pain (CIPN) mechanisms in adults have been extensively studied, those in early life remain unexplored. In the CIPN literature there is a growing body of evidence for tumour derived factors altering pain processing. Our study aims to understand how tumour derived extracellular vesicles (EVs) interact with the developing nervous system and impact pain processing in childhood brain tumour survivors. METHODS We determined dose-response curves and IC50 values for several medulloblastoma (MB) cell lines using standard-of-care chemotherapy drugs: vincristine, etoposide, cisplatin, and lomustine. EVs were isolated via size exclusion chromatography and characterised through western blotting, flow cytometry and electron microscopy. We quantified the effects of chemotherapy on EV release using ZetaView analysis. We then tested whether EVs from relevant MB cell lines could influence axon development in primary mouse embryonic day 16.5 dorsal root ganglion (DRG) neurons in vitro after pre-treatment with chemotherapy-treated EVs. RESULTS EVs from chemotherapy-treated and untreated cells exhibited similar size and shape. However, a combination of vincristine, etoposide, and cisplatin at low concentrations significantly increased EV secretion by MB cell lines. Moreover, 24-hours following treatment with chemotherapeutic exposed EVs there was complete cell death of embryonic DRG neurons compared to the PBS or control EV treated neurons. CONCLUSION Standard chemotherapy drugs substantially enhanced EV release from MB cells, and co-culture of embryonic DRG neurons with chemotherapy exposed EVs resulted in neuronal death. Our next step is to investigate whether administering these selected EVs in healthy animals alters pain responses and pain maturation. We also aim to study the biodistribution of these EVs within the nervous system and body, after infusion into the cerebrospinal fluid as well as how EV cargo is altered by chemotherapeutics.

The NR4A2/VGF pathway fuels inflammation-induced neurodegeneration via promoting neuronal glycolysis.

A disturbed balance between excitation and inhibition (E/I balance) is increasingly recognized as a key driver of neurodegeneration in multiple sclerosis (MS), a chronic inflammatory disease of the central nervous system. To understand how chronic hyperexcitability contributes to neuronal loss in MS, we transcriptionally profiled neurons from mice lacking inhibitory metabotropic glutamate signaling with shifted E/I balance and increased vulnerability to inflammation-induced neurodegeneration. This revealed a prominent induction of the nuclear receptor NR4A2 in neurons. Mechanistically, NR4A2 increased susceptibility to excitotoxicity by stimulating continuous VGF secretion leading to glycolysis-dependent neuronal cell death. Extending these findings to people with MS (pwMS), we observed increased VGF levels in serum and brain biopsies. Notably, neuron-specific deletion of Vgf in a mouse model of MS ameliorated neurodegeneration. These findings underscore the detrimental effect of a persistent metabolic shift driven by excitatory activity as a fundamental mechanism in inflammation-induced neurodegeneration.

A2AR antagonists triggered the AMPK/m-TOR autophagic pathway to reverse the calcium-dependent cell damage in 6-OHDA induced model of PD

Calcium dyshomeostasis, oxidative stress, autophagy and apoptosis are the pathogenesis of selective dopaminergic neuronal loss in Parkinson's disease (PD). Earlier, we reported that A2A R modulates IP3-dependent intracellular Ca2+ signalling via PKA. Moreover, A2A R antagonist has been reported to reduce oxidative stress and apoptosis in PD models, however intracellular Ca2+ ([Ca2+]i) dependent autophagy regulation in the 6-OHDA model of PD has not been explored. In the present study, we investigated the A2A R antagonists mediated neuroprotective effects in 6-OHDA-induced primary midbrain neuronal (PMN) cells and unilateral lesioned rat model of PD. 6-OHDA-induced oxidative stress (ROS and superoxide) and [Ca2+]i was measured using Fluo4AM, DCFDA and DHE dye respectively. Furthermore, autophagy was assessed by Western blot of p-m-TOR/mTOR, p-AMPK/AMPK, LC3I/II, Beclin and β-actin. Apoptosis was measured by Annexin V-APC-PI detection and Western blot of Bcl2, Bax, caspase3 and β-actin. Dopamine levels were measured by Dopamine ELISA kit and Western blot of tyrosine hydroxylase. Our results suggest that 6-OHDA-induced PMN cell death occurred due to the interruption of [Ca2+]i homeostasis, accompanied by activation of autophagy and apoptosis. A2A R antagonists prevented 6-OHDA-induced neuronal cell death by decreasing [Ca2+]i overload and oxidative stress. In addition, we found that A2A R antagonists upregulated mTOR phosphorylation and downregulated AMPK phosphorylation thereby reducing autophagy and apoptosis both in 6-OHDA induced PMN cells and 6-OHDA unilateral lesioned rat model. In conclusion, A2A R antagonists alleviated 6-OHDA toxicity by modulating [Ca2+]i signalling to inhibit autophagy mediated by the AMPK/mTOR pathway.

