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1.
Traffic ; 23(5): 238-269, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35343629

RESUMO

Since the discovery of lysosomes more than 70 years ago, much has been learned about the functions of these organelles. Lysosomes were regarded as exclusively degradative organelles, but more recent research has shown that they play essential roles in several other cellular functions, such as nutrient sensing, intracellular signalling and metabolism. Methodological advances played a key part in generating our current knowledge about the biology of this multifaceted organelle. In this review, we cover current methods used to analyze lysosome morphology, positioning, motility and function. We highlight the principles behind these methods, the methodological strategies and their advantages and limitations. To extract accurate information and avoid misinterpretations, we discuss the best strategies to identify lysosomes and assess their characteristics and functions. With this review, we aim to stimulate an increase in the quantity and quality of research on lysosomes and further ground-breaking discoveries on an organelle that continues to surprise and excite cell biologists.


Assuntos
Lisossomos , Redes e Vias Metabólicas , Lisossomos/metabolismo , Transdução de Sinais
2.
Mol Ther ; 30(4): 1432-1450, 2022 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-35121108

RESUMO

Mucopolysaccharidosis type IIIA (MPS-IIIA) is an autosomal recessive disorder caused by mutations in SGSH involved in the degradation of heparan sulfate. MPS-IIIA presents severe neurological symptoms such as progressive developmental delay and cognitive decline, for which there is currently no treatment. Brain targeting represents the main challenge for therapeutics to treat MPS-IIIA, and the development of small-molecule-based treatments able to reach the CNS could be a relevant advance for therapy. Using cell-based high content imaging to survey clinically approved drugs in MPS-IIIA cells, we identified fluoxetine, a selective serotonin reuptake inhibitor. Fluoxetine increases lysosomal and autophagic functions via TFEB activation through a RagC-dependent mechanism. Mechanistically, fluoxetine increases lysosomal exocytosis in mouse embryonic fibroblasts from MPS-IIIA mice, suggesting that this process may be responsible for heparan sulfate clearance. In vivo, fluoxetine ameliorates somatic and brain pathology in a mouse model of MPS-IIIA by decreasing the accumulation of glycosaminoglycans and aggregated autophagic substrates, reducing inflammation, and slowing down cognitive deterioration. We repurposed fluoxetine for potential therapeutics to treat human MPS-IIIA disease.


Assuntos
Mucopolissacaridose III , Animais , Modelos Animais de Doenças , Fibroblastos/metabolismo , Fluoxetina/farmacologia , Fluoxetina/uso terapêutico , Heparitina Sulfato/metabolismo , Hidrolases/genética , Camundongos , Mucopolissacaridose III/tratamento farmacológico , Mucopolissacaridose III/genética
3.
Mol Ther ; 28(4): 1167-1176, 2020 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-32087148

RESUMO

Lysosomal storage diseases (LSDs) are inherited disorders caused by lysosomal deficiencies and characterized by dysfunction of the autophagy-lysosomal pathway (ALP) often associated with neurodegeneration. No cure is currently available to treat neuropathology in LSDs. By studying a mouse model of mucopolysaccharidosis (MPS) type IIIA, one of the most common and severe forms of LSDs, we found that multiple amyloid proteins including α-synuclein, prion protein (PrP), Tau, and amyloid ß progressively aggregate in the brain. The amyloid deposits mostly build up in neuronal cell bodies concomitantly with neurodegeneration. Treating MPS-IIIA mice with CLR01, a "molecular tweezer" that acts as a broad-spectrum inhibitor of amyloid protein self-assembly reduced lysosomal enlargement and re-activates autophagy flux. Restoration of the ALP was associated with reduced neuroinflammation and amelioration of memory deficits. Together, these data provide evidence that brain deposition of amyloid proteins plays a gain of neurotoxic function in a severe LSD by affecting the ALP and identify CLR01 as new potent drug candidate for MPS-IIIA and likely for other LSDs.


