Cystatin B Involvement in Synapse Physiology of Rodent Brains and Human Cerebral Organoids

Abstract

Cystatin B (CSTB) is a ubiquitous protein belonging to a superfamily of protease inhibitors. CSTB may play a critical role in brain physiology because its mutations cause progressive myoclonic epilepsy-1A (EPM1A), the most common form of progressive myoclonic epilepsy. However, the molecular mechanisms underlying the role of CSTB in the central nervous system (CNS) are largely unknown. To investigate the possible involvement of CSTB in the synaptic plasticity, we analyzed its expression in synaptosomes as a model system in studying the physiology of the synaptic regions of the CNS. We found that CSTB is not only present in the synaptosomes isolated from rat and mouse brain cortex, but also secreted into the medium in a depolarization-controlled manner. In addition, using biorthogonal noncanonical amino acid tagging (BONCAT) procedure, we demonstrated, for the first time, that CSTB is locally synthesized in the synaptosomes. The synaptic localization of CSTB was confirmed in a human 3D model of cortical development, namely cerebral organoids. Altogether, these results suggest that CSTB may play a role in the brain plasticity and open a new perspective in studying the involvement of CSTB deregulation in neurodegenerative and neuropsychiatric diseases.

Keywords: CSTB; EPM1A; cerebral organoids; local protein synthesis; synaptic plasticity; synaptosomes.

Figure 1

Differential distribution of cystatin B (CSTB), synaptophysin (SYP) and β-actin in the homogenate and synaptosomal fraction of rodent brains. Proteins obtained from homogenate and synaptosomes of rat and mouse brains were subjected to western blot analysis and the signals for CSTB, SYP and β-actin were quantified by densitometry; the signal ratio between synaptosomes (syn) and homogenate (hom) was plotted for each protein. (A) Homogenate and synaptosomal fraction from rat brain cortex. (B) Homogenate and synaptosomal fraction from rat cerebellum. (C) Homogenate and synaptosomal fraction from mouse brain cortex. Data are presented as means ± standard deviation (n = 4 rats, n = 3 mice). ANOVA statistical analysis indicated significantly different ratio syn/hom of each protein analyzed, *p < 0.05, **p < 0.01, ***p < 0.001.Representative images of the corresponding signals in the western blot were shown below each graph.

Figure 2

CSTB is locally synthesized in the synaptosomal fraction from rat cerebral cortex. Local synthesis of CSTB was assessed by metabolic labeling of synaptosomal proteins incubated with Homopropargylglycine (HPG) for 2 h as described in “Materials and Methods” section. The newly synthesized proteins, label with HPG (green), were biotinylated and isolated by affinity purification using streptavidin beads (yellow). Western blot analysis (representative image from two biological replicates), using CSTB antibody, was performed on synaptosomal proteins (SYN, lane 1), proteins newly synthesized in synaptosomal fraction (lane 2), negative control (incubation without HPG; lane 3). While 5 μg of proteins were loaded in lane 1, 50% of the pulldown was loaded in lanes 2 and 3 of the SDS gel for the western blot analysis.

Figure 3

Secretion of CSTB from rat cortex synaptosomes. (A) Coomassie staining of proteins from synaptosomal pellet at incubation time 0 in Ringer medium (lane 1), incubation time 2 h in Ringer medium (lane 2), incubation time 2 h in depolarizing medium (lane 3); coomassie staining of proteins from incubation medium at time 0 (Ringer medium, lane 4), time 2 h (Ringer medium, lane 5) and time 2 h (depolarizing medium, lane 6). (B) Representative image of western blot analysis using β-actin, SYP and CSTB antibodies on the samples indicated in panel (A). (C) The histograms shows the expression level of CSTB and SYP normalized to that of β-actin in synaptosomal pellet at incubation time 2 h in Ringer medium (lane 2 in panel B), incubation time 2 h in depolarizing medium (lane 3 in panel B) compared to incubation time 0 (lane 1 in panel B). Data are expressed as mean ± SEM from four independent experiments. (D) The histograms show the expression level of CSTB in Ringer incubation medium at time 2 h (lane 5 in panel B), and depolarizing medium at time 2 h (lane 6 in panel B). Data are expressed as mean ± SEM from three independent experiments. *Significantly different from Ringer medium by paired t-test (p < 0.05). Rm, Ringer medium; Dm, Depolarizing medium.

Figure 4

Mass spectrometry analysis of the proteins secreted by synaptosomes. (A) Venn diagram of the secreted proteins after 2 h incubation of the synaptosomes in Ringer medium (sample-R), and in the depolarizing medium (sample-D), identified by mass-spectrometry analysis. (B) Representative image of Western blot analysis using superoxide dismutase 1 (SOD1) and cathepsin B antibodies on proteins from synaptosomal fraction (lane 1), from Ringer incubation medium at time 2 h (lane 2) and from depolarizing medium at time 2 h (lane 3). The presence of SOD1 and the absence of cathepsin B in incubation media was verified, in four biological replicates, by Exact binomial test statistics. (C) Gene ontology (GO) analysis to classify the 125 common proteins based on biological processes determined by Panther (http://pantherdb.org), using Rattus norvegicus as reference species. Shown here are only terms related to synaptic activity with p-value < 1 × 10⁻⁶.

Figure 5

Presence of CSTB in the synaptic regions of cerebral human organoids. (A–E’) Confocal microscopic images of human brain organoids (70 days old) immunostained using antibodies against beta-catenin (CTNNB; red), doublecortin (DCX; white), and SYP (green). The cell nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI; blue). In panels (A–E’) the white dashed circle indicates the ventricle-like area. (E’) Higher magnification of the boxed area in panel (E). (F,G) Confocal microscopic images of sections of human brain organoids (70 days old) immunostained using antibodies against SYP (green), CSTB (red) and DAPI. The arrowheads in (F,G) indicate the colocalized (or very closely apposed) signals of SYP and CSTB. Scale bars, 30 μm in (A–E), 20 μm in (E’), 10 μm in (F,G). (H) Western blot analysis of homogenate (hom) and crude synaptosomal fraction (P2) prepared from a pool of 20–40 cerebral human organoids 35 days, 60 days and 70 days old. Proteins (20 μg) from each fraction were loaded in the gel and the western blot analysis was performed using antibodies specific for β-actin, SYP, and CSTB.