RESUMO
Planning and execution of voluntary movement depend on the contribution of distinct classes of neurons in primary motor and premotor areas. However, timing and pattern of activation of GABAergic cells during specific motor behaviors remain only partly understood. Here, we directly compared the response properties of putative pyramidal neurons (PNs) and GABAergic fast-spiking neurons (FSNs) during spontaneous licking and forelimb movements in male mice. Recordings centered on the face/mouth motor field of the anterolateral motor cortex (ALM) revealed that FSNs fire longer than PNs and earlier for licking, but not for forelimb movements. Computational analysis revealed that FSNs carry vastly more information than PNs about the onset of movement. While PNs differently modulate their discharge during distinct motor acts, most FSNs respond with a stereotyped increase in firing rate. Accordingly, the informational redundancy was greater among FSNs than PNs. Finally, optogenetic silencing of a subset of FSNs reduced spontaneous licking movement. These data suggest that a global rise of inhibition contributes to the initiation and execution of spontaneous motor actions.SIGNIFICANCE STATEMENT Our study contributes to clarifying the causal role of fast-spiking neurons (FSNs) in driving initiation and execution of specific, spontaneous movements. Within the face/mouth motor field of mice premotor cortex, FSNs fire before pyramidal neurons (PNs) with a specific activation pattern: they reach their peak of activity earlier than PNs during the initiation of licking, but not of forelimb, movements; duration of FSNs activity is also greater and exhibits less selectivity for the movement type, as compared with that of PNs. Accordingly, FSNs appear to carry more redundant information than PNs. Optogenetic silencing of FSNs reduced spontaneous licking movement, suggesting that FSNs contribute to the initiation and execution of specific spontaneous movements, possibly by sculpting response selectivity of nearby PNs.
Assuntos
Córtex Motor , Masculino , Camundongos , Animais , Córtex Motor/fisiologia , Interneurônios/fisiologia , Células Piramidais/fisiologia , Movimento/fisiologia , Neurônios GABAérgicosRESUMO
Krabbe disease is a rare, childhood lysosomal storage disorder caused by a deficiency of galactosylceramide beta-galactosidase (GALC). The major effect of GALC deficiency is the accumulation of psychosine in the nervous system and widespread degeneration of oligodendrocytes and Schwann cells, causing rapid demyelination. The molecular mechanisms of Krabbe disease are not yet fully elucidated and a definite cure is still missing. Here we report the first in-depth characterization of the proteome of the Twitcher mouse, a spontaneous mouse model of Krabbe disease, to investigate the proteome changes in the Central and Peripheral Nervous System. We applied a TMT-based workflow to compare the proteomes of the corpus callosum, motor cortex and sciatic nerves of littermate homozygous Twitcher and wild-type mice. More than 400 protein groups exhibited differences in expression and included proteins involved in pathways that can be linked to Krabbe disease, such as inflammatory and defense response, lysosomal proteins accumulation, demyelination, reduced nervous system development and cell adhesion. These findings provide new insights on the molecular mechanisms of Krabbe disease, representing a starting point for future functional experiments to study the molecular pathogenesis of Krabbe disease. Data are available via ProteomeXchange with identifier PXD010594.
Assuntos
Sistema Nervoso Central/metabolismo , Leucodistrofia de Células Globoides/metabolismo , Sistema Nervoso Periférico/metabolismo , Proteômica/métodos , Animais , Sistema Nervoso Central/patologia , Modelos Animais de Doenças , Feminino , Ontologia Genética , Masculino , Camundongos , Sistema Nervoso Periférico/patologia , Análise de Componente Principal , Proteoma/metabolismoRESUMO
Krabbe disease (KD) is a childhood leukodystrophy with no cure currently available. KD is due to a deficiency of a lysosomal enzyme called galactosyl-ceramidase (GALC) and is characterized by the accumulation in the nervous system of the sphingolipid psychosine (PSY), whose cytotoxic molecular mechanism is not fully known yet. Here, we study the expression of some fundamental autophagy markers (LC3, p62, and Beclin-1) in a KD murine model [the twitcher (TWI) mouse] by immunohistochemistry and Western blot. Moreover, the autophagy molecular process is also shown in primary fibroblasts from TWI and WT mice, with and without PSY treatment. Data demonstrate that large p62 cytoplasmic aggregates are present in the brain of both early and late symptomatic TWI mice. p62 expression is also upregulated in TWI sciatic nerves compared to that measured for WT nerves. In vitro data suggest that this effect might not be fully PSY-driven. Finally, we investigate in vitro the capability of autophagy inducers (Rapamycin, RAP and Resveratrol, RESV) to reinstate the WT phenotype in TWI cells. We show that RAP administration can partially restore the autophagy markers levels, while RESV cannot, indicating a line along which new therapeutic approaches can be developed.
