Synucleinopathies including Parkinson's disease (PD) and Dementia with Lewy bodies (DLB) are characterized by the accumulation of abnormal α-synuclein in intraneuronal inclusions, named Lewy bodies. ...Mutations in GBA1, the gene encoding the lysosomal hydrolase glucocerebrosidase, have been identified as the most common genetic risk factor for PD and DLB. However, despite extensive research, the mechanism by which glucocerebrosidase dysfunction increases the risk for PD or DLB still remains elusive.
In our study we expand the toolbox for PD-DLB post-mortem studies by introducing new quantitative biochemical assays for glucocerebrosidase and α-synuclein. Applying causal modelling, we determine how these parameters are interrelated and ultimately impact disease manifestation.
We developed quantitative immuno-based assays for glucocerebrosidase and α-synuclein (total and phosphorylated at Serine 129) protein levels, as well as a liquid chromatography–mass spectrometry method for the detection of the glucocerebrosidase lipid substrate glucosylsphingosine. These assays were applied on tissue samples from frontal cortex, putamen and substantia nigra of PD (n = 15) and DLB (n = 15) patients and age-matched non-demented controls (n = 15).
Our results confirm elevated p-129 over total α-synuclein levels in the insoluble fraction of PD and DLB post-mortem brain tissue and we found significantly increased α-synuclein levels in the soluble fractions in PD and DLB. Furthermore, we identified an inverse correlation between reduced glucocerebrosidase enzyme activity and protein levels with increased glucosylsphingosine levels. In the substantia nigra, a brain region particularly vulnerable in Parkinson's disease, we found a significant correlation between glucocerebrosidase protein reduction and increased p129/total α-synuclein ratios.
We assessed the direction and strength of the interrelation between all measured parameters by confirmatory path analysis. Interestingly, we found that glucocerebrosidase dysfunction impacts the PD-DLB status by increasing α-synuclein ratios in the substantia nigra, which was partly mediated by increasing glucosylsphingosine levels.
In conclusion, we show that the introduced immuno-based assays enable the quantitative assessment of glucocerebrosidase and α-synuclein parameters in post-mortem brain. In the substantia nigra, reduced glucocerebrosidase levels contribute to the increase in α-synuclein levels and to PD-DLB disease manifestation partly by increasing its glycolipid substrate glucosylsphingosine. This interrelation between glucocerebrosidase, glucosylsphingosine and α-synuclein parameters supports the hypothesis that glucocerebrosidase acts as a modulator of PD-DLB.
•Path analysis - a new tool to understand the GBA/ a-syn axis•Ser129 phosphorylated a-syn levels are increased in LBD•In the SN reduced glucocerebrosidase protein correlates with increased α-synuclein•Glucocerebrosidase contributes to LBD partly by increasing glucosylsphingosine
Ocular hypertension due to impaired aqueous humor (AH) drainage through the trabecular meshwork (TM) is a major risk factor for glaucoma, a leading cause of irreversible blindness. However, the ...etiology of ocular hypertension remains unclear. Although autotaxin, a secreted lysophospholipase D and its catalytic product lysophosphatidic acid (LPA) have been shown to modulate AH drainage through TM, we do not have a complete understanding of their role and regulation in glaucoma patients, TM and AH outflow. This study reports a significant increase in the levels of autotaxin, lysophosphatidylcholine (LPC), LPA and connective tissue growth factor (CTGF) in the AH of Caucasian and African American open angle glaucoma patients relative to age-matched non-glaucoma patients. Treatment of human TM cells with dexamethasone, tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) increased the levels of autotaxin protein, a response that was mitigated by inhibitors of glucocorticoid receptor, NF-kB and SMAD3. Dexamethasone, TNF-α, IL-1β and LPC treatment of TM cells also led to an increase in the levels of CTGF, fibronectin and collagen type 1 in an autotaxin dependent manner. Additionally, in perfused enucleated mouse eyes, autotaxin and LPC were noted to decrease, while inhibition of autotaxin was increased aqueous outflow through the TM. Taken together, these results provide additional evidence for dysregulation of the autotaxin-LPA axis in the AH of glaucoma patients, reveal molecular insights into the regulation of autotaxin expression in TM cells and the consequences of autotaxin inhibitors in suppressing the fibrogenic response and resistance to AH outflow through the TM.
