Expression Of Septin Research Articles

OP38 Modulation of the septin cytoskeleton ameliorates intestinal fibrogenesis

Abstract Background Fibrosis-induced intestinal strictures leading to obstruction are a common and serious complication of Crohn’s disease (CD) with currently no available specific anti-fibrotic therapies. Cytoskeletal dynamics in mesenchymal cells are associated with organ fibrogenesis. Septin filaments are a novel cytoskeletal element, but the functional role of septins in regulating intestinal inflammation and fibrosis remain unknown. We examined the involvement of the septin cytoskeleton in modulating fibrogenic signaling in intestinal fibrogenesis in vitro and in vivo. Methods Expression of different septins in human intestinal resection tissues, primary human intestinal myofibroblasts (HIMF) and immortalized colonic myofibroblast (CCD 18co cells) were examined by real-time PCR and immunoblotting. Functional inhibition of the septin cytoskeleton was achieved using siRNA for septin 7 and the cell-permeable small molecule forchlorfenuron (FCF). Migration and proliferation of HIMF were examined by wound healing and thymidine incorporation assays, respectively. Levels of secreted cytokines were measured using cytometric bead array. Roles of septins in the development intestinal fibrosis in vivo was studied using a chronic DSS colitis model in mice with administration of FCF or vehicle. Results All septin genes were expressed in intestinal tissues with a predominant expression of septin 7. Pro-fibrotic transforming growth factor-β1 (TGFβ1) triggered HIMF activation manifested by the increased production of ECM proteins such as fibronectin and collagen 1 and enhanced expression of the myofibroblast markers α-smooth muscle actin, L-caldesmon and tropomyosin. Septin 7 knockdown resulted in loss of other septin proteins thereby disrupting the entire septin cytoskeleton. Either pharmacological, or genetic disruption of the septin cytoskeleton reversed the effect of TGFβ1 by attenuating production of ECM proteins and reducing expression of myofibroblast markers. Interestingly, these effects were only found on protein level and were not due to changes in gene expression, suggesting inhibition of their expression at a post-transcriptional level. Moreover, pharmacological or genetic disruptions of the septin cytoskeleton resulted in reduced migration, and attenuated cytokine secretion by activated myofibroblasts. FCF administration did not affect intestinal inflammation, but decreased the fibrosis score during chronic DSS colitis in mice. Conclusion Our data highlights the septin cytoskeleton as a novel regulator of intestinal myofibroblast activation and function. This for the first time implicates the cytoskeleton as an active participant in intestinal fibrosis and as a potential novel target for the development of anti-fibrotic therapies in IBD patients.

Septin 9 and phosphoinositides regulate lysosome localization and their association with lipid droplets

SummaryThe accumulation of lipid droplets (LDs) in the liver is a hallmark of steatosis, which is often associated with lysosomal dysfunction. Nevertheless, the underlying mechanisms remain unclear. Here, using Huh7 cells loaded with oleate as a model to study LD metabolism, we show that cellular content and distribution of LDs are correlated with those of the lysosome and regulated by oleate and septin 9. High expression of septin 9 promotes perinuclear clustering of lysosomes which co-localized with Golgi and not with their surrounding LDs. On the other hand, knockdown of septin 9 disperses the two organelles which colocalize at the cell periphery. The Rab7 is present around these peripheral LDs. PtdIns5P which binds septin 9 and MTMR3 which converts PtdIns(3,5)P2 into PtdIns(5) recapitulates the effects of septin 9. By contrast, PtdIns(3,5)P2 promotes LD/lysosome co-localization. Overall, our data reveal a phosphoinositide/septin 9-dependent mechanism that regulates LD behavior through the control of their association with lysosomes.

Expression of Septin 2 and Her2/neu in Colorectal Cancer.

