Hyperactivity and cortical disinhibition in mice with restricted expression of mutant huntingtin to parvalbumin-positive cells.

Recent evidence suggests that interneurons are involved in the pathophysiology of Huntington Disease (HD). Abnormalities in the function of interneurons expressing the calcium buffer parvalbumin (PV) have been observed in multiple mouse models of HD, although it is not clear how PV-positive interneuron dysfunction contributes to behavioral and synaptic deficits. Here, we use the cre-lox system to drive expression of mutant huntingtin (mthtt) in parvalbumin (PV)-positive neurons and find that mutant mice exhibit diffuse mthtt immunoreactivity in PV-rich areas at 10months of age and mthtt aggregates in PV-positive processes at 24months of age. At midlife, mutant mice are hyperactive and display impaired GABA release in the motor cortex, characterized by reduced miniature inhibitory events and severely blunted responses to gamma frequency stimulation, without a loss of PV-positive interneurons. In contrast, 24month-old mutant mice show normalized behavior and responses to gamma frequency stimulation, possibly due to compensatory changes in pyramidal neurons or the formation of inclusions with age. These data indicate that mthtt expression in PV-positive neurons is sufficient to drive a hyperactive phenotype and suggest that mthtt-mediated dysfunction in PV-positive neuronal populations could be a key factor in the hyperkinetic behavior observed in HD. Further clarification of the roles for specific PV-positive populations in this phenotype is warranted to definitively identify cellular targets for intervention.

Purkinje cell dysfunction and loss in a knock-in mouse model of Huntington disease.

Huntington Disease (HD) is an autosomal dominant neurological disorder characterized by motor, psychiatric and cognitive disturbances. Recent evidence indicates that the viability and function of cerebellar Purkinje cells (PCs) are compromised in an aggressive mouse model of HD. Here we investigate whether this is also the case in the HdhQ200 knock-in mouse model of HD. Using quantitative-real time-PCR and immunofluorescence, we observed a loss of the PC marker and calcium buffer calbindin in 50week-old symptomatic mice. Reductions were also observed in parvalbumin and glutamic acid decarboxylase protein expression, most markedly in the molecular cell layer. Stereological analysis revealed an overall reduction in the PC population in HdhQ200/Q200 mice by nearly 40%, and loose patch electrophysiology of remaining PCs indicated a reduction in firing rate in HD mice compared to control littermates. Taken together, these data demonstrate that PC survival and function are compromised in a mouse model of adult-onset HD and suggest that further experiments should investigate the contribution of PC death and dysfunction to HD-associated motor impairment.

Disruption of Purkinje cell function prior to huntingtin accumulation and cell loss in an animal model of Huntington disease.

Huntington Disease (HD) is a devastating neurological disorder characterized by progressive deterioration of psychiatric, motor, and cognitive function. Purkinje cells (PCs), the output neurons of the cerebellar cortex, have been found to be vulnerable in multiple CAG repeat disorders, but little is known about the involvement of PC dysfunction in HD. To investigate possible PC abnormalities, we performed quantitative real time PCR, Western blot analysis, and immunohistochemistry experiments to explore the changes in PC markers in the R6/2 mouse model of severe HD. There were reductions in the transcript and protein levels of the calcium-binding proteins parvalbumin and calbindin, as well as the enzyme glutamic acid decarboxylase 67. Immunohistochemistry supported these results, with the most substantial changes occurring in the PC layer. To determine whether the reductions in PC marker expression were due to cell loss, we performed stereology on both presymptomatic and end-stage R6/2 mice. Stereological counts indicated a significant reduction in PC number by end-stage but no change in presymptomatic animals (4 weeks of age). To assess cellular function prior to cell loss and symptom onset, we measured spontaneous firing in PCs from 4-week old animals and found a striking deficit in PC firing as indicated by a 57% decrease in spike rate. Interestingly, huntingtin inclusions were not widely observed in PCs until 12 weeks of age, indicating that soluble huntingtin and/or abnormalities in other cell types may contribute to PC dysfunction. Considering the roles for PCs in motor control, these data suggest that early PC dysfunction potentially contributes to motor impairment in this model of HD.

Tissue transglutaminase selectively modifies proteins associated with truncated mutant huntingtin in intact cells.

