RESUMO
In the absence of disease, humans produce smooth and accurate movement trajectories. Despite such 'macroscopic' aspect, the 'microscopic' structure of movements reveals recurrent (quasi-rhythmic) discontinuities. To date, it is unclear how the sensorimotor system contributes to the macroscopic and microscopic architecture of movement. Here, we investigated how corticospinal excitability changes in relation to microscopic fluctuations that are naturally embedded within larger macroscopic variations in motor output. Participants performed a visuomotor tracking task. In addition to the 0.25 Hz modulation that is required for task fulfilment (macroscopic scale), the motor output shows tiny but systematic fluctuations at â¼2 and 8 Hz (microscopic scales). We show that motor-evoked potentials (MEPs) elicited by transcranial magnetic stimulation (TMS) during task performance are consistently modulated at all (time) scales. Surprisingly, MEP modulation covers a similar range at both micro- and macroscopic scales, even though the motor output differs by several orders of magnitude. Thus, corticospinal excitability finely maps the multiscale temporal patterning of the motor output, but it does so according to a principle of scale invariance. These results suggest that corticospinal excitability indexes a relatively abstract level of movement encoding that may reflect the hierarchical organisation of sensorimotor processes. KEY POINTS: Motor behaviour is organised on multiple (time)scales. Small but systematic ('microscopic') fluctuations are engrained in larger and slower ('macroscopic') variations in motor output, which are instrumental in deploying the desired motor plan. Corticospinal excitability is modulated in relation to motor fluctuations on both macroscopic and microscopic (time)scales. Corticospinal excitability obeys a principle of scale invariance, that is, it is modulated similarly at all (time)scales, possibly reflecting hierarchical mechanisms that optimise motor encoding.
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Córtex Motor , Humanos , Córtex Motor/fisiologia , Tratos Piramidais/fisiologia , Estimulação Magnética Transcraniana/métodos , Movimento , Potencial Evocado Motor/fisiologia , Músculo Esquelético/fisiologia , EletromiografiaRESUMO
Autism spectrum disorder (ASD) is a neurodevelopmental disorder that impairs normal brain development and socio-cognitive abilities. The pathogenesis of this condition points out the involvement of genetic and environmental factors during in-utero life. Placenta, as an interface tissue between mother and fetus, provides developing fetus requirements and exposes it to maternal environment as well. Therefore, the alteration of DNA methylation as epigenetic consequence of gene-environmental interaction in the placenta could shed light on ASD pathogenesis. In this study, we reviewed the current findings on placental methylation status and its association with ASD. Differentially methylated regions (DMRs) in ASD-developing placenta were found to be mainly enriched in ASD gene loci affecting synaptogenesis, microtubule dynamics, neurogenesis and neuritogenesis. In addition, non-genic DMRs in ASD-placenta proposes an alternative contributing mechanism for ASD development. Our study highlights the importance of placental DNA methylation signature as a biomarker for ASD prediction.
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Transtorno do Espectro Autista , Humanos , Feminino , Gravidez , Transtorno do Espectro Autista/diagnóstico , Transtorno do Espectro Autista/genética , Transtorno do Espectro Autista/metabolismo , Metilação de DNA , Placenta/metabolismo , Epigênese Genética , Biomarcadores/metabolismoRESUMO
Movements are naturally composed of submovements, i.e. recurrent speed pulses (2-3 Hz), possibly reflecting intermittent feedback-based motor adjustments. In visuomotor (unimanual) synchronization tasks, partners alternate submovements over time, indicating mutual coregulation. However, it is unclear whether submovement coordination is organized differently between and within individuals. Indeed, different types of information may be variably exploited for intrapersonal and interpersonal coordination. Participants performed a series of bimanual tasks alone or in pairs, with or without visual feedback (solo task only). We analysed the relative timing of submovements between their own hands or between their own hands and those of their partner. Distinct coordinative structures emerged at the submovement level depending on the relevance of visual feedback. Specifically, the relative timing of submovements (between partners/effectors) shifts from alternation to simultaneity and a mixture of both when coordination is achieved using vision (interpersonal), proprioception/efference-copy only (intrapersonal, without vision) or all information sources (intrapersonal, with vision), respectively. These results suggest that submovement coordination represents a behavioural proxy for the adaptive weighting of different sources of information within action-perception loops. In sum, the microstructure of movement reveals common principles governing the dynamics of sensorimotor control to achieve both intra- and interpersonal coordination.
