Degeneration Of Dopamine Neurons Research Articles

Nilotinib: from animal-based studies to clinical investigation in Alzheimer's disease patients.

Nilotinib: from animal-based studies to clinical investigation in Alzheimer's disease patients.

Upregulation of Ca2+-binding proteins contributes to VTA dopamine neuron survival in the early phases of Alzheimer’s disease in Tg2576 mice

BackgroundRecent clinical and experimental studies have highlighted the involvement of Ventral Tegmental Area (VTA) dopamine (DA) neurons for the early pathogenesis of Alzheimer’s Disease (AD). We have previously described a progressive and selective degeneration of these neurons in the Tg2576 mouse model of AD, long before amyloid-beta plaque formation. The degenerative process in DA neurons is associated with an autophagy flux impairment, whose rescue can prevent neuronal loss. Impairments in autophagy can be the basis for accumulation of damaged mitochondria, leading to disturbance in calcium (Ca2+) homeostasis, and to functional and structural deterioration of DA neurons.MethodsIn Tg2576 mice, we performed amperometric recordings of DA levels and analysis of dopaminergic fibers in the Nucleus Accumbens – a major component of the ventral striatum precociously affected in AD patients – together with retrograde tracing, to identify the most vulnerable DA neuron subpopulations in the VTA. Then, we focused on these neurons to analyze mitochondrial integrity and Apoptosis-inducing factor (AIF) localization by electron and confocal microscopy, respectively. Stereological cell count was also used to evaluate degeneration of DA neuron subpopulations containing the Ca2+-binding proteins Calbindin-D28K and Calretinin. The expression levels for these proteins were analyzed by western blot and confocal microscopy. Lastly, using electrophysiology and microfluorometry we analyzed VTA DA neuron intrinsic properties and cytosolic free Ca2+ levels.ResultsWe found a progressive degeneration of mesolimbic DA neurons projecting to the ventral striatum, located in the paranigral nucleus and parabrachial pigmented subnucleus of the VTA. At the onset of degeneration (3 months of age), the vulnerable DA neurons in the Tg2576 accumulate damaged mitochondria, while AIF translocates from the mitochondria to the nucleus. Although we describe an age-dependent loss of the DA neurons expressing Calbindin-D28K or Calretinin, we observed that the remaining cells upregulate the levels of Ca2+-binding proteins, and the free cytosolic levels of Ca2+ in these neurons are significantly decreased. Coherently, TUNEL-stained Tg2576 DA neurons express lower levels of Calbindin-D28K when compared with non-apoptotic cells.ConclusionOverall, our results suggest that the overexpression of Ca2+-binding proteins in VTA DA neurons might be an attempt of cells to survive by increasing their ability to buffer free Ca2+. Exploring strategies to overexpress Ca2+-binding proteins could be fundamental to reduce neuronal suffering and improve cognitive and non-cognitive functions in AD.

Neuroprotective potential of plant derived parenchymal stem cells extract on environmental and genetic models of Parkinson disease through attenuation of oxidative stress and neuroinflammation.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by both motor and non-motor features. The current treatment regimen for PD are dopamine enhancers which have been reported to worsen the disease prognosis after long term treatment, thus, the need for better treatment options. This study sought to investigate the protective action of Double Stem Cell® (DSC), a blend of stem cells extracts from Swiss apples (Malus Domestica) and Burgundy grapes (Vitis vinifera) on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinsonism in mice and genetic model of PD in Drosophila melanogaster. Male albino mice were pretreated with MPTP (4 × 20mg/kg, i.p., two hourly in 8h), twelve hours before administration of DSC (8, 40, or 200mg/kg, p.o.). Thereafter, behavioural, biochemical and immunohistochemical assays were carried out. The impact of vehicle or DSC supplementation on α-synuclein aggregation was evaluated in Drosophila melanogaster using the UAS-Gal4 system, female DDC-Gal4 flies were crossed with male UAS-α-synuclein, the progenies were examined for fecundity, locomotion, memory, and lifespan. MPTP-induced motor deficits in open field test (OFT), working memory impairment (Y-maze test (YMT)) and muscle incoordination (rotarod test) were ameliorated by DSC (8, 40 or 200mg/kg) through dose-dependent and significant improvements in motor, cognitive and motor coordination. Moreso, MPTP exposure caused significant increase in lipid peroxidation and decrease in antioxidant enzymes activities (glutathione, catalase and superoxide dismutase) in the midbrain which were attenuated by DSC. MPTP-induced expression of microglia (iba-1), astrocytes (glia fibrillary acidic protein; GFAP) as well as degeneration of dopamine neurons (tyrosine hydroxylase positive neurons) in the substantia nigra (SN) were reversed by DSC. Supplementation of flies feed with graded concentration of DSC (0.8, 4 or 20mg/ml) did not affect fecundity but improved climbing activity and lifespan. Findings from this study showed that Double Stem Cell improved motor and cognitive functions in both mice and Drosophila through attenuation of neurotoxin-induced oxidative stress and neuroinflammation.