The cause of Huntington's disease (HD) is a pathological expansion of the polyglutamine domain within the N-terminal region of huntingtin. Neuronal intranuclear inclusions and cytoplasmic aggregates composed of the mutant huntingtin within certain neuronal populations are a characteristic hallmark of HD. However, how the expanded polyglutamine repeats of mutant huntingtin cause HD is not known. Because in vitro expanded polyglutamine repeats are excellent glutaminyl-donor substrates of tissue transglutaminase (tTG), it has been hypothesized that tTG may contribute to the formation of these aggregates in HD. However, an association between huntingtin and tTG or modification of huntingtin by tTG has not been demonstrated in cells. To examine the interactions between tTG and huntingtin human neuroblastoma SH-SY5Y cells were stably transfected with full-length huntingtin containing 23 (FL-Q23) (wild type) or 82 (FL-Q82) (mutant) glutamine repeats or a truncated N-terminal huntingtin construct containing 23 (Q23) (wild type) or 62 (Q62) (mutant) glutamine repeats. Aggregates were rarely observed in the cells expressing full-length mutant huntingtin, and no specific colocalization of full-length huntingtin and tTG was observed. In contrast, in cells expressing truncated mutant huntingtin (Q62) there were numerous complexes of truncated mutant huntingtin and many of these complexes co-localized with tTG. However, the complexes were not insoluble structures. Further, truncated huntingtin coimmunoprecipitated with tTG, and this association increased when tTG was activated. Activation of tTG did not result in the modification of either truncated or full-length huntingtin, however proteins that were associated with truncated mutant huntingtin were selectively modified by tTG. This study is the first to demonstrate that tTG specifically interacts with a truncated form of huntingtin, and that activated tTG selectively modifies mutant huntingtin-associated proteins. These data suggest that proteolysis of full-length mutant huntingtin likely precedes its interaction with tTG and this process may facilitate the modification of huntingtin-associated proteins and thus contribute to the etiology of HD.

Cholinergic- and stress-induced signaling activities in cells overexpressing wild-type and mutant presenilin-1.

This study examined the effects of overexpression of presenilin-1 wild-type (PS1wt) or mutant L286V (PS1m) in human neuroblastoma SH-SY5Y cells on signal transduction systems. Oxotremorine-M-induced activation of AP-1 was 40--53% lower in PS1wt than control cells, and further impaired (63--76%) in PS1m cells. Heat shock (45 degrees C) activated Akt, increased heat shock factor-1 (HSF-1) DNA binding activity, and increased levels of heat shock protein 70, and these responses were not altered by overexpression of PS1wt or PS1m. H(2)O(2) also caused a time-dependent increase in HSF-1 DNA binding activity which was similar in all cell lines. Thus, overexpression of PS1wt reduced muscarinic receptor-mediated activation of AP-1, and PS1m overexpression caused greater inhibition, but stress-induced activation of Akt and HSF-1 was unaffected by either PS1wt or PS1m.

Tissue transglutaminase does not contribute to the formation of mutant huntingtin aggregates.

The cause of Huntington's disease (HD) is a pathological expansion of the polyglutamine domain within the NH(2)-terminal region of huntingtin. Neuronal intranuclear inclusions and cytoplasmic aggregates composed of the mutant huntingtin within certain neuronal populations are a characteristic hallmark of HD. Because in vitro expanded polyglutamine repeats are glutaminyl-donor substrates of tissue transglutaminase (tTG), it has been hypothesized that tTG may contribute to the formation of these aggregates in HD. Therefore, it is of fundamental importance to establish whether tTG plays a significant role in the formation of mutant huntingtin aggregates in the cell. Human neuroblastoma SH-SY5Y cells were stably transfected with truncated NH(2)-terminal huntingtin constructs containing 18 (wild type) or 82 (mutant) glutamines. In the cells expressing the mutant truncated huntingtin construct, numerous SDS-resistant aggregates were present in the cytoplasm and nucleus. Even though numerous aggregates were present in the mutant huntingtin-expressing cells, tTG did not coprecipitate with mutant truncated huntingtin. Further, tTG was totally excluded from the aggregates, and significantly increasing tTG expression had no effect on the number of aggregates or their intracellular localization (cytoplasm or nucleus). When a YFP-tagged mutant truncated huntingtin construct was transiently transfected into cells that express no detectable tTG due to stable transfection with a tTG antisense construct, there was extensive aggregate formation. These findings clearly demonstrate that tTG is not required for aggregate formation, and does not facilitate the process of aggregate formation. Therefore, in HD, as well as in other polyglutamine diseases, tTG is unlikely to play a role in the formation of aggregates.

Tissue transglutaminase is essential for neurite outgrowth in human neuroblastoma SH-SY5Y cells.