Assuntos
Autofagia/fisiologia , Encéfalo/patologia , Leucodistrofia de Células Globoides/patologia , Nervo Isquiático/patologia , Animais , Autofagia/efeitos dos fármacos , Biomarcadores/análise , Encéfalo/metabolismo , Leucodistrofia de Células Globoides/metabolismo , Camundongos , Resveratrol/farmacologia , Nervo Isquiático/metabolismo , Sirolimo/farmacologiaRESUMO
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/farmacologiaRESUMO
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.
RESUMO
Krabbe disease (KD; or globoid cell leukodystrophy) is an autosomal recessive lysosomal storage disorder caused by deficiency of the galactosylceramidase (GALC) enzyme. No cure is currently available for KD. Clinical applied treatments are supportive only. Recently, we demonstrated that two differently acting autophagy inducers (lithium and rapamycin) can improve some KD hallmarks in-vitro, laying the foundation for their in-vivo pre-clinical testing. Here, we test lithium carbonate in-vivo, in the spontaneous mouse model for KD, the Twitcher (TWI) mouse. The drug is administered ad libitum via drinking water (600 mg/L) starting from post natal day 20. We longitudinally monitor the mouse motor performance through the grip strength, the hanging wire and the rotarod tests, and a set of biochemical parameters related to the KD pathogenesis [i.e., GALC enzymatic activity, psychosine (PSY) accumulation and astrogliosis]. Additionally, we investigate the expression of some crucial markers related to the two pathways that could be altered by lithium: the autophagy and the ß-catenin-dependent pathways. Results demonstrate that lithium has not a significant rescue effect on the TWI phenotype, although it can slightly and transiently improves muscle strength. We also show that lithium, with this administration protocol, is unable to stimulate autophagy in the TWI mice central nervous system, whereas results suggest that it can restore the ß-catenin activation status in the TWI sciatic nerve. Overall, these data provide intriguing inputs for further evaluations of lithium treatment in TWI mice.
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PURPOSE: The SARS-CoV-2 spread has impacted Healthcare systems. COVID-19 pandemic has had consequences for patients with cancer, being associated with delays in diagnosis, in treatment And follow-up care, increase in overall infection rates and higher mortality. A survey on COVID-19 and a vaccination-questionnaire were developed at different times of the outbreak, to evaluate cancer patient-reported experience measures (PREMs) on the policies implemented to reduce the infection from SARS-CoV-2 and on the timing and methods of COVID-19 vaccination. PATIENTS AND METHODS: The survey was distributed to all patients accessing the Institute during the "first-wave" Of the pandemic, evaluating patients' concerns about the pandemic, the pandemics' consequences on their cancer care, and their perception Of the measures adopted to limit the infection spread. The vaccination-questionnaire was proposed to 10% of the first 5297 cancer patients vaccinated with two doses of the Pfizer-BioNTechCOVID-19 vaccine. This questionnaire aimed at assessing the degree Of satisfaction with the Institutional vaccination campaign and vaccination-related adverse events. RESULTS: From May 18th 2020 to June 15th 2020 the survey was completed by 3238 patients. Most of the responders expressed concern on the pandemic yet acknowledging their oncological disease as a priority. Measures implemented were appreciated by patients. Telemedicine was positively evaluated and the absence of the caregiver during the visit did not determine discomfort for two thirds of patients. From March 6th 2021 to May 8th 2021 the vaccination-questionnaire was completed by 357 patients. The 98.8% were satisfied with the vaccination campaign. No serious vaccination-correlated adverse events were reported. No patient had to delay/discontinue chemotherapy due to vaccination. CONCLUSION: PREMs during COVID-19 pandemic and related vaccination can provide important information to help reorganization of the health care systems for cancer care. Patients' feedback on the organizational changes implemented in the emergency period are essential for healthcare improvement and to help informed choices that are consistent with patients' needs.
