Site-specific phosphorylation of tau impacts mitochondrial function and response to stressors.

Phosphorylation of tau at sites associated with Alzheimer's disease (AD) likely plays a role in the disease progression. Mitochondrial impairment, correlating with increased presence of phosphorylated tau, has been identified as a contributing factor to neurodegenerative processes in AD. However, how tau phosphorylated at specific sites impacts mitochondrial function has not been fully defined. We examined how AD-relevant phosphomimetics of tau impact selected aspects of mitochondrial biology. To mimic phosphorylation at AD-associated sites, the serine/threonine (Ser/Thr) sites in wild-type green fluorescent protein (GFP)-tagged tau (T4) were converted to glutamic acid (E) to make pseudo-phosphorylated GFP-tagged Ser-396/404 (2EC) and GFP-tagged Thr-231/Ser-235 (2EM) constructs. These constructs were expressed in immortalized mouse hippocampal neuronal cell lines, and their impact on specific mitochondrial functions and responses to stressors were measured. Phosphomimetic tau altered mitochondrial distribution. Specifically, mitochondria accumulated in the soma of cells expressing either 2EC or 2EM and neurite-like extensions in 2EC cells were shorter. Additionally, adenosine triphosphate levels were reduced in both 2EC- and 2EM-expressing cells, and reactive oxygen species (ROS) production increased in 2EC cells during oxidation of succinate when compared to T4-expressing cells. Thapsigargin reduced mitochondrial membrane potential and increased ROS production in both 2EC and 2EM cells relative to T4 cells, with no significant difference in the effects of rotenone. These results show that tau phosphorylation at specific AD-relevant epitopes negatively affects mitochondria, with the extent of dysfunction and stress response varying according to the sites of phosphorylation. Altogether, these findings show that phosphorylated tau increases mitochondrial susceptibility to stressors and extend our understanding of potential mechanisms whereby phosphorylated tau promotes mitochondria dysfunction in tauopathies, including AD.

Deletion of Transglutaminase 2 from Mouse Astrocytes Significantly Improves Their Ability to Promote Neurite Outgrowth on an Inhibitory Matrix.

Astrocytes are the primary support cells of the central nervous system (CNS) that help maintain the energetic requirements and homeostatic environment of neurons. CNS injury causes astrocytes to take on reactive phenotypes with an altered overall function that can range from supportive to harmful for recovering neurons. The characterization of reactive astrocyte populations is a rapidly developing field, and the underlying factors and signaling pathways governing which type of reactive phenotype that astrocytes take on are poorly understood. Our previous studies suggest that transglutaminase 2 (TG2) has an important role in determining the astrocytic response to injury. Selectively deleting TG2 from astrocytes improves functional outcomes after CNS injury and causes widespread changes in gene regulation, which is associated with its nuclear localization. To begin to understand how TG2 impacts astrocytic function, we used a neuron-astrocyte co-culture paradigm to compare the effects of TG2-/- and wild-type (WT) mouse astrocytes on neurite outgrowth and synapse formation. Neurons were grown on a control substrate or an injury-simulating matrix comprised of inhibitory chondroitin sulfate proteoglycans (CSPGs). Compared to WT astrocytes, TG2-/- astrocytes supported neurite outgrowth to a significantly greater extent only on the CSPG matrix, while synapse formation assays showed mixed results depending on the pre- and post-synaptic markers analyzed. We hypothesize that TG2 regulates the supportive functions of astrocytes in injury conditions by modulating gene expression through interactions with transcription factors and transcription complexes. Based on the results of a previous yeast two-hybrid screen for TG2 interactors, we further investigated the interaction of TG2 with Zbtb7a, a ubiquitously expressed transcription factor. Co-immunoprecipitation and colocalization analyses confirmed the interaction of TG2 and Zbtb7a in the nucleus of astrocytes. Overexpression or knockdown of Zbtb7a levels in WT and TG2-/- astrocytes revealed that Zbtb7a robustly influenced astrocytic morphology and the ability of astrocytes to support neuronal outgrowth, which was significantly modulated by the presence of TG2. These findings support our hypothesis that astrocytic TG2 acts as a transcriptional regulator to influence astrocytic function, with greater influence under injury conditions that increase its expression, and Zbtb7a likely contributes to the overall effects observed with astrocytic TG2 deletion.

