RESUMEN
Christianson syndrome (CS), an X-linked neurological disorder characterized by postnatal attenuation of brain growth (postnatal microcephaly), is caused by mutations in SLC9A6, the gene encoding endosomal Na+/H+ exchanger 6 (NHE6). To hasten treatment development, we established induced pluripotent stem cell (iPSC) lines from patients with CS representing a mutational spectrum, as well as biologically related and isogenic control lines. We demonstrated that pathogenic mutations lead to loss of protein function by a variety of mechanisms: The majority of mutations caused loss of mRNA due to nonsense-mediated mRNA decay; however, a recurrent, missense mutation (the G383D mutation) had both loss-of-function and dominant-negative activities. Regardless of mutation, all patient-derived neurons demonstrated reduced neurite growth and arborization, likely underlying diminished postnatal brain growth in patients. Phenotype rescue strategies showed mutation-specific responses: A gene transfer strategy was effective in nonsense mutations, but not in the G383D mutation, wherein residual protein appeared to interfere with rescue. In contrast, application of exogenous trophic factors (BDNF or IGF-1) rescued arborization phenotypes across all mutations. These results may guide treatment development in CS, including gene therapy strategies wherein our data suggest that response to treatment may be dictated by the class of mutation.
Asunto(s)
Células Madre Pluripotentes Inducidas , Microcefalia , Ataxia , Epilepsia , Enfermedades Genéticas Ligadas al Cromosoma X , Humanos , Discapacidad Intelectual , Microcefalia/genética , Mutación/genética , Neuronas , Trastornos de la Motilidad OcularRESUMEN
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.
Asunto(s)
Histona Desacetilasa 6/genética , Mitocondrias/metabolismo , Neuronas/metabolismo , Proteínas de Unión al GTP rho/genética , Quinasas Asociadas a rho/genética , Acetilación/efectos de los fármacos , Animales , Transporte Axonal/efectos de los fármacos , Transporte Axonal/genética , Calcio/metabolismo , Proteoglicanos Tipo Condroitín Sulfato/farmacología , Femenino , Ganglios Espinales/citología , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/metabolismo , Regulación de la Expresión Génica , Histona Desacetilasa 6/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Mitocondrias/efectos de los fármacos , Glicoproteína Asociada a Mielina/farmacología , Neuronas/citología , Neuronas/efectos de los fármacos , Cultivo Primario de Células , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Proteínas de Unión al GTP rho/metabolismo , Quinasas Asociadas a rho/metabolismoRESUMEN
High mobility group (HMG) proteins concentrate in the nucleus, interacting with chromatin. Amphoterin is an HMG protein (HMGB1) that has been shown to have extranuclear functions and can be secreted from some cell types. Exogenous amphoterin can increase neurite growth, suggesting that the secreted protein may have growth promoting activities in neurons. Consistent with this, we show that depletion of amphoterin mRNA from cultured adult rat DRG neurons attenuates neurite outgrowth, pointing to autocrine or paracrine mechanisms for its growth-promoting effects. The mRNA encoding amphoterin localizes to axonal processes and we showed recently that its 3'-UTR is sufficient for axonal localization of heterologous transcripts (Donnelly et al., 2013). Here, we show that amphoterin mRNA is transported constitutively into axons of adult DRG neurons. A preconditioning nerve injury increases the levels of amphoterin protein in axons without a corresponding increase in amphoterin mRNA in the axons. A 60 nucleotide region of the amphoterin mRNA 3'-UTR is necessary and sufficient for its localization into axons of cultured sensory neurons. Amphoterin mRNA 3'-UTR is also sufficient for axonal localization in distal axons of DRG neurons in vivo. Overexpression of axonally targeted amphoterin mRNA increases axon outgrowth in cultured sensory neurons, but axon growth is not affected when the overexpressed mRNA is restricted to the cell body.
Asunto(s)
Axones/metabolismo , Regulación de la Expresión Génica/genética , Proteína HMGB1/genética , Biosíntesis de Proteínas/genética , ARN Mensajero/metabolismo , Células Receptoras Sensoriales/citología , Regiones no Traducidas 3'/genética , Animales , Axones/efectos de los fármacos , Transporte Biológico/genética , Células Cultivadas , Ganglios Espinales/citología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteína HMGB1/metabolismo , Masculino , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Fotoblanqueo , ARN Interferente Pequeño/farmacología , Ratas , Ratas Sprague-Dawley , Células Receptoras Sensoriales/efectos de los fármacos , Transducción GenéticaRESUMEN
Anabolic androgenic steroids (AAS) are often misused by adolescents and athletes. Their effects vary according to chemical structure and metabolism, route of administration, and AAS regimen. In this study, adult C57Bl/6 male mice were systemically exposed to testosterone propionate (TP), nandrolone or 17alpha-methyltestosterone (17alpha-meT), type I, type II and type III AAS, respectively, in order to determine the hedonic or aversive properties of each drug. For this purpose, the conditioned place preference (CPP) test was employed at three different AAS doses (0.075, 0.75 and 7.5 mg/kg). Other behavioral domains monitored were light-dark transitions (side changes) and general activity. TP shifted place preference at all doses tested, and nandrolone shifted place preference at 0.75 and 7.5 mg/kg, but not at 0.075 mg/kg, the lower dose tested. Conversely, mice receiving 17alpha-meT did not show alteration in the preference score. The lower dose of nandrolone did modify exploratory-based anxiety showing a decrease in light-dark transitions if compared to vehicle-treated animals, while mice treated with TP or 17alpha-meT were not affected. Our data suggest that when studying hedonic and rewarding properties of synthetic androgens, distinction has to be made based on type of AAS and metabolism.