•AH derived from the POAG patients contains elevated levels of ATX, LPA and LPC.•Dexamethasone, TNFα and IL-1β increase ATX protein levels in TM cells.•ATX plays a crucial role in dexamethasone, TNFα and IL-1β induced fibrogenic response in TM cells.•LPC induces fibrogenic response in TM cells in an ATX-dependent manner.•ATX inhibitor increases aqueous outflow facility in mouse eyes.
Chronic inflammation and blood-brain barrier dysfunction are key pathological hallmarks of neurological disorders such as multiple sclerosis, Alzheimer's disease and Parkinson's disease. Major ...drivers of these pathologies include pro-inflammatory stimuli such as prostaglandins, which are produced in the central nervous system by the oxidation of arachidonic acid in a reaction catalyzed by the cyclooxygenases COX1 and COX2. Monoacylglycerol lipase hydrolyzes the endocannabinoid signaling lipid 2-arachidonyl glycerol, enhancing local pools of arachidonic acid in the brain and leading to cyclooxygenase-mediated prostaglandin production and neuroinflammation. Monoacylglycerol lipase inhibitors were recently shown to act as effective anti-inflammatory modulators, increasing 2-arachidonyl glycerol levels while reducing levels of arachidonic acid and prostaglandins, including PGE.sub.2 and PGD.sub.2 . In this study, we characterized a novel, highly selective, potent and reversible monoacylglycerol lipase inhibitor (MAGLi 432) in a mouse model of lipopolysaccharide-induced blood-brain barrier permeability and in both human and mouse cells of the neurovascular unit: brain microvascular endothelial cells, pericytes and astrocytes. We confirmed the expression of monoacylglycerol lipase in specific neurovascular unit cells in vitro, with pericytes showing the highest expression level and activity. However, MAGLi 432 did not ameliorate lipopolysaccharide-induced blood-brain barrier permeability in vivo or reduce the production of pro-inflammatory cytokines in the brain. Our data confirm monoacylglycerol lipase expression in mouse and human cells of the neurovascular unit and provide the basis for further cell-specific analysis of MAGLi 432 in the context of blood-brain barrier dysfunction caused by inflammatory insults.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Mutations in glucocerebrosidase cause the lysosomal storage disorder Gaucher's disease and are the most common risk factor for Parkinson's disease. Therapies to restore the enzyme's function in the ...brain hold great promise for treating the neurological implications. Thus, we developed blood-brain barrier penetrant therapeutic molecules by fusing transferrin receptor-binding moieties to β-glucocerebrosidase (referred to as GCase-BS). We demonstrate that these fusion proteins show significantly increased uptake and lysosomal efficiency compared to the enzyme alone. In a cellular disease model, GCase-BS rapidly rescues the lysosomal proteome and lipid accumulations beyond known substrates. In a mouse disease model, intravenous injection of GCase-BS leads to a sustained reduction of glucosylsphingosine and can lower neurofilament-light chain plasma levels. Collectively, these findings demonstrate the potential of GCase-BS for treating GBA1-associated lysosomal dysfunction, provide insight into candidate biomarkers, and may ultimately open a promising treatment paradigm for lysosomal storage diseases extending beyond the central nervous system.
