The Effect of Sleep Deprivation and Subsequent Recovery Period on the Synaptic Proteome of Rat Cerebral Cortex

Sleep deprivation (SD) is commonplace in the modern way of life and has a substantial social, medical, and human cost. Sleep deprivation induces cognitive impairment such as loss of executive attention, working memory decline, poor emotion regulation, increased reaction times, and higher cognitive functions are particularly vulnerable to sleep loss. Furthermore, SD is associated with obesity, diabetes, cardiovascular diseases, cancer, and a vast majority of psychiatric and neurodegenerative disorders are accompanied by sleep disturbances. Despite the widespread scientific interest in the effect of sleep loss on synaptic function, there is a lack of investigation focusing on synaptic transmission on the proteome level. In the present study, we report the effects of SD and recovery period (RP) on the cortical synaptic proteome in rats. Synaptosomes were isolated after 8 h of SD performed by gentle handling and after 16 h of RP. The purity of synaptosome fraction was validated with western blot and electron microscopy, and the protein abundance alterations were analyzed by mass spectrometry. We observed that SD and RP have a wide impact on neurotransmitter-related proteins at both the presynaptic and postsynaptic membranes. The abundance of synaptic proteins has changed to a greater extent in consequence of SD than during RP: we identified 78 proteins with altered abundance after SD and 39 proteins after the course of RP. Levels of most of the altered proteins were upregulated during SD, while RP showed the opposite tendency, and three proteins (Gabbr1, Anks1b, and Decr1) showed abundance changes with opposite direction after SD and RP. The functional cluster analysis revealed that a majority of the altered proteins is related to signal transduction and regulation, synaptic transmission and synaptic assembly, protein and ion transport, and lipid and fatty acid metabolism, while the interaction network analysis revealed several connections between the significantly altered proteins and the molecular processes of synaptic plasticity or sleep. Our proteomic data implies suppression of SNARE-mediated synaptic vesicle exocytosis and impaired endocytic processes after sleep deprivation. Both SD and RP altered GABA neurotransmission and affected protein synthesis, several regulatory processes and signaling pathways, energy homeostatic processes, and metabolic pathways. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12035-021-02699-x

Autophagy is Required for Spermatogonial Differentiation in the Drosophila Testis

Autophagy is a conserved, lysosome-dependent catabolic process of eukaryotic cells which is involved in cellular differentiation. Here, we studied its specific role in the differentiation of spermatogonial cells in the Drosophila testis. In the apical part of the Drosophila testis, there is a niche of germline stem cells (GSCs), which are connected to hub cells. Hub cells emit a ligand for bone morhphogenetic protein (BMP)-mediated signalling that represses Bam (bag of marbles) expression in GSCs to maintain them in an undifferentiated state. GSCs divide asymmetrically, and one of the daughter cells differentiates into a gonialblast, which eventually generates a cluster of spermatogonia (SG) by mitoses. Bam is active in SG, and defects in Bam function arrest these cells at mitosis. We show that BMP signalling represses autophagy in GSCs, but upregulates the process in SG. Inhibiting autophagy in SG results in an overproliferating phenotype similar to that caused by bam mutations. Furthermore, Bam deficiency leads to a failure in downstream mechanisms of the autophagic breakdown. These results suggest that the BMP-Bam signalling axis regulates developmental autophagy in the Drosophila testis, and that acidic breakdown of cellular materials is required for spermatogonial differentiation

Chronic Cerebral Hypoperfusion Induced Synaptic Proteome Changes in the rat Cerebral Cortex