Assuntos
Autofagia/efeitos dos fármacos , Hidrocarbonetos Aromáticos com Pontes/administração & dosagem , Mucopolissacaridose III/tratamento farmacológico , Doenças Neurodegenerativas/tratamento farmacológico , Organofosfatos/administração & dosagem , Amiloide/antagonistas & inibidores , Amiloide/metabolismo , Animais , Encéfalo/metabolismo , Hidrocarbonetos Aromáticos com Pontes/farmacologia , Corpo Celular/metabolismo , Modelos Animais de Doenças , Masculino , Camundongos , Mucopolissacaridose III/complicações , Mucopolissacaridose III/metabolismo , Doenças Neurodegenerativas/etiologia , Organofosfatos/farmacologia , Resultado do Tratamento
4.
Brain ; 142(5): 1365-1385, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30927362

RESUMO

Parkinson's disease is a progressive neurodegenerative disorder characterized by altered striatal dopaminergic signalling that leads to motor and cognitive deficits. Parkinson's disease is also characterized by abnormal presence of soluble toxic forms of α-synuclein that, when clustered into Lewy bodies, represents one of the pathological hallmarks of the disease. However, α-synuclein oligomers might also directly affect synaptic transmission and plasticity in Parkinson's disease models. Accordingly, by combining electrophysiological, optogenetic, immunofluorescence, molecular and behavioural analyses, here we report that α-synuclein reduces N-methyl-d-aspartate (NMDA) receptor-mediated synaptic currents and impairs corticostriatal long-term potentiation of striatal spiny projection neurons, of both direct (D1-positive) and indirect (putative D2-positive) pathways. Intrastriatal injections of α-synuclein produce deficits in visuospatial learning associated with reduced function of GluN2A NMDA receptor subunit indicating that this protein selectively targets this subunit both in vitro and ex vivo. Interestingly, this effect is observed in spiny projection neurons activated by optical stimulation of either cortical or thalamic glutamatergic afferents. We also found that treatment of striatal slices with antibodies targeting α-synuclein prevents the α-synuclein-induced loss of long-term potentiation and the reduced synaptic localization of GluN2A NMDA receptor subunit suggesting that this strategy might counteract synaptic dysfunction occurring in Parkinson's disease.


Assuntos
Corpo Estriado/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Memória Espacial/fisiologia , Sinapses/fisiologia , Percepção Visual/fisiologia , alfa-Sinucleína/toxicidade , Animais , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/patologia , Humanos , Potenciação de Longa Duração/efeitos dos fármacos , Potenciação de Longa Duração/fisiologia , Masculino , Camundongos , Camundongos Transgênicos , Técnicas de Cultura de Órgãos , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/metabolismo , Ratos , Ratos Wistar , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Memória Espacial/efeitos dos fármacos , Sinapses/efeitos dos fármacos , Percepção Visual/efeitos dos fármacos , alfa-Sinucleína/administração & dosagem
5.
Brain ; 141(2): 505-520, 2018 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-29281030

RESUMO

Nigro-striatal dopamine transmission is central to a wide range of neuronal functions, including skill learning, which is disrupted in several pathologies such as Parkinson's disease. The synaptic plasticity mechanisms, by which initial motor learning is stored for long time periods in striatal neurons, to then be gradually optimized upon subsequent training, remain unexplored. Addressing this issue is crucial to identify the synaptic and molecular mechanisms involved in striatal-dependent learning impairment in Parkinson's disease. In this study, we took advantage of interindividual differences between outbred rodents in reaching plateau performance in the rotarod incremental motor learning protocol, to study striatal synaptic plasticity ex vivo. We then assessed how this process is modulated by dopamine receptors and the dopamine active transporter, and whether it is impaired by overexpression of human α-synuclein in the mesencephalon; the latter is a progressive animal model of Parkinson's disease. We found that the initial acquisition of motor learning induced a dopamine active transporter and D1 receptors mediated long-term potentiation, under a protocol of long-term depression in striatal medium spiny neurons. This effect disappeared in animals reaching performance plateau. Overexpression of human α-synuclein reduced striatal dopamine active transporter levels, impaired motor learning, and prevented the learning-induced long-term potentiation, before the appearance of dopamine neuronal loss. Our findings provide evidence of a reorganization of cellular plasticity within the dorsolateral striatum that is mediated by dopamine receptors and dopamine active transporter during the acquisition of a skill. This newly identified mechanism of cellular memory is a form of metaplasticity that is disrupted in the early stage of synucleinopathies, such as Parkinson's disease, and that might be relevant for other striatal pathologies, such as drug abuse.


