Activity-dependent localization in spines of the F-actin capping protein CapZ screened in a rat model of dementia.

Actin reorganization in dendritic spines is hypothesized to underlie neuronal plasticity. Actin-related proteins, therefore, might serve as useful markers of plastic changes in dendritic spines. Here, we utilized memory deficits induced by fimbria-fornix transection (FFT) in rats as a dementia model to screen candidate memory-associated molecules by using a two-dimensional gel method. Comparison of protein profiles between the transected and control sides of hippocampi after unilateral FFT revealed a reduction in the F-actin capping protein (CapZ) signal on the FFT side. Subsequent immunostaining of brain sections and cultured hippocampal neurons revealed that CapZ localized in dendritic spines and the signal intensity in each spine varied widely. The CapZ content decreased after suppression of neuronal firing by tetrodotoxin treatment in cultured neurons, indicating rapid and activity-dependent regulation of CapZ accumulation in spines. To test input specificity of CapZ accumulation in vivo, we delivered high-frequency stimuli to the medial perforant path unilaterally in awake rats. This path selectively inputs to the middle molecular layer of the dentate gyrus, where CapZ immunoreactivity increased. We conclude that activity-dependent, synapse-specific regulation of CapZ redistribution might be important in both maintenance and remodeling of synaptic connections in neurons receiving specific spatial and temporal patterns of inputs.

A missense mutation in the Capza3 gene and disruption of F-actin organization in spermatids of repro32 infertile male mice.

Males homozygous for the repro32 ENU-induced mutation produced by the Reproductive Genomics program at The Jackson Laboratory are infertile, have low epididymal sperm concentrations, and produce sperm with abnormally shaped heads and poor motility. The purpose of the present study was to identify the mutated gene in repro32 mice and to define the structural and functional changes causing infertility and the aberrant sperm phenotype. In repro32/repro32 mice, we discovered a failure to shed excess cytoplasm and disorganization of the middle piece of the flagellum at spermiation, resulting in the outer dense fibers being wrapped around the sperm head within a bag of cytoplasm. Using a candidate-gene approach, a mutation was identified in the spermatid-specific "capping protein (actin filament) muscle Z-line, alpha 3" gene (Capza3). CAPZA3 protein localization was altered in spermatids concurrent with altered localization of a unique CAPZB variant isoform and disruption of the filamentous actin (F-actin) network. These observations strongly suggest the missense mutation in Capza3 is responsible for the mutant phenotype of repro32/repro32 sperm and regulation of F-actin dynamics by a spermatogenic cell-specific CAPZ heterodimer is essential for removal of the cytoplasm and maintenance of midpiece integrity during spermiation in the mouse.

Modifications of trout (Oncorhynchus mykiss) muscle proteins by preslaughter activity.

The effect of two different preslaughter procedures (limited or 15-min intense muscular activity) on muscle trout proteins was investigated. Muscle was sampled 45 min and 24 h post-mortem, proteins were separated using two-dimensional electrophoresis, and spots of interest were tentatively identified by MALDI-TOF spectrometry. Twenty-nine and 4 spots were differentially represented between the two groups of fish at 45 min and 24 h post-mortem, respectively. Spots that could be identified corresponded mainly to proteins involved in energy-producing pathways (triosephosphate isomerase, enolase, pyruvate dehydrogenase) or to structural proteins (desmin, cap-Z, myosin heavy chain fragment). Persistent under-representation of desmin, a key cytoskeletal protein, in fish submitted to intense muscular activity suggests that such a preslaughter treatment can have an effect on post-mortem muscle integrity.

Proteomics-based identification of spontaneous regression-associated proteins in neuroblastoma.

BACKGROUND: Spontaneous regression is usually found in stage 4s neuroblastoma, whereas the elucidation of the underlying molecular mechanism(s) is still limited. PURPOSE: Our study aims to investigate the pathogenesis of spontaneous regression at the protein level. METHODS AND MATERIALS: Differential expression of proteins in stage 4s neuroblastoma tissue, in stage 4 neuroblastoma tissue, and in normal adrenal tissue was investigated by use of 2-dimensional difference gel electrophoresis (2D-DIGE). RESULTS: Twenty-four protein spots were found to have significant changes among the different tissues, in which 16 proteins were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Among these proteins, 7 proteins (RhoGDP-dissociation inhibitor 1, phosphatidylethanolamine-binding protein, prohibitin, etc) were up-regulated and 2 proteins (F-actin capping protein 1 subunit and aldose reductase) were down-regulated in stage 4s neuroblastoma compared with stage 4 neuroblastoma. The differential expression of selected candidate protein (RhoGDP-dissociation inhibitor 1 and CAPZA1) was further validated by western blotting. CONCLUSION: Some proteins are differentially expressed between stage 4s and stage 4 neuroblastoma tissue, including those associated with differentiation and proliferation as well as apoptosis. RhoGDP-dissociation inhibitor 1 is highly expressed in stage 4s neuroblastoma tissue, whereas CAPZA1 is down-regulated.

Molecular-assisted immunohistochemical optimization.

Immunohistochemistry (IHC) is an essential tool in diagnostic surgical pathology, allowing analysis of protein subcellular localization. The use of IHC by different laboratories has lead to inconsistencies in published literature for several antibodies, due to either interpretative (inter-observer variation) or technical reasons. These disparities have major implications in both clinical and research settings. In this study, we report our experience conducting an IHC optimization of antibodies against five proteins previously identified by proteomic analysis to be breast cancer biomarkers, namely 6PGL (PGLS), CAZ2 (CAPZA2), PA2G4 (EBP1) PSD2 and TKT. Large variations in the immunolocalizations and intensities were observed when manipulating the antigen retrieval method and primary antibody incubation concentration. However, the use of an independent molecular analysis method provided a clear indication in choosing the appropriate biologically and functionally relevant "staining pattern". Without this latter step, each of these contradictory results would have been a priori "technically acceptable" and would have led to different biological and functional interpretations of these proteins and potentially different applications in a routine pathology setting. Thus, we conclude that full validation of immunohistochemical protocols for scientific and clinical use will require the incorporation of biological knowledge of the biomarker and the disease in question.

Novel breast cancer biomarkers identified by integrative proteomic and gene expression mapping.

Proteomic and transcriptomic platforms both play important roles in cancer research, with differing strengths and limitations. Here, we describe a proteo-transcriptomic integrative strategy for discovering novel cancer biomarkers, combining the direct visualization of differentially expressed proteins with the high-throughput scale of gene expression profiling. Using breast cancer as a case example, we generated comprehensive two-dimensional electrophoresis (2DE)/mass spectrometry (MS) proteomic maps of cancer (MCF-7 and HCC-38) and control (CCD-1059Sk) cell lines, identifying 1724 expressed protein spots representing 484 different protein species. The differentially expressed cell-line proteins were then mapped to mRNA transcript databases of cancer cell lines and primary breast tumors to identify candidate biomarkers that were concordantly expressed at the gene expression level. Of the top nine selected biomarker candidates, we reidentified ANX1, a protein previously reported to be differentially expressed in breast cancers and normal tissues, and validated three other novel candidates, CRAB, 6PGL, and CAZ2, as differentially expressed proteins by immunohistochemistry on breast tissue microarrays. In total, close to half (4/9) of our protein biomarker candidates were successfully validated. Our study thus illustrates how the systematic integration of proteomic and transcriptomic data from both cell line and primary tissue samples can prove advantageous for accelerating cancer biomarker discovery.