sirolimus and Parkinson-Disease

The role of autophagy in cell survival and suppression of neurodegeneration was considered. We discussed its involvement in Alzheimer's, Parkinson's, and Huntington's diseases connected with accumulation of amy- loid-β, α-synuclein, and huntingtin, respectively. Autophagy is reduced in these diseases and in aging as well to various extent. Elimination of accumulated toxic proteins and structures is performed by autophagy mech- anisms (chaperon-mediated autophagy, macroautophagy, selected autophagy) in an interaction with ubiqui- tin-proteasome system. In many cases activation of mTOR-dependent autophagy and mTOR-independent regulatory pathways lead to the therapeutic effect of inhibition of neurodegeneration in cell cultures and an- imal models. Some autophagy enhancers such as resveratrol, metformin, rilmenidine, lithium, and curcumin are tested now in clinical trials.

Topics: alpha-Synuclein; Alzheimer Disease; Amyloid beta-Peptides; Animals; Autophagy; Clinical Trials as Topic; Gene Expression Regulation; Humans; Huntingtin Protein; Huntington Disease; Metformin; Molecular Chaperones; Molecular Targeted Therapy; Neuroprotective Agents; Parkinson Disease; Proteasome Endopeptidase Complex; Sirolimus; TOR Serine-Threonine Kinases; Ubiquitin

Parkinson's disease (PD), a common neurodegenerative disorder caused by the loss of the dopaminergic input to the basal ganglia, is commonly treated with l-DOPA. Use of this drug, however, is severely limited by the development of dystonic and choreic motor complications, or dyskinesia. This chapter describes the molecular mechanisms implicated in the emergence and manifestation of l-DOPA-induced dyskinesia (LID). Particular emphasis is given to the role played in this condition by abnormalities in signal transduction at the level of the medium spiny neurons (MSNs) of the striatum, which are the principal target of l-DOPA. Recent evidence pointing to pre-synaptic dysregulation is also discussed.

Topics: Amantadine; Animals; Antiparkinson Agents; Basal Ganglia; Dyskinesia, Drug-Induced; Genes, Immediate-Early; Humans; Levodopa; Neurons; Parkinson Disease; Receptor, Cannabinoid, CB1; Receptors, Dopamine; Receptors, N-Methyl-D-Aspartate; Receptors, Serotonin; Signal Transduction; Sirolimus

After kidney transplantation neurologic manifestations may develop, including Parkinson's disease (PD). An enlarged substantia nigra (SN) by transcranial sonography has been recognized as a marker of PD.. In renal transplant recipients (RTRs = 95) and controls (n = 20), measurement of mesencephalon, SN, third ventricle, spleen and carotid intima-media thickness (cIMT) and middle cerebral artery (MCA), kidney and spleen arteries Doppler resistive index (RI) were performed.. RTRs had larger SN, third ventricle and cIMT and higher renal RI than controls. The SN was larger in the CNIs group than in controls and rapamycin group, while the third ventricle was similar between patients but larger than in controls. In RTRs, SN showed a direct linear correlation with spleen and the third ventricle with age, cIMT and RI of the MCA, kidney and spleen. In CNIs group the SN correlated positively with age and cIMT, while the third ventricle reproduced RTRs correlations. Rapamycin group showed a direct linear relationship between the third ventricle and age and RI of the MCA, kidney and spleen; SN showed no correlations.. RTRs on CNIs present a larger SN area than on rapamycin, probably due to the antiproliferative effect of rapamycin. This finding might be relevant when interpreting TCS in RTRs.

Topics: Calcineurin Inhibitors; Carotid Intima-Media Thickness; Humans; Kidney Transplantation; Parkinson Disease; Sirolimus; Substantia Nigra; Ultrasonography, Doppler, Transcranial

We studied the possibilities of inhibition of neurodegeneration in MPTP-induced model of Parkinson's disease (PD) in C57Bl/6J mice and transgenic model of early PD stage (5-monthold B6.Cg-Tg(Prnp-SNCA*A53T)23Mkle/J mice) by autophagy activation through mTOR-dependent and mTOR-independent pathways with rapamycin and trehalose, respectively. Therapy with autophagy inducers in a "postponed" mode (7 days after MPTP intoxication) restored the expression of the dopaminergic neuron marker tyrosine hydroxylase and markedly improved cognitive function in the conditioned passive avoidance response (CPAR; fear memory). The transgenic model also showed an increase in the expression of tyrosine hydroxylase in the nigrostriatal system of the brain. An enhanced therapeutic effect of the combined treatment with the drugs was revealed on the expression of tyrosine hydroxylase, but not in the CPAR test. Thus, activation of both pathways of autophagy regulation in PD models with weakened neuroinflammation can restore the dopaminergic function of neurons and cognitive activity in mice.

