The GBA variant E326K is associated with alpha-synuclein aggregation and lipid droplet accumulation in human cell lines.

Sequence variants or mutations in the GBA gene are numerically the most important risk factor for Parkinson disease (PD). The GBA gene encodes for the lysosomal hydrolase enzyme, glucocerebrosidase (GCase). GBA mutations often reduce GCase activity and lead to the impairment of the autophagy-lysosomal pathway, which is important in the turnover of alpha-synuclein, accumulation of which is a key pathological hallmark of PD. Although the E326K variant is one of the most common GBA variants associated with PD, there is limited understanding of its biochemical effects. We have characterized homozygous and heterozygous E326K variants in human fibroblasts. We found that E326K variants did not cause a significant loss of GCase protein or activity, endoplasmic reticulum (ER) retention or ER stress, in contrast to the L444P GBA mutation. This was confirmed in human dopaminergic SH-SY5Y neuroblastoma cell lines overexpressing GCase with either E326K or L444P protein. Despite no loss of the GCase activity, a significant increase in insoluble alpha-synuclein aggregates in E326K and L444P mutants was observed. Notably, SH-SY5Y overexpressing E326K demonstrated a significant increase in the lipid droplet number under basal conditions, which was exacerbated following treatment with the fatty acid oleic acid. Similarly, a significant increase in lipid droplet formation following lipid loading was observed in heterozygous and homozygous E326K fibroblasts. In conclusion, the work presented here demonstrates that the E326K mutation behaves differently to the common loss of function GBA mutations; however, lipid dyshomeostasis and alpha-synuclein pathology are still evident.

Glucocerebrosidase 1 and leucine-rich repeat kinase 2 in Parkinson disease and interplay between the two genes.

The glucocerebrosidase 1 gene (GBA1), bi-allelic variants of which cause Gaucher disease (GD), encodes the lysosomal enzyme glucocerebrosidase (GCase) and is a risk factor for Parkinson Disease (PD). GBA1 variants are linked to a reduction in GCase activity in the brain. Variants in Leucine-Rich Repeat Kinase 2 (LRRK2), such as the gain-of-kinase-function variant G2019S, cause the most common familial form of PD. In patients without GBA1 and LRRK2 mutations, GCase and LRRK2 activity are also altered, suggesting that these two genes are implicated in all forms of PD and that they may play a broader role in PD pathogenesis. In this review, we review the proposed roles of GBA1 and LRRK2 in PD, focussing on the endolysosomal pathway. In particular, we highlight the discovery of Ras-related in brain (Rab) guanosine triphosphatases (GTPases) as LRRK2 kinase substrates and explore the links between increased LRRK2 activity and Rab protein function, lysosomal dysfunction, alpha-synuclein accumulation and GCase activity. We also discuss the discovery of RAB10 as a potential mediator of LRRK2 and GBA1 interaction in PD. Finally, we discuss the therapeutic implications of these findings, including current approaches and future perspectives related to novel drugs targeting LRRK2 and GBA1.

Mitochondrial and lysosomal biogenesis are activated following PINK1/parkin-mediated mitophagy.