CXCR4 knockout induces neuropathological changes in the MPTP-lesioned model of Parkinson's disease

C-X-C chemokine receptor type 4 (CXCR4) is highly expressed in Parkinson's disease (PD) mice's brains and is related to astrocyte signaling and microglial activation. This makes CXCR4 related to neuroinflammation and also makes CXCR4 considered to be the PD development mechanism and possible therapeutic targets. Therefore, it is worth studying the effect of CXCR4 on neuropathological changes and its potential therapeutic value for PD. This study aimed to investigate the effect of CXCR4 knockout on neuropathological changes in the mouse model of PD and its mechanism. In this study, CXCR4-WT and CXCR4+/− C57BL mice were used to make Parkinson's model. Behavioral experiments, dopaminergic neuron markers, neuroinflammation, and blood-brain barrier damage were detected to verify the effect of CXCR4 knockout on neuropathological changes. CXCR4 knockout improved the behavioral results and tyrosine hydroxylase (TH) expression of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned mice. In the substantia nigra (SN) area of the brain of PD mouse model, the number of Iba1-positive (p = 0.0004) and GFAP-positive cells (p = 0.0349) was significantly lower in CXCR4 knockout group than CXCR4-WT group. CXCR4 knockout reduced MPTP-induced infiltration of peripheral immune cells and the expression of pro-inflammatory cytokines. CXCR4 knockout also protected blood-brain barrier (BBB) from MPTP-induced damage. In conclusion, CXCR4 knockout inhibits the degeneration of dopamine neurons, microglial and astrocyte activation, neuroinflammation, and BBB damages in the MPTP-lesioned PD mice.

Protective Effects of Ursodeoxycholic Acid Against Oxidative Stress and Neuroinflammation Through Mitogen-Activated Protein Kinases Pathway in MPTP-Induced Parkinson Disease.

Parkinson disease (PD) is the second most common neurodegenerative disorder, and no disease-modifying medications are available. Ursodeoxycholic acid (UDCA) has been shown to prevent neuronal damage; however, the effect of UDCA on PD is unclear. This study aimed to the role of UDCA on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model of PD. Mice were divided into 3 experimental groups: the control group, MPTP group, and UDCA-treat group. Mice were tested for behavioral impairments, and slices at the level of the ventral midbrain were collected to perform hematoxylin and eosin and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining and immunohistochemistry. To evaluate the levels of dopamine (DA), serotonin (5-HT), antioxidant markers, and inflammatory cytokines, enzyme-linked immunoassays were carried out. The protein (α-synuclein, p38, phospho-p38, c-Jun N-terminal kinase [JNK], and phospho-JNK) expression was examined adopting Western blot. We found that UDCA reduced the MPTP-induced degeneration of DA neurons, improved behavioral impairments, and decreased the protein level of α-synuclein, accompanied with increases of DA and 5-HT. In the present study, UDCA prevented DA neurons from MPTP toxicity with increased superoxide dismutase, catalase, glutathione, and decreased malondialdehyde levels. Ursodeoxycholic acid prevented DA neurons from MPTP toxicity with decreased levels of tumor necrosis factor α, interferon γ, and interleukin (IL)-1β, IL-6, and IL-10. Our results demonstrated that UDCA inhibited the phosphorylation of JNK and p38MAPK. This study revealed protective effects of UDCA against oxidative stress and neuroinflammation through mitogen-activated protein kinases pathways in MPTP-induced PD, suggesting that UDCA may be a novel therapeutic candidate for PD.