Topics: Adenine; Animals; Autophagy; Disease Models, Animal; Mice; Mice, Inbred C57BL; Mice, Transgenic; MTOR Inhibitors; Neuroinflammatory Diseases; Neuroprotective Agents; Parkinson Disease; Parkinson Disease, Secondary; Signal Transduction; Sirolimus; Substantia Nigra; TOR Serine-Threonine Kinases; Trehalose

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the progressive loss of dopaminergic neurons in the substantia nigra (SN) pars compacta region of the brain. The main pathological hallmark involves cytoplasmic inclusions of α-synuclein and mitochondrial dysfunction, which is observed in other part of the central nervous system other than SN suggesting the spread of pathogenesis to bystander neurons. The inter-neuronal communication through exosomes may play an important role in the spread of the disease; however, the mechanisms are not well elucidated. Mitochondria and its role in inter-organellar crosstalk with multivesicular body (MVB) and lysosome and its role in modulation of exosome release in PD is not well understood. In the current study, we investigated the mitochondria-lysosome crosstalk modulating the exosome release in neuronal and glial cells. We observed that PD stress showed enhanced release of exosomes in dopaminergic neurons and glial cells. The PD stress condition in these cells showed fragmented network and mitochondrial dysfunction which further leads to functional deficit of lysosomes and hence inhibition of autophagy flux. Neuronal and glial cells treated with rapamycin showed enhanced autophagy and inhibited the exosomal release. The results here suggest that maintenance of mitochondrial function is important for the lysosomal function and hence exosomal release which is important for the pathogenesis of PD.

Topics: Autophagy; Cell Line, Tumor; Exosomes; Humans; Lysosomes; Mitochondria; Parkinson Disease; Sirolimus; Stress, Physiological

Parkinson's disease is the second major neurodegenerative diseases secondarily to Alzheimer's disease. Rapamycin is a fermentation product, which derived from Streptomyces hygroscopius. The aim of this study is to investigate the effect of rapamycin and its potential mechanisms on the acute attack of 1-methyl-4-phenyl-1,2,3,6-four hydrogen pyridine (MPTP) induced Parkinson's disease (PD) in mice.. PD model was established by intraperitoneal injection of MPTP for 5 days. The effect of intraperitoneal injection of rapamycin for treating the symptoms caused by PD was evaluated by behavior observation and HE pathological section. In order to understand the possible mechanism, immunofluorescence and immune precipitation mainly analyzes were used to measure the expression of critical protein p-4ebp1 in mammalian target of rapamycin (mTOR) signaling pathways in the striatum and substantia nigra.. Rapamycin can effectively alleviate symptoms of PD. The levels of key protein p-4EBP1 in the striatum and substantia nigra were both significantly higher in PD group compared with control group (P<0.01), while being pretreated with rapamycin, the expression of p-4EBP1 in the striatum and substantia nigra were both decreased obviously (P<0.01).. p-4EBP1 protein may be involved in the pathogenesis of PD via mTOR signaling pathway. Inhibited mTOR-4EBP1 pathways could make a certain protective effect for the acute attack of PD induced by MPTP.

Topics: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; Animals; Mice; Mice, Inbred C57BL; Parkinson Disease; Sirolimus; Substantia Nigra

The neuroprotective effect of autophagy activation by rapamycin and trehalose was studied in a mouse model of Parkinson's disease (PD) induced by neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Both rapamycin (10 mg/kg/day, 7 days) and trehalose (2% in drinking water, 7 days) increased the expression of LC3-II (a marker of autophagy activation) in the frontal cortex and striatum of normal C57Bl/6J mice, with signs of an additive effect. Autophagy stimulation in the striatum was confirmed by a lysosomal osmotic test. In the model of MPTP-induced PD, the two drugs were applied starting from the 2nd day after subchronic daily MPTP administration (20 mg/kg/day, 4 days). A marked increase in LC3-II expression in the striatum was detected under the action of trehalose and in the S. nigra after combined treatment with rapamycin and trehalose. The drugs had a positive effect for recovery of dopaminergic neurons and neuroprotection after MPTP-induced PD-like injury. The therapeutic effect was proven by active restoration of tyrosine hydroxylase (TH) content in the striatum and S. nigra and by improved cognition measured by the passive avoidance learning task. The results revealed the additive effect of the combined treatment with rapamycin and trehalose on dopaminergic deficits (according to the levels of TH expression in the nigrostriatal system) but not on the behavioral performance in the mouse PD model. Thus, the autophagy activation through different pathways by the combination of rapamycin and trehalose reverses both neuronal dopaminergic and behavioral deficits in vivo and seems to be a promising therapy for PD-like pathology.

