RESUMO
BACKGROUND: Lipid droplets (LD), lipid-storing organelles containing neutral lipids like glycerolipids and cholesterol, are increasingly accepted as hallmarks of inflammation. The nuclear paraspeckle assembly transcript 1 (NEAT1), a long non-coding RNA with over 200 nucleotides, exerts an indispensable impact on regulating both LD agglomeration and autophagy in multiple neurological disorders. However, knowledge as to how NEAT1 modulates the formation of LD and associated signaling pathways is limited. METHODS: In this study, primary microglia were isolated from newborn mice and exposed to oxygen-glucose-deprivation/reoxygenation (OGD/R). To further explore NEAT1-dependent mechanisms, an antisense oligonucleotide (ASO) was adopted to silence NEAT1 under in vitro conditions. Studying NEAT1-dependent interactions with regard to autophagy and LD agglomeration under hypoxic conditions, the inhibitor and activator of autophagy 3-methyladenine (3-MA) and rapamycin (RAPA) were used, respectively. In a preclinical stroke model, mice received intraventricular injections of ASO NEAT1 or control vectors in order to yield NEAT1 knockdown. Analysis of readout parameters included qRT-PCR, immunofluorescence, western blot assays, and behavioral tests. RESULTS: Microglia exposed to OGD/R displayed a temporal pattern of NEAT1 expression, peaking at four hours of hypoxia followed by six hours of reoxygenation. After effectively silencing NEAT1, LD formation and autophagy-related proteins were significantly repressed in hypoxic microglia. Stimulating autophagy in ASO NEAT1 microglia under OGD/R conditions by means of RAPA reversed the downregulation of LD agglomeration and perilipin 2 (PLIN2) expression. On the contrary, application of 3-MA promoted repression of both LD agglomeration and expression of the LD-associated protein PLIN2. Under in vivo conditions, NEAT1 was significantly increased in mice at 24 h post-stroke. Knockdown of NEAT1 significantly alleviated LD agglomeration and inhibited autophagy, resulting in improved cerebral perfusion, reduced brain injury and increased neurological recovery. CONCLUSION: NEAT1 is a key player of LD agglomeration and autophagy stimulation, and NEAT1 knockdown provides a promising therapeutic value against stroke.
Assuntos
RNA Longo não Codificante , Acidente Vascular Cerebral , Animais , Camundongos , Apoptose/genética , Autofagia/genética , Gotículas Lipídicas/metabolismo , Microglia/metabolismo , Oxigênio/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Transdução de Sinais , Acidente Vascular Cerebral/genética , Acidente Vascular Cerebral/metabolismoRESUMO
Mutations in PARK15, which encodes for the F-box protein FBXO7 have been associated with Parkinsonian Pyramidal syndrome, a rare and complex movement disorder with Parkinsonian symptoms, pyramidal tract signs and juvenile onset. Our previous study showed that systemic loss of Fbxo7 in mice causes motor defects and premature death. We have also demonstrated that FBXO7 has a crucial role in neurons as the specific deletion in tyrosine hydroxylase-positive or glutamatergic forebrain neurons leads to late-onset or early-onset motor dysfunction, respectively. In this study, we examined NEX-Cre;Fbxo7fl/fl mice, in which Fbxo7 was specifically deleted in glutamatergic projection neurons. The effects of FBXO7 deficiency on striatal integrity were investigated with HPLC and histological analyses. NEX-Cre;Fbxo7fl/fl mice revealed an increase in striatal dopamine concentrations, changes in the glutamatergic, GABAergic and dopaminergic pathways, astrogliosis and microgliosis and little or no neuronal loss in the striatum. To determine the effects on the integrity of the synapse, we purified synaptic membranes, subjected them to quantitative mass spectrometry analysis and found alterations in the complement system, endocytosis and exocytosis pathways. These neuropathological changes coincide with alterations in spontaneous home cage behavior. Taken together, our findings suggest that FBXO7 is crucial for corticostriatal projections and the synaptic integrity of the striatum, and consequently for proper motor control.
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Regional iron accumulation and α-synuclein (α-syn) spreading pathology within the central nervous system are common pathological findings in Parkinson's disease (PD). Whereas iron is known to bind to α-syn, facilitating its aggregation and regulating α-syn expression, it remains unclear if and how iron also modulates α-syn spreading. To elucidate the influence of iron on the propagation of α-syn pathology, we investigated α-syn spreading after stereotactic injection of α-syn preformed fibrils (PFFs) into the striatum of mouse brains after neonatal brain iron enrichment. C57Bl/6J mouse pups received oral gavage with 60, 120, or 240 mg/kg carbonyl iron or vehicle between postnatal days 10 and 17. At 12 weeks of age, intrastriatal injections of 5-µg PFFs were performed to induce seeding of α-syn aggregates. At 90 days post-injection, PFFs-injected mice displayed long-term memory deficits, without affection of motor behavior. Interestingly, quantification of α-syn phosphorylated at S129 showed reduced α-syn pathology and attenuated spreading to connectome-specific brain regions after brain iron enrichment. Furthermore, PFFs injection caused intrastriatal microglia accumulation, which was alleviated by iron in a dose-dependent way. In primary cortical neurons in a microfluidic chamber model in vitro, iron application did not alter trans-synaptic α-syn propagation, possibly indicating an involvement of non-neuronal cells in this process. Our study suggests that α-syn PFFs may induce cognitive deficits in mice independent of iron. However, a redistribution of α-syn aggregate pathology and reduction of striatal microglia accumulation in the mouse brain may be mediated via iron-induced alterations of the brain connectome.
