RESUMEN
Bi-allelic mutations in GBA1, the gene that encodes ß-glucocerebrosidase (GCase), cause Gaucher disease (GD), whereas mono-allelic mutations do not cause overt pathology. Yet mono- or bi-allelic GBA1 mutations are the highest known risk factor for Parkinson's disease (PD). GCase deficiency results in the accumulation of glucosylceramide (GluCer) and its deacylated metabolite glucosylsphingosine (GluSph). Brains from patients with neuronopathic GD have high levels of GluSph, and elevation of this lipid in GBA1-associated PD has been reported. To uncover the mechanisms involved in GBA1-associated PD, we used human induced pluripotent stem cell-derived dopaminergic (DA) neurons from patients harboring heterozygote mutations in GBA1 (GBA1/PD-DA neurons). We found that compared with gene-edited isogenic controls, GBA1/PD-DA neurons exhibit mammalian target of rapamycin complex 1 (mTORC1) hyperactivity, a block in autophagy, an increase in the levels of phosphorylated α-synuclein (129) and α-synuclein aggregation. These alterations were prevented by incubation with mTOR inhibitors. Inhibition of acid ceramidase, the lysosomal enzyme that deacylates GluCer to GluSph, prevented mTOR hyperactivity, restored autophagic flux and lowered α-synuclein levels, suggesting that GluSph was responsible for these alterations. Incubation of gene-edited wild type (WT) controls with exogenous GluSph recapitulated the mTOR/α-synuclein abnormalities of GBA1/PD neurons, and these phenotypic alterations were prevented when GluSph treatment was in the presence of mTOR inhibitors. We conclude that GluSph causes an aberrant activation of mTORC1, suppressing normal lysosomal functions, including the clearance of pathogenic α-synuclein species. Our results implicate acid ceramidase in the pathogenesis of GBA1-associated PD, suggesting that this enzyme is a potential therapeutic target for treating synucleinopathies caused by GCase deficiency.
Asunto(s)
Enfermedad de Gaucher , Células Madre Pluripotentes Inducidas , Enfermedad de Parkinson , Humanos , Enfermedad de Parkinson/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Inhibidores mTOR , Ceramidasa Ácida/genética , Ceramidasa Ácida/metabolismo , Glucosilceramidasa/genética , Glucosilceramidasa/metabolismo , Enfermedad de Gaucher/metabolismo , Neuronas Dopaminérgicas/metabolismo , Serina-Treonina Quinasas TOR/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Mutación , Lisosomas/metabolismoRESUMEN
Neurons are dependent on proper trafficking of lipids to neighboring glia for lipid exchange and disposal of potentially lipotoxic metabolites, producing distinct lipid distribution profiles among various cell types of the central nervous system. Little is known of the cellular distribution of neutral lipids in the substantia nigra (SN) of Parkinson's disease (PD) patients and its relationship to inflammatory signaling. This study aimed to determine human PD SN neutral lipid content and distribution in dopaminergic neurons, astrocytes, and microglia relative to age-matched healthy subject controls. The results show that while total neutral lipid content was unchanged relative to age-matched controls, the levels of whole SN triglycerides were correlated with inflammation-attenuating glycoprotein non-metastatic melanoma protein B (GPNMB) signaling in human PD SN. Histological localization of neutral lipids using a fluorescent probe (BODIPY) revealed that dopaminergic neurons and midbrain microglia significantly accumulated intracellular lipids in PD SN, while adjacent astrocytes had a reduced lipid load overall. This pattern was recapitulated by experimental in vivo inhibition of glucocerebrosidase activity in mice. Agents or therapies that restore lipid homeostasis among neurons, astrocytes, and microglia could potentially correct PD pathogenesis and disease progression.
Asunto(s)
Glucolípidos/metabolismo , Enfermedad de Parkinson/patología , Sustancia Negra/patología , Triglicéridos/metabolismo , Anciano , Anciano de 80 o más Años , Animales , Astrocitos/metabolismo , Astrocitos/patología , Estudios de Casos y Controles , Estudios de Cohortes , Modelos Animales de Enfermedad , Neuronas Dopaminérgicas/metabolismo , Neuronas Dopaminérgicas/patología , Femenino , Glucosilceramidasa/genética , Glucosilceramidasa/metabolismo , Voluntarios Sanos , Humanos , Masculino , Glicoproteínas de Membrana/metabolismo , Ratones , Microglía/metabolismo , Microglía/patología , Persona de Mediana Edad , Sustancia Negra/citología , Sustancia Negra/metabolismo , alfa-Sinucleína/metabolismoRESUMEN
It is well established that lysosomal glucocerebrosidase gene (GBA) variants are a risk factor for Parkinson's disease (PD), with increasing evidence suggesting a loss of function mechanism. One question raised by this genetic association is whether variants of genes involved in other aspects of sphingolipid metabolism are also associated with PD. Recent studies in sporadic PD have identified variants in multiple genes linked to diseases of glycosphingolipid (GSL) metabolism to be associated with PD. GSL biosynthesis is a complex pathway involving the coordinated action of multiple enzymes in the Golgi apparatus. GSL catabolism takes place in the lysosome and is dependent on the action of multiple acid hydrolases specific for certain substrates and glycan linkages. The finding that variants in multiple GSL catabolic genes are over-represented in PD in a heterozygous state highlights the importance of GSLs in the healthy brain and how lipid imbalances and lysosomal dysfunction are associated with normal ageing and neurodegenerative diseases. In this article we will explore the link between lysosomal storage disorders and PD, the GSL changes seen in both normal ageing, lysosomal storage disorders (LSDs) and PD and the mechanisms by which these changes can affect neurodegeneration.