The role of BAG3 in health and disease: A "Magic BAG of Tricks".

The multi-domain structure of Bcl-2-associated athanogene 3 (BAG3) facilitates its interaction with many different proteins that participate in regulating a variety of biological pathways. After revisiting the BAG3 literature published over the past ten years with Citespace software, we classified the BAG3 research into several clusters, including cancer, cardiomyopathy, neurodegeneration, and viral propagation. We then highlighted recent key findings in each cluster. To gain greater insight into the roles of BAG3, we analyzed five different published mass spectrometry data sets of proteins that co-immunoprecipitate with BAG3. These data gave us insight into universal, as well as cell-type-specific BAG3 interactors in cancer cells, cardiomyocytes, and neurons. Finally, we mapped variable BAG3 SNPs and also mutation data from previous publications to further explore the link between the domains and function of BAG3. We believe this review will provide a better understanding of BAG3 and direct future studies towards understanding BAG3 function in physiological and pathological conditions.

The role of transglutaminase 2 in mediating glial cell function and pathophysiology in the central nervous system.

The ubiquitously expressed transglutaminase 2 (TG2) has diverse functions in virtually all cell types, with its role depending not only on cell type, but also on specific subcellular localization. In the central nervous system (CNS) different types of glial cells, such as astrocytes, microglia, and oligodendrocytes and their precursor cells (OPCs), play pivotal supportive functions. This review is focused on what is currently known about the role of TG2 in each type of glial cell, in the context of normal function and pathophysiology. For example, astrocytic TG2 facilitates their migration and proliferation, but hinders their ability to protect neurons after CNS injury. The review also examines the interactions between glial cell types, and how TG2 in one cell type may affect another, as well as implications for specific TG2 populations as therapeutic targets in CNS pathology.

Tissue Transglutaminase-Mediated AT1 Receptor Sensitization Underlies Pro-inflammatory Cytokine LIGHT-Induced Hypertension.