Assuntos
Química Encefálica , Ferro/farmacologia , Sinucleinopatias/metabolismo , Sinucleinopatias/patologia , alfa-Sinucleína/metabolismo , alfa-Sinucleína/toxicidade , Animais , Animais Recém-Nascidos , Conectoma , Corpo Estriado , Relação Dose-Resposta a Droga , Feminino , Humanos , Ferro/administração & dosagem , Masculino , Transtornos da Memória/induzido quimicamente , Transtornos da Memória/psicologia , Camundongos Endogâmicos C57BL , Microglia/patologia , Microinjeções , Atividade Motora/efeitos dos fármacos , alfa-Sinucleína/administração & dosagemRESUMO
Mutations in the FBXO7 (PARK15) gene have been implicated in a juvenile form of parkinsonism termed parkinsonian pyramidal syndrome (PPS), characterized by Parkinsonian symptoms and pyramidal tract signs. FBXO7 (F-box protein only 7) is a subunit of the SCF (SKP1/cullin-1/F-box protein) E3 ubiquitin ligase complex, but its relevance and function in neurons remain to be elucidated. Here, we report that the E3 ligase FBXO7-SCF binds to and ubiquitinates the proteasomal subunit PSMA2. In addition, we show that FBXO7 is a proteasome-associated protein involved in proteasome assembly. In FBXO7 knockout mice, we find reduced proteasome activity and early-onset motor deficits together with premature death. In addition, we demonstrate that NEX (neuronal helix-loop-helix protein-1)-Cre-induced deletion of the FBXO7 gene in forebrain neurons or the loss of FBXO7 in tyrosine hydroxylase (TH)-positive neurons results in motor defects, reminiscent of the phenotype in PARK15 patients. Taken together, our study establishes a vital role for FBXO7 in neurons, which is required for proper motor control and accentuates the importance of FBXO7 in proteasome function.
Assuntos
Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Técnicas de Inativação de Genes , Transtornos Parkinsonianos/patologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Animais , Camundongos Knockout , Processamento de Proteína Pós-Traducional , UbiquitinaçãoRESUMO
Parkinson's disease (PD) is a neurodegenerative disorder with prominent neuronal cell death in the substantia nigra (SN) and other parts of the brain. Previous studies in models of traumatic and neurodegenerative CNS disease showed that pharmacological inhibition of Rho-associated kinase (ROCK), a molecule involved in inhibitory signaling in the CNS, by small-molecule inhibitors improves neuronal survival and increases regeneration. Most small-molecule inhibitors, however, offer only limited target specificity and also inhibit other kinases, including both ROCK isoforms. To establish the role of the predominantly brain-expressed ROCK2 isoform in models of regeneration and PD, we used adeno-associated viral vectors (AAV) to specifically knockdown ROCK2 in neurons. Rat primary midbrain neurons (PMN) were transduced with AAV expressing short-hairpin-RNA (shRNA) against ROCK2 and LIM-domain kinase 1 (LIMK1), one of the downstream targets of ROCK2. While knock-down of ROCK2 and LIMK1 both enhanced neurite regeneration in a traumatic scratch lesion model, only ROCK2-shRNA protected PMN against 1-methyl-4-phenylpyridinium (MPP+) toxicity. Moreover, AAV.ROCK2-shRNA increased levels of the pro-survival markers Bcl-2 and phospho-Erk1. In vivo, AAV.ROCK2-shRNA vectors were injected into the ipsilateral SN and a unilateral 6-OHDA striatal lesion was performed. After four weeks, behavioral, immunohistochemical and biochemical alterations were investigated. Downregulation of ROCK2 protected dopaminergic neurons in the SN from 6-OHDA-induced degeneration and resulted in significantly increased TH-positive neuron numbers. This effect, however, was confined to nigral neuronal somata as striatal terminal density, dopamine and metabolite levels were not significantly preserved. Interestingly, motor behavior was improved in the ROCK2-shRNA treated animals compared to control after four weeks. Our studies thus confirm ROCK2 as a promising therapeutic target in models of PD and demonstrate that neuron-specific inhibition of ROCK2 promotes survival of lesioned dopaminergic neurons.