Reelin protects against pathological α-synuclein accumulation and dopaminergic neurodegeneration after environmental enrichment in Parkinson's disease

Two of the primary features of Parkinson's disease (PD) are the accumulation of α-synuclein (α-Syn) and the depletion of lysosomal-associated membrane protein 1 (LAMP1) in the brain. Beneficial effects of environmental enrichment (EE) have been reported on the activation of lysosomal function and the amelioration of PD symptoms. Furthermore, Reelin could be a novel therapeutic target in PD. Hence, in this study, we validated the effects of EE on the activation of LAMP1 via Reelin in PD. Heterogeneous α-Syn (A53T)-overexpressing transgenic mice (age 6 and 16 months) were exposed to EE for 8 weeks. After motor and cognitive tests, brain tissues were obtained from mice and subjected to immunohistochemistry and molecular analyses. EE ameliorated motor and non-motor symptoms, protected dopamine neurons, and reduced pathological α-Syn accumulation in the early stage of PD. Striatal Reelin levels were altered depending on the disease stage and regulated by EE in PD mice. To elucidate the underlying mechanism of the effect of EE on PD, we performed further molecular and cellular analyses using activated preformed fibril (PFF)-induced SH-SY5Y cells, an in vitro model of PD, which were treated with recombinant Reelin protein and a Reelin blocker, CR-50. The CR-50 increased pathological α-Syn accumulation and accelerated dopamine neuronal degeneration by decreasing LAMP1 in the PFF-induced PD model. Our results showed that Reelin increased LAMP1 after EE and decreased pathological α-Syn accumulation, thus protecting dopamine neurons from degeneration in the striatum and substantia nigra, and ameliorating neurobehavioral deficits. These results suggest that Reelin is a promising target in treating histopathological changes and improving behavioral symptoms associated with PD.

Uric Acid in Parkinson's Disease: What Is the Connection?

Numerous studies have linked Parkinson's disease (PD) with low levels of uric acid (UA). Low UA has been associated with the risk of developing PD, and its progression and severity. The biological mechanisms underlying these relationships have never been firmly established. The most frequently proposed mechanism is that UA is an antioxidant. Low UA is thought to predispose to oxidative stress, which contributes to dopamine neuron degeneration, and leads to initial appearance of symptoms of PD and its worsening over time. Several recent studies have questioned this explanation. In this review, we describe the biology of UA, its many links with PD, evidence regarding UA as an antioxidant, and we questionwhether UA causes PD or contributes to its progression. We also address the possibility that something about PD causes low UA (reverse causation) or that low UA is a biomarker of some other more relevant mechanism in PD. We hope the evidence provided here will stimulate additional studies to better understand the links between UA and PD. Elucidating these mechanisms remains important, because they may provide new insights into the pathogenesis of PD or novel approaches to treatments. © 2022 International Parkinson and Movement Disorder Society.

Generation of Human Ventral Midbrain Organoids Derived from Pluripotent Stem Cells.

