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Phosphoproteomic analysis of mouse neurons with SRPK2 overexpression or knockdown

ABSTRACT: SRPK2 was identified as a presynaptic protein kinase involved in the regulation of neurotransmitter release. The phosphoproteome of mouse primary neurons was screened, under the conditions of SRPK2 overexpression and SRPK2 knockdown, to identify candidate substrates of SRPK2.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Brain, Primary Neuron, Neuron Of Cerebral Cortex, Cell Culture

DISEASE(S): Neurological Dysfunction

SUBMITTER: Mark Graham

LAB HEAD: Mark Evan Graham

PROVIDER: PXD016636 | Pride | 2022-02-14

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	MaxQuant_txt_files.zip	Other
	Mus-uniprot-Iso18072017.fasta	Fasta
	SRPK2.zip	Other
	shSRPK2.zip	Other

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Publications

A presynaptic phosphosignaling hub for lasting homeostatic plasticity.

Müller Johannes Alexander JA Betzin Julia J Santos-Tejedor Jorge J Mayer Annika A Oprişoreanu Ana-Maria AM Engholm-Keller Kasper K Paulußen Isabelle I Gulakova Polina P McGovern Terrence Daniel TD Gschossman Lena Johanna LJ Schönhense Eva E Wark Jesse R JR Lamprecht Alf A Becker Albert J AJ Waardenberg Ashley J AJ Graham Mark E ME Dietrich Dirk D Schoch Susanne S

Cell reports 20220401 3

Stable function of networks requires that synapses adapt their strength to levels of neuronal activity, and failure to do so results in cognitive disorders. How such homeostatic regulation may be implemented in mammalian synapses remains poorly understood. Here we show that the phosphorylation status of several positions of the active-zone (AZ) protein RIM1 are relevant for synaptic glutamate release. Position RIMS1045 is necessary and sufficient for expression of silencing-induced homeostatic p ...[more]

PMID: 35443170

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Phosphoproteomic and proteomic analysis of early epileptogenesis in mice at 4 and 24 h post-status epilepticus

Project description:The phosphorylation-based signalling and protein changes occurring in the early phases after a pathophysiological insult, like status epilepticus, have not been detailed. To this end, the hippocampi of mice treated with pilocarpine and diazepam were examined by mass spectrometry at 4 and 24 h post-status epilepticus at vast depth using the tandem mass tag 11-plex system. In the accompanying article, the results implicated posttranscriptional regulatory proteins as early targets of increased phosphorylation. Also, the major targets of decreased phosphorylation at 4 h and 24 h were a subset of post synaptic density scaffold proteins, ion channels and neurotransmitter receptors. Many proteins targeted by dephosphorylation at 4 h also had decreased protein abundance at 24 h, indicating a phosphatase-mediated weakening of synapses.

2024-02-22 | PXD038241 | Pride

Proteomics and phosphoproteomics of the diurnal cycle in chow and high fat high sucrose feed rats

Project description:There is growing evidence that energy metabolism and insulin action are regulated by mechanisms that follow a diurnal rhythm and it has been proposed that defects in Akt signalling are associated with the pathophysiology of metabolic disease. It is therefore important to investigate these parameters under physiology of the free-living state. We therefore examined the insulin action in muscle of chow or high fat, high sucrose diet-fed (HFHS) rats during the normal diurnal cycle. HFHS animals displayed hyperinsulinemia, however had reduced systemic glucose disposal and impaired muscle glucose uptake during the feeding period. Proteomics and phosphoproteomics was performed over the diurnal cycle in chow and HFHS rats.

2019-05-20 | PXD010242 | Pride

The temporal profile of activity-dependent presynaptic phospho-signalling reveals long lasting patterns of post-stimulus regulation

Project description:Depolarization of presynaptic terminals stimulates calcium influx, which evokes neurotransmitter release and activates phosphorylation-based signalling. Here, we present the first global temporal profile of presynaptic activity-dependent phospho-signalling, which includes two KCl stimulation levels and analysis of the post-stimulus period. We profiled 1,917 regulated phosphopeptides and bioinformatically identified six temporal patterns of co-regulated proteins. The presynaptic proteins with large changes in phospho-status were again prominently regulated in the analysis of 7,070 activity-dependent phosphopeptides from KCl-stimulated cultured hippocampal neurons. Active zone scaffold proteins showed a high level of activity-dependent phospho-regulation that far exceeded the response from postsynaptic density scaffold proteins. Accordingly, bassoon was identified as the major target of neuronal phospho-signalling. We developed a probabilistic computational method, KinSwing, which matched protein kinase substrate motifs to regulated phosphorylation sites to reveal underlying protein kinase activity. This approach allowed us to link protein kinases to profiles of co-regulated presynaptic protein networks. CaMKIIα responded rapidly, scaled with stimulus strength and had long-lasting activity. MAPK/ERK was the main protein kinase predicted to control a distinct and significant pattern of post-stimulus up-regulation of phosphorylation. This work provides a unique resource of activity-dependent phosphorylation sites of synaptosomes and neurons, the vast majority of which have not been investigated with regard to their functional impact. This resource will enable detailed characterization of the phospho-regulated mechanisms impacting the plasticity of neurotransmitter release.