Topics: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; Animals; Autophagy; Behavior, Animal; Cognition; Corpus Striatum; Disease Models, Animal; Dopamine; Dopaminergic Neurons; Drug Therapy, Combination; Male; Mice; Mice, Inbred C57BL; Microtubule-Associated Proteins; MPTP Poisoning; Neuroprotective Agents; Neurotoxins; Parkinson Disease; Sirolimus; Substantia Nigra; Trehalose; Tyrosine 3-Monooxygenase

Cell-to-cell propagation of aggregated alpha synuclein (aSyn) has been suggested to play an important role in the progression of alpha synucleinopathies. A critical step for the propagation process is the accumulation of extracellular aSyn within recipient cells. Here, we investigated the trafficking of distinct exogenous aSyn forms and addressed the mechanisms influencing their accumulation in recipient cells. The aggregated aSyn species (oligomers and fibrils) exhibited more pronounced accumulation within recipient cells than aSyn monomers. In particular, internalized extracellular aSyn in the aggregated forms was able to seed the aggregation of endogenous aSyn. Following uptake, aSyn was detected along endosome-to-lysosome and autophagosome-to-lysosome routes. Intriguingly, aggregated aSyn resulted in lysosomal activity impairment, accompanied by the accumulation of dilated lysosomes. Moreover, analysis of autophagy-related protein markers suggested decreased autophagosome clearance. In contrast, the endocytic pathway, proteasome activity, and mitochondrial homeostasis were not substantially affected in recipient cells. Our data suggests that extracellularly added aggregated aSyn primarily impairs lysosomal activity, consequently leading to aSyn accumulation within recipient cells. Importantly, the autophagy inducer trehalose prevented lysosomal alterations and attenuated aSyn accumulation within aSyn-exposed cells. Our study underscores the importance of lysosomes for the propagation of aSyn pathology, thereby proposing these organelles as interventional targets.

Topics: alpha-Synuclein; Animals; Autophagy; Cell Line, Tumor; Escherichia coli; Glioma; Humans; Lysosomes; Neurons; Parkinson Disease; Protein Aggregation, Pathological; Rats; Rats, Wistar; Recombinant Proteins; Sirolimus; Trehalose

Post-translational modification on protein Ser/Thr residues by O-linked attachment of ß-N-acetyl-glucosamine (O-GlcNAcylation) is a key mechanism integrating redox signaling, metabolism and stress responses. One of the most common neurodegenerative diseases that exhibit aberrant redox signaling, metabolism and stress response is Parkinson's disease, suggesting a potential role for O-GlcNAcylation in its pathology. To determine whether abnormal O-GlcNAcylation occurs in Parkinson's disease, we analyzed lysates from the postmortem temporal cortex of Parkinson's disease patients and compared them to age matched controls and found increased protein O-GlcNAcylation levels. To determine whether increased O-GlcNAcylation affects neuronal function and survival, we exposed rat primary cortical neurons to thiamet G, a highly selective inhibitor of the enzyme which removes the O-GlcNAc modification from target proteins, O-GlcNAcase (OGA). We found that inhibition of OGA by thiamet G at nanomolar concentrations significantly increased protein O-GlcNAcylation, activated MTOR, decreased autophagic flux, and increased α-synuclein accumulation, while sparing proteasomal activities. Inhibition of MTOR by rapamycin decreased basal levels of protein O-GlcNAcylation, decreased AKT activation and partially reversed the effect of thiamet G on α-synuclein monomer accumulation. Taken together we have provided evidence that excessive O-GlcNAcylation is detrimental to neurons by inhibition of autophagy and by increasing α-synuclein accumulation.

