Project description:Metabolic dysfunction occurs in many age-related neurodegenerative diseases, yet its role in disease etiology remains poorly understood. We recently discovered a potential causal link between the branched-chain amino acid transferase BCAT-1 and the neurodegenerative movement disorder Parkinson's disease (PD). RNAi-mediated knockdown of Caenorhabditis elegans bcat-1 is known to recapitulate PD-like features, including progressive motor deficits and neurodegeneration with age, yet the underlying mechanisms have remained unknown. Using transcriptomic, metabolomic, and imaging approaches, we show here that bcat-1 knockdown increases mitochondrial respiration and induces oxidative damage in neurons through mammalian target of rapamycin-independent mechanisms. Increased mitochondrial respiration, or "mitochondrial hyperactivity," is required for bcat-1(RNAi) neurotoxicity. Moreover, we show that post-disease-onset administration of the type 2 diabetes medication metformin reduces mitochondrial respiration to control levels and significantly improves both motor function and neuronal viability. Taken together, our findings suggest that mitochondrial hyperactivity may be an early event in the pathogenesis of PD, and that strategies aimed at reducing mitochondrial respiration may constitute a surprising new avenue for PD treatment.
Project description:Rationale: Retinal ganglion cell (RGC) degeneration is extremely hard to repair or regenerate and is often coupled with mitochondrial dysfunction. Mesenchymal stem cells (MSCs)-based treatment has been demonstrated beneficial for RGC against degeneration. However, underlying mechanisms of MSC-provided RGC protection are largely unknown other than neuroprotective paracrine actions. In this study, we sought to investigate whether mitochondrial donation from induced pluripotent stem cell-derived MSC (iPSC-MSCs) could preserve RGC survival and restore retinal function. Methods: iPSC-MSCs were injected into the vitreous cavity of one eye in NADH dehydrogenase (ubiquinone) Fe-S protein 4 (Ndufs4) knockout (KO) and wild type mice. Phosphate buffer saline (PBS) or rotenone treated iPSC-MSCs were injected as control groups. Retinal function was detected by flash electroretinogram (ERG). Whole-mount immunofluorescence (IF), morphometric analysis, confocal microscopy imaging, polymerase chain reaction (PCR) of the retinas were conducted to investigate mitochondrial transfer from human iPSC-MSCs to mouse retina. Quantitative mouse cytokine arrays were carried out to measure retinal inflammatory response under difference treatments. Results: RGC survival in the iPSC-MSC injected retina of Ndufs4 KO mice was significantly increased with improved retinal function. GFP labelled human mitochondria from iPSC-MSC were detected in the RGCs in the retina of Ndufs4 KO mice starting from 96 hours post injection. PCR result showed only human mitochondrial DNA without human nuclear DNA could be detected in the mouse retinas after iPSC-MSC treatment in Ndufs4 KO mice eye. Quantitative cytokine array analysis showed pro-inflammatory cytokines was also downregulated by this iPSC-MSC treatment. Conclusion: Intravitreal transplanted iPSC-MSCs can effectively donate functional mitochondria to RGCs and protect against mitochondrial damage-induced RGC loss.
Project description:Slit-Robo GTPase-activating protein 2 (SRGAP2) plays important roles in axon guidance, neuronal migration, synapse formation, and nerve regeneration. However, the role of SRGAP2 in neuroretinal degenerative disease remains unclear. In this study, we found that SRGAP2 protein was first expressed in the retina of normal mice at the embryonic stage and was mainly located in the mature retinal ganglion cell layer and the inner nuclear layer. SRGAP2 protein in the retina and optic nerve increased after optic nerve crush. Then, we established a heterozygous knockout (Srgap2+/-) mouse model of optic nerve crush and found that Srgap2 suppression increased retinal ganglion cell survival, lowered intraocular pressure, inhibited glial cell activation, and partially restored retinal function. In vitro experiments showed that Srgap2 suppression activated the mammalian target of rapamycin signaling pathway. RNA sequencing results showed that the expression of small heat shock protein genes (Cryaa, Cryba4, and Crygs) related to optic nerve injury were upregulated in the retina of Srgap2+/- mice. These results suggest that Srgap2 suppression reduced the robust activation of glial cells, activated the mammalian target of rapamycin signaling pathway related to nerve protein, increased the expression of small heat shock protein genes, inhibited the degeneration of retinal ganglion cells, and partially restored optic nerve function.
