GCH1 Deficiency Activates Brain Innate Immune Response and Impairs Tyrosine Hydroxylase Homeostasis.

The Parkinson's disease (PD) risk gene GTP cyclohydrolase 1 (GCH1) catalyzes the rate-limiting step in tetrahydrobiopterin (BH4) synthesis, an essential cofactor in the synthesis of monoaminergic neurotransmitters. To investigate the mechanisms by which GCH1 deficiency may contribute to PD, we generated a loss of function zebrafish gch1 mutant (gch1-/-), using CRISPR/Cas technology. gch1-/- zebrafish develop marked monoaminergic neurotransmitter deficiencies by 5 d postfertilization (dpf), movement deficits by 8 dpf and lethality by 12 dpf. Tyrosine hydroxylase (Th) protein levels were markedly reduced without loss of ascending dopaminergic (DAergic) neurons. L-DOPA treatment of gch1-/- larvae improved survival without ameliorating the motor phenotype. RNAseq of gch1-/- larval brain tissue identified highly upregulated transcripts involved in innate immune response. Subsequent experiments provided morphologic and functional evidence of microglial activation in gch1-/- The results of our study suggest that GCH1 deficiency may unmask early, subclinical parkinsonism and only indirectly contribute to neuronal cell death via immune-mediated mechanisms. Our work highlights the importance of functional validation for genome-wide association studies (GWAS) risk factors and further emphasizes the important role of inflammation in the pathogenesis of PD.SIGNIFICANCE STATEMENT Genome-wide association studies have now identified at least 90 genetic risk factors for sporadic Parkinson's disease (PD). Zebrafish are an ideal tool to determine the mechanistic role of genome-wide association studies (GWAS) risk genes in a vertebrate animal model. The discovery of GTP cyclohydrolase 1 (GCH1) as a genetic risk factor for PD was counterintuitive, GCH1 is the rate-limiting enzyme in the synthesis of dopamine (DA), mutations had previously been described in the non-neurodegenerative movement disorder dopa-responsive dystonia (DRD). Rather than causing DAergic cell death (as previously hypothesized by others), we now demonstrate that GCH1 impairs tyrosine hydroxylase (Th) homeostasis and activates innate immune mechanisms in the brain and provide evidence of microglial activation and phagocytic activity.

The Tyrosine Phosphatase hPTPRß Controls the Early Signals and Dopaminergic Cells Viability via the P2X7 Receptor.

ATP, one of the signaling molecules most commonly secreted in the nervous system and capable of stimulating multiple pathways, binds to the ionotropic purinergic receptors, in particular, the P2X7 receptor (P2X7R) and stimulates neuronal cell death. Given this effect of purinergic receptors on the viability of dopaminergic neurons model cells and that Ras GTPases control Erk1/2-regulated mitogen-activated cell proliferation and survival, we have investigated the role of the small GTPases of the Ras superfamily, together with their regulatory and effector molecules as the potential molecular intermediates in the P2X7R-regulated cell death of SN4741 dopaminergic neurons model cells. Here, we demonstrate that the neuronal response to purinergic stimulation involves the Calmodulin/RasGRF1 activation of the small GTPase Ras and Erk1/2. We also demonstrate that tyrosine phosphatase PTPRß and other tyrosine phosphatases regulate the small GTPase activation pathway and neuronal viability. Our work expands the knowledge on the intracellular responses of dopaminergic cells by identifying new participating molecules and signaling pathways. In this sense, the study of the molecular circuitry of these neurons is key to understanding the functional effects of ATP, as well as considering the importance of these cells in Parkinson's Disease.

The Parkinson's-disease-associated mutation LRRK2-G2019S alters dopaminergic differentiation dynamics via NR2F1.

