scholarly journals SerThr-PhosphoProteome of Brain from Aged PINK1-KO+A53T-SNCA Mice Reveals pT1928-MAP1B and pS3781-ANK2 Deficits, as Hub between Autophagy and Synapse Changes

2019 ◽  
Vol 20 (13) ◽  
pp. 3284 ◽  
Author(s):  
Georg Auburger ◽  
Suzana Gispert ◽  
Sylvia Torres-Odio ◽  
Marina Jendrach ◽  
Nadine Brehm ◽  
...  
Keyword(s):  
Posttranslational Modifications ◽  
Alpha Synuclein ◽  
Synaptic Function ◽  
Label Free ◽  
Similar Reduction ◽  
C2 Domains ◽  
Brain Hemispheres ◽  
Locomotor Deficits ◽  
As Stress ◽  
Transmission Factors

Hereditary Parkinson’s disease (PD) can be triggered by an autosomal dominant overdose of alpha-Synuclein (SNCA) as stressor or the autosomal recessive deficiency of PINK1 Serine/Threonine-phosphorylation activity as stress-response. We demonstrated the combination of PINK1-knockout with overexpression of SNCAA53T in double mutant (DM) mice to exacerbate locomotor deficits and to reduce lifespan. To survey posttranslational modifications of proteins underlying the pathology, brain hemispheres of old DM mice underwent quantitative label-free global proteomic mass spectrometry, focused on Ser/Thr-phosphorylations. As an exceptionally strong effect, we detected >300-fold reductions of phosphoThr1928 in MAP1B, a microtubule-associated protein, and a similar reduction of phosphoSer3781 in ANK2, an interactor of microtubules. MAP1B depletion is known to trigger perturbations of microtubular mitochondria trafficking, neurite extension, and synaptic function, so it was noteworthy that relevantly decreased phosphorylation was also detected for other microtubule and microfilament factors, namely MAP2S1801, MARK1S394, MAP1AT1794, KIF1AS1537, 4.1NS541, 4.1GS86, and ADD2S528. While the MAP1B heavy chain supports regeneration and growth cones, its light chain assists DAPK1-mediated autophagy. Interestingly, relevant phosphorylation decreases of DAPK2S299, VPS13DS2429, and VPS13CS2480 in the DM brain affected regulators of autophagy, which are implicated in PD. Overall, significant downregulations were enriched for PFAM C2 domains, other kinases, and synaptic transmission factors upon automated bioinformatics, while upregulations were not enriched for selective motifs or pathways. Validation experiments confirmed the change of LC3 processing as reflection of excessive autophagy in DM brain, and dependence of ANK2/MAP1B expression on PINK1 levels. Our new data provide independent confirmation in a mouse model with combined PARK1/PARK4/PARK6 pathology that MAP1B/ANK2 phosphorylation events are implicated in Parkinsonian neurodegeneration. These findings expand on previous observations in Drosophila melanogaster that the MAP1B ortholog futsch in the presynapse is a primary target of the PARK8 protein LRRK2, and on a report that MAP1B is a component of the pathological Lewy body aggregates in PD patient brains. Similarly, ANK2 gene locus variants are associated with the risk of PD, ANK2 interacts with PINK1/Parkin-target proteins such as MIRO1 or ATP1A2, and ANK2-derived peptides are potent inhibitors of autophagy.

scholarly journals SerThr-PhosphoProteome of Brain from Aged PINK1-KO+A53T-SNCA Mice Reveals pT1928-MAP1B and pS3781-ANK2 Deficits, as Hub between Autophagy and Synapse Changes

2019 ◽  
Author(s):  
Georg Auburger ◽  
Suzana Gispert ◽  
Sylvia Torres-Odio ◽  
Marina Jendrach ◽  
Nadine Brehm ◽  
...  
Keyword(s):  
Posttranslational Modifications ◽  
Alpha Synuclein ◽  
Synaptic Function ◽  
Label Free ◽  
Similar Reduction ◽  
C2 Domains ◽  
Brain Hemispheres ◽  
Locomotor Deficits ◽  
As Stress ◽  
Transmission Factors