Impairment of the autophagy-lysosome pathway is implicated with the changes in α-synuclein and mitochondrial dysfunction observed in Parkinson's disease (PD). Damaged mitochondria accumulate PINK1, which then recruits parkin, resulting in ubiquitination of mitochondrial proteins. These can then be bound by the autophagic proteins p62/SQSTM1 and LC3, resulting in degradation of mitochondria by mitophagy. Mutations in PINK1 and parkin genes are a cause of familial PD. We found a significant increase in the expression of p62/SQSTM1 mRNA and protein following mitophagy induction in human neuroblastoma SH-SY5Y cells. p62 protein not only accumulated on mitochondria, but was also greatly increased in the cytosol. Increased p62/SQSMT1 expression was prevented in PINK1 knock-down cells, suggesting increased p62 expression was a consequence of mitophagy induction. The transcription factors Nrf2 and TFEB, which play roles in mitochondrial and lysosomal biogenesis, respectively, can regulate p62/SQSMT1. We report that both Nrf2 and TFEB translocate to the nucleus following mitophagy induction and that the increase in p62 mRNA levels was significantly impaired in cells with Nrf2 or TFEB knockdown. TFEB translocation also increased expression of itself and lysosomal proteins such as glucocerebrosidase and cathepsin D following mitophagy induction. We also report that cells with increased TFEB protein have significantly higher PGC-1α mRNA levels, a regulator of mitochondrial biogenesis, resulting in increased mitochondrial content. Our data suggests that TFEB is activated following mitophagy to maintain autophagy-lysosome pathway and mitochondrial biogenesis. Therefore, strategies to increase TFEB may improve both the clearance of α-synuclein and mitochondrial dysfunction in PD. Damaged mitochondria are degraded by the autophagy-lysosome pathway and is termed mitophagy. Following mitophagy induction, the transcription factors Nrf2 and TFEB translocate to the nucleus, inducing the transcription of genes encoding for autophagic proteins such as p62, as well as lysosomal and mitochondrial proteins. We propose that these events maintain autophagic flux, replenish lysosomes and replace mitochondria.

Resistance to the most common optic neuropathy is associated with systemic mitochondrial efficiency.

Glaucomatous optic neuropathy, an important neurodegenerative condition and the commonest optic neuropathy in humans, is the leading cause of irreversible blindness worldwide. Its prevalence and incidence increase exponentially with ageing and raised intraocular pressure (IOP). Using glaucomatous optic neuropathy as an exemplar for neurodegeneration, this study investigates putative factors imparting resistance to neurodegeneration. Systemic mitochondrial function, oxidative stress and vascular parameters were compared from isolated lymphocytes, whole blood and urine samples between 30 patients who have not developed the neuropathy despite being exposed for many years to very high IOP ('resistant'), 30 fast deteriorating glaucoma patients despite having low IOP ('susceptible'), and 30 age-similar controls. We found that 'resistant' individuals showed significantly higher rates of ADP phosphorylation by mitochondrial respiratory complexes I, II and IV, hyperpolarised mitochondrial membrane potential, higher levels of mitochondrial DNA, and enhanced capacity to deal with cytosolic calcium overload and exogenous oxidative stress, as compared to both controls and glaucoma patients. While it has been known for some years that mitochondrial dysfunction is implicated in neurodegeneration, this study provides a fresh perspective to the field of neurodegeneration by providing, for the first time, evidence that systemic mitochondrial efficiency above normal healthy levels is associated with an enhanced ability to withstand optic nerve injury. These results demonstrate the importance of cellular bioenergetics in glaucomatous disease progression, with potential relevance for other neurodegenerative disorders, and raise the possibility for new therapeutic targets in the field of neurodegeneration.

Ambroxol improves lysosomal biochemistry in glucocerebrosidase mutation-linked Parkinson disease cells.

