Mouse Brain Proteome Research Articles

Molecular Signatures of Neurodegenerative Diseases Identified by Proteomic and Phosphoproteomic Analyses in Aging Mouse Brain

A central hallmark of neurodegenerative diseases is theirreversible accumulation of misfolded proteins in the brain by aberrant phosphorylation. Understanding the mechanisms underlying protein phosphorylation and its role in pathological protein aggregation within the context of aging is crucial for developing therapeutic strategies aimed at preventing or reversing such diseases. Here, we applied multi-protease digestion and quantitative mass spectrometry to compare and characterize dysregulated proteins and phosphosites in the mouse brain proteome using three different age groups: young-adult (3-4months), middle-age (10months), and old mice (19-21months). Proteins associated with senescence, neurodegeneration, inflammation, cell cycle regulation, the p53 hallmark pathway, and cytokine signaling showed significant age-dependent changes in abundances and level of phosphorylation. Several proteins implicated in Alzheimer's disease (AD) and Parkinson's disease (PD) including tau (Mapt), Nefh, and Dpysl2 (also known as Crmp2) were hyperphosphorylated in old mice brain suggesting their susceptibility to the diseases. Cdk5 and Gsk3b, which are known to phosphorylate Dpysl2 at multiple specific sites, had also increased phosphorylation levels in old mice suggesting a potential crosstalk between them to contribute to AD. Hapln2, which promotes α-synuclein aggregation in patients with PD, was one of the proteins with highest abundance in old mice. CD9, which regulates senescence through the PI3K-AKT-mTOR-p53 signaling was upregulated in old mice and its regulation was correlated with the activation of phosphorylated AKT1. Overall, the findings identify a significant association between aging and the dysregulation of proteins involved in various pathways linked to neurodegenerative diseases with potential therapeutic implications.

Proteomic dissection of vanishing white matter pathogenesis

Vanishing white matter (VWM) is a leukodystrophy caused by biallelic pathogenic variants in eukaryotic translation initiation factor 2B. To date, it remains unclear which factors contribute to VWM pathogenesis. Here, we investigated the basis of VWM pathogenesis using the 2b5ho mouse model. We first mapped the temporal proteome in the cerebellum, corpus callosum, cortex, and brainstem of 2b5ho and wild-type (WT) mice. Protein changes observed in 2b5ho mice were then cross-referenced with published proteomic datasets from VWM patient brain tissue to define alterations relevant to the human disease. By comparing 2b5ho mice with their region- and age-matched WT counterparts, we showed that the proteome in the cerebellum and cortex of 2b5ho mice was already dysregulated prior to pathology development, whereas proteome changes in the corpus callosum only occurred after pathology onset. Remarkably, protein changes in the brainstem were transient, indicating that a compensatory mechanism might occur in this region. Importantly, 2b5ho mouse brain proteome changes reflect features well-known in VWM. Comparison of the 2b5ho mouse and VWM patient brain proteomes revealed shared changes. These could represent changes that contribute to the disease or even drive its progression in patients. Taken together, we show that the 2b5ho mouse brain proteome is affected in a region- and time-dependent manner. We found that the 2b5ho mouse model partly replicates the human disease at the protein level, providing a resource to study aspects of VWM pathogenesis by highlighting alterations from early to late disease stages, and those that possibly drive disease progression.

Unbiased insights into the multiplicity of the CYP46A1 brain effects in 5XFAD mice treated with low dose-efavirenz

