RESUMO
L-dopa is the most effective drug used to date for management of Parkinson's disease symptoms. Unfortunately, long-term administration of L-dopa often results in development of motor disorders, including dyskinesias. Despite extensive research on L-dopa-induced dyskinesia, its pathogenesis remains poorly understood. We demonstrated previously that L-dopa can be post-translationally incorporated into the C-terminus of α-tubulin in living cells. In the present study, we investigated the effect of the presence of L-dopa-tubulin-enriched microtubules on mitochondrial traffic mediated by molecular motor KIF5B. Using biochemical approaches in combination with experiments on neuronal cell lines and mouse hippocampal primary cultures, we demonstrated that L-dopa incorporation into tubulin is irreversible. Transport of mitochondria along the axon was altered after L-dopa treatment of cells. In L-dopa-treated cells, mitochondria had reduced ability to reach the distal segment of the axon, spent more time in pause, and showed reduced velocity of anterograde movement. KIF5B motor, a member of the kinesin family involved in mitochondrial transport in neurons, showed reduced affinity for Dopa-tubulin-containing microtubules. Our findings, taken together, suggest that tyrosination state of tubulin (and microtubules) is altered by L-dopa incorporation into tubulin; the gradual increase in amount of altered microtubules affects microtubule functioning, impairs mitochondrial traffic and distribution, and this could be relevant in Parkinson's disease patients chronically treated with L-dopa.
Assuntos
Transporte Axonal/efeitos dos fármacos , Cinesinas/metabolismo , Levodopa/toxicidade , Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Tubulina (Proteína)/efeitos dos fármacos , Animais , Axônios/efeitos dos fármacos , Axônios/metabolismo , Linhagem Celular , Humanos , Camundongos , Ratos , Tubulina (Proteína)/metabolismoRESUMO
Inhibition of histone deacetylase 6 (HDAC6) was shown to support axon growth on the nonpermissive substrates myelin-associated glycoprotein (MAG) and chondroitin sulfate proteoglycans (CSPGs). Though HDAC6 deacetylates α-tubulin, we find that another HDAC6 substrate contributes to this axon growth failure. HDAC6 is known to impact transport of mitochondria, and we show that mitochondria accumulate in distal axons after HDAC6 inhibition. Miro and Milton proteins link mitochondria to motor proteins for axon transport. Exposing neurons to MAG and CSPGs decreases acetylation of Miro1 on Lysine 105 (K105) and decreases axonal mitochondrial transport. HDAC6 inhibition increases acetylated Miro1 in axons, and acetyl-mimetic Miro1 K105Q prevents CSPG-dependent decreases in mitochondrial transport and axon growth. MAG- and CSPG-dependent deacetylation of Miro1 requires RhoA/ROCK activation and downstream intracellular Ca2+ increase, and Miro1 K105Q prevents the decrease in axonal mitochondria seen with activated RhoA and elevated Ca2+ These data point to HDAC6-dependent deacetylation of Miro1 as a mediator of axon growth inhibition through decreased mitochondrial transport.
Assuntos
Desacetilase 6 de Histona/genética , Mitocôndrias/metabolismo , Neurônios/metabolismo , Proteínas rho de Ligação ao GTP/genética , Quinases Associadas a rho/genética , Acetilação/efeitos dos fármacos , Animais , Transporte Axonal/efeitos dos fármacos , Transporte Axonal/genética , Cálcio/metabolismo , Proteoglicanas de Sulfatos de Condroitina/farmacologia , Feminino , Gânglios Espinais/citologia , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Regulação da Expressão Gênica , Desacetilase 6 de Histona/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/efeitos dos fármacos , Glicoproteína Associada a Mielina/farmacologia , Neurônios/citologia , Neurônios/efeitos dos fármacos , Cultura Primária de Células , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Proteínas rho de Ligação ao GTP/metabolismo , Quinases Associadas a rho/metabolismoRESUMO
In this work, we have immunohistochemically analyzed the effects of single injections of apotransferrin (aTf) on the expression of myelin (myelin basic proteins [MBPs]) and axonal (protein gene product 9.5 [PGP 9.5] and beta(III)-tubulin [beta(III)-tub]) proteins in colchicine-injected and crushed sciatic nerves of adult rats. A protein redistribution was seen in the distal stump of injured nerves, with the appearance of MBP- and PGP 9.5-immunoreactive (IR) clusters which occurred earlier in crushed nerves (3 days post-injury [PI]) as compared to colchicine-injected nerves (7 days PI). beta(III)-tub-IR clusters appeared at 1 day PI preceding the PGP 9.5- and MBP-IR clusters in colchicine-injected nerves. With image analysis, the peak of clustering formation was found at 14 days PI for MBP and at 3 days PI for beta(III)-tub in colchicine-injected nerves. At 28 days of survival, the protein distribution patterns were almost normal. The intraneural application of aTf, at different concentrations (0.0005 mg/ml, 0.005 mg/ml, 0.05 mg/ml, 0.5 mg/ml), prevented nerve degeneration produced by colchicine, with the appearance of only a small number of MBP- and beta(III)-tub-IR clusters. However, aTf was not able to prevent clustering formation when the nerve was crushed, a kind of injury that also involves necrosis and blood flow alterations. The results suggest that aTf could prevent the colchicine effects by stabilizing the cytoskeleton proteins of the nerve fibers, avoiding the disruption of the axonal transport and thus the myelin degeneration. Transferrin is proposed as a complementary therapeutic avenue for treatment of cytotoxic nerve injuries.