Hereditary Parkinson’s disease (PD) can be triggered by an autosomal dominant overdose of alpha-Synuclein (SNCA) as stressor or the autosomal recessive deficiency of PINK1 Serine/Threonine-phosphorylation activity as stress-response. We demonstrated the combination of PINK1-knockout with overexpression of SNCAA53T in double mutant (DM) mice to exacerbate locomotor deficits and to reduce lifespan. To survey posttranslational modifications of proteins underlying the pathology, brain hemispheres of old DM mice underwent quantitative label-free global proteomic mass spectrometry, focused on Ser/Thr-phosphorylations. As exceptionally strong effect, we detected >300-fold reductions of phosphoThr1928 in MAP1B, a microtubule-associated protein, and a similar reduction of phosphoSer3781 in ANK2, an interactor of microtubules. MAP1B depletion is known to trigger perturbations of microtubular mitochondria trafficking, neurite extension and synaptic function, so it was noteworthy that relevantly decreased phosphorylation was detected also for other microtubule and microfilament factors, namely MAP2S1801, MARK1S394, MAP1AT1794, KIF1AS1537, 4.1NS541, 4.1GS86 and ADD2S528. While the MAP1B heavy chain supports regeneration and growth cones, its light-chain assists DAPK1-mediated autophagy. Interestingly, relevant phosphorylation decreases of DAPK2S299, VPS13DS2429 and VPS13CS2480 in the DM brain affected regulators of autophagy, which are implicated in PD. Overall, significant downregulations were enriched for PFAM C2 domains, other kinases, and synaptic transmission factors upon automated bioinformatics, while upregulations were not enriched for selective motifs or pathways. Validation experiments confirmed the change of LC3 processing as reflection of excessive autophagy in DM brain, and dependence of ANK2/MAP1B expression on PINK1 levels. Our new data provide independent confirmation in a mouse model with combined PARK1/PARK4/PARK6 pathology that MAP1B/ANK2 phosphorylation events are implicated in Parkinsonian neurodegeneration. These findings expand on previous observations in D. melanogaster that the MAP1B ortholog futsch in the presynapse is a primary target of the PARK8 protein LRRK2, and on a report that MAP1B is a component of the pathological Lewy body aggregates in PD patient brains. Similarly, ANK2 gene locus variants are associated with the risk of PD, ANK2 interacts with PINK1/Parkin-target proteins such as MIRO1 or ATP1A2, and ANK2-derived peptides are potent inhibitors of autophagy.

scholarly journals Methyl-Arginine Profile of Brain from Aged PINK1-KO+A53T-SNCA Mice Suggests Altered Mitochondrial Biogenesis

2016 ◽  
Vol 2016 ◽  
pp. 1-13 ◽  
Author(s):  
Georg Auburger ◽  
Suzana Gispert ◽  
Nadine Brehm
Keyword(s):  
Mitochondrial Biogenesis ◽  
Posttranslational Modifications ◽  
Stress Responses ◽  
Autosomal Recessive ◽  
Oxygen Sensor ◽  
Alpha Synuclein ◽  
Label Free ◽  
Brain Hemispheres ◽  
Mrna Decapping ◽  
Dopaminergic Differentiation

Hereditary Parkinson’s disease can be triggered by an autosomal dominant overdose of alpha-Synuclein (SNCA) or the autosomal recessive deficiency of PINK1. We recently showed that the combination of PINK1-knockout with overexpression of A53T-SNCA in double mutant (DM) mice potentiates phenotypes and reduces survival. Now we studied brain hemispheres of DM mice at age of 18 months in a hypothesis-free approach, employing a quantitative label-free global proteomic mass spectrometry scan of posttranslational modifications focusing on methyl-arginine. The strongest effects were documented for the adhesion modulator CMAS, the mRNA decapping/deadenylation factor PATL1, and the synaptic plasticity mediator CRTC1/TORC1. In addition, an intriguing effect was observed for the splicing factor PSF/SFPQ, known to interact with the dopaminergic differentiation factor NURR1 as well as with DJ-1, the protein responsible for the autosomal recessive PARK7 variant of PD. CRTC1, PSF, and DJ-1 are modulators of PGC1alpha and of mitochondrial biogenesis. This pathway was further stressed by dysregulations of oxygen sensor EGLN3 and of nuclear TMPO. PSF and TMPO cooperate with dopaminergic differentiation factors LMX1B and NURR1. Further dysregulations concerned PRR18, TRIO, HNRNPA1, DMWD, WAVE1, ILDR2, DBNDD1, and NFM. Thus, we report selective novel endogenous stress responses in brain, which highlight early dysregulations of mitochondrial homeostasis and midbrain vulnerability.

scholarly journals Development of a Robust High-Throughput Screening Platform for Inhibitors of the Striatal-Enriched Tyrosine Phosphatase (STEP)

