Transglutaminase 6 antibodies in gluten neuropathy

BACKGROUND: TG6 antibodies have been shown to be a marker of gluten ataxia but their presence in the context of other neurological manifestations of gluten sensitivity has not been explored. We investigated the presence of TG6 antibodies in gluten neuropathy (GN), defined as as an otherwise idiopathic peripheral neuropathy associated with serological markers of gluten sensitivity (one or more of antigliadin IgG and/or IgA, endomysial and transglutaminase-2 antibodies). METHODS: This was a cross-sectional study conducted at the Sheffield Institute of Gluten Related Diseases, Royal Hallamshire Hospital, Sheffield, UK. Blood samples were collected whilst the patients were on a gluten containing diet. Duodenal biopsies were performed to establish the presence of enteropathy. RESULTS: Twenty-eight patients were recruited (mean age 62.5±13.7 years). Fifteen (53.6%) had sensory ganglionopathy, 12 (42.9%) had symmetrical axonal neuropathy and 1 had mononeuritis multiplex. The prevalence of TG6 antibodies was 14 of 28 (50%) compared to 4% in the healthy population. TG6 antibodies were found in 5/15 (33.3%) patients with sensory ganglionopathy and in 8/12 (66.7%) with symmetrical axonal neuropathy. Twenty-four patients underwent duodenal biopsy 11 (45.8%) of which had enteropathy. The prevalence of TG6 was not significantly different when comparing those with or without enteropathy. CONCLUSIONS: We found a high prevalence of antibodies against TG6 in patients with GN. This suggests that TG6 involvement is not confined to the central nervous system. The role of transglutaminase 6 in peripheral nerve function remains to be determined but TG6 antibodies may be helpful in the diagnosis of GN

Phenytoin-related ataxia in patients with epilepsy: clinical and radiological characteristics

Purpose Phenytoin is an effective anticonvulsant for focal epilepsy. Its use can be associated with long-term adverse effects including cerebellar ataxia. Whilst phenytoin is toxic to Purkinje cells in vitro; the clinical and radiological phenotype and mechanism of cerebellar degeneration in vivo remain unclear. We describe the prevalence, clinical and radiological characteristics of phenytoin-related ataxia. Methods Patients with epilepsy receiving treatment with phenytoin were recruited from the Epilepsy clinics at Royal Hallamshire Hospital, Sheffield, UK. Neurological examination was performed on all patients after recruitment. Patients were categorised into those with and without ataxia. We determined the severity of ataxia clinically (SARA score) and the pattern of cerebellar involvement by neuroimaging (MRI volumetry and MR spectroscopy). Results Forty-seven patients were recruited. Median duration of epilepsy was 24 years, median duration of phenytoin treatment was 15 years and current median phenytoin daily dose was 325 mg. Fifty-five percent of patients complained of poor balance. Clinical evidence of ataxia was seen in 40% patients. Gait, stance and heel-shin slide were the predominant features of cerebellar dysfunction. MRI demonstrated structural, volumetric and functional deficits of the cerebellum. Only one patient with ataxia had phenytoin levels above the normal range. Conclusions Cerebellar ataxia is present in 40% of patients with epilepsy and chronic exposure to phenytoin. Patients on long-term phenytoin have reduced cerebellar volume even if they have no clinical evidence of ataxia. Evidence of structural deficits on imaging suggests a predilection for vermian involvement

Mutations in TGM6 induce the unfolded protein response in SCA35

Spinocerebellar ataxia type 35 (SCA35) is a rare autosomal-dominant neurodegenerative disease caused by mutations in the TGM6 gene, which codes for transglutaminase 6 (TG6). Mutations in TG6 induce cerebellar degeneration by an unknown mechanism. We identified seven patients bearing new mutations in TGM6. To gain insights into the molecular basis of mutant TG6-induced neurotoxicity, we analyzed all of the seven new TG6 mutants and the five TG6 mutants previously linked to SCA35. We found that wild-type (TG6-WT) mainly localized to the nucleus and perinuclear area, whereas five TG6 mutations showed nuclear depletion, increased accumulation in the perinuclear area, insolubility and loss of enzymatic function. Aberrant accumulation of these TG6 mutants in the perinuclear area led to activation of the unfolded protein response (UPR), suggesting that specific TG6 mutants elicit an endoplasmic reticulum (ER) stress response. Mutations associated with activation of the UPR caused death of primary neurons and reduced the survival of novel D. melanogaster models of SCA35. These results indicate that mutations differently impacting on TG6 function cause neuronal dysfunction and death through diverse mechanisms and highlight the UPR as a potential therapeutic target for patient treatment

Transglutaminase 6: a protein associated with central nervous system development and motor function.