Asunto(s)
Enfermedades por Almacenamiento Lisosomal , Enfermedad de Parkinson , Envejecimiento , Glucosilceramidasa/genética , Glucosilceramidasa/metabolismo , Glicoesfingolípidos/metabolismo , Humanos , Enfermedades por Almacenamiento Lisosomal/metabolismo , Lisosomas/metabolismo , Mutación , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismoRESUMEN
This study utilized human fibroblasts as a preclinical discovery and diagnostic platform for identification of cell biological signatures specific for the LRRK2 G2019S mutation producing Parkinson's disease (PD). Using live cell imaging with a pH-sensitive Rosella biosensor probe reflecting lysosomal breakdown of mitochondria, mitophagy rates were found to be decreased in fibroblasts carrying the LRRK2 G2019S mutation compared to cells isolated from healthy subject (HS) controls. The mutant LRRK2 increased kinase activity was reduced by pharmacological inhibition and targeted antisense oligonucleotide treatment, which normalized mitophagy rates in the G2019S cells and also increased mitophagy levels in HS cells. Detailed mechanistic analysis showed a reduction of mature autophagosomes in LRRK2 G2019S fibroblasts, which was rescued by LRRK2 specific kinase inhibition. These findings demonstrate an important role for LRRK2 protein in regulation of mitochondrial clearance by the lysosomes, which is hampered in PD with the G2019S mutation. The current results are relevant for cell phenotypic diagnostic approaches and potentially for stratification of PD patients for targeted therapy.
Asunto(s)
Autofagosomas/metabolismo , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/metabolismo , Mutación , Enfermedad de Parkinson/genética , Adulto , Anciano , Autofagosomas/efectos de los fármacos , Femenino , Fibroblastos/metabolismo , Regulación de la Expresión Génica , Humanos , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/antagonistas & inhibidores , Masculino , Persona de Mediana Edad , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Oligonucleótidos Antisentido/farmacología , Enfermedad de Parkinson/metabolismoRESUMEN
This article describes pathogenic concepts and factors, in particular glycolipid abnormalities, that create cell dysfunction and synaptic loss in neurodegenerative diseases. By phenocopying lysosomal storage disorders, such as Gaucher disease and related disorders, age- and dose-dependent changes in glycolipid cell metabolism can lead to Parkinson's disease and related dementias. Recent results show that perturbation of sphingolipid metabolism can precede or is a part of abnormal protein handling in both genetic and idiopathic Parkinson's disease and Lewy body dementia. In aging and genetic predisposition with lipid disturbance, α-synuclein's normal vesicular and synaptic role may be detrimentally shifted toward accommodating and binding such lipids. Specific neuronal glycolipid, protein, and vesicular interactions create potential pathophysiology that is amplified by astroglial and microglial immune mechanisms resulting in neurodegeneration. This perspective provides a new logic for therapeutic interventions that do not focus on protein aggregation, but rather provides a guide to the complex biology and the common sequence of events that lead to age-dependent neurodegenerative disorders.