Cytochrome P450 46A1 (CYP46A1) is the CNS-specific cholesterol 24-hydroxylase that controls cholesterol elimination and turnover in the brain. In mouse models, pharmacologic CYP46A1 activation with low-dose efavirenz or by gene therapy mitigates the manifestations of various brain disorders, neurologic, and nonneurologic, by affecting numerous, apparently unlinked biological processes. Accordingly, CYP46A1 is emerging as a promising therapeutic target; however, the mechanisms underlying the multiplicity of the brain CYP46A1 activity effects are currently not understood. We proposed the chain reaction hypothesis, according to which CYP46A1 is important for the three primary (unifying) processes in the brain (sterol flux through the plasma membranes, acetyl-CoA, and isoprenoid production), which in turn affect a variety of secondary processes. We already identified several processes secondary to changes in sterol flux and herein undertook a multiomics approach to compare the brain proteome, acetylproteome, and metabolome of 5XFAD mice (an Alzheimer’s disease model), control and treated with low-dose efavirenz. We found that the latter had increased production of phospholipids from the corresponding lysophospholipids and a globally increased protein acetylation (including histone acetylation). Apparently, these effects were secondary to increased acetyl-CoA production. Signaling of small GTPases due to their altered abundance or abundance of their regulators could be affected as well, potentially via isoprenoid biosynthesis. In addition, the omics data related differentially abundant molecules to other biological processes either reported previously or new. Thus, we obtained unbiased mechanistic insights and identified potential players mediating the multiplicity of the CYP46A1 brain effects and further detailed our chain reaction hypothesis.

Genetic background influences the 5XFAD Alzheimer's disease mouse model brain proteome.

There is an urgent need to improve the translational validity of Alzheimer's disease (AD) mouse models. Introducing genetic background diversity in AD mouse models has been proposed as a way to increase validity and enable the discovery of previously uncharacterized genetic contributions to AD susceptibility or resilience. However, the extent to which genetic background influences the mouse brain proteome and its perturbation in AD mouse models is unknown. In this study, we crossed the 5XFAD AD mouse model on a C57BL/6J (B6) inbred background with the DBA/2J (D2) inbred background and analyzed the effects of genetic background variation on the brain proteome in F1 progeny. Both genetic background and 5XFAD transgene insertion strongly affected protein variance in the hippocampus and cortex (n = 3,368 proteins). Protein co-expression network analysis identified 16 modules of highly co-expressed proteins common across the hippocampus and cortex in 5XFAD and non-transgenic mice. Among the modules strongly influenced by genetic background were those related to small molecule metabolism and ion transport. Modules strongly influenced by the 5XFAD transgene were related to lysosome/stress responses and neuronal synapse/signaling. The modules with the strongest relationship to human disease-neuronal synapse/signaling and lysosome/stress response-were not significantly influenced by genetic background. However, other modules in 5XFAD that were related to human disease, such as GABA synaptic signaling and mitochondrial membrane modules, were influenced by genetic background. Most disease-related modules were more strongly correlated with AD genotype in the hippocampus compared with the cortex. Our findings suggest that the genetic diversity introduced by crossing B6 and D2 inbred backgrounds influences proteomic changes related to disease in the 5XFAD model, and that proteomic analysis of other genetic backgrounds in transgenic and knock-in AD mouse models is warranted to capture the full range of molecular heterogeneity in genetically diverse models of AD.

Matrix-assisted laser desorption/ionization analysis of the brain proteome of microgravity-exposed mice from the International Space Station

Manned spaceflight exposes humans to extreme environmental conditions, including microgravity exposures. The effects of microgravity during spaceflight could lead to changes in brain structure, gene expression, and vascular physiology. Given the known physiological effects, it is highly likely that there are microgravity-initiated proteomic differentials in the brain, possibly domain specific. MALDI-TOF (matrix-assisted laser desorption/ionization time of flight) Imaging Mass Spectrometry allows the visualization of the spatial distribution of highly abundant intact proteins in tissue specimens. This study utilized this technique to visualize global proteomic changes induced by microgravity exposure in brain tissue received from the Rodent Research-1 Center for the Advancement of Science in Space (CASIS)/National Aeronautics and Space Administration (NASA). Proteome profiles were obtained from isolated whole brain tissue from microgravity exposed, Habitat control, and baseline. While a total of 135 mass peaks equating to individual proteins were identified, statistical analysis determined that there were no significant differences in the spectra profiles from the three test groups utilizing this methodology, possibly due to sample collection logistics rather than lack of cellular response.