Colorectal cancer (CRC) is a common and lethal disease. Septin 2 belongs to the same class of GTPases as the RAS oncogenes influence the invasion and metastasis of many types of tumor cells. Furthermore, HER2/neu is involved in the tumor genesis and progression of various types of tumors. The role of both molecules is still questionable in CRC. The aim of the study is to examine the expression of septin 2 and Her2/neu in patients with CRC. The study was conducted on 2 groups; the first group consisted of 70 paraffin blocks for CRC patients and the second group was formed of 24 blocks from patients diagnosed as colorectal adenoma. For each adenoma and carcinoma case, a section was immunohistochemically stained using antihuman SEPT2 polyclonal antibody. For each carcinoma case, another section was immunostained using monoclonal anti-HER2/neu. The results were statistically analyzed and compared with the collected clinicopathologic data of the cases. For the carcinoma patients, there was a significant association between SEPT2 staining intensity and histologic type (P = 0.001) and grade (P < 0.001), tumor T (P = 0.001) and N (P = 0.011) stages and the presence of lymphovascular invasion (P < 0.001) and a significant association between Her2/neu immunoreactivity scores (IRSs) and histologic grade (P = 0.048), tumor T (P < 0.001) and N (P = 0.019) stages and the presence of perineural (P = 0.004) and lymphovascular (P = 0.003) invasion. In colonic adenoma patients, there was a significant relation between septin 2 IRSs and the grade of dysplasia in the adenoma (P < 0.001) and significant relation with its expression in carcinoma group (P < 0.001). A potential prognostic role of septin 2 and Her2/neu for patients with CRC is suggested as expression of both markers was associated with many important prognostic clinicopathologic variables in patients of CRC.

Septin 9 Methylation in Nasopharyngeal Swabs: A Potential Minimally Invasive Biomarker for the Early Detection of Nasopharyngeal Carcinoma.

Nasopharyngeal carcinoma (NPC) is highly prevalent in Southeast Asia, and an unfavorable outcome is usually attributed to advanced stage NPC. Current methods for the early diagnosis of NPC have limitations in clinical practice. The aim of this study was to investigate the diagnostic ability of Septin 9 methylation for NPC. A quantitative methylation-sensitive PCR (qMS-PCR) assay was developed to measure the methylation status and levels of Septin 9 in nasopharyngeal tissues and paired swabs from patients with NPC, chronic nasopharyngitis, and healthy donors. Methylated Septin 9 was detected in 92% (23/25) of NPC tissues and 25% (4/16) of nasopharyngitis controls (p < 0.05). High-frequency hypermethylation with decreased mRNA expression of Septin 9 in NPC was also identified. Further, Septin 9 methylation was identified in 90.5% (19/21) of NPC biopsies and 71.4% (15/21) of paired swabs, indicating a good concordance between the two sample types. In addition, methylated Septin 9 was found in 16 (72.7%) nasal swabs from 22 NPC patients, 2 of 19 (10.5%) nasopharyngitis, but not in any of the healthy controls (p < 0.01). The methylation score in nasal swabs of the NPC group was also significantly higher than that of non-NPC controls (p < 0.001). Moreover, receiver operating characteristic (ROC) curve analysis showed an area under the curve (AUC) of 0.882 of Septin 9 methylation tests to discriminate NPC from non-NPC subjects. Our study demonstrated that frequent methylation of Septin 9 was present in NPC. Its detection in nasopharyngeal swabs may provide a minimally invasive and informative method for identifying early NPC cases.

Expression and localization of Septin 14 gene and protein in infertile men testis

An increasing body of data implicates the Septin family in the pathology of several diseases, including male fertility. The objective of this study was to evaluate the gene and protein expression pattern of Septin 14 in the testis tissue of azoospermic men. In addition, Septin 14 localization was also assessed in the sperm. Testicular tissues were obtained from biopsies of non-obstrutive azoospermic men who underwent diagnostic testicular biopsy in Royan institute and were divided into two groups: TESE + with positive result in testicular sperm extraction (with hypospermatogenesis pathology) and TESE- with negative result (included patients with Sertoli cell only syndrome and maturation arrest pathologies). Total RNA and protein was extracted using trizol reagent. Septin 14 gene and protein expression level were assessed by real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blot techniques, respectively. The localization of Septin 14 protein was also studied by Immunocytochemistry. The expression of Septin 14 was significantly lower (p < 0. 05) in TESE- group than TESE + in both mRNA and protein levels. The localization of Septin 14 protein was detected in the head to tail of normal sperms with high localization in front of the acrosome and the neck. This is a novel localization report on Septin 14 in sperm. Regarding the presence of this protein in the sperm acrosome and neck, it can be concluded that decreasing of Septin 14 protein expression may be associated with the pathogenesis of male infertility and therefore Septin 14 expression level maybe critical for human spermatogenesis.