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Movimento , Desempenho Psicomotor , Humanos , Desempenho Psicomotor/fisiologia , Movimento/fisiologia , MãosRESUMO
Increasing evidence suggests that the peroxisome proliferator-activated receptor γ (PPARγ), a member of the nuclear receptor superfamily, plays an important role in physiological processes in the central nervous system (CNS) and is involved in cellular metabolism and repair. Cellular damage caused by acute brain injury and long-term neurodegenerative disorders is associated with alterations of these metabolic processes leading to mitochondrial dysfunction, oxidative stress, and neuroinflammation. PPARγ agonists have demonstrated the potential to be effective treatments for CNS diseases in preclinical models, but to date, most drugs have failed to show efficacy in clinical trials of neurodegenerative diseases including amyotrophic lateral sclerosis, Parkinson's disease, and Alzheimer's disease. The most likely explanation for this lack of efficacy is the insufficient brain exposure of these PPARγ agonists. Leriglitazone is a novel, blood-brain barrier (BBB)-penetrant PPARγ agonist that is being developed to treat CNS diseases. Here, we review the main roles of PPARγ in physiology and pathophysiology in the CNS, describe the mechanism of action of PPARγ agonists, and discuss the evidence supporting the use of leriglitazone to treat CNS diseases.
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Doenças do Sistema Nervoso Central , Doenças Neurodegenerativas , Humanos , Doenças do Sistema Nervoso Central/metabolismo , Doenças Neurodegenerativas/metabolismo , Doenças Neuroinflamatórias , PPAR gama/metabolismoRESUMO
LRRK2 is a highly phosphorylated multidomain protein and mutations in the gene encoding LRRK2 are a major genetic determinant of Parkinson's disease (PD). Dephosphorylation at LRRK2's S910/S935/S955/S973 phosphosite cluster is observed in several conditions including in sporadic PD brain, in several disease mutant forms of LRRK2 and after pharmacological LRRK2 kinase inhibition. However, the mechanism of LRRK2 dephosphorylation is poorly understood. We performed a phosphatome-wide reverse genetics screen to identify phosphatases involved in the dephosphorylation of the LRRK2 phosphosite S935. Candidate phosphatases selected from the primary screen were tested in mammalian cells, Xenopus oocytes and in vitro. Effects of PP2A on endogenous LRRK2 phosphorylation were examined via expression modulation with CRISPR/dCas9. Our screening revealed LRRK2 phosphorylation regulators linked to the PP1 and PP2A holoenzyme complexes as well as CDC25 phosphatases. We showed that dephosphorylation induced by different kinase inhibitor triggered relocalisation of phosphatases PP1 and PP2A in LRRK2 subcellular compartments in HEK-293 T cells. We also demonstrated that LRRK2 is an authentic substrate of PP2A both in vitro and in Xenopus oocytes. We singled out the PP2A holoenzyme PPP2CA:PPP2R2 as a powerful phosphoregulator of pS935-LRRK2. Furthermore, we demonstrated that this specific PP2A holoenzyme induces LRRK2 relocalization and triggers LRRK2 ubiquitination, suggesting its involvement in LRRK2 clearance. The identification of the PPP2CA:PPP2R2 complex regulating LRRK2 S910/S935/S955/S973 phosphorylation paves the way for studies refining PD therapeutic strategies that impact LRRK2 phosphorylation.