Assuntos
Neurônios Dopaminérgicos/metabolismo , Regulação para Baixo/fisiologia , Degeneração Neural/etiologia , Degeneração Neural/patologia , Doença de Parkinson/complicações , Quinases Associadas a rho/metabolismo , Ácido 3,4-Di-Hidroxifenilacético/metabolismo , Adrenérgicos/toxicidade , Animais , Dependovirus/genética , Modelos Animais de Doenças , Regulação para Baixo/genética , Vetores Genéticos/fisiologia , Ácido Homovanílico , Quinases Lim/genética , Quinases Lim/metabolismo , Oxidopamina/toxicidade , Doença de Parkinson/etiologia , Desempenho Psicomotor , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Wistar , Substância Negra/patologia , Tirosina 3-Mono-Oxigenase , Quinases Associadas a rho/genéticaRESUMO
Glypicans are biomarkers for various pathologies, including cardiovascular disease, cancer and diabetes. Increasing evidence suggests that glypicans also play a role in the context of neurodegenerative disorders. Initially described as supporting functionality of synapses via glutamate receptors during CNS development, Glypican 4 (GPC-4) also plays a role in the context of dementia via tau hyperphosphorylation in Alzheimer's disease, which is also a co-pathology in Parkinson's disease dementia. However, clinical evidence of circulating GPC-4 in Parkinson's disease (PD) is missing so far. We therefore investigated GPC-4 in biofluids of PD patients. We analyzed GPC-4 levels in cerebrospinal fluid (CSF, n = 140), serum (n = 80), and tear fluid samples (n = 70) of PD patients and control subjects in a similar age range by ELISA (serum, CSF) and western blot (tear fluid). Expression of circulating GPC-4 was confirmed in all three biofluids, with highest levels in serum. Interestingly, GPC-4 levels were age-dependent, and multiple regression analysis revealed a significant association between GPC-4 serum levels and MoCA score, suggesting an involvement of GPC-4 in PD-associated cognitive decline. Furthermore, stratification of PD patients for vascular risk factors revealed a significant increase of GPC-4 serum levels in PD patients with vascular risk factors. Our results suggest GPC-4 as a clinical biomarker for vascular risk stratification in order to identify PD patients with increased risk of developing dementia.
Assuntos
Doença de Alzheimer , Disfunção Cognitiva , Demência , Doença de Parkinson , Humanos , Doença de Alzheimer/complicações , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Biomarcadores/líquido cefalorraquidiano , Disfunção Cognitiva/complicações , Demência/complicações , Glipicanas , Doença de Parkinson/complicações , Doença de Parkinson/líquido cefalorraquidiano , Fatores de Risco , Proteínas tau/líquido cefalorraquidianoRESUMO
Triggering receptor expressed on myeloid cells 2 (TREM2) is a transmembrane receptor protein predominantly expressed in microglia within the central nervous system (CNS). TREM2 regulates multiple microglial functions, including lipid metabolism, immune reaction, inflammation, and microglial phagocytosis. Recent studies have found that TREM2 is highly expressed in activated microglia after ischemic stroke. However, the role of TREM2 in the pathologic response after stroke remains unclear. Herein, TREM2-deficient microglia exhibit an impaired phagocytosis rate and cholesteryl ester (CE) accumulation, leading to lipid droplet formation and upregulation of Perilipin-2 (PLIN2) expression after hypoxia. Knockdown of TREM2 results in increased lipid synthesis (PLIN2, SOAT1) and decreased cholesterol clearance and lipid hydrolysis (LIPA, ApoE, ABCA1, NECH1, and NPC2), further impacting microglial phenotypes. In these lipid droplet-rich microglia, the TGF-ß1/Smad2/3 signaling pathway is downregulated, driving microglia towards a pro-inflammatory phenotype. Meanwhile, in a neuron-microglia co-culture system under hypoxic conditions, we found that microglia lost their protective effect against neuronal injury and apoptosis when TREM2 was knocked down. Under in vivo conditions, TREM2 knockdown mice express lower TGF-ß1 expression levels and a lower number of anti-inflammatory M2 phenotype microglia, resulting in increased cerebral infarct size, exacerbated neuronal apoptosis, and aggravated neuronal impairment. Our work suggests that TREM2 attenuates stroke-induced neuroinflammation by modulating the TGF-ß1/Smad2/3 signaling pathway. TREM2 may play a direct role in the regulation of inflammation and also exert an influence on the post-ischemic inflammation and the stroke pathology progression via regulation of lipid metabolism processes. Thus, underscoring the therapeutic potential of TREM2 agonists in ischemic stroke and making TREM2 an attractive new clinical target for the treatment of ischemic stroke and other inflammation-related diseases.
