DDAH-1 maintains endoplasmic reticulum-mitochondria contacts and protects dopaminergic neurons in Parkinson's disease.

The loss of dopaminergic neurons in the substantia nigra is a hallmark of pathology in Parkinson's disease (PD). Dimethylarginine dimethylaminohydrolase-1 (DDAH-1) is the critical enzyme responsible for the degradation of asymmetric dimethylarginine (ADMA) which inhibits nitric oxide (NO) synthase and has been implicated in neurodegeneration. Mitochondrial dysfunction, particularly in the mitochondria-associated endoplasmic reticulum membrane (MAM), plays a critical role in this process, although the specific molecular target has not yet been determined. This study aims to examine the involvement of DDAH-1 in the nigrostriatal dopaminergic pathway and PD pathogenesis. The distribution of DDAH-1 in the brain and its colocalization with dopaminergic neurons were observed. The loss of dopaminergic neurons and aggravated locomotor disability after rotenone (ROT) injection were showed in the DDAH-1 knockout rat. L-arginine (ARG) and NO donors were employed to elucidate the role of NO respectively. In vitro, we investigated the effects of DDAH-1 knockdown or overexpression on cell viability and mitochondrial functions, as well as modulation of ADMA/NO levels using ADMA or ARG. MAM formation was assessed by the Mitofusin2 oligomerization and the mitochondrial ubiquitin ligase (MITOL) phosphorylation. We found that DDAH-1 downregulation resulted in enhanced cell death and mitochondrial dysfunctions, accompanied by elevated ADMA and reduced NO levels. However, the recovered NO level after the ARG supplement failed to exhibit a protective effect on mitochondrial functions and partially restored cell viability. DDAH-1 overexpression prevented ROT toxicity, while ADMA treatment attenuated these protective effects. The declines of MAM formation in ROT-treated cells were exacerbated by DDAH-1 downregulation via reduced MITOL phosphorylation, which was reversed by DDAH-1 overexpression. Together, the abundant expression of DDAH-1 in nigral dopaminergic neurons may exert neuroprotective effects by maintaining MAM formation and mitochondrial function probably via ADMA, indicating the therapeutic potential of targeting DDAH-1 for PD.

Presequence protease reverses mitochondria-specific amyloid-ß-induced mitophagy to protect mitochondria.

Amyloid-ß (Aß) peptide is accumulated in the mitochondria and has been shown to play a central role in the development of Alzheimer's disease (AD). It has been shown that exposure of neurons to aggregated Aß can result in damaged mitochondria and dysregulated mitophagy, indicating that changes in the Aß content of mitochondria may affect the levels of mitophagy and interfere with the progression of AD. However, the direct influence of mitochondrial Aß on mitophagy has not been elucidated. In the present study, the effect of the mitochondria-specific Aß was assessed following a direct change of Aß content in the mitochondria. We directly change mitochondrial Aß by transfecting cells with mitochondria-associated plasmids, including the mitochondrial outer membrane protein translocase 22 (TOMM22) and 40 (TOMM40) or presequence protease (PreP) overexpression plasmids. The changes in the levels of mitophagy were assessed by TEM, Western blot, mito-Keima construct, organelle tracker, and probe JC-1 assay. We demonstrated that increased mitochondrial Aß content enhance mitophagy levels; overexpression of PreP could reverse the mitochondrial Aß-induced mitophagy levels in vivo and in vitro by reversing the levels of reactive oxygen species (ROS) and the mitochondrial membrane potential. The data provide novel insight into the role of mitochondria-specific Aß in the progression of AD pathophysiology.

Activation of TRPV4 induces intraneuronal tau hyperphosphorylation via cholesterol accumulation.