Mitochondrial Organization is Severly Modified in Skeletal Muscles of Septin 7 Knockdown Animals

Components of the cytoskeleton have essential role in the organization and appropriate distribution of ion channels and proteins involved in calcium homeostasis, signal transduction, and contractile function of striated muscle. Our goal was to identify the role of septin 7, as extra-sarcomeric cytoskeletal protein in the organization and regeneration of the skeletal muscle. As knocking out septin 7 is lethal at the embryonic stage, a tissue specific, tamoxifen-induced Cre-Lox system was used to downregulate (KD) the expression of septin 7 in adult mouse skeletal muscle.

Essential Role of Septin 7 in Skeletal Muscle Structure and Function

Septins as components of the cytoskeleton have been shown to play an important role in various cellular processes, including cell mobility, apoptosis, and determining cell shape within a wide range of organisms. 13 human septins have been classified into 4 subgroups, where septin 7 is the only member of its group and is crucial in the formation of higher-order structures as filaments and rings. As limited information is available on septins in skeletal muscle, here the effect of downregulating septin 7 expression on skeletal muscle was examined. Cytoplasmic distribution of septin 7 in cultured myoblast, myotubes, and enzimatically isolated individual muscle fibers was investigated and its distribution was compared to the pattern of well-known proteins as α-actinin and ryanodine receptors. Septin 7 expression was modified both in cell culture and in mouse muscle fibers. In septin 7 knockdown (KD) C2C12 cells the parameters describing cellular shape and size were highly modified and cell proliferation and differentiation were suppressed. The reduced septin 7 expression caused severe vertebra deformity, similar to human Scheuermann's disease. In vivo and in vitro force measurements were carried out on septin 7 KD mice and muscles and significant reduction in the contractile parameters (Grip force, twitch, and tetanus) were observed. Voltage activation of calcium transients was examined on enzymatically isolated single FDB fibers under whole cell voltage clamp. Results indicate that the reduced expression of septin 7 does not alter the release channel activation significantly and the ECC machinery remains unaltered and fully operational in the mutant. These finding in skeletal muscle indicate that septin 7 has essential role in the development and differentiation process of myotubes and in proper function of adult skeletal muscle fibers. The research was supported by NKFIH K-115461 and GINOP-2.3.2-15-2016-00044.

Expression of septin 2 and association with clinicopathological parameters in colorectal cancer.

Septin 2 (SEPT2) is a tumor-related gene belonging to the SEPT family that affects the cellular processes of hepatoma carcinoma cells, glioblastoma cells and mesangial cells and is highly expressed in breast cancer, biliary tract cancer and acute myeloid leukemia. Colorectal cancer (CRC) is the third most common type of malignancy in humans. In the present study, Oncomine database was used to compare the expression pattern of SEPT2 mRNA between CRC and normal tissues. Additionally, protein expression in 90 pairs of CRC and paracancerous tissues was analyzed by western blotting and immunohistochemistry (IHC). The results showed that SEPT2 was highly expressed in CRC tissues at the mRNA and protein levels. SEPT2 expression quantified by IHC was associated with lymph node metastasis, the degree of differentiation and TNM staging. Increased SEPT2 wass associated with reduced overall survival (OS) according to Kaplan-Meier analysis. COX proportional hazard analysis indicated that SEPT2 was an independent factor that influenced the OS of patients with CRC. Therefore, SEPT2 was associated with the occurrence, progression and prognosis of CRC and thus, may be a marker and prognostic indicator of CRC.