Deoxynivalenol Induces Drp-1-Mediated Mitochondrial Dysfunction via Elevating Oxidative Stress.

Mitochondrial dysfunction is often linked to neurotoxicity and neurological diseases and stems from oxidative stress, yet effective therapies are lacking. Deoxynivalenol (DON or vomitoxin) is one of the most common and hazardous type-B trichothecene mycotoxins, which contaminates crops used for food and animal feed. Despite the abundance of preliminary reports, comprehensive investigations are scarce to explore the relationship between these fungal metabolites and neurodegenerative disorders. The present study aimed to elucidate the precise role of DON in mitochondrial dynamics and cell death in neuronal cells. Excessive mitochondrial fission is associated with the pathology of several neurodegenerative diseases. Human SH-SY5Y cells were treated with different concentrations of DON (250-1000 ng/mL). Post 24 and 48 h DON treatment, the indexes were measured as follows: generation of reactive oxygen species (ROS), ATP levels, mitochondrial membrane potential, calcium levels, and cytotoxicity in SH-SY5Y cells. The results showed that cytotoxicity, intracellular calcium levels, and ROS in the DON-treated group increased, while the ATP levels and mitochondrial membrane potential decreased in a dose-dependent manner. With increasing DON concentrations, the expression levels of P-Drp-1, mitochondrial fission proteins Mff, and Fis-1 were elevated with reduced activities of MFN1, MFN2, and OPA1, further resulting in an increased expression of autophagic marker LC3 and beclin-1. The reciprocal relationship between mitochondrial damage and ROS generation is evident as ROS can instigate structural and functional deficiencies within the mitochondria. Consequently, the impaired mitochondria facilitate the release of ROS, thereby intensifying the cycle of damage and exacerbating the overall process. Using specific hydroxyl, superoxide inhibitors, and calcium chelators, our study confirmed that ROS and Ca2+-mediated signaling pathways played essential roles in DON-induced Drp1 phosphorylation. Therefore, ROS and mitochondrial fission inhibitors could provide critical research tools for drug development in mycotoxin-induced neurodegenerative diseases.

STING orchestrates the neuronal inflammatory stress response in multiple sclerosis

Inflammation-induced neurodegeneration is a defining feature of multiple sclerosis (MS), yet the underlying mechanisms remain unclear. By dissecting the neuronal inflammatory stress response, we discovered that neurons in MS and its mouse model induce the stimulator of interferon genes (STING). However, activation of neuronal STING requires its detachment from the stromal interaction molecule 1 (STIM1), a process triggered by glutamate excitotoxicity. This detachment initiates non-canonical STING signaling, which leads to autophagic degradation of glutathione peroxidase 4 (GPX4), essential for neuronal redox homeostasis and thereby inducing ferroptosis. Both genetic and pharmacological interventions that target STING in neurons protect against inflammation-induced neurodegeneration. Our findings position STING as a central regulator of the detrimental neuronal inflammatory stress response, integrating inflammation with glutamate signaling to cause neuronal cell death, and present it as a tractable target for treating neurodegeneration in MS.

Apoptosis, Autophagy, and Mitophagy Genes in the CA3 Area in an Ischemic Model of Alzheimer's Disease with 2-Year Survival.

Currently, no evidence exists on the expression of apoptosis (CASP3), autophagy (BECN1), and mitophagy (BNIP3) genes in the CA3 area after ischemia with long-term survival. The goal of the paper was to study changes in above genes expression in CA3 area after ischemia in the period of 6-24 months. In this study, using quantitative RT-PCR, we present the expression of genes associated with neuronal death in a rat ischemic model of Alzheimer's disease. First time, we demonstrated overexpression of the CASP3 gene in CA3 area after ischemia with survival ranging from 0.5 to 2 years. Overexpression of the CASP3 gene was accompanied by a decrease in the activity level of the BECN1 and BNIP3 genes over a period of 0.5 year. Then, during 1-2 years, BNIP3 gene expression increased significantly and coincided with an increase in CASP3 gene expression. However, BECN1 gene expression was variable, increased significantly at 1 and 2 years and was below control values 1.5 years post-ischemia. Our observations suggest that ischemia with long-term survival induces neuronal death in CA3 through activation of caspase 3 in cooperation with the pro-apoptotic gene BNIP3. This study also suggests that the BNIP3 gene regulates caspase-independent pyramidal neuronal death post-ischemia. Thus, caspase-dependent and -independent death of neuronal cells occur post-ischemia in the CA3 area. Our data suggest new role of the BNIP3 gene in the regulation of post-ischemic neuronal death in CA3. This suggests the involvement of the BNIP3 together with the CASP3 in the CA3 in neuronal death post-ischemia.