Asunto(s)
Encéfalo/patología , Inflamación/patología , Degeneración Nerviosa/patología , Neuronas/patología , Enfermedad de Parkinson/patología , Animales , Encéfalo/inmunología , Encéfalo/metabolismo , Humanos , Inflamación/inmunología , Inflamación/metabolismo , Degeneración Nerviosa/inmunología , Degeneración Nerviosa/metabolismo , Neuronas/inmunología , Neuronas/metabolismo , Enfermedad de Parkinson/inmunología , Enfermedad de Parkinson/metabolismo , alfa-Sinucleína/metabolismo , Proteínas tau/metabolismoRESUMEN
GPNMB is a glycoprotein observed upon tissue damage and inflammation and is associated with astrocytes, microglia, and macrophages. Gene variations in GPNMB are linked with Parkinson's disease (PD) risk, and changes in protein levels of GPNMB have been found in lysosomal storage disorders, including Gaucher's disease with glucocerebrosidase (GCase) deficiency. In the current study, GPNMB increases were seen in the substantia nigra (SN) of PD patients compared to age-matched controls. Such PD patients have a decrease in GCase activity and corresponding elevation of glycosphingolipids in the SN (Rocha et al., 2015a). Interestingly, transgenic mice modelling synucleinopathy did not show GPNMB elevations or altered GCase activity levels compared to wild-type mice. However, upon CBE-induced GCase lysosomal dysfunction with elevated glycosphingolipids in wild-type mice, there were similar changes in GPNMB levels in the brain as seen in PD patient brains. These results indicate that GPNMB levels do not depend on alpha-synuclein load per se but relate directly to the lipidopathy changes induced by CBE-mediated GCase inhibition. The experimental modelling of elevating glycolipids resulted in GPNMB elevations with glial activation in several brain regions in mice. This is the first demonstration of region-specific elevations of GPNMB protein in Parkinson's disease. The presence of GPNMB in PD patient substantia nigra, the induction of GPNMB after experimental glycosphingolipid increases, but not with pure alpha-synucleinopathy, point towards the potential for primary lipid-induced degeneration in PD.
Asunto(s)
Lisosomas/metabolismo , Glicoproteínas de Membrana/biosíntesis , Estrés Oxidativo/fisiología , Enfermedad de Parkinson/metabolismo , Sustancia Negra/metabolismo , Anciano , Anciano de 80 o más Años , Animales , Estudios de Cohortes , Femenino , Humanos , Lisosomas/patología , Masculino , Ratones Transgénicos , Persona de Mediana Edad , Enfermedad de Parkinson/patología , Sustancia Negra/patologíaRESUMEN
A long-term goal of modeling Huntington's disease (HD) is to recapitulate the cardinal features of the disease in mice that express both mutant and wild-type (WT) huntingtin (Htt), as HD commonly manifests as a heterozygous condition in humans, and loss of WT Htt is associated with loss-of-function. In a new heterozygous Q175 knock-in (KI) mouse model, we performed an extensive evaluation of motor and cognitive functional deficits, neuropathological and biochemical changes and levels of proteins involved in synaptic function, the cytoskeleton and axonal transport, at 1-16 months of age. Motor deficits were apparent at 6 months of age in Q175 KI mice and at that time, postmortem striatal gamma-aminobutyric acid (GABA) levels were elevated and mutant Htt inclusions were present throughout the brain. From 6 months of age, levels of proteins associated with synaptic function, including SNAP-25, Rab3A and PSD-95, and with axonal transport and microtubules, including KIF3A, dynein and dynactin, were altered in the striatum, motor cortex, prefrontal cortex and hippocampus of Q175 KI mice, compared with WT levels. At 12-16 months of age, Q175 KI mice displayed motor and cognitive deficits, which were paralleled at postmortem by striatal atrophy, cortical thinning, degeneration of medium spiny neurons, dense mutant Htt inclusion formation, decreased striatal dopamine levels and loss of striatal brain-derived neurotrophic factor (BDNF). Data from this study indicate that the heterozygous Q175 KI mouse represents a realistic model for HD and also provides new insights into the specific and progressive synaptic, cytoskeletal and axonal transport protein abnormalities that may accompany the disease.
Asunto(s)
Transporte Axonal , Conducta Animal , Enfermedad de Huntington/genética , Enfermedad de Huntington/patología , Sinapsis/metabolismo , Envejecimiento/patología , Animales , Atrofia/genética , Atrofia/patología , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Citoesqueleto/metabolismo , Modelos Animales de Enfermedad , Técnicas de Sustitución del Gen , Heterocigoto , Cuerpos de Inclusión/metabolismo , Metaboloma , Ratones , Ratones Endogámicos C57BL , Mutación/genética , Neostriado/metabolismo , Neostriado/patología , Neurotransmisores/metabolismo , Receptor trkB/metabolismo , Proteínas de Transporte de Serotonina en la Membrana Plasmática/metabolismoRESUMEN
Diminished lysosomal function can lead to abnormal cellular accumulation of specific proteins, including α-synuclein, contributing to disease pathogenesis of vulnerable neurons in Parkinson's disease (PD) and related α-synucleinopathies. GBA1 encodes for the lysosomal hydrolase glucocerebrosidase (GCase), and mutations in GBA1 are a prominent genetic risk factor for PD. Previous studies showed that in sporadic PD, and in normal aging, GCase brain activity is reduced and levels of corresponding glycolipid substrates are increased. The present study tested whether increasing GCase through AAV-GBA1 intra-cerebral gene delivery in two PD rodent models would reduce the accumulation of α-synuclein and protect midbrain dopamine neurons from α-synuclein-mediated neuronal damage. In the first model, transgenic mice overexpressing wildtype α-synuclein throughout the brain (ASO mice) were used, and in the second model, a rat model of selective dopamine neuron degeneration was induced by AAV-A53T mutant α-synuclein. In ASO mice, intra-cerebral AAV-GBA1 injections into several brain regions increased GCase activity and reduced the accumulation of α-synuclein in the substantia nigra and striatum. In rats, co-injection of AAV-GBA1 with AAV-A53T α-synuclein into the substantia nigra prevented α-synuclein-mediated degeneration of nigrostriatal dopamine neurons by 6 months. These neuroprotective effects were associated with altered protein expression of markers of autophagy. These experiments demonstrate, for the first time, the neuroprotective effects of increasing GCase against dopaminergic neuron degeneration, and support the development of therapeutics targeting GCase or other lysosomal genes to improve neuronal handling of α-synuclein.