Understanding global changes of the mouse brain proteome after vaginal infection with HSV-2 using a label-free shotgun approach.

Herpes simplex virus type 2 (HSV-2) is a common human pathogen that establishes lifelong latency in neurons of the nervous system. The number of severe central nervous system infections caused by the virus has increased recently. However, the pathogenesis of HSV-2 infection in the nervous system is not fully understood. Here, we demonstrated global proteomic changes in the brain tissue in BALB/c mice vaginally infected with HSV-2. Data are available via ProteomeXchange with identifier PXD034186. A total of 249 differentially expressed proteins were identified in infected brain tissue. The GO and KEGG enrichment analysis of these proteins indicated that they were mainly involved in the regulation of synapse formation and synaptic excitability. In addition, genes affecting autophagy, the development of other neurodegenerative diseases, and signaling pathways relevant to other neurologic diseases were identified. Additional experiments, comparing the brain tissue of asymptomatic and symptomatic mice showed a differential expression of proteins involved in synapse formation and synaptic transmission. Others were involved in autophagy, addiction, and signaling pathways of other neurologic diseases. These results suggest that changes in synaptic structure and function, as well as autophagy, may be related to the development of neurologic abnormalities that follow HSV-2 infection. We also identified a protein GluN2A encoded by Grin2a was continuously expressed at high levels after infection. We propose that GluN2A may be a key molecule in the pathogenesis of HSV-2-induced neurologic diseases.

Genetic architecture of protein expression and its regulation in the mouse brain

BackgroundNatural variation in protein expression is common in all organisms and contributes to phenotypic differences among individuals. While variation in gene expression at the transcript level has been extensively investigated, the genetic mechanisms underlying variation in protein expression have lagged considerably behind. Here we investigate genetic architecture of protein expression by profiling a deep mouse brain proteome of two inbred strains, C57BL/6 J (B6) and DBA/2 J (D2), and their reciprocal F1 hybrids using two-dimensional liquid chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS) technology.ResultsBy comparing protein expression levels in the four mouse strains, we observed 329 statistically significant differentially expressed proteins between the two parental strains and characterized the genetic basis of protein expression. We further applied a proteogenomic approach to detect variant peptides and define protein allele-specific expression (pASE), identifying 33 variant peptides with cis-effects and 17 variant peptides showing trans-effects. Comparison of regulation at transcript and protein levels show a significant divergence.ConclusionsThe results provide a comprehensive analysis of genetic architecture of protein expression and the contribution of cis- and trans-acting regulatory differences to protein expression.

Hippocampal disruptions of synaptic and astrocyte metabolism are primary events of early amyloid pathology in the 5xFAD mouse model of Alzheimer\u2019s disease

Alzheimer’s disease (AD) is an unremitting neurodegenerative disorder characterized by cerebral amyloid-β (Aβ) accumulation and gradual decline in cognitive function. Changes in brain energy metabolism arise in the preclinical phase of AD, suggesting an important metabolic component of early AD pathology. Neurons and astrocytes function in close metabolic collaboration, which is essential for the recycling of neurotransmitters in the synapse. However, this crucial metabolic interplay during the early stages of AD development has not been sufficiently investigated. Here, we provide an integrative analysis of cellular metabolism during the early stages of Aβ accumulation in the cerebral cortex and hippocampus of the 5xFAD mouse model of AD. Our electrophysiological examination revealed an increase in spontaneous excitatory signaling in the 5xFAD hippocampus. This hyperactive neuronal phenotype coincided with decreased hippocampal tricarboxylic acid (TCA) cycle metabolism mapped by stable 13C isotope tracing. Particularly, reduced astrocyte TCA cycle activity and decreased glutamine synthesis led to hampered neuronal GABA synthesis in the 5xFAD hippocampus. In contrast, the cerebral cortex of 5xFAD mice displayed an elevated capacity for oxidative glucose metabolism, which may suggest a metabolic compensation in this brain region. We found limited changes when we explored the brain proteome and metabolome of the 5xFAD mice, supporting that the functional metabolic disturbances between neurons and astrocytes are early primary events in AD pathology. In addition, synaptic mitochondrial and glycolytic function was selectively impaired in the 5xFAD hippocampus, whereas non-synaptic mitochondrial function was maintained. These findings were supported by ultrastructural analyses demonstrating disruptions in mitochondrial morphology, particularly in the 5xFAD hippocampus. Collectively, our study reveals complex regional and cell-specific metabolic adaptations in the early stages of amyloid pathology, which may be fundamental for the progressing synaptic dysfunctions in AD.