Topics: alpha-Synuclein; Animals; Autophagy; Cells, Cultured; Glucosamine; Glycosylation; Homeostasis; Humans; Models, Biological; Neurons; Parkinson Disease; Phosphorylation; Postmortem Changes; Proto-Oncogene Proteins c-akt; Pyrans; Rats, Sprague-Dawley; Signal Transduction; Sirolimus; Thiazoles; TOR Serine-Threonine Kinases

Rapamycin protects mice against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced loss of dopaminergic neurons, which is an established model for Parkinson's disease. We demonstrated that rapamycin preserves astrocytic expression of glutamate transporters and glutamate reuptake. The protective effect was also observed in astrocyte cultures, indicating that rapamycin acts directly on astrocytes. In the MPTP model, rapamycin caused reduced expression of the E3 ubiquitin ligase Nedd4-2 (neuronal precursor cell expressed developmentally downregulated 4-2) and reduced colocalization of glutamate transporters with ubiquitin. Rapamycin increased interleukin-6 (IL-6) expression, which was associated with reduced expression of inflammatory cytokines, indicating anti-inflammatory properties of IL-6 in the MPTP model. NF-κB was shown to be a key mediator for rapamycin, whereas Janus kinase 2, signal transducer and activator of transcription 3, phosphoinositide 3-kinase, and Akt partially mediated rapamycin effects in astrocytes. These results demonstrate for the first time in a Parkinson's disease animal model that the neuroprotective effects of rapamycin are associated with glial and anti-inflammatory effects.

Topics: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; Amino Acid Transport System X-AG; Animals; Astrocytes; Cytokines; Disease Models, Animal; Down-Regulation; Glutamates; Inflammation; Interleukin-6; Janus Kinase 2; Male; Mice; Mice, Inbred C57BL; Neuroprotective Agents; NF-kappa B; Parkinson Disease; Phosphatidylinositol 3-Kinases; Sirolimus; STAT3 Transcription Factor; Up-Regulation

Parkinson's disease (PD) is the second most prevalent neurodegenerative disease among aging individuals, affecting greatly the quality of their life. However, the pathogenesis of Parkinson's disease is still incompletely understood to date. Increasing experimental evidence suggests that cell cycle reentry of postmitotic neurons precedes many instances of neuronal death. Since cell cycle dysfunction is not restricted to neurons, we investigated this issue in peripheral cells from patients suffering from sporadic PD and age-matched control individuals. Here, we describe increased cell cycle activity in immortalized lymphocytes from PD patients that is associated to enhanced activity of the cyclin D3/CDK6 complex, resulting in higher phosphorylation of the pRb family protein and thus, in a G1/S regulatory failure. Decreased degradation of cyclin D3, together with increased p21 degradation, as well as elevated levels of CDK6 mRNA and protein were found in PD lymphoblasts. Inhibitors of cyclin D3/CDK6 activity like sodium butyrate, PD-332991, and rapamycin were able to restore the response of PD cells to serum stimulation. We conclude that lymphoblasts from PD patients are a suitable model to investigate cell biochemical aspects of this disease. It is suggested that cyclin D3/CDK6-associated kinase activity could be potentially a novel therapeutic target for the treatment of PD.

Topics: Aged; Aged, 80 and over; Butyric Acid; Case-Control Studies; Cell Cycle Checkpoints; Cell Cycle Proteins; Cell Proliferation; Cyclin-Dependent Kinases; DNA-Binding Proteins; G1 Phase; Humans; Lymphocytes; Middle Aged; Parkinson Disease; Phosphorylation; Piperazines; Proteolysis; Pyridines; RNA, Messenger; S Phase; Sirolimus; Subcellular Fractions; Time Factors