Asunto(s)
Neuronas Dopaminérgicas/enzimología , Terapia Genética/métodos , Glucosilceramidasa/genética , Mesencéfalo/enzimología , Enfermedades Neurodegenerativas/terapia , alfa-Sinucleína/metabolismo , Animales , Dependovirus/genética , Modelos Animales de Enfermedad , Dopamina/metabolismo , Neuronas Dopaminérgicas/patología , Femenino , Vectores Genéticos , Glucosilceramidasa/metabolismo , Humanos , Masculino , Mesencéfalo/patología , Ratones Transgénicos , Enfermedades Neurodegenerativas/enzimología , Enfermedades Neurodegenerativas/patología , Ratas Sprague-Dawley , alfa-Sinucleína/genéticaRESUMEN
Parkinson's disease is a neurodegenerative disorder, characterized by accumulation and misfolding of α-synuclein. Although the level of α-synuclein in neurons is fundamentally linked to the onset of neurodegeneration, multiple pathways have been implicated in its degradation, and it remains unclear which are the critical ubiquitination enzymes that protect against α-synuclein accumulation in vivo. The ubiquitin ligase Nedd4 targets α-synuclein to the endosomal-lysosomal pathway in cultured cells. Here we asked whether Nedd4-mediated degradation protects against α-synuclein-induced toxicity in the Drosophila and rodent models of Parkinson's disease. We show that overexpression of Nedd4 can rescue the degenerative phenotype from ectopic expression of α-synuclein in the Drosophila eye. Overexpressed Nedd4 in the Drosophila brain prevented the α-synuclein-induced locomotor defect whereas reduction in endogenous Nedd4 by RNAi led to worsening motor function and increased loss of dopaminergic neurons. Accordingly, AAV-mediated expression of wild-type but not the catalytically inactive Nedd4 decreased the α-synuclein-induced dopaminergic cell loss in the rat substantia nigra and reduced α-synuclein accumulation. Collectively, our data in two evolutionarily distant model organisms strongly suggest that Nedd4 is a modifier of α-synuclein pathobiology and thus a potential target for neuroprotective therapies.
Asunto(s)
Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Trastornos Parkinsonianos/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , alfa-Sinucleína/metabolismo , Animales , Animales Modificados Genéticamente , Encéfalo/metabolismo , Encéfalo/patología , Neuronas Dopaminérgicas/metabolismo , Neuronas Dopaminérgicas/patología , Drosophila , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Ojo/metabolismo , Ojo/patología , Femenino , Humanos , Locomoción/fisiología , Masculino , Mutación , Ubiquitina-Proteína Ligasas Nedd4 , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/patología , Trastornos Parkinsonianos/patología , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Sustancia Negra/metabolismo , Sustancia Negra/patología , Ubiquitina-Proteína Ligasas/genética , alfa-Sinucleína/genéticaRESUMEN
The main motor symptoms of Parkinson's disease are due to the loss of dopaminergic (DA) neurons in the ventral midbrain (VM). For the future treatment of Parkinson's disease with cell transplantation it is important to develop efficient differentiation methods for production of human iPSCs and hESCs-derived midbrain-type DA neurons. Here we describe an efficient differentiation and sorting strategy for DA neurons from both human ES/iPS cells and non-human primate iPSCs. The use of non-human primate iPSCs for neuronal differentiation and autologous transplantation is important for preclinical evaluation of safety and efficacy of stem cell-derived DA neurons. The aim of this study was to improve the safety of human- and non-human primate iPSC (PiPSC)-derived DA neurons. According to our results, NCAM(+) /CD29(low) sorting enriched VM DA neurons from pluripotent stem cell-derived neural cell populations. NCAM(+) /CD29(low) DA neurons were positive for FOXA2/TH and EN1/TH and this cell population had increased expression levels of FOXA2, LMX1A, TH, GIRK2, PITX3, EN1, NURR1 mRNA compared to unsorted neural cell populations. PiPSC-derived NCAM(+) /CD29(low) DA neurons were able to restore motor function of 6-hydroxydopamine (6-OHDA) lesioned rats 16 weeks after transplantation. The transplanted sorted cells also integrated in the rodent brain tissue, with robust TH+/hNCAM+ neuritic innervation of the host striatum. One year after autologous transplantation, the primate iPSC-derived neural cells survived in the striatum of one primate without any immunosuppression. These neural cell grafts contained FOXA2/TH-positive neurons in the graft site. This is an important proof of concept for the feasibility and safety of iPSC-derived cell transplantation therapies in the future.