Gaucher disease is caused by mutations in the glucocerebrosidase gene, which encodes the lysosomal hydrolase glucosylceramidase. Patients with Gaucher disease and heterozygous glucocerebrosidase mutation carriers are at increased risk of developing Parkinson's disease. Indeed, glucocerebrosidase mutations are the most frequent risk factor for Parkinson's disease in the general population. Therefore there is an urgent need to understand the mechanisms by which glucocerebrosidase mutations predispose to neurodegeneration to facilitate development of novel treatments. To study this we generated fibroblast lines from skin biopsies of five patients with Gaucher disease and six heterozygous glucocerebrosidase mutation carriers with and without Parkinson's disease. Glucosylceramidase protein and enzyme activity levels were assayed. Oxidative stress was assayed by single cell imaging of dihydroethidium. Glucosylceramidase enzyme activity was significantly reduced in fibroblasts from patients with Gaucher disease (median 5% of controls, P = 0.0001) and heterozygous mutation carriers with (median 59% of controls, P = 0.001) and without (56% of controls, P = 0.001) Parkinson's disease compared with controls. Glucosylceramidase protein levels, assessed by western blot, were significantly reduced in fibroblasts from Gaucher disease (median glucosylceramidase levels 42% of control, P < 0.001) and heterozygous mutation carriers with (median 59% of control, P < 0.001) and without (median 68% of control, P < 0.001) Parkinson's disease. Single cell imaging of dihydroethidium demonstrated increased production of cytosolic reactive oxygen species in fibroblasts from patients with Gaucher disease (dihydroethidium oxidation rate increased by a median of 62% compared to controls, P < 0.001) and heterozygous mutation carriers with (dihydroethidium oxidation rate increased by a median of 68% compared with controls, P < 0.001) and without (dihydroethidium oxidation rate increased by a median of 70% compared with controls, P < 0.001) Parkinson's disease. We hypothesized that treatment with the molecular chaperone ambroxol hydrochloride would improve these biochemical abnormalities. Treatment with ambroxol hydrochloride increased glucosylceramidase activity in fibroblasts from healthy controls, Gaucher disease and heterozygous glucocerebrosidase mutation carriers with and without Parkinson's disease. This was associated with a significant reduction in dihydroethidium oxidation rate of ∼50% (P < 0.05) in fibroblasts from controls, Gaucher disease and heterozygous mutation carriers with and without Parkinson's disease. In conclusion, glucocerebrosidase mutations are associated with reductions in glucosylceramidase activity and evidence of oxidative stress. Ambroxol treatment significantly increases glucosylceramidase activity and reduces markers of oxidative stress in cells bearing glucocerebrosidase mutations. We propose that ambroxol hydrochloride should be further investigated as a potential treatment for Parkinson's disease.

G2019S leucine-rich repeat kinase 2 causes uncoupling protein-mediated mitochondrial depolarization.

The G2019S leucine rich repeat kinase 2 (LRRK2) mutation is the most common genetic cause of Parkinson's disease (PD), clinically and pathologically indistinguishable from idiopathic PD. Mitochondrial abnormalities are a common feature in PD pathogenesis and we have investigated the impact of G2019S mutant LRRK2 expression on mitochondrial bioenergetics. LRRK2 protein expression was detected in fibroblasts and lymphoblasts at levels higher than those observed in the mouse brain. The presence of G2019S LRRK2 mutation did not influence LRRK2 expression in fibroblasts. However, the expression of the G2019S LRRK2 mutation in both fibroblast and neuroblastoma cells was associated with mitochondrial uncoupling. This was characterized by decreased mitochondrial membrane potential and increased oxygen utilization under basal and oligomycin-inhibited conditions. This resulted in a decrease in cellular ATP levels consistent with compromised cellular function. This uncoupling of mitochondrial oxidative phosphorylation was associated with a cell-specific increase in uncoupling protein (UCP) 2 and 4 expression. Restoration of mitochondrial membrane potential by the UCP inhibitor genipin confirmed the role of UCPs in this mechanism. The G2019S LRRK2-induced mitochondrial uncoupling and UCP4 mRNA up-regulation were LRRK2 kinase-dependent, whereas endogenous LRRK2 levels were required for constitutive UCP expression. We propose that normal mitochondrial function was deregulated by the expression of G2019S LRRK2 in a kinase-dependent mechanism that is a modification of the normal LRRK2 function, and this leads to the vulnerability of selected neuronal populations in PD.

Recharging mitochondrial batteries in old eyes. Near infra-red increases ATP.