Assuntos
Apoproteínas/farmacologia , Axônios/efeitos dos fármacos , Proteínas do Tecido Nervoso/efeitos dos fármacos , Neuropatia Ciática/tratamento farmacológico , Transferrina/farmacologia , Degeneração Walleriana/tratamento farmacológico , Degeneração Walleriana/prevenção & controle , Animais , Transporte Axonal/efeitos dos fármacos , Transporte Axonal/fisiologia , Axônios/metabolismo , Colchicina/antagonistas & inibidores , Colchicina/toxicidade , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Citoesqueleto/patologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Imuno-Histoquímica , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Microtúbulos/patologia , Proteína Básica da Mielina/efeitos dos fármacos , Proteína Básica da Mielina/metabolismo , Bainha de Mielina/efeitos dos fármacos , Bainha de Mielina/metabolismo , Bainha de Mielina/patologia , Proteínas do Tecido Nervoso/metabolismo , Fármacos Neuroprotetores/farmacologia , Neurotoxinas/antagonistas & inibidores , Neurotoxinas/toxicidade , Ratos , Ratos Wistar , Neuropatia Ciática/metabolismo , Neuropatia Ciática/fisiopatologia , Resultado do Tratamento , Tubulina (Proteína)/efeitos dos fármacos , Tubulina (Proteína)/metabolismo , Ubiquitina Tiolesterase/efeitos dos fármacos , Ubiquitina Tiolesterase/metabolismo , Degeneração Walleriana/fisiopatologiaRESUMO
The Wobbler mouse, a mutant characterized by motoneuron degeneration in the cervical spinal cord, has been used to test the efficacy of novel treatments for human motoneuron diseases (HMD). Previous reports have shown that slow axonal transport is impaired in Wobblers and other models of HMD. Since progesterone (PROG) corrects some morphological, molecular, and functional abnormalities of Wobbler mice, we studied if steroid exposure for 8 weeks restored retrograde axonal transport by measuring motoneuron labeling after injection of fluorogold into the limb muscles. The dye was injected into forelimb biceps bracchii and flexor or into the rearlimb gastrocnemius muscles; 6 days later, the number of fluorescent motoneurons and the total number of cresyl violet stained motoneurons were counted in the cervical (C5-T1) or lumbar (L3-L5) spinal cord regions. A pronounced reduction (- 42.2%) of the percent of fluorescent motoneurons in Wobbler mice cervical cord was noted, which was significantly corrected after PROG treatment. In contrast, labeling of lumbar motoneurons was not reduced in Wobbler mice and was not affected by PROG treatment. In no case PROG showed an effect in control mice. Concomitantly, PROG slightly but significantly increased biceps weight of Wobbler mice. Behaviorally, PROG-treated Wobblers performed better on a motor test (hanging time from a horizontal rope) compared to untreated counterparts. We postulate a dual role for PROG in the Wobbler mouse, in part by prevention of motoneuron degeneration and also by enhancement of axonal transport. The latter mechanism could improve the traffic of neurotrophic factors from the forelimb muscles into the ailing motoneurons, improving neuromuscular function in this murine model of HMD.
