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1.
Neurobiol Dis ; 191: 106411, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38228253

RESUMO

Parkinson's disease (PD) pathology is characterized by alpha-synuclein (α-syn) aggregates, degeneration of dopamine neurons in the substantia nigra pars compacta (SNpc), and neuroinflammation. The presence of reactive glia correlates with deposition of pathological α-syn in early-stage PD. Thus, understanding the neuroinflammatory response of microglia and astrocytes to synucleinopathy may identify therapeutic targets. Here we characterized the neuroinflammatory gene expression profile of reactive microglia and astrocytes in the SNpc during early synucleinopathy in the rat α-syn pre-formed fibril (PFF) model. Rats received intrastriatal injection of α-syn PFFs and expression of immune genes was quantified with droplet digital PCR (ddPCR), after which fluorescent in situ hybridization (FISH) was used to localize gene expression to microglia or astrocytes in the SNpc. Genes previously associated with reactive microglia (Cd74, C1qa, Stat1, Axl, Casp1, Il18, Lyz2) and reactive astrocytes (C3, Gbp2, Serping1) were significantly upregulated in the SN of PFF injected rats. Localization of gene expression to SNpc microglia near α-syn aggregates identified a unique α-syn aggregate microglial gene expression profile characterized by upregulation of Cd74, Cxcl10, Rt-1a2, Grn, Csf1r, Tyrobp, C3, C1qa, Serping1 and Fcer1g. Importantly, significant microglial upregulation of Cd74 and C3 were only observed following injection of α-syn PFFs, not α-syn monomer, confirming specificity to α-syn aggregation. Serping1 expression also localized to astrocytes in the SNpc. Interestingly, C3 expression in the SNpc localized to microglia at 2- and 4-months post-PFF, but to astrocytes at 6-months post-PFF. We also observed expression of Rt1-a2 and Cxcl10 in SNpc dopamine neurons. Cumulatively our results identify a dynamic, yet reproducible gene expression profile of reactive microglia and astrocytes associated with early synucleinopathy in the rat SNpc.


Assuntos
Doença de Parkinson , Sinucleinopatias , Animais , Ratos , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , Proteína Inibidora do Complemento C1/genética , Proteína Inibidora do Complemento C1/metabolismo , Neurônios Dopaminérgicos/metabolismo , Hibridização in Situ Fluorescente , Neuroglia/metabolismo , Doenças Neuroinflamatórias , Doença de Parkinson/metabolismo , Substância Negra/metabolismo , Sinucleinopatias/patologia , Transcriptoma
2.
Eur J Neurosci ; 59(7): 1585-1603, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38356120

RESUMO

Parkinson's disease (PD) is characterized by the accumulation of misfolded alpha-synuclein (α-syn) protein, forming intraneuronal Lewy body (LB) inclusions. The α-syn preformed fibril (PFF) model of PD recapitulates α-syn aggregation, progressive nigrostriatal degeneration and motor dysfunction; however, little is known about the time course of PFF-induced alterations in basal and evoked dopamine (DA). In vivo microdialysis is well suited for identifying small changes in neurotransmitter levels over extended periods. In the present study, adult male Fischer 344 rats received unilateral, intrastriatal injections of either α-syn PFFs or phosphate-buffered saline (PBS). At 4 or 8 months post-injection (p.i.), animals underwent in vivo microdialysis to evaluate basal extracellular striatal DA and metabolite levels, local KCl-evoked striatal DA release and the effects of systemic levodopa (l-DOPA). Post-mortem analysis demonstrated equivalent PFF-induced reductions in tyrosine hydroxylase (TH) immunoreactive nigral neurons (~50%) and striatal TH (~20%) at both time points. Compared with reduction in striatal TH, reduction in striatal dopamine transporter (DAT) was more pronounced and progressed between the 4- and 8-month p.i. intervals (36% âž” 46%). Significant PFF-induced deficits in basal and evoked striatal DA, as well as deficits in motor performance, were not observed until 8 months p.i. Responses to l-DOPA did not differ regardless of PBS or PFF treatment. These results suggest that basal and evoked striatal DA are maintained for several months following PFF injection, with loss of both associated with motor dysfunction. Our studies provide insight into the time course and magnitude of PFF-induced extracellular dopaminergic deficits in the striatum.


Assuntos
Doença de Parkinson , alfa-Sinucleína , Ratos , Masculino , Animais , alfa-Sinucleína/metabolismo , Dopamina/metabolismo , Levodopa/farmacologia , Microdiálise , Substância Negra/metabolismo , Doença de Parkinson/metabolismo
3.
J Neuroinflammation ; 21(1): 108, 2024 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-38664840

