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.

Role of OCT3 and DRP1 in the Transport of Paraquat in Astrocytes: A Mouse Study.

BACKGROUND: Paraquat (PQ) is a pesticide, exposure to which has been associated with an increased risk of Parkinson's disease; however, PQ transport mechanisms in the brain are still unclear. Our previous studies indicated that the organic cation transporter 3 (OCT3) expressed on astrocytes could uptake PQ and protect the dopaminergic (DA) neurons from a higher level of extracellular PQ. At present, it is unknown how OCT3 levels are altered during chronic PQ exposure or aging, nor is it clear how the compensatory mechanisms are triggered by OCT3 deficiency. Dynamic related protein 1 (DRP1) was previously reported to ameliorate the loss of neurons during Parkinson's disease. Nowadays, mounting studies have revealed the functions of astrocyte DRP1, prompting us to hypothesize that DRP1 could regulate the PQ transport capacity of astrocytes. OBJECTIVES: The present study aimed to further explore PQ transport mechanisms in the nigrostriatal system and identify pathways involved in extracellular PQ clearance. METHODS: Models of PQ-induced neurodegeneration were established by intraperitoneal (i.p.) injection of PQ in wild-type (WT) and organic cation transporter-3-deficient (Oct3-/-) mice. DRP1 knockdown was achieved by viral tools in vivo and small interfering RNA (siRNA) in vitro. Extracellular PQ was detected by in vivo microdialysis. In vitro transport assays were used to directly observe the functions of different transporters. PQ-induced neurotoxicity was evaluated by tyrosine hydroxylase immunohistochemistry, in vivo microdialysis for striatal DA and behavior tests. Western blotting analysis or immunofluorescence was used to evaluate the expression levels and locations of proteins in vitro or in vivo. RESULTS: Older mice and those chronically exposed to PQ had a lower expression of brain OCT3 and, following exposure to a 10-mg/kg i.p. PQ2+ loading dose, a higher concentration of extracellular PQ. DRP1 levels were higher in astrocytes and neurons of WT and Oct3-/- mice after chronic exposure to PQ; this was supported by finding higher levels of DRP1 after PQ treatment of dopamine transporter-expressing neurons with and without OCT3 inhibition and in primary astrocytes of WT and Oct3-/- mice. Selective astrocyte DRP1 knockdown ameliorated the PQ2+-induced neurotoxicity in Oct3-/- mice but not in WT mice. GL261 astrocytes with siRNA-mediated DRP1 knockdown had a higher expression of alanine-serine-cysteine transporter 2 (ASCT2), and transport studies suggest that extracellular PQ was transported into astrocytes by ASCT2 when OCT3 was absent. DISCUSSION: The present study mainly focused on the transport mechanisms of PQ between the dopaminergic neurons and astrocytes. Lower OCT3 levels were found in the older or chronically PQ-treated mice. Astrocytes with DRP1 inhibition (by viral tools or mitochondrial division inhibitor-1) had higher levels of ASCT2, which we hypothesize served as an alternative transporter to remove extracellular PQ when OCT3 was deficient. In summary, our data suggest that OCT3, ASCT2 located on astrocytes and the dopamine transporter located on DA terminals may function in a concerted manner to mediate striatal DA terminal damage in PQ-induced neurotoxicity. https://doi.org/10.1289/EHP9505.

Dl-3-n-Butylphthalide Alleviates Demyelination and Improves Cognitive Function by Promoting Mitochondrial Dynamics in White Matter Lesions.