Assuntos
Corpo Estriado/citologia , Aprendizagem/fisiologia , Atividade Motora/fisiologia , Plasticidade Neuronal/fisiologia , Neurônios/fisiologia , Animais , Benzazepinas/farmacologia , Antagonistas de Dopamina/farmacologia , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Inibidores da Captação de Dopamina/farmacologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Aprendizagem/efeitos dos fármacos , Potenciação de Longa Duração/efeitos dos fármacos , Potenciação de Longa Duração/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Atividade Motora/efeitos dos fármacos , Destreza Motora/efeitos dos fármacos , Piperazinas/farmacologia , Tempo de Reação/fisiologia , Sinapsinas/genética , Sinapsinas/metabolismo , Sinaptofisina/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismo , alfa-Sinucleína/metabolismo , alfa-Sinucleína/farmacologia
7.
NPJ Parkinsons Dis ; 9(1): 92, 2023 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-37328503

RESUMO

Cognitive deficits, including working memory, and visuospatial deficits are common and debilitating in Parkinson's disease. α-synucleinopathy in the hippocampus and cortex is considered as the major risk factor. However, little is known about the progression and specific synaptic mechanisms underlying the memory deficits induced by α-synucleinopathy. Here, we tested the hypothesis that pathologic α-Synuclein (α-Syn), initiated in different brain regions, leads to distinct onset and progression of the pathology. We report that overexpression of human α-Syn in the murine mesencephalon leads to late onset memory impairment and sensorimotor deficits accompanied by reduced dopamine D1 expression in the hippocampus. In contrast, human α-Syn overexpression in the hippocampus leads to early memory impairment, altered synaptic transmission and plasticity, and decreased expression of GluA1 AMPA-type glutamate receptors. These findings identify the synaptic mechanisms leading to memory impairment induced by hippocampal α-synucleinopathy and provide functional evidence of the major neuronal networks involved in disease progression.

8.
Nat Commun ; 12(1): 6137, 2021 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-34675233

RESUMO

The mammalian brain stores and distinguishes among episodic memories, i.e. memories formed during the personal experience, through a mechanism of pattern separation computed in the hippocampal dentate gyrus. Decision-making for food-related behaviors, such as the choice and intake of food, might be affected in obese subjects by alterations in the retrieval of episodic memories. Adult neurogenesis in the dentate gyrus regulates the pattern separation. Several molecular factors affect adult neurogenesis and exert a critical role in the development and plasticity of newborn neurons. Orexin-A/hypocretin-1 and downstream endocannabinoid 2-arachidonoylglycerol signaling are altered in obese mice. Here, we show that excessive orexin-A/2-arachidonoylglycerol/cannabinoid receptor type-1 signaling leads to the dysfunction of adult hippocampal neurogenesis and the subsequent inhibition of plasticity and impairment of pattern separation. By inhibiting orexin-A action at orexin-1 receptors we rescued both plasticity and pattern separation impairment in obese mice, thus providing a molecular and functional mechanism to explain alterations in episodic memory in obesity.


Assuntos
Endocanabinoides/metabolismo , Hipocampo/crescimento & desenvolvimento , Neurogênese , Plasticidade Neuronal , Obesidade/metabolismo , Obesidade/psicologia , Orexinas/metabolismo , Animais , Feminino , Hipocampo/metabolismo , Hipocampo/fisiopatologia , Humanos , Masculino , Memória Episódica , Camundongos , Camundongos Obesos , Neurônios/citologia , Neurônios/metabolismo , Obesidade/genética , Obesidade/fisiopatologia , Receptores de Orexina/genética , Receptores de Orexina/metabolismo , Receptor CB1 de Canabinoide/genética , Receptor CB1 de Canabinoide/metabolismo , Transdução de Sinais
9.
Nat Commun ; 12(1): 3495, 2021 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-34108486

RESUMO

Lysosomal storage disorders characterized by altered metabolism of heparan sulfate, including Mucopolysaccharidosis (MPS) III and MPS-II, exhibit lysosomal dysfunctions leading to neurodegeneration and dementia in children. In lysosomal storage disorders, dementia is preceded by severe and therapy-resistant autistic-like symptoms of unknown cause. Using mouse and cellular models of MPS-IIIA, we discovered that autistic-like behaviours are due to increased proliferation of mesencephalic dopamine neurons originating during embryogenesis, which is not due to lysosomal dysfunction, but to altered HS function. Hyperdopaminergia and autistic-like behaviours are corrected by the dopamine D1-like receptor antagonist SCH-23390, providing a potential alternative strategy to the D2-like antagonist haloperidol that has only minimal therapeutic effects in MPS-IIIA. These findings identify embryonic dopaminergic neurodevelopmental defects due to altered function of HS leading to autistic-like behaviours in MPS-II and MPS-IIIA and support evidence showing that altered HS-related gene function is causative of autism.