Parkinson's disease is the most common movement disorder, characterized by a progressive and extensive loss of dopaminergic neurons in the substantia nigra pars compacta and their terminals in the striatum. So far, only symptomatic treatment is available, and no cure or disease-modifying drugs exist. The present study was designed to investigate the neuroprotective effect of rapamycin, an autophagy inducer, on dopaminergic neurons against rotenone-induced cell death in primary mesencephalic cell culture.. Primary mesencephalic cell cultures were prepared from embryonic mouse mesencephala (OFI/SPF, Vienna, Austria) at gestation day 14. Four sets of cultures were treated as follows: one was run as an untreated control, a second one was treated with 20 nM rotenone on the 10th day in vitro (DIV) for 48 h, a third one was co-treated with 20 nM rotenone and rapamycin (1, 10, 100, 1000 nM) on the 10th DIV for 48 h, and a fourth one was treated with rapamycin alone (1, 10, 100, 1000 nM) on the 10th DIV for 48 h. On the 12th DIV, cultures were subjected to immunohistochemistry against tyrosine hydroxylase and to fluorescence staining using LysoTracker Deep Red, JC-1 and DAPI stains.. Exposure of such cultures to 20 nM rotenone on the 10th DIV for 48 h reduced the number of dopaminergic neurons by 41% and increased the release of lactate dehydrogenase (LDH) by 178% above untreated controls. Rapamycin (1, 10, 100, 1000 nM) added together with rotenone from the 10th to 12th DIV spared dopaminergic neurons by 17% and reduced the release of LDH by 64% at the concentration of 100 nM compared to rotenone-treated cultures. Activation of an autophagic process by rapamycin was demonstrated by LysoTracker Deep Red fluorescent dye, as indicated by a shift to increased red fluorescence. Rapamycin also significantly elevated the mitochondrial membrane potential (Δψm), as shown by an increase of the red:green fluorescence ratio of JC-1. Increased apoptotic cell death due to rotenone was lowered by rapamycin, as shown by the blue-fluorescent DAPI nucleic acid stain.. Our study indicates for the first time that rapamycin, known as an autophagy inducer, protected dopaminergic neurons against rotenone-induced cell death in primary mesencephalic cell culture.

Topics: Animals; Apoptosis; Cells, Cultured; Dopaminergic Neurons; Immunosuppressive Agents; Mesencephalon; Mice; Neuroprotective Agents; Parkinson Disease; Rotenone; Sirolimus; Uncoupling Agents

Parkinson's disease (PD) is a neurodegenerative disease, in which oxidative stress and mitochondrial dysfunction are responsible for neuronal apoptosis. Rapamycin plays a crucial role in reducing oxidative stress and protecting the mitochondria. However, its protective role in PD has not yet been fully elucidated. In this study, we report that pre-treatment with rapamycin provides behavioral improvements, protects against the loss of dopaminergic neurons, and alleviates mitochondrial ultrastructural injuries in a rat model of PD. Peroxide levels were lower and antioxidant activities were higher in PD rats pre-treated with rapamycin compared to the PD rats pre-treated with the vehicle. Furthermore, pre-treatment with rapamycin significantly elevated the expression of anti-apoptotic markers and reduced the levels of pro-apoptotic markers compared to pre-treatment with the vehicle. In conclusion, our results demonstrated that rapamycin reduced oxidative stress and alleviated mitochondrial injuries in the 6-hydroxydopamine (6-OHDA)-induced rat model of PD, which may subsequently contribute to its anti-apoptotic effects. The ability of rapamycin to exhibit neuroprotection in a rat model of PD may be related to its antioxidant capabilities.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Disease Models, Animal; Dopaminergic Neurons; Female; Mitochondria; Neuroprotective Agents; Oxidative Stress; Parkinson Disease; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Sprague-Dawley; Sirolimus

The development of dyskinesia upon chronic L-DOPA treatment is a major complication for the management of the motor symptoms in Parkinson's disease (PD) patients. Efforts are made to understand the underlying mechanisms and identify targets for the pharmacological alleviation of dyskinesia without affecting the therapeutic effect of L-DOPA. Previous studies have shown that the mTOR pathway is hyperactive in dyskinesia as a consequence of D1 receptor hypersensitivity. We investigated the effect of the FDA-approved mTOR inhibitor Temsirolimus (CCI-779), currently used in the clinic, on the development of LID and on the severity of already established LID in hemi-parkinsonian rats. Systemic delivery of CCI-779 prevented the development of LID and significantly alleviated the severity of dyskinesia in L-DOPA-primed animals. This was associated with a reduced activation of the mTOR pathway in striatal medium spiny neurons. Drugs with mTOR inhibiting activity that are actively developed in cancer research may be of interest for the management of LID in PD patients.

Topics: Animals; Antiparkinson Agents; Dyskinesia, Drug-Induced; Female; Levodopa; Parkinson Disease; Protein Kinase Inhibitors; Rats; Rats, Sprague-Dawley; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Mutations in LRRK2 are associated with familial and sporadic Parkinson's disease (PD). Subjects with PD caused by LRRK2 mutations show pleiotropic pathology that can involve inclusions containing α-synuclein, tau or neither protein. The mechanisms by which mutations in LRRK2 lead to this pleiotropic pathology remain unknown.. To investigate mechanisms by which LRRK2 might cause PD.. We used systems biology to investigate the transcriptomes from human brains, human blood cells and Caenorhabditis elegans expressing wild-type LRRK2. The role of autophagy was tested in lines of C. elegans expressing LRRK2, V337M tau or both proteins. Neuronal function was measured by quantifying thrashing.. Genes regulating autophagy were coordinately regulated with LRRK2. C. elegans expressing V337M tau showed reduced thrashing, as has been noted previously. Coexpressing mutant LRRK2 (R1441C or G2019S) with V337M tau increased the motor deficits. Treating the lines of C. elegans with an mTOR inhibitor that enhances autophagic flux, ridaforolimus, increased the thrashing behavior to the same level as nontransgenic nematodes.. These data support a role for LRRK2 in autophagy, raise the possibility that deficits in autophagy contribute to the pathophysiology of LRRK2, and point to a potential therapeutic approach addressing the pathophysiology of LRRK2 in PD.

