RESUMO
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.
Assuntos
Autofagossomos/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Mutação , Doença de Parkinson/genética , Adulto , Idoso , Autofagossomos/efeitos dos fármacos , Feminino , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/antagonistas & inibidores , Masculino , Pessoa de Meia-Idade , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Oligonucleotídeos Antissenso/farmacologia , Doença de Parkinson/metabolismoRESUMO
Repulsive guidance molecule member a (RGMa) is a membrane-associated or released guidance molecule that is involved in axon guidance, cell patterning, and cell survival. In our previous work, we showed that RGMa is significantly upregulated in the substantia nigra of patients with Parkinson's disease. Here we demonstrate the expression of RGMa in midbrain human dopaminergic (DA) neurons. To investigate whether RGMa might model aspects of the neuropathology of Parkinson's disease in mouse, we targeted RGMa to adult midbrain dopaminergic neurons using adeno-associated viral vectors. Overexpression of RGMa resulted in a progressive movement disorder, including motor coordination and imbalance, which is typical for a loss of DA release in the striatum. In line with this, RGMa induced selective degeneration of dopaminergic neurons in the substantia nigra (SN) and affected the integrity of the nigrostriatal system. The degeneration of dopaminergic neurons was accompanied by a strong microglia and astrocyte activation. The behavioral, molecular, and anatomical changes induced by RGMa in mice are remarkably similar to the clinical and neuropathological hallmarks of Parkinson's disease. Our data indicate that dysregulation of RGMa plays an important role in the pathology of Parkinson's disease, and antibody-mediated functional interference with RGMa may be a disease modifying treatment option.SIGNIFICANCE STATEMENT Parkinson's disease (PD) is a neurodegenerative disease characterized by severe motor dysfunction due to progressive degeneration of mesencephalic dopaminergic (DA) neurons in the substantia nigra. To date, there is no regenerative treatment available. We previously showed that repulsive guidance molecule member a (RGMa) is upregulated in the substantia nigra of PD patients. Adeno-associated virus-mediated targeting of RGMa to mouse DA neurons showed that overexpression of this repulsive axon guidance and cell patterning cue models the behavioral and neuropathological characteristics of PD in a remarkable way. These findings have implications for therapy development as interfering with the function of this specific axon guidance cue may be beneficial to the survival of DA neurons.
Assuntos
Proteínas do Tecido Nervoso/genética , Doença de Parkinson/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Linhagem Celular Tumoral , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Feminino , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/metabolismo , Microglia/patologia , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/patologia , Doença de Parkinson/fisiopatologia , Substância Negra/metabolismo , Substância Negra/patologiaRESUMO
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.
RESUMO
There are many indications that neurogenesis is impaired in Parkinson's disease, which might be due to a lack of dopamine in the subventricular zone. An impairment in neurogenesis may have negative consequences for the development of new therapeutic approaches in Parkinson's disease, as neural stem cells are a potential source for endogenous repair. In this study, we examined the subventricular zone of 10 patients with Parkinson's disease and 10 age- and sex-matched controls for proliferation and neural stem cell numbers. We also included five cases with incidental Lewy body disease, which showed Parkinson's disease pathology but no clinical symptoms and thus did not receive dopaminergic treatment. We quantified the neural stem cell number and proliferative capacity in the subventricular zone of these three donor groups. We found subventricular neural stem cells in each donor, with a high variation in number. We did not observe significant differences in neural stem cell number or in proliferation between the groups. Additionally, we were able to culture neural stem cells from post-mortem brain of several patients with Parkinson's disease, confirming the presence of viable neural stem cells in these brains. We have also examined the subventricular zone of a chronic, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Parkinson's disease mouse model, and again found no effect of dopaminergic denervation on precursor proliferation. Lastly, we investigated the proliferation capacity of two different human neural stem cell lines in response to dopamine. Both cell lines did not respond with a change in proliferation to treatment with dopamine agonists and an antagonist. In summary, the adult neural stem cell pool in the subventricular zone was not clearly affected in the human parkinsonian brain or a Parkinson's disease mouse model. Furthermore, we did not find evidence that dopamine has a direct effect on human neural stem cell proliferation in vitro. Thus, we conclude that the number of adult neural stem cells is probably not diminished in the parkinsonian brain and that dopamine depletion most likely has no effect on human neural stem cells.