Assuntos
Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Proteína Fosfatase 1/metabolismo , Proteína Fosfatase 2/metabolismo , Animais , Células HEK293 , Holoenzimas/metabolismo , Humanos , Técnicas In Vitro , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Proteínas do Tecido Nervoso/metabolismo , Oócitos/metabolismo , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Transporte Proteico/efeitos dos fármacos , Proteínas de Xenopus/metabolismo , Xenopus laevisRESUMO
Dopamine (DA) controls many vital physiological functions and is critically involved in several neuropsychiatric disorders such as schizophrenia and attention deficit hyperactivity disorder. The major function of the plasma membrane dopamine transporter (DAT) is the rapid uptake of released DA into presynaptic nerve terminals leading to control of both the extracellular levels of DA and the intracellular stores of DA. Here, we present a newly developed strain of rats in which the gene encoding DAT knockout Rats (DAT-KO) has been disrupted by using zinc finger nuclease technology. Male and female DAT-KO rats develop normally but weigh less than heterozygote and wild-type rats and demonstrate pronounced spontaneous locomotor hyperactivity. While striatal extracellular DA lifetime and concentrations are significantly increased, the total tissue content of DA is markedly decreased demonstrating the key role of DAT in the control of DA neurotransmission. Hyperactivity of DAT-KO rats can be counteracted by amphetamine, methylphenidate, the partial Trace Amine-Associated Receptor 1 (TAAR1) agonist RO5203648 ((S)-4-(3,4-Dichloro-phenyl)-4,5-dihydro-oxazol-2-ylamine) and haloperidol. DAT-KO rats also demonstrate a deficit in working memory and sensorimotor gating tests, less propensity to develop obsessive behaviors and show strong dysregulation in frontostriatal BDNF function. DAT-KO rats could provide a novel translational model for human diseases involving aberrant DA function and/or mutations affecting DAT or related regulatory mechanisms.SIGNIFICANCE STATEMENT Here, we present a newly developed strain of rats in which the gene encoding the dopamine transporter (DAT) has been disrupted (Dopamine Transporter Knockout rats [DAT-KO rats]). DAT-KO rats display functional hyperdopaminergia accompanied by pronounced spontaneous locomotor hyperactivity. Hyperactivity of DAT-KO rats can be counteracted by amphetamine, methylphenidate, and a few other compounds exerting inhibitory action on dopamine-dependent hyperactivity. DAT-KO rats also demonstrate cognitive deficits in working memory and sensorimotor gating tests, less propensity to develop compulsive behaviors, and strong dysregulation in frontostriatal BDNF function. These observations highlight the key role of DAT in the control of brain dopaminergic transmission. DAT-KO rats could provide a novel translational model for human diseases involving aberrant dopamine functions.
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Fator Neurotrófico Derivado do Encéfalo/metabolismo , Disfunção Cognitiva/etiologia , Modelos Animais de Doenças , Proteínas da Membrana Plasmática de Transporte de Dopamina/deficiência , Hipercinese/etiologia , Animais , Disfunção Cognitiva/metabolismo , Feminino , Técnicas de Inativação de Genes , Hipercinese/metabolismo , Masculino , Ratos , Ratos WistarRESUMO
Transcranial magnetic stimulation was used to investigate corticospinal output changes in 10 professional piano players during motor imagery of triad chords in C major to be "mentally" performed with three fingers of the right hand (thumb, index, and little finger). Five triads were employed in the task; each composed by a stable 3rd interval (C4-E4) and a varying third note that could generate a 5th (G4), a 6th (A4), a 7th (B4), a 9th (D5), or a 10th (E5) interval. The 10th interval chord was thought to be impossible in actual execution for biomechanical reasons, as long as the thumb and the index finger remained fixed on the 3rd interval. Chords could be listened from loudspeakers, read on a staff, or listened and read at the same time while performing the imagery task. The corticospinal output progressively increased along with task demands in terms of mental representation of hand extension. The effects of audio, visual, or audiovisual musical stimuli were generally similar, unless motor imagery of kinetically impossible triads was required. A specific three-effector motor synergy was detected, governing the representation of the progressive mental extension of the hand. Results demonstrate that corticospinal facilitation in professional piano players can be modulated according to the motor plan, even if simply "dispatched" without actual execution. Moreover, specific muscle synergies, usually encoded in the motor cortex, emerge along the cross-modal elaboration of musical stimuli and in motor imagery of musical performances.