Progressive accumulation of age related mitochondrial DNA mutations reduce ATP production and increase reactive oxygen species output, leading to oxidative stress, inflammation and degradation. The pace of this is linked to metabolic demand. The retina has the greatest metabolic demand and mitochondrial density in the body and displays progressive age related inflammation and marked cell loss. Near infra-red (670 nm) is thought to be absorbed by cytochrome c oxidase (COX), a key element in mitochondrial respiration and it has been demonstrated that it improves mitochondrial membrane potentials in aged eyes. It also significantly reduces the impact of experimental pathology and ameliorates age related retinal inflammation. We show ATP decline with ageing in mouse retina and brain. Also, in these tissues that ATP is significantly increased by 670 nm exposure in old mice. In the retina this was associated with increased COX and reduced acrolein expression. Acrolein, being a free radical marker of retinal oxidative stress, is up regulated in Alzheimer's and retinal degeneration. This is the first demonstration of ATP manipulation in vivo and may provide a simple non-invasive route to combating age related tissue decline.

Peripheral blood mononuclear cell respiratory function is associated with progressive glaucomatous vision loss.

Intraocular pressure (IOP) is currently the only modifiable risk factor for glaucoma and all licensed treatments lower IOP. However, many patients continue to lose vision despite IOP-lowering treatment. Identifying biomarkers for progressive vision loss would have considerable clinical utility. We demonstrate that lower peripheral blood mononuclear cell (PBMC) oxygen consumption rate (OCR) is strongly associated with faster visual field (VF) progression in patients treated by lowering IOP (P < 0.001, 229 eyes of 139 participants), explaining 13% of variance in the rate of progression. In a separate reference cohort of untreated patients with glaucoma (213 eyes of 213 participants), IOP explained 16% of VF progression variance. OCR is lower in patients with glaucoma (n = 168) than in controls (n = 50; P < 0.001) and is lower in patients with low baseline IOP (n = 99) than those with high baseline IOP (n = 69; P < 0.01). PBMC nicotinamide adenine dinucleotide (NAD) levels are lower in patients with glaucoma (n = 29) compared to controls (n = 25; P < 0.001) and strongly associated with OCR (P < 0.001). Our results support PBMC OCR and NAD levels as new biomarkers for progressive glaucoma.

The Role of Mitophagy in Glaucomatous Neurodegeneration.

This review aims to provide a better understanding of the emerging role of mitophagy in glaucomatous neurodegeneration, which is the primary cause of irreversible blindness worldwide. Increasing evidence from genetic and other experimental studies suggests that mitophagy-related genes are implicated in the pathogenesis of glaucoma in various populations. The association between polymorphisms in these genes and increased risk of glaucoma is presented. Reduction in intraocular pressure (IOP) is currently the only modifiable risk factor for glaucoma, while clinical trials highlight the inadequacy of IOP-lowering therapeutic approaches to prevent sight loss in many glaucoma patients. Mitochondrial dysfunction is thought to increase the susceptibility of retinal ganglion cells (RGCs) to other risk factors and is implicated in glaucomatous degeneration. Mitophagy holds a vital role in mitochondrial quality control processes, and the current review explores the mitophagy-related pathways which may be linked to glaucoma and their therapeutic potential.

α-Synuclein expression in response to bacterial ligands and metabolites in gut enteroendocrine cells: an in vitro proof of concept study.