Assuntos
Lesões Encefálicas , AVC Isquêmico , Acidente Vascular Cerebral , Animais , Camundongos , Lesões Encefálicas/metabolismo , Ésteres do Colesterol/metabolismo , Inflamação/metabolismo , AVC Isquêmico/metabolismo , Gotículas Lipídicas/metabolismo , Microglia/metabolismo , Acidente Vascular Cerebral/genética , Acidente Vascular Cerebral/metabolismo , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Axonal degeneration is one of the earliest features of Parkinson's disease pathology, which is followed by neuronal death in the substantia nigra and other parts of the brain. Inhibition of axonal degeneration combined with cellular neuroprotection therefore seem key to targeting an early stage in Parkinson's disease progression. Based on our previous studies in traumatic and neurodegenerative disease models, we have identified rho kinase as a molecular target that can be manipulated to disinhibit axonal regeneration and improve survival of lesioned central nervous system neurons. In this study, we examined the neuroprotective potential of pharmacological rho kinase inhibition mediated by fasudil in the in vitro 1-methyl-4-phenylpyridinium cell culture model and in the subchronic in vivo 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson's disease. Application of fasudil resulted in a significant attenuation of dopaminergic cell loss in both paradigms. Furthermore, dopaminergic terminals were preserved as demonstrated by analysis of neurite network in vitro, striatal fibre density and by neurochemical analysis of the levels of dopamine and its metabolites in the striatum. Behavioural tests demonstrated a clear improvement in motor performance after fasudil treatment. The Akt survival pathway was identified as an important molecular mediator for neuroprotective effects of rho kinase inhibition in our paradigm. We conclude that inhibition of rho kinase using the clinically approved small molecule inhibitor fasudil may be a promising new therapeutic strategy for Parkinson's disease.
Assuntos
1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , 1-Metil-4-fenilpiridínio/toxicidade , Neurônios Dopaminérgicos/fisiologia , Degeneração Neural/tratamento farmacológico , Fármacos Neuroprotetores/farmacologia , Doença de Parkinson Secundária/enzimologia , Quinases Associadas a rho/fisiologia , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/uso terapêutico , Animais , Axônios/efeitos dos fármacos , Axônios/patologia , Comportamento Animal/efeitos dos fármacos , Comportamento Animal/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Corpo Estriado/metabolismo , Modelos Animais de Doenças , Dopamina/metabolismo , Neurônios Dopaminérgicos/enzimologia , Neurônios Dopaminérgicos/patologia , Intoxicação por MPTP/tratamento farmacológico , Intoxicação por MPTP/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Degeneração Neural/induzido quimicamente , Degeneração Neural/enzimologia , Neuritos/patologia , Fármacos Neuroprotetores/metabolismo , Fármacos Neuroprotetores/uso terapêutico , Doença de Parkinson Secundária/induzido quimicamente , Doença de Parkinson Secundária/tratamento farmacológico , Doença de Parkinson Secundária/patologia , Proteínas Proto-Oncogênicas c-akt/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Wistar , Substância Negra/efeitos dos fármacos , Substância Negra/enzimologia , Quinases Associadas a rho/antagonistas & inibidoresRESUMO
Parkinson's disease (PD), multiple system atrophy (MSA), and dementia with Lewy bodies (DLB) are neurodegenerative disorders with alpha-synuclein (α-syn) aggregation pathology. Different strains of α-syn with unique properties are suggested to cause distinct clinical and pathological manifestations resulting in PD, MSA, or DLB. To study individual α-syn spreading patterns, we injected α-syn fibrils amplified from brain homogenates of two MSA patients and two PD patients into the brains of C57BI6/J mice. Antibody staining against pS129-α-syn showed that α-syn fibrils amplified from the brain homogenates of the four different patients caused different levels of α-syn spreading. The strongest α-syn pathology was triggered by α-syn fibrils of one of the two MSA patients, followed by comparable pS129-α-syn induction by the second MSA and one PD patient material. Histological analysis using an antibody against Iba1 further showed that the formation of pS129-α-syn is associated with increased microglia activation. In contrast, no differences in dopaminergic neuron numbers or co-localization of α-syn in oligodendrocytes were observed between the different groups. Our data support the spreading of α-syn pathology in MSA, while at the same time pointing to spreading heterogeneity between different patients potentially driven by individual patient immanent factors.