2021 ◽  
Vol 22 (9) ◽  
pp. 4417
Author(s):  
Lester J Lambert ◽  
Stefan Grotegut ◽  
Maria Celeridad ◽  
Palak Gosalia ◽  
Laurent JS De Backer ◽  
...  
Keyword(s):  
Drug Discovery ◽  
High Throughput ◽  
Tyrosine Kinases ◽  
High Throughput Screening ◽  
Kinase Inhibitors ◽  
Tyrosine Phosphatase ◽  
Protein Tyrosine Phosphatases ◽  
Synaptic Function ◽  
Label Free ◽  
Protein Tyrosine

Many human diseases are the result of abnormal expression or activation of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). Not surprisingly, more than 30 tyrosine kinase inhibitors (TKIs) are currently in clinical use and provide unique treatment options for many patients. PTPs on the other hand have long been regarded as “undruggable” and only recently have gained increased attention in drug discovery. Striatal-enriched tyrosine phosphatase (STEP) is a neuron-specific PTP that is overactive in Alzheimer’s disease (AD) and other neurodegenerative and neuropsychiatric disorders, including Parkinson’s disease, schizophrenia, and fragile X syndrome. An emergent model suggests that the increase in STEP activity interferes with synaptic function and contributes to the characteristic cognitive and behavioral deficits present in these diseases. Prior efforts to generate STEP inhibitors with properties that warrant clinical development have largely failed. To identify novel STEP inhibitor scaffolds, we developed a biophysical, label-free high-throughput screening (HTS) platform based on the protein thermal shift (PTS) technology. In contrast to conventional HTS using STEP enzymatic assays, we found the PTS platform highly robust and capable of identifying true hits with confirmed STEP inhibitory activity and selectivity. This new platform promises to greatly advance STEP drug discovery and should be applicable to other PTP targets.

scholarly journals Alterations in Sub-Axonal Architecture Between Normal Aging and Parkinson’s Diseased Human Brains Using Label-Free Cryogenic X-ray Nanotomography

2020 ◽  
Vol 14 ◽  
Author(s):  
Hung Tri Tran ◽  
Esther H. R. Tsai ◽  
Amanda J. Lewis ◽  
Tim Moors ◽  
J. G. J. M. Bol ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Human Brain ◽  
Brain Tissue ◽  
Multispectral Imaging ◽  
Alpha Synuclein ◽  
Label Free ◽  
Myelinated Axons ◽  
X Ray ◽  
X Ray Imaging ◽  
Non Destructive

Gaining insight to pathologically relevant processes in continuous volumes of unstained brain tissue is important for a better understanding of neurological diseases. Many pathological processes in neurodegenerative disorders affect myelinated axons, which are a critical part of the neuronal circuitry. Cryo ptychographic X-ray computed tomography in the multi-keV energy range is an emerging technology providing phase contrast at high sensitivity, allowing label-free and non-destructive three dimensional imaging of large continuous volumes of tissue, currently spanning up to 400,000 μm3. This aspect makes the technique especially attractive for imaging complex biological material, especially neuronal tissues, in combination with downstream optical or electron microscopy techniques. A further advantage is that dehydration, additional contrast staining, and destructive sectioning/milling are not required for imaging. We have developed a pipeline for cryo ptychographic X-ray tomography of relatively large, hydrated and unstained biological tissue volumes beyond what is typical for the X-ray imaging, using human brain tissue and combining the technique with complementary methods. We present four imaged volumes of a Parkinson’s diseased human brain and five volumes from a non-diseased control human brain using cryo ptychographic X-ray tomography. In both cases, we distinguish neuromelanin-containing neurons, lipid and melanic pigment, blood vessels and red blood cells, and nuclei of other brain cells. In the diseased sample, we observed several swellings containing dense granular material resembling clustered vesicles between the myelin sheaths arising from the cytoplasm of the parent oligodendrocyte, rather than the axoplasm. We further investigated the pathological relevance of such swollen axons in adjacent tissue sections by immunofluorescence microscopy for phosphorylated alpha-synuclein combined with multispectral imaging. Since cryo ptychographic X-ray tomography is non-destructive, the large dataset volumes were used to guide further investigation of such swollen axons by correlative electron microscopy and immunogold labeling post X-ray imaging, a possibility demonstrated for the first time. Interestingly, we find that protein antigenicity and ultrastructure of the tissue are preserved after the X-ray measurement. As many pathological processes in neurodegeneration affect myelinated axons, our work sets an unprecedented foundation for studies addressing axonal integrity and disease-related changes in unstained brain tissues.