Brain Acetyl-CoA Production and Phosphorylation of Cytoskeletal Proteins Are Targets of CYP46A1 Activity Modulation and Altered Sterol Flux

Cholesterol and 24-hydroxycholesterol are the most abundant brain sterols and represent the substrate and product, respectively, of cytochrome P450 46A1 (CYP46A1), a CNS-specific enzyme. CYP46A1 controls cholesterol elimination and turnover in the brain, the two processes that determine the rate of brain sterol flux through the plasma membranes and thereby the properties of these membranes. Brain sterol flux is decreased in Cyp46a1−/− mice compared to wild-type mice and increased in 5XFAD mice (a model of Alzheimer’s disease) when they are treated with a small dose of efavirenz, a CYP46A1 activator. Herein, we first assessed the brain proteome (synaptosomal fractions) and phospho-proteome (synaptosomal fractions and brain homogenates) of efavirenz-treated and control 5XFAD mice. Then, based on the pattern of protein abundance change, we conducted acetyl-CoA measurements (brain homogenates and mitochondria) and metabolic profiling (brain homogenates). The phospho-proteomics datasets were used for comparative analyses with the datasets obtained by us previously on mice with the same changes (efavirenz-treated and control 5XFAD mice from a different treatment paradigm) or with changes in the opposite direction (Cyp46a1−/− vs wild-type mice) in brain sterol flux. We found that CYP46A1 activity or the rate of brain sterol flux affects acetyl-CoA-related metabolic pathways as well as phosphorylation of cytoskeletal and other proteins. Knowledge of the key roles of acetyl-CoA and cytoskeletal phosphorylation in cell biology expands our understanding of the significance of CYP46A1-mediated cholesterol 24-hydroxylation in the brain and provides an additional explanation for why CYP46A1 activity modulations are beneficial in mouse models of different brain diseases.

Complement and Coagulation Cascades are Potentially Involved in Dopaminergic Neurodegeneration in α-Synuclein-Based Mouse Models of Parkinson's Disease.

Parkinson's disease (PD) is the second most common neurodegenerative disorder that results in motor dysfunction and, eventually, cognitive impairment. α-Synuclein protein is known as a central protein to the pathophysiology of PD, but the underlying pathological mechanism still remains to be elucidated. In an effort to understand how α-synuclein underlies the pathology of PD, various PD mouse models with α-synuclein overexpression have been developed. However, systemic analysis of the brain proteome of those mouse models is lacking. In this study, we established two mouse models of PD by injecting α-synuclein preformed fibrils (PFF) or by inducing overexpression of human A53T α-synuclein to investigate common pathways in the two different types of the PD mouse models. For more accurate quantification of mouse brain proteome, the proteins were quantified using the method of stable isotope labeling with amino acids in mammals . We identified a total of 8355 proteins from the two mouse models; ∼6800 and ∼7200 proteins from α-synuclein PFF-injected mice and human A53T α-synuclein transgenic mice, respectively. Through pathway analysis of the differentially expressed proteins common to both PD mouse models, it was discovered that the complement and coagulation cascade pathways were enriched in the PD mice compared to control animals. Notably, a validation study demonstrated that complement component 3 (C3)-positive astrocytes were increased in the ventral midbrain of the intrastriatal α-synuclein PFF-injected mice and C3 secreted from astrocytes could induce the degeneration of dopaminergic neurons. This is the first study that highlights the significance of the complement and coagulation pathways in the pathogenesis of PD through proteome analyses with two sophisticated mouse models of PD.