Caudo-rostral migration of pathological forms of α-synuclein from the gut to the brain is proposed as an early feature in Parkinson's disease pathogenesis, but the underlying mechanisms remain unknown. Intestinal epithelial enteroendocrine cells sense and respond to numerous luminal signals, including bacterial factors, and transmit this information to the brain via the enteric nervous system and vagus nerve. There is evidence that gut bacteria composition and their metabolites change in Parkinson's disease patients, and these alterations can trigger α-synuclein pathology in animal models of the disorder. Here, we investigated the effect of toll-like receptor and free fatty acid receptor agonists on the intracellular level of α-synuclein and its release using mouse secretin tumour cell line 1 enteroendocrine cells. Secretin tumour cell line 1 enteroendocrine cells were treated for 24 or 48 h with toll-like receptor agonists (toll-like receptor 4 selective lipopolysaccharide; toll-like receptor 2 selective Pam3CysSerLys4) and the free fatty acid receptor 2/3 agonists butyrate, propionate and acetate. The effect of selective receptor antagonists on the agonists' effects after 24 hours was also investigated. The level of α-synuclein protein was measured in cell lysates and cell culture media by western blot and enzyme-linked immunosorbent assay. The level of α-synuclein and tumour necrosis factor messenger RNA was measured by quantitative reverse transcription real-time polymerase chain reaction. Stimulation of secretin tumour cell line 1 enteroendocrine cells for 24 and 48 hours with toll-like receptor and free fatty acid receptor agonists significantly increased the amount of intracellular α-synuclein and the release of α-synuclein from the cells into the culture medium. Both effects were significantly reduced by antagonists selective for each receptor. Toll-like receptor and free fatty acid receptor agonists also significantly increased tumour necrosis factor transcription, and this was effectively inhibited by corresponding antagonists. Elevated intracellular α-synuclein increases the likelihood of aggregation and conversion to toxic forms. Factors derived from bacteria induce α-synuclein accumulation in secretin tumour cell line 1 enteroendocrine cells. Here, we provide support for a mechanism by which exposure of enteroendocrine cells to specific bacterial factors found in Parkinson's disease gut dysbiosis might facilitate accumulation of α-synuclein pathology in the gut.

Mitofusin 1 and mitofusin 2 are ubiquitinated in a PINK1/parkin-dependent manner upon induction of mitophagy.

Mitochondrial dysfunction and perturbed degradation of proteins have been implicated in Parkinson's disease (PD) pathogenesis. Mutations in the Parkin and PINK1 genes are a cause of familial PD. PINK1 is a putative kinase associated with mitochondria, and loss of PINK1 expression leads to mitochondrial dysfunction, which increases with time. Parkin is suggested to be downstream of PINK1 and also mediates the removal of damaged mitochondria by macroautophagy (mitophagy). We investigated whether mitochondrial dysfunction in dopaminergic SH-SY5Y cells following decreased PINK1 expression by RNAi may in part be due to the inhibition of mitophagy. Reduced flux through the macroautophagy pathway was found to be coincident with the inhibition of ATP synthesis following 12 days of PINK1 silencing. Overexpression of parkin in these cells restored both autophagic flux and ATP synthesis. Overexpression and RNAi studies also indicated that PINK1 and parkin were required for mitophagy following CCCP-induced mitochondrial damage. The ubiquitination of several mitochondrial proteins, including mitofusin 1 and mitofusin 2, were detected within 3 h of CCCP treatment. These post-translational modifications were reduced following the silencing of parkin or PINK1. The ubiquitination of mitochondrial proteins appears to identify mitochondria for degradation and facilitate mitophagy. PINK1 and parkin are thus required for the removal of damaged mitochondria in dopaminergic cells, and inhibition of this pathway may lead to the accumulation of defective mitochondria which may contribute to PD pathogenesis.

Neuroprotection in Glaucoma: NAD+/NADH Redox State as a Potential Biomarker and Therapeutic Target.

Glaucoma is the leading cause of irreversible blindness worldwide. Its prevalence and incidence increase exponentially with age and the level of intraocular pressure (IOP). IOP reduction is currently the only therapeutic modality shown to slow glaucoma progression. However, patients still lose vision despite best treatment, suggesting that other factors confer susceptibility. Several studies indicate that mitochondrial function may underlie both susceptibility and resistance to developing glaucoma. Mitochondria meet high energy demand, in the form of ATP, that is required for the maintenance of optimum retinal ganglion cell (RGC) function. Reduced nicotinamide adenine dinucleotide (NAD+) levels have been closely correlated to mitochondrial dysfunction and have been implicated in several neurodegenerative diseases including glaucoma. NAD+ is at the centre of various metabolic reactions culminating in ATP production-essential for RGC function. In this review we present various pathways that influence the NAD+(H) redox state, affecting mitochondrial function and making RGCs susceptible to degeneration. Such disruptions of the NAD+(H) redox state are generalised and not solely induced in RGCs because of high IOP. This places the NAD+(H) redox state as a potential systemic biomarker for glaucoma susceptibility and progression; a hypothesis which may be tested in clinical trials and then translated to clinical practice.