Septin 7 mediates high glucose-induced podocyte apoptosis

Podocyte depletion is a central pathological mechanism of diabetic nephropathy (DN). Hyperglycemia induced podocyte apoptosis, resulting in podocyte depletion. However, the crucial mechanism of hyperglycemia-induced podocyte apoptosis remains poorly understood. In this study, we evaluated the expression of septin 7, a GTP-binding protein, in glomerular podocytes of patients and mice with DN, and investigated the pro-apoptotic effect of septin 7 on high glucose (HG) induced podocyte apoptosis in vitro. We found septin 7 expression was markedly increased not only in glomerular podocytes of patients and db/db mice with DN but also in cultured podocytes with HG stimulation. Knocking down septin 7 with siRNA could attenuate HG induced podocytes apoptosis and excessive intracellular Ca2+ concentration. This study revealed septin7 may potentially play a proapoptotic role in podocyte under diabetic conditions and may provide a potential target for preventing podocyte apoptosis in DN.

Septin 6 regulates engraftment and lymphoid differentiation potential of murine long-term hematopoietic stem cells

Septins are a family of filament-forming GTP-binding proteins that serve as scaffolds and diffusion barriers in various cellular processes. Septin 6 is known as a fusion partner of mixed-lineage leukemia in infant acute myeloid leukemia. The occurrence of the fusion gene is associated with a reduced expression of septin 6 itself. The role of septin 6 in hematopoiesis and whether it is involved in scaffolds within hematopoietic cells is not known. Septin 6-deficient hematopoietic stem cells (HSCs) present with an increased engraftment potential but altered lymphoid differentiation with a reduced contribution to the T-cell compartment and an increased B-cell contribution. Although multipotent progenitor cells showed a very distinct septin 6 filament organization and intracellular distribution, their function was not impaired by septin 6 deficiency. Our data therefore suggest a regulatory role for septin 6 in long-term HSC function and lymphoid lineage differentiation.

Adenovirus-Mediated Overexpression of Septin 2 Attenuates α-Smooth Muscle Actin Expression and Adventitial Myofibroblast Migration Induced by Angiotensin II

Phenotypic transformation from adventitial fibroblasts (AFs) to myofibroblasts (MFs) is critical for vascular remodeling. Septin 2 was found to be downregulated during the differentiation of AFs to MFs induced by angiotensin II (Ang II); however, the role of septin 2 in this process is still unknown. In this study, we investigate whether septin 2 contributes to the adventitial MF phenotypic modulation caused by Ang II. The decreased level of septin 2 and the increased expression of α-smooth muscle actin (α-SMA), a marker of MFs, were readily observed in Ang II-stimulated MF differentiation. After gene transfer of septin 2, the expression of α-SMA was markedly decreased and the MF migration response to Ang II was inhibited. Furthermore, the inhibition of RhoA, another molecule involved in MF phenotypic modulation, decreased the motility of MFs and the expression of septin 2 triggered in Ang II. Finally, transfection of septin 2 rescued the level of acetyl-α-tubulin in MFs. These findings demonstrate that, as a downstream molecule of RhoA, septin 2 blunted the responses of AFs to Ang II by protecting α-tubulin acetylation, which suggests that septin 2 may serve as a potential therapeutic target for vascular injury.

A critical role of solute carrier 22a14 in sperm motility and male fertility in mice.

We previously identified solute carrier 22a14 (Slc22a14) as a spermatogenesis-associated transmembrane protein in mice. Although Slc22a14 is a member of the organic anion/cation transporter family, its expression profile and physiological role have not been elucidated. Here, we show that Slc22a14 is crucial for sperm motility and male fertility in mice. Slc22a14 is expressed specifically in male germ cells, and mice lacking the Slc22a14 gene show severe male infertility. Although the overall differentiation of sperm was normal, Slc22a14−/− cauda epididymal spermatozoa showed reduced motility with abnormal flagellar bending. Further, the ability to migrate into the female reproductive tract and fertilise the oocyte were also impaired in Slc22a14−/− spermatozoa. The abnormal flagellar bending was thought to be partly caused by osmotic cell swelling since osmotic challenge or membrane permeabilisation treatment alleviated the tail abnormality. In addition, we found structural abnormalities in Slc22a14−/− sperm cells: the annulus, a ring-like structure at the mid-piece–principal piece junction, was disorganised, and expression and localisation of septin 4, an annulus component protein that is essential for the annulus formation, was also impaired. Taken together, our results demonstrated that Slc22a14 plays a pivotal role in normal flagellar structure, motility and fertility in mouse spermatozoa.