Tissue transglutaminase is a normal constituent of the central and peripheral nervous systems and in rats transglutaminase activity in brain and spinal cord is highest during fetal stages when axonal outgrowth is occurring. Further, treatment of human neuroblastoma SH-SY5Y cells with retinoic acid results in the cells withdrawing from the cell cycle and extending neurites, in the same time frame that tissue transglutaminase expression significantly increases. Considering these and other previous findings, this study was carried out to determine whether tissue transglutaminase is involved in neuronal differentiation of SH-SY5Y cells. For these studies SH-SY5Y cells stably overexpressing wild-type tissue transglutaminase, an inactive tissue transglutaminase mutant (C277S) or an antisense tissue transglutaminase construct (which decreased endogenous tissue transglutaminase below detectable levels) were used. SH-SY5Y cells overexpressing wild-type tissue transglutaminase spontaneously differentiated into a neuronal phenotype when grown in low-serum media. In contrast, cells overexpressing inactive tissue transglutaminase or the antisense tissue transglutaminase continued to proliferate and exhibit a flat polygenic morphology even when maintained in low-serum conditions. In addition, increased tissue transglutaminase expression in response to retinoic acid was abolished in the antisense tissue transglutaminase cells, and antisense and mutant tissue transglutaminase expressing cells did not extend neurites in response to retinoic acid. Moreover, wild-type and inactive tissue transglutaminase exhibited differential intracellular localization. These data indicate that tissue transglutaminase is necessary and sufficient for neuronal differentiation of human neuroblastoma SH-SY5Y cells.

Impaired mitochondrial function results in increased tissue transglutaminase activity in situ.

Tissue transglutaminase (tTG) is a transamidating enzyme that is elevated in Huntington's disease (HD) brain and may be involved in the etiology of the disease. Further, there is evidence of impaired mitochondrial function in HD. Therefore, in this study, we examined the effects of mitochondrial dysfunction on the transamidating activity of tTG. Neuroblastoma SH-SY5Y cells stably overexpressing human tTG or mutated inactive tTG were treated with 3-nitropropionic acid (3-NP), an irreversible inhibitor of succinate dehydrogenase. 3-NP treatment of tTG-expressing cells resulted in a significant increase of TG activity in situ. In vitro measurements demonstrated that 3-NP had no direct effect on tTG activity. However, 3-NP treatment resulted in a significant decrease of the levels of GTP and ATP, two potent inhibitors of the transamidating activity of tTG. No significant changes in the intracellular levels of calcium were observed in 3-NP-treated cells. Treatment with 3-NP in combination with antioxidants significantly reduced the 3-NP-induced increase in in situ TG activity, demonstrating that oxidative stress is a contributing factor to the increase of TG activity. This study demonstrates for the first time that impairment of mitochondrial function significantly increases TG activity in situ, a finding that may have important relevance to the etiology of HD.

Insulin-like growth factor-1 and insulin mediate transient site-selective increases in tau phosphorylation in primary cortical neurons.

The modulation of tau phosphorylation and localization in response to insulin-like growth factor-1 or insulin was examined in primary cultures of rat cortical neurons. Insulin and insulin-like growth factor-1 treatment resulted in a rapid and transient increase in tau phosphorylation at specific epitopes. These effects were completely inhibited by lithium, revealing that the insulin and insulin-like growth factor-1 induced changes in tau phosphorylation were mediated by glycogen synthase kinase-3beta. In addition, the increase in tau phosphorylation directly correlated with a transient dissociation of tau from the cytoskeleton, indicating that insulin and insulin-like growth factor-1 treatment resulted in a change in tau localization. Using immunocytochemistry, it was also demonstrated that treatment of neurons with insulin-like growth factor-1 for 3 min resulted in a redistribution of tau to the growth cone and the distal segment of the axons. Further, insulin-like growth factor-1 treatment resulted in an increased immunoreactivity with the phospho-dependent antibody AT8 in the same areas of the axons. Thus, the phosphorylation state and distribution of tau can be modulated by insulin and insulin-like growth factor-1 signaling pathways involving glycogen synthase kinase-3beta. We propose that by transiently increasing tau phosphorylation, insulin and insulin-like growth factor-1 may contribute to the reorganization of the cytoskeleton necessary for the development and growth of the neurites.

Tissue transglutaminase: a possible role in neurodegenerative diseases.