RESUMO

BACKGROUND: Parkinson's disease (PD) is a neurodegenerative disorder that is characterized by the presence of proteinaceous alpha-synuclein (α-syn) inclusions (Lewy bodies), markers of neuroinflammation and the progressive loss of nigrostriatal dopamine (DA) neurons. These pathological features can be recapitulated in vivo using the α-syn preformed fibril (PFF) model of synucleinopathy. We have previously determined that microglia proximal to PFF-induced nigral α-syn inclusions increase in soma size, upregulate major-histocompatibility complex-II (MHC-II) expression, and increase expression of a suite of inflammation-associated transcripts. This microglial response is observed months prior to degeneration, suggesting that microglia reacting to α-syn inclusion may contribute to neurodegeneration and could represent a potential target for novel therapeutics. The goal of this study was to determine whether colony stimulating factor-1 receptor (CSF1R)-mediated microglial depletion impacts the magnitude of α-syn aggregation, nigrostriatal degeneration, or the response of microglial in the context of the α-syn PFF model. METHODS: Male Fischer 344 rats were injected intrastriatally with either α-syn PFFs or saline. Rats were continuously administered Pexidartinib (PLX3397B, 600 mg/kg), a CSF1R inhibitor, to deplete microglia for a period of either 2 or 6 months. RESULTS: CSF1R inhibition resulted in significant depletion (~ 43%) of ionized calcium-binding adapter molecule 1 immunoreactive (Iba-1ir) microglia within the SNpc. However, CSF1R inhibition did not impact the increase in microglial number, soma size, number of MHC-II immunoreactive microglia or microglial expression of Cd74, Cxcl10, Rt-1a2, Grn, Csf1r, Tyrobp, and Fcer1g associated with phosphorylated α-syn (pSyn) nigral inclusions. Further, accumulation of pSyn and degeneration of nigral neurons was not impacted by CSF1R inhibition. Paradoxically, long term CSF1R inhibition resulted in increased soma size of remaining Iba-1ir microglia in both control and PFF rats, as well as expression of MHC-II in extranigral regions. CONCLUSIONS: Collectively, our results suggest that CSF1R inhibition does not impact the microglial response to nigral pSyn inclusions and that CSF1R inhibition is not a viable disease-modifying strategy for PD.


Assuntos
Microglia , Ratos Endogâmicos F344 , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos , alfa-Sinucleína , Animais , Microglia/metabolismo , Microglia/efeitos dos fármacos , alfa-Sinucleína/metabolismo , Ratos , Masculino , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/antagonistas & inibidores , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Pirróis/farmacologia , Aminopiridinas/farmacologia , Corpos de Inclusão/metabolismo , Corpos de Inclusão/patologia , Substância Negra/metabolismo , Substância Negra/patologia , Substância Negra/efeitos dos fármacos , Modelos Animais de Doenças
4.
J Pharmacol Exp Ther ; 390(2): 250-259, 2024 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-38866563

RESUMO

Mutations in the GNAO1 gene, which encodes the abundant brain G-protein Gα o, result in neurologic disorders characterized by developmental delay, epilepsy, and movement abnormalities. There are over 50 mutant alleles associated with GNAO1 disorders; the R209H mutation results in dystonia, choreoathetosis, and developmental delay without seizures. Mice heterozygous for the human mutant allele (Gnao1 +/R209H) exhibit hyperactivity in open field tests but no seizures. We developed self-complementary adeno-associated virus serotype 9 (scAAV9) vectors expressing two splice variants of human GNAO1 Gα o isoforms 1 (GoA, GNAO1.1) and 2 (GoB, GNAO1.2). Bilateral intrastriatal injections of either scAAV9-GNAO1.1 or scAAV9-GNAO1.2 significantly reversed mutation-associated hyperactivity in open field tests. GNAO1 overexpression did not increase seizure susceptibility, a potential side effect of GNAO1 vector treatment. This represents the first report of successful preclinical gene therapy for GNAO1 encephalopathy applied in vivo. Further studies are needed to uncover the molecular mechanism that results in behavior improvements after scAAV9-mediated Gα o expression and to refine the vector design. SIGNIFICANCE STATEMENT: GNAO1 mutations cause a spectrum of developmental, epilepsy, and movement disorders. Here we show that intrastriatal delivery of scAAV9-GNAO1 to express the wild-type Gα o protein reduces the hyperactivity of the Gnao1 +/R209H mouse model, which carries one of the most common movement disorder-associated mutations. This is the first report of a gene therapy for GNAO1 encephalopathy applied in vivo on a patient-allele model.


Assuntos
Dependovirus , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP , Heterozigoto , Animais , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Camundongos , Dependovirus/genética , Humanos , Masculino , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Hipercinese/genética , Mutação , Terapia Genética/métodos , Camundongos Endogâmicos C57BL , Locomoção/genética
5.
J Neurosci ; 41(9): 2039-2052, 2021 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-33472823