Hippocampal Disruptions of Synaptic and Astrocyte Metabolism are Primary Events of Early Amyloid Pathology in the 5xFAD Mouse Model of Alzheimer's Disease

Alzheimer’s disease (AD) is an unremitting neurodegenerative disorder characterized by cerebral amyloid-β (Aβ) accumulation and gradual decline in cognitive function. Changes in brain energy metabolism arise in the preclinical phase of AD, suggesting an important metabolic component of early AD pathology. Neurons and astrocytes function in close metabolic collaboration, which is essential for the recycling of neurotransmitters in the synapse. However, this crucial metabolic interplay during the early stages of AD development has not been sufficiently investigated. Here, we provide an integrative analysis of cellular metabolism during the early stages of Aβ accumulation in the cerebral cortex and hippocampus of the 5xFAD mouse model of AD. Our electrophysiological examination revealed an increase in spontaneous excitatory signaling in the 5xFAD hippocampus. This hyperactive neuronal phenotype coincided with decreased hippocampal TCA cycle metabolism mapped by stable 13C isotope tracing. Particularly, reduced astrocyte TCA cycle activity led to decreased glutamine synthesis, in turn hampering neuronal GABA synthesis in the 5xFAD hippocampus. In contrast, the cerebral cortex of 5xFAD mice displayed an elevated capacity for oxidative glucose metabolism suggesting a metabolic compensation. We found limited changes when we explored the brain proteome and metabolome of the 5xFAD mice, supporting that the functional metabolic disturbances between neurons and astrocytes are early primary events in AD pathology. In addition, synaptic mitochondrial and glycolytic function was impaired selectively in the 5xFAD hippocampus, whereas non-synaptic mitochondrial function was maintained. These findings were supported by ultrastructural analyses demonstrating disruptions in mitochondrial morphology, particularly in the 5xFAD hippocampus. Collectively, our study reveals complex regional and cell-specific metabolic adaptations, in the early stages of amyloid pathology, which may be fundamental for the progressing synaptic dysfunction in AD.

PKC downregulation upon rapamycin treatment attenuates mitochondrial disease.

Leigh syndrome is a fatal neurometabolic disorder caused by defects in mitochondrial function. mTOR inhibition with rapamycin attenuates disease progression in a mouse model of Leigh syndrome (Ndufs4 KO mouse); however, the mechanism of rescue is unknown. Here we identify PKC downregulation as a key event mediating the beneficial effects of rapamycin treatment of Ndufs4 KO mice. Assessing the impact of rapamycin on the brain proteome and phosphoproteome of Ndufs4 KO mice we find that rapamycin restores mitochondrial protein levels, inhibits signaling through both mTOR complexes, and reduces the abundance and activity of multiple protein kinase C (PKC) isoforms. Administration of PKC inhibitors increases survival, delays neurological deficits, prevents hair loss, and decreases inflammation in Ndufs4 KO mice. Thus, PKC may be a viable therapeutic target for treating severe mitochondrial disease.Reporting SummaryFurther information on research design is available in the Nature Research Reporting Summary linked to this article.

Network analysis of the progranulin-deficient mouse brain proteome reveals pathogenic mechanisms shared in human frontotemporal dementia caused by GRN mutations