Monoacylglycerol lipase (MAGL) regulates endocannabinoid 2-arachidonoylglycerol (2-AG) and eicosanoid signalling. MAGL inhibition provides therapeutic opportunities but clinical potential is limited ...by central nervous system (CNS)-mediated side effects. Here, we report the discovery of LEI-515, a peripherally restricted, reversible MAGL inhibitor, using high throughput screening and a medicinal chemistry programme. LEI-515 increased 2-AG levels in peripheral organs, but not mouse brain. LEI-515 attenuated liver necrosis, oxidative stress and inflammation in a CCl
-induced acute liver injury model. LEI-515 suppressed chemotherapy-induced neuropathic nociception in mice without inducing cardinal signs of CB
activation. Antinociceptive efficacy of LEI-515 was blocked by CB
, but not CB
, antagonists. The CB
antagonist rimonabant precipitated signs of physical dependence in mice treated chronically with a global MAGL inhibitor (JZL184), and an orthosteric cannabinoid agonist (WIN55,212-2), but not with LEI-515. Our data support targeting peripheral MAGL as a promising therapeutic strategy for developing safe and effective anti-inflammatory and analgesic agents.
The endocannabinoid system (ECS) is critically involved in the pathophysiology of Multiple Sclerosis (MS), a neuroinflammatory and neurodegenerative disease of the central nervous system (CNS). Over ...the past decade, researchers have extensively studied the neuroprotective and anti-inflammatory effects of the ECS. Inhibiting the degradation of the endocannabinoid 2-arachidonoylglycerol (2-AG) has emerged as a promising strategy to mitigate brain damage in MS.
In this study, we investigated the effects of a novel reversible MAGL inhibitor (MAGLi 432) on C57/BL6 female mice with experimental autoimmune encephalomyelitis (EAE), a model of MS. We assessed its implications on motor disability, neuroinflammation, and synaptic dysfunction. Systemic in vivo treatment with MAGLi 432 resulted in a less severe EAE disease, accompanied by increased 2-AG levels and decreased levels of arachidonic acid (AA) and prostaglandins (PGs) in the brain. Additionally, MAGLi 432 reduced both astrogliosis and microgliosis, as evidenced by decreased microglia/macrophage density and a less reactive morphology. Flow cytometry analysis further revealed fewer infiltrating CD45+ and CD3+ cells in the brains of MAGLi 432-treated EAE mice. Finally, MAGLi treatment counteracted the striatal synaptic hyperexcitability promoted by EAE neuroinflammation.
In conclusion, MAGL inhibition significantly ameliorated EAE clinical disability and striatal inflammatory synaptopathy through potent anti-inflammatory effects. These findings provide new mechanistic insights into the neuroprotective role of the ECS during neuroinflammation and highlight the therapeutic potential of MAGLi-based drugs in mitigating MS-related inflammatory and neurodegenerative brain damage.
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•Intraperitoneal treatment with MAGLi 432 (inhibitor of 2-AG degradation) induces the increase of 2-AG levels into the brain.•In vivo treatment with MAGLi 432 ameliorates the clinical score and the synaptic hyperexcitability typical of EAE.•MAGLi 432 ameliorates striatal inflammation and attenuates the infiltration of CD45+ and CD3+ cells in the brain of EAE mice.
Colonization with Staphylococci is widely distributed among patients with end-stage renal disease who are receiving hemodialysis (HD). In addition to more intensive care and use of artificial ...devices, the incidence of methicillin-resistant Staphylococcus aureus (MRSA) colonization and infection has increased. Such colonization has recently been associated with a more than doubled mortality rate in HD patients. However, it is not clear whether the (presumably increasing) incidence of methicillin-sensitive Staphylococcus aureus (MSSA) colonization is associated with MRSA and/or morbidity and mortality. We therefore established a screening program in our HD population (n=156) and followed these patients over 10 years. We discovered eighty-eight MSSA-colonized patients and one MRSA-colonized patient by cross-sectional and admission-related screenings between 2000 and 2010. The morbidity and mortality of the HD patients was not related to MSSA colonization. The MSSA colonization rate decreased slightly during the 10-year observation period. We conclude that the incidence of MRSA colonization in our unit was lower compared to that reported in the literature. The reasons for this finding are complex and require further investigation. The incidence of MSSA colonization was frequent but did not impact morbidity or mortality.