2019-01-28 | PXD010007 | Pride

Interferon stimulated changes in the brain phosphoproteome

Project description:Type I interferons (IFN-Is) are immunoregulatory cytokines that are essential for normal host antiviral responses. The current understanding is that IFN-Is mediate their effects through the expression of several hundred interferon-regulated genes. Here we identified a novel IFN-I response mechanism that relies on widespread changes in protein phosphorylation. IFN-I-induced phosphorylation in primary murine microglia and astrocytes – key IFN-I-responding cells of the central nervous system – was rapid and thus largely independent of gene expression. Based on in silico analysis, this mechanism relies predominantly on a small number of kinase families. Further, functional analysis suggested that this rapid response induces an immediate reactive state in cells and prepares them for the subsequent transcriptomic response. Similar extensive phosphoproteome changes were also present in a mouse model for IFN-I-induced neuroinflammatory diseases. Strikingly, the altered phosphoproteome in these transgenic mice predicted the clinical and pathological outcome of neuroinflammatory disease. These findings demonstrate for the first time a novel mechanism by which IFN-Is mediate cellular responses.

2021-11-02 | PXD014443 | Pride

The temporal profile of activity-dependent presynaptic phospho-signalling reveals a PP1-dependent reduction in vesicle release

Project description:Depolarization of presynaptic terminals stimulates calcium influx, which evokes neurotransmitter release and activates phosphorylation-based signalling. Here, we present the first global temporal profile of presynaptic proteins undergoing post-stimulus phospho-signalling to reveal long-lasting changes, key substrates and master regulators. A total of 5,715 unique phosphopeptides from 1,817 proteins were profiled and 1,917 phosphopeptides had activity-dependent phosphorylation sites. A new computational method, KinSwing, combining protein kinase substrate prediction and activity across time, enabled the prediction of effector protein kinase activity. CaMKII responded rapidly to depolarization. MAPK, CDK5 and GSK3β had a post-stimulus role in compensating for initial dephosphorylation. Despite this, the post-stimulus period was dominated by down-regulation of phosphorylation and was exacerbated by conditions stimulating increased calcium influx. Down-regulated phosphorylation correlated with perturbed phospho-signalling to protein phosphatase 1 (PP1) regulatory molecules and reduced glutamate and fluorescent dye release. Inhibition PP1 prevented reduction in dye release, identifying PP1 as a major signalling target and mediator of post-stimulus presynaptic phospho-signalling.

2019-01-24 | PXD008495 | Pride

Phosphoproteome of five-minute KCl-depolarised mouse cultured hippocampal neurons

Project description:Cultured hippocampal neurons from mice were treated with high concentration KCl for five minutes to depolarise the plasma membrane and model sustained brain activity. A high KCl condition was compared to low KCl using phosphoproteomics. Phosphopeptides were enriched from trypsin digested bio-replicates of the high and low KCl conditions and analysed by mass spectrometry. Approximately 20,000 phosphopeptides were identified enabling a comparison of the relative level of phosphorylation in response to depolarisation.

2023-10-15 | PXD020232 | Pride

An integrated omics approach reveals synaptic plasticity changes in an APP/PS1 Alzheimer´s mouse model