Assuntos
Transporte Axonal/efeitos dos fármacos , Doença dos Neurônios Motores/tratamento farmacológico , Doença dos Neurônios Motores/fisiopatologia , Neurônios Motores/efeitos dos fármacos , Progestinas/uso terapêutico , Medula Espinal/patologia , Animais , Comportamento Animal , Vértebras Cervicais/patologia , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Camundongos Mutantes Neurológicos , Progestinas/sangue , Estilbamidinas/metabolismo , Extremidade Superior/fisiopatologiaRESUMO
1. The injection of acetylcholine (ACh) into the medial prefrontal cortex (MPFC) caused marked hypotensive response in either unanesthetized or anesthetized rats. 2. The present experiment was designed to investigate anatomical connections of the ACh injection site in the MPFC with putative autonomic-related brain nuclei, as well as their possible involvement in the mediation of the hypotensive response to ACh. 3. For the above purpose, the bidirectional neuronal tracer biotinylated dextran amine (BDA) was injected into Cg1 and Cg3 areas, within the MPFC of male Wistar rats. Five days later the animals were sacrificed and brain slices were processed and analyzed to determine neuronal projections efferent from as well afferent to the MPFC. 4. Neuronal staining was more prominent in regions ipsilateral to the BDA injection site. Prominent efferent projections of the MPFC were observed in the contralateral MPFC: ipsi- and contralateral amygdala and hypothalamus; ipsilateral septal area, diagonal band, and zona incerta. 5. Similar but not equal patterns of neuronal labeling were observed when BDA injections were performed within the two adjacent MPFC areas. BDA injections centered in the ACh injection site in the Cg3 area caused strong labeling in the septal area and diagonal band as well as an overall hypothalamic labeling. Within the hypothalamus an intense cortical projection was observed in the lateral hypothalamus (LH). BDA injections into the Cg1 area caused a more evident labeling of the amygdaloid complex. 6. Neuronal cell bodies were evident throughout the MPFC as well as in the sensory-motor cortex when BDA was injected into the LH, thus indicating a massive ipsilateral cortical projection from the Cg3 to the LH. 7. Bilateral NMDA-induced lesions within the LH caused a significant attenuation of the depressor responses to ACh injection in the MPFC, whereas unilateral lesions were marginally effective. These results indicate the involvement of the LH in the mediation of the hypotensive response to ACh injection into the MPFC as well as the bilateral distribution of the hypotensive pathway.
Assuntos
Biotina/análogos & derivados , Colinérgicos/farmacologia , Giro do Cíngulo/fisiologia , Hipotensão/fisiopatologia , Região Hipotalâmica Lateral/fisiologia , Córtex Pré-Frontal/fisiologia , Acetilcolina/farmacologia , Animais , Transporte Axonal/efeitos dos fármacos , Transporte Axonal/fisiologia , Biotina/administração & dosagem , Dextranos/administração & dosagem , Corantes Fluorescentes/administração & dosagem , Giro do Cíngulo/efeitos dos fármacos , Hipotensão/induzido quimicamente , Região Hipotalâmica Lateral/efeitos dos fármacos , Injeções Intraventriculares , Masculino , N-Metilaspartato/toxicidade , Vias Neurais/efeitos dos fármacos , Vias Neurais/fisiologia , Córtex Pré-Frontal/efeitos dos fármacos , Ratos , Ratos WistarRESUMO
Parâmetros entre o transporte anterógrado e os níveis de cálcio foram demonstrados em neurônios isolados, e somam-se informaçoes que o fluxo axoplasmático pode estar relacionado com microtúbulos, miosina e actina em neurônios secretores hipotalâmicos. Neste estágio, procuramos verificar se há suscetilidade dos núcleos paraventrículares à açao do bloqueador de canais de cálcio (verapamil) e a decorrência do efeito sobre o transporte axoplasmático da neurossecreçao em Gallus domesticus aos 25 dias após eclosao. Os resultados da análise morfométrica com integralizador de imagem realizada após processamento segundo Bargmann e os da avaliaçao do teor proteico hipotalâmico pelo método de Lowry, foram analisados estatisticamente registrando consistência na hipótese de ser cálcio-dependente o transporte da neurossecreçao dos núcleos paraventriculares, nesta condiçao experimental.
Assuntos
Animais , Transporte Axonal/efeitos dos fármacos , Núcleo Hipotalâmico Paraventricular , Aves Domésticas , Verapamil/farmacologia , NeurossecreçãoRESUMO
Brain slices obtained from young rats were incubated with different radioactive precursors, in the presence and absence of L-cycloserine (an inhibitor of the synthesis of sphingosine) in order to explore the possibility that transport of proteolipids--and specifically of the major myelin proteolipid PLP--to the myelin membrane could be coupled to the transport of cerebrosides or sulfatides. At a concentration of 0.15 mM L-cycloserine, the incorporation of [3H] glycine into total proteins, proteolipid apoproteins (APL), PLP, and myelin basic proteins (MBP) of the total homogenate was unaffected by the presence of the inhibitor, whereas the incorporation of [3H] serine into glycosphingolipids decreased markedly. Under similar incubation conditions, the entry of labeled APL and of PLP into the myelin membranes in the presence of L-cycloserine decreased markedly (50%) in comparison to controls. Entry of MBP was not affected by the inhibitor. These results indicate that when synthesis of glycosphingolipids is inhibited by L-cycloserine, thus decreasing the availability of cerebrosides and sulfatides, the translocation of PLP to myelin is disrupted, suggesting that its transport through the oligodendroglial cell could be coupled to the transport of glycosphingolipids and, most probably, of sulfatides.