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TRIM32 is a E3 ubiquitin -ligase containing RING, B-box, coiled-coil and six C-terminal NHL domains. Mutations involving NHL and coiled-coil domains result in a pure myopathy (LGMD2H/STM) while the only described mutation in the B-box domain is associated with a multisystemic disorder without myopathy (Bardet-Biedl syndrome type11), suggesting that these domains are involved in distinct processes. Knock-out (T32KO) and knockin mice carrying the c.1465G > A (p.D489N) involving the NHL domain (T32KI) show alterations in muscle regrowth after atrophy and satellite cells senescence. Here, we present phenotypical description and functional characterization of mutations in the RING, coiled-coil and NHL domains of TRIM32 causing a muscle dystrophy. Reduced levels of TRIM32 protein was observed in all patient muscle studied, regardless of the type of mutation (missense, single amino acid deletion, and frameshift) or the mutated domain. The affected patients presented with variable phenotypes but predominantly proximal weakness. Two patients had symptoms of both muscular dystrophy and Bardet-Biedl syndrome. The muscle magnetic resonance imaging (MRI) pattern is highly variable among patients and families. Primary myoblast culture from these patients demonstrated common findings consistent with reduced proliferation and differentiation, diminished satellite cell pool, accelerated senescence of muscle, and signs of autophagy activation.

Skeletal muscle regeneration by muscle satellite cells is a physiological mechanism activated upon muscle damage and regulated by Notch signaling. In a family with autosomal recessive limbgirdle muscular dystrophy, we identified a missense mutation in POGLUT1 (protein O-glucosyltransferase 1), an enzyme involved in Notch posttranslational modification and function. In vitro and in vivo experiments demonstrated that the mutation reduces Oglucosyltransferase activity on Notch and impairs muscle development. Muscles from patients revealed decreased Notch signaling, dramatic reduction in satellite cell pool and a muscle-specific adystroglycan hypoglycosylation not present in patients’ fibroblasts. Primary myoblasts from patients showed slow proliferation, facilitated differentiation, and a decreased pool of quiescent PAX7+ cells. A robust rescue of the myogenesis was demonstrated by increasing Notch signaling. None of these alterations were found in muscles from secondary dystroglycanopathy patients. These data suggest that a key pathomechanism for this novel form of muscular dystrophy is Notch-dependent loss of satellite cells.

Las neurexinas son proteínas de membrana sináptica con cientos de isoformas que participan en la formación o establecimiento de las sinapsis. La interacción de neurexinas presinápticas con sus receptores postsinápticos, como las neuroliguinas, media un mecanismo bidireccional de señalización que regula la función de las sinapsis. Se han identificado mutaciones en genes de neurexinas y neuroliguinas en pacientes con autismo, retraso mental y esquizofrenia, sugiriendo que defectos en el sistema de señalización de neurexinas y neuroliguinas podrían subyacer algunas sinaptopatías. Por otro lado, la enfermedad de Alzheimer es un trastorno neurodegenerativo que se caracteriza por déficit sináptico, pérdida progresiva de memoria y neurodegeneración. La mayoría de las formas familiares de la enfermedad de Alzheimer (FAD) son causadas por mutaciones en los genes de presenilinas (PS), subunidades catalíticas del complejo gamma-secretasa. El objetivo de la presente Tesis consistió en indagar si la función de neurexinas está regulada por presenilinas (PS), el componente activo del complejo gamma-secretasa encargado de proteolizar proteínas de membrana. Mutaciones en PS son las responsables de la mayoría de los casos de FAD. Hemos identificado un procesamiento secuencial de neurexinas por metaloproteasas y PS/gamma-secretasa. Tras un primer corte proteolítico en el dominio extracelular mediado por metaloproteasas se libera el ectodominio soluble y se generan los fragmentos C-terminales (CTFs) unidos a membrana. Estos CTFs son posteriormente procesados proteolíticamente por el complejo PS/gamma-secretasa, como muestra el hecho de que la inhibición de la actividad PS/gamma-secretasa induce la acumulación de los CTFs de neurexinas in vivo e in vitro. Posteriormente se demostró que PS se recluta junto con neurexinas a los terminales glutamatérgicos en formación mediados por neuroliguinas en cultivos de neuronas de hipocampo y que los CTFs de neurexinas se acumulan principalmente en

Neurexins are a large family of neuronal plasma membrane proteins, which function as trans-synaptic receptors during synaptic differentiation. The binding of presynaptic neurexins to postsynaptic partners, such as neuroligins, has been proposed to participate in a signaling pathway that regulates synapse formation/stabilization. The identification of mutations in neurexin genes associated with autism and mental retardation suggests that dysfunction of neurexins may underlie synaptic defects associated with brain disorders. However, the mechanisms that regulate neurexin function at synapses are still unclear. Here, we show that neurexins are proteolytically processed by presenilins (PS), the catalytic components of the c-secretase complex that mediates the intramembraneous cleavage of several type I membrane proteins. Inhibition of PS/c-secretase by using pharmacological and genetic approaches induces a drastic accumulation of neurexin C-terminal fragments (CTFs) in cultured rat hippocampal neurons and mouse brain. Neurexin-CTFs accumulate mainly at the presynaptic terminals of PS conditional double knockout (PS cDKO) mice lacking both PS genes in glutamatergic neurons of the forebrain. The fact that loss of PS function enhances neurexin accumulation at glutamatergic terminals mediated by neuroligin-1 suggests that PS regulate the processing of neurexins at glutamatergic synapses. Interestingly, presenilin 1 (PS1) is recruited to glutamatergic terminals mediated by neuroligin-1, thus concentrating PS1 at terminals containing b-neurexins. Furthermore, familial Alzheimer’s disease (FAD)-linked PS1 mutations differentially affect b-neurexin-1 processing. Expression of PS1 M146L and PS1 H163R mutants in PS2/2 cells rescues the processing of bneurexin-1, whereas PS1 C410Y and PS1 DE9 fail to rescue the processing defect. These results suggest that PS regulate the synaptic function and processing of neurexins at glutamatergic synapses, and that impaired neurexin processing