Extracellular vesicle sorting of α-Synuclein is regulated by sumoylation

Extracellular α-Synuclein has been implicated in interneuronal propagation of disease pathology in Parkinson’s Disease. How α-Synuclein is released into the extracellular space is still unclear. Here, we show that α-Synuclein is present in extracellular vesicles in the central nervous system. We find that sorting of α-Synuclein in extracellular vesicles is regulated by sumoylation and that sumoylation acts as a sorting factor for targeting of both, cytosolic and transmembrane proteins, to extracellular vesicles. We provide evidence that the SUMO-dependent sorting utilizes the endosomal sorting complex required for transport (ESCRT) by interaction with phosphoinositols. Ubiquitination of cargo proteins is so far the only known determinant for ESCRT-dependent sorting into the extracellular vesicle pathway. Our study reveals a function of SUMO protein modification as a Ubiquitin-independent ESCRT sorting signal, regulating the extracellular vesicle release of α-Synuclein. We deciphered in detail the molecular mechanism which directs α-Synuclein into extracellular vesicles which is of highest relevance for the understanding of Parkinson’s disease pathogenesis and progression at the molecular level. We furthermore propose that sumo-dependent sorting constitutes a mechanism with more general implications for cell biology.Instituto de Investigaciones Bioquímicas de La Plat

Mapping the Molecular Determinants of Positive Allosteric Modulators of the mGlu2 Receptor

Dissertação de mestrado em em Biologia Celular e Molecular, especialização em Neurociências, apresentada ao Departamento de Ciências da Vida da Faculdade de Ciências e Tecnologia da Universidade de Coimbra.Glutamato é o principal neurotransmissor excitatório no cérebro, sendo bastante importante em várias funções do sistema nervoso central. Alterações no sistema glutamatérgico estão envolvidas em doenças como esquizofrenia e ansiedade. Tem vindo a ser demonstrado que a activação de receptores mGlu2 reduz a transmissão glutamatérgica nas regiões cerebrais associadas a estas doenças. Por esta mesma razão, a activação destes receptores tem sido alvo de investigação para desenvolvimento de novas técnicas terapêuticas, especialmente com o uso de modeladores alostericos positivos (PAMs); estes modeladores ligam-se a uma zona do receptor diferente do local de ligação do glutamato. Dado que a área de estudo dos PAM do receptor mGlu2 se encontra em expansão e o primeiro ensaio clínico está a decorrer é bastante importante obter mais informacão sobre o correcto local de ligacão destes ligandos. Esta informacão também pode suportar e facilitar os esforços da investigação química. Com o objectivo de identificar os aminoácidos responsáveis pela interação entre os PAMs e o receptor mGlu2, foi efectuada modulação molecular e docking de PAMs de receptores mGlu2 em paralelo com mutagénese dirigida. Nos receptores mGlu2 mutantes foi avaliado o impacto das mutações na actividade e afinidade dos PAMs. Este estudo confima a importância de aminoácidos previamente demonstrados como importantes na actividade de PAMs estruturalmente diferentes nestes receptores. É tambem demonstrado que adicionais aminoácidos seleccionados com base na comparação de sequencias entre mGlu2/3 parecem não ser importantes na actividade dos PAMs. A informação obtida neste estudo tambem demonstra que a actividade dos modeladores testados é reduzida devido à diminuição da afinidade de ligação. Toda esta informação oferece um melhor entendimento sobre o ‘binding pocket’ para PAMs do receptor mGlu2.Glutamate is the major excitatory neurotransmitter in the brain and plays an important role in a wide variety of central nervous system functions. Alterations in the glutamatergic system are involved in disorders like schizophrenia and anxiety. It has been shown that activation of the metabotropic glutamate 2 receptor reduces the glutamatergic transmission in brain regions associated with these disorders. Therefore, activation of mGlu2 receptor is being pursued as a novel therapeutic approach, specially using positive allosteric modulators (PAMs), which bind to a site other than that of the endogenous mGlu2 receptor agonist glutamate. Since the field of mGlu2 PAMs is expanding and the first clinical studies are ongoing with mGlu2 PAMs, it will be important to get more insight into the actual binding site of these ligands. This knowledge may also facilitate and support future chemistry endeavors. In order to identify the amino acids important for the activity of mGlu2 PAMs, homology modeling and docking of mGlu2 receptor PAMs were performed in parallel with site-directed mutagenesis. Mutant mGlu2 receptors were generated and the impact of these mutations on activity and affinity of PAMs was evaluated. This study confirms the importance of several amino acids previously shown as crucial for the activity of structurally diverse mGlu2 receptor PAMs. It furthermore demonstrates that additional amino acids that were selected based on mGlu2/3 comparison did not seem to be important for PAM activity. Our data also suggest that their activity is reduced due to lower binding affinity. All this sheds further light on the mGlu2 PAM binding pocket

Extracellular vesicle sorting of α-Synuclein is regulated by sumoylation.

Extracellular α-Synuclein has been implicated in interneuronal propagation of disease pathology in Parkinson's Disease. How α-Synuclein is released into the extracellular space is still unclear. Here, we show that α-Synuclein is present in extracellular vesicles in the central nervous system. We find that sorting of α-Synuclein in extracellular vesicles is regulated by sumoylation and that sumoylation acts as a sorting factor for targeting of both, cytosolic and transmembrane proteins, to extracellular vesicles. We provide evidence that the SUMO-dependent sorting utilizes the endosomal sorting complex required for transport (ESCRT) by interaction with phosphoinositols. Ubiquitination of cargo proteins is so far the only known determinant for ESCRT-dependent sorting into the extracellular vesicle pathway. Our study reveals a function of SUMO protein modification as a Ubiquitin-independent ESCRT sorting signal, regulating the extracellular vesicle release of α-Synuclein. We deciphered in detail the molecular mechanism which directs α-Synuclein into extracellular vesicles which is of highest relevance for the understanding of Parkinson's disease pathogenesis and progression at the molecular level. We furthermore propose that sumo-dependent sorting constitutes a mechanism with more general implications for cell biology.peerReviewe