Transient receptor potential vanilloid 4 (TRPV4) is a non-selective cation channel, whose aberrant function in neurons has been reported to participate in the progression of brain disorders, including Alzheimer's disease (AD). However, the influence of TRPV4 activation on tau hyperphosphorylation in AD has not yet been elucidated. Since disturbed brain cholesterol homeostasis is considered to be related to excessive tau phosphorylation, this study aimed to explore whether dysregulation of TRPV4 affects tau phosphorylation and whether it involves cholesterol unbalance. Our data indicated that TRPV4 activation increased tau phosphorylation in the cortex and hippocampus of P301S tauopathy mouse model and aggravated its cognitive decline. In addition, we detected that TRPV4 activation upregulated cholesterol levels in primary neurons, and the elevation of cholesterol promoted hyperphosphorylation of tau. TRPV4 knockdown improved tau hyperphosphorylation by reducing intracellular cholesterol accumulation. Our results suggest that activation of TRPV4 may take part in the pathological mechanism of AD by promoting intraneuronal tau hyperphosphorylation in a cholesterol-dependent manner.

DDAH1/ADMA Regulates Adiponectin Resistance in Cerebral Ischemia via the ROS/FOXO1/APR1 Pathway.

Dimethylarginine dimethylaminohydrolase 1 (DDAH1) protects against cerebral ischemia injury via regulating the level of asymmetric dimethylarginine (ADMA). This study is aimed at exploring the effect of adiponectin resistance on ADMA-induced neuronal loss in ischemic stroke (IS) and the underlying mechanism. DDAH1 knockout (DDAH1-/-) and wild-type (DDAH1+/+) rats underwent middle cerebral artery occlusion/reperfusion (MCAO/R). Plasma and brain adiponectin levels and the expressions of adiponectin receptor 1 (APR1), adaptor protein, phosphotyrosine interacting with PH domain and leucine zipper 1 (APPL1), adenosine monophosphate-activated protein kinase (AMPK), and phosphorylated AMPK were determined after 24 h, 3 days, and 7 days. Neurological behavior, infarct volume, and adiponectin signaling were evaluated using adiponectin peptide or AdipoRon. The levels of reactive oxygen species (ROS) and Forkhead box O1 (FOXO1) (a transcription factor for APR1) were also assessed. An oxygen-glucose deprivation/reoxygenation (OGD/R) model was established in primary neurons. DDAH1 was overexpressed in neurons, after which FOXO1 expression, ROS production, adiponectin resistance, and cell viability were detected. DDAH1-/- rats showed no significant difference in adiponectin level in either plasma or brain after MCAO/R in DDAH1+/+ rats, but downregulated APR1 expression and suppressed adiponectin signaling were observed. AdipoRon, but not adiponectin peptide, attenuated the neurological deficits and adiponectin resistance in DDAH1-/- rats. ROS accumulation and phosphorylated FOXO1 expression also increased with DDAH1 depletion. Following DDAH1 overexpression, decreased cell viability and inhibited adiponectin signaling induced by OGD/R were alleviated in primary neurons, accompanied by reduced ROS production and phosphorylated FOXO1 expression. Our study elucidated that in IS, DDAH1 protected against adiponectin resistance in IS via the ROS/FOXO1/APR1 pathway.

Neurofilament Light Chain (NF-L) Stimulates Lipid Peroxidation to Neuronal Membrane through Microglia-Derived Ferritin Heavy Chain (FTH) Secretion.

A part of the axonal cytoskeleton protein complex, neurofilament light chain (NF-L) has been suggested as a pathological hallmark in various neurological disorders, including hemorrhagic stroke, vascular dementia, and cerebral small vessel disease. Neuroaxonal debris are mainly engulfed and phagocytosed by microglia, while the effects of NF-L on microglia have not been elucidated. Ferritin heavy chain (FTH) not only reflects the age-related status of microglia but may also be secreted into the extracellular space. After treatment of microglia with varying concentrations of NF-L (0-3 µg/ml), we found robust increases in the number of secretory FTH-containing exosomes in the medium. Induction of the FTH-containing exosomes secreted from microglia stimulates neuronal loss and membrane lipid peroxidation, as assessed by CKK8 and C11-Bodipy581/591, respectively. However, this oxidative stress damage was attenuated by blocking Fth1 expression. Our results suggest that NF-L, as a biomarker of axonal injury itself, could participate in neuronal ferroptosis in a nonclassical manner by secreting FTH-containing exosomes from microglia into the extracellular matrix.