Chronic cerebral hypoperfusion (CCH) evokes mild cognitive impairment (MCI) and contributes to the progression of vascular dementia and Alzheimer’s disease (AD). How CCH induces these neurodegenerative processes that may spread along the synaptic network and whether they are detectable at the synaptic proteome level of the cerebral cortex remains to be established. In the present study, we report the synaptic protein changes in the cerebral cortex after stepwise bilateral common carotid artery occlusion (BCCAO) induced CCH in the rat. The occlusions were confirmed with magnetic resonance angiography 5 weeks after the surgery. Synaptosome fractions were prepared using sucrose gradient centrifugation from cerebral cortex dissected 7 weeks after the occlusion. The synaptic protein differences between the sham operated and CCH groups were analyzed with label-free nanoUHPLC-MS/MS. We identified 46 proteins showing altered abundance due to CCH. In particular, synaptic protein and lipid metabolism, as well as GABA shunt-related proteins showed increased while neurotransmission and synaptic assembly-related proteins showed decreased protein level changes in CCH rats. Protein network analysis of CCH-induced protein alterations suggested the importance of increased synaptic apolipoprotein E (APOE) level as a consequence of CCH. Therefore, the change in APOE level was confirmed with Western blotting. The identified synaptic protein changes would precede the onset of dementia-like symptoms in the CCH model, suggesting their importance in the development of vascular dementia. © 2017 Springer Science+Business Media New Yor

Sex-specific parenting and depression evoked by preoptic inhibitory neurons

Summary: The role of preoptic GABAergic inhibitory neurons was addressed in parenting, anxiety and depression. Pup exposure and forced swimming resulted in similar c-Fos activation pattern in neurons expressing vesicular GABA transporter in the preoptic area with generally stronger labeling and different distributional pattern in females than in males. Chemogenetic stimulation of preoptic GABAergic cells resulted in elevated maternal motivation and caring behavior in females and mothers but aggression toward pups in males. Behavioral effects were the opposite following inhibition of preoptic GABAergic neurons suggesting their physiological relevance. In addition, increased anxiety-like and depression-like behaviors were found following chemogenetic stimulation of the same neurons in females, whereas previous pup exposure increased only anxiety-like behavior suggesting that not the pups, but overstimulation of the cells can lead to depression-like behavior. A sexually dimorphic projection pattern of preoptic GABAergic neurons was also identified, which could mediate sex-dependent parenting and associated emotional behaviors

Proteomic comparison of different synaptosome preparation procedures

Synaptosomes are frequently used research objects in neurobiology studies focusing on synaptic transmission as they mimic several aspects of the physiological synaptic functions. They contain the whole apparatus for neurotransmission, the presynaptic nerve ending with synaptic vesicles, synaptic mitochondria and often a segment of the postsynaptic membrane along with the postsynaptic density is attached to its outer surface. As being artificial functional organelles, synaptosomes are viable for several hours, retain their activity, membrane potential, and capable to store, release, and reuptake neurotransmitters. Synaptosomes are ideal subjects for proteomic analysis. The recently available separation and protein detection techniques can cope with the reduced complexity of the organelle and enable the simultaneous qualitative and quantitative analysis of thousands of proteins shaping the structural and functional characteristics of the synapse. Synaptosomes are formed during the homogenization of nervous tissue in the isoosmotic milieu and can be isolated from the homogenate by various approaches. Each enrichment method has its own benefits and drawbacks and there is not a single method that is optimal for all research purposes. For a proper proteomic experiment, it is desirable to preserve the native synaptic structure during the isolation procedure and keep the degree of contamination from other organelles or cell types as low as possible. In this article, we examined five synaptosome isolation methods from a proteomic point of view by the means of electron microscopy, Western blot, and liquid chromatography-mass spectrometry to compare their efficiency in the isolation of synaptosomes and depletion of contaminating subcellular structures. In our study, the different isolation procedures led to a largely overlapping pool of proteins with a fairly similar distribution of presynaptic, active zone, synaptic vesicle, and postsynaptic proteins; however, discrete differences were noticeable in individual postsynaptic proteins and in the number of identified transmembrane proteins. Much pronounced variance was observed in the degree of contamination with mitochondrial and glial structures. Therefore, we suggest that in selecting the appropriate isolation method for any neuroproteomics experiment carried out on synaptosomes, the degree and sort/source of contamination should be considered as a primary aspect