Project description:Synaptic dysfunction occurs early in Alzheimer´s disease. However, efforts to protect against these detriments have not been possible due to the inadequate understanding of synaptic signalling pathways involved in the neuropathological disease process. The aim of this study was to elucidate the molecular mechanisms behind Alzheimer´s and synaptic plasticity-related signalling using a multi-omics and in situ imaging approach. We used an Alzheimer´s mouse model (APPswe/PSN1dE9, age of 12 month) (APP/PS1) and compared the changes on the proteome including global phosphorylation and N-linked glycosylation pattern, pathway-focused transcriptome and neurological disease-associated miRNAome with age-matched control mice in the neocortex, hippocampus, olfactory bulb and brainstem. Our analysis showed that signalling pathways related to synaptic functions associated to dendritic spine morphology, neurite outgrowth, long-term potentiaiton, CREB signalling and cytoskeletal dynamics were altered in the APP/PS1 transgenic mice, particular in the neocortex and olfactory bulb. This was associated with Aβ plaques and neurofibrillary tangle formation as well as microglial clustering in all brain regions except brainstem. The responses may be epigenetically modulated by the interaction with a number of miRNAs. We suggest that the alterations in synaptic plasticity-related signalling are associated to neurocognitive dysfunctions that resemble the situtation in human AD patients.

2016-05-11 | PXD003312 | Pride

db/db - Proteomic Analysis of Restored Insulin Production and Trafficking in Obese Diabetic Mouse Pancreatic Islets Following Euglycemia

Project description:the phosphoproteomic landscape of beta-cell adaptive flexibility after exposure to glucose

2019-07-22 | PXD010265 | Pride

Combined assessment of global proteome, phosphoproteome and N-terminal protein cleavage to characterize altered platelet functions in human Scott syndrome

Project description:The Scott syndrome is a rare bleeding disorder associated with a mutation in the gene encoding anoctamin-6 (TMEM16F). After stimulation of Ca2+-mobilizing agonists, syndromatic platelets show a reduced phosphatidylserine exposure and do not form membrane blebs. Given the central role of anoctamin-6 in the platelet procoagulant response, we used quantitative proteomics to understand the underlying molecular mechanisms and the complex phenotypic changes in Scott platelets compared to control platelets. Therefore, we applied an iTRAQ-based multi-pronged strategy to quantify changes in (i) the global proteome, (ii) the phosphoproteome and (iii) proteolytic events between resting and stimulated Scott and control platelets. Our data indicate a limited number of proteins with decreased (70) or increased (64) expression in Scott platelets, among those we observed the absence of anoctamin-6 and the strong up-regulation of aquaporin-1. Furthermore, the quantification of 1,566 phosphopeptides revealed major differences between Scott and control platelets after stimulation with thrombin/convulxin or ionomycin. Finally, we quantified 1,596 N-terminal peptides in activated Scott and control platelets, 180 of which we identified as calpain-regulated, whereas a distinct set of 23 neo-N-termini was caspase-regulated. In Scott platelets, calpain-induced cleavage of cytoskeleton-linked and signaling proteins was down-regulated, in accordance with an increased phosphorylation state. Thus, multi-pronged proteomic profiling of Scott platelets provides detailed insight into their protection against detrimental Ca2+-dependent changes that are normally associated with phosphatidylserine exposure.

2016-08-24 | PXD002883 | Pride

Chronic low-dose ionising radiation rates affect the hippocampal phospho-proteome of synaptogenesis in an ApoE-/--Alzheimer mouse model over 300 days of exposure

Project description:Cognitive impairments are an important side effect after radiation exposure. Yet, it comes more and more into scientific focus that ionising radiation can resemble pathologies of neurodegenerative diseases such as Alzheimer´s. Here, we used a multidisciplinary approach to elucidate the effect of chronic low-dose irradiation exposure (1 mGy/day and 20 mGy/day) over 300 days (cumulative dose of 0.3 Gy and 6.0 Gy) on the murine ApoE-/- hippocampus as an Alzheimer´s model. By using mass spectrometry to quantify global expression levels of unmodified proteins, phospho-proteins and N-linked sialylated glycoproteins, we found nearly exclusive changes on the phospho-proteome at both doses. These proteins regulate signalling of synaptic plasticity and calcium-dependent signalling. We used synaptic plasticity-targeted transcriptomics, immunoblotting and ELISA to validate our findings and could identify that memory-related CREB signalling is reduced at both doses whereas Rac1-Cofilin signalling is only altered at 1 mGy/day. Interestingly, we noted a reduction in the number of activated microglia in the molecular layer of the hippocampus only at 1 mGy/day paralleled with reduced levels of TNFα mRNA and lipid peroxidation at this dose. Adult neurogenesis (Ki67, GFAP and NeuN as markers) and cell death (activated caspase-3) were not changed / initiated at both doses. Our analysis showed that several molecular targets induced by chronic low-dose radiation overlap with molecular targets of Alzheimer´s pathology. We suggest that chronic low-dose ionising radiation can be a potential confounder in Alzheimer´s disease.

2017-07-25 | PXD003969 | Pride