Pathological deficit of Cystatin B impairs synaptic plasticity in EPM1 human cerebral organoids

ABSTRACT: Cystatin B (CSTB) is a small protease inhibitor involved in cell proliferation and migration, and composition of extracellular matrix during brain development. Loss-of-function mutations in the gene encoding CSTB cause progressive myoclonic epilepsy 1 (EPM1). We previously demonstrated that CSTB is locally synthesized in the synaptosomes from rat brain and secreted into the media, indicating its role in synaptic plasticity. In this work, we further investigate the involvement of CSTB in synaptic plasticity, using synaptosomes from rodents’ brain, as well as from human Cerebral Organoids (hCOs). Our data demonstrated that CSTB is released by synaptosomes in two ways: as a soluble protein, and in extracellular vesicles-mediated manner. Synaptosomes isolated from hCOs are enriched in pre-synaptic proteins, as expected for an in vitro model of human synapses, and contain CSTB at all developmental stages analysed. However, only at late maturation stages synapse becomes physiologically active, i.e. particularly enriched with exocytosis-associated proteins. CSTB presence in the synaptic territories was confirmed also by immunostaining on human neurons in vitro. To investigate if the depletion of CSTB in EPM1 is affecting synaptic plasticity, we used synaptosomes from EPM1 hCOs. The synaptosomal levels of presynaptic proteins and of an initiation factor linked to local protein synthesis, were reduced in EPM1 hCOs compared to controls, and the extracellular vesicles trafficking was impaired. Moreover, morphological alteration was detected in neurons from EPM1 patients, indicating a connectivity deficit. In conclusion, our data indicate a significant role of synaptic CSTB in cell-to-cell communication in physiological and pathological conditions, and demonstrated that CSTB-dependent altered synaptic plasticity is one of the mechanisms underlying EPM1.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Brain

SUBMITTER: Angela Chambery  

LAB HEAD: Angela Chambery

PROVIDER: PXD041125 | Pride | 2024-08-10

REPOSITORIES: Pride