Assuntos
Percepção Auditiva/fisiologia , Imaginação/fisiologia , Córtex Motor/fisiologia , Destreza Motora/fisiologia , Tratos Piramidais/fisiologia , Percepção Visual/fisiologia , Adulto , Eletromiografia , Potencial Evocado Motor , Feminino , Humanos , Masculino , Músculo Esquelético/fisiologia , Estimulação Magnética Transcraniana , Adulto JovemRESUMO
Four different classes of new 17ß-hydroxysteroid dehydrogenase type 2 (17ß-HSD2) inhibitors were synthesized, in order to lower the cytotoxicity exhibited by the lead compound A, via disrupting the linearity and the aromaticity of the biphenyl moiety. Compounds 3, 4, 7a and 8 displayed comparable or better inhibitory activity and selectivity, as well as a lower cytotoxic effect, compared to the reference compound A. The best compound 4 (IC50=160nM, selectivity factor=168, LD50≈25µM) turned out as new lead compound for inhibition of 17ß-HSD2.
Assuntos
Amidas/farmacologia , Compostos de Bifenilo/farmacologia , Citotoxinas/farmacologia , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Estradiol Desidrogenases/antagonistas & inibidores , Amidas/síntese química , Amidas/química , Compostos de Bifenilo/síntese química , Compostos de Bifenilo/química , Sobrevivência Celular/efeitos dos fármacos , Citotoxinas/síntese química , Citotoxinas/química , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Estradiol Desidrogenases/metabolismo , Células HEK293 , Humanos , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
α-Synuclein is thought to regulate neurotransmitter release through multiple interactions with presynaptic proteins, cytoskeletal elements, ion channels, and synaptic vesicles membrane. α-Synuclein is abundant in the presynaptic compartment, and its release from neurons and glia has been described as responsible for spreading of α-synuclein-derived pathology. α-Synuclein-dependent dysregulation of neurotransmitter release might occur via its action on surface-exposed calcium channels. Here, we provide electrophysiological and biochemical evidence to show that α-synuclein, applied to rat neurons in culture or striatal slices, selectively activates Cav2.2 channels, and said activation correlates with increased neurotransmitter release. Furthermore, in vivo perfusion of α-synuclein into the striatum also leads to acute dopamine release. We further demonstrate that α-synuclein reduces the amount of plasma membrane cholesterol and alters the partitioning of Cav2.2 channels, which move from raft to cholesterol-poor areas of the plasma membrane. We provide evidence for a novel mechanism through which α-synuclein acts from the extracellular milieu to modulate neurotransmitter release and propose a unifying hypothesis for the mechanism of α-synuclein action on multiple targets: the reorganization of plasma membrane microdomains.