Asunto(s)
Neuronas Dopaminérgicas/citología , Células Madre Embrionarias/citología , Células Madre Pluripotentes Inducidas/citología , Neuronas/metabolismo , Enfermedad de Parkinson/terapia , Células Madre Pluripotentes/citología , Trasplante de Células Madre/métodos , Adulto , Animales , Diferenciación Celular/fisiología , Modelos Animales de Enfermedad , Células Madre Embrionarias/trasplante , Femenino , Expresión Génica , Humanos , Células Madre Pluripotentes Inducidas/trasplante , Macaca fascicularis , Masculino , Neuronas/citología , Enfermedad de Parkinson/patología , Células Madre Pluripotentes/trasplante , Distribución Aleatoria , RatasRESUMEN
Alternative splicing is a complex post-transcriptional process that can be regulated by cis-acting elements located within genomic non-coding regions. Recent studies have identified that polymorphic variations in non-coding regions of the α-synuclein gene (SNCA) locus are associated with an increased risk for developing Parkinson's disease (PD). The underlying mechanism(s) for this susceptibility may involve changes in α-synuclein mRNA expression and alternative splicing. As a first step towards understanding the biology of α-synuclein splice variants in PD, we characterized the levels of the full-length SNCA-140 mRNA transcript and SNCA-126, -112, and -98 alternatively spliced variants in different neuronal regions from PD patients or transgenic mice overexpressing human α-synuclein (ASO). In human post-mortem tissue, α-synuclein spliced transcripts were expressed in a region-specific manner in the cortex, substantia nigra, and cerebellum. We observed increased nigral SNCA-140 and SNCA-126 transcript levels in PD patients when compared to neurologically unaffected cases. Human α-synuclein splicing changes were also found to occur in a region-specific manner in ASO mice. Here, SNCA-126, -112, and -98 transcript levels did not increase proportionally with SNCA-140 levels, or parallel the region-specific mouse transcript ratios seen in wild-type (WT) littermates. While most transcripts were elevated in ASO mice when compared to WT mice, the most prominent increase was found in the ventral midbrain of 15-month-old ASO mice. These results demonstrate region-specific human α-synuclein transcript level abnormalities in PD patients and in a transgenic mouse model of α-synucleinopathy. This study is relevant to understanding the normal, adaptive, or pathological role(s) of α-synuclein splice variants.
Asunto(s)
Empalme Alternativo/genética , Enfermedad de Parkinson/genética , Sustancia Negra/metabolismo , alfa-Sinucleína/metabolismo , Secuencia de Aminoácidos , Animales , Cerebelo/metabolismo , Modelos Animales de Enfermedad , Expresión Génica , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Datos de Secuencia Molecular , Neuronas/metabolismo , alfa-Sinucleína/genéticaRESUMEN
Recent advances in deriving induced pluripotent stem (iPS) cells from patients offer new possibilities for biomedical research and clinical applications, as these cells could be used for autologous transplantation. We differentiated iPS cells from patients with Parkinson's disease (PD) into dopaminergic (DA) neurons and show that these DA neurons can be transplanted without signs of neurodegeneration into the adult rodent striatum. The PD patient iPS (PDiPS) cell-derived DA neurons survived at high numbers, showed arborization, and mediated functional effects in an animal model of PD as determined by reduction of amphetamine- and apomorphine-induced rotational asymmetry, but only a few DA neurons projected into the host striatum at 16 wk after transplantation. We next applied FACS for the neural cell adhesion molecule NCAM on differentiated PDiPS cells before transplantation, which resulted in surviving DA neurons with functional effects on amphetamine-induced rotational asymmetry in a 6-OHDA animal model of PD. Morphologically, we found that PDiPS cell-derived non-DA neurons send axons along white matter tracts into specific close and remote gray matter target areas in the adult brain. Such findings establish the transplantation of human PDiPS cell-derived neurons as a long-term in vivo method to analyze potential disease-related changes in a physiological context. Our data also demonstrate proof of principle of survival and functional effects of PDiPS cell-derived DA neurons in an animal model of PD and encourage further development of differentiation protocols to enhance growth and function of implanted PDiPS cell-derived DA neurons in regard to potential therapeutic applications.