BACKGROUND: Although numerous recent studies have shown a strong link between inflammation and hypertension, the underlying mechanisms by which inflammatory cytokines induce hypertension remain to be fully elucidated. Hypertensive disorders are also associated with elevated pressor sensitivity. Tissue transglutaminase (TG2), a potent cross-linking enzyme, is known to be transcriptionally activated by inflammatory cytokines and stabilize angiotensin II (Ang II) receptor AT1 (AT1R) via ubiquitination-preventing posttranslational modification. Here we sought to investigate the TG2-mediated AT1R stabilization in inflammation-induced hypertension and its functional consequences with a focus on receptor abundance and Ang II responsiveness. METHODS AND RESULTS: Using an experimental model of inflammation-induced hypertension established by introducing the pro-inflammatory tumor necrosis factor cytokine LIGHT, we provide pharmacologic and genetic evidence that TG2 is required for LIGHT-induced hypertension (systolic pressure on day 6: LIGHT = 152.3 ± 7.4 vs. LIGHT+ERW1041E [TG2 inhibitor] = 105.8 ± 13.1 or LIGHT+TG2-/- = 114.3 ± 4.3 mm Hg, P < 0.05, n = 4-5) and renal compromise (urine albumin/creatinine: LIGHT = 0.17 ± 0.05 vs. LIGHT+ERW1041E = 0.03 ± 0.01 or LIGHT+TG2-/- = 0.06 ± 0.01 mg/mg; plasma creatinine: LIGHT = 1.11 ± 0.04 vs. LIGHT+ERW1041E = 0.94 ± 0.04 or LIGHT+TG2-/- = 0.88 ± 0.09 mg/dl; urine volume: LIGHT = 0.23 ± 0.1 vs. LIGHT+ERW1041E = 0.84 ± 0.13 or LIGHT+TG2-/- = 1.02 ± 0.09 ml/24 hour on day 14, P < 0.05, n = 4-5). Our mechanistic studies showed that the TG2-mediated AT1R modification and accumulation (relative renal AT1R level: phosphate-buffered saline [PBS] = 1.23 ± 0.22, LIGHT = 3.49 ± 0.37, and LIGHT+ERW1041E = 1.77 ± 0.46, P < 0.05, n = 3; LIGHT+TG2+/+ = 85.28 ± 36.11 vs. LIGHT+TG2-/- = 7.01 ± 5.68, P < 0.05, n = 3) induced by LIGHT is associated with abrogated ß-arrestin binding (AT1R/associated ß-arrestin ratio: PBS = 2.62 ± 1.07, LIGHT = 38.60 ± 13.91, and LIGHT+ERW1041E = 6.97 ± 2.91, P < 0.05, n = 3; LIGHT+TG2+/+ = 66.43 ± 44.81 vs. LIGHT+TG2-/- = 2.45 ± 1.78, P < 0.01, n = 3) and could be found in renal medulla tubules of kidneys (relative tubular AT1R level: PBS = 5.91 ± 2.93, LIGHT = 92.82 ± 19.54, LIGHT+ERW1041E = 28.49 ± 11.65, and LIGHT+TG2-/- = 0.14 ± 0.10, P < 0.01, n = 5) and the blood vasculature (relative vascular AT1R level: PBS = 0.70 ± 0.30, LIGHT = 13.75 ± 2.49, and LIGHT+ERW1041E = 3.28 ± 0.87, P < 0.01, n = 3), 2 of the tissues highly related to the genesis of hypertension. Our in vitro cellular assays showed that LIGHT stimulation triggered a rapid TG2-dependent increase in the abundance of AT1Rs (relative AT1R level after 2-hour LIGHT treatment: AT1R (WT)+TG2 = 2.21 ± 0.23, AT1R (Q315A)+TG2 = 0.18 ± 0.23, P < 0.05 vs. starting point = 1, n = 2) and downstream calcium signaling (fold increase in NFAT-driven luciferase activity: Saline = 0.02 ± 0.03, Ang II = 0.17 ± 0.08, LIGHT = 0.05 ± 0.04, LIGHT+Ang II = 0.90 ± 0.04 (P < 0.01 vs. Ang II), and LIGHT+Ang II+ERW1041E = 0.15 ± 0.15 (P < 0.01 vs. LIGHT+Ang II), n = 3). CONCLUSIONS: Our data indicate an essential and systemic role for TG2 in bridging inflammation to hypertension via its posttranslational modifications stabilizing AT1 receptor and sensitizing Ang II. Our findings also suggest that TG2 inhibitors could be used as a novel group of cardiovascular agents.

Transglutaminase 2: Friend or foe? The discordant role in neurons and astrocytes.

Members of the transglutaminase family catalyze the formation of isopeptide bonds between a polypeptide-bound glutamine and a low molecular weight amine (e.g., spermidine) or the ɛ-amino group of a polypeptide-bound lysine. Transglutaminase 2 (TG2), a prominent member of this family, is unique because in addition to being a transamidating enzyme, it exhibits numerous other activities. As a result, TG2 plays a role in many physiological processes, and its function is highly cell type specific and relies upon a number of factors, including conformation, cellular compartment location, and local concentrations of Ca2+ and guanine nucleotides. TG2 is the most abundant transglutaminase in the central nervous system (CNS) and plays a pivotal role in the CNS injury response. How TG2 affects the cell in response to an insult is strikingly different in astrocytes and neurons. In neurons, TG2 supports survival. Overexpression of TG2 in primary neurons protects against oxygen and glucose deprivation (OGD)-induced cell death and in vivo results in a reduction in infarct volume subsequent to a stroke. Knockdown of TG2 in primary neurons results in a loss of viability. In contrast, deletion of TG2 from astrocytes results in increased survival following OGD and improved ability to protect neurons from injury. Here, a brief overview of TG2 is provided, followed by a discussion of the role of TG2 in transcriptional regulation, cellular dynamics, and cell death. The differing roles TG2 plays in neurons and astrocytes are highlighted and compared to how TG2 functions in other cell types.

The complex role of transglutaminase 2 in glioblastoma proliferation.