Heterozygous, loss-of-function mutations in the granulin gene (GRN) encoding progranulin (PGRN) are a common cause of frontotemporal dementia (FTD). Homozygous GRN mutations cause neuronal ceroid lipofuscinosis-11 (CLN11), a lysosome storage disease. PGRN is a secreted glycoprotein that can be proteolytically cleaved into seven bioactive 6 kDa granulins. However, it is unclear how deficiency of PGRN and granulins causes neurodegeneration. To gain insight into the mechanisms of FTD pathogenesis, we utilized Tandem Mass Tag isobaric labeling mass spectrometry to perform an unbiased quantitative proteomic analysis of whole-brain tissue from wild type (Grn+/+) and Grn knockout (Grn−/−) mice at 3- and 19-months of age. At 3-months lysosomal proteins (i.e. Gns, Scarb2, Hexb) are selectively increased indicating lysosomal dysfunction is an early consequence of PGRN deficiency. Additionally, proteins involved in lipid metabolism (Acly, Apoc3, Asah1, Gpld1, Ppt1, and Naaa) are decreased; suggesting lysosomal degradation of lipids may be impaired in the Grn−/− brain. Systems biology using weighted correlation network analysis (WGCNA) of the Grn−/− brain proteome identified 26 modules of highly co-expressed proteins. Three modules strongly correlated to Grn deficiency and were enriched with lysosomal proteins (Gpnmb, CtsD, CtsZ, and Tpp1) and inflammatory proteins (Lgals3, GFAP, CD44, S100a, and C1qa). We find that lysosomal dysregulation is exacerbated with age in the Grn−/− mouse brain leading to neuroinflammation, synaptic loss, and decreased markers of oligodendrocytes, myelin, and neurons. In particular, GPNMB and LGALS3 (galectin-3) were upregulated by microglia and elevated in FTD-GRN brain samples, indicating common pathogenic pathways are dysregulated in human FTD cases and Grn−/− mice. GPNMB levels were significantly increased in the cerebrospinal fluid of FTD-GRN patients, but not in MAPT or C9orf72 carriers, suggesting GPNMB could be a biomarker specific to FTD-GRN to monitor disease onset, progression, and drug response. Our findings support the idea that insufficiency of PGRN and granulins in humans causes neurodegeneration through lysosomal dysfunction, defects in autophagy, and neuroinflammation, which could be targeted to develop effective therapies.

Normal Mouse Brain Proteome II: Analysis of Brain Regions by High-resolution Mass Spectrometry.

Proteomics technologies provide fundamental insights into the high organizational complexity and diversity of the central nervous system. In the present study, high-resolution mass spectrometry (MS) was applied in order to identify whole-proteome content of anatomically distinct and functionally specific mouse brain regions. Brains from eight 8-week-old C57BL/6N normal male mice were separated into seven anatomically district regions. The protein content of each region was analyzed by high-throughput nano-liquid chromatography-MS/MS Orbitrap elite technology. A total of 16,574 proteins were identified: 2,795 in cerebral cortex, 2,311 in olfactory bulb, 2,246 in hippocampus, 2,247 in hypothalamus, 2,250 in mid brain, 2,334 in cerebellum and 2,391 in medulla. Of these proteins, 534 were uniquely expressed in cerebral cortex, 323 in olfactory bulb, 230 in hippocampus, 272 in hypothalamus, 1,326 in mid brain, 320 in cerebellum and 268 in medulla. These data represent the most comprehensive proteomic map of the normal mouse brain and they might further be used in studies related to brain diseases, including cancer and neurodegenerative diseases.

From Analysis of Ischemic Mouse Brain Proteome to Identification of Human Serum Clusterin as a Potential Biomarker for Severity of Acute Ischemic Stroke.

Ischemic stroke is a devastating neurological disease that can cause permanent brain damage, but to date, few biomarkers are available to reliably assess the severity of injury during acute onset. In this study, quantitative proteomic analysis of ischemic mouse brain detected the increase in expression levels of clusterin (CLU) and cystatin C (CST3). Since CLU is a secretary protein, serum samples (n = 70) were obtained from acute ischemic stroke (AIS) patients within 24h of stroke onset and together with 70 matched health controls. Analysis of CLU levels indicated significantly higher levels in AIS patients than healthy controls (14.91 ± 4.03 vs. 12.79 ± 2.22ng/L; P = 0.0004). Analysis of serum CST3 also showed significant increase in AIS patients as compared with healthy controls (0.90 ± 0.19 vs. 0.84 ± 0.12ng/L; P = 0.0064). The serum values of CLU were also positively correlated with the NIH Stroke Scale (NIHSS) scores, the time interval after stroke onset, as well as major stroke risk factors associated with lipid profile. These data demonstrate that elevated levels of serum CLU and CST3 are independently associated with AIS and may serve as peripheral biomarkers to aid clinical assessment of AIS and its severity. This pilot study thus contributes to progress toward preclinical proteomic screening by using animal models and allows translation of results from bench to bedside.