Asunto(s)
Enfermedad de Parkinson/cirugía , Células Madre Pluripotentes/citología , Animales , Humanos , Enfermedad de Parkinson/patología , RatasRESUMEN
Tightly regulated and highly adaptive lipid metabolic and transport pathways are critical to maintaining brain cellular lipid homeostasis and responding to lipid and inflammatory stress to preserve brain function and health. Deficits in the lipid handling genes APOE and GBA1 are the most significant genetic risk factors for Lewy body dementia and related dementia syndromes. Parkinson's disease patients who carry both APOE4 and GBA1 variants have accelerated cognitive decline compared to single variant carriers. To investigate functional interactions between brain ApoE and GBA1, in vivo GBA1 inhibition was tested in WT versus ApoE-deficient mice. The experiments demonstrated glycolipid stress caused by GBA1 inhibition in WT mice induced ApoE expression in several brain regions associated with movement and dementia disorders. The absence of ApoE in ApoE-KO mice amplified complement C1q elevations, reactive microgliosis and astrocytosis after glycolipid stress. Mechanistically, GBA1 inhibition triggered increases in cell surface and intracellular lipid transporters ABCA1 and NPC1, respectively. Interestingly, the absence of NPC1 in mice also triggered elevations of brain ApoE levels. These new data show that brain ApoE, GBA1 and NPC1 functions are interconnected in vivo, and that the removal or reduction of ApoE would likely be detrimental to brain function. These results provide important insights into brain ApoE adaptive responses to increased lipid loads.
Asunto(s)
Encéfalo , Glucosilceramidasa , Humanos , Ratones , Animales , Glucosilceramidasa/genética , Glucosilceramidasa/metabolismo , Encéfalo/metabolismo , Lisosomas/metabolismo , Apolipoproteínas E , Glucolípidos/metabolismoRESUMEN
Inflammatory processes and mechanisms are of central importance in neurodegenerative diseases. In the brain, α-synucleinopathies such as Parkinson's disease (PD) and Lewy body dementia (LBD) show immune cytokine network activation and increased toll like receptor 3 (TLR3) levels for viral double-stranded RNA (dsRNA). Brain inflammatory reactions caused by TLR3 activation are also relevant to understand pathogenic cascades by viral SARS-CoV-2 infection causing post- COVID-19 brain-related syndromes. In the current study, following regional brain TLR3 activation induced by dsRNA in mice, an acute complement C3 response was seen at 2 days. A C3 splice-switching antisense oligonucleotide (ASO) that promotes the splicing of a non-productive C3 mRNA, prevented downstream cytokines, such as IL-6, and α-synuclein changes. This report is the first demonstration that α-synuclein increases occur downstream of complement C3 activation. Relevant to brain dysfunction, post-COVID-19 syndromes and pathological changes leading to PD and LBD, viral dsRNA TLR3 activation in the presence of C3 complement blockade further revealed significant interactions between complement systems, inflammatory cytokine networks and α-synuclein changes.
Asunto(s)
COVID-19 , Enfermedad por Cuerpos de Lewy , Enfermedad de Parkinson , Animales , Ratones , alfa-Sinucleína/genética , Encéfalo , Complemento C3/genética , Citocinas , ARN Bicatenario , SARS-CoV-2 , Síndrome , Receptor Toll-Like 3/genéticaRESUMEN
Dopa-responsive dystonia (DRD) and Parkinson's disease (PD) are movement disorders caused by the dysfunction of nigrostriatal dopaminergic neurons. Identifying druggable pathways and biomarkers for guiding therapies is crucial due to the debilitating nature of these disorders. Recent genetic studies have identified variants of GTP cyclohydrolase-1 (GCH1), the rate-limiting enzyme in tetrahydrobiopterin (BH4) synthesis, as causative for these movement disorders. Here, we show that genetic and pharmacological inhibition of BH4 synthesis in mice and human midbrain-like organoids accurately recapitulates motor, behavioral and biochemical characteristics of these human diseases, with severity of the phenotype correlating with extent of BH4 deficiency. We also show that BH4 deficiency increases sensitivities to several PD-related stressors in mice and PD human cells, resulting in worse behavioral and physiological outcomes. Conversely, genetic and pharmacological augmentation of BH4 protects mice from genetically- and chemically induced PD-related stressors. Importantly, increasing BH4 levels also protects primary cells from PD-affected individuals and human midbrain-like organoids (hMLOs) from these stressors. Mechanistically, BH4 not only serves as an essential cofactor for dopamine synthesis, but also independently regulates tyrosine hydroxylase levels, protects against ferroptosis, scavenges mitochondrial ROS, maintains neuronal excitability and promotes mitochondrial ATP production, thereby enhancing mitochondrial fitness and cellular respiration in multiple preclinical PD animal models, human dopaminergic midbrain-like organoids and primary cells from PD-affected individuals. Our findings pinpoint the BH4 pathway as a key metabolic program at the intersection of multiple protective mechanisms for the health and function of midbrain dopaminergic neurons, identifying it as a potential therapeutic target for PD.
