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Cystatin B (CSTB) is a small protease inhibitor protein being involved in cell proliferation and neuronal differentiation. Loss-of-function mutations in CSTB gene cause progressive myoclonic epilepsy 1 (EPM1). We previously demonstrated that CSTB is locally synthesized in synaptic nerve terminals from rat brain and secreted into the media, indicating its role in synaptic plasticity. In this work, we have further investigated the involvement of CSTB in synaptic plasticity, using synaptosomes from human cerebral organoids (hCOs) as well as from rodents' brain. Our data demonstrate that CSTB is released from synaptosomes in two ways: (i) as a soluble protein and (ii) in extracellular vesicles-mediated pathway. Synaptosomes isolated from hCOs are enriched in pre-synaptic proteins and contain CSTB at all developmental stages analyzed. CSTB presence in the synaptic territories was also confirmed by immunostaining on human neurons in vitro. To investigate if the depletion of CSTB affects synaptic plasticity, we characterized the synaptosomes from EPM1 hCOs. We found that the levels of presynaptic proteins and of an initiation factor linked to local protein synthesis were both reduced in EPM1 hCOs and that the extracellular vesicles trafficking pathway was impaired. Moreover, EPM1 neurons displayed anomalous morphology with longer and more branched neurites bearing higher number of intersections and nodes, suggesting connectivity alterations. In conclusion, our data strengthen the idea that CSTB plays a critical role in the synapse physiology and reveal that pathologically low levels of CSTB may affect synaptic plasticity, leading to synaptopathy and altered neuronal morphology., (© 2023. The Author(s).)

While protein synthesis in neurons is largely attributed to cell body and dendrites, the capability of synaptic regions to synthesize new proteins independently of the cell body has been widely demonstrated as an advantageous mechanism subserving synaptic plasticity. Thus, the contribution that local protein synthesis at synapses makes to physiology and pathology of brain plasticity may be more prevalent than initially thought. In this study, we tested if local protein synthesis at synapses is deregulated in the brains of TgCRND8 mice, an animal model for Alzheimer's disease (AD) overexpressing mutant human amyloid precursor protein (APP). To this end, we used synaptosomes as a model system to study the functionality of the synaptic regions in mouse brains. Our results showed that, while TgCRND8 mice exhibit early signs of brain inflammation and deficits in learning, the electrophoretic profile of newly synthesized proteins in their synaptosomes was subtly different from that of the control mice. Interestingly, APP itself was, in part, locally synthesized in the synaptosomes, underscoring the potential importance of local translation at synapses. More importantly, after the contextual fear conditioning, de novo synthesis of some individual proteins was significantly enhanced in the synaptosomes of control animals, but the TgCRND8 mice failed to display such synaptic modulation by training. Taken together, our results demonstrate that synaptic synthesis of proteins is impaired in the brain of a mouse model for AD, and raise the possibility that this deregulation may contribute to the early progression of the pathology.

miRNAs are master regulators of gene expression in diverse biological processes, including the modulation of neuronal cytoarchitecture. The identification of their physiological target genes remains one of the outstanding challenges. Recently, it has been demonstrated that the activation of serotonin receptor 7 (5-HT7R) plays a key role in regulating the neuronal structure, synaptogenesis, and synaptic plasticity during embryonic and early postnatal development of the central nervous system (CNS). In order to identify putative miRNAs targeting the 3'UTR of 5-HT7R mouse transcript, we used a computational prediction tool and detected the miR-29 family members as the only candidates. Thus, since miR-29a is more expressed than other members in the brain, we investigated its possible involvement in the regulation of neuronal morphology mediated by 5-HT7R. By luciferase assay, we show that miR-29a can act as a post-transcriptional regulator of 5-HT7R mRNA. Indeed, it downregulates 5-HT7R gene expression in cultured hippocampal neurons, while the expression of other serotonin receptors is not affected. From a functional point of view, miR-29a overexpression in hippocampal primary cultures impairs the 5HT7R-dependent neurite elongation and remodeling through the inhibition of the ERK intracellular signaling pathway. In vivo, the upregulation of miR-29a in the developing hippocampus parallels with the downregulation of 5-HT7R expression, supporting the hypothesis that this miRNA is a physiological modulator of 5-HT7R expression in the CNS.

The synthesis of brain metabolic DNA (BMD) is modulated by learning and circadian oscillations and is not involved in cell division or DNA repair. Data from rats have highlighted its prevalent association with the mitochondrial fraction and its lack of identity with mtDNA. These features suggested that BMD could be localized in synaptosomes that are the major contaminants of brain mitochondrial fractions. The hypothesis has been examined by immunochemical analyses of the large synaptosomes of squid optic lobes that are readily prepared and identified. Optic lobe slices were incubated with 5-bromo-2-deoxyuridine (BrdU) and the isolated synaptosomal fraction was exposed to the green fluorescent anti-BrdU antibody. This procedure revealed that newly synthesized BrdU-labeled BMD is present in a significant percent of the large synaptosomes derived from the nerve terminals of retinal photoreceptor neurons and in synaptosomal bodies of smaller size. Synaptosomal BMD synthesis was strongly inhibited by actinomycin D. In addition, treatment of the synaptosomal fraction with Hoechst 33258, a blue fluorescent dye specific for dsDNA, indicated that native DNA was present in all synaptosomes. The possible role of synaptic BMD is briefly discussed.

Squid giant axon has been an excellent model system for studying fundamental topics in neurobiology such as neuronal signaling. It has been also useful in addressing the questions of local protein synthesis in the axons. Incubation of isolated squid giant axons with [ 35 S]methionine followed by immunoprecipitation with a rabbit antibody against all squid neurofilament (NF) proteins demonstrates the local synthesis of a major 180 kDa NF protein and of several NF proteins of lower molecular weights. Their identification as NF proteins is based on their absence in the preimmune precipitates. Immunoprecipitates washed with more stringent buffers confirmed these results. Our data are at variance with a recent study based on the same experimental procedure that failed to visualize the local synthesis of NF proteins by the giant axon and thereby suggested their exclusive derivation from nerve cell bodies (as reported by Gainer et al. in Cell Mol Neurobiol 37:475-486, 2017). By reviewing the pertinent literature, we confute the claims that mRNA translation is absent in mature axons because of a putative translation block and that most proteins of mature axons are synthesized in the surrounding glial cells. Given the intrinsic axonal capacity to synthesize proteins, we stress the glial derivation of axonal and presynaptic RNAs and the related proposal that these neuronal domains are endowed with largely independent gene expression systems (as reported by Giuditta et al. in Physiol Rev 88:515-555, 2008).