Detailed information on the metabolic fate of lead compounds can be a powerful tool for an informed approach to the stabilization of metabolically labile compounds in the lead optimization phase. The ...combination of high performance liquid chromatography (HPLC) with nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) has been used to give comprehensive structural data on metabolites of novel drugs in development. Recently, increased automation and the embedding of on‐line solid‐phase extraction (SPE) into a integrated LC‐SPE‐NMR‐MS system have improved enormously the detection limits of this approach. The new technology platform allows the analysis of complex mixtures from microsome incubations, combining low material requirements with relatively high throughput. Such characteristics make it possible to thoroughly characterize metabolites of selected compounds at earlier phases along the path to lead identification and clinical candidate selection, thus providing outstanding guidance in the process of eliminating undesired metabolism and detecting active or potentially toxic metabolites. Such an approach was applied at the lead identification stage of a backup program on metabotropic glutamate receptor 5 (mGlu5) allosteric inhibition. The major metabolites of a lead 5‐aminothiazole‐4‐carboxylic acid amide 1 were synthesized and screened, revealing significant in vitro activity and possible involvement in the overall pharmacodynamic behavior of 1. The information collected on the metabolism of the highly active compound 1 was pivotal to the synthesis of related compounds with improved microsomal stability.
LC‐SPE‐NMR‐MS. The combination of high performance liquid chromatography, on‐line solid‐phase extraction, NMR and MS spectroscopy into an integrated LC‐SPE‐NMR‐MS system has been used to give comprehensive structural data on the major metabolites of a lead metabotropic glutamate receptor 5 (mGlu5) allosteric antagonist.
We experimentally tested the costs of deep torpor at low temperatures by comparing telomere dynamics in two species of rodents hibernating at either 3 or 14°C. Our data show that hibernators kept at ...the warmer temperature had higher arousal frequencies, but maintained longer telomeres than individuals hibernating at the colder temperature. We suggest that the high-energy demand of frequent arousals is counteracted by a lower temperature differential between torpid and euthermic body temperature and that telomere length is restored during arousals when the body temperature is returned to normothermic values. Taken together, our study shows that hibernation at low body temperatures comes with costs on a cellular level and that hibernators need to actively counterbalance the shortening of telomeres.
Background: Previous research reveals an association between traumatic stress and an increased risk for numerous diseases, including cancer. At the molecular level, stress may increase carcinogenesis ...via increased DNA damage and impaired DNA repair mechanisms. We assessed DNA breakage in peripheral blood mononuclear cells from individuals with post-traumatic stress disorder (PTSD) and measured the cellular capacity to repair single-strand breaks after exposure to ionizing X-radiation. We also investigated the effect of psychotherapy on both DNA breakage and DNA repair. Methods: In a first study we investigated DNA breakage and repair in 34 individuals with PTSD and 31 controls. Controls were subdivided into 11 trauma-exposed subjects and 20 individuals without trauma exposure. In a second study, we analysed the effect of psychotherapy (Narrative Exposure Therapy) on DNA breakage and repair. Thirty-eight individuals with PTSD were randomly assigned to either a treatment or a waitlist control condition. Follow-up was performed 4 months and 1 year after therapy. Results: In study 1 we found higher levels of basal DNA breakage in individuals with PTSD and trauma-exposed subjects than in controls, indicating that traumatic stress is associated with DNA breakage. However, single-strand break repair was unimpaired in individuals with PTSD. In study 2, we found that psychotherapy reversed not only PTSD symptoms, but also DNA strand break accumulation. Conclusion: Our results show - for the first time in vivo - an association between traumatic stress and DNA breakage; they also demonstrate changes at the molecular level, i.e., the integrity of DNA, after psychotherapeutic interventions.
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