scholarly journals Subcellular orchestration of alpha-synuclein variants in Parkinson’s disease brains revealed by 3D multicolor STED microscopy

2018 ◽  
Author(s):  
Tim E. Moors ◽  
Christina A. Maat ◽  
Daniel Niedieker ◽  
Daniel Mona ◽  
Dennis Petersen ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Lewy Bodies ◽  
Distribution Patterns ◽  
Alpha Synuclein ◽  
Label Free ◽  
Post Translational Modifications ◽  
Sted Microscopy ◽  
C Terminus

AbstractPost-translational modifications of alpha-synuclein (aSyn), particularly phosphorylation at Serine 129 (Ser129-p) and truncation of its C-terminus (CTT), have been implicated in Parkinson’s disease (PD) pathology. To gain more insight in the relevance of Ser129-p and CTT aSyn under physiological and pathological conditions, we investigated their subcellular distribution patterns in normal aged and PD brains using highly-selective antibodies in combination with 3D multicolor STED microscopy. We show that CTT aSyn localizes in mitochondria in PD patients and controls, whereas the organization of Ser129-p in a cytoplasmic network is strongly associated with pathology. Nigral Lewy bodies show an onion skin-like architecture, with a structured framework of Ser129-p aSyn and neurofilaments encapsulating CTT aSyn in their core, which displayed high content of proteins and lipids by label-free CARS microscopy. The subcellular phenotypes of antibody-labeled pathology identified in this study provide evidence for a crucial role of Ser129-p aSyn in Lewy body formation.

scholarly journals Protein Prenylation in Plant Stress Responses

Molecules ◽  
2019 ◽  
Vol 24 (21) ◽  
pp. 3906 ◽  
Author(s):  
Michal Hála ◽  
Viktor Žárský
Keyword(s):  
Posttranslational Modifications ◽  
Stress Responses ◽  
Lipid Membrane ◽  
Plant Stress ◽  
Small Gtpases ◽  
Plant Responses ◽  
Protein Prenylation ◽  
Biotic Stresses ◽  
Plant Stress Responses ◽  
As Stress

Protein prenylation is one of the most important posttranslational modifications of proteins. Prenylated proteins play important roles in different developmental processes as well as stress responses in plants as the addition of hydrophobic prenyl chains (mostly farnesyl or geranyl) allow otherwise hydrophilic proteins to operate as peripheral lipid membrane proteins. This review focuses on selected aspects connecting protein prenylation with plant responses to both abiotic and biotic stresses. It summarizes how changes in protein prenylation impact plant growth, deals with several families of proteins involved in stress response and highlights prominent regulatory importance of prenylated small GTPases and chaperons. Potential possibilities of these proteins to be applicable for biotechnologies are discussed.

scholarly journals Stress-induced Changes in the S-palmitoylation and S-nitrosylation of Synaptic Proteins

2019 ◽  
Vol 18 (10) ◽  
pp. 1916-1938 ◽  
Author(s):  
Monika Zareba-Koziol ◽  
Anna Bartkowiak-Kaczmarek ◽  
Izabela Figiel ◽  
Adam Krzystyniak ◽  
Tomasz Wojtowicz ◽  
...  
Keyword(s):  
Chronic Stress ◽  
Posttranslational Modifications ◽  
Protein Localization ◽  
Network Connectivity ◽  
Covalent Modification ◽  
Synaptic Proteins ◽  
Postsynaptic Density Proteins ◽  
Protein Functions ◽  
Reversible Covalent ◽  
As Stress

The precise regulation of synaptic integrity is critical for neuronal network connectivity and proper brain function. Essential aspects of the activity and localization of synaptic proteins are regulated by posttranslational modifications. S-palmitoylation is a reversible covalent modification of the cysteine with palmitate. It modulates affinity of the protein for cell membranes and membranous compartments. Intracellular palmitoylation dynamics are regulated by crosstalk with other posttranslational modifications, such as S-nitrosylation. S-nitrosylation is a covalent modification of cysteine thiol by nitric oxide and can modulate protein functions. Therefore, simultaneous identification of endogenous site-specific proteomes of both cysteine modifications under certain biological conditions offers new insights into the regulation of functional pathways. Still unclear, however, are the ways in which this crosstalk is affected in brain pathology, such as stress-related disorders. Using a newly developed mass spectrometry-based approach Palmitoylation And Nitrosylation Interplay Monitoring (PANIMoni), we analyzed the endogenous S-palmitoylation and S-nitrosylation of postsynaptic density proteins at the level of specific single cysteine in a mouse model of chronic stress. Among a total of 813 S-PALM and 620 S-NO cysteine sites that were characterized on 465 and 360 proteins, respectively, we sought to identify those that were differentially affected by stress. Our data show involvement of S-palmitoylation and S-nitrosylation crosstalk in the regulation of 122 proteins including receptors, scaffolding proteins, regulatory proteins and cytoskeletal components. Our results suggest that atypical crosstalk between the S-palmitoylation and S-nitrosylation interplay of proteins involved in synaptic transmission, protein localization and regulation of synaptic plasticity might be one of the main events associated with chronic stress disorder, leading to destabilization in synaptic networks.