Transglutaminases (TG) form a family of enzymes that catalyse various post-translational modifications of glutamine residues in proteins and peptides including intra- and intermolecular isopeptide bond formation, esterification and deamidation. We have characterized a novel member of the mammalian TG family, TG6, which is expressed in a human carcinoma cell line with neuronal characteristics and in mouse brain. Besides full-length protein, alternative splicing results in a short variant lacking the second β-barrel domain in man and a variant with truncated β-sandwich domain in mouse. Biochemical data show that TG6 is allosterically regulated by Ca(2+) and guanine nucleotides. Molecular modelling indicates that TG6 could have Ca(2+) and GDP-binding sites related to those of TG3 and TG2, respectively. Localization of mRNA and protein in the mouse identified abundant expression of TG6 in the central nervous system. Analysis of its temporal and spatial pattern of induction in mouse development indicates an association with neurogenesis. Neuronal expression of TG6 was confirmed by double-labelling of mouse forebrain cells with cell type-specific markers. Induction of differentiation in mouse Neuro 2a cells with NGF or dibutyryl cAMP is associated with an upregulation of TG6 expression. Familial ataxia has recently been linked to mutations in the TGM6 gene. Autoantibodies to TG6 were identified in immune-mediated ataxia in patients with gluten sensitivity. These findings suggest a critical role for TG6 in cortical and cerebellar neurons

TG6 ATAXIA

We have demonstrated that autoantibodies in gluten ataxia recognise a novel neuronal transglutaminase, TG6. TG6 is primarily expressed in neural tissue. We have postulated that anti-TG6 antibodies may prove to be a more sensitive and specific marker for gluten related neurological dysfunction. We investigated if TG6 antibodies can be the sole marker of gluten related neurological dysfunction. We tested 12 consecutive patients with sporadic idiopathic ataxia and no serological evidence of gluten sensitivity (negative for TG2, EMA and IgG and IgA anti-gliadin antibodies) for the presence of anti-TG6. All 12 patients underwent duodenal biopsy for routine histological examination as well as for evidence of IgA deposits against TG in the gut (a specific test for gluten sensitivity). Seven out of the 12 patients had antibodies against TG6. Four had both IgG and IgA, two had only IgG and one had only IgA antibodies. In 3 out of the 7 patients there was evidence of small bowel involvement (one had enteropathy, 2 had increased intraepithelial lymphocytes and all 3 had deposits against TG). These results suggest that anti-TG6 may be the only marker of gluten sensitivity in patients presenting with ataxia even in the presence of an enteropathy

The impact of probiotic bacteria on intestinal barrier function

It has been widely reported that probiotics have a variety of health benefits, however, the underlining mechanisms for these are mainly unknown. This KESS project was a collaboration with Cultech Ltd., a company that produces such a probiotic formulation, called Lab4, which has been shown in several clinical trials to have such benefits for gut health. Our hypothesis was that the health benefits reported, are related to augmentation of epithelial barrier integrity. To evaluate this, we established in vitro models of varying complexity to assess molecular responses of probiotic application to a mature epithelium. My results revealed, that conditioned medium (metabolites) from Lab4 improved barrier integrity and importantly, that this had a protective effect when a subsequent inflammatory stimulus (IL-22) was applied. Furthermore, I found, that inclusion of mucus producing cells altered the epithelial responses to the inflammatory challenge. The main probiotic-mediated change observed, was in the pattern of activation of the MAPK signalling pathway, which is known to control epithelial proliferation. Based on our findings we propose, that short-chain fatty acids (SCFA) are a key active constituent of Lab4 probiotic conditioned media, and that these modulate ERK1/2 activation, and thereby modulate the responses of the epithelial lining and ultimately control barrier integrity. These novel findings provide important insights on actions in the host, driven by probiotic strains of Bifidobacterium and Lactobacillus on a mechanistic level, that significantly advance our current understanding, and may explain how probiotics mediate their health benefits

Transglutaminase gene products

The invention provides a nucleotide sequence comprising at least a portion of the nucleotide sequence of FIG. 10A, FIG. 6B or FIG. 10A or FIG. 10B; nucleotides which hybridise to the nucleotide sequences of FIG. 6A, FIG. 6B or FIG. 10A or FIG. 10B; nucleotides which are degenerate to the nucleotide sequences of FIG. 6A, FIG. 6B or FIG. 10A or FIG. 10B; all of which nucleotides encode a polypeptide having transglutaminase activity