RESUMO

Preclinical studies show a link between subthalamic nucleus (STN) deep brain stimulation (DBS) and neuroprotection of nigrostriatal dopamine (DA) neurons, potentially through brain-derived neurotrophic factor (BDNF) signaling. However, the question of whether DBS of the STN can be disease-modifying in Parkinson's disease (PD) remains unanswered. In particular, the impact of STN DBS on α-synuclein (α-syn) aggregation, inclusion-associated neuroinflammation, and BDNF levels has yet to be examined in the context of synucleinopathy. To address this, we examined the effects of STN DBS on BDNF using the α-syn preformed fibril (PFF) model in male rats. While PFF injection resulted in accumulation of phosphorylated α-syn (pSyn) inclusions in the substantia nigra pars compacta (SNpc) and cortical areas, STN DBS did not impact PFF-induced accumulation of pSyn inclusions in the SNpc. In addition, nigral pSyn inclusions were associated with increased microgliosis and astrogliosis; however, the magnitude of these processes was not altered by STN DBS. Total BDNF protein was not impacted by pSyn inclusions, but the normally positive association of nigrostriatal and corticostriatal BDNF was reversed in rats with PFF-induced nigrostriatal and corticostriatal inclusions. Despite this, rats receiving both STN DBS and PFF injection showed increased BDNF protein in the striatum, which partially restored the normal corticostriatal relationship. Our results suggest that pathologic α-syn inclusions disrupt anterograde BDNF transport within nigrostriatal and corticostriatal circuitry. Further, STN DBS has the potential to exert protective effects by modifying the long-term neurodegenerative consequences of synucleinopathy.SIGNIFICANCE STATEMENT An increase in brain-derived neurotrophic factor (BDNF) has been linked to the neuroprotection elicited by subthalamic nucleus (STN) deep brain stimulation (DBS) in neurotoxicant models of Parkinson's disease (PD). However, whether STN DBS can similarly increase BDNF in nigrostriatal and corticostriatal circuitry in the presence of α-synuclein (α-syn) inclusions has not been examined. We examined the impact of STN DBS on rats in which accumulation of α-syn inclusions is induced by injection of α-syn preformed fibrils (PFFs). STN DBS significantly increased striatal BDNF protein in rats seeded with α-syn inclusions and partially restored the normal corticostriatal BDNF relationship. These findings suggest that STN DBS can drive BDNF in the parkinsonian brain and retains the potential for neuroprotection in PD.


Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Estimulação Encefálica Profunda , Parte Compacta da Substância Negra/metabolismo , Parte Compacta da Substância Negra/patologia , Sinucleinopatias/metabolismo , Sinucleinopatias/patologia , Animais , Modelos Animais de Doenças , Masculino , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Ratos , Ratos Endogâmicos F344 , Núcleo Subtalâmico/fisiologia
6.
Mov Disord ; 37(8): 1739-1748, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35524682

RESUMO

BACKGROUND: Positron emission tomography (PET) imaging in early Parkinson's disease (PD) subjects reveals that increased dopamine (DA) turnover and reduced dopamine transporter (DAT) density precede decreases in DA synthesis and storage. The rat α-synuclein preformed fibril (α-syn PFF) model provides a platform to investigate DA dynamics during multiple stages of α-syn inclusion-triggered nigrostriatal degeneration. OBJECTIVES: We investigated multiple aspects of in vivo dopaminergic deficits longitudinally and similarities to human PD using translational PET imaging readouts. METHODS: Longitudinal imaging was performed every 2 months in PFF and control rats for 7 months. [18 F]-Fluoro-3,4-dihydroxyphenyl-L-alanine (FDOPA) imaging was performed to investigate DA synthesis and storage (Kocc ) and DA turnover, estimated by its inverse, the effective distribution volume ratio (EDVR). 11 C-Methylphenidate (MP) was used to estimate DAT density (BPND ). RESULTS: Early DA turnover increases and DAT binding decreases were observed in the ipsilateral striatum of PFF rats, progressing longitudinally. EDVR decreased 26%, 38%, and 47%, and BPND decreased 36%, 50%, and 65% at the 2-, 4-, and 6-month time points, respectively, compared to ipsilateral control striatum. In contrast, deficits in DA synthesis and storage were not observed in the ipsilateral striatum of PFF rats compared to control injections and were relatively preserved up to 6 months (Kocc decreased 20% at 6 months). CONCLUSIONS: The relative preservation of DA synthesis and storage compared to robust progressive deficits in DAT density and increases in DA turnover in the rat α-syn PFF model display remarkable face validity to dopaminergic alterations in human PD. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.


Assuntos
Doença de Parkinson , alfa-Sinucleína , Animais , Corpo Estriado/metabolismo , Dopamina/metabolismo , Neurônios Dopaminérgicos/metabolismo , Humanos , Doença de Parkinson/diagnóstico por imagem , Doença de Parkinson/metabolismo , Tomografia por Emissão de Pósitrons , Ratos , alfa-Sinucleína/metabolismo
7.
Neurobiol Dis ; 141: 104947, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32422283

