Craig D. C. Bailey

Cystamine treatment is neuroprotective in the YAC128 mouse model of Huntington disease

Huntington disease (HD) is an adult onset neurodegenerative disorder characterized by selective atrophy and cell loss within the striatum. There is currently no treatment that can prevent the striatal neuropathology. Transglutaminase (TG) activity is increased in HD patients, is associated with cell death, and has been suggested to contribute to striatal neuronal loss in HD. This work assesses the therapeutic potential of cystamine, an inhibitor of TG activity with additional potentially beneficial effects. Specifically, we examine the effect of cystamine on striatal neuronal loss in the YAC128 mouse model of HD. We demonstrate here for the first time that YAC128 mice show a forebrain‐specific increase in TG activity compared with wild‐type (WT) littermates which is decreased by oral delivery of cystamine. Treatment of symptomatic YAC128 mice with cystamine starting at 7 months prevented striatal neuronal loss. Cystamine treatment also ameliorated the striatal volume loss and striatal neuronal atrophy observed in these animals, but was unable to prevent motor dysfunction or the down‐regulation of dopamine and cyclic adenosine monophsophate‐regulated phosphoprotein (DARPP‐32) expression in the striatum. While the exact mechanism responsible for the beneficial effects of cystamine in YAC128 mice is uncertain, our findings suggest that cystamine is neuroprotective and may be beneficial in the treatment of HD.

Tissue transglutaminase contributes to disease progression in the R6/2 Huntington's disease mouse model via aggregate‐independent mechanisms

Huntingtons disease (HD) is caused by an expansion of CAG repeats within the huntingtin gene and is characterized by intraneuronal mutant huntingtin protein aggregates. In order to determine the role of tissue transglutaminase (tTG) in HD aggregate formation and disease progression, we cross‐bred the R6/2 HD mouse model with a tTG knockout mouse line. R6/2 mice that were tTG heterozygous knockouts (R6/2 : tTG+/–) and tTG homozygous knockouts (R6/2 : tTG–/–) showed a very similar increase in aggregate number within the striatum compared with R6/2 mice that were wild‐type with respect to tTG (R6/2 : tTG+/+). Interestingly, a significant delay in the onset of motor dysfunction and death occurred in R6/2 : tTG–/– mice compared with both R6/2 : tTG+/+ and R6/2 : tTG+/– mice. As aggregate number was similarly increased in the striatum of both R6/2 : tTG+/– and R6/2 : tTG–/– mice, whereas only R6/2 : tTG–/– mice showed delayed disease progression, these data suggest that the contribution of tTG towards motor dysfunction and death in the R6/2 mouse is independent of its ability to negatively regulate aggregate formation. Moreover, the combined results from this study suggest that the formation of striatal huntingtin aggregates does not directly influence motor dysfunction or death in this HD mouse model.

Transglutaminase 2 protects against ischemic insult, interacts with HIF1β, and attenuates HIF1 signaling

Transglutaminase 2 (TG2) is a multifunctional enzyme that has been implicated in the pathogenesis of neurodegenerative diseases, ischemia, and stroke. The mechanism by which TG2 modulates disease progression have not been elucidated. In this study we investigate the role of TG2 in the cellular response to ischemia and hypoxia. TG2 is up‐regulated in neurons exposed to oxygen and glucose deprivation (OGD), and increased TG2 expression protects neurons against OGD‐induced cell death independent of its transamidating activity. We identified hypoxia inducible factor 1β (HIF1β) as a TG2 binding partner. HIF1β and HIF1α together form the heterodimeric transcription factor hypoxia inducible factor 1 (HIF1). TG2 and the transaminase‐inactive mutant C277S‐TG2 inhibited a HIF‐dependent transcription reporter assay under hypoxic conditions without affecting nuclear protein levels for HIF1α or HIF1β, their ability to form the HIF1 heterodimeric transcription factor, or HIF1 binding to its DNA response element. Interestingly, TG2 attenuates the up‐regulation of the HIF‐dependent proapoptotic gene Bnip3 in response to OGD but had no effect on the expression of VEGF, which has been linked to prosurvival processes. This study demonstrates for the first time that TG2 protects against OGD, interacts with HIF1β, and attenuates the HIF1 hypoxic response pathway. These results indicate that TG2 may play an important role in protecting against the delayed neuronal cell death in ischemia and stroke.—Filiano, A. J., Bailey, C. D. C., Tucholski, J., Gundemir, S., Johnson, G. V. W. Transglutaminase 2 protects against ischemic insult, interacts with HIF1β, and attenuates HIF1 signaling. FASEB J. 22, 2662–2675 (2008)