Septin 9 isoform expression, localization and epigenetic changes during human and mouse breast cancer progression

IntroductionAltered expression of Septin 9 (SEPT9), a septin coding for multiple isoform variants, has been observed in several carcinomas, including colorectal, head and neck, ovarian and breast, compared to normal tissues. The mechanisms regulating its expression during tumor initiation and progression in vivo and the oncogenic function of its different isoforms remain elusive.MethodsUsing an integrative approach, we investigated SEPT9 at the genetic, epigenetic, mRNA and protein levels in breast cancer. We analyzed a panel of breast cancer cell lines, human primary tumors and corresponding tumor-free areas, normal breast tissues from reduction mammoplasty patients, as well as primary mammary gland adenocarcinomas derived from the polyoma virus middle T antigen, or PyMT, mouse model. MCF7 clones expressing individual GFP-tagged SEPT9 isoforms were used to determine their respective intracellular distributions and effects on cell migration.ResultsAn overall increase in gene amplification and altered expression of SEPT9 were observed during breast tumorigenesis. We identified an intragenic alternative promoter at which methylation regulates SEPT9_v3 expression. Transfection of specific GFP-SEPT9 isoforms in MCF7 cells indicates that these isoforms exhibit differential localization and affect migration rates. Additionally, the loss of an uncharacterized SEPT9 nucleolar localization is observed during tumorigenesis.ConclusionsIn this study, we found conserved in vivo changes of SEPT9 gene amplification and overexpression during human and mouse breast tumorigenesis. We show that DNA methylation is a prominent mechanism responsible for regulating differential SEPT9 isoform expression and that breast tumor samples exhibit distinctive SEPT9 intracellular localization. Together, these findings support the significance of SEPT9 as a promising tool in breast cancer detection and further emphasize the importance of analyzing and targeting SEPT9 isoform-specific expression and function.

Schizophrenia is associated with dysregulation of a Cdk5 activator that regulates synaptic protein expression and cognition

Cyclin-dependent kinase 5 is activated by small subunits, of which p35 is the most abundant. The functions of cyclin-dependent kinase 5 signalling in cognition and cognitive disorders remains unclear. Here, we show that in schizophrenia, a disorder associated with impaired cognition, p35 expression is reduced in relevant brain regions. Additionally, the expression of septin 7 and OPA1, proteins downstream of truncated p35, is decreased in schizophrenia. Mimicking a reduction of p35 in heterozygous knockout mice is associated with cognitive endophenotypes. Furthermore, a reduction of p35 in mice results in protein changes similar to schizophrenia post-mortem brain. Hence, heterozygous p35 knockout mice model both cognitive endophenotypes and molecular changes reminiscent of schizophrenia. These changes correlate with reduced acetylation of the histone deacetylase 1 target site H3K18 in mice. This site has previously been shown to be affected by truncated p35. By restoring H3K18 acetylation with the clinically used specific histone deacetylase 1 inhibitor MS-275 both cognitive and molecular endophenotypes of schizophrenia can be rescued in p35 heterozygous knockout mice. In summary, we suggest that reduced p35 expression in schizophrenia has an impact on synaptic protein expression and cognition and that these deficits can be rescued, at least in part, by the inhibition of histone deacetylase 1.

The Influence of Methylated Septin 9 Gene on RNA and Protein Level in Colorectal Cancer

Colorectal cancer is one of the leading death causes in the world. Specificity and sensitivity of the present screening methods are unsuitable and their compliance is too low. Nowadays the most effective method is the colonoscopy, because it gives not only macroscopic diagnosis but therapeutic possibility as well, however the compliance of the patients is very low. Hence development of new diagnostic methods is needed. Altered expression of septin 9 was found in several tumor types including colorectal cancer. The aim of this study was to detect the methylation related mRNA and protein expression changes of septin 9 in colorectal adenoma-dysplasia-carcinoma sequence and to analyze its reversibility by demethylation treatment. Septin 9 protein expression showed significant difference between normal and colorectal cancer (CRC) samples (p < 0,001). According to biopsy microarray results, septin 9 mRNA expression decreased in the progression of colon neoplastic disease (p < 0,001). In laser microdissected epithelial cells, septin 9 significantly underexpressed in CRC compared to healthy controls (p < 0,001). The expression of septin9_v1 region was higher in the healthy samples, while septin9_v2, v4, v4*, v5 overexpression were detected in cancer epithelial cells compared to normal. The septin 9 mRNA and protein levels of HT29 cells increased after demethylation treatment. The increasing methylation of septin 9 gene during colorectal adenoma-dysplasia-carcinoma sequence progression is reflected in the decreasing mRNA and protein expression, especially in the epithelium. These changes can be reversed by demethylation agents converting this screening marker gene into therapeutic target.