Insulin Resistance Promotes Parkinson's Disease through Aberrant Expression of α-Synuclein, Mitochondrial Dysfunction, and Deregulation of the Polo-Like Kinase 2 Signaling.

Background: Insulin resistance (IR), considered a hallmark of diabetes at the cellular level, is implicated in pre-diabetes, results in type 2 diabetes, and negatively affects mitochondrial function. Diabetes is increasingly associated with enhanced risk of developing Parkinson's disease (PD); however, the underlying mechanism remains unclear. This study investigated the probable culpability of IR in the pathogenesis of PD. Methods: Using MitoPark mice in vivo models, diabetes was induced by a high-fat diet in the in vivo models, and IR was induced by protracted pulse-stimulation with 100 nM insulin treatment of neuronal cells, in vitro to determine the molecular mechanism(s) underlying altered cellular functions in PD, including mitochondrial dysfunction and α-synuclein (SNCA) aberrant expression. Findings: We observed increased SNCA expression in the dopaminergic (DA) neurons of both the wild-type and diabetic MitoPark mice, coupled with enhanced degeneration of DA neurons in the diabetic MitoPark mice. Ex vivo, in differentiated human DA neurons, IR was associated with increased SNCA and reactive oxygen species (ROS) levels, as well as mitochondrial depolarization. Moreover, we demonstrated concomitant hyperactivation of polo-like kinase-2 (PLK2), and upregulated p-SNCA (Ser129) and proteinase K-resistant SNCA proteins level in IR SH-SY5Y cells, however the inhibition of PLK2 reversed IR-related increases in phosphorylated and total SNCA. Similarly, the overexpression of peroxisome proliferator-activated receptor-γ coactivator 1-alpha (PGC)-1α suppressed ROS production, repressed PLK2 hyperactivity, and resulted in downregulation of total and Ser129-phosphorylated SNCA in the IR SH-SY5Y cells. Conclusions: These findings demonstrate that IR-associated diabetes promotes the development and progression of PD through PLK2-mediated mitochondrial dysfunction, upregulated ROS production, and enhanced SNCA signaling, suggesting the therapeutic targetability of PLK2 and/or SNCA as potential novel disease-modifying strategies in patients with PD.

Relationship between alpha synuclein phosphorylation, proteasomal inhibition and cell death: relevance to Parkinson's disease pathogenesis.

Alpha synuclein can be phosphorylated at serine129 (P-S129), and the presence of highly phosphorylated alpha-synuclein in Lewy bodies suggests changes to its phosphorylation status has an important pathological role. We demonstrate that the kinase(s) responsible for alpha-synuclein S129 phosphorylation is constitutively active in SH-SY5Y cells and involves casein kinase 2 activity. Increased oxidative stress or proteasomal inhibition caused significant elevation of P-S129 alpha-synuclein levels. Under these conditions, similar increases in P-S129 alpha-synuclein were found in both sodium dodecyl sulphate lysates and Triton extracts indicating the phosphorylated protein was soluble and did not lead to aggregation. The rate of S129 phosphorylation was increased in response to proteasomal inhibition indicating a higher activity of the relevant kinase. Cells expressing the phosphorylation mimic, S129D alpha-synuclein increased cell death and enhanced sensitivity to epoxomycin exposure. Proteasomal inhibition markedly decreased S129D alpha-synuclein turnover suggesting proteasomal inhibition leads to the accumulation of P-S129 alpha-synuclein through an increase in the kinase activity and a decrease in protein turnover resulting in increased cell death. We conclude that S129 phosphorylation is toxic to dopaminergic cells and both the levels of S129 phosphorylated protein and its toxicity are increased with proteasomal inhibition emphasising the interdependence of these pathways in Parkinson's disease pathogenesis.