Impact of diet-induced obesity on the mouse brain phosphoproteome

Obesity is closely associated to several diseases such as type 2 diabetes, cardiovascular disease, hepatic steatosis, airway disease, neurodegeneration, biliary diseases and certain cancers. It is, therefore, of importance to assess the role of nutrition in disease prevention as well as its effect in the course of such pathologies. In the present study, we addressed the impact of the exposure to different obesogenic diets in the mice brains phosphoproteome. To analyze if the obesity could be able to modify the protein pattern expression of brain neurons, obesity was induced in two different groups of mice. One group of mice was fed with hyperglycemic diet (HGD) and the other one was fed with high-fat diet (HFD), both for 12 weeks. A control group of lean mice was fed with a standard diet (SD). Metabolic parameters were measured before sacrifice, and brains were harvested for label-free phosphoproteomic analysis. Mice brains were analyzed to find differences, if any, in protein phosphorylation. Interestingly, the changes were independent of the obesogenic diet as no changes were detected between the two obese groups. Dephosphorylation of proteins involved in neuronal development (among others SYNGAP1 and PPP1R9B), in vesicle trafficking (for example SNAP91 and AMPH) and in cytoskeletal functions (for example, CLASP2 and GSK3B) was identified, while increased phosphorylation was detected for microtubule proteins (such as MAP2 and MAPT). Phospho site analysis of the mouse brain proteome reveals important changes that point to a connection between diet-induced obesity and impairment of neuronal functions and signaling.

Top-Down Proteomics Enables Comparative Analysis of Brain Proteoforms Between Mouse Strains.

Over the past decade, advances in mass spectrometry-based proteomics have accelerated brain proteome research aimed at studying the expression, dynamic modification, interaction and function of proteins in the nervous system that are associated with physiological and behavioral processes. With the latest hardware and software improvements in top-down mass spectrometry, the technology has expanded from mere protein profiling to high-throughput identification and quantification of intact proteoforms. Murine systems are broadly used as models to study human diseases. Neuroscientists specifically study the mouse brain from inbred strains to help understand how strain-specific genotype and phenotype affect development, functioning, and disease progression. This work describes the first application of label-free quantitative top-down proteomics to the analysis of the mouse brain proteome. Operating in discovery mode, we determined physiochemical differences in brain tissue from four healthy inbred strains, C57BL/6J, DBA/2J, FVB/NJ, and BALB/cByJ, after probing their intact proteome in the 3.5-30 kDa mass range. We also disseminate these findings using a new tool for top-down proteomics, TDViewer and cataloged them in a newly established Mouse Brain Proteoform Atlas. The analysis of brain tissues from the four strains identified 131 gene products leading to the full characterization of 343 of the 593 proteoforms identified. Within the results, singly and doubly phosphorylated ARPP-21 proteoforms, known to inhibit calmodulin, were differentially expressed across the four strains. Gene ontology (GO) analysis for detected differentially expressed proteoforms also helps to illuminate the similarities and dissimilarities in phenotypes among these inbred strains.