Assuntos
Encéfalo/patologia , Proliferação de Células , Ventrículos Cerebrais/patologia , Intoxicação por MPTP/patologia , Neurogênese/fisiologia , Doença de Parkinson/patologia , Idoso , Idoso de 80 Anos ou mais , Animais , Encéfalo/metabolismo , Encéfalo/fisiopatologia , Células Cultivadas , Ventrículos Cerebrais/metabolismo , Ventrículos Cerebrais/fisiopatologia , Feminino , Humanos , Intoxicação por MPTP/metabolismo , Intoxicação por MPTP/fisiopatologia , Masculino , Camundongos , Células-Tronco Neurais , Doença de Parkinson/metabolismo , Doença de Parkinson/fisiopatologiaRESUMO
Sandhoff disease (SD) is a lysosomal storage disease, caused by loss of ß-hexosaminidase (HEX) activity resulting in the accumulation of ganglioside GM2. There are shared features between SD and Parkinson's disease (PD). α-synuclein (aSYN) inclusions, the diagnostic hallmark sign of PD, are frequently found in the brain in SD patients and HEX knockout mice, and HEX activity is reduced in the substantia nigra in PD. In this study, we biochemically demonstrate that HEX deficiency in mice causes formation of high-molecular weight (HMW) aSYN and ubiquitin in the brain. As expected from HEX enzymatic function requirements, overexpression in vivo of HEXA and B combined, but not either of the subunits expressed alone, increased HEX activity as evidenced by histochemical assays. Biochemically, such HEX gene expression resulted in increased conversion of GM2 to its breakdown product GM3. In a neurodegenerative model of overexpression of aSYN in rats, increasing HEX activity by AAV6 gene transfer in the substantia nigra reduced aSYN embedding in lipid compartments and rescued dopaminergic neurons from degeneration. Overall, these data are consistent with a paradigm shift where lipid abnormalities are central to or preceding protein changes typically associated with PD.
Assuntos
Neurônios Dopaminérgicos/patologia , Gangliosídeos/metabolismo , alfa-Sinucleína/metabolismo , beta-N-Acetil-Hexosaminidases/metabolismo , Animais , Feminino , Lipídeos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Doença de Parkinson/metabolismo , Ratos , Ratos Sprague-Dawley , Doença de Sandhoff/metabolismo , Regulação para CimaRESUMO
Parkinson's disease (PD) is a progressive neurological disorder estimated to affect 7-10 million people worldwide. There is no treatment available that cures or slows the progression of PD. Elevated leucine-rich repeat kinase 2 (LRRK2) activity has been associated with genetic and sporadic forms of PD and, thus, reducing LRRK2 function is a promising therapeutic strategy. We have previously reported that an antisense oligonucleotide (ASO) that blocks splicing of LRRK2 exon 41, which encodes part of the kinase domain, reverses aberrant endoplasmic reticulum (ER) calcium levels and mitophagy defects in PD patient-derived cell lines harboring the LRRK2 G2019S mutation. In this study, we show that treating transgenic mice expressing human wild-type or G2019S LRRK2 with a single intracerebroventricular injection of ASO induces exon 41 skipping and results in a decrease in phosphorylation of the LRRK2 kinase substrate RAB10. Exon 41 skipping also reverses LRRK2 kinase-dependent changes in LC3B II/I ratios, a marker for the autophagic process. These results demonstrate the potential of LRRK2 exon 41 skipping as a possible therapeutic strategy to modulate pathogenic LRRK2 kinase activity associated with PD development.
RESUMO
The Parkinson disease (PD) genetic LRRK2 gain-of-function mutations may relate to the ER pathological changes seen in PD patients at postmortem. Human induced pluripotent stem cell (iPSC)-derived neurons with the PD pathogenic LRRK2 G2019S mutation exhibited neurite collapse when challenged with the ER Ca2+ influx sarco/ER Ca2+-ATPase inhibitor thapsigargin (THP). Baseline ER Ca2+ levels measured with the ER Ca2+ indicator CEPIA-ER were lower in LRRK2 G2019S human neurons, including in differentiated midbrain dopamine neurons in vitro. After THP challenge, PD patient-derived neurons displayed increased Ca2+ influx and decreased intracellular Ca2+ buffering upon membrane depolarization. These effects were reversed following LRRK2 mutation correction by antisense oligonucleotides and gene editing. Gene expression analysis in LRRK2 G2019S neurons identified modified levels of key store-operated Ca2+ entry regulators, with no alterations in ER Ca2+ efflux. These results demonstrate PD gene mutation LRRK2 G2019S ER calcium-dependent pathogenic effects in human neurons.
Assuntos
Sinalização do Cálcio , Células-Tronco Pluripotentes Induzidas/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Neuritos/metabolismo , Doença de Parkinson/metabolismo , Células Cultivadas , Retículo Endoplasmático/metabolismo , Humanos , Mutação de Sentido Incorreto , Neuritos/efeitos dos fármacos , Neuritos/patologia , Doença de Parkinson/genética , Tapsigargina/farmacologiaRESUMO
Adeno-associated viral vectors have numerous applications in neuroscience, including the study of gene function in health and disease, targeting of light-sensitive proteins to anatomically distinct sets of neurons to manipulate neuronal activity (optogenetics), and the delivery of fluorescent protein to study anatomical connectivity in the brain. Moreover several phase I/II clinical trials for gene therapy of eye and brain diseases with adeno-associated viral vectors have shown that these vectors are well tolerated by human patients. In this chapter we describe a detailed protocol for the small scale production of recombinant adeno-associated viral vectors. This protocol can be executed by investigators with experience in cell culture and molecular biological techniques in any well-equipped molecular neurobiology laboratory. With this protocol we typically obtain research batches of 100-200 µL that range in titer from 5 × 1012 to 2 × 1013 genomic copies/mL.