RESUMO

Human and animal studies have shown that exposure to the organochlorine pesticide dieldrin is associated with increased risk of Parkinson's disease (PD). Previous work showed that developmental dieldrin exposure increased neuronal susceptibility to MPTP toxicity in male C57BL/6 mice, possibly via changes in dopamine (DA) packaging and turnover. However, the relevance of the MPTP model to PD pathophysiology has been questioned. We therefore studied dieldrin-induced neurotoxicity in the α-synuclein (α-syn)-preformed fibril (PFF) model, which better reflects the α-syn pathology and toxicity observed in PD pathogenesis. Specifically, we used a "two-hit" model to determine whether developmental dieldrin exposure increases susceptibility to α-syn PFF-induced synucleinopathy. Dams were fed either dieldrin (0.3 mg/kg, every 3-4 days) or vehicle corn oil starting 1 month prior to breeding and continuing through weaning of pups at postnatal day 22. At 12 weeks of age, male and female offspring received intrastriatal α-syn PFF or control saline injections. Consistent with the male-specific increased susceptibility to MPTP, our results demonstrate that developmental dieldrin exposure exacerbates PFF-induced toxicity in male mice only. Specifically, in male offspring, dieldrin exacerbated PFF-induced motor deficits on the challenging beam and increased DA turnover in the striatum 6 months after PFF injection. However, male offspring showed neither exacerbation of phosphorylated α-syn aggregation (pSyn) in the substantia nigra (SN) at 1 or 2 months post-PFF injection, nor exacerbation of PFF-induced TH and NeuN loss in the SN 6 months post-PFF injection. Collectively, these data indicate that developmental dieldrin exposure produces a male-specific exacerbation of synucleinopathy-induced behavioral and biochemical deficits. This sex-specific result is consistent with both previous work in the MPTP model, our previously reported sex-specific effects of this exposure paradigm on the male and female epigenome, and the higher prevalence and more severe course of PD in males. The novel two-hit environmental toxicant/PFF exposure paradigm established in this project can be used to explore the mechanisms by which other PD-related exposures alter neuronal vulnerability to synucleinopathy in sporadic PD.


Assuntos
Dieldrin/toxicidade , Atividade Motora/efeitos dos fármacos , Transtornos Parkinsonianos/induzido quimicamente , Transtornos Parkinsonianos/patologia , Praguicidas/toxicidade , Agregação Patológica de Proteínas , alfa-Sinucleína/toxicidade , Animais , Dopamina/metabolismo , Feminino , Masculino , Camundongos Endogâmicos C57BL , Agregação Patológica de Proteínas/induzido quimicamente , Agregação Patológica de Proteínas/metabolismo , Fatores Sexuais , Substância Negra/metabolismo , Substância Negra/patologia , alfa-Sinucleína/administração & dosagem
8.
Genesis ; 57(2): e23267, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30489010

RESUMO

A key aspect of development in all metazoans is remodeling at the cellular level. During the development of gametes, remodeling occurs throughout the germ line. When Caenorhabditis elegans hermaphrodites become depleted of sperm after 4 days of adulthood, significant cellular remodeling occurs within the meiotically-arrested oocytes, including the formation of ribonucleoprotein granules. Since major remodeling of the endoplasmic reticulum (ER) occurs in early embryos, we investigated the extent of ER remodeling in meiotically-arrested oocytes. We found, using a combination of fluorescence reporters and transmission electron microscopy, that the ER in arrested oocytes accumulates in patches and sheets that are enriched at the cortex. Our findings suggest this remodeling is not due to simple displacement by large amounts of yolk that accumulate in arrested oocytes, and instead may be genetically regulated. We further identified the Ddx6 RNA helicase, CGH-1, as a key regulator of ER in the germ line. In cgh-1(tn691) oocytes, we detected cortical ER patches as well as aberrant granules of the RNA-binding proteins, PAB-1, MEX-3, and CGH-1. Taken together, our results suggest the possibility that the spatial organization of RNA binding proteins may regulate the translation of mRNAs associated with the ER that in turn, controls the organization of the ER in the adult germ line.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Retículo Endoplasmático/metabolismo , Oócitos/metabolismo , RNA Nucleotidiltransferases/metabolismo , Animais , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Proteína I de Ligação a Poli(A)/metabolismo , RNA Nucleotidiltransferases/genética , Proteínas de Ligação a RNA/metabolismo
9.
Neurobiol Dis ; 130: 104525, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31276792

RESUMO

Animal models that accurately recapitulate the accumulation of alpha-synuclein (α-syn) inclusions, progressive neurodegeneration of the nigrostriatal system and motor deficits can be useful tools for Parkinson's disease (PD) research. The preformed fibril (PFF) synucleinopathy model in rodents generally displays these PD-relevant features, however, the magnitude and predictability of these events is far from established. We therefore sought to optimize the magnitude of α-syn accumulation and nigrostriatal degeneration, and to understand the time course of both. Rats were injected unilaterally with different quantities of α-syn PFFs (8 or 16 µg of total protein) into striatal sites selected to concentrate α-syn inclusion formation in the substantia nigra pars compacta (SNpc). Rats displayed an α-syn PFF quantity-dependent increase in the magnitude of ipsilateral SNpc inclusion formation at 2 months and bilateral loss of nigral dopamine neurons at 6 months. Unilateral 16 µg PFF injection also resulted in modest sensorimotor deficits in forelimb adjusting steps associated with degeneration at 6 months. Bilateral injection of 16 µg α-syn PFFs resulted in symmetric bilateral degeneration equivalent to the ipsilateral nigral degeneration observed following unilateral 16 µg PFF injection (~50% loss). Bilateral PFF injections additionally resulted in alterations in several gait analysis parameters. These α-syn PFF parameters can be applied to generate a reproducible synucleinopathy model in rats with which to study pathogenic mechanisms and vet potential disease-modifying therapies.