Assuntos
Atrofia de Múltiplos Sistemas , Doença de Parkinson , Sinucleinopatias , Animais , Camundongos , alfa-Sinucleína/metabolismo , Anticorpos , Encéfalo/patologia , Atrofia de Múltiplos Sistemas/patologia , Doença de Parkinson/patologia , Sinucleinopatias/patologiaRESUMO
Background: Stroke stimulates reactive astrogliosis, aquaporin 4 (AQP4) depolarization and neuroinflammation. Preconditioned extracellular vesicles (EVs) from microglia exposed to hypoxia, in turn, reduce poststroke brain injury. Nevertheless, the underlying mechanisms of such effects are elusive, especially with regards to inflammation, AQP4 polarization, and cerebrospinal fluid (CSF) flow. Methods: Primary microglia and astrocytes were exposed to oxygen-glucose deprivation (OGD) injury. For analyzing the role of AQP4 expression patterns under hypoxic conditions, a co-culture model of astrocytes and microglia was established. Further studies applied a stroke model, where some mice also received an intracisternal tracer infusion of rhodamine B. As such, these in vivo studies involved the analysis of AQP4 polarization, CSF flow, astrogliosis, and neuroinflammation as well as ischemia-induced brain injury. Results: Preconditioned EVs decreased periinfarct AQP4 depolarization, brain edema, astrogliosis, and inflammation in stroke mice. Likewise, EVs promoted postischemic CSF flow and cerebral blood perfusion, and neurological recovery. Under in vitro conditions, hypoxia stimulated M2 microglia polarization, whereas EVs augmented M2 microglia polarization and repressed M1 microglia polarization even further. In line with this, astrocytes displayed upregulated AQP4 clustering and proinflammatory cytokine levels when exposed to OGD, which was reversed by preconditioned EVs. Reduced AQP4 depolarization due to EVs, however, was not a consequence of unspecific inflammatory regulation, since LPS-induced inflammation in co-culture models of astrocytes and microglia did not result in altered AQP4 expression patterns in astrocytes. Conclusions: These findings show that hypoxic microglia may participate in protecting against stroke-induced brain damage by regulating poststroke inflammation, astrogliosis, AQP4 depolarization, and CSF flow due to EV release.
Assuntos
Aquaporina 4 , Lesões Encefálicas , Vesículas Extracelulares , Acidente Vascular Cerebral , Animais , Camundongos , Aquaporina 4/metabolismo , Lesões Encefálicas/metabolismo , Vesículas Extracelulares/metabolismo , Gliose/metabolismo , Hipóxia/metabolismo , Inflamação/metabolismo , Microglia/metabolismo , Doenças Neuroinflamatórias , Oxigênio/metabolismo , Acidente Vascular Cerebral/metabolismoRESUMO
α-synuclein (αSyn) is the main protein component of Lewy bodies, intracellular inclusions found in the brain of Parkinson's disease (PD) patients. Neurotoxic αSyn species are broadly modified post-translationally and, in patients with genetic forms of PD, carry genetically encoded amino acid substitutions. Mutations and C-terminal truncation can increase αSyn oligomerization and fibrillization. Although several genetic mouse models based on αSyn mutations and/or truncations exist, there is still a lack of mouse models for synucleinopathies not relying on overexpression. We report here two synucleinopathy mouse models, which are based on a triple alanine to proline mutation and a C-terminal truncation of αSyn, but do not overexpress the mutant protein when compared to the endogenous mouse protein. We knocked hαSyn TP or hαSynΔ119 (h stands for "human") into the murine αSyn locus. hαSynTP is a structure-based mutant with triple alanine to proline substitutions that favors oligomers, is neurotoxic and evokes PD-like symptoms in Drosophila melanogaster. hαSynΔ119 lacks 21 amino acids at the C-terminus, favors fibrillary aggregates and occurs in PD. Knocking-in of hαSyn TP or hαSynΔ119 into the murine αSyn locus places the mutant protein under the control of the endogenous regulatory elements while simultaneously disrupting the mαSyn gene. Mass spectrometry revealed that hαSyn TP and hαSynΔ119 mice produced 12 and 10 times less mutant protein, compared to mαSyn in wild type mice. We show phenotypes in 1 and 1.5 years old hαSyn TP and hαSynΔ119 mice, despite the lower levels of hαSynTP and hαSynΔ119 expression. Direct comparison of the two mouse models revealed many commonalities but also aspects unique to each model. Commonalities included strong immunoactive state, impaired olfaction and motor coordination deficits. Neither model showed DAergic neuronal loss. Impaired climbing abilities at 1 year of age and a deviant gait pattern at 1.5 years old were specific for hαSynΔ119 mice, while a compulsive behavior was exclusively detected in hαSyn TP mice starting at 1 year of age. We conclude that even at very moderate levels of expression the two αSyn variants evoke measurable and progressive deficiencies in mutant mice. The two transgenic mouse models can thus be suitable to study αSyn-variant-based pathology in vivo and test new therapeutic approaches.
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Axonal damage is an early step in traumatic and neurodegenerative disorders of the central nervous system (CNS). Damaged axons are not able to regenerate sufficiently in the adult mammalian CNS, leading to permanent neurological deficits. Recently, we showed that inhibition of the autophagic protein ULK1 promotes neuroprotection in different models of neurodegeneration. Moreover, we demonstrated previously that axonal protection improves regeneration of lesioned axons. However, whether axonal protection mediated by ULK1 inhibition could also improve axonal regeneration is unknown. Here, we used an adeno-associated viral (AAV) vector to express a dominant-negative form of ULK1 (AAV.ULK1.DN) and investigated its effects on axonal regeneration in the CNS. We show that AAV.ULK1.DN fosters axonal regeneration and enhances neurite outgrowth in vitro. In addition, AAV.ULK1.DN increases neuronal survival and enhances axonal regeneration after optic nerve lesion, and promotes long-term axonal protection after spinal cord injury (SCI) in vivo. Interestingly, AAV.ULK1.DN also increases serotonergic and dopaminergic axon sprouting after SCI. Mechanistically, AAV.ULK1.DN leads to increased ERK1 activation and reduced expression of RhoA and ROCK2. Our findings outline ULK1 as a key regulator of axonal degeneration and regeneration, and define ULK1 as a promising target to promote neuroprotection and regeneration in the CNS.