BACKGROUND: Glioblastomas (GBMs) are a heterogeneous group of primary brain tumors. These tumors are resistant to therapeutic interventions and invariably recur after surgical resection. The multifunctional protein transglutaminase 2 (TG2) has been shown to promote cell survival in a number of different tumors. There is also evidence that TG2 may be a pro-survival factor in GBMs. However, the roles that TG2 plays in facilitating GBM survival and proliferation have not yet been clearly delineated . METHODS: The functions of TG2 are often cell- and context-specific. Therefore, in this study we examined the ability of TG2 to facilitate GBM proliferation using colony formation assays and 5-ethynyl-2'-deoxyuridine (EdU) incorporation in several different GBM cell lines as well as neurospheres derived from patient tumors representing the 3 major subtypes of GBM tumors (mesenchymal, proneural, and classical) and maintained in the absence of serum. TG2 knockdown or selective TG2 inhibitors were used to modulate TG2 expression and activity. RESULTS: We show that TG2 plays differential roles in the proliferative process depending on the cell type. In most, but not all, GBM models TG2 plays a crucial role in the proliferative process, and some but not all TG2 inhibitors were highly effective at reducing proliferation in a large subset of the GBM models. CONCLUSION: Our results show that TG2 plays an important-but notoriously context-specific-role in GBM cell biology. Nonetheless, as future studies unravel the genetic "fingerprints" that make TG2 inhibitors effective, this information could be exploited to develop TG2 inhibitors into personalized GBM therapies.

Epigallocatechin-3-gallate enhances clearance of phosphorylated tau in primary neurons.

OBJECTIVES: Alzheimer's disease (AD) is a neurodegenerative disorder characterized by intracellular accumulations of phosphorylated forms of the microtubule binding protein tau. This study aimed to explore a novel mechanism for enhancing the clearance of these pathological tau species using the green tea flavonoid epigallocatechin-3-gallate (EGCG). EGCG is a potent antioxidant and an activator of the Nrf2 transcriptional pathway. Nrf2 activators including EGCG have shown promise in mitigating amyloid pathology in vitro and in vivo. This study assessed whether EGCG could also alter tau clearance. METHODS: Rat primary cortical neuron cultures were treated on day in vitro 8 with EGCG and analyzed for changes in gene and protein expression using luciferase assay, q-PCR, and western blotting. RESULTS: EGCG treatment led to a significant decrease in the protein levels of three AD-relevant phospho-tau epitopes. Unexpectedly, EGCG does not appear to be facilitating this effect through the Nrf2 pathway or by increasing autophagy in general. However, EGCG did significantly increase mRNA expression of the key autophagy adaptor proteins NDP52 and p62. DISCUSSION: In this study, we show that EGCG enhances the clearance of AD-relevant phosphorylated tau species in primary neurons. Interestingly, this result appears to be independent of both Nrf2 activation and enhanced autophagy - two previously reported mechanisms of phytochemical-induced tau clearance. EGCG did significantly increase expression of two autophagy adaptor proteins. Taken together, these results demonstrate that EGCG has the ability to increase the clearance of phosphorylated tau species in a highly specific manner, likely through increasing adaptor protein expression.

Sulforaphane induces autophagy through ERK activation in neuronal cells.

Sulforaphane (SFN), an activator of nuclear factor E2-related factor 2 (Nrf2), has been reported to induce autophagy in several cells. However, little is known about its signaling mechanism of autophagic induction. Here, we provide evidence that SFN induces autophagy with increased levels of LC3-II through extracellular signal-regulated kinase (ERK) activation in neuronal cells. Pretreatment with NAC (N-acetyl-l-cysteine), a well-known antioxidant, completely blocked the SFN-induced increase in LC3-II levels and activation of ERK. Knockdown or overexpression of Nrf2 did not affect autophagy. Together, the results suggest that SFN-mediated generation of reactive oxygen species (ROS) induces autophagy via ERK activation, independent of Nrf2 activity in neuronal cells.

Transglutaminase regulation of cell function.

Transglutaminases (TGs) are multifunctional proteins having enzymatic and scaffolding functions that participate in regulation of cell fate in a wide range of cellular systems and are implicated to have roles in development of disease. This review highlights the mechanism of action of these proteins with respect to their structure, impact on cell differentiation and survival, role in cancer development and progression, and function in signal transduction. We also discuss the mechanisms whereby TG level is controlled and how TGs control downstream targets. The studies described herein begin to clarify the physiological roles of TGs in both normal biology and disease states.