Topics: Animals; Animals, Genetically Modified; Autophagy; Caenorhabditis elegans; Disease Models, Animal; Gene Expression Regulation; Humans; Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; Methionine; Mutation; Parkinson Disease; Protein Serine-Threonine Kinases; Sirolimus; tau Proteins; Valine

Parkinson's disease (PD) is a common neurodegenerative disorder caused by genetic and environmental factors that results in degeneration of the nigrostriatal dopaminergic pathway in the brain. We analyzed neural cells generated from induced pluripotent stem cells (iPSCs) derived from PD patients and presymptomatic individuals carrying mutations in the PINK1 (PTEN-induced putative kinase 1) and LRRK2 (leucine-rich repeat kinase 2) genes, and compared them to those of healthy control subjects. We measured several aspects of mitochondrial responses in the iPSC-derived neural cells including production of reactive oxygen species, mitochondrial respiration, proton leakage, and intraneuronal movement of mitochondria. Cellular vulnerability associated with mitochondrial dysfunction in iPSC-derived neural cells from familial PD patients and at-risk individuals could be rescued with coenzyme Q(10), rapamycin, or the LRRK2 kinase inhibitor GW5074. Analysis of mitochondrial responses in iPSC-derived neural cells from PD patients carrying different mutations provides insight into convergence of cellular disease mechanisms between different familial forms of PD and highlights the importance of oxidative stress and mitochondrial dysfunction in this neurodegenerative disease.

Topics: Humans; Indoles; Induced Pluripotent Stem Cells; Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; Mitochondria; Neurons; Parkinson Disease; Phenols; Protein Serine-Threonine Kinases; Sirolimus; Ubiquinone

Heterozygous mutations in the GBA1 gene elevate the risk of Parkinson disease and dementia with Lewy bodies; both disorders are characterized by misprocessing of α-synuclein (SNCA). A loss in lysosomal acid-β-glucosidase enzyme (GCase) activity due to biallelic GBA1 mutations underlies Gaucher disease. We explored mechanisms for the gene's association with increased synucleinopathy risk.. We analyzed the effects of wild-type (WT) and several GBA mutants on SNCA in cellular and in vivo models using biochemical and immunohistochemical protocols.. We observed that overexpression of all GBA mutants examined (N370S, L444P, D409H, D409V, E235A, and E340A) significantly raised human SNCA levels to 121 to 248% of vector control (p < 0.029) in neural MES23.5 and PC12 cells, but without altering GCase activity. Overexpression of WT GBA in neural and HEK293-SNCA cells increased GCase activity, as expected (ie, to 167% in MES-SNCA, 128% in PC12-SNCA, and 233% in HEK293-SNCA; p < 0.002), but had mixed effects on SNCA. Nevertheless, in HEK293-SNCA cells high GCase activity was associated with SNCA reduction by ≤32% (p = 0.009). Inhibition of cellular GCase activity (to 8-20% of WT; p < 0.0017) did not detectably alter SNCA levels. Mutant GBA-induced SNCA accumulation could be pharmacologically reversed in D409V-expressing PC12-SNCA cells by rapamycin, an autophagy-inducer (≤40%; 10μM; p < 0.02). Isofagomine, a GBA chaperone, showed a related trend. In mice expressing two D409Vgba knockin alleles without signs of Gaucher disease (residual GCase activity, ≥20%), we recorded an age-dependent rise of endogenous Snca in hippocampal membranes (125% vs WT at 52 weeks; p = 0.019). In young Gaucher disease mice (V394Lgba+/+//prosaposin[ps]-null//ps-transgene), which demonstrate neurological dysfunction after age 10 weeks (GCase activity, ≤10%), we recorded no significant change in endogenous Snca levels at 12 weeks of age. However, enhanced neuronal ubiquitin signals and axonal spheroid formation were already present. The latter changes were similar to those seen in three week-old cathepsin D-deficient mice.. Our results demonstrate that GBA mutants promote SNCA accumulation in a dose- and time-dependent manner, thereby identifying a biochemical link between GBA1 mutation carrier status and increased synucleinopathy risk. In cell culture models, this gain of toxic function effect can be mitigated by rapamycin. Loss in GCase activity did not immediately raise SNCA concentrations, but first led to neuronal ubiquitinopathy and axonal spheroids, a phenotype shared with other lysosomal storage disorders.