Tissue transglutaminase is a multifunctional protein that is likely to play a role in numerous processes in the nervous system. Tissue transglutaminase posttranslationally modifies proteins by transamidation of specific polypeptide bound glutamines. This action results in the formation of protein crosslinks or the incorporation of polyamines into substrate proteins, modifications that likely have significant effects on neural function. Tissue transglutaminase is a unique member of the transglutaminase family as in addition to catalyzing the calcium-dependent transamidation reaction, it also binds and hydrolyzes ATP and Guanosine 5'-triphosphate and may play a role in signal transduction. Tissue transglutaminase is a highly regulated and inducible enzyme that is developmentally regulated in the nervous system. In vitro, numerous substrates of tissue transglutaminase have been identified, and several of these proteins have been shown to be in situ substrates as well. Several specific roles for tissue transglutaminase have been described and there is evidence that tissue transglutaminase may also play a role in apoptosis. Recent findings have provided evidence that dysregulation of tissue transglutaminase may contribute to the pathology of several neurodegenerative conditions including Alzheimer's disease and Huntington's disease. In both of these diseases tissue transglutaminase and transglutaminase activity are elevated compared to age-matched controls. Further, immunohistochemical studies have demonstrated that there is an increase in tissue transglutaminase reactivity in affected neurons in both Alzheimer's and Huntington's disease. Although intriguing, many issues remain to be addressed to definitively establish a role for tissue transglutaminase in these neurodegenerative diseases.

Tissue transglutaminase is increased in Huntington's disease brain.

The polyglutamine-expanded N-terminal region of mutant huntingtin causes neurodegeneration in Huntington's disease (HD). Neuronal intranuclear and cytosolic inclusions composed of mutant huntingtin are found in brains of HD patients. Because tissue transglutaminase cross-links proteins into filamentous aggregates and polypeptide-bound glutamines are primary determining factors for tissue transglutaminase-catalyzed reactions, it has been hypothesized that tissue transglutaminase may contribute to the formation of these aggregates. In this report immunohistochemical and biochemical methods were used to demonstrate that tissue transglutaminase expression and transglutaminase activity are elevated in HD brains in a grade-dependent manner. In the striatum, tissue transglutaminase activity was significantly increased in the grade 3 HD cases compared with controls. When normalized to the neuronal marker calbindin D28k, immunoblot analysis revealed that in the striatum the levels of tissue transglutaminase were significantly increased in all HD cases compared with controls. Immunohistochemical staining of the HD striatum revealed that tissue transglutaminase immunoreactivity was markedly increased in all grades as compared with controls. In the superior frontal cortex, tissue transglutaminase activity was significantly higher in all HD cases as compared with controls. Quantitative analysis of immunoblots demonstrated that tissue transglutaminase levels were elevated in HD grades 2 and 3 cases. Tissue transglutaminase immunoreactivity within the superior frontal neocortex was also greater in all the HD cases compared with controls. These data clearly indicate that tissue transglutaminase is elevated in HD brain and may play a role in the disease process.

Novel bimodal effects of the G-protein tissue transglutaminase on adrenoreceptor signalling.

Tissue transglutaminase (tTG) is a novel G-protein that previous studies showed can couple ligand-bound activated alpha(1B) adrenoreceptors to phospholipase C-delta, resulting in phosphoinositide (PI) hydrolysis. In human neuroblastoma SH-SY5Y cells we found that although endogenous tTG can facilitate alpha(1B) adrenoreceptor-stimulated PI hydrolysis, its contribution is minor compared with the classical heterotrimeric G-protein G(q/11). Further, we show that the alpha(1B) adrenoreceptor recruits tTG to the membrane and that this recruitment is enhanced by agonist occupancy of the receptor. In addition, the effects of tTG on signalling are bimodal. At low expression levels, tTG enhanced alpha(1B) adrenoreceptor-stimulated PI hydrolysis, whereas at higher expression levels tTG attenuated significantly this response. These findings are the first to demonstrate that a protein can both facilitate and attenuate receptor-stimulated PI hydrolysis.

Toxic neuronal apoptosis and modifications of tau and APP gene and protein expressions.