Increasing evidence suggests that neurodevelopmental alterations might contribute to increase the susceptibility to develop neurodegenerative diseases. We investigate the occurrence of developmental abnormalities in dopaminergic neurons in a model of Parkinson's disease (PD). We monitor the differentiation of human patient-specific neuroepithelial stem cells (NESCs) into dopaminergic neurons. Using high-throughput image analyses and single-cell RNA sequencing, we observe that the PD-associated LRRK2-G2019S mutation alters the initial phase of neuronal differentiation by accelerating cell-cycle exit with a concomitant increase in cell death. We identify the NESC-specific core regulatory circuit and a molecular mechanism underlying the observed phenotypes. The expression of NR2F1, a key transcription factor involved in neurogenesis, decreases in LRRK2-G2019S NESCs, neurons, and midbrain organoids compared to controls. We also observe accelerated dopaminergic differentiation in vivo in NR2F1-deficient mouse embryos. This suggests a pathogenic mechanism involving the LRRK2-G2019S mutation, where the dynamics of dopaminergic differentiation are modified via NR2F1.

The LRRK2 G2019S mutation alters astrocyte-to-neuron communication via extracellular vesicles and induces neuron atrophy in a human iPSC-derived model of Parkinson's disease.

Astrocytes are essential cells of the central nervous system, characterized by dynamic relationships with neurons that range from functional metabolic interactions and regulation of neuronal firing activities, to the release of neurotrophic and neuroprotective factors. In Parkinson's disease (PD), dopaminergic neurons are progressively lost during the course of the disease, but the effects of PD on astrocytes and astrocyte-to-neuron communication remain largely unknown. This study focuses on the effects of the PD-related mutation LRRK2 G2019S in astrocytes generated from patient-derived induced pluripotent stem cells. We report the alteration of extracellular vesicle (EV) biogenesis in astrocytes and identify the abnormal accumulation of key PD-related proteins within multivesicular bodies (MVBs). We found that dopaminergic neurons internalize astrocyte-secreted EVs and that LRRK2 G2019S EVs are abnormally enriched in neurites and fail to provide full neurotrophic support to dopaminergic neurons. Thus, dysfunctional astrocyte-to-neuron communication via altered EV biological properties may participate in the progression of PD.

Extract of Polygala tenuifolia, Angelica tenuissima, and Dimocarpus longan Reduces Behavioral Defect and Enhances Autophagy in Experimental Models of Parkinson's Disease.

The 20% ethanol extract of Polygala tenuifolia, Angelica tenuissima, and Dimocarpus longan (WIN-1001X) was derived from a modified version of Korean traditional herbal formula 'Chungsimyeolda-tang' which has been used for the treatment of cerebrovascular disorders. The Parkinson's disease presents with impaired motor functions and loss of dopaminergic neurons. However, the treatment for Parkinson's disease is not established until now. This study aims to elucidate the therapeutic advantages of WIN-1001X on animal models of Parkinson's disease. WIN-1001X administration successfully relieved the Parkinsonism symptoms in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinson's disease mice tested by rota-rod and pole tests. The loss of tyrosine hydroxylase activities in substantia nigra and striatum was also attenuated by administration of WIN-1001X. In mice with sub-chronical MPTP injections, autophagy-related proteins, such as LC3, beclin-1, mTOR, and p62, were measured using the immunoblot assay. The results were favorable to induction of autophagy after the WIN-1001X administration. WIN-1001X treatment on 6-hydroxydopamine-injected rats also exhibited protective effects against striatal neuronal damage and loss of dopaminergic cells. Such protection is expected to be due to the positive regulation of autophagy by administration of WIN-1001X with confirmation both in vivo and in vitro. In addition, an active compound, onjisaponin B was isolated and identified from WIN-1001X. Onjisaponin B also showed significant autophagosome-inducing effect in human neuroblastoma cell line. Our study suggests that relief of Parkinsonism symptoms and rescue of tyrosine hydroxylase activity in dopaminergic neurons are affected by autophagy enhancing effect of WIN-1001X which the onjisaponin B is one of the major components of activity.

Tat­aldose reductase prevents dopaminergic neuronal cell death by inhibiting oxidative stress and MAPK activation.