Assuntos
Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Axônios/metabolismo , Dependovirus/genética , Técnicas de Transferência de Genes , Vetores Genéticos , Regeneração Nervosa , Traumatismos do Nervo Óptico/terapia , Nervo Óptico/metabolismo , Traumatismos da Medula Espinal/terapia , Medula Espinal/metabolismo , Animais , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Axônios/patologia , Células Cultivadas , Modelos Animais de Doenças , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Regulação para Baixo , Feminino , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Crescimento Neuronal , Nervo Óptico/patologia , Traumatismos do Nervo Óptico/genética , Traumatismos do Nervo Óptico/metabolismo , Traumatismos do Nervo Óptico/patologia , Ratos Wistar , Neurônios Serotoninérgicos/metabolismo , Neurônios Serotoninérgicos/patologia , Medula Espinal/patologia , Traumatismos da Medula Espinal/genética , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/patologia , Fatores de Tempo , Proteínas rho de Ligação ao GTP/metabolismo , Quinases Associadas a rho/metabolismoRESUMO
Loss of nigrostriatal projections by axonal degeneration is a key early event in Parkinson's disease (PD) pathophysiology, being accountable for the lack of dopamine in the nigrostriatal system and resulting in motor symptoms such as bradykinesia, rigidity, and tremor. Since autophagy is an important mechanism contributing to axonal degeneration, we aimed to evaluate the effects of competitive autophagy inhibition in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD in vivo. Adeno-associated viral vector (AAV)-mediated overexpression of a dominant-negative form of the unc-51 like autophagy-initiating kinase (ULK1.DN) in the substantia nigra was induced 3 weeks before MPTP treatment. Analysis of motor behavior demonstrated a significant improvement of ULK1.DN expressing mice after MPTP treatment. Immunohistochemical analyses of dopaminergic nigral neurons and nigrostriatal projections revealed a significant protection from MPTP-induced neurotoxicity after ULK1.DN expression. Western blot analysis linked these findings to an activation of mTOR signaling. Taken together, our results indicate that expression of ULK1.DN can attenuate MPTP-induced axonal neurodegeneration, suggesting that ULK1 could be a promising novel target in the treatment of PD.
Assuntos
Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Dependovirus/metabolismo , Genes Dominantes , Atividade Motora , Neurônios/enzimologia , Neurônios/patologia , Doença de Parkinson/enzimologia , Doença de Parkinson/fisiopatologia , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina , Animais , Comportamento Animal , Sobrevivência Celular , Modelos Animais de Doenças , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Células HEK293 , Humanos , Masculino , Metaboloma , Camundongos Endogâmicos C57BL , Substância Negra/patologia , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
The objective of the study was to estimate if altered levels of alpha-synuclein can be detected in tear fluid of patients with Parkinson's disease (PD). Therefore, tear fluid samples of 75 PD patients, 75 control subjects and 31 atypical Parkinsonian patients were collected and analyzed in triplicates using an ultra-sensitive single molecule array (SIMOA) system and applying a human alpha-synuclein immunoassay. In PD, levels of total soluble alpha-synuclein were significantly increased compared to control subjects (p = 0.03; AUC PD vs. controls 0.60). There was no difference comparing PD patients stratified by Hoehn & Yahr stages and atypical Parkinsonian syndromes stratified by tauopathies and non-PD-synucleinopathies against each other (p > 0.05). In conclusion, alpha-synuclein can be detected and quantified in tear fluid, revealing small but significant differences in total alpha-synuclein levels between PD and control subjects. Tear fluid can be collected non-invasively and risk-free, therefore presenting a promising source for further biomarker research.
Assuntos
Biomarcadores/metabolismo , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Lágrimas/metabolismo , alfa-Sinucleína/metabolismo , Idoso , Estudos de Casos e Controles , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , PrognósticoRESUMO
Axonal degeneration is a key and early pathological feature in traumatic and neurodegenerative disorders of the CNS. Following a focal lesion to axons, extended axonal disintegration by acute axonal degeneration (AAD) occurs within several hours. During AAD, the accumulation of autophagic proteins including Unc-51 like autophagy activating kinase 1 (ULK1) has been demonstrated, but its role is incompletely understood. Here, we study the effect of ULK1 inhibition in different models of lesion-induced axonal degeneration in vitro and in vivo. Overexpression of a dominant negative of ULK1 (ULK1.DN) in primary rat cortical neurons attenuates axotomy-induced AAD in vitro. Both ULK1.DN and the ULK1 inhibitor SBI-0206965 protect against AAD after rat optic nerve crush in vivo. ULK1.DN additionally attenuates long-term axonal degeneration after rat spinal cord injury in vivo. Mechanistically, ULK1.DN decreases autophagy and leads to an mTOR-mediated increase in translational proteins. Consistently, treatment with SBI-0206965 results in enhanced mTOR activation. ULK1.DN additionally modulates the differential splicing of the degeneration-associated genes Kif1b and Ddit3. These findings uncover ULK1 as an important mediator of axonal degeneration in vitro and in vivo, and elucidate its function in splicing, defining it as a putative therapeutic target.