Strong cation exchange-reversed phase liquid chromatography-capillary zone electrophoresis-tandem mass spectrometry platform with high peak capacity for deep bottom-up proteomics

Two-dimensional (2D) liquid chromatography (LC)-tandem mass spectrometry (MS/MS) are typically employed for deep bottom-up proteomics, and the state-of-the-art 2D-LC-MS/MS has approached over 8000 protein identifications (IDs) from mammalian cell lines or tissues in 1–3 days of mass spectrometer time. Capillary zone electrophoresis (CZE)-MS/MS has been suggested as an alternative to LC-MS/MS for bottom-up proteomics. CZE-MS/MS and LC-MS/MS are complementary in protein/peptide ID from complex proteome digests because CZE and LC are orthogonal for peptide separation. In addition, the migration time of peptides from CZE-MS can be predicted accurately, which is invaluable for evaluating the confidence of peptide ID from the database search and even guiding the database search. However, the number of protein IDs from complex proteomes using CZE-MS/MS is still much lower than the state of the art using 2D-LC-MS/MS. In this work, for the first time, we established a strong cation exchange (SCX)-reversed phase LC (RPLC)-CZE-MS/MS platform for deep bottom-up proteomics. The platform identified around 8200 protein groups and 65,000 unique peptides from a mouse brain proteome digest in 70 h. The data represents the largest bottom-up proteomics dataset using CZE-MS/MS and provides a valuable resource for further improving the tool for prediction of peptide migration time in CZE. The peak capacity of the orthogonal SCX-RPLC-CZE platform was estimated to be around 7000. SCX-RPLC-CZE-MS/MS produced comparable numbers of protein and peptide IDs with 2D-LC-MS/MS (8200 vs. 8900 protein groups, 65,000 vs. 70,000 unique peptides) from the mouse brain proteome digest using comparable instrument time. This is the first time that CZE-MS/MS showed its capability to approach comparable performance to the state-of-the-art 2D-LC-MS/MS for deep proteomic sequencing. SCX-RPLC-CZE-MS/MS and 2D-LC-MS/MS showed good complementarity in protein and peptide IDs and combining those two methods improved the number of protein group and unique peptide IDs by nearly 10% and over 40%, respectively, compared with 2D-LC-MS/MS alone.

An Anatomically Resolved Mouse Brain Proteome Reveals Parkinson Disease-relevant Pathways

Here, we present a mouse brain protein atlas that covers 17 surgically distinct neuroanatomical regions of the adult mouse brain, each less than 1 mm3 in size. The protein expression levels are determined for 6,500 to 7,500 gene protein products from each region and over 12,000 gene protein products for the entire brain, documenting the physiological repertoire of mouse brain proteins in an anatomically resolved and comprehensive manner. We explored the utility of our spatially defined protein profiling methods in a mouse model of Parkinson's disease. We compared the proteome from a vulnerable region (substantia nigra pars compacta) of wild type and parkinsonian mice with that of an adjacent, less vulnerable, region (ventral tegmental area) and identified several proteins that exhibited both spatiotemporal- and genotype-restricted changes. We validated the most robustly altered proteins using an alternative profiling method and found that these modifications may highlight potential new pathways for future studies. This proteomic atlas is a valuable resource that offers a practical framework for investigating the molecular intricacies of normal brain function as well as regional vulnerability in neurological diseases. All of the mouse regional proteome profiling data are published on line at http://mbpa.bprc.ac.cn/.

Low-temperature exothermic transitions in brain proteome of mice, effect of scopolamine

Differential scanning calorimetry (DSC) has been employed to examine the thermal behavior of brain tissues affected by a drug-induced neurodegenerative disorder. An animal (mouse) model of scopolamine-induced dementia was used. The DSC measurements performed on supernatants of brain tissue homogenates revealed large differences between the heat capacity profiles for healthy animals and for animals with scopolamine-induced dementia. The heat capacity profiles of the supernatants from healthy animals displayed well expressed exothermic transitions peaking in the range 35–45°C, thus preceding in temperature the endothermic denaturational transitions. No such exothermic transitions were found in other samples from the same animals, e.g., centrifugation sediments of brain homogenates, liver homogenates, blood plasma. Remarkably, the low-temperature exotherms were completely abolished by the scopolamine treatment. The exothermic events may possibly reflect a process of aggregation of specific protein fractions in the brain supernatants. The reported findings may be important for the elucidation of the molecular mechanisms of cognitive impairment.