Assuntos
Canais de Cálcio Tipo N/metabolismo , Dopamina/metabolismo , Microdomínios da Membrana/efeitos dos fármacos , Neurônios/citologia , alfa-Sinucleína/farmacologia , Compostos de Anilina/metabolismo , Animais , Anticorpos/farmacologia , Canais de Cálcio Tipo N/imunologia , Células Cultivadas , Córtex Cerebral/citologia , Embrião de Mamíferos , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/genética , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Neurônios/efeitos dos fármacos , Ratos , Ratos Wistar , Bloqueadores dos Canais de Sódio/farmacologia , Gânglio Cervical Superior/citologia , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/genética , Sinaptofisina/metabolismo , Xantenos/metabolismoRESUMO
This preliminary study introduces a novel action observation therapy (AOT) protocol associated with electroencephalographic (EEG) monitoring to be used in the future as a rehabilitation strategy for the upper limb in patients with subacute stroke. To provide initial evidence on the usefulness of this method, we compared the outcome of 11 patients who received daily AOT for three weeks with that of patients who undertook two other approaches recently investigated by our group, namely intensive conventional therapy (ICT), and robot-assisted therapy combined with functional electrical stimulation (RAT-FES). The three rehabilitative interventions showed similar arm motor recovery as indexed by Fugl-Meyer's assessment of the upper extremity (FMA_UE) and box and block test (BBT). The improvement in the FMA_UE was yet more favourable in patients with mild/moderate motor impairments who received AOT, in contrast with patients carrying similar disabilities who received the other two treatments. This suggests that AOT might be more effective in this subgroup of patients, perhaps because the integrity of their mirror neurons system (MNS) was more preserved, as indexed by EEG recording from central electrodes during action observation. In conclusion, AOT may reveal an effective rehabilitative tool in patients with subacute stroke; the EEG evaluation of MNS integrity may help to select patients who could maximally benefit from this intervention.
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High cholesterol levels are a risk factor for the development of Alzheimer's disease. Experiments investigating the influence of cholesterol on the proteolytic processing of the amyloid precursor protein (APP) by the ß-secretase Bace1 and on their proximity in cells have led to conflicting results. By using a fluorescence bioassay coupled with flow cytometry we found a direct correlation between the increase in membrane cholesterol amount and the degree of APP shedding in living human neuroblastoma cells. Analogue results were obtained for cells overexpressing an APP mutant that cannot be processed by α-secretase, highlighting the major influence of cholesterol enrichment on the cleavage of APP carried out by Bace1. By contrast, the cholesterol content was not correlated with changes in membrane dynamics of APP and Bace1 analyzed with single molecule tracking, indicating that the effect of cholesterol enrichment on APP processing by Bace1 is uncoupled from changes in their lateral diffusion.
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Mutations in leucine-rich repeat kinase 2 (LRRK2) are a common cause of inherited and sporadic Parkinson's disease (PD) and previous work suggests that dephosphorylation of LRRK2 at a cluster of heterologous phosphosites is associated to disease. We have previously reported subunits of the PP1 and PP2A classes of phosphatases as well as the PAK6 kinase as regulators of LRRK2 dephosphorylation. We therefore hypothesized that PAK6 may have a functional link with LRRK2's phosphatases. To investigate this, we used PhosTag gel electrophoresis with purified proteins and found that PAK6 phosphorylates the PP2A regulatory subunit PPP2R2C at position S381. While S381 phosphorylation did not affect PP2A holoenzyme formation, a S381A phosphodead PPP2R2C showed impaired binding to LRRK2. Also, PAK6 kinase activity changed PPP2R2C subcellular localization in a S381 phosphorylation-dependent manner. Finally, PAK6-mediated dephosphorylation of LRRK2 was unaffected by phosphorylation of PPP2R2C at S381, suggesting that the previously reported mechanism whereby PAK6-mediated phosphorylation of 14-3-3 proteins promotes 14-3-3-LRRK2 complex dissociation and consequent exposure of LRRK2 phosphosites for dephosphorylation is dominant. Taken together, we conclude that PAK6-mediated phosphorylation of PPP2R2C influences the recruitment of PPP2R2C to the LRRK2 complex and PPP2R2C subcellular localization, pointing to an additional mechanism in the fine-tuning of LRRK2 phosphorylation.
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During Paracentrotus lividus sea urchin embryo development one alpha and one beta tubulin genes are expressed specifically in the neural cells and they are early end output of the gene regulatory network that specifies the neural commitment. In this paper we have used a comparative genomics approach to identify conserved regulatory elements in the P. lividus neural alpha tubulin gene. To this purpose, we have first isolated a genomic clone containing the entire gene plus 4.5 Kb of 5' upstream sequences. Then, we have shown by gene transfer experiments that its non-coding region drives the spatio-temporal gene expression corresponding substantially to that of the endogenous gene. In addition, we have identified by genome and EST sequence analysis the S. purpuratus alpha tubulin orthologous gene and we propose a revised annotation of some tubulin family members. Moreover, by computational techniques we delineate at least three putative regulatory regions located both in the upstream region and in the first intron containing putative binding sites for Forkhead and Nkx transcription factor families.