Neurotransmitter-stimulated neuron-derived sEVs have opposite effects on amyloid ß-induced neuronal damage.

The ratio of excitatory to inhibitory neurotransmitters is essential for maintaining the firing patterns of neural networks, and is strictly regulated within individual neurons and brain regions. Excitatory to inhibitory (E/I) imbalance has been shown to participate in the progression of neurodegenerative diseases, including Alzheimer's disease (AD). Glutamate excitotoxicity and GABAergic neuron dysfunction appear to be key components of the neuronal cell death that takes place in AD. Since extracellular vesicles (EVs) are now explored as an important vehicle in transmitting signals between cells, we hypothesized that the function of neuron-derived small EVs (sEVs) might be regulated by the status of neurotransmitter balance and that sEVs might affect amyloid ß (Aß) toxicity on neurons. This study aimed to reveal the effects of sEVs from unbalanced neurotransmitter-stimulated neurons on Aß-induced toxicity. We demonstrated the opposite effects of the two groups of sEVs isolated from neurons stimulated by glutamate or GABA on Aß toxicity in vivo and in vitro. The sEVs released from GABA-treated neurons alleviated Aß-induced damage, while those released from glutamate-treated neurons aggravated Aß toxicity. Furthermore, we compared the microRNA (miRNA) composition of sEVs isolated from glutamate/GABA/PBS-treated neurons. Our results showed that glutamate and GABA oppositely regulated miR-132 levels in sEVs, resulting in the opposite destiny of recipient cells challenged with Aß. Our results indicated that manipulating the function of sEVs by different neurotransmitters may reveal the mechanisms underlying the pathogenesis of AD and provide a promising strategy for AD treatment.

MiR-132 down-regulates high glucose-induced ß-dystroglycan degradation through Matrix Metalloproteinases-9 up-regulation in primary neurons.

Cognitive dysfunction is one of the complications of diabetes. Unfortunately, there is no effective methods to block its progression currently. One of the pathophysiological mechanisms is synaptic protein damage and neuronal signal disruption because of glucose metabolism disorder. Dystroglycan protein, located in the post-synaptic membrane of neurons, links the intracellular cytoskeleton with extracellular matrix. Abnormal expression of dystroglycan protein affects neuronal biological functions and leads to cognitive impairment. However, there are no relevant studies to observe the changes of ß-dystroglycan protein in diabetes rat brain and in primary neurons under high glucose exposure. Our data demonstrated the alterations of cognitive abilities in the diabetic rats; ß-dystroglycan protein degradation occurred in hippocampal and cortical tissues in diabetic rat brain. We further explored the mechanisms underlying of this phenomenon. When neurons are exposed to high glucose environment in long-term period, microRNA-132 (miR-132) would be down-regulated in neurons. Matrix Metalloproteinases-9 (MMP-9) mRNA, as a target of miR-132, could be up-regulated; higher expression and overlay activity of MMP-9 protein could increase ß-DG protein degradation. In this way, ß-DG degradation may affect structure and functions among the synapses, which related to cognition decline. It may provide some theoretical basis for elucidating the molecular mechanism of diabetes-induced cognitive dysfunction.

Repressor Element 1 Silencing Transcription Factor (REST) Governs Microglia-Like BV2 Cell Migration via Progranulin (PGRN).