Assuntos
Corpo Estriado/metabolismo , Substância Negra/metabolismo , Sinucleinopatias/metabolismo , alfa-Sinucleína/metabolismo , Animais , Corpo Estriado/patologia , Modelos Animais de Doenças , Neurônios Dopaminérgicos/metabolismo , Corpos de Inclusão/metabolismo , Corpos de Inclusão/patologia , Masculino , Ratos , Ratos Endogâmicos F344 , Substância Negra/patologia , Sinucleinopatias/patologia
10.
Neurobiol Dis ; 112: 106-118, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29341898

RESUMO

Alpha-Synuclein (α-syn) is by far the most highly vetted pathogenic and therapeutic target in Parkinson's disease. Aggregated α-syn is present in sporadic Parkinson's disease, both in the central nervous system (CNS) and peripheral nervous system (PNS). The enteric division of the PNS is of particular interest because 1) gastric dysfunction is a key clinical manifestation of Parkinson's disease, and 2) Lewy pathology in myenteric and submucosal neurons of the enteric nervous system (ENS) has been referred to as stage zero in the Braak pathological staging of Parkinson's disease. The presence of Lewy pathology in the ENS and the fact that patients often experience enteric dysfunction before the onset of motor symptoms has led to the hypothesis that α-syn pathology starts in the periphery, after which it spreads to the CNS via interconnected neural pathways. Here we sought to directly test this hypothesis in rodents and non-human primates (NHP) using two distinct models of α-syn pathology: the α-syn viral overexpression model and the preformed fibril (PFF) model. Subjects (rat and NHP) received targeted enteric injections of PFFs or adeno-associated virus overexpressing the Parkinson's disease associated A53T α-syn mutant. Rats were evaluated for colonic motility monthly and sacrificed at 1, 6, or 12 months, whereas NHPs were sacrificed 12 months following inoculation, after which the time course and spread of pathology was examined in all animals. Rats exhibited a transient GI phenotype that resolved after four months. Minor α-syn pathology was observed in the brainstem (dorsal motor nucleus of the vagus and locus coeruleus) 1 month after PFF injections; however, no pathology was observed at later time points (nor in saline or monomer treated animals). Similarly, a histopathological analysis of the NHP brains revealed no pathology despite the presence of robust α-syn pathology throughout the ENS which persisted for the entirety of the study (12 months). Our study shows that induction of α-syn pathology in the ENS is sufficient to induce GI dysfunction. Moreover, our data suggest that sustained spread of α-syn pathology from the periphery to the CNS and subsequent propagation is a rare event, and that the presence of enteric α-syn pathology and dysfunction may represent an epiphenomenon.


Assuntos
Doenças do Sistema Nervoso Central/metabolismo , Sistema Nervoso Entérico/metabolismo , Gastroenteropatias/metabolismo , Motilidade Gastrointestinal/fisiologia , alfa-Sinucleína/biossíntese , Animais , Doenças do Sistema Nervoso Central/patologia , Sistema Nervoso Entérico/patologia , Gastroenteropatias/patologia , Humanos , Macaca fascicularis , Masculino , Camundongos , Primatas , Ratos , Ratos Sprague-Dawley
11.
J Neuroinflammation ; 15(1): 129, 2018 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-29716614

RESUMO

BACKGROUND: Converging evidence suggests a role for microglia-mediated neuroinflammation in Parkinson's disease (PD). Animal models of PD can serve as a platform to investigate the role of neuroinflammation in degeneration in PD. However, due to features of the previously available PD models, interpretations of the role of neuroinflammation as a contributor to or a consequence of neurodegeneration have remained elusive. In the present study, we investigated the temporal relationship of neuroinflammation in a model of synucleinopathy following intrastriatal injection of pre-formed alpha-synuclein fibrils (α-syn PFFS). METHODS: Male Fischer 344 rats (N = 114) received unilateral intrastriatal injections of α-syn PFFs, PBS, or rat serum albumin with cohorts euthanized at monthly intervals up to 6 months. Quantification of dopamine neurons, total neurons, phosphorylated α-syn (pS129) aggregates, major histocompatibility complex-II (MHC-II) antigen-presenting microglia, and ionized calcium-binding adaptor molecule-1 (Iba-1) immunoreactive microglial soma size was performed in the substantia nigra. In addition, the cortex and striatum were also examined for the presence of pS129 aggregates and MHC-II antigen-presenting microglia to compare the temporal patterns of pSyn accumulation and reactive microgliosis. RESULTS: Intrastriatal injection of α-syn PFFs to rats resulted in widespread accumulation of phosphorylated α-syn inclusions in several areas that innervate the striatum followed by significant loss (~ 35%) of substantia nigra pars compacta dopamine neurons within 5-6 months. The peak magnitudes of α-syn inclusion formation, MHC-II expression, and reactive microglial morphology were all observed in the SN 2 months following injection and 3 months prior to nigral dopamine neuron loss. Surprisingly, MHC-II immunoreactivity in α-syn PFF injected rats was relatively limited during the later interval of degeneration. Moreover, we observed a significant correlation between substantia nigra pSyn inclusion load and number of microglia expressing MHC-II. In addition, we observed a similar relationship between α-syn inclusion load and number of microglia expressing MHC-II in cortical regions, but not in the striatum. CONCLUSIONS: Our results demonstrate that increases in microglia displaying a reactive morphology and MHC-II expression occur in the substantia nigra in close association with peak numbers of pSyn inclusions, months prior to nigral dopamine neuron degeneration, and suggest that reactive microglia may contribute to vulnerability of SNc neurons to degeneration. The rat α-syn PFF model provides an opportunity to examine the innate immune response to accumulation of pathological α-syn in the context of normal levels of endogenous α-syn and provides insight into the earliest neuroinflammatory events in PD.