Assuntos
Embrião não Mamífero , Regulação da Expressão Gênica/genética , Proteínas do Tecido Nervoso/genética , Paracentrotus/genética , Regiões Promotoras Genéticas/genética , Tubulina (Proteína)/genética , Animais , Sítios de Ligação/genética , Biologia Computacional , Pegada de DNA , Primers do DNA/genética , Etiquetas de Sequências Expressas , Técnicas de Transferência de Genes , Genes Reporter/genética , Genômica , Proteínas de Fluorescência Verde/metabolismo , Microinjeções , Anotação de Sequência MolecularRESUMO
Developmental coordination disorder (DCD) is a common neurodevelopmental condition characterized by disabling motor impairments being visible from the first years of life. Over recent decades, research in this field has gained important results, showing alterations in several processes involved in the regulation of motor behavior (e.g., planning and monitoring of actions, motor learning, action imitation). However, these studies mostly pursued a behavioral approach, leaving relevant questions open concerning the neural correlates of this condition. In this narrative review, we first survey the literature on motor control and sensorimotor impairments in DCD. Then, we illustrate the contributions to the field that may be achieved using transcranial magnetic stimulation (TMS) of the motor cortex. While still rarely employed in DCD research, this approach offers several opportunities, ranging from the clarification of low-level cortical electrophysiology to the assessment of the motor commands transmitted throughout the corticospinal system. We propose that TMS may help to investigate the neural correlates of motor impairments reported in behavioral studies, thus guiding DCD research toward a brain-oriented acknowledgment of this condition. This effort would help translational research to provide novel diagnostic and therapeutic tools.
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Most animal species group together and coordinate their behavior in quite sophisticated manners for mating, hunting, or defense purposes. In humans, coordination at a macroscopic level (the pacing of movements) is evident both in daily life (e.g., walking) and skilled (e.g., music and dance) behaviors. By examining the fine structure of movement, we here show that interpersonal coordination is established also at a microscopic - submovement - level. Natural movements appear as marked by recurrent (2-3 Hz) speed breaks, i.e., submovements, that are traditionally considered the result of intermittency in (visuo)motor feedback-based control. In a series of interpersonal coordination tasks, we show that submovements produced by interacting partners are not independent but alternate tightly over time, reflecting online mutual adaptation. These findings unveil a potential core mechanism for behavioral coordination that is based on between-persons synchronization of the intrinsic dynamics of action-perception cycles.
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BACKGROUND: Allergic contact dermatitis involving the hands is a common occupational skin disease for hairdressers and the potent sensitizers p -phenylenediamine (PPD) and toluene-2,5-diamine (PTD) are associated with the development of occupational allergic contact dermatitis. OBJECTIVE: The aim of the study was to analyze whether the use of the moderate sensitizer 2-methoxymethyl-PPD (ME-PPD) in professional hair dyes is a suitable tool to reduce the occupational contact allergy risk for hairdressers. METHODS: Hand exposure of hairdressers (N = 11) to ME-PPD was analyzed under routine hair coloring conditions in commercial salons. By accounting for wet work and uneven hand exposure, the daily hand exposure was derived and compared with the occupational acceptable exposure level (AEL), that is, the sensitization induction threshold of ME-PPD adjusted for interindividual variability among workers. RESULTS: The daily hand exposure to ME-PPD was 1.6 µg/cm 2 , and the occupational AEL was 215 µg/cm 2 . The ratio of hand exposure to AEL was calculated as the margin of safety (MOS) against occupational sensitization. For ME-PPD, the MOS of 134 indicates a low likelihood of sensitization versus PPD and PTD with MOS values of 2.7 and 5.9, respectively. CONCLUSIONS: Our data predict that the use of ME-PPD in professional hair color products improves the protection of hairdressers against hair dye-related contact allergy versus the use of PPD and PTD.