Septin 11 Is Present in GABAergic Synapses and Plays a Functional Role in the Cytoarchitecture of Neurons and GABAergic Synaptic Connectivity

Mass spectrometry and immunoblot analysis of a rat brain fraction enriched in type-II postsynaptic densities and postsynaptic GABAergic markers showed enrichment in the protein septin 11. Septin 11 is expressed throughout the brain, being particularly high in the spiny branchlets of the Purkinje cells in the molecular layer of cerebellum and in the olfactory bulb. Immunofluorescence of cultured hippocampal neurons showed that 54 +/- 4% of the GABAergic synapses and 25 +/- 2% of the glutamatergic synapses had colocalizing septin 11 clusters. Similar colocalization numbers were found in the molecular layer of cerebellar sections. In cultured hippocampal neurons, septin 11 clusters were frequently present at the base of dendritic protrusions and at the bifurcation points of the dendritic branches. Electron microscopy immunocytochemistry of the rat brain cerebellum revealed the accumulation of septin 11 at the neck of dendritic spines, at the bifurcation of dendritic branches, and at some GABAergic synapses. Knocking down septin 11 in cultured hippocampal neurons with septin 11 small hairpin RNAs showed (i) reduced dendritic arborization; (ii) decreased density and increased length of dendritic protrusions; and (iii) decreased GABAergic synaptic contacts that these neurons receive. The results indicate that septin 11 plays important roles in the cytoarchitecture of neurons, including dendritic arborization and dendritic spines, and that septin 11 also plays a role in GABAergic synaptic connectivity.

Expression of Lewy body protein septin 4 in postmortem brain of Parkinson's disease and control subjects

In Parkinson's disease (PD) neuronal degeneration is associated with abnormal protein aggregation in various forms including Lewy bodies (LBs). A major component of LBs is alpha-synuclein; septin 4 (SEPT4), a polymerizing GTP-binding protein that serves as scaffold for diverse molecules has been found to colocalize with alpha-synuclein in LBs. The central role of SEPT4 in the etiopathogenesis of PD has been suggested since SEPT4 also shows a physiological association with alpha-synuclein and serves as a substrate for parkin. To this end, we studied the expression of septin 4 and alpha-synuclein in postmortem human substantia nigra (SN) and amygdala from patients with PD and healthy controls. Twenty patients (14 men : 6 women, onset 63.0 +/- 11.4 years, age 77.3 +/- 7.6 years, Hoehn and Yahr 4.05/5) and 9 neurologically healthy controls (4 men/5 women, age at death 80.1 +/- 8.6 years) were studied. Sporadic PD cases showed a statistically significant decrease of the fold change (FC) of SNCA (FC = 0.31, P = 0.00001) and SEPT4 (FC = 0.67, P = 0.054) gene expressions in the SN and the amygdala (SNCA: FC = 0.49, P = 0.02; SEPT4: FC = 0.32, P = 0.007) versus healthy controls. However, an increase of both proteins in PD versus control subjects was observed with immunoblotting. The semi-quantitative protein ratio calculations revealed more than 10-fold increases for both SEPT4 and alpha-synuclein in PD versus control subjects. We present for the first time similar signal expression patterns and parallel accumulation of SEPT4 and alpha-synuclein in well-characterized postmortem PD brain. Considering the heterogeneous etiology of sporadic PD and the variability of individual human samples, SEPT4 accumulation may be regarded as one of the common pathological changes in PD and should therefore be further explored.