Evolution of transglutaminase genes: identification of a transglutaminase gene cluster on human chromosome 15q15. Structure of the genes encoding transglutaminase X and a novel gene family member, Transglutaminase Z

We isolated and characterized the gene encoding human transglutaminase (TG)X (TGM5) and mapped it to the 15q15.2 region of chromosome 15 by fluorescence in situ hybridization. The gene consists of 13 exons separated by 12 introns and spans about 35 kilobases. Further sequence analysis and mapping showed that this locus contained three transglutaminase genes arranged in tandem: EPB42 (band 4.2 protein), TGM5, and a novel gene (TGM7). A full-length cDNA for the novel transglutaminase (TGZ) was obtained by anchored polymerase chain reaction. The deduced amino acid sequence encoded a protein with 710 amino acids and a molecular mass of 80 kDa. Northern blotting showed that the three genes are differentially expressed in human tissues. Band 4.2 protein expression was associated with hematopoiesis, whereas TGX and TGZ showed widespread expression in different tissues. Interestingly, the chromosomal segment containing the human TGM5, TGM7, and EPB42 genes and the segment containing the genes encoding TGC,TGE, and another novel gene (TGM6) on chromosome 20q11 are in mouse all found on distal chromosome 2 as determined by radiation hybrid mapping. This finding suggests that in evolution these six genes arose from local duplication of a single gene and subsequent redistribution to two distinct chromosomes in the human genome

Crosslinking and G-protein functions of transglutaminase 2 contribute differentially to fibroblast wound healing responses

Tissue transglutaminase (TG2) affects cell-matrix interactions in cell spreading, migration and extracellular matrix (ECM) reorganisation. Using fibroblasts deficient in TG2 or overexpressing normal or crosslinking-deficient enzyme, we show that the extracellular crosslinking activity and intracellular G-protein function in signal transduction contribute differentially to regulation of cell-matrix interactions. TG2-deficient cells displayed normal attachment but delayed spreading on ECM substrata and defects in motility unrelated to crosslinking. Blocking antibodies to TG2 failed to induce similar defects in normal fibroblasts. TG2-deficient fibroblasts had defects in focal adhesion turnover and stress fibre formation, showed changes in focal adhesion kinase (FAK) phosphorylation and failed to activate protein kinase C (PKC). Phospholipase C (PLC) and PKC inhibitors blocked spreading of normal fibroblasts whilst PKC activators induced spreading in TG2-deficient cells. In contrast, ECM remodelling was not only compromised by TG2 deficiency but also by overexpression of dominant negative enzyme and TG inhibitors. TG2 activity increased matrix tension and was required for membrane type 1-MMP (MT1-MMP)-dependent activation of MMP-2. Our results demonstrate that TG2 is involved in the control of dynamic adhesion formation in cell spreading and migration via regulation of phospholipase C activity. By virtue of its crosslinking activity, the enzyme plays a central role in regulating ECM remodelling

Transglutaminase 6 antibodies in the diagnosis of gluten ataxia

Objectives: The previous finding of an immunologic response primarily directed against transglutaminase (TG)6 in patients with gluten ataxia (GA) led us to investigate the role of TG6 antibodies in diagnosing GA. Methods: This was a prospective cohort study. We recruited patients from the ataxia, gluten/neurology, celiac disease (CD), and movement disorder clinics based at Royal Hallamshire Hospital (Sheffield, UK) and the CD clinic, Tampere University Hospital (Tampere, Finland). The groups included patients with idiopathic sporadic ataxia, GA, and CD, and neurology and healthy controls. All were tested for TG6 antibodies. Duodenal biopsies were performed in patients with positive serology. In addition, biopsies from 15 consecutive patients with idiopathic sporadic ataxia and negative serology for gluten-related disorders were analyzed for immunoglobulin A deposits against TG. Results: The prevalence of TG6 antibodies was 21 of 65 (32%) in idiopathic sporadic ataxia, 35 of 48 (73%) in GA, 16 of 50 (32%) in CD, 4 of 82 (5%) in neurology controls, and 2 of 57 (4%) in healthy controls. Forty-two percent of patients with GA had enteropathy as did 51% of patients with ataxia and TG6 antibodies. Five of 15 consecutive patients with idiopathic sporadic ataxia had immunoglobulin A deposits against TG2, 4 of which subsequently tested positive for TG6 antibodies. After 1 year of gluten-free diet, TG6 antibody titers were significantly reduced or undetectable. Conclusions: Antibodies against TG6 are gluten-dependent and appear to be a sensitive and specific marker of GA