Assuntos
Transtorno do Espectro Autista/metabolismo , Dopamina/metabolismo , Heparitina Sulfato/metabolismo , Doenças por Armazenamento dos Lisossomos/metabolismo , Animais , Transtorno do Espectro Autista/tratamento farmacológico , Transtorno do Espectro Autista/patologia , Benzazepinas/uso terapêutico , Proliferação de Células , Células Cultivadas , Modelos Animais de Doenças , Antagonistas de Dopamina/uso terapêutico , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Heparitina Sulfato/farmacologia , Doenças por Armazenamento dos Lisossomos/tratamento farmacológico , Doenças por Armazenamento dos Lisossomos/patologia , Mesencéfalo/efeitos dos fármacos , Mesencéfalo/embriologia , Mesencéfalo/patologia , Camundongos , Mucopolissacaridose III/tratamento farmacológico , Mucopolissacaridose III/metabolismo , Mucopolissacaridose III/patologia , Receptores de Dopamina D1/antagonistas & inibidores , Receptores de Dopamina D1/metabolismo
10.
Aging Cell ; 19(9): e13189, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32729663

RESUMO

Autophagy agonists have been proposed to slow down neurodegeneration. Spermidine, a polyamine that acts as an autophagy agonist, is currently under clinical trial for the treatment of age-related memory decline. How Spermidine and other autophagy agonists regulate memory and synaptic plasticity is under investigation. We set up a novel mouse model of mild cognitive impairment (MCI), in which middle-aged (12-month-old) mice exhibit impaired memory capacity, lysosomes engulfed with amyloid fibrils (ß-amyloid and α-synuclein) and impaired task-induced GluA1 hippocampal post-translation modifications. Subchronic treatment with Spermidine as well as the autophagy agonist TAT-Beclin 1 rescued memory capacity and GluA1 post-translational modifications by favouring the autophagy/lysosomal-mediated degradation of amyloid fibrils. These findings provide new mechanistic evidence on the therapeutic relevance of autophagy enhancers which, by improving the degradation of misfolded proteins, slow down age-related memory decline.


Assuntos
Autofagia/genética , Disfunção Cognitiva/genética , Memória/efeitos dos fármacos , Envelhecimento , Animais , Modelos Animais de Doenças , Camundongos
11.
Sci Rep ; 10(1): 9619, 2020 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-32541823

RESUMO

The presence of α-synuclein aggregates in the retina of Parkinson's disease patients has been associated with vision impairment. In this study we sought to determine the effects of α-synuclein overexpression on the survival and function of dopaminergic amacrine cells (DACs) in the retina. Adult mice were intravitreally injected with an adeno-associated viral (AAV) vector to overexpress human wild-type α-synuclein in the inner retina. Before and after systemic injections of levodopa (L-DOPA), retinal responses and visual acuity-driven behavior were measured by electroretinography (ERG) and a water maze task, respectively. Amacrine cells and ganglion cells were counted at different time points after the injection. α-synuclein overexpression led to an early loss of DACs associated with a decrease of light-adapted ERG responses and visual acuity that could be rescued by systemic injections of L-DOPA. The data show that α-synuclein overexpression affects dopamine neurons in the retina. The approach provides a novel accessible method to model the underlying mechanisms implicated in the pathogenesis of synucleinopathies and for testing novel treatments.


Assuntos
Células Amácrinas/metabolismo , Neurônios Dopaminérgicos/metabolismo , Retina/metabolismo , Degeneração Retiniana/metabolismo , Transtornos da Visão/metabolismo , alfa-Sinucleína/metabolismo , Células Amácrinas/patologia , Animais , Neurônios Dopaminérgicos/patologia , Feminino , Imunofluorescência , Levodopa/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Retina/efeitos dos fármacos , Retina/patologia , Degeneração Retiniana/patologia , Transtornos da Visão/patologia , Acuidade Visual
12.
Behav Brain Res ; 359: 197-205, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30391393