Protection against paraquat and A53T alpha-synuclein toxicity by cabergoline is partially mediated by dopamine receptors.

Both genetic and environmental factors are thought to be involved in the aetiology of Parkinson's disease (PD). Oxidative damage, mitochondrial and proteasomal dysfunction, and inflammatory change are considered to participate in PD pathogenesis. Dopamine agonists are used in the symptomatic treatment of PD but attention has recently also been focussed on their potential for use in slowing disease progression. We have studied the protective actions of the D2 dopamine agonist cabergoline in toxin (paraquat) and genetic (wild-type and mutant [A53T] alpha-synuclein) models of PD using SHSY-5Y cells. Cabergoline increased glutathione content, reduced free radical production and caspase-3 activation, increased mitochondrial membrane potential and ameliorated cell death in SHSY-5Y cells exposed to paraquat and this action was inhibited in part by D2 receptor blockade. Cabergoline also reduced the toxicity of wild-type and mutant alpha-synuclein expression following paraquat exposure by similar mechanisms. These results confirm the protective action of cabergoline in reducing cell death in two separate genetic and environmental model systems of PD.

GBA mutation promotes early mitochondrial dysfunction in 3D neurosphere models.

Glucocerebrosidase (GBA) mutations are the most important genetic risk factor for the development of Parkinson disease (PD). GBA encodes the lysosomal enzyme glucocerebrosidase (GCase). Loss-of-GCase activity in cellular models has implicated lysosomal and mitochondrial dysfunction in PD disease pathogenesis, although the exact mechanisms remain unclear. We hypothesize that GBA mutations impair mitochondria quality control in a neurosphere model.We have characterized mitochondrial content, mitochondrial function and macroautophagy flux in 3D-neurosphere-model derived from neural crest stem cells containing heterozygous and homozygous N370SGBA mutations, under carbonyl cyanide-m-chlorophenyl-hydrazine (CCCP)- induced mitophagy.Our findings on mitochondrial markers and ATP levels indicate that mitochondrial accumulation occurs in mutant N370SGBA neurospheres under basal conditions, and clearance of depolarised mitochondria is impaired following CCCP-treatment. A significant increase in TFEB-mRNA levels, the master regulator of lysosomal and autophagy genes, may explain an unchanged macroautophagy flux in N370SGBA neurospheres. PGC1α-mRNA levels were also significantly increased following CCCP-treatment in heterozygote, but not homozygote neurospheres, and might contribute to the increased mitochondrial content seen in cells with this genotype, probably as a compensatory mechanism that is absent in homozygous lines.Mitochondrial impairment occurs early in the development of GCase-deficient neurons. Furthermore, impaired turnover of depolarised mitochondria is associated with early mitochondrial dysfunction.In summary, the presence of GBA mutation may be associated with higher levels of mitochondrial content in homozygous lines and lower clearance of damaged mitochondria in our neurosphere model.

A Human Neural Crest Stem Cell-Derived Dopaminergic Neuronal Model Recapitulates Biochemical Abnormalities in GBA1 Mutation Carriers.

Numerically the most important risk factor for the development of Parkinson's disease (PD) is the presence of mutations in the glucocerebrosidase GBA1 gene. In vitro and in vivo studies show that GBA1 mutations reduce glucocerebrosidase (GCase) activity and are associated with increased α-synuclein levels, reflecting similar changes seen in idiopathic PD brain. We have developed a neural crest stem cell-derived dopaminergic neuronal model that recapitulates biochemical abnormalities in GBA1 mutation-associated PD. Cells showed reduced GCase protein and activity, impaired macroautophagy, and increased α-synuclein levels. Advantages of this approach include easy access to stem cells, no requirement to reprogram, and retention of the intact host genome. Treatment with a GCase chaperone increased GCase protein levels and activity, rescued the autophagic defects, and decreased α-synuclein levels. These results provide the basis for further investigation of GCase chaperones or similar drugs to slow the progression of PD.