Assuntos
Proteína Homóloga à Proteína-1 Relacionada à Autofagia , Axônios , Sistema Nervoso Central , Degeneração Neural , Doenças Neurodegenerativas , Animais , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/antagonistas & inibidores , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/fisiologia , Axônios/metabolismo , Axônios/patologia , Células Cultivadas , Sistema Nervoso Central/lesões , Sistema Nervoso Central/metabolismo , Feminino , Degeneração Neural/tratamento farmacológico , Degeneração Neural/metabolismo , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Cultura Primária de Células , RatosRESUMO
Glioblastoma represent the most common primary brain tumor in adults and are currently considered incurable. We investigated antiproliferative and anti-invasive mechanisms of 6-OH-11-O-hydroxyfenantrene (IIF), a retinoid X receptor ligand, and pioglitazone (PGZ), a peroxisome proliferator-activated receptor gamma activator, in three different glioblastoma cell lines. A dose-dependent reduction of tumor invasion and strong decrease of matrix metalloproteinases 2 and 9 expression was observed, especially when a combination therapy of IIF and PGZ was administered. Combined treatment also markedly reduced proliferation and induced apoptosis in all glioma cell lines tested. This was in particular accompanied by decrease of antiapoptotic proteins Bcl2 and p53, while simultaneously pro-apoptotic cytochrome c, cleaved caspase 3, Bax and Bad levels increased. These in vitro findings were further substantiated in a murine glioma model in vivo, where oral administration of PGZ and IIF resulted in significantly reduced tumor volume and proliferation. Of note, treatment with nuclear receptor ligands was not only effective when the treatment was initiated shortly after the intraparenchymal seeding of the glioma cells, but even when initiated in the last third of the observation period. Collectively, our results demonstrate the effectiveness of a combined treatment of ligands of proliferator-activated receptor and retinoid X receptor against glioblastoma.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioma/tratamento farmacológico , PPAR gama/uso terapêutico , Tretinoína/análogos & derivados , Análise de Variância , Animais , Anexina A5/metabolismo , Bromodesoxiuridina/metabolismo , Caspase 3/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Citocromos c/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Humanos , Ligantes , Metaloproteinases da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Invasividade Neoplásica/fisiopatologia , PPAR gama/metabolismo , Pioglitazona , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Receptor X Retinoide gama/metabolismo , Sais de Tetrazólio , Tiazóis , Tiazolidinedionas/uso terapêutico , Transfecção/métodos , Tretinoína/uso terapêutico , Ensaio Tumoral de Célula-Tronco/métodos , Proteína X Associada a bcl-2/metabolismoRESUMO
Neurodegenerative disorders like Parkinson's disease, Alzheimer's disease, or amyotrophic lateral sclerosis are affecting a rapidly increasing population worldwide. While common pathomechanisms such as protein aggregation, axonal degeneration, dysfunction of protein clearing and an altered immune response have been characterized, no disease-modifying therapies have been developed so far. Interestingly, a significant involvement of the Rho kinase (ROCK) signaling pathway has been described in all of these mechanisms making it a promising target for new therapeutic approaches. In this article, we first review current knowledge of the involvement of ROCK in neurodegenerative disorders and the utility of its inhibition as a disease-modifying therapy in different neurodegenerative disorders. After a detailed description of the biochemical characteristics of ROCK and its molecular interactors, differences of ROCK-expression under physiological and pathological conditions are compared. Next, different pharmacological and molecular-genetic strategies to inhibit ROCK-function are discussed, focusing on pharmacological ROCK-inhibitors. The role of the ROCK-pathway in cellular processes that are central in neurodegenerative disorders pathology like axonal degeneration, autophagy, synaptic and glial function is explained in detail. Finally, all available data on ROCK-inhibition in different animal models of neurodegenerative disorders is reviewed and first approaches for translation into human patients are discussed. Taken together, there is now extensive evidence from preclinical studies in several neurodegenerative disorders that characterize ROCK as a promising drug target for further translational research in neurodegenerative disorders.