Parkinson's disease (PD) is the second most common neurodegenerative disorder worldwide and is caused by the degeneration and loss of dopamine (DA) neurons in the ventral midbrain (VM). The focal and progressive degeneration of DA neurons in the VM makes PD a particularly attractive target for cell-based therapies. Human pluripotent stem cells (hPSCs) offer unprecedented opportunities to model the development and functional properties of human DA neurons in a dish. The use of human in vitro models based on hPSCs has empowered studies of VM development and provided access to neurons expressing a particular disease-specific phenotype. Currently, hPSC differentiation is most routinely carried out in monolayer cultures, which do not properly recapitulate cell-cell interactions and the structural complexity of the brain. Moreover, 2D cultures are challenging to maintain long term, as the cells tend to detach from the plate and lose their functional characteristics. This precludes the possibility of mimicking later phases of DA neurogenesis and recreating the complexity of functional neural circuitries. Here, we describe protocols showing how to maintain hPSCs in an undifferentiated state and how to then drive these hPSCs into 3D regionalized VM organoids. After long-term culture, these VM organoids exhibit mature and post-mitotic molecular features, including neuromelanin pigments similar to those released in primate VMs. We also report a protocol describing how to efficiently perform immunohistochemistry and how to detect neuromelanin-containing DA neurons in VM organoids. Together, these protocols provide a 3D in vitro platform that can be used to better understand the molecular mechanisms underlying DA neuron function and disease and may serve as a powerful tool for designing more targeted disease-modifying therapies. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Human pluripotent stem cell culture Basic Protocol 2: hPS cell differentiation for the generation of human ventral midbrain organoids Basic Protocol 3: Characterization of ventral midbrain organoids.

Dopamine plays a critical role in the olfactory adaptive learning pathway in Caenorhabditis elegans.

Encoding and consolidating information through learning and memory is vital in adaptation and survival. Dopamine (DA) is a critical neurotransmitter that modulates behavior. However, the role of DA in learning and memory processes is not well defined. Herein, we used the olfactory adaptive learning paradigm in Caenorhabditis elegans to elucidate the role of DA in the memory pathway. Cat-2 mutant worms with low DA synthesis showed a significant reduction in chemotaxis index (CI) compared to the wild type (WT) after short-term conditioning. In dat-1::ICE worms, having degeneration of DA neurons, there was a significant reduction in adaptive learning and memory. When the worms were trained in the presence of exogenous DA (10 mM) instead of food, a substantial increase in CI value was observed. Furthermore, our results suggest that both dop-1 and dop-3 DA receptors are involved in memory retention. The release of DA during conditioning is essential to initiate the learning pathway. We also noted an enhanced cholinergic receptor activity in the absence of dopaminergic neurons. The strains expressing GCaMP6 in DA neurons (pdat-1::GCaMP-6::mCherry) showed a rise in intracellular calcium influx in the presence of the conditional stimulus after training, suggesting DA neurons are activated during memory recall. These results reveal the critical role of DA in adaptive learning and memory, indicating that DA neurons play a crucial role in the effective processing of cognitive function.

Heparan sulfate proteoglycans mediate prion-like α-synuclein toxicity in Parkinson's in vivo models.

Parkinson's disease (PD) is a debilitating neurodegenerative disorder characterized by progressive motor decline and the aggregation of α-synuclein protein. Growing evidence suggests that α-synuclein aggregates may spread from neurons of the digestive tract to the central nervous system in a prion-like manner, yet the mechanisms of α-synuclein transmission and neurotoxicity remain poorly understood. Animal models that are amenable to high-throughput investigations are needed to facilitate the discovery of disease mechanisms. Here we describe the first Caenorhabditis elegans models in which feeding with α-synuclein preformed fibrils (PFFs) induces dopaminergic neurodegeneration, prion-like seeding of aggregation of human α-synuclein expressed in the host, and an associated motor decline. RNAi-mediated knockdown of the C. elegans syndecan sdn-1, or other enzymes involved in heparan sulfate proteoglycan synthesis, protected against PFF-induced α-synuclein aggregation, motor dysfunction, and dopamine neuron degeneration. This work offers new models by which to investigate gut-derived α-synuclein spreading and propagation of disease.

Insights Into the Role of Mortalin in Alzheimer's Disease, Parkinson's Disease, and HIV-1-Associated Neurocognitive Disorders.