Aldose reductase (AR) is known to detoxify aldehydes and prevent oxidative stress. Although AR exerts antioxidant effects, the role of AR in Parkinson's disease (PD) remains unclear. The objective of the present study was to investigate the protective effects of AR protein against 1­methyl­4­phenylpyridinium (MPP+)­induced SH­SY5Y cell death and 1­methyl­4­phenyl­1,2,3,6­tetrahydropyridine (MPTP)­induced PD in a mouse model using the cell permeable Tat­AR fusion protein. The results revealed that when Tat­AR protein was transduced into SH­SY5Y cells, it markedly protected the cells against MPP+­induced death and DNA fragmentation. It also reduced the activation of mitogen-activated protein kinase (MAPKs) and regulated the expression levels of Bcl­2, Bax and caspase­3. Immunohistochemical analysis revealed that when Tat­AR protein was transduced into the substantia nigra (SN) of mice with PD, it markedly inhibited dopaminergic neuronal cell death. Therefore, Tat­AR may be useful as a therapeutic protein for PD.

Cell-type-specific asynchronous modulation of PKA by dopamine in learning.

Reinforcement learning models postulate that neurons that release dopamine encode information about action and action outcome, and provide a teaching signal to striatal spiny projection neurons in the form of dopamine release1. Dopamine is thought to guide learning via dynamic and differential modulation of protein kinase A (PKA) in each class of spiny projection neuron2. However, the real-time relationship between dopamine and PKA in spiny projection neurons remains untested in behaving animals. Here we monitor the activity of dopamine-releasing neurons, extracellular levels of dopamine and net PKA activity in spiny projection neurons in the nucleus accumbens of mice during learning. We find positive and negative modulation of dopamine that evolves across training and is both necessary and sufficient to explain concurrent fluctuations in the PKA activity of spiny projection neurons. Modulations of PKA in spiny projection neurons that express type-1 and type-2 dopamine receptors are dichotomous, such that these neurons are selectively sensitive to increases and decreases, respectively, in dopamine that occur at different phases of learning. Thus, PKA-dependent pathways in each class of spiny projection neuron are asynchronously engaged by positive or negative dopamine signals during learning.

Imbalance of Lysine Acetylation Contributes to the Pathogenesis of Parkinson's Disease.

Parkinson's disease (PD) is one of the most common neurodegenerative disorders. The neuropathological features of PD are selective and progressive loss of dopaminergic neurons in the substantia nigra pars compacta, deficiencies in striatal dopamine levels, and the presence of intracellular Lewy bodies. Interactions among aging and genetic and environmental factors are considered to underlie the common etiology of PD, which involves multiple changes in cellular processes. Recent studies suggest that changes in lysine acetylation and deacetylation of many proteins, including histones and nonhistone proteins, might be tightly associated with PD pathogenesis. Here, we summarize the changes in lysine acetylation of both histones and nonhistone proteins, as well as the related lysine acetyltransferases (KATs) and lysine deacetylases (KDACs), in PD patients and various PD models. We discuss the potential roles and underlying mechanisms of these changes in PD and highlight that restoring the balance of lysine acetylation/deacetylation of histones and nonhistone proteins is critical for PD treatment. Finally, we discuss the advantages and disadvantages of different KAT/KDAC inhibitors or activators in the treatment of PD models and emphasize that SIRT1 and SIRT3 activators and SIRT2 inhibitors are the most promising effective therapeutics for PD.

Tristetraprolin destabilizes NOX2 mRNA and protects dopaminergic neurons from oxidative damage in Parkinson's disease.

Tristetraprolin (TTP), an RNA-binding protein encoded by the ZFP36 gene, is vital for neural differentiation; however, its involvement in neurodegenerative diseases such as Parkinson's disease (PD) remains unclear. To explore the role of TTP in PD, an in vitro 1-methyl-4-phenylpyridinium (MPP+ ) cell model and an in vivo 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) of PD were used. Transfection of small interfering (si)-TTP RNA upregulated pro-oxidative NOX2 expression and ROS formation, downregulated anti-oxidative GSH and SOD activity；si-TTP upregulated pro-apoptotic cleaved-caspase-3 expression, and downregulated antiapoptotic Bcl-2 expression; while overexpression (OE)-TTP lentivirus caused opposite effects. Through database prediction, luciferase experiment, RNA immunoprecipitation (RIP), and mRNA stability analysis, we evaluated the potential binding sites of TTP to 3'-untranslated regions (3'-UTR) of NOX2 mRNA. TTP affected the NOX2 luciferase activity by binding to two sites in the NOX2 3'-UTR. RIP-qPCR confirmed TTP binding to both sites, with a higher affinity for site-2. In addition, TTP reduced the NOX2 mRNA stability. si-NOX2 and antioxidant N-acetyl cysteine (NAC) reversed si-TTP-induced cell apoptosis. In MPTP-treated mice, TTP expression increased and was co-located with dopaminergic neurons. TTP also inhibited NOX2 and decreased the oxidative stress in vivo. In conclusion, TTP protects against dopaminergic oxidative injury by promoting NOX2 mRNA degradation in the MPP+ /MPTP model of PD, suggesting that TTP could be a potential therapeutic target for regulating the oxidative stress in PD.