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Dermatite Alérgica de Contato , Dermatite Ocupacional , Tinturas para Cabelo , Exposição Ocupacional , Fenilenodiaminas , Dermatite Alérgica de Contato/diagnóstico , Dermatite Alérgica de Contato/etiologia , Dermatite Alérgica de Contato/prevenção & controle , Dermatite Ocupacional/etiologia , Dermatite Ocupacional/prevenção & controle , Tinturas para Cabelo/efeitos adversos , Humanos , Exposição Ocupacional/efeitos adversos , Exposição Ocupacional/prevenção & controle , Fenilenodiaminas/efeitos adversos , Medição de RiscoRESUMO
The Leucine Rich Repeat Kinase 2 (LRRK2) gene is a major genetic determinant of Parkinson's disease (PD), encoding a homonymous multi-domain protein with two catalytic activities, GTPase and Kinase, involved in intracellular signaling and trafficking. LRRK2 is phosphorylated at multiple sites, including a cluster of autophosphorylation sites in the GTPase domain and a cluster of heterologous phosphorylation sites at residues 860 to 976. Phosphorylation at these latter sites is found to be modified in brains of PD patients, as well as for some disease mutant forms of LRRK2. The main aim of this study is to investigate the functional consequences of LRRK2 phosphorylation or dephosphorylation at LRRK2's heterologous phosphorylation sites. To this end, we generated LRRK2 phosphorylation site mutants and studied how these affected LRRK2 catalytic activity, neurite outgrowth and lysosomal physiology in cellular models. We show that phosphorylation of RAB8a and RAB10 substrates are reduced with phosphomimicking forms of LRRK2, while RAB29 induced activation of LRRK2 kinase activity is enhanced for phosphodead forms of LRRK2. Considering the hypothesis that PD pathology is associated to increased LRRK2 kinase activity, our results suggest that for its heterologous phosphorylation sites LRRK2 phosphorylation correlates to healthy phenotypes and LRRK2 dephosphorylation correlates to phenotypes associated to the PD pathological processes.
Assuntos
Doença de Parkinson , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Lisossomos/metabolismo , Doença de Parkinson/metabolismo , Fosforilação/fisiologia , Transdução de SinaisRESUMO
(R)-ND-336-designated as compound (R)-5-is a highly selective inhibitor of matrix metalloproteinase (MMP)-9 with efficacy in accelerating diabetic wound healing in murine models. (R)-ND-336 belongs to the class of thiirane inhibitors of MMPs and it is currently undergoing Investigation New Drug (IND)-enabling studies. We investigated the in vitro metabolism of (R)-ND-336 using S9 fractions obtained from mice, rats, dogs, minipigs, monkeys, and humans in order to select the rodent and nonrodent species for toxicology studies. Three metabolites were observed. One metabolite, M3, was observed across all species. Metabolite M2 was found in rats, monkeys, and humans. Metabolite M1 was observed only in rats. The identities of the metabolites were suggested by liquid chromatography/tandem mass spectroscopy (LC/MS-MS) analyses, which were authenticated by comparison to synthetic samples. Metabolites M2 and M3 arise from oxidative deamination of (R)-ND-336 by monoamine oxidase to give the arylaldehyde as a transient (and unobserved) intermediate. Reductive metabolism of this aldehyde gives the alcohol metabolite M2, while further oxidative metabolism of the aldehyde produces the carboxylate metabolite M3. A minor route of metabolism, seen only in rats, is N-acetylation of (R)-ND-336 to give the acetamide M1. The metabolism of (R)-ND-336 is distinctly different from that of the prototype member of this thiirane class ((±)-1, lacking the 4-aminomethyl aryl substituent) which is metabolized primarily by oxidation α to the sulfone to lead to a benzenesulfinate metabolite. All three metabolites are poorer MMP-9 inhibitors, compared to (R)-ND-336 (MMP-9, K i = 19 nM): M3, MMP-9 IC50 > 100 µM; M2, K i = 390 nM; and M1, IC50 > 100 µM). The rat and the minipig were selected as the rodent and nonrodent species, respectively, for toxicology studies.