scholarly journals Label-Free Quantitation and Mapping of the ErbB2 Tumor Receptor by Multiple Protease Digestion with Data-Dependent (MS1) and Data-Independent (MS2) Acquisitions

2013 ◽  
Vol 2013 ◽  
pp. 1-11 ◽  
Author(s):  
Jason M. Held ◽  
Birgit Schilling ◽  
Alexandria K. D'Souza ◽  
Tara Srinivasan ◽  
Jessica B. Behring ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Resolution ◽  
Posttranslational Modifications ◽  
Development Time ◽  
Assay Development ◽  
Selected Reaction Monitoring ◽  
Label Free ◽  
Data Independent Acquisition ◽  
Key Indicator ◽  
Biomarker Quantitation

The receptor tyrosine kinase ErbB2 is a breast cancer biomarker whose posttranslational modifications (PTMs) are a key indicator of its activation. Quantifying the expression and PTMs of biomarkers such as ErbB2 by selected reaction monitoring (SRM) mass spectrometry has several limitations, including minimal coverage and extensive assay development time. Therefore, we assessed the utility of two high resolution, full scan mass spectrometry approaches, MS1 Filtering and SWATH MS2, for targeted ErbB2 proteomics. Endogenous ErbB2 immunoprecipitated from SK-BR-3 cells was in-gel digested with trypsin, chymotrypsin, Asp-N, or trypsin plus Asp-N in triplicate. Data-dependent acquisition with an AB SCIEX TripleTOF 5600 and MS1 Filtering data processing was used to assess peptide and PTM coverage as well as the reproducibility of enzyme digestion. Data-independent acquisition (SWATH) was also performed for MS2 quantitation. MS1 Filtering and SWATH MS2 allow quantitation of all detected analytes after acquisition, enabling the use of multiple proteases for quantitative assessment of target proteins. Combining high resolution proteomics with multiprotease digestion enabled quantitative mapping of ErbB2 with excellent reproducibility, improved amino acid sequence and PTM coverage, and decreased assay development time compared to typical SRM assays. These results demonstrate that high resolution quantitative proteomic approaches are an effective tool for targeted biomarker quantitation.

The influence of glucocorticoids on neuronal survival and synaptic function

2012 ◽  
Vol 3 (6) ◽  
pp. 495-504 ◽  
Author(s):  
Tadahiro Numakawa ◽  
Naoki Adachi ◽  
Misty Richards ◽  
Shuichi Chiba ◽  
Hiroshi Kunugi
Keyword(s):  
Current Knowledge ◽  
Neuronal Survival ◽  
Synaptic Function ◽  
Multiple Roles ◽  
Neuronal Populations ◽  
Animal Models Of Depression ◽  
The Central Nervous System ◽  
As Stress ◽  
And Function ◽  
The Impact

AbstractGlucocorticoids, recognized as stress-related steroid hormones secreted from adrenal glands, have multiple roles in brain function. The concentration of glucocorticoids is regulated by the hypothalamic-pituitary-adrenal axis, and chronically elevated levels of glucocorticoids are putatively involved in the pathophysiology of mental disorders, such as depression. As corticosteroids are also widely used as medical drugs (e.g., for chronic lung disease in infants), the developmental influence of glucocorticoids on neuronal survival and synaptic plasticity is a critical concern. Although many reports suggest a biological effect of glucocorticoids on neuronal populations of the central nervous system (CNS), some reports suggest a possibility that glial responses (including regulation of neurotrophic factor expression) to glucocorticoids are different from that of neurons. In the present review, we show an overview of the current knowledge concerning the impact of glucocorticoids on behavior in animal models of depression, and on cell survival and function in the CNS.

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