Gail V. W. Johnson

Akt is a serine/threonine protein kinase that plays a vital role in promoting cellular survival. Predominantly cytosolic, upon stimulation with growth‐factors or stress, active Akt translocates into mitochondria, but the functions of Akt in mitochondria are not yet fully understood. Mitochondria play a central role in apoptotic pathways and given Akt's functions in the cytoplasm, Akt in mitochondria may help preserve mitochondrial integrity during cellular stress. To test if the translocation of Akt into mitochondria is neuroprotective, adenoviral vectors expressing a constitutively active Akt, Ad‐HA‐Akt (DD), and a constitutively active Akt with a mitochondrial targeting signal, Ad‐Mito‐HA‐Akt (DD), were generated. Human SH‐SY5Y neuroblastoma cells expressing the adenoviral constructs were treated with staurosporine to initiate intrinsic apoptotic cell death and several aspects of the mitochondrial apoptotic pathway were evaluated. Expression of active Akt targeted to mitochondria was found to be sufficient to significantly reduce staurosporine‐induced activation of caspase‐3 and caspase‐9, the release of cytochrome c from mitochondria, and Bax oligomerization at mitochondria. These findings demonstrate that intramitochondrial active Akt results in efficient protection against apoptotic signaling. J. Cell. Biochem. 102: 196–210, 2007.

Low‐density lipoprotein receptor‐related protein 6 (LRP6) is a member of low‐density lipoprotein receptor (LDLR) family which cooperates with Frizzled receptors to transduce the canonical Wnt signal. As a critical component of the canonical Wnt pathway, LRP6 is essential for appropriate brain development, however, the mechanism by which LRP6 facilitates Wnt canonical signaling has not been fully elucidated. Interestingly, LRP6 which lacks its extracellular domain can constitutively activate TCF/LEF and potentiate the Wnt signal. Further, the free cytosolic tail of LRP6 interacts directly with glycogen synthase kinase (GSK3) and inhibits GSK3’s activity in the Wnt canonical pathway which results in increased TCF/LEF activation. However, whether these truncated forms of LRP6 are physiologically relevant is unclear. Recent studies have shown that other members of the LDLR family undergo γ‐secretase dependent regulated intramembrane proteolysis (RIP). Using independent experimental approaches, we show that LRP6 also undergoes RIP. The extracellular domain of LRP6 is shed and released into the surrounding milieu and the cytoplasmic tail is cleaved by γ‐secretase‐like activity to release the intracellular domain. Furthermore, protein kinase C, Wnt 3a and Dickkopf‐1 modulate this process. These findings suggest a novel mechanism for LRP6 in Wnt signaling: induction of ectodomain shedding of LRP6, followed by the γ‐secretase involved proteolytic releasing its intracellular domain (ICD) which then binds to GSK3 inhibiting its activity and thus activates the canonical Wnt signaling pathway.

Huntington’s disease (HD), which is caused by an expanded polyglutamine tract in huntingtin (htt), is characterized by extensive loss of striatal neurons. The dysregulation of type 2 transglutaminase (TG2) has been proposed to contribute to the pathogenesis in HD as TG2 is up‐regulated in HD brain and knocking out TG2 in mouse models of HD ameliorates the disease process. To understand the role of TG2 in the pathogenesis of HD, immortalized striatal cells established from mice in which mutant htt with a polyglutamine stretch of 111 Gln had been knocked‐in and wild type (WT) littermates, were stably transfected with human TG2 in a tetracycline inducible vector. Overexpression of TG2 in the WT striatal cells resulted in significantly greater cell death under basal conditions as well as in response to thapsigargin treatment, which causes increased intracellular calcium concentrations. Furthermore, in WT striatal cells TG2 overexpression potentiated mitochondrial membrane depolarization, intracellular reactive oxygen species production, and apoptotic cell death in response to thapsigargin. In contrast, in mutant striatal cells, TG2 overexpression did not increase cell death, nor did it potentiate thapsigargin‐induced mitochondrial membrane depolarization or intracellular reactive oxygen species production. Instead, TG2 overexpression in mutant striatal cells attenuated the thapsigargin‐activated apoptosis. Whenin situtransglutaminase activity was quantitatively analyzed in these cell lines, we found that in response to thapsigargin treatment TG2 was activated in WT, but not mutant striatal cells. These data suggest that mutant htt alters the activation of TG2 in response to certain stimuli and therefore differentially modulates how TG2 contributes to cell death processes.