Novel Drosophila model for parkinsonism by targeting phosphoglycerate kinase.

Patients with Parkinson's disease (PD) show a common progressive neurodegenerative movement disorder characterized by rigidity, tremors, postural instability, and bradykinesia due to the loss of dopaminergic neurons in the substantia nigra, and is often accompanied by several non-motor symptoms, called parkinsonism. Several lines of recent evidence support the hypothesis that mutations in the gene encoding phosphoglycerate kinase (PGK) play an important role in the PD mechanism. PGK is a key enzyme in the glycolytic pathway that catalyzes the reaction from 1,3-diphosphoglycerate to 3-phosphoglycerate. We herein established a parkinsonism model targeting Drosophila Pgk. Dopaminergic (DA) neuron-specific Pgk knockdown lead to locomotive defects in both young and aged adult flies and was accompanied by progressive DA neuron loss with aging. Pgk knockdown in DA neurons decreased dopamine levels in the central nervous system (CNS) of both young and aged adult flies. These phenotypes are similar to the defects observed in human PD patients, suggesting that the Pgk knockdown flies established herein are a promising model for parkinsonism. Furthermore, pan-neuron-specific Pgk knockdown induced low ATP levels and the accumulation of reactive oxygen species (ROS) in the CNS of third instar larvae. Collectively, these results indicate that a failure in the energy production system of Pgk knockdown flies causes locomotive defects accompanied by neuronal dysfunction and degeneration in DA neurons.

Intrastriatal administration of coenzyme Q10 enhances neuroprotection in a Parkinson's disease rat model.

Parkinson's disease is a neurodegenerative disorder, and no treatment has been yet established to prevent disease progression. Coenzyme Q10, an antioxidant, has been considered a promising neuroprotective agent; however, conventional oral administration provides limited efficacy due to its very low bioavailability. In this study, we hypothesised that continuous, intrastriatal administration of a low dose of Coenzyme Q10 could effectively prevent dopaminergic neuron degeneration. To this end, a Parkinson's disease rat model induced by 6-hydroxydopamine was established, and the treatment was applied a week before the full establishment of this disease model. Behavioural tests showed a dramatically decreased number of asymmetric rotations in the intrastriatal Coenzyme Q10 group compared with the no treatment group. Rats with intrastriatal Coenzyme Q10 exposure also exhibited a larger number of dopaminergic neurons, higher expression of neurogenetic and angiogenetic factors, and less inflammation, and the effects were more prominent than those of orally administered Coenzyme Q10, although the dose of intrastriatal Coenzyme Q10 was 17,000-times lower than that of orally-administered Coenzyme Q10. Therefore, continuous, intrastriatal delivery of Coenzyme Q10, especially when combined with implantable devices for convection-enhanced delivery or deep brain stimulation, can be an effective strategy to prevent neurodegeneration in Parkinson's disease.

Schizandrol A protects against Aß1-42-induced autophagy via activation of PI3K/AKT/mTOR pathway in SH-SY5Y cells and primary hippocampal neurons.

Autophagy, a lysosomal degradative pathway, is crucial for the pathogenesis of Alzheimer's disease (AD). Schizandrol A (SchA) shows multiple pharmacological effects. However, the potential effects and mechanisms of SchA on amyloid-ß (Aß)-induced autophagy remain unclear. In this study, differentiated SH-SY5Y cells or primary hippocampal neurons were pretreated with SchA (2 µg/ml) for 1 h before subjected to Aß1-42 (10 µM) for 24 h to test its effects on cell viability, apoptosis, oxidative stress, and autophagy. Then an mTOR inhibitor (rapamycin) and a PI3K inhibitor (LY294002) were employed to explore the role of PI3K/AKT/mTOR pathway. The results showed that SchA significantly inhibited Aß1-42-triggered reduction of viable cells, increases of apoptotic cell number and pro-apoptotic protein expressions, as well as alterations of oxidative stress markers. In addition, the increases of LC3-II/LC3-I and Beclin-1 and decrease of p62 were suppressed by SchA. At the molecular level, we found that the inactivation of PI3K/AKT/mTOR pathway was ameliorated by SchA. Inhibition of PI3K/AKT/mTOR pathway deteriorated the protective effects of SchA against Aß1-42-induced autophagy activation, cell death, and apoptosis. In conclusion, we demonstrate that SchA attenuates Aß1-42-induced autophagy through activating PI3K/AKT/mTOR signaling pathway. SchA may be a novel drug for the prevention and treatment of AD.

Phosphodiesterase 7 Regulation in Cellular and Rodent Models of Parkinson's Disease.

Parkinson's disease is characterized by a loss of dopaminergic neurons in the ventral midbrain. This disease is diagnosed when around 50% of these neurons have already died; consequently, therapeutic treatments start too late. Therefore, an urgent need exists to find new targets involved in the onset and progression of the disease. Phosphodiesterase 7 (PDE7) is a key enzyme involved in the degradation of intracellular levels of cyclic adenosine 3', 5'-monophosphate in different cell types; however, little is known regarding its role in neurodegenerative diseases, and specifically in Parkinson's disease. We have previously shown that chemical as well as genetic inhibition of this enzyme results in neuroprotection and anti-inflammatory activity in different models of neurodegenerative disorders, including Parkinson's disease. Here, we have used in vitro and in vivo models of Parkinson's disease to study the regulation of PDE7 protein levels. Our results show that PDE7 is upregulated after an injury both in the human dopaminergic cell line SH-SY5Y and in primary rat mesencephalic cultures and after lipopolysaccharide or 6-hidroxydopamine injection in the Substantia nigra pars compacta of adult mice. PDE7 increase takes place mainly in degenerating dopaminergic neurons and in microglia cells. This enhanced expression appears to be direct since 6-hydroxydopamine and lipopolysaccharide increase the expression of a 962-bp fragment of its promoter. Taking together, these results reveal an essential function for PDE7 in the pathways leading to neurodegeneration and inflammatory-mediated brain damage and suggest novel roles for PDE7 in neurodegenerative diseases, specifically in PD, opening the door for new therapeutic interventions.

LRRK2 kinase activity regulates lysosomal glucocerebrosidase in neurons derived from Parkinson's disease patients.

Mutations in LRRK2 and GBA1 are common genetic risk factors for Parkinson's disease (PD) and major efforts are underway to develop new therapeutics that target LRRK2 or glucocerebrosidase (GCase). Here we describe a mechanistic and therapeutic convergence of LRRK2 and GCase in neurons derived from patients with PD. We find that GCase activity was reduced in dopaminergic (DA) neurons derived from PD patients with LRRK2 mutations. Inhibition of LRRK2 kinase activity results in increased GCase activity in DA neurons with either LRRK2 or GBA1 mutations. This increase is sufficient to partially rescue accumulation of oxidized dopamine and alpha-synuclein in PD patient neurons. We have identified the LRRK2 substrate Rab10 as a key mediator of LRRK2 regulation of GCase activity. Together, these results suggest an important role of mutant LRRK2 as a negative regulator of lysosomal GCase activity.

Suppressing nNOS Enzyme by Small-Interfering RNAs Protects SH-SY5Y Cells and Nigral Dopaminergic Neurons from 6-OHDA Injury.

Nitric oxide (NO) has chemical properties that make it uniquely suitable as an intracellular and intercellular messenger. NO is produced by the activity of the enzyme nitric oxide synthases (NOS). There is substantial and mounting evidence that slight abnormalities of NO may underlie a wide range of neurodegenerative disorders. NO participates of the oxidative stress and inflammatory processes that contribute to the progressive dopaminergic loss in Parkinson's disease (PD). The present study aimed to evaluate in vitro and in vivo the effects of neuronal NOS-targeted siRNAs on the injury caused in dopaminergic neurons by the toxin 6-hidroxydopamine (6-OHDA). First, we confirmed (immunohistochemistry and Western blotting) that SH-SY5Y cell lineage expresses the dopaminergic marker tyrosine hydroxylase (TH) and the protein under analysis, neuronal NOS (nNOS). We designed four siRNAs by using the BIOPREDsi algorithm choosing the one providing the highest knockdown of nNOS mRNA in SH-SY5Y cells, as determined by qPCR. siRNA 4400 carried by liposomes was internalized into cells, caused a concentration-dependent knockdown on nNOS, and reduced the toxicity induced by 6-OHDA (p < 0.05). Regarding in vivo action in the dopamine-depleted animals, intra-striatal injection of siRNA 4400 at 4 days prior 6-OHDA produced a decrease in the rotational behavior induced by apomorphine. Finally, siRNA 4400 mitigated the loss of TH(+) cells in substantia nigra dorsal and ventral part. In conclusion, the suppression of nNOS enzyme by targeted siRNAs modified the progressive death of dopaminergic cells induced by 6-OHDA and merits further pre-clinical investigations as a neuroprotective approach for PD.

NADPH oxidase regulates paraquat and maneb-induced dopaminergic neurodegeneration through ferroptosis.

The activation of NADPH oxidase contributes to dopaminergic neurodegeneration induced by paraquat and maneb, two concurrently used pesticides in agriculture. However, the mechanisms remain unclear. Ferroptosis, a recently recognized form of regulated cell death, has been implicated in the pathogenesis of multiple neurodegenerative diseases. This study is designed to investigate whether ferroptosis is involved in NADPH oxidase-regulated dopaminergic neurotoxicity. In vitro study showed that paraquat and maneb exposure induced ferroptosis in SHSY5Y dopaminergic cells, which was associated with activation of NADPH oxidase. Inhibition of NADPH oxidase by apocynin or diphenyleneiodonium (DPI), two widely used NADPH oxidase inhibitors mitigated paraquat and maneb-induced ferroptotic cell death. Consistently, stimulating activation of NADPH oxidase by phorbol myristate acetate (PMA) or supplementation of H2O2 exacerbated ferroptosis in paraquat and maneb-treated SHSY5Y cells. Mechanistic inquiry revealed that NADPH oxidase activation elicited lipid peroxidation, a main driving force for ferroptosis, since both apocynin and DPI greatly reduced MDA contents and simultaneously recovered levels of glutathione and glutathione peroxidase 4 (GPX4) in paraquat and maneb-treated SHSY5Y cells. The contribution of NADPH oxidase on ferroptosis of dopaminergic neurons was further verified in vivo by showing reduced iron content, lipid peroxidation, neuroinflammation and dopaminergic neurodegeneration, which are all involved in ferroptosis, in combined apocynin and paraquat and maneb-treated mice compared with paraquat and maneb alone group. Altogether, our findings showed that NADPH oxidase contributed to paraquat and maneb-induced dopaminergic neurodegeneration through ferroptosis, providing a novel mechanism for pesticide-induced dopaminergic neurotoxicity.

Hyperactive Innate Immunity Causes Degeneration of Dopamine Neurons upon Altering Activity of Cdk5.

Innate immunity is central to the pathophysiology of neurodegenerative disorders, but it remains unclear why immunity is altered in the disease state and whether changes in immunity are a cause or a consequence of neuronal dysfunction. Here, we identify a molecular pathway that links innate immunity to age-dependent loss of dopaminergic neurons in Drosophila. We find, first, that altering the expression of the activating subunit of the Cdk5 protein kinase (Cdk5α) causes severe disruption of autophagy. Second, this disruption of autophagy is both necessary and sufficient to cause the hyperactivation of innate immunity, particularly expression of anti-microbial peptides. Finally, it is the upregulation of immunity that induces the age-dependent death of dopaminergic neurons. Given the dysregulation of Cdk5 and innate immunity in human neurodegeneration and the conserved role of the kinase in the regulation of autophagy, this sequence is likely to have direct application to the chain of events in human neurodegenerative disease.

Lentiviral-mediated knock-down of GD3 synthase protects against MPTP-induced motor deficits and neurodegeneration.

Converging evidence demonstrates an important role for gangliosides in brain function and neurodegenerative diseases. Exogenous GM1 is broadly neuroprotective, including in rodent, feline, and primate models of Parkinson's disease, and has shown positive effects in clinical trials. We and others have shown that inhibition of the ganglioside biosynthetic enzyme GD3 synthase (GD3S) increases endogenous levels GM1 ganglioside. We recently reported that targeted deletion of St8sia1, the gene that codes for GD3S, prevents motor impairments and significantly attenuates neurodegeneration induced by 1-methy-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The current study investigated the effects of GD3S inhibition on the neurotoxicity and parkinsonism induced by MPTP. Mice were injected intrastriatally with a lentiviral-vector-mediated shRNA construct targeting GD3S (shGD3S) or a scrambled-sequence control (scrRNA). An MPTP regimen of 18 mg/kg x 5 days reduced tyrosine-hydroxylase-positive neurons in the substantia nigra pars compacta of scrRNA-treated mice by nearly two-thirds. In mice treated with shGD3S the MPTP-induced lesion was approximately half that size. MPTP induced bradykinesia and deficits in fine motor skills in mice treated with scrRNA. These deficits were absent in shGD3S-treated mice. These results suggest that inhibition of GD3S protects against the nigrostriatal damage, bradykinesia, and fine-motor-skill deficits associated with MPTP administration.

Molecular Association of Glia Maturation Factor with the Autophagic Machinery in Rat Dopaminergic Neurons: a Role for Endoplasmic Reticulum Stress and MAPK Activation.

Parkinson's disease (PD) is one of the several neurodegenerative diseases where accumulation of aggregated proteins like α-synuclein occurs. Dysfunction in autophagy leading to this protein build-up and subsequent dopaminergic neurodegeneration may be one of the causes of PD. The mechanisms that impair autophagy remain poorly understood. 1-Methyl-4-phenylpiridium ion (MPP+) is a neurotoxin that induces experimental PD in vitro. Our studies have shown that glia maturation factor (GMF), a brain-localized inflammatory protein, induces dopaminergic neurodegeneration in PD and that suppression of GMF prevents MPP+-induced loss of dopaminergic neurons. In the present study, we demonstrate a molecular action of GMF on the autophagic machinery resulting in dopaminergic neuronal loss and propose GMF-mediated autophagic dysfunction as one of the contributing factors in PD progression. Using dopaminergic N27 neurons, primary neurons from wild type (WT), and GMF-deficient (GMF-KO) mice, we show that GMF and MPP+ enhanced expression of MAPKs increased the mammalian target of rapamycin (mTOR) activation and endoplasmic reticulum stress markers such as phospho-eukaryotic translation initiation factor 2 alpha kinase 3 (p-PERK) and inositol-requiring enzyme 1α (IRE1α). Further, GMF and MPP+ reduced Beclin 1, focal adhesion kinase (FAK) family-interacting protein of 200 kD (FIP200), and autophagy-related proteins (ATGs) 3, 5, 7, 16L, and 12. The combined results demonstrate that GMF affects autophagy through autophagosome formation with significantly reduced lysosomal-associated membrane protein 1/2, and the number of autophagic acidic vesicles. Using primary neurons, we show that MPP+ treatment leads to differential expression and localization of p62/sequestosome and in GMF-KO neurons, there was a marked increase in p62 staining implying autophagy deficiency with very little co-localization of α-synuclein and p62 as compared with WT neurons. Collectively, this study provides a bidirectional role for GMF in executing dopaminergic neuronal death mediated by autophagy that is relevant to PD.