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Pathogenic NR2F1 variants cause a rare autosomal dominant neurodevelopmental disorder referred to as the Bosch-Boonstra-Schaaf Optic Atrophy Syndrome. Although visual loss is a prominent feature seen in affected individuals, the molecular and cellular mechanisms contributing to visual impairment are still poorly characterized. We conducted a deep phenotyping study on a cohort of 22 individuals carrying pathogenic NR2F1 variants to document the neurodevelopmental and ophthalmological manifestations, in particular the structural and functional changes within the retina and the optic nerve, which have not been detailed previously. The visual impairment became apparent in early childhood with small and/or tilted hypoplastic optic nerves observed in 10 cases. High-resolution optical coherence tomography imaging confirmed significant loss of retinal ganglion cells with thinning of the ganglion cell layer, consistent with electrophysiological evidence of retinal ganglion cells dysfunction. Interestingly, for those individuals with available longitudinal ophthalmological data, there was no significant deterioration in visual function during the period of follow-up. Diffusion tensor imaging tractography studies showed defective connections and disorganization of the extracortical visual pathways. To further investigate how pathogenic NR2F1 variants impact on retinal and optic nerve development, we took advantage of an Nr2f1 mutant mouse disease model. Abnormal retinogenesis in early stages of development was observed in Nr2f1 mutant mice with decreased retinal ganglion cell density and disruption of retinal ganglion cell axonal guidance from the neural retina into the optic stalk, accounting for the development of optic nerve hypoplasia. The mutant mice showed significantly reduced visual acuity based on electrophysiological parameters with marked conduction delay and decreased amplitude of the recordings in the superficial layers of the visual cortex. The clinical observations in our study cohort, supported by the mouse data, suggest an early neurodevelopmental origin for the retinal and optic nerve head defects caused by NR2F1 pathogenic variants, resulting in congenital vision loss that seems to be non-progressive. We propose NR2F1 as a major gene that orchestrates early retinal and optic nerve head development, playing a key role in the maturation of the visual system.
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Transplantation of human bone marrow mesenchymal stromal cells (hBM-MSC) promotes functional recovery after stroke in animal models, but the mechanisms underlying these effects remain incompletely understood. We tested the efficacy of Good Manufacturing Practices (GMP) compliant hBM-MSC, injected intravenously 3.5 hours after injury in mice subjected to transient middle cerebral artery occlusion (tMCAo). We addressed whether hBM-MSC are efficacious and if this efficacy is associated with cortical circuit reorganization using neuroanatomical analysis of GABAergic neurons (parvalbumin; PV-positive cells) and perineuronal nets (PNN), a specialized extracellular matrix structure which acts as an inhibitor of neural plasticity. tMCAo mice receiving hBM-MSC, showed early and lasting improvement of sensorimotor and cognitive functions compared to control tMCAo mice. Furthermore, 5 weeks post-tMCAo, hBM-MSC induced a significant rescue of ipsilateral cortical neurons; an increased proportion of PV-positive neurons in the perilesional cortex, suggesting GABAergic interneurons preservation; and a lower percentage of PV-positive cells surrounded by PNN, indicating an enhanced plastic potential of the perilesional cortex. These results show that hBM-MSC improve functional recovery and stimulate neuroprotection after stroke. Moreover, the downregulation of "plasticity brakes" such as PNN suggests that hBM-MSC treatment stimulates plasticity and formation of new connections in the perilesional cortex.