Project description:Glaucoma is a prevalent cause of blindness globally, characterized by the progressive degeneration of retinal ganglion cells (RGCs). Among various factors, glutamate excitotoxicity stands out as a significant contributor of RGCs loss in glaucoma. Our study focused on Ripa-56 and its protective effect against NMDA-induced retinal damage in mice, aiming to delve into the potential underlying mechanism. The R28 cells were categorized into four groups: glutamate (Glu), Glu + Ripa-56, Ripa-56 and Control group. After 24 h of treatment, cell death was assessed by PI / Hoechst staining. Mitochondrial membrane potential changes, apoptosis and reactive oxygen species (ROS) production were analyzed using flow cytometry. The alterations in the expression of RIP-1, p-MLKL, Bcl-2, BAX, Caspase-3, Gpx4 and SLC7A11 were examined using western blot analysis. C57BL/6j mice were randomly divided into NMDA, NMDA + Ripa-56, Ripa-56 and control groups. Histological changes in the retina were evaluated using hematoxylin and eosin (H&E) staining. RGCs survival and the protein expression changes of RIP-1, Caspase-3, Bcl-2, Gpx4 and SLC7A11 were observed using immunofluorescence. Ripa-56 exhibited a significant reduction in the levels of RIP-1, p-MLKL, Caspase-3, and BAX induced by glutamate, while promoting the expression of Bcl-2, Gpx-4, and SLC7A1 in the Ripa-56-treated group. In our study, using an NMDA-induced normal tension glaucoma mice model, we employed immunofluorescence and H&E staining to observe that Ripa-56 treatment effectively ameliorated retinal ganglion cell loss, mitigating the decrease in retinal ganglion cell layer and bipolar cell layer thickness caused by NMDA. In this study, we have observed that Ripa-56 possesses remarkable anti- necroptotic, anti-apoptotic and anti-ferroptosis properties. It demonstrates the ability to combat not only glutamate-induced excitotoxicity in R28 cells, but also NMDA-induced retinal excitotoxicity in mice. Therefore, Ripa-56 could be used as a potential retinal protective agent.
Project description:In the central nervous system, neurons and the vasculature influence each other. While it is well described that a functional vascular system is trophic to neurons and that vascular damage contributes to neurodegeneration, the opposite scenario in which neural damage might impact the microvasculature is less defined. In this study, using an in vivo excitotoxic approach in adult mice as a tool to cause specific damage to retinal ganglion cells, we detected subsequent damage to endothelial cells in retinal capillaries. Furthermore, we detected decreased expression of vascular endothelial growth factor D (VEGFD) in retinal ganglion cells. In vivo VEGFD supplementation via neuronal-specific viral-mediated expression or acute intravitreal delivery of the mature protein preserved the structural and functional integrity of retinal ganglion cells against excitotoxicity and, additionally, spared endothelial cells from degeneration. Viral-mediated suppression of expression of the VEGFD-binding receptor VEGFR3 in retinal ganglion cells revealed that VEGFD exerts its protective capacity directly on retinal ganglion cells, while protection of endothelial cells is the result of upheld neuronal integrity. These findings suggest that VEGFD supplementation might be a novel, clinically applicable approach for neuronal and vascular protection.
Project description:It is often said that substantial retinal ganglion cells are lost before glaucomatous damage is detected by standard automated perimetry. There are 4 key articles referenced to support this belief. To test the hypothesis that the 4 key articles are incorrectly cited, the publications in the first 6 months of 2019 that reference 1 or more of these 4 articles were examined. In particular, the degree to which the quotes from these 2019 publications accurately reflected the evidence in the 4 key articles was assessed. These quotes are inadequately supported by the data, and in some cases even by the conclusions found in the abstracts of the key articles. This is despite several review articles that have questioned the evidence in these key articles. Further, a case can be made that the evidence in the key articles better supports the opposite conclusion. That is, the data suggest that sensitivity loss can be seen on standard automated perimetry before retinal ganglion cells are missing.
Project description:This study investigated the effect of low-intensity blue light on the albino Wistar rat retina, including intrinsically photosensitive retinal ganglion cells (ipRGCs). Three groups of nine albino Wistar rats were used. One group was continuously exposed to blue light (150 lx) for 2 d (STE); one was exposed to 12 h of blue light and 12 h of darkness for 10 d (LTE); one was maintained in 12 h of white light (150 lx) and 12 h of darkness for 10 d (control). Melanopsin (Opn4) was immunolabelled on retinal whole-mounts. To count and measure Opn4-positive ipRGC somas and dendrites (including Sholl profiles), Neuron J was used. Retinal cryosections were immunolabeled for glial fibrillary acid protein (GFAP) and with terminal deoxynucleotidyl transferase dUTP nick-end labelling for apoptosis detection. LTE reduced the length of Opn4-positive ipRGC dendrites (p = 0.03) and decreased Opn4-immunoreactivity in ipRGC outer stratifying dendrites. LTE and STE decreased the complexity of dendritic arborization (Sholl profile; p < 0.001, p = 0.03, respectively), increased retinal GFAP immunoreactivity (p < 0.001, p = 0.002, respectively), and caused outer segment vesiculation and outer nuclear layer apoptosis. Ultrastructural analysis showed that LTE damaged mitochondria in retinal ganglion cells and in the inner plexiform layer. Thus, LTE to low-intensity blue light harms the retinas of albino Wistar rats.