Assuntos
Doenças Neurodegenerativas/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Quinases Associadas a rho/antagonistas & inibidores , Animais , Modelos Animais de Doenças , Humanos , Doenças Neurodegenerativas/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Inibidores de Proteínas Quinases/farmacologia , Quinases Associadas a rho/fisiologiaRESUMO
Parkinson's disease (PD) is the second-most-frequent neurodegenerative disorder worldwide. One major hallmark of PD is the degeneration of dopaminergic (DA) neurons in the substantia nigra. Glial cell line-derived neurotrophic factor (GDNF) potently increases DA neuron survival in models of PD; however, the underlying mechanisms are incompletely understood. MicroRNAs (miRNAs) are small, non-coding RNAs that are important for post-transcriptional regulation of gene expression. Using small RNA sequencing, we show that GDNF specifically increases the expression of miR-182-5p and miR-183-5p in primary midbrain neurons (PMNs). Transfection of synthetic miR-182-5p and miR-183-5p mimics leads to increased neurite outgrowth and mediates neuroprotection of DA neurons in vitro and in vivo, mimicking GDNF effects. This is accompanied by decreased expression of FOXO3 and FOXO1 transcription factors and increased PI3K-Akt signaling. Inhibition of endogenous miR-182-5p or miR-183-5p in GDNF-treated PMNs attenuated the pro-DA effects of GDNF. These findings unveil an unknown miR-mediated mechanism of GDNF action and suggest that targeting miRNAs is a new therapeutic avenue to PD phenotypes.
RESUMO
Parkinson's disease (PD) is the most common neurodegenerative movement disorder, and its causes remain unknown. A major hallmark of the disease is the increasing presence of aggregated alpha-synuclein (aSyn). Furthermore, there is a solid consensus on iron (Fe) accumulation in several regions of PD brains during disease progression. In our study, we focused on the interaction of Fe and aggregating aSyn in vivo in a transgenic mouse model overexpressing human aSyn bearing the A53T mutation (prnp.aSyn.A53T). We utilized a neonatal iron-feeding model to exacerbate the motor phenotype of the transgenic mouse model. Beginning from day 100, mice were treated with deferiprone (DFP), a ferric chelator that is able to cross the blood-brain barrier and is currently used in clinics as treatment for hemosiderosis. Our paradigm resulted in an impairment of the learning abilities in the rotarod task and the novel object recognition test. DFP treatment significantly improved the performance in both tasks. Although this was not accompanied by alterations in aSyn aggregation, our results support DFP as possible therapeutic option in PD.
Assuntos
Transtornos Neurológicos da Marcha/tratamento farmacológico , Quelantes de Ferro/uso terapêutico , Ferro/toxicidade , Deficiências da Aprendizagem/tratamento farmacológico , Transtornos Parkinsonianos/tratamento farmacológico , Piridonas/uso terapêutico , alfa-Sinucleína/genética , Animais , Contagem de Células , Deferiprona , Avaliação Pré-Clínica de Medicamentos , Feminino , Transtornos Neurológicos da Marcha/etiologia , Transtornos Neurológicos da Marcha/metabolismo , Humanos , Ferro/metabolismo , Deficiências da Aprendizagem/etiologia , Deficiências da Aprendizagem/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Neurônios/patologia , Transtornos Parkinsonianos/metabolismo , Transtornos Parkinsonianos/psicologia , Agregação Patológica de Proteínas/tratamento farmacológico , Agregação Patológica de Proteínas/metabolismo , Reconhecimento Psicológico/efeitos dos fármacos , Teste de Desempenho do Rota-Rod , alfa-Sinucleína/metabolismoRESUMO
To identify specific markers for the diagnosis of choroid plexus tumors, gene expression profiles of choroid plexus epithelial cells (n = 8) and ependymal cells (n = 6) microdissected from human autopsy brains as well as choroid plexus papilloma tissue were investigated using DNA microarrays. Protein expression of genes overexpressed in choroid plexus was evaluated in normal choroid plexus, choroid plexus papilloma, choroid plexus carcinoma, other primary brain tumors, and cerebral metastases. Forty-six genes found to be overexpressed in normal choroid plexus epithelial cells were also present in choroid plexus papilloma. Among those, 11 were further analyzed by immunohistochemistry. Expression of inward rectifier potassium channel Kir7.1 was confirmed in normal choroid plexus (34 of 35), choroid plexus papilloma (12 of 18), and choroid plexus carcinoma (5 of 5) but was not found in 100 other primary brain tumors and cerebral metastases. Similarly, stanniocalcin-1 stained normal choroid plexus (32 of 35), choroid plexus papilloma (16 of 18), and choroid plexus carcinoma (3 of 5), whereas staining was seen in only 2 of 100 other primary brain tumors and cerebral metastases. Transthyretin stained choroid plexus (33 of 35), choroid plexus papilloma (14 of 18), and plexus carcinoma (2 of 5), but its specificity was significantly lower. Antibodies directed against coagulation factor V, glutathione peroxidase 3, pigment epithelium derived factor, serotonin receptor 5-HTR2C, lumican, fibulin-1, plastin-1, and cytokeratin 18 revealed varying degrees of specificity and sensitivity. Our data suggest that antibodies directed against Kir7.1 and stanniocalcin-1 might serve as sensitive and specific diagnostic markers for choroid plexus tumors.