Systemic PTEN-Akt1-mTOR pathway activity in patients with normal tension glaucoma and ocular hypertension: A case series.

Glaucoma is the most common optic neuropathy in humans and the leading cause of irreversible blindness worldwide. Its prevalence and incidence increase exponentially with ageing and raised intraocular pressure (IOP), while increasing evidence suggests that systemic mitochondrial abnormalities may also be implicated in its pathogenesis. We have recently shown that patients who have not developed glaucoma despite being exposed for many years to high IOP (ocular hypertension - OHT) have more efficient mitochondria, measured in peripheral blood lymphocytes, when compared to age-similar controls and fast progressing normal tension glaucoma (NTG) patients. In this prospective case series we aimed to explore some of the molecular pathways involved in mitochondrial efficiency in glaucoma resistance by measuring the systemic activity (in peripheral blood) of key mitochondrial regulators: the mammalian target of rapamycin (mTOR) and its major upstream regulators and downstream effectors that form the PTEN-Akt1-mTOR signalling pathway. We found no statistically significant difference in the systemic mTOR activity between the three groups (control, NTG and OHT). In line with the mTOR results, there was no significant difference in the activity of both the two major upstream mTOR regulators (PTEN and Akt1) and its two main downstream effectors (S6K and 4EBP1). In a single NTG patient, with history of Raynaud's, significantly higher mTOR activity was noted. We conclude that the PTEN-Akt1-mTOR pathway does not appear to play a central role in mitochondrial efficiency in OHT.

Meclizine-induced enhanced glycolysis is neuroprotective in Parkinson disease cell models.

Meclizine is a well-tolerated drug routinely used as an anti-histamine agent in the management of disequilibrium. Recently, meclizine has been assessed for its neuroprotective properties in ischemic stroke and Huntington disease models. We found that meclizine protected against 6-hydroxydopamine-induced apoptosis and cell death in both SH-SY5Y cells and rat primary cortical cultures. Meclizine increases the level of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), which activates phosphofructokinase, a rate-determining enzyme of glycolysis. This protection is therefore mediated by meclizine's ability to enhance glycolysis and increase mitochondrial hyperpolarization. Meclizine represents an interesting candidate for further investigation to re-purpose for its potential to be neuroprotective in Parkinson disease.

The Cytomegalovirus protein pUL37×1 targets mitochondria to mediate neuroprotection.

There is substantial evidence that mitochondrial dysfunction plays a significant role in the pathogenesis of Parkinson disease (PD). This contribution probably encompasses defects of oxidative phosphorylation, mitochondrial turnover (mitophagy), mitochondrial derived oxidative stress, and apoptotic signalling. Human cytomegalovirus immediate-early protein pUL37 × 1 induces Bax mitochondrial translocation and inactivation to prevent apoptosis. Over-expressing pUL37 × 1 in neuronal cells protects against staurosporin and 6-hydroxydopamine induced apoptosis and cell death. Protection is not enhanced by bax silencing in pUL37 × 1 over-expressing cells, suggesting a bax-dependent mechanism of action. pUL37 × 1 increases glycolysis and induces mitochondrial hyperpolarization, a bax independent anti-apoptotic action. pUL37 × 1 increases glycolysis through activation of phosphofructokinase by a calcium-dependent pathway. The dual anti-apoptotic mechanism of pUL37 × 1 may be considered a novel neuroprotective strategy in diseases where mitochondrial dysfunction and apoptotic pathways are involved.