Topics: alpha-Synuclein; Animals; Cathepsin D; Cell Line; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Gaucher Disease; Gene Expression Regulation; Glucosylceramidase; Green Fluorescent Proteins; Humans; Immunosuppressive Agents; Lewy Body Disease; Mice; Mice, Knockout; Mutagenesis, Site-Directed; Mutation; Parkinson Disease; Rats; Sirolimus; Transfection

We report that rapamycin, an allosteric inhibitor of certain but not all actions of the key cellular kinase mammalian target of rapamycin (mTOR), protects neurons from death in both cellular and animal toxin models of Parkinson's disease (PD). This protective action appears to be attributable to blocked translation of RTP801/REDD1/Ddit4, a protein that is induced in cell and animal models of PD and in affected neurons of PD patients and that causes neuron death by leading to dephosphorylation of the survival kinase Akt. In support of this mechanism, in PD models, rapamycin spares phosphorylation of Akt at a site critical for maintenance of its survival-promoting activity. The capacity of rapamycin to provide neuroprotection in PD models appears to arise from its selective suppression of some but not all actions of mTOR, as indicated by the contrasting finding that Torin1, a full catalytic mTOR inhibitor, is not protective and induces Akt dephosphorylation and neuron death.

Topics: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; Analysis of Variance; Animals; Cell Death; Cycloheximide; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Administration Schedule; Enzyme Inhibitors; Gene Expression Regulation; Green Fluorescent Proteins; Humans; Intracellular Signaling Peptides and Proteins; Male; Mice; Mice, Inbred C57BL; Nerve Growth Factor; Neurons; Neuroprotective Agents; Oxidopamine; Parkinson Disease; Parkinsonian Disorders; PC12 Cells; Protein Serine-Threonine Kinases; Rats; Serine; Sirolimus; Time Factors; TOR Serine-Threonine Kinases; Transcription Factors; Transfection; Tyrosine 3-Monooxygenase

Parkinson disease is caused by the progressive loss of dopamine innervation to the basal ganglia and is commonly treated with the dopamine precursor, L-DOPA. Prolonged administration of L-DOPA results in the development of severe motor complications or dyskinesia, which seriously hamper its clinical use. Recent evidence indicates that L-DOPA-induced dyskinesia (LID) is associated with persistent activation of the mammalian target of rapamycin complex 1 (mTORC1) in the medium spiny neurons (MSNs) of the striatum, the main component of the basal ganglia. This phenomenon is secondary to the development of a strong sensitization at the level of dopamine D1 receptors, which are abundantly expressed in a subset of MSNs. Such sensitization confers to dopaminergic drugs (including L-DOPA) the ability to activate the extracellular signal-regulated protein kinases 1/2, which, in turn promote mTORC1 signaling. Using a mouse model of LID, we recently showed that administration of the allosteric mTORC1 inhibitor, rapamycin, reduces dyskinesia. This finding is discussed with respect to underlying mechanisms and potential significance for the development of future therapeutic interventions.

Topics: Animals; Anti-Bacterial Agents; Antiparkinson Agents; Basal Ganglia; Dyskinesia, Drug-Induced; Levodopa; Mechanistic Target of Rapamycin Complex 1; Mice; Multiprotein Complexes; Parkinson Disease; Proteins; Receptors, Dopamine D1; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