In 1992 little was known about the specific protein kinases that phosphorylate tau and the proteases that regulate tau turnover. Although we had already demonstrated that tau was a substrate of the calcium-activated protease calpain (Johnson et al. (

Tissue transglutaminase (tTG) post‐translationally modifies proteins in a calcium‐dependent manner by incorporation of polyamines, deamination or crosslinking. Moreover, tTG can also bind and hydrolyze GTP. tTG is the major transglutaminase in the mammalian nervous system, localizing predominantly in neurons. Although tTG has been clearly demonstrated to be elevated in neurodegenerative diseases and in response to acute CNS injury, its role in these pathogenic processes remains unclear. Transgenic mice that overexpress human tTG (htTG) primarily in CNS neurons were generated to explore the role of tTG in the nervous system and its contribution to neuropathological processes. tTG transgenic mice were phenotypically normal and were born with the expected Mendelian frequency. However, when challenged systemically with kainic acid, tTG transgenic mice, in comparison to wild‐type (WT) mice, developed more extensive hippocampal neuronal damage. This was evidenced by a decreased number of healthy neurons, and increased terminal deoxynucleotidyl dUTP nick end labeling (TUNEL) labeling as an indicator of neuronal cell death in the kainic acid‐treated transgenic mice. Moreover, the duration and severity of seizures developed by htTG transgenics in response to kainic acid administration were significantly more pronounced than those observed in WT mice. These data indicate for the first time that tTG may play an active role in excitatory amino acid‐induced neuronal cell death, which has been postulated to be an important component of acute CNS injury and chronic CNS neurodegenerative conditions.

LDL‐receptor related proteins 5 and 6 (LRP5/6) are co‐receptors of Frizzled receptors that mediate Wnt‐induced activation of the transcription factor family TCF/LEF‐1. Even though LRP5 and LRP6 are highly homologous, LRP6, but not LRP5, is expressed primarily in the nervous system and deletion of the LRP6 gene results in significant brain abnormalities, while deletion of LRP5 results in primarily decreased bone density. Additionally, the exact function of LRP5 and LRP6 have not been clearly defined, although it is clear that they both play key roles in the Wnt canonical pathway. In this study the role of the intracellular domains of LRP5/6 in mediating Wnt signaling was examined. In the absence of exogenous Wnt 3a, full‐length (FL) LRP6, but not LRP5, increased TCF/LEF‐1 transcriptional activity, however both significantly potentiated Wnt 3a‐induced TCF/LEF‐1 activation. In contrast to the findings with the FL constructs, the intracellular domains (membrane‐anchored and cytosolic) of both LRP5 and LRP6 significantly increased TCF/LEF‐1 activation in the absence of Wnt 3a, and potentiated the Wnt 3a‐induced decrease in β‐catenin phosphorylation, increase in free β‐catenin levels and the increase in TCF/LEF‐1 activity. These findings demonstrate that: (1) LRP5 and LRP6 differentially modulate TCF/LEF‐1 activation in the absence of Wnt 3a and (2) the intracellular C‐terminal domains of LRP5/6 potentiate Wnt 3a‐induced TCF/LEF‐1 activation whether or not they are membrane‐anchored. These findings provide significant new insights into the roles of LRP5/6 in modulating canonical Wnt signaling.

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 occurin vivoduring 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.

Tau is a group of neuronal microtubule-associated proteins that are formed by alternative mRNA splicing and accumulate in neurofibrillary tangles in Alzheimer's disease (AD) brain. Tau plays a key role in regulating microtubule dynamics, axonal transport and neurite outgrowth, and all these functions of tau are modulated by site-specific phosphorylation. There is significant evidence that a disruption of normal phosphorylation events results in tau dysfunction in neurodegenerative diseases, such as AD, and is a contributing factor to the pathogenic processes. Indeed, the abnormal tau phosphorylation that occurs in neurodegenerative conditions not only results in a toxic loss of function (e.g. decreased microtubule binding) but probably also a toxic gain of function (e.g. increased tau-tau interactions). Although tau is phosphorylated in vitro by numerous protein kinases, how many of these actually phosphorylate tau in vivo is unclear. Identification of the protein kinases that phosphorylate tau in vivo in both physiological and pathological processes could provide potential therapeutic targets for the treatment of AD and other neurodegenerative diseases in which there is tau pathology.