RESUMEN
While studying transgenic mice that overexpress human wildtype alpha-synuclein (Thy1-ASO, ASO) for typical brain alpha-synucleinopathy and central nervous system neuropathology, we observed progressive functional changes in the gastrointestinal and other peripheral organs. A more systematic study revealed that the gastrointestinal tract in ASO mice showed severe distension and blockage of the large intestine by 9-12 months of age. Functional assessments demonstrated a reduction in fecal water content and fecal pellet output, and increased whole gut transit time, in ASO mice compared to wildtype littermates, indicative of constipation, a symptom commonly reported by Parkinson's disease (PD) patients. Food intake was increased and body weight was decreased in 12 month old ASO mice, suggestive of metabolic abnormalities. Post-mortem histological analyses showed that human alpha-synuclein protein was robustly expressed in axonal fibers and in occasional cell bodies of the enteric nervous system, and in the heart of ASO mice. Accumulation of proteinase-K insoluble alpha-synuclein, reminiscent of neurodegenerative processes in PD was also observed. The functional and pathological changes we document here in ASO mice could relate to the autonomic deficits also seen in idiopathic and alpha-synuclein-mediated genetic forms of PD. These experimental data provide a foundation for therapeutic modeling of autonomic changes in PD and related alpha-synucleinopathies.
Asunto(s)
Enfermedades del Sistema Nervioso Autónomo/genética , Enfermedades del Sistema Nervioso Autónomo/metabolismo , Tracto Gastrointestinal/patología , Regulación de la Expresión Génica , Vejiga Urinaria/patología , alfa-Sinucleína/biosíntesis , Animales , Enfermedades del Sistema Nervioso Autónomo/patología , Tracto Gastrointestinal/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Transgénicos , Vejiga Urinaria/metabolismo , alfa-Sinucleína/genéticaRESUMEN
Immune rejection and risk of tumor formation are perhaps the greatest hurdles in the field of stem cell transplantation. Here, we report the generation of several lines of induced pluripotent stem cells (iPSCs) from cynomolgus macaque (CM) skin fibroblasts carrying specific major histocompatibility complex (MHC) haplotypes. To develop a collection of MHC-matched iPSCs, we genotyped the MHC locus of 25 CMs by microsatellite polymerase chain reaction analysis. Using retroviral infection of dermal skin fibroblasts, we generated several CM-iPSC lines carrying different haplotypes. We characterized the immunological properties of CM-iPSCs and demonstrated that CM-iPSCs can be induced to differentiate in vitro along specific neuronal populations, such as midbrain dopaminergic (DA) neurons. Midbrain-like DA neurons generated from CM-iPSCs integrated into the striatum of a rodent model of Parkinson's disease and promoted behavioral recovery. Importantly, neither tumor formation nor inflammatory reactions were observed in the transplanted animals up to 6 months after transplantation. We believe that the generation and characterization of such histocompatible iPSCs will allow the preclinical validation of safety and efficacy of iPSCs for neurodegenerative diseases and several other human conditions in the field of regenerative medicine.
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Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/trasplante , Neuronas/trasplante , Trasplante de Células Madre/métodos , Animales , Diferenciación Celular/fisiología , Cuerpo Estriado/cirugía , Femenino , Humanos , Células Madre Pluripotentes Inducidas/inmunología , Macaca fascicularis , Ratones , Ratones Endogámicos NOD , Ratones SCID , Neuronas/inmunología , Enfermedad de Parkinson/inmunología , Enfermedad de Parkinson/cirugía , Ratas , Ratas Sprague-DawleyRESUMEN
In Parkinson's disease (PD), dopaminergic (DA) neurons in the substantia nigra (SN, A9) are particularly vulnerable, compared to adjacent DA neurons within the ventral tegmental area (VTA, A10). Here, we show that in rat and human, one RAB3 isoform, RAB3B, has higher expression levels in A10 compared to A9 neurons. RAB3 is a monomeric GTPase protein that is highly enriched in synaptic vesicles and is involved in synaptic vesicle trafficking and synaptic transmission, disturbances of which have been implicated in several neurodegenerative diseases, including PD. These findings prompted us to further investigate the biology and neuroprotective capacity of RAB3B both in vitro and in vivo. RAB3B overexpression in human dopaminergic BE (2)-M17 cells increased neurotransmitter content, [(3)H] dopamine uptake, and levels of presynaptic proteins. AAV-mediated RAB3B overexpression in A9 DA neurons of the rat SN increased striatal dopamine content, number and size of synaptic vesicles, and levels of the presynaptic proteins, confirming in vitro findings. Measurement of extracellular DOPAC, a dopamine metabolite, following l-DOPA injection supported a role for RAB3B in enhancing the dopamine storage capacity of synaptic terminals. RAB3B overexpression in BE (2)-M17 cells was protective against toxins that simulate aspects of PD in vitro, including an oxidative stressor 6-hydroxydopamine (6-OHDA) and a proteasome inhibitor MG-132. Furthermore, RAB3B overexpression in rat SN both protected A9 DA neurons and resulted in behavioral improvement in a 6-OHDA retrograde lesion model of PD. These results suggest that RAB3B improves dopamine handling and storage capacity at presynaptic terminals, and confers protection to vulnerable DA neurons.