Physiochemical and protein datasets related to citrus juice sac cells-derived nanovesicles and microvesicles

Qualitative and quantitative data obtained on micro and nanovesicle enriched fractions isolated from four citrus species, C. sinensis, C. limon, C. paradisi and C. aurantium are presented. It includes physiochemical characterization by transmission electron microscopy (TEM) and dynamic laser scattering (DLS); and molecular characterization of the biocargo of citrus vesicles by quantitative label-free proteomics. Vesicular transport related proteins of C. sinensis were predicted by (i) finding orthologues based on previously described vesicular transport proteins and (ii) GO term enrichment analysis. Based on the protein content different types of intra and intercellular vesicles were dissected and the distribution of different Enzyme classes (ECs) were determined. This data article is related to “Protein biocargo of citrus fruit-derived vesicles reveals heterogeneous transport and extracellular vesicle populations” (Pocsfalvi et al., 2018)

Protein biocargo of citrus fruit-derived vesicles reveals heterogeneous transport and extracellular vesicle populations

Cell-derived vesicles are membrane-enclosed organelles that transport material inside and outside the cell. Plant-derived vesicles are receiving more and more attention due to their potential as nanovectors for the delivery of biologically active substances. Here, we studied the heterogeneity and protein biocargo in citrus fruit juice sac cell-derived vesicles populations. Micro- and nano-sized vesicle fractions were isolated from four citrus species, C. sinensis, C. limon, C. paradisi and C. aurantium, characterized using physicochemical methods and protein cargos were compared using label-free quantitative shotgun proteomics. In each sample approximately 600–800 proteins were identified. Orthologues of most of the top-ranking proteins have previously been reported in extracellular vesicles of mammalian origin. High expression levels of patellin-3-like, clathrin heavy chain, heat shock proteins, 14-3-3 protein, glyceraldehyde-3-phosphate dehydrogenase and fructose-bisphosphate aldolase 6 were measured in all samples while aquaporin was highly expressed only in the nanovesicle fractions. Bioinformatics revealed more than hundred protein orthologues potentially implicated in vesicular trafficking. In particular, the presence of CCV, COPI and COPII coat proteins indicates the presence of heterogeneous populations of intracellular transport vesicles. Moreover, a high number of different enzymes including hydrolases and oxidoreductases are ubiquities in citrus fruit sac cell-derived vesicles

Intracellular processing of disease-associated α-synuclein in the human brain suggests prion-like cell-to-cell spread

Dementia with Lewy bodies (DLB), Parkinson’s disease (PD) and multiple system atrophy are characterized by the deposition of disease-associated α-synuclein. In the present study we 1) examined the molecular specificity of the novel anti-α-synuclein 5G4 antibody; 2) evaluated immunoreactivity patterns and their correlation in human brain tissue with micro- and astrogliosis in 57 cases with PD or DLB; and 3) performed a systematic immunoelectron microscopical mapping of subcellular localizations. 5G4 strongly binds to the high molecular weight fraction of β-sheet rich oligomers, while no binding to primarily disordered oligomers or monomers was observed. We show novel localizations of disease-associated α-synuclein including perivascular macrophages, ependyma and cranial nerves. α-Synuclein immunoreactive neuropil dots and thin threads associate more with glial reaction than Lewy bodies alone. Astrocytic α-synuclein is an important component of the pathology. Furthermore, we document ultrastructurally the pathway of processing of disease-associated α-synuclein within neurons and astroglial cells. Interaction of mitochondria and disease-associated α-synuclein plays a key role in the molecular–structural cytopathogenesis of disorders with Lewy bodies. We conclude that 1) the 5G4 antibody has strong selectivity for β-sheet rich α-synuclein oligomers; 2) Lewy bodies themselves are not the most relevant morphological substrate that evokes tissue lesioning; 3) both neurons and astrocytes internalize disease-associated α-synuclein in the human brain, suggesting prion-like cell-to-cell spread of α-synuclein by uptake from surrounding structures, as shown previously in experimental observations