Assuntos
Corpos de Lewy/patologia , Microglia/patologia , Degeneração Neural/induzido quimicamente , Degeneração Neural/patologia , Substância Negra/patologia , alfa-Sinucleína/toxicidade , Animais , Injeções Intraventriculares , Corpos de Lewy/efeitos dos fármacos , Corpos de Lewy/metabolismo , Masculino , Microglia/efeitos dos fármacos , Microglia/metabolismo , Degeneração Neural/metabolismo , Ratos , Ratos Endogâmicos F344 , Substância Negra/efeitos dos fármacos , Substância Negra/metabolismo , alfa-Sinucleína/administração & dosagem
12.
J Neuroinflammation ; 15(1): 169, 2018 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-29843738

RESUMO

After publication of the original article [1] it was noted that the name of author, D. Luke Fisher, was erroneously typeset in both the PDF and online formats of the manuscript as Luke D. Fisher.

13.
J Cell Sci ; 126(Pt 3): 850-9, 2013 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-23264733

RESUMO

Large cytoplasmic ribonucleoprotein germ granule complexes are a common feature in germ cells. In C. elegans these are called P granules and for much of the life-cycle they associate with nuclear pore complexes in germ cells. P granules are rich in proteins that function in diverse RNA pathways. Here we report that the C. elegans homolog of the eIF4E-transporter IFET-1 is required for oogenesis but not spermatogenesis. We show that IFET-1 is required for translational repression of several maternal mRNAs in the distal gonad and functions in conjunction with the broad-scale translational regulators CGH-1, CAR-1 and PATR-1 to regulate germ cell sex determination. Furthermore we have found that IFET-1 localizes to P granules throughout the gonad and in the germ cell lineage in the embryo. Interestingly, IFET-1 is required for the normal ultrastructure of P granules and for the localization of CGH-1 and CAR-1 to P granules. Our findings suggest that IFET-1 is a key translational regulator and is required for normal P granule formation.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiologia , Grânulos Citoplasmáticos/metabolismo , Células Germinativas/fisiologia , Poro Nuclear/metabolismo , Proteínas Repressoras/metabolismo , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Células Cultivadas , Fator de Iniciação 4E em Eucariotos/genética , Mutação/genética , Oogênese/genética , Biossíntese de Proteínas , Transporte Proteico , RNA Nucleotidiltransferases/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/genética , Homologia de Sequência de Aminoácidos , Processos de Determinação Sexual
14.
NPJ Parkinsons Dis ; 10(1): 7, 2024 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-38172128

RESUMO

Examination of early phases of synucleinopathy when inclusions are present, but long before neurodegeneration occurs, is critical to both understanding disease progression and the development of disease modifying therapies. The rat alpha-synuclein (α-syn) preformed fibril (PFF) model induces synchronized synucleinopathy that recapitulates the pathological features of Parkinson's disease (PD) and can be used to study synucleinopathy progression. In this model, phosphorylated α-syn (pSyn) inclusion-containing neurons and reactive microglia (major histocompatibility complex-II immunoreactive) peak in the substantia nigra pars compacta (SNpc) months before appreciable neurodegeneration. However, it remains unclear which specific genes are driving these phenotypic changes. To identify transcriptional changes associated with early synucleinopathy, we used laser capture microdissection of the SNpc paired with RNA sequencing (RNASeq). Precision collection of the SNpc allowed for the assessment of differential transcript expression in the nigral dopamine neurons and proximal glia. Transcripts upregulated in early synucleinopathy were mainly associated with an immune response, whereas transcripts downregulated were associated with neurotransmission and the dopamine pathway. A subset of 29 transcripts associated with neurotransmission/vesicular release and the dopamine pathway were verified in a separate cohort of males and females to confirm reproducibility. Within this subset, fluorescent in situ hybridization (FISH) was used to localize decreases in the Syt1 and Slc6a3 transcripts to pSyn inclusion-containing neurons. Identification of transcriptional changes in early synucleinopathy provides insight into the molecular mechanisms driving neurodegeneration.