RESUMO

Memory capacity (MC) refers to the limited capacity of working memory and is defined as the number of elements that an individual can remember for a short retention interval. MC is impaired in many human pathologies, such as schizophrenia and ageing. Fronto-striatal dopamine regulates working memory, through its action on dopamine D1- and D2-like receptors. Human and rodent studies have suggested that MC is improved by D2 dopamine receptor agonists. Although D1 receptors have been crucially involved in the maintenance of working memory during delay, their role in regulating the capacity of WM remains poorly explored. In this study, we tested the effects of systemic injection of the D1-like and D2-like receptor antagonists, SCH 23390 and Haloperidol respectively, on MC in mice. For this, we used a modified version of the object recognition task, the Different/Identical Objects Task (DOT/IOT), which allows the evaluation of MC in rodents. The results showed a negative interaction between the dose of both drugs and the number of objects that could be remembered. The doses of SCH 23390 and Haloperidol that impaired novel object discrimination in the highest memory load condition were about 4 and 3 time lower, respectively, of those impairing performance in the lowest memory load condition. However, while SCH 23390 specifically impaired memory load capacity, the effects of Haloperidol were associated to impairment in exploratory behaviors. These findings may help to predict the cognitive side effects induced by Haloperidol in healthy subjects.


Assuntos
Benzazepinas/farmacologia , Antagonistas de Dopamina/farmacologia , Haloperidol/farmacologia , Memória de Curto Prazo/efeitos dos fármacos , Animais , Animais não Endogâmicos , Relação Dose-Resposta a Droga , Masculino , Memória de Curto Prazo/fisiologia , Camundongos , Receptores de Dopamina D1/antagonistas & inibidores , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D2/metabolismo
13.
Mol Ther Methods Clin Dev ; 15: 333-342, 2019 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-31788497

RESUMO

Mucopolysaccharidosis type IIIA (MPS-IIIA) is a lysosomal storage disorder (LSD) caused by inherited defect of sulfamidase, a lysosomal sulfatase. MPS-IIIA is one of the most common and severe forms of LSDs with CNS involvement. Presently there is no cure. Here we have developed a new gene delivery approach for the treatment of MPS-IIIA based on the use of a modified version of sulfamidase expression cassette. This cassette encodes both a chimeric sulfamidase containing an alternative signal peptide (sp) to improve enzyme secretion and sulfatase-modifying factor 1 (SUMF1) to increase sulfamidase post-translational activation rate. We demonstrate that improved secretion and increased activation of sulfamidase act synergistically to enhance enzyme biodistribution in wild-type (WT) pigs upon intrathecal adeno-associated virus serotype 9 (AAV9)-mediated gene delivery. Translating such gene delivery strategy to a mouse model of MPS-IIIA results in a rescue of brain pathology, including memory deficit, as well as improvement in somatic tissues. These data may pave the way for developing effective gene delivery replacement protocols for the treatment of MPS-IIIA patients.

14.
EMBO Mol Med ; 9(1): 112-132, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27881461

RESUMO

Lysosomal storage disorders (LSDs) are inherited diseases characterized by lysosomal dysfunction and often showing a neurodegenerative course. There is no cure to treat the central nervous system in LSDs. Moreover, the mechanisms driving neuronal degeneration in these pathological conditions remain largely unknown. By studying mouse models of LSDs, we found that neurodegeneration develops progressively with profound alterations in presynaptic structure and function. In these models, impaired lysosomal activity causes massive perikaryal accumulation of insoluble α-synuclein and increased proteasomal degradation of cysteine string protein α (CSPα). As a result, the availability of both α-synuclein and CSPα at nerve terminals strongly decreases, thus inhibiting soluble NSF attachment receptor (SNARE) complex assembly and synaptic vesicle recycling. Aberrant presynaptic SNARE phenotype is recapitulated in mice with genetic ablation of one allele of both CSPα and α-synuclein. The overexpression of CSPα in the brain of a mouse model of mucopolysaccharidosis type IIIA, a severe form of LSD, efficiently re-established SNARE complex assembly, thereby ameliorating presynaptic function, attenuating neurodegenerative signs, and prolonging survival. Our data show that neurodegenerative processes associated with lysosomal dysfunction may be presynaptically initiated by a concomitant reduction in α-synuclein and CSPα levels at nerve terminals. They also demonstrate that neurodegeneration in LSDs can be slowed down by re-establishing presynaptic functions, thus identifying synapse maintenance as a novel potentially druggable target for brain treatment in LSDs.


Assuntos
Proteínas de Choque Térmico HSP40/análise , Doenças por Armazenamento dos Lisossomos/patologia , Proteínas de Membrana/análise , Doenças Neurodegenerativas/patologia , Terminações Pré-Sinápticas/patologia , alfa-Sinucleína/análise , Animais , Modelos Animais de Doenças , Camundongos , Proteólise , Proteínas SNARE/metabolismo , Vesículas Sinápticas/metabolismo
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