Neuroproteomics: a biochemical means to discriminate the extent and modality of brain injury

Abstract

Diagnosis and treatment of stroke and traumatic brain injury remain significant health care challenges to society. Patient care stands to benefit from an improved understanding of the interactive biochemistry underlying neurotrauma pathobiology. In this study, we assessed the power of neuroproteomics to contrast biochemical responses following ischemic and traumatic brain injuries in the rat. A middle cerebral artery occlusion (MCAO) model was employed in groups of 30-min and 2-h focal neocortical ischemia with reperfusion. Neuroproteomes were assessed via tandem cation-anion exchange chromatography-gel electrophoresis, followed by reversed-phase liquid chromatography-tandem mass spectrometry. MCAO results were compared with those from a previous study of focal contusional brain injury employing the same methodology to characterize homologous neocortical tissues at 2 days post-injury. The 30-min MCAO neuroproteome depicted abridged energy production involving pentose phosphate, modulated synaptic function and plasticity, and increased chaperone activity and cell survival factors. The 2-h MCAO data indicated near complete loss of ATP production, synaptic dysfunction with degraded cytoarchitecture, more conservative chaperone activity, and additional cell survival factors than those seen in the 30-min MCAO model. The TBI group exhibited disrupted metabolism, but with retained malate shuttle functionality. Synaptic dysfunction and cytoarchitectural degradation resembled the 2-h MCAO group; however, chaperone and cell survival factors were more depressed following TBI. These results underscore the utility of neuroproteomics for characterizing interactive biochemistry for profiling and contrasting the molecular aspects underlying the pathobiological differences between types of brain injuries.

FIG. 1.

Histology of modeled injuries. Triphenyltetrazolium chloride (TTC)-stained coronal sections are shown to illustrate the extent of injury within the tissues prepared for the 30-min middle cerebral artery occlusion (MCAO) (A), 2-h MCAO (B), and 1.6-mm controlled cortical impact (CCI) (C) experimental groups. The presented sections are proximate to the rostral-caudal center of the dissected region prepared for proteomic and Western blot experiments (near bregma −2.6 mm). Histopathology was assessed 2 days after injury. An overlay of the target dissection area is shown.

FIG. 2.

Comparison of middle cerebral artery occlusion (MCAO) and traumatic brain injury (TBI) neuroproteomes. Numbers of protein products with an increased (A) or decreased (B) abundance, and the overlap of the MCAO and TBI neuroproteomes at 2 days following neocortical insults. (C) Thirty-two proteins from the MCAO differential neuroproteome that have a reported pathobiological association with brain ischemia in previous literature. The proteins are illustrated a top their native cell compartment: membrane depiction defines extracellular, intermembrane, and intracellular proteins; nuclear, mitochondrial, and golgi-associated proteins appear atop their respective cartoons; the remaining intracellular proteins are common to the cytosol (SYN1, synaptin 1; TF, transferrin; PYGB, glycogen phosphorylase (brain form); SNAP25, synaptosomal-associated protein 25; P4HB, protein disulfide-isomerase; MAP2, microtubule-associated protein 2; HSPE1, heat shock protein 10; ALB, albumin; TKT, transketolase; HSPH1, heat shock protein 105; ALDH 9A1, 4-trimethylaminobutyraldehyde dehydrogenase; GPT, alanine aminotransferase; PPP3CA, calcineurin (PP2B); PGK1, phosphoglycerate kinase 1; MAP1B, microtubule-associated protein 1B; SPTAN1, spectrin α2; AHCY, adenosylhomocysteinase; THOP1, thimet oligopeptidase; gad, ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1); ARF1, ADP-ribosylation factor 1; ALDOC, aldolase C; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PKM2, pyruvate kinase M2; HSPCB, heat shock protein 90; ENO2, neuron-specific enolase; ACO2, aconatase 2; ACO1, aconatase 1; SOD1, superoxide dismutase 1; HSPA 8, heat shock protein 70; PREP, prolyl endopeptidase; STIP1, stress-induced phosphoprotein 1).

FIG. 3.

Comparison of brain-specific glycogen phosphorylase (PYGB) neuroproteomic and immunoblot data. (A) Protein quantity as assessed by gel band density measurement and peptide count for PYGB. (B) PYGB immunoblot of MCAO and TBI neocortical tissue lysates and matched controls. (C) Histogram of densitometric data for the intact 97-kDa band of PYGB among the MCAO and TBI groups normalized to controls. (D) Histogram of densitometric data for degraded PYGB (67-kDa band). All samples were of neocortical tissue collected 2 days post-injury (n = 4 per group). Load was controlled by co-immunoblot with β-actin. Values are presented as mean ± standard deviation as a percentage of control. MCAO data were evaluated by one-way analysis of variance (Holm-Sidak test), and TBI data by t-test (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; TBI, traumatic brain injury; MCAO, middle cerebral artery occlusion).

FIG. 4.

Comparison of amphiphysin neuroproteomic and immunoblot data. (A) Protein quantity assessed by gel band density measurement and peptide count for amphiphysin. (B) Amphiphysin immunoblot of MCAO and TBI neocortical tissue lysates and matched controls. (C) Histogram of densitometric data for the 125-kDa isoform of amphiphysin among the MCAO and TBI groups normalized to controls. (D) Histogram of densitometric data for the 110-kDa isoform of amphiphysin. All samples were of neocortical tissue collected 2 days post-injury (n = 4 per group). Load was controlled by co-immunoblot with β-actin. Values are presented as mean ± standard deviation as a percentage of control. MCAO data were evaluated by one-way analysis of variance (Holm-Sidak test), and TBI data by t-test (*p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; TBI, traumatic brain injury; MCAO, middle cerebral artery occlusion).

FIG. 5.

Classification of neuroproteomic data by cellular process. Proportions of proteins identified in the MCAO neuroproteome (A) and the TBI neuroproteome (B) divided among the seven most prominent processes identified. The indicated percentages are out of the total number of differential proteins with known biological processes from the MCAO (54) and TBI (45) neuroproteomes. Bar graphs depict the proportion of increased, decreased, or degraded proteins associated with a given process (TBI, traumatic brain injury; MCAO, middle cerebral artery occlusion).

FIG. 6.

Loss of synaptic vesicle cycle elements following middle cerebral artery occlusion (MCAO). The 2-h MCAO neuroproteome included the loss of many key synaptic proteins (in bold red type) associated with the synaptic vesicle cycle, from neurotransmitter (NT) uptake on through docking, exocytosis, endocytosis, and vesicle recycling. The data correlated with the observed degradation of axonal cytoskeletal components: actin microfilaments, αII-spectrin membrane scaffold, and tau-supported tubulin microtubules were all reduced or degraded. The combined 2-h MCAO results depict prominent axonal degradation and loss of synaptic function in accord with the massive cell loss associated with the infarcted pathobiology. In contrast, only vesicle ATPase was decreased in the 30-min MCAO group (ATP, adenosine triphosphate; SNAP25, synaptosomal-associated protein 25; AP2, adapter protein complex AP-2; Rab3, ras-related protein-3; PP2B, protein phosphatase 2B).