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Human genetic studies have recently suggested that the postsynaptic activity-regulated cytoskeleton-associated protein (Arc) complex is a convergence signal for several genes implicated in schizophrenia. However, the functional significance of Arc in schizophrenia-related neurobehavioral phenotypes and brain circuits is unclear. Here, we find that, consistent with schizophrenia-related phenotypes, disruption of Arc in mice produces deficits in sensorimotor gating, cognitive functions, social behaviors, and amphetamine-induced psychomotor responses. Furthermore, genetic disruption of Arc leads to concomitant hypoactive mesocortical and hyperactive mesostriatal dopamine pathways. Application of a D1 agonist to the prefrontal cortex or a D2 antagonist in the ventral striatum rescues Arc-dependent cognitive or psychomotor abnormalities, respectively. Our findings demonstrate a role for Arc in the regulation of dopaminergic neurotransmission and related behaviors. The results also provide initial biological support implicating Arc in dopaminergic and behavioral abnormalities related to schizophrenia.
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Disfunção Cognitiva/genética , Proteínas do Citoesqueleto/genética , Dopamina/metabolismo , Proteínas do Tecido Nervoso/genética , Transtornos Psicomotores/genética , Esquizofrenia/genética , 2,3,4,5-Tetra-Hidro-7,8-Di-Hidroxi-1-Fenil-1H-3-Benzazepina/farmacologia , Anfetamina/farmacologia , Animais , Disfunção Cognitiva/metabolismo , Disfunção Cognitiva/fisiopatologia , Disfunção Cognitiva/prevenção & controle , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Corpo Estriado/fisiopatologia , Proteínas do Citoesqueleto/deficiência , Modelos Animais de Doenças , Agonistas de Dopamina/farmacologia , Antagonistas de Dopamina/farmacologia , Feminino , Expressão Gênica , Masculino , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/deficiência , Fenótipo , Córtex Pré-Frontal/efeitos dos fármacos , Córtex Pré-Frontal/metabolismo , Córtex Pré-Frontal/fisiopatologia , Transtornos Psicomotores/metabolismo , Transtornos Psicomotores/fisiopatologia , Transtornos Psicomotores/prevenção & controle , Receptores de Dopamina D1/genética , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D2/genética , Receptores de Dopamina D2/metabolismo , Salicilamidas/farmacologia , Esquizofrenia/metabolismo , Esquizofrenia/fisiopatologia , Esquizofrenia/prevenção & controle , Filtro Sensorial/efeitos dos fármacos , Filtro Sensorial/genética , Transmissão SinápticaRESUMO
Alpha-synuclein (αSyn) interferes with multiple steps of synaptic activity at pre-and post-synaptic terminals, however the mechanism/s by which αSyn alters neurotransmitter release and synaptic potentiation is unclear. By atomic force microscopy we show that human αSyn, when incubated with reconstituted membrane bilayer, induces lipid rafts' fragmentation. As a consequence, ion channels and receptors are displaced from lipid rafts with consequent changes in their activity. The enhanced calcium entry leads to acute mobilization of synaptic vesicles, and exhaustion of neurotransmission at later stages. At the post-synaptic terminal, an acute increase in glutamatergic transmission, with increased density of PSD-95 puncta, is followed by disruption of the interaction between N-methyl-d-aspartate receptor (NMDAR) and PSD-95 with ensuing decrease of long term potentiation. While cholesterol loading prevents the acute effect of αSyn at the presynapse; inhibition of casein kinase 2, which appears activated by reduction of cholesterol, restores the correct localization and clustering of NMDARs.