BCAT1 alleviates early brain injury by inhibiting ferroptosis through PI3K/AKT/mTOR/GPX4 pathway after subarachnoid hemorrhage

Regulation of the redox system by branched-chain amino acid transferase 1 (BCAT1) is of great significance in the occurrence and development of diseases, but the relationship between BCAT1 and subarachnoid hemorrhage (SAH) is still unknown. Ferroptosis, featured by iron-dependent lipid peroxidation accompanied by the depletion of glutathione peroxidase 4 (GPX4), has been implicated in the pathological process of early brain injury after subarachnoid hemorrhage. This study established SAH model by endovascular perforation and adding oxyhemoglobin (Hb) to HT22 cells and delved into the mechanism of BCAT1 in SAH-induced ferroptotic neuronal cell death. It was found that SAH-induced neuronal ferroptosis could be inhibited by BCAT1 overexpression (OE) in rats and HT22 cells, and BCAT1 OE alleviated neurological deficits and cognitive dysfunction in rats after SAH. In addition, the effect of BCAT1 could be reversed by the Ly294002, a specific inhibitor of the PI3K pathway. In summary, our present study indicated that BCAT1 OE alleviated early brain injury EBI after SAH by inhibiting neuron ferroptosis via activation of PI3K/AKT/mTOR pathway and the elevation of GPX4. These results suggested that BCAT1 was a promising therapeutic target for subarachnoid hemorrhage.

Blood brain barrier-targeted lipid nanoparticles improved the neuroprotection of Ferrostatin-1 against cerebral ischemic damage in an experimental stroke model

Cerebral ischemic stroke is a serious disease with high mortality and disability rates. However, few neuroprotective drugs have been used for ischemic stroke in the clinic. Two main reasons may be responsible for this failure: difficulty in penetrating the blood-brain barrier (BBB) and easily inactivated in the blood circulation. Ferroptosis, a lipid oxidation-related cell death, plays significant roles in cerebral ischemia-reperfusion injury. We utilized RVG29, a peptide derived from Rabies virus glycoprotein, to obtain BBB-targeted lipid nanoparticles (T-LNPs) in order to investigate whether T-LNPs improved the neuroprotective effects of Ferrostatin-1 (Fer1, an inhibitor of ferroptosis) against cerebral ischemic damage. T-LNPs significantly increased BBB penetration following oxygen/glucose deprivation exposure in an in vitro BBB model and enhanced the fluorescence distribution in brain tissues at 6 h post-administration in a cerebral ischemic murine model. Moreover, T-LNPs encapsulated Fer1 (T-LNPs-Fer1) significantly enhanced the inhibitory effects of Fer1 on ferroptosis by maintaining the homeostasis of NADPH oxidase 4 (NOX4) and glutathione peroxidase 4 (GPX4) signals in neuronal cells after cerebral ischemia. T-LNPs-Fer1 significantly suppressed oxidative stress [heme oxygenase-1 expression and malondialdehyde (the product of lipid ROS reaction)] in neurons and alleviated ischemia-induced neuronal cell death, compared to Fer1 alone without encapsulation. Furthermore, T-LNPs-Fer1 significantly reduced cerebral infarction and improved behavior functions compared to Fer1-treated cerebral ischemic mice after 45-min ischemia/24-h reperfusion. These findings showed that the T-LNPs helped Fer1 penetrate the BBB and improved the neuroprotection of Fer1 against cerebral ischemic damage in experimental stroke, providing a feasible translational strategy for the development of clinical drugs for the treatment of ischemic stroke.

SARS-CoV-2 Viroporin E Induces Ca2+ Release and Neuron Cell Death in Primary Cultures of Rat Hippocampal Cells Aged In Vitro.

The COVID-19 pandemic was caused by infection with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which may lead to serious respiratory, vascular and neurological dysfunctions. The SARS-CoV-2 envelope protein (E protein) is a structural viroporin able to form ion channels in cell membranes, which is critical for viral replication. However, its effects in primary neurons have not been addressed. Here we used fluorescence microscopy and calcium imaging to study SARS-CoV-2 viroporin E localization and the effects on neuron damage and intracellular Ca2+ homeostasis in a model of rat hippocampal neurons aged in vitro. We found that the E protein quickly enters hippocampal neurons and colocalizes with the endoplasmic reticulum (ER) in both short-term (6-8 days in vitro, DIV) and long-term (20-22 DIV) cultures resembling young and aged neurons, respectively. Strikingly, E protein treatment induces apoptosis in aged neurons but not in young neurons. The E protein induces variable increases in cytosolic Ca2+ concentration in hippocampal neurons. Ca2+ responses to the E protein are due to Ca2+ release from intracellular stores at the ER. Moreover, E protein-induced Ca2+ release is very small in young neurons and increases dramatically in aged neurons, consistent with the enhanced Ca2+ store content in aged neurons. We conclude that the SARS-CoV-2 E protein quickly translocates to ER endomembranes of rat hippocampal neurons where it releases Ca2+, probably acting like a viroporin, thus producing Ca2+ store depletion and neuron apoptosis in aged neurons and likely contributing to neurological damage in COVID-19 patients.