Mortalin is a chaperone protein that regulates physiological functions of cells. Its multifactorial role allows cells to survive pathological conditions. Pharmacological, chemical, and siRNA-mediated downregulation of mortalin increases oxidative stress, mitochondrial dysfunction leading to unregulated inflammation. In addition to its well-characterized function in controlling oxidative stress, mitochondrial health, and maintaining physiological balance, recent evidence from human brain autopsies and cell culture–based studies suggests a critical role of mortalin in attenuating the damage seen in several neurodegenerative diseases. Overexpression of mortalin provides an important line of defense against accumulated proteins, inflammation, and neuronal loss, a key characteristic feature observed in neurodegeneration. Neurodegenerative diseases are a group of progressive disorders, sharing pathological features in Alzheimer’s disease, Parkinson’s disease, multiple sclerosis, and HIV-associated neurocognitive disorder. Aggregation of insoluble amyloid beta-proteins and neurofibrillary tangles in Alzheimer’s disease are among the leading cause of neuropathology in the brain. Parkinson’s disease is characterized by the degeneration of dopamine neurons in substantia nigra pars compacta. A substantial synaptic loss leading to cognitive decline is the hallmark of HIV-associated neurocognitive disorder (HAND). Brain autopsies and cell culture studies showed reduced expression of mortalin in Alzheimer’s, Parkinson’s, and HAND cases and deciphered the important role of mortalin in brain cells. Here, we discuss mortalin and its regulation and describe how neurotoxic conditions alter the expression of mortalin and modulate its functions. In addition, we also review the neuroprotective role of mortalin under neuropathological conditions. This knowledge showcases the importance of mortalin in diverse brain functions and offers new opportunities for the development of therapeutic targets that can modulate the expression of mortalin using chemical compounds.

Targeting NAAA counters dopamine neuron loss and symptom progression in mouse models of parkinsonism

The lysosomal cysteine hydrolase N-acylethanolamine acid amidase (NAAA) deactivates palmitoylethanolamide (PEA), a lipid-derived PPAR-α agonist that is critically involved in the control of pain and inflammation. In this study, we asked whether NAAA-regulated PEA signaling might contribute to dopamine neuron degeneration and parkinsonism induced by the mitochondrial neurotoxins, 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). In vitro experiments showed that 6-OHDA and MPTP enhanced NAAA expression and lowered PEA content in human SH-SY5Y cells. A similar effect was observed in mouse midbrain dopamine neurons following intra-striatal 6-OHDA injection. Importantly, deletion of the Naaa gene or pharmacological inhibition of NAAA activity substantially attenuated both dopamine neuron death and parkinsonian symptoms in mice treated with 6-OHDA or MPTP. Moreover, NAAA expression was elevated in postmortem brain cortex and premortem blood-derived exosomes from persons with Parkinson’s disease compared to age-matched controls. The results identify NAAA-regulated PEA signaling as a molecular control point for dopaminergic neuron survival and a potential target for neuroprotective intervention.

Neuronal NLRP3 is a parkin substrate that drives neurodegeneration in Parkinson’s disease

Parkinson's disease (PD) is mediated, in part, by intraneuronal accumulation of α-synuclein aggregates andsubsequent death of dopamine (DA) neurons in the substantia nigra pars compacta (SNpc). Microglial hyperactivation of the NOD-like receptor protein 3 (NLRP3) inflammasome has been well-documented in various neurodegenerative diseases, including PD. We show here that loss of parkin activity in mouse and human DA neurons results in spontaneous neuronal NLRP3 inflammasome assembly, leading to DA neuron death. Parkin normally inhibits inflammasome priming by ubiquitinating and targeting NLRP3 for proteasomal degradation. Loss of parkin activity also contributes to the assembly of an active NLRP3 inflammasome complex via mitochondrial-derived reactive oxygen species (mitoROS) generation through the accumulation of another parkin ubiquitination substrate, ZNF746/PARIS. Inhibition of neuronal NLRP3 inflammasome assembly prevents degeneration of DA neurons in familial and sporadic PD models. Strategies aimed at limiting neuronal NLRP3 inflammasome activation hold promise as a disease-modifying therapy for PD.