The causes and the mechanisms of neuronal death in Alzheimer's disease are not elucidated, although some new insights have been proposed over the past years, including free-radical toxicity, beta-amyloid toxicity, excitotoxicity, and disturbed cellular calcium metabolism. Some authors have also pointed out that apoptosis could play a role in neuronal degeneration, but it is still largely debated. Here, we review some recent data linking the induction of experimental neuronal apoptosis in vitro and the molecular pathology of the tau protein and amyloid precursor protein (APP). In cultures exposed to mild glutamate toxicity, tau mRNA expression, not beta-actin, is enhanced in stressed neurons. The Guam cycad toxin metabolite methylazoxymethanol also produces an increase of tau gene transcription that exacerbates changes induced by glutamate. In serum-deprived cultures or glutamate-exposed cultures, neurons committed to apoptosis have a reduced tau gene expression, whereas resistant neurons display a stable or even augmented tau mRNA expression accompanied by a persistent tau phosphorylation near serine 202. In the same conditions, stressed neurons produce membrane blebbings strongly immunopositive for APP and putative amyloidogenic fragments that are subsequently released in the extracellular space. Experimental apoptosis in neurons can recapitulate tau and APP modifications that could be associated with a selective vulnerability and a progression of cellular degeneration along the neuronal network.

Insulin transiently increases tau phosphorylation: involvement of glycogen synthase kinase-3beta and Fyn tyrosine kinase.

The modulation of tau phosphorylation in response to insulin was examined in human neuroblastoma SH-SY5Y cells. Insulin treatment resulted in a transient increase in tau phosphorylation followed by a decrease in tau phosphorylation that correlated directly with a sequential activation and deactivation of glycogen synthase kinase-3beta (GSK-3beta). The insulin-induced increase in tau phosphorylation and concurrent activation of GSK-3beta was rapid (<2 min) and transient, and was associated with increased tyrosine phosphorylation of GSK-3beta. The increase in GSK-3beta tyrosine phosphorylation corresponded directly to an increase in the association of Fyn tyrosine kinase with GSK-3beta, and Fyn immunoprecipitated from cells treated with insulin for 1 min phosphorylated GSK-3beta to a significantly greater extent than Fyn immunoprecipitated from control cells. Subsequent to the increase in GSK-3beta activation and tau phosphorylation, treatment of cells with insulin for 60 min resulted in a dephosphorylation of tau and a decrease in GSK-3beta activity. Thus, insulin rapidly and transiently activated GSK-3beta and modulated tau phosphorylation, alterations that may contribute to neuronal plasticity.

The Guam cycad toxin methylazoxymethanol damages neuronal DNA and modulates tau mRNA expression and excitotoxicity.

As in Alzheimer's disease, brains of Guam Chamorros with amyotrophic lateral sclerosis (ALS) and Parkinsonism-dementia complex (PDC) contain intraneuronal-paired helical filaments composed of accumulated phosphorylated tau protein. Tau mRNA expression in rat neuronal cultures-normally modulated by glutamate-increases after treatment with the aglycone of cycasin, a cycad-derived toxin whose concentration in Chamorro food varies with disease incidence. Elevated Tau gene expression in vitro is coincident with increased cycasin-related DNA adducts and reduced DNA repair. Cycasin and endogenous glutamate may together promote the accumulation of tau protein and neuronal degeneration in Western Pacific ALS/PDC.

Glycogen synthase kinase-3beta, beta-catenin, and tau in postmortem bipolar brain.

Therapeutic concentrations of the anti-bipolar drug lithium inhibit the activity of glycogen synthase kinase-3beta, which raises the possibility that this enzyme and its substrates may be altered in the brain of subjects with bipolar disorder. Therefore, in prefrontal cortical samples from subjects with bipolar disorder and age-matched control subjects, we examined the levels of glycogen synthase kinase 3beta and of two proteins modified by it, beta-catenin and the microtubule associated protein tau. There were no significant differences between subject groups among these measurements, but there was a tendency for the tau isoform profile to be modified in bipolar tissue. Thus, while there are no differences between bipolars and controls in prefrontal cortical levels of glycogen synthase kinase-3beta, beta-catenin, or tau, tau isoform levels or phosphorylation states may be modified in bipolar disorder.

Characterization of human presenilin 1 transgenic rats: increased sensitivity to apoptosis in primary neuronal cultures.

Mutations in the gene for presenilin 1 are causative for the majority of cases of early onset familial Alzheimer's disease. Yet, the physiological function of presenilin 1 and the pathological mechanisms of the mutations leading to Alzheimer's disease are still unknown. To analyse potential pathological effects of presenilin 1 over-expression, we have generated transgenic rats which express high levels of human presenilin 1 protein in the brain. The over-expression of presenilin 1 leads to saturation of its normal processing and to the appearance of full-length protein in the transgenic rat brain. The transgenic protein is expressed throughout the brain and is predominantly found in neuronal cells. Cultured primary cortical neurons derived from these transgenic rats are significantly more sensitive than non-transgenic controls to apoptosis induced by standard culture conditions and to apoptosis induced by trophic factor withdrawal. Furthermore, the observed apoptosis is directly correlated with the expression of the transgenic protein. The results further emphasize the role of presenilin 1 in apoptotic cell death in native neuronal cultures.