Assuntos
Encefalopatias/terapia , Dependovirus/genética , Técnicas de Transferência de Genes , Terapia Genética/métodos , Vetores Genéticos , Encefalopatias/genética , Oftalmopatias/genética , Oftalmopatias/terapia , Células HEK293 , Humanos , Injeções Intraoculares/métodos , Sistema Nervoso/metabolismo , PlasmídeosRESUMO
PURPOSE: Parkinson's disease (PD) is a movement disorder mainly characterized by progressive neurodegeneration of dopaminergic (DAergic) neurons in the substantia nigra (SN). As yet, unknown molecular changes contribute to the development of PD leading to a great need for in vivo models that herald this disorder. Here we characterize an animal model presenting early PD pathology. METHODS: Young, adult C57/BL6 mice were treated for five weeks twice a week with 15 mg/kg 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP) in combination with 250 mg/kg probenecid. During the treatment mice were tested on their dopamine dependent movement skills. The integrity of their nigrostriatal system was examined through immunohistochemical studies. RESULTS: During the treatment, mice developed dopamine-dependent movement deficits induced by loss of tyrosine hydroxylase (TH) positive nigrostriatal axon terminals. Immunohistochemical study identified astrogliosis and microgliosis in the SN but no decrease of TH immunostaining, demonstrating lack of DAergic neuron degeneration. We also observed formation of α-synuclein inclusion bodies in the SN. CONCLUSIONS: The combined features of this MPTP model appear to represent an early neurotoxic cellular stress to the SN neurons bearing a striking resemblance to the early stages of PD neuropathology. This model might prove very useful to investigate early neurodegenerative events in the nigrostriatal DAergic system and to study the effects of potential treatment strategies counteracting the early PD cellular changes.
Assuntos
Modelos Animais de Doenças , Intoxicação por MPTP/patologia , Movimento/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Substância Negra/efeitos dos fármacos , Animais , Intoxicação por MPTP/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Degeneração Neural/metabolismo , Degeneração Neural/patologia , Neurônios/metabolismo , Neurônios/patologia , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Substância Negra/metabolismo , Substância Negra/patologia , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Multiple genetic and environmental factors play a role in the development and progression of Parkinson's disease (PD). The main neuropathological hallmark of PD is the degeneration of dopaminergic (DAergic) neurons in the substantia nigra pars compacta. To study genetic and molecular contributors to the disease process, there is a great need for readily accessible cells with prominent DAergic features that can be used for reproducible in vitro cellular screening. Here, we investigated the molecular phenotype of retinoic acid (RA) differentiated SH-SY5Y cells using genome wide transcriptional profiling combined with gene ontology, transcription factor and molecular pathway analysis. We demonstrated that RA induces a general neuronal differentiation program in SH-SY5Y cells and that these cells develop a predominantly mature DAergic-like neurotransmitter phenotype. This phenotype is characterized by increased dopamine levels together with a substantial suppression of other neurotransmitter phenotypes, such as those for noradrenaline, acetylcholine, glutamate, serotonin and histamine. In addition, we show that RA differentiated SH-SY5Y cells express the dopamine and noradrenalin neurotransmitter transporters that are responsible for uptake of MPP(+), a well known DAergic cell toxicant. MPP(+) treatment alters mitochondrial activity according to its proposed cytotoxic effect in DAergic neurons. Taken together, RA differentiated SH-SY5Y cells have a DAergic-like phenotype, and provide a good cellular screening tool to find novel genes or compounds that affect cytotoxic processes that are associated with PD.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Perfilação da Expressão Gênica , Tretinoína/farmacologia , 1-Metil-4-fenilpiridínio/toxicidade , Biomarcadores/metabolismo , Linhagem Celular , Dopamina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Neurônios Dopaminérgicos/citologia , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Neurotransmissores/metabolismo , Proteínas da Membrana Plasmática de Transporte de Norepinefrina/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Substância Negra/citologia , Substância Negra/efeitos dos fármacos , Substância Negra/metabolismoRESUMO
Parkinson's disease and Alzheimer's disease are the most common neurodegenerative diseases in the elderly population. Given that age is the most important risk factor in these diseases, the number of patients is expected to rise dramatically in the coming years. Therefore, an effective therapy for these diseases is highly sought. Current treatment brings only temporary symptomatic relief and does not result in halting the progression of these diseases. The increasing knowledge on the molecular mechanisms that underlie these diseases enables the design of novel therapies, targeted at degenerating neurons by creating an optimal regenerative cellular environment. Here, we review the progress made in the field of cell-replacement and gene-therapy strategies. New developments in the application of embryonic stem cells and adult neuronal progenitors are discussed. We also discuss the use of genetically engineered cells in neuronal rescuing strategies that have recently advanced into the clinic. The first trials for the treatment of Alzheimer's disease and Parkinson's disease with this approach are ongoing.