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Dopamina/metabolismo , Terminales Presinápticos/metabolismo , Proteínas de Unión al GTP rab3/genética , Proteínas de Unión al GTP rab3/metabolismo , Ácido 3,4-Dihidroxifenilacético/metabolismo , Animales , Línea Celular , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/metabolismo , Femenino , Expresión Génica , Perfilación de la Expresión Génica , Humanos , Técnicas In Vitro , Leupeptinas/toxicidad , Levodopa/farmacología , Modelos Neurológicos , Oxidopamina/toxicidad , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Trastornos Parkinsonianos/genética , Trastornos Parkinsonianos/metabolismo , Trastornos Parkinsonianos/patología , Terminales Presinápticos/efectos de los fármacos , Terminales Presinápticos/ultraestructura , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Sustancia Negra/metabolismo , Vesículas Sinápticas/metabolismo , Área Tegmental Ventral/metabolismoRESUMEN
In Parkinson's disease (PD), loss of striatal dopaminergic (DA) terminals and degeneration of DA neurons in the substantia nigra (SN) are associated with glial reactions. Such inflammatory processes are commonly considered an epiphenomenon of neuronal degeneration. However, there is increasing recognition of the role of neuroinflammation as an initiation factor of DA neuron degeneration. To investigate this issue, we established a new model of brain inflammation by injecting the Toll-like receptor 3 (TLR-3) agonist polyinosinic:polycytidylic acid [poly(I:C)] in the SN of adult rats. Poly(I:C) injection induced a sustained inflammatory reaction in the SN and in the dorsolateral striatum. Significant changes were detected in proteins relevant to synaptic transmission and axonal transport. In addition, cytoplasmic mislocalization of neuronal TAR DNA binding protein TDP-43 was observed. Poly(I:C) injection increased the susceptibility of midbrain DA neurons to a subsequent neurotoxic trigger (low-dose 6-hydroxydopamine). Systemic delivery of interleukin-1 receptor antagonist protected SN DA neurons exposed to combined poly(I:C) induced inflammatory and neurotoxic oxidative stress. These data indicate that viral-like neuroinflammation induces predegenerative changes in the DA system, which lowers the set point toward neuronal dysfunction and degeneration. New powerful neuroprotective therapies for PD might be considered by targeting critical inflammatory mechanisms, including cytokine-induced neurotoxicity.
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Cuerpo Estriado/patología , Dopamina , Degeneración Nerviosa/patología , Poli I-C/toxicidad , Sustancia Negra/patología , Receptor Toll-Like 3/agonistas , Animales , Cuerpo Estriado/efectos de los fármacos , Dopamina/fisiología , Femenino , Inyecciones Intraventriculares , Degeneración Nerviosa/inducido químicamente , Poli I-C/administración & dosificación , Ratas , Ratas Sprague-Dawley , Sustancia Negra/efectos de los fármacos , Receptor Toll-Like 3/fisiologíaRESUMEN
Lysosomal dysfunction is a central pathway associated with Parkinson's disease (PD) pathogenesis. Haploinsufficiency of the lysosomal hydrolase GBA (encoding glucocerebrosidase (GCase)) is one of the largest genetic risk factors for developing PD. Deficiencies in the activity of the GCase enzyme have been observed in human tissues from both genetic (harboring mutations in the GBA gene) and idiopathic forms of the disease. To understand the mechanisms behind the deficits of lysosomal GCase enzyme activity in idiopathic PD, this study utilized a large cohort of fibroblast cells from control subjects and PD patients with and without mutations in the GBA gene (N370S mutation) (control, n = 15; idiopathic PD, n = 31; PD with GBA N370S mutation, n = 6). The current data demonstrates that idiopathic PD fibroblasts devoid of any mutations in the GBA gene also exhibit reduction in lysosomal GCase activity, similar to those with the GBA N370S mutation. This reduced GCase enzyme activity in idiopathic PD cells was accompanied by decreased expression of the GBA trafficking receptor, LIMP2, and increased ER retention of the GBA protein in these cells. Importantly, in idiopathic PD fibroblasts LIMP2 protein levels correlated significantly with GCase activity, which was not the case in control subjects or in genetic PD GBA N370S cells. In conclusion, idiopathic PD fibroblasts have decreased GCase activity primarily driven by altered LIMP2-mediated transport of GBA to lysosome and the reduced GCase activity exhibited by the genetic GBA N370S derived PD fibroblasts occurs through a different mechanism.