The protective effects of cystamine in the R6/2 Huntington's disease mouse involve mechanisms other than the inhibition of tissue transglutaminase

Tissue transglutaminase (tTG) is a multifunctional enzyme that contributes to disease progression in mouse models of Huntingtons disease (HD), an inherited neurodegenerative disease that shows an age-related onset. Moreover, administration of the transglutaminase inhibitor cystamine delays the onset of pathology in the R6/2 HD mouse model. However, the contribution of tTG inhibition towards the therapeutic effects of cystamine has not been determined, as this compound likely has multiple mechanisms of action in the R6/2 mouse. In this study, we found that administration of cystamine in drinking water delayed the age of onset for motor dysfunction and extended lifespan to a similar extent in R6/2 mice that had a normal genetic complement of tTG compared with R6/2 mice that did not express tTG. Since the magnitude of cystamines therapeutic effects was not affected by the genetic deletion of tTG, these results suggest that the mechanism of action for cystamine in this HD mouse model involves targets other than tTG inhibition.

Developmental regulation of tissue transglutaminase in the mouse forebrain

Tissue transglutaminase (tTG) is a multifunctional enzyme that catalyzes both transamidation and GTPase reactions. In cell culture models tTG‐mediated transamidation positively regulates many processes that occur in vivo during the mammalian brain growth spurt (BGS), including neuronal differentiation, neurite outgrowth, synaptogenesis and cell death mechanisms. However, little is known about the levels of tTG expression and transglutaminase (TG) activity during mammalian brain development. In this study, C57BL/6 mouse forebrains were collected at embryonic day (E) 12, E14, E17, postnatal day (P) 0, P7 and P56 and analyzed for tTG expression and TG activity. RT–PCR analysis demonstrated that tTG mRNA content increases during mouse forebrain development, whereas immunoblot analysis demonstrated that tTG protein content decreases during this time. TG activity was low in prenatal mouse forebrain but increased fivefold to peak at P0, which corresponds with the beginning of the mouse BGS. Further analysis demonstrated that the lack of temporal correlation between tTG protein content and TG activity is the result of an endogenous inhibitor of tTG that is present in prenatal but not postnatal mouse forebrain. These results demonstrate for the first time that tTG enzymatic activity in the mammalian forebrain is developmentally regulated by post‐translational mechanisms.

Validity of mouse models for the study of tissue transglutaminase in neurodegenerative diseases

Tissue transglutaminase (tTG) is a multifunctional enzyme that catalyzes peptide cross-linking and polyamination reactions, and also is a signal-transducing GTPase. tTG protein content and enzymatic activity are upregulated in the brain in Huntingtons disease and in other neurological diseases and conditions. Since mouse models are currently being used to study the role of tTG in Huntingtons disease and other neurodegenerative diseases, it is critical that the level of its expression in the mouse forebrain be determined. In contrast to human forebrain where tTG is abundant, tTG can only be detected in mouse forebrain by immunoblotting a GTP-binding-enriched protein fraction. tTG mRNA content and transamidating activity are approximately 70% lower in mouse than in human forebrain. However, tTG contributes to the majority of transglutaminase activity within mouse forebrain. Thus, although tTG is expressed at lower levels in mouse compared with human forebrain, it likely plays important roles in neuronal function.

Axin negatively affects tau phosphorylation by glycogen synthase kinase 3 beta

Glycogen synthase kinase 3β (GSK3β) is an essential protein kinase that regulates numerous functions within the cell. One critically important substrate of GSK3β is the microtubule‐associated protein tau. Phosphorylation of tau by GSK3β decreases tau–microtubule interactions. In addition to phosphorylating tau, GSK3β is a downstream regulator of the wnt signaling pathway, which maintains the levels of β‐catenin. Axin plays a central role in regulating β‐catenin levels by bringing together GSK3β and β‐catenin and facilitating the phosphorylation of β‐catenin, targeting it for ubiquitination and degradation by the proteasome. Although axin clearly facilitates the phosphorylation of β‐catenin, its effects on the phosphorylation of other GSK3β substrates are unclear. Therefore in this study the effects of axin on GSK3β‐mediated tau phosphorylation were examined. The results clearly demonstrate that axin is a negative regulator of tau phosphorylation by GSK3β. This negative regulation of GSK3β‐mediated tau phosphorylation is due to the fact that axin efficiently binds GSK3β but not tau and thus sequesters GSK3β away from tau, as an axin mutant that does not bind GSK3β did not inhibit tau phosphorylation by GSK3β. This is the first demonstration that axin negatively affects the phosphorylation of a GSK3β substrate, and provides a novel mechanism by which tau phosphorylation and function can be regulated within the cell.