White matter lesions (WMLs) are a type of cerebrovascular disorder accompanied by demyelination and cognitive decline. Dl-3-n-butylphthalide (D1-NBP) is a neuroprotective drug used for the treatment of ischemic cerebrovascular diseases, although the function of DI-NBP on WML is still not clear. This study aims to investigate whether DI-NBP affects cognitive function and ameliorates demyelination in a model of WML. The bilateral carotid artery stenosis (BCAS) mouse model and in vitro brain slice cultures with low glucose and low oxygen (LGLO) treatment were adopted. The Dl-NBP was administered intragastrically for 28 days after BCAS or added at a dose of 50 µm for 48 h after LGLO. Spatial learning and memory were evaluated by an eight-arm radial maze. Demyelination was detected using a TEM. Mitochondrial dynamics were assessed by time-lapse imaging in the cultured brain slices. The function of the synapse was evaluated by the patch clamp technique. In BCAS mice, obvious demyelination and cognitive decline were observed, while both were significantly relieved by a high-dose D1-NBP treatment (100 mg/kg). Along with demyelination, mitochondrial accumulation in the axons was significantly increased in the BCAS mice model, but with the treatment of a high-dose D1-NBP, mitochondrial accumulation was mitigated, and the anterograde/retrograde transport of mitochondria was increased. Following the improved anterograde/retrograde transport of mitochondria, the synapse activity was significantly upregulated while the reactive oxygen species (ROS) generation was remarkably decreased in the cultured brain slices. In addition, we identified syntaphilin (SNPH) as the downstream target of D1-NBP. The overexpression of SNPH mediated the effects of D1-NBP in mitigating axonal mitochondrial accumulation. In conclusion, the D1-NBP treatment significantly relieved demyelination and improved spatial learning and memory in the WML model by promoting mitochondrial dynamics. These neuroprotective effects of D1-NBP were mediated by inhibiting the mitochondrial arching protein, SNPH, which provided a potential therapeutic target for WML.

Mesenchymal-Stem-Cell-Derived Extracellular Vesicles Mitigate Trained Immunity in the Brain.

Trained immunity was recently discovered in innate immune cells and shown to facilitate the clearance of pathogens at the time of occurrence of the second insult. However, it exacerbates several aspects of neuropathologies, and proper therapy is needed to rectify this abnormal immune reaction. Mesenchymal-stem cells (MSCs) exhibit a distinct capability for brain repair but are associated with safety concerns. Extracellular vesicles derived from MSCs are a promising alternative therapy. In this study, we used lipopolysaccharides to activate trained immunity in the brain and examined the therapeutic potential of MSC-derived extracellular vesicles in mitigating the trained-immunity-induced exacerbated neuropathology. We found that MSC-derived extracellular vesicles showed comparable effects to those of MSCs in the mitigation of trained immunity in the brain. Moreover, the administration of MCS-derived extracellular vesicles mitigated the aggregated inflammatory responses in the acute stage of stroke and alleviated the trained-immunity-induced increased load of amyloid-ß in APP/PS1 mice. We further investigated the molecular machinery of MSC-derived extracellular vesicles and found that IL-10 is important for the mediation of the therapeutic potential of MSC-derived extracellular vesicles toward the alleviation of trained immunity. Our study indicates that extracellular-vesicle-based regenerative strategies might be useful to mitigate trained immunity in the brain.

The FAM171A2 gene is a key regulator of progranulin expression and modifies the risk of multiple neurodegenerative diseases.

Progranulin (PGRN) is a secreted pleiotropic glycoprotein associated with the development of common neurodegenerative diseases. Understanding the pathophysiological role of PGRN may help uncover biological underpinnings. We performed a genome-wide association study to determine the genetic regulators of cerebrospinal fluid (CSF) PGRN levels. Common variants in region of FAM171A2 were associated with lower CSF PGRN levels (rs708384, P = 3.95 × 10-12). This was replicated in another independent cohort. The rs708384 was associated with increased risk of Alzheimer's disease, Parkinson's disease, and frontotemporal dementia and could modify the expression of the FAM171A2 gene. FAM171A2 was considerably expressed in the vascular endothelium and microglia, which are rich in PGRN. The in vitro study further confirmed that the rs708384 mutation up-regulated the expression of FAM171A2, which caused a decrease in the PGRN level. Collectively, genetic, molecular, and bioinformatic findings suggested that FAM171A2 is a key player in regulating PGRN production.

Microglial exosomes facilitate α-synuclein transmission in Parkinson's disease.