Project description:Glaucoma is characterized by the loss of retinal ganglion cells (RGC), and accordingly the preservation of RGCs and their axons has recently attracted significant attention to improve therapeutic outcomes in the disease. Here, we report that Src homology region 2-containing protein tyrosine phosphatase 2 (Shp2) undergoes activation in the RGCs, in animal model of glaucoma as well as in the human glaucoma tissues and that Shp2 dephosphorylates tropomyosin receptor kinase B (TrkB) receptor, leading to reduced BDNF/TrkB neuroprotective survival signaling. This was elucidated by specifically modulating Shp2 expression in the RGCs in vivo, using adeno-associated virus serotype 2 (AAV2) constructs. Shp2 upregulation promoted endoplasmic reticulum (ER) stress and apoptosis, along with functional and structural deficits in the inner retina. In contrast, loss of Shp2 decelerated the loss of RGCs, preserved their function, and suppressed ER stress and apoptosis in glaucoma. This report constitutes the first identification of Shp2-mediated TrkB regulatory mechanisms in the RGCs that can become a potential therapeutic target in both glaucoma and other neurodegenerative disorders.
Project description:Retinal ganglion cell (RGC) degeneration is an important cause of visual impairment, and results in part from microglia-mediated inflammation. Numerous experimental studies have focused on identifying drug targets to rescue these neurons. We recently showed that K(V)1.1 and K(V)1.3 channels are expressed in adult rat RGCs and that siRNA-mediated knockdown of either channel reduces RGC death after optic nerve transection. Earlier we found that K(V)1.3 channels also contribute to microglial activation and neurotoxicity; raising the possibility that these channels contribute to neurodegeneration through direct roles in RGCs and through inflammatory mechanisms. Here, RGC survival was increased by combined siRNA-mediated knockdown of K(V)1.1 and K(V)1.3 in RGCs, but survival was much greater when knockdown of either channel was combined with intraocular injection of a K(V)1.3 channel blocker (agitoxin-2 or margatoxin). After axotomy, increased expression of several inflammation-related molecules preceded RGC loss and, consistent with a dual mechanism, their expression was differentially affected when channel knockdown in RGCs was combined with K(V)1.3 blocker injection. K(V)1.3 blockers reduced activation of retinal microglia and their tight apposition along RGC axon fascicles after axotomy, but did not prevent their migration from the inner plexiform to the damaged ganglion cell layer. Expression of several growth factors increased after axotomy; and again, there were differences following blocker injection compared with RGC-selective channel knockdown. These results provide evidence that K(V)1.3 channels play important roles in apoptotic degeneration of adult RGCs through cell-autonomous mechanisms mediated by channels in the neurons, and nonautonomous mechanisms mediated by microglia and inflammation.
Project description:Optic neuropathy including glaucoma is one of the leading causes of irreversible vision loss, and there are currently no effective therapies. The hallmark of pathophysiology of optic neuropathy is oxidative stress and apoptotic death of retinal ganglion cells (RGCs), a population of neurons in the central nervous system with their soma in the inner retina and axons in the optic nerve. We here tested that an anti-apoptotic protein stanniocalcin-1 (STC-1) can prevent loss of RGCs in the rat retina with optic nerve transection (ONT) and in cultures of RGC-5 cells with CoCl2 injury. We found that intravitreal injection of STC-1 increased the number of RGCs in the retina at days 7 and 14 after ONT, and decreased apoptosis and oxidative damage. In cultures, treatment with STC-1 dose-dependently increased cell viability, and decreased apoptosis and levels of reactive oxygen species in RGC-5 cells that were exposed to CoCl2. The expression of HIF-1? that was up-regulated by injury was significantly suppressed in the retina and in RGC-5 cells by STC-1 treatment. The results suggested that intravitreal injection of STC-1 might be a useful therapy for optic nerve diseases in which RGCs undergo apoptosis through oxidative stress.