FK506 antagonizes apoptosis and c-jun protein expression in neuronal cultures.

FK506 is an immunosuppressive drug that binds to FK506 binding protein (FKBPs), a subgroup of cytosolic proteins called immunophillins. Previous works have revealed that FK506 protects neural cells from ischemia or excitotoxicity. Here we report that FK506 (10(-6) M) and not cyclosporine A (10(-6) M) blocks neuronal apoptosis induced by serum deprivation in rat neuronal cultures. In addition the immunohistochemical staining of C-jun protein in deprived cultures is markedly attenuated by FK506. The proportion of C-jun-positive neurons in control cultures, in serum-deprived cultures (48 h) and in serum-deprived cultures exposed to FK506 (10(-6) M) were 12.5%, 56.5% and 16.5%, respectively. The down-regulation of C-jun could play a major role in the anti-apoptotic action of FK506 in stressed neuronal cultures.

Distinct nuclear localization and activity of tissue transglutaminase.

Tissue transglutaminase is a calcium-dependent transamidating enzyme that has been postulated to play a role in the pathology of expanded CAG repeat disorders with polyglutamine expansions expressed within the affected proteins. Because intranuclear inclusions have recently been shown to be a common feature of many of these codon reiteration diseases, the nuclear localization and activity of tissue transglutaminase was examined. Subcellular fractionation of human neuroblastoma SH-SY5Y cells demonstrated that 93% of tissue transglutaminase is localized to the cytosol. Of the 7% found in the nucleus, 6% copurified with the chromatin-associated proteins, and the remaining 1% was in the nuclear matrix fraction. In situ transglutaminase activity was measured in the cytosolic and nuclear compartments of control cells, as well as cells treated with the calcium-mobilizing agent maitotoxin to increase endogenous tissue transglutaminase activity. These studies revealed that tissue transglutaminase was activated in the nucleus, a finding that was further supported by cytochemical analysis. Immunofluorescence studies revealed that nuclear proteins modified by transglutaminase exhibited a discrete punctate, as well as a diffuse staining pattern. Furthermore, different proteins were modified by transglutaminase in the nucleus compared with the cytosol. The results of these experiments clearly demonstrate localization of tissue transglutaminase in the nucleus that can be activated. These findings may have important implications in the formation of the insoluble nuclear inclusions, which are characteristic of codon reiteration diseases such as Huntington's disease and the spinocerebellar ataxias.

Modulation of the in situ activity of tissue transglutaminase by calcium and GTP.

Tissue transglutaminase (tTG) is a calcium-dependent enzyme that catalyzes the posttranslational modification of proteins by transamidation of specific polypeptide-bound glutamine residues. Previous in vitro studies have demonstrated that the transamidating activity of tTG requires calcium and is inhibited by GTP. To investigate the endogenous regulation of tTG, a quantitative in situ transglutaminase (TG) activity assay was developed. Treatment of human neuroblastoma SH-SY5Y cells with retinoic acid (RA) resulted in a significant increase in tTG levels and in vitro TG activity. In contrast, basal in situ TG activity did not increase concurrently with RA-induced increased tTG levels. However, stimulation of cells with the calcium-mobilizing drug maitotoxin (MTX) resulted in increases in in situ TG activity that correlated (r2 = 0.76) with increased tTG levels. To examine the effects of GTP on in situ TG activity, tiazofurin, a drug that selectively decreases GTP levels, was used. Depletion of GTP resulted in a significant increase in in situ TG activity; however, treatment of SH-SY5Y cells with a combination of MTX and tiazofurin resulted in significantly less in situ TG activity compared with treatment with MTX alone. This raised the possibility of calcium-dependent proteolysis due to the effects of tiazofurin, because in vitro GTP protects tTG against proteolysis by trypsin. Studies with a selective membrane permeable calpain inhibitor indicated that tTG is likely to be an endogenous substrate of calpain, and that depletion of GTP increases tTG degradation after elevation of intracellular calcium levels. TG activity was also increased in response to activation of muscarinic cholinergic receptors, which increases intracellular calcium through inositol 1,4,5-trisphosphate generation. The results of these experiments demonstrate that selective changes in calcium and GTP regulate the activity and levels of tTG in situ.