Post-Translational Protein Modifications involved in Exo- and Endocytosis of Synaptic Vesicles

Neurotransmitter release is a key step that enables information flow between the pre- and post-synapse. However, regulation of the neurotransmitter release remains an intricate and widely unexplored matter despite recent advances in the understanding of the neurotransmitter release machinery and the analysis of the synaptic proteome and protein modifications. Indeed, post-translational protein modifications such as phosphorylation are suitable to quickly fine-tune the neurotransmitter release “in place” via affecting tertiary protein structures and protein-protein interactions, and globally, via modulating signaling pathways. Here, the investigations were focused on the dependence of protein phosphorylation in synaptosomes on the synaptic vesicle (SV) cycling, determining kinase-substrate interactions, and modulatory effects of selected sites on exo- and endocytosis. The analysis of synaptic phosphoproteome was conducted using TiO2-based enrichment of phosphorylated peptides with subsequent chemical labeling by isobaric mass tags (TMT) and a mass spectrometry-based quantification. Synaptosomes were employed as a functional model of a synapse as they contain the required neurotransmitter release machinery and respond to stimulation. First, the applicability of electrical stimulation was tested. The field- stimulation evoked reproducible glutamate release that was significantly suppressed in the absence of Ca2+, though it remained uncertain, to which degree the release is governed by exocytosis. Therefore, another approach using a KCl-induced depolarization and treatment with botulinum neurotoxins (BoNTs) was used to identify phosphorylation events that depend on SV cycling. BoNTs cleave specifically SNARE proteins and thus block exocytosis and SV cycling, but do not impede Ca2+-influx evoked by the plasma membrane depolarization. Comparison of phosphorylation events in synaptosomes stimulated in the presence of Ca2+, EGTA (0 net Ca2+) or pre-treated with BoNTs identified sites that were differentially phosphorylated following BoNT treatment, i.e., SV-cycling-dependent sites, and sites that were differentially phosphorylated when comparing Ca and EGTA conditions, but did not change under BoNT treatment, i.e., primarily Ca2+-dependent sites. Further differential expression analysis revealed that BoNT-treatment mostly caused de-phosphorylation of synaptic proteins. A kinase-substrate analysis showed that >25% of BoNT-responsive sites are predicted MAPK substrates and 20% of primarily Ca2+-dependent sites are presumably regulated by CaMKII, which corroborates Ca2+- dependence of these phosphorylation events. SV-cycling-dependent phosphorylation sites on syntaxin-1 (T21/T23-Stx1), synaptobrevin (S75-Vamp2), and cannabinoid receptor-1 (S314/T322-Cnr1) were further investigated for their impact on exo- and endocytosis. In collaboration with Dr. Eugenio Fornasiero and Prof. Dr. Silvio O. Rizzoli, corresponding phosphomimetic and non-phosphorylatable variants of the proteins were expressed in cultured hippocampal neurons. Imaging of the pH-sensor pHluorine coupled to synaptobrevin-2 revealed that the expression of phosphomimetic and non-phosphorylatable sites affected exo- and endocytosis in neurons. This work is first to investigate the electrical stimulation in relation to the Ca2+-dependent neurotransmitter release and exocytosis in synaptosomes. It further provides a comprehensive draft of synaptosomal phosphoproteome and is first to demonstrate its global dependence on an active SV cycling. The analysis of cultured hippocampal neurons expressing non-phosphorylatable and phosphomimetic mutants of pre-synaptic proteins syntaxin-1, synaptobrevin-2, and cannabinoid receptor-1 further demonstrates that the identified SV-cycling-dependent sites affect exo- and endocytosis.2021-11-0

Proteomic analysis of the human hippocampus identifies neuronal pentraxin 1 (NPTX1) as synapto-axonal target in late-stage Parkinson's disease

Parkinson's disease (PD) affects a significant proportion of the population over the age of 60 years, and its prevalence is increasing. While symptomatic treatment is available for motor symptoms of PD, non-motor complications such as dementia result in diminished life quality for patients and are far more difficult to treat. In this study, we analyzed PD-associated alterations in the hippocampus of PD patients, since this brain region is strongly affected by PD dementia. We focused on synapses, analyzing the proteome of post-mortal hippocampal tissue from 16 PD cases and 14 control subjects by mass spectrometry. Whole tissue lysates and synaptosomal fractions were analyzed in parallel. Differential analysis combined with bioinformatic network analyses identified neuronal pentraxin 1 (NPTX1) to be significantly dysregulated in PD and interacting with proteins of the synaptic compartment. Modulation of NPTX1 protein levels in primary hippocampal neuron cultures validated its role in synapse morphology. Our analysis suggests that NPTX1 contributes to synaptic pathology in late-stage PD and represents a putative target for novel therapeutic strategies

Metabolic switch from fatty acid oxidation to glycolysis in knock‐in mouse model of Barth syndrome

Abstract Mitochondria are central for cellular metabolism and energy supply. Barth syndrome (BTHS) is a severe disorder, due to dysfunction of the mitochondrial cardiolipin acyl transferase tafazzin. Altered cardiolipin remodeling affects mitochondrial inner membrane organization and function of membrane proteins such as transporters and the oxidative phosphorylation (OXPHOS) system. Here, we describe a mouse model that carries a G197V exchange in tafazzin, corresponding to BTHS patients. TAZG197V mice recapitulate disease‐specific pathology including cardiac dysfunction and reduced oxidative phosphorylation. We show that mutant mitochondria display defective fatty acid‐driven oxidative phosphorylation due to reduced levels of carnitine palmitoyl transferases. A metabolic switch in ATP production from OXPHOS to glycolysis is apparent in mouse heart and patient iPSC cell‐derived cardiomyocytes. An increase in glycolytic ATP production inactivates AMPK causing altered metabolic signaling in TAZG197V. Treatment of mutant cells with AMPK activator reestablishes fatty acid‐driven OXPHOS and protects mice against cardiac dysfunction