Accumulation of neuronal α-synuclein is a prominent feature in Parkinson's disease. More recently, such abnormal protein aggregation has been reported to spread from cell to cell and exosomes are considered as important mediators. The focus of such research, however, has been primarily in neurons. Given the increasing recognition of the importance of non-cell autonomous-mediated neurotoxicity, it is critical to investigate the contribution of glia to α-synuclein aggregation and spread. Microglia are the primary phagocytes in the brain and have been well-documented as inducers of neuroinflammation. How and to what extent microglia and their exosomes impact α-synuclein pathology has not been well delineated. We report here that when treated with human α-synuclein preformed fibrils, exosomes containing α-synuclein released by microglia are fully capable of inducing protein aggregation in the recipient neurons. Additionally, when combined with microglial proinflammatory cytokines, these exosomes further increased protein aggregation in neurons. Inhibition of exosome synthesis in microglia reduced α-synuclein transmission. The in vivo significance of these exosomes was demonstrated by stereotaxic injection of exosomes isolated from α-synuclein preformed fibrils treated microglia into the mouse striatum. Phosphorylated α-synuclein was observed in multiple brain regions consistent with their neuronal connectivity. These animals also exhibited neurodegeneration in the nigrostriatal pathway in a time-dependent manner. Depleting microglia in vivo dramatically suppressed the transmission of α-synuclein after stereotaxic injection of preformed fibrils. Mechanistically, we report here that α-synuclein preformed fibrils impaired autophagy flux by upregulating PELI1, which in turn, resulted in degradation of LAMP2 in activated microglia. More importantly, by purifying microglia/macrophage derived exosomes in the CSF of Parkinson's disease patients, we confirmed the presence of α-synuclein oligomer in CD11b+ exosomes, which were able to induce α-synuclein aggregation in neurons, further supporting the translational aspect of this study. Taken together, our study supports the view that microglial exosomes contribute to the progression of α-synuclein pathology and therefore, they may serve as a promising therapeutic target for Parkinson's disease.

In chronic hypoxia, glucose availability and hypoxic severity dictate the balance between HIF-1 and HIF-2 in astrocytes.

Astrocyte function is an important contributor to cellular viability during brain hypoxia and ischemia. Levels of the hypoxia-inducible transcription factors (HIFs) HIF-1 and HIF-2 are increased in hypoxic conditions and impact the neuroprotective properties of astrocytes. For example, HIF-2 induces levels of erythropoietin (EPO), a neuroprotectant, by astrocytes. In contrast, HIF-1 activity in astrocytes diminishes the viability of neurons in cocultures during hypoxia. Thus, HIF-1 and HIF-2 may have opposing effects on astrocytes. In this study, we explore the balance of HIF-1 and HIF-2 signaling in astrocytes during chronic (1-7 d) hypoxia while altering the degree of hypoxia and glucose availability. In addition, we investigate the effects of these conditions on neuron apoptosis. During exposure to chronic moderate hypoxia (2% O2) and plentiful glucose (10 mM), HIF-2 and EPO abundance increases from d 1 to 7. Similarly, pretreatment with moderate hypoxia markedly increases the abundance of HIF-2 and EPO when astrocytes are subsequently exposed to severe hypoxia (0.5% O2; 24 h) in 10 mM glucose, which inhibits neuron apoptosis in coculture. Although HIF-1 targets the expression increase during the 7 d in chronic moderate hypoxia (2% O2) and limited glucose (2 mM), further exposure to severe hypoxia (0.5% O2; 24 h) induces a decrease of most HIF-1 targets in astrocytes. Notably, in astrocyte exposure to 2% O2 prior to 0.5% O2, the expression of iNOS, an HIF-1-regulated protein, keeps increasing when glucose is limited, whereas EPO and VEGF abundance is suppressed, inducing increased apoptosis of neurons in coculture under limited glucose (2 mM). Thus, both hypoxic severity and glucose abundance regulate the balance of HIF-1 and HIF-2 activity in astrocytes, leading to diverse effects on neurons. These results could have important implications on the adaptive or pathologic role of astrocytes during chronic hypoxia and ischemia.-Guo, M., Ma, X., Feng, Y., Han, S., Dong, Q., Cui, M., Zhao, Y. In chronic hypoxia, glucose availability and hypoxic severity dictate the balance between HIF-1 and HIF-2 in astrocytes.