15.
Res Sq ; 2023 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-37205534

RESUMO

Background: Parkinson's disease (PD) is a neurodegenerative disorder that is characterized by the presence of proteinaceous alpha-synuclein (α-syn) inclusions (Lewy bodies), markers of neuroinflammation and the progressive loss of nigrostriatal dopamine (DA) neurons. These pathological features can be recapitulated in vivo using the α-syn preformed fibril (PFF) model of synucleinopathy. We have previously described the time course of microglial major-histocompatibility complex-II (MHC-II) expression and alterations in microglia morphology in the PFF model in rats. Specifically, the peaks of α-syn inclusion formation, MHC-II expression, and reactive morphology in the substantia nigra pars compacta (SNpc) all occur two months post PFF injection, months before neurodegeneration occurs. These results suggest that activated microglia may contribute to neurodegeneration and could represent a potential target for novel therapeutics. The goal of this study was to determine whether microglial depletion could impact the magnitude of α-syn aggregation, nigrostriatal degeneration, or related microglial activation during the α-syn PFF model. Methods: Male Fischer 344 rats were injected intrastriatally with either α-syn PFFs or saline. Rats were continuously administered Pexidartinib (PLX3397B, 600mg/kg), a colony stimulating factor-1 receptor (CSF1R) inhibitor, to deplete microglia for a period of either two or six months. Results: PLX3397B administration resulted in significant depletion (45-53%) of ionized calcium-binding adapter molecule 1 immunoreactive (Iba-1ir) microglia within the SNpc. Microglial depletion did not impact accumulation of phosphorylated α-syn (pSyn) within SNpc neurons and did not alter pSyn associated microglial reactivity or expression of MHC-II. Further, microglial depletion did not impact SNpc neuron degeneration. Paradoxically, long term microglial depletion resulted in increased soma size of remaining microglia in both control and PFF rats, as well as expression of MHC-II in extranigral regions. Conclusions: Collectively, our results suggest that microglial depletion is not a viable disease-modifying strategy for PD and that partial microglial depletion can induce a heightened proinflammatory state in remaining microglia.

16.
Toxicol Sci ; 196(1): 99-111, 2023 10 30.
Artigo em Inglês | MEDLINE | ID: mdl-37607008

RESUMO

Parkinson's disease (PD) is the fastest-growing neurological disease worldwide, with increases outpacing aging and occurring most rapidly in recently industrialized areas, suggesting a role of environmental factors. Epidemiological, post-mortem, and mechanistic studies suggest that persistent organic pollutants, including the organochlorine pesticide dieldrin, increase PD risk. In mice, developmental dieldrin exposure causes male-specific exacerbation of neuronal susceptibility to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and synucleinopathy. Specifically, in the α-synuclein (α-syn) pre-formed fibril (PFF) model, exposure leads to increased deficits in striatal dopamine (DA) turnover and motor deficits on the challenging beam. Here, we hypothesized that alterations in DA handling contribute to the observed changes and assessed vesicular monoamine transporter 2 (VMAT2) function and DA release in this dieldrin/PFF 2-hit model. Female C57BL/6 mice were exposed to 0.3 mg/kg dieldrin or vehicle every 3 days by feeding, starting at 8 weeks of age and continuing throughout breeding, gestation, and lactation. Male offspring from independent litters underwent unilateral, intrastriatal injections of α-syn PFFs at 12 weeks of age, and vesicular 3H-DA uptake assays and fast-scan cyclic voltammetry were performed 4 months post-PFF injection. Dieldrin-induced an increase in DA release in striatal slices in PFF-injected animals, but no change in VMAT2 activity. These results suggest that developmental dieldrin exposure increases a compensatory response to synucleinopathy-triggered striatal DA loss. These findings are consistent with silent neurotoxicity, where developmental exposure to dieldrin primes the nigrostriatal striatal system to have an exacerbated response to synucleinopathy in the absence of observable changes in typical markers of nigrostriatal dysfunction and degeneration.


Assuntos
Doença de Parkinson , Praguicidas , Sinucleinopatias , Camundongos , Animais , Masculino , Feminino , alfa-Sinucleína/metabolismo , Dopamina , Dieldrin/toxicidade , Camundongos Endogâmicos C57BL , Praguicidas/toxicidade , Proteínas Vesiculares de Transporte de Monoamina , Transmissão Sináptica , Substância Negra/metabolismo
17.
Dev Biol ; 353(2): 173-85, 2011 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-21382369

RESUMO

Protective cellular responses to stress and aging in the germline are essential for perpetuation of a species; however, relatively few studies have focused on how germ cells respond to stress and aging. We have previously shown that large ribonucleoprotein (RNP) complexes assemble in oocytes of Caenorhabditis during extended meiotic arrest or after environmental stress. Here we explore the regulation of these dynamic RNPs and demonstrate their assembly is coordinated with dramatic, nuclear membrane blebbing in oocytes. Our ultrastructural analyses reveal distinct changes in the endoplasmic reticulum, and the first evidence for the assembly of stacked annulate lamellae in Caenorhabditis. We further show several nucleoporins are required for the complete assembly of RNP granules, and a disruption in RNP granule assembly coupled with a low frequency of nuclear blebbing in arrested oocytes negatively impacts embryonic viability. Our observations support a model where nuclear membrane blebbing is required to increase the trafficking of nucleoporins to the cell cortex in stressed or meiotically arrested cells and to facilitate the recruitment of RNA and protein components of RNPs into large complexes. These new insights may have general implications for better understanding how germ cells preserve their integrity when fertilization is delayed and how cells respond to stress.