Localization of septin 8 in murine retina, and spatiotemporal expression of septin 8 in a murine model of photoreceptor cell degeneration

The septins, which form a conserved family of cytoskeletal GTP-binding proteins in mammals, comprise stable heteromeric complexes and have diverse roles in protein scaffolding, cytokinesis, vesicle trafficking and plasma membrane integrity following cell division. The goal of this study was to determine the localization of septin 8 in murine adult retina, and analyze the spatiotemporal expression of septin 8 in a murine model of photoreceptor cell degeneration. Expression of septin 8 in the normal retina of mouse and rat was observed by using immunohistochemistry and Western blotting. Furthermore, time course of the expression of septin 8 in mouse photoreceptor cell degeneration were examined by immunohistochemistry combined with hematoxylin and eosin staining, and in situ DNA fragment labeling method. In normal mouse and rat retina, localization of septin 8 is restricted in nuclei of photoreceptor cells. 96 h after intravitreal injection of cobalt chloride most photoreceptor cells lost septin 8 immunostaining at the same time as nuclear DNA fragmentation. The results of this study show that septin 8 protein is present in the specific location within the retina. Furthermore, the disappearance of septin 8 in the nuclei of photoreceptor cells is concomitant with nuclear DNA fragmentation. This suggests that loss of septin 8 could be a useful prognostic indicator for photoreceptor cell degeneration.

Proteomic identification of a role for the von Hippel Lindau tumour suppressor in changes in the expression of mitochondrial proteins and septin 2 in renal cell carcinoma

The von Hippel Lindau (VHL) tumour suppressor gene, VHL, plays a central role in development of sporadic conventional renal cell carcinomas (RCCs). Studying VHL function may, therefore, increase understanding of the pathogenesis of RCC and identify markers/therapeutic targets. Comparison of 2-DE protein profiles of VHL-defective RCC cells (UMRC2) transfected with control vector or wild-type VHL showed differences in 30 proteins, including several novel changes. One of the findings confirmed by Western blotting was up-regulation of the mitochondrial protein ubiquinol cytochrome c reductase complex core protein 2 following VHL transfection, a change that was also observed in two other cell line backgrounds. A marked decrease in expression of this and several other mitochondrial proteins was demonstrated in RCC tissues and using VHL-transfectants, several were shown to exhibit VHL-dependent regulation. Thus, VHL may contribute to the decreased mitochondrial function seen in RCC. A form of septin 2 down-regulated following VHL transfection was also identified. Septin 2 was up-regulated in 12/16 RCCs, while alteration of the form present was also observed in 1/3 tumours analysed. Thus, increased expression of septin 2 is a common event in RCC and protein modification may also alter septin 2 function in a subset of tumours.

Cerebellar protein expression in three different mouse strains and their relevance for motor performance

The present study uses a proteomic approach to link motor function to cerebellar protein expression in 129X1/SvJ, C57BL/6J and nNOS WT mice. Poor performance on the Rota rod, the standard test for motor coordination, was detected in 129X1/SvJ mice. No gross impairments of neurological, cognitive and behavioural functions were observed. Identification and quantification of 48 proteins revealed reduced expression of calbindin, septin 5 and syntaxin binding protein 1 in 129X1/SvJ. In nNos WT glucose-6-phosphate 1 dehydrogenase X was decreased whereas dihydropyrimidinase-related protein-4 was increased. In C57BL/6J stress-70 protein, alpha enolase, NAD-dependent deacetylase sirtuin 2, septin 2, dihydropyrimidinase-related protein-2 and brain derived neurotrophic factor showed elevated levels. Neurological examination, Rota rod test, Morris Water Maze, Multiple-T-Maze, Open field and Elevated plus-maze were employed to study motor, cognitive and behavioural function. Mice were sacrificed and cerebellar tissue was homogenized. Proteins were extracted and separated on two-dimensional gel electrophoresis with subsequent in-gel digestion followed by mass spectrometrical analysis of peptides (MALDI-TOF/TOF-TOF). Quantification of spots was carried out by specific software. A strong association of impaired motor function with altered cerebellar protein expression of calbindin, septin 5 and syntaxin binding protein 1in 129X1/SvJ was observed and is in agreement with previous observations of motor deficiencies in a calbindin knock-out mouse. These results have to be taken into account when using 129X1/SvJ for biochemical, toxicological or gene targeting experiments as well as when studying the above-mentioned proteins or corresponding pathways and cascades in this mouse strain.