Microglia activation contributes to Alzheimer's disease (AD) etiology, and microglia migration is a fundamental function during microglia activation. The repressor element-1 silencing transcription factor (REST), a powerful transcriptional factor, was found to play a neuroprotective role in AD. Despite its possible role in disease progression, little is known about whether REST participates in microglia migration. In this study, we aimed to explore the function of REST and its molecular basis during microglia migration under Aß 1-42-treated pathological conditions. When treated by Aß 1-42 REST was upregulated through JAK2/STAT3 signal pathway in BV2 cells. And transwell coculture system was used to evaluate cell migration function of microglia-like BV2. Small interfering RNA (siRNA) targeting progranulin (PGRN) were delivered into BV2 cells, and results showed that PGRN functions to promote BV2 migration. REST expression was inhibited by sh-RNA, which induced BV2 cell migration obviously. On the contrary, REST was overexpressed by REST recombinant plasmid transfection, which repressed BV2 cell migration, indicating that REST may act as a repressor of cell migration. To more comprehensively examine the molecular basis, we analyzed the promoter sequence of PGRN and found that it has the potential binding site of REST. Moreover, knocking-down of REST can increase the expression of PGRN, which confirms the inhibiting effect of REST on PGRN expression. Further detection of double luciferase reporter gene also confirmed the inhibition of REST on the activity of PGRN promoter, indicating that REST may be an inhibitory transcription factor of PGRN which governs microglia-like BV2 cell migration. In conclusion, the present study demonstrates that transcription factor REST may act as a repressor of microglia migration through PGRN.

Aß-Induced Repressor Element 1-Silencing Transcription Factor (REST) Gene Delivery Suppresses Activation of Microglia-Like BV-2 Cells.

Compelling evidence from basic molecular biology has demonstrated the crucial role of microglia in the pathogenesis of Alzheimer's disease (AD). Microglia were believed to play a dual role in both promoting and inhibiting Alzheimer's disease progression. It is of great significance to regulate the function of microglia and make them develop in a favorable way. In the present study, we investigated the function of repressor element 1-silencing transcription factor (REST) in Aß 1-42-induced BV-2 cell dysfunction. We concluded that Aß 1-42 could promote type I activation of BV-2 cells and induce cell proliferation, migration, and proinflammation cytokine TNF-α, IL-1ß, and IL-6 expression. Meanwhile, REST was upregulated, and nuclear translocalization took place due to Aß 1-42 stimulation. When REST was knocked down by a specific short hairpin RNA (sh-RNA), BV-2 cell proliferation, migration, and proinflammation cytokine expression and secretion induced by Aß 1-42 were increased, demonstrating that REST may act as a repressor of microglia-like BV-2 cell activation.

Osteocalcin improves outcome after acute ischemic stroke.

BACKGROUND: Osteocalcin is related to energy metabolism, memory and the acute stress response, suggesting a relationship between bone and the brain. The need to explore the effect of osteocalcin on acute ischemic stroke is therefore urgent. RESULTS: Patients with better outcomes had higher serum osteocalcin levels than those whose NIHSS scores did not improve. Multivariable logistic regression analysis showed acceptable performance (area under the curve = 0.766). The effect of osteocalcin on the promotion of neuron survival was confirmed by Cell Counting Kit-8 experiments. In addition, osteocalcin could decrease proline hydroxylase 1 and inhibit the degradation of gasdermin D. CONCLUSIONS: We propose that osteocalcin can improve outcome after acute ischemic stroke in the acute period. By downregulating proline hydroxylase 1, osteocalcin leads glucose metabolism to the pentose phosphate pathway and therefore promotes neuronal survival through inhibiting pyroptosis. METHODS: Demographic data and laboratory results were obtained from patients with ischemic stroke in the acute period for analysis. A receiver operating characteristic curve was used to assess the discrimination of the prediction model. The potential effect of osteocalcin on cerebral ischemia and osteocalcin mechanism were explored in cultured primary rat cerebral cortical neurons treated with oxygen-glucose deprivation and reoxygenation.