Assuntos
Caenorhabditis/citologia , Caenorhabditis/metabolismo , Proteínas de Helminto/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Oócitos/metabolismo , Ribonucleoproteínas/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Animais Geneticamente Modificados , Caenorhabditis/genética , Senescência Celular , Feminino , Resposta ao Choque Térmico , Masculino , Microscopia Eletrônica de Transmissão , Modelos Biológicos , Membrana Nuclear/metabolismo , Membrana Nuclear/ultraestrutura , Oócitos/citologia , Estresse Fisiológico
18.
NPJ Parkinsons Dis ; 8(1): 61, 2022 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-35610264

RESUMO

ß2-adrenoreceptor (ß2AR) agonists have been associated with a decreased risk of developing Parkinson's disease (PD) and are hypothesized to decrease expression of both alpha-synuclein mRNA (Snca) and protein (α-syn). Effects of ß2AR agonist clenbuterol on the levels of Snca mRNA and α-syn protein were evaluated in vivo (rats and mice) and in rat primary cortical neurons by two independent laboratories. A modest decrease in Snca mRNA in the substantia nigra was observed after a single acute dose of clenbuterol in rats, however, this decrease was not maintained after multiple doses. In contrast, α-syn protein levels remained unchanged in both single and multiple dosing paradigms. Furthermore, clenbuterol did not decrease Snca in cultured rat primary cortical neurons, or decrease Snca or α-syn in mice. Additionally, compared to the single-dose paradigm, repeat dosing resulted in substantially lower levels of clenbuterol in plasma and brain tissue in rodents. Based on our observations of a transient decrease in Snca and no effect on α-syn protein in this preclinical study, these data support the conclusion that clenbuterol is not likely a viable disease-modifying strategy for PD.

19.
J Vis Exp ; (184)2022 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-35815999

RESUMO

Identification and isolation of contagious individuals along with quarantine of close contacts, is critical for slowing the spread of COVID-19. Large-scale testing in a surveillance or screening capacity for asymptomatic carriers of COVID-19 provides both data on viral spread and the follow-up ability to rapidly test individuals during suspected outbreaks. The COVID-19 early detection program at Michigan State University has been utilizing large-scale testing in a surveillance or screening capacity since fall of 2020. The methods adapted here take advantage of the reliability, large sample volume, and self-collection benefits of saliva, paired with a cost-effective, reagent conserving two-dimensional pooling scheme. The process was designed to be adaptable to supply shortages, with many components of the kits and the assay easily substituted. The processes outlined for collecting and processing SARS-CoV-2 samples can be adapted to test for future viral pathogens reliably expressed in saliva. By providing this blueprint for universities or other organizations, preparedness plans for future viral outbreaks can be developed.


Assuntos
COVID-19 , SARS-CoV-2 , COVID-19/diagnóstico , Teste para COVID-19 , Humanos , Reprodutibilidade dos Testes , Saliva , Manejo de Espécimes
20.
Parkinsonism Relat Disord ; 89: 41-47, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34218047

RESUMO

BACKGROUND: Alpha-synuclein (α-syn) preformed fibril (PFF)-induced pathology can be used to study the features and progression of synucleinopathies, such as Parkinson's disease. Intrastriatal injection of mouse α-syn PFFs produce accumulation of α-syn pathology in both mice and rats. Previous studies in mice have revealed that greater sequence homology between the α-syn amino acid sequence used to produce PFFs with that of the endogenous host α-syn increases α-syn pathology in vivo. NEW METHODS: Based on the prediction that greater sequence homology will result in more α-syn pathology, PFFs generated from recombinant rat α-syn (rPFFs) were used instead of PFFs produced from recombinant mouse α-syn (mPFFs), which are normally used in the model. Rats received unilateral intrastriatal injections of either rPFFs or mPFFs and accumulation of α-syn phosphorylated at serine 129 (pSyn) was examined at 1-month post-surgery. RESULTS: Rats injected with mPFFs exhibited abundant accumulation of α-syn inclusions in the substantia nigra and cortical regions, whereas in rats injected with rPFFs had significantly fewer SNpc neurons containing pSyn inclusions (≈60% fewer) and little, if any, pSyn inclusions were observed in the cortex. CONCLUSIONS: Our results suggest that additional factors beyond the degree of sequence homology between host α-syn and injected recombinant α-syn impact efficiency of seeding and subsequent inclusion formation. More practically, these findings caution against the use of rPFFs in the rat preformed fibril model.


Assuntos
Sinucleinopatias/metabolismo , Sinucleinopatias/patologia , alfa-Sinucleína , Animais , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Modelos Animais de Doenças , Camundongos , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Ratos , Proteínas Recombinantes , Substância Negra/metabolismo , Substância Negra/patologia
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