The ubiquitin-proteasome system (UPS) and the autophagy-lysosomal pathway (ALP) are the two most important cellular mechanisms for protein degradation. To investigate the role of autophagy in reversing neuronal injury, the proteasome inhibitor lactacystin was used to cause UPS dysfunction in differentiated PC12 cells and in C57BL/6 mice and rapamycin was used as an autophagy enhancer. The results showed that rapamycin pre-treatment attenuated lactacystin-induced apoptosis and reduced lactacystin-induced ubiquitinated protein aggregation in differentiated PC12 cells. The observed protection was partially blocked by the autophagy inhibitor 3-methyladenine. Furthermore, post-treatment of mice with rapamycin significantly attenuated lactacystin-induced loss of nigral DA neurons and the reduction of striatal DA levels. The lactacystin-induced high molecular ubiquitinated proteins were also attenuated by rapamycin treatment in vivo. In addition, as a chemical compound, rapamycin caused an increase of bcl2 protein level and blocked the release of cytochrome c from mitochondria to cytosal. We concluded that the neuroprotective effect of rapamycin is partially mediated by autophagy enhancement through enhanced degradation of misfolded proteins and autophagy enhancement may be considered to be a promising strategy to prevent diseases associated with misfolded/aggregated proteins, such as Parkinson's disease.

Topics: Acetylcysteine; Animals; Autophagy; Cells, Cultured; Male; Mice; Mice, Inbred C57BL; Nerve Degeneration; Neuroprotective Agents; Parkinson Disease; PC12 Cells; Protein Folding; Rats; Sirolimus

Trehalose, a disaccharide present in many non-mammalian species, protects cells against various environmental stresses. Whereas some of the protective effects may be explained by its chemical chaperone properties, its actions are largely unknown. Here we report a novel function of trehalose as an mTOR-independent autophagy activator. Trehalose-induced autophagy enhanced the clearance of autophagy substrates like mutant huntingtin and the A30P and A53T mutants of alpha-synuclein, associated with Huntington disease (HD) and Parkinson disease (PD), respectively. Furthermore, trehalose and mTOR inhibition by rapamycin together exerted an additive effect on the clearance of these aggregate-prone proteins because of increased autophagic activity. By inducing autophagy, we showed that trehalose also protects cells against subsequent pro-apoptotic insults via the mitochondrial pathway. The dual protective properties of trehalose (as an inducer of autophagy and chemical chaperone) and the combinatorial strategy with rapamycin may be relevant to the treatment of HD and related diseases, where the mutant proteins are autophagy substrates.

Topics: alpha-Synuclein; Animals; Antibiotics, Antineoplastic; Autophagy; Chlorocebus aethiops; COS Cells; HeLa Cells; Humans; Huntingtin Protein; Huntington Disease; Mice; Molecular Chaperones; Mutation; Nerve Tissue Proteins; Nuclear Proteins; Parkinson Disease; Protein Kinases; Sirolimus; TOR Serine-Threonine Kinases; Trehalose

The immunosuppressive drugs tacrolimus (TAC) and rapamycin (RAPA) have both been found to have neuroprotective effects on dopaminergic neurons. The purpose of the present study was to investigate whether liposomal formulations of these drugs administered directly into the brain improve cell survival and fiber outgrowth. Rats with unilateral 6-hydroxydopamine lesions were transplanted with 800,000 fetal rat ventral mesencephalic cells and randomly divided to one of four groups. Group 1 received a transplant containing cells only; group 2 received a cell suspension containing 0.68 microM liposomal RAPA (LRAPA); group 3 received a cell suspension containing 2.0 microM liposomal TAC (LTAC); and group 4 received a cell suspension containing a liposomal formulation of both 0.68 microM RAPA and 2.0 microM TAC (LRAPATAC). Rats were sacrificed after 6 weeks, and cell survival and fiber outgrowth were assessed using tyrosine hydroxylase (TH) immunohistochemistry. The animals receiving a cell suspension containing either LTAC or LRAPATAC were found to have significantly more surviving TH-immunoreactive (TH-ir) cells than the control group receiving cells only. The group receiving LTAC had significantly longer fibers, the group receiving LRAPA had significantly more fibers close to the graft, and the group receiving LRAPATAC had significantly more fibers at all distances. This study shows the feasibility of using liposomal formulations of neuroimmunophilins directly in the brain at the time of implantation to improve graft survival and fiber outgrowth. Furthermore, we have shown that the combination of LTAC and LRAPA has a synergistic effect. These compounds may play an important role in optimizing graft survival and host reinnervation in cell-mediated brain repair strategies for the treatment of neurological conditions.

Topics: Adrenergic Agents; Animals; Brain; Cell Growth Processes; Cell Survival; Cell Transplantation; Dopamine; Female; Immunohistochemistry; Immunosuppressive Agents; Liposomes; Mesencephalon; Neurons; Neuroprotective Agents; Oxidopamine; Parkinson Disease; Rats; Rats, Wistar; Sirolimus; Tacrolimus; Tyrosine 3-Monooxygenase; Water