Nrf2 reduces levels of phosphorylated tau protein by inducing autophagy adaptor protein NDP52.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a pivotal transcription factor in the defence against oxidative stress. Here we provide evidence that activation of the Nrf2 pathway reduces the levels of phosphorylated tau by induction of an autophagy adaptor protein NDP52 (also known as CALCOCO2) in neurons. The expression of NDP52, which we show has three antioxidant response elements (AREs) in its promoter region, is strongly induced by Nrf2, and its overexpression facilitates clearance of phosphorylated tau in the presence of an autophagy stimulator. In Nrf2-knockout mice, phosphorylated and sarkosyl-insoluble tau accumulates in the brains concurrent with decreased levels of NDP52. Moreover, NDP52 associates with phosphorylated tau from brain cortical samples of Alzheimer disease cases, and the amount of phosphorylated tau in sarkosyl-insoluble fractions is inversely proportional to that of NDP52. These results suggest that NDP52 plays a key role in autophagy-mediated degradation of phosphorylated tau in vivo.

Transglutaminase and polyamination of tubulin: posttranslational modification for stabilizing axonal microtubules.

Neuronal microtubules support intracellular transport, facilitate axon growth, and form a basis for neuronal morphology. While microtubules in nonneuronal cells are depolymerized by cold, Ca(2+), or antimitotic drugs, neuronal microtubules are unusually stable. Such stability is important for normal axon growth and maintenance, while hyperstability may compromise neuronal function in aging and degeneration. Though mechanisms for stability are unclear, studies suggest that stable microtubules contain biochemically distinct tubulins that are more basic than conventional tubulins. Transglutaminase-catalyzed posttranslational incorporation of polyamines is one of the few modifications of intracellular proteins that add positive charges. Here we show that neuronal tubulin can be polyaminated by transglutaminase. Endogenous brain transglutaminase-catalyzed polyaminated tubulins have the biochemical characteristics of neuronal stable microtubules. Inhibiting polyamine synthesis or transglutaminase activity significantly decreases microtubule stability in vitro and in vivo. Together, these findings suggest that transglutaminase-catalyzed polyamination of tubulins stabilizes microtubules essential for unique neuronal structures and functions.

Vena cava and aortic smooth muscle cells express transglutaminases 1 and 4 in addition to transglutaminase 2.

Transglutaminase (TG) function facilitates several vascular processes and diseases. Although many of these TG-dependent vascular processes have been ascribed to the function of TG2, TG2 knockout mice have a mild vascular phenotype. We hypothesized that TGs besides TG2 exist and function in the vasculature. Biotin-pentylamide incorporation, a measure of general TG activity, was similar in wild-type and TG2 knockout mouse aortae, and the general TG inhibitor cystamine reduced biotin-pentylamine incorporation to a greater extent than the TG2-specific inhibitor Z-DON, indicating the presence of other functional TGs. Additionally, 5-hydroxytryptamine-induced aortic contraction, a TG-activity-dependent process, was decreased to a greater extent by general TG inhibitors vs. Z-DON (maximum contraction: cystamine = abolished, monodansylcadaverine = 28.6 ± 14.9%, and Z-DON = 60.2 ± 15.2% vehicle), providing evidence for the importance of TG2-independent activity in the vasculature. TG1, TG2, TG4, and Factor XIII (FXIII) mRNA in rat aortae and vena cavae was detected by RT-PCR. Western analysis detected TG1 and TG4, but not FXIII, in rat aortae and vena cavae and in TG2 knockout and wild-type mouse aortae. Immunostaining confirmed the presence of TG1, TG2, and TG4 in rat aortae and vena cavae, notably in smooth muscle cells; FXIII was absent. K5 and T26, FITC-labeled peptide substrates specific for active TG1 and TG2, respectively, were incorporated into rat aortae and vena cavae and wild-type, but not TG2 knockout, mouse aortae. These studies demonstrate that TG2-independent TG activity exists in the vasculature and that TG1 and TG4 are expressed in vascular tissues.

Complete transglutaminase 2 ablation results in reduced stroke volumes and astrocytes that exhibit increased survival in response to ischemia.