α-synuclein enfolds tyrosine hydroxylase and dopamine ß-hydroxylase, potentially reducing dopamine and norepinephrine synthesis.

Parkinson's disease (PD) results from degeneration of dopamine and norepinephrine neurons due to α-synuclein aggregates that likely have their origin in the gut. Tyrosine hydroxylase (TH) catalyses the formation of L-DOPA, the rate-limiting step in the biosynthesis of dopamine. A second enzyme, DOPA decarboxylase (DDC), catalyzes the conversion of L-DOPA to dopamine. A third enzyme, dopamine ß-hydroxylase (DBH), catalyzes the conversion of dopamine to norepinephrine. To analyze possible interactions of α-synuclein with TH, DDC and DBH, we performed in silico protein-protein docking. Protein data bank (pdb) entries were searched on the RCSB Protein Data Bank. We identified four structures that allowed us to examine the relationship of α-synuclein with TH, DDC, and DBH: (1) Human micelle-bound alpha-synuclein, (2) solution structure of the regulatory domain of tyrosine hydroxylase (Rattus norvegicus), (3) crystal structure of human aromatic L-amino acid decarboxylase (DOPA decarboxylase) in the apo form and (4) crystal structure of human dopamine ß-hydroxylase at 2.9 angstrom resolution. We used the ClusPro server (https://cluspro.org) for protein-protein docking. The protein structures were visualized with PyMOL v 2.3.4. α-synuclein partially enfolds tyrosine hydroxylase and dopamine ß-hydroxylase, potentially reducing dopamine and norepinephrine synthesis. α-synuclein may dock too far away from DOPA decarboxylase to affect its function directly. Our in silico finding of α-synuclein partly enfolding tyrosine hydroxylase and dopamine ß-hydroxylase suggests that α-synuclein docking inhibition could increase dopamine and norepinephrine biosynthesis, ameliorating PD symptoms. Small molecules that bind to α-synuclein have already been identified. Further studies may lead to new small molecule drugs that block α-synuclein enfolding of tyrosine hydroxylase and dopamine ß-hydroxylase.

Parkinsonian Consequences in Patients Treated with Psychotropics-Why Does It Affect the Elderly More so than the Young?

Introduction: Psychotropics may provoke medication induced parkinsonism (MIP). Age is its most common risk factor and predictor for idiopathic PD. This paper reviews these relationships. Background: MIP may result from a trigger evoking subclinical idiopathic PD and idiopathic PD may represent an expression of aging. Discussion: Aging may play a substantive role in the pathogenesis of idiopathic PD, involving nondopaminergic structures, causing a cascade of stressors within the substantia nigra, reducing capacity to respond to insults. Mitochondrial dysfunction and altered protein degradation may be more detrimental to these neurons than elsewhere within the brain. The neurons, degenerating in idiopathic PD, are the same as in aging. Despite epidemiology identifying aging, as the PD risk factor, biological correlates remain elusive. Midbrain dopamine neurons, from aging nonhuman primates, show markers of known correlates of dopamine neuron degeneration, found in PD. Pang et al claim most octogenarian do not have PD, challenging age in its aetiology. Idiopathic PD remains a clinical diagnosis, questioning how detailed was their physical examination. Raghunathan et al evaluated glycosaminoglycans and proteins in PD brains, compared with controls, finding idiopathic PD differed from controls, casting doubt that PD is an expression of aging. Environmental or genetic factors may be important but age remains pivotal with treatment started as soon as the diagnosis of PD is made. Conclusions: Age is more relevant to the prognosis of idiopathic PD than is disease duration. MIP may

GM1 ganglioside modifies microglial and neuroinflammatory responses to α-synuclein in the rat AAV-A53T α-synuclein model of Parkinson's disease

Among the pathological events associated with the dopaminergic neurodegeneration characteristic of Parkinson's disease (PD) are the accumulation of toxic forms of α-synuclein and microglial activation associated with neuroinflammation. Although numerous other processes may participate in the pathogenesis of PD, the two factors mentioned above may play critical roles in the initiation and progression of dopamine neuron degeneration in PD. In this study, we employed a slowly progressing model of PD using adeno-associated virus-mediated expression of human A53T α-synuclein into the substantia nigra on one side of the brain and examined the microglial response in the striatum on the injected side compared to the non-injected (control) side. We further examined the extent to which administration of the neuroprotective ganglioside GM1 influenced α-synuclein-induced glial responses. Changes in a number of microglial morphological measures (i.e., process length, number of endpoints, fractal dimension, lacunarity, density, and cell perimeter) were indicative of the presence of activated microglial and an inflammatory response on the injected side of the brain, compared to the control side. In GM1-treated animals, no significant differences in microglial morphology were observed between the injected and control striata. Follow-up studies showed that mRNA expression for several inflammation-related genes was increased on the A53T α-synuclein injected side vs. the non-injected side in saline-treated animals and that such changes were not observed in GM1-treated animals. These data show that inhibition of microglial activation and potentially damaging neuroinflammation by GM1 ganglioside administration may be among the many factors that contribute to the neuroprotective effects of GM1 in this model and possibly in human PD.

In Vivo Dopamine Neuron Imaging-Based Small Molecule Screen Identifies Novel Neuroprotective Compounds and Targets.

Parkinson’s disease (PD) is the second most common neurodegenerative disorder with prominent dopamine (DA) neuron degeneration. PD affects millions of people worldwide, but currently available therapies are limited to temporary relief of symptoms. As an effort to discover disease-modifying therapeutics, we have conducted a screen of 1,403 bioactive small molecule compounds using an in vivo whole organism screening assay in transgenic larval zebrafish. The transgenic model expresses the bacterial enzyme nitroreductase (NTR) driven by the tyrosine hydroxylase (th) promotor. NTR converts the commonly used antibiotic pro-drug metronidazole (MTZ) to the toxic nitroso radical form to induce DA neuronal loss. 57 compounds were identified with a brain health score (BHS) that was significantly improved compared to the MTZ treatment alone after FDR adjustment (padj<0.05). Independently, we curated the high throughput screening (HTS) data by annotating each compound with pharmaceutical classification, known mechanism of action, indication, IC50, and target. Using the Reactome database, we performed pathway analysis, which uncovered previously unknown pathways in addition to validating previously known pathways associated with PD. Non-topology-based pathway analysis of the screening data further identified apoptosis, estrogen hormone, dipeptidyl-peptidase 4, and opioid receptor Mu1 to be potentially significant pathways and targets involved in neuroprotection. A total of 12 compounds were examined with a secondary assay that imaged DA neurons before and after compound treatment. The z’-factor of this secondary assay was determined to be 0.58, suggesting it is an excellent assay for screening. Etodolac, nepafenac, aloperine, protionamide, and olmesartan showed significant neuroprotection and was also validated by blinded manual DA neuronal counting. To determine whether these compounds are broadly relevant for neuroprotection, we tested them on a conduritol-b-epoxide (CBE)-induced Gaucher disease (GD) model, in which the activity of glucocerebrosidase (GBA), a commonly known genetic risk factor for PD, was inhibited. Aloperine, olmesartan, and nepafenac showed significant protection of DA neurons in this assay. Together, this work, which combines high content whole organism in vivo imaging-based screen and bioinformatic pathway analysis of the screening dataset, delineates a previously uncharted approach for identifying hit-to-lead candidates and for implicating previously unknown pathways and targets involved in DA neuron protection.

LRRK2-G2019S mice display alterations in glutamatergic synaptic transmission in midbrain dopamine neurons.

The progressive degeneration of dopamine (DA) neurons in the substantia nigra compacta (SNc) leads to the emergence of motor symptoms in patients with Parkinson's disease (PD). To propose neuroprotective therapies able to slow or halt the progression of the disease, it is necessary to identify cellular alterations that occur before DA neurons degenerate and before the onset of the motor symptoms that characterize PD. Using electrophysiological, histochemical, and biochemical approaches, we have examined if glutamatergic synaptic transmission in DA neurons in the SNc and in the adjacent ventral tegmental area (VTA) was altered in middle‐aged (10–12 months old) mice with the hG2019S point mutation (G2019S) in the leucine‐rich repeat kinase 2 (LRRK2) gene. G2019S mice showed increased locomotion and exploratory behavior compared with wildtype (WT) littermates, and intact DA neuron integrity. The intrinsic membrane properties and action potential characteristics of DA neurons recorded in brain slices were similar in WT and G2019S mice. Initial glutamate release probability onto SNc‐DA neurons, but not VTA‐DA neurons, was reduced in G2019S mice. We also found reduced protein amounts of the presynaptic marker of glutamatergic terminals, VGLUT1, and of the GluA1 and GluN1 subunits of AMPA and NMDA receptors, respectively, in the ventral midbrain of G2019S mice. These results identify alterations in glutamatergic synaptic transmission in DA neurons of the SNc and VTA before the onset of motor impairments in the LRRK2‐G2019S mouse model of PD.

CDNF and MANF regulate ER stress in a tissue-specific manner

Cerebral dopamine neurotrophic factor (CDNF) and mesencephalic astrocyte-derived neurotrophic factor (MANF) display cytoprotective effects in animal models of neurodegenerative diseases. These endoplasmic reticulum (ER)-resident proteins belong to the same protein family and function as ER stress regulators. The relationship between CDNF and MANF function, as well as their capability for functional compensation, is unknown. We aimed to investigate these questions by generating mice lacking both CDNF and MANF. Results showed that CDNF-deficient Manf−/− mice presented the same phenotypes of growth defect and diabetes as Manf−/− mice. In the muscle, CDNF deficiency resulted in increased activation of unfolded protein response (UPR), which was aggravated when MANF was ablated. In the brain, the combined loss of CDNF and MANF did not exacerbate UPR activation caused by the loss of MANF alone. Consequently, CDNF and MANF deficiency in the brain did not cause degeneration of dopamine neurons. In conclusion, CDNF and MANF present functional redundancy in the muscle, but not in the other tissues examined here. Thus, they regulate the UPR in a tissue-specific manner.

Identification of Altered Blood MicroRNAs and Plasma Proteins in a Rat Model of Parkinson's Disease.

Parkinson's disease (PD) is the age-related neurological disorder characterized by the degeneration of dopamine (DA) neurons in the substantia nigrapars compacta (SNpc). PD is based on motor deficits which start to appear when up to 80% of the DA neurons of SNpc have been lost. Effective management of PD requires the development of novel biomarkers. Therefore, the present study aimed to characterize biomarkers of PD using miRNomics, proteomics, and bioinformatics approaches. Rats exposed to rotenone (2.5mg/kg b.wt) for 2months were used as an animal model to identify the unbiased set of miRNAs and proteins deregulated in blood samples. OpenArray, a real-time PCR-based array, is used for high-throughput profiling of miRNAs, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to carry out the global protein profiling. Systematic bioinformatics analysis of miRNAs and proteins was also performed, including annotation, functional classification and functional enrichment, network analysis, and miRNA-protein interaction analysis. Expression of 19 miRNAs and 96 proteins was significantly upregulated in the blood, while 22 proteins were significantly downregulated in blood samples of rotenone-exposed rats. In silico pathway analysis of deregulated proteins and miRNAs in rotenone-exposed rats has identified multiple pathways leading to PD. In summary, we have identified a set of miRNAs (miR-144, miR-96, and miR-29a) and proteins (PLP1, TUBB4A, and TUBA1C), which can be used as a potential biomarker of PD, while further validation required large human population studies.