Transglutaminase 2 (TG2) is a very multifunctional protein that is ubiquitously expressed in the body. It is a Ca(2+)-dependent transamidating enzyme, a GTPase, as well as a scaffolding protein. TG2 is the predominant form of transglutaminase expressed in the mammalian nervous system. Previously, it was shown that TG2 can affect both cell death and cell survival mechanisms depending on the cell type and the stressor. In the case of ischemic stress, TG2 was previously shown to play a protective role in the models used. For example in hTG2 transgenic mice, where TG2 is overexpressed only in neurons, middle cerebral artery ligation (MCAL) resulted in smaller infarct volumes compared to wild type mice. In this study TG2 knock out mice were used to determine how endogenous TG2 affected stroke volumes. Intriguingly, infarct volumes in TG2 knock out mice were significantly smaller compared to wild type mice. As expected, primary neurons isolated from TG2 knock out mice showed decreased viability in response to oxygen-glucose deprivation. However, primary astrocytes that were isolated from TG2 knock out mice were resistant to oxygen-glucose deprivation in situ. Both wild type and knock out neurons were protected against oxygen glucose deprivation when they were co-cultured with astrocytes from TG2 knockout mice. Therefore, the decreased stroke volumes observed in TG2 knock out mice after MCAL, can be correlated with the protective effects of TG2 knock out in astrocytes in response to oxygen glucose deprivation in situ. These findings suggest that neuron-astrocyte crosstalk plays a significant role in mediating ischemic cell death and that TG2 differentially impacts cell survival depending on cell context.

Decreases in valosin-containing protein result in increased levels of tau phosphorylated at Ser262/356.

VCP/p97 is a multifunctional AAA+-ATPase involved in vesicle fusion, proteasomal degradation, and autophagy. Reported dysfunctions of these processes in Alzheimer disease (AD), along with the linkage of VCP/p97 to inclusion body myopathy with Paget's disease and frontotemporal dementia (IBMPFD) led us to examine the possible linkage of VCP to the AD-relevant protein, tau. VCP levels were reduced in AD brains, but not in the cerebral cortex of an AD mouse model, suggesting that VCP reduction occurs upstream of tau pathology. Genetic reduction of VCP in a primary neuronal model led to increases in the levels of tau phosphorylated at Ser(262/356), indicating that VCP may be involved in regulating post-translational processing of tau in AD, demonstrating a possible functional linkage between tau and VCP.

The application of permanent middle cerebral artery ligation in the mouse.

Focal cerebral ischemia is among the most common type of stroke seen in patients. Due to the clinical significance there has been a prolonged effort to develop suitable animal models to study the events that unfold during ischemic insult. These techniques include transient or permanent, focal or global ischemia models using many different animal models, with the most common being rodents. The permanent MCA ligation method which is also referred as pMCAo in the literature is used extensively as a focal ischemia model in rodents. This method was originally described for rats by Tamura et al. in 1981. In this protocol a craniotomy was used to access the MCA and the proximal regions were occluded by electrocoagulation. The infarcts involve mostly cortical and sometimes striatal regions depending on the location of the occlusion. This technique is now well established and used in many laboratories. Early use of this technique led to the definition and description of "infarct core" and "penumbra", and it is often used to evaluate potential neuroprotective compounds. Although the initial studies were performed in rats, permanent MCA ligation has been used successfully in mice with slight modifications. This model yields reproducible infarcts and increased post-survival rates. Approximately 80% of the ischemic strokes in humans happen in the MCA area and thus this is a very relevant model for stroke studies. Currently, there is a paucity of effective treatments available to stroke patients, and thus there is a need for good models to test potential pharmacological compounds and evaluate physiological outcomes. This method can also be used for studying intracellular hypoxia response mechanisms in vivo. Here, we present the MCA ligation surgery in a C57/BL6 mouse. We describe the pre-surgical preparation, MCA ligation surgery and 2,3,5 Triphenyltetrazolium chloride (TTC) staining for quantification of infarct volumes.

Transglutaminase 2 and its role in pulmonary fibrosis.

RATIONALE: Idiopathic pulmonary fibrosis (IPF) is a deadly progressive disease with few treatment options. Transglutaminase 2 (TG2) is a multifunctional protein, but its function in pulmonary fibrosis is unknown. OBJECTIVES: To determine the role of TG2 in pulmonary fibrosis. METHODS: The fibrotic response to bleomycin was compared between wild-type and TG2 knockout mice. Transglutaminase and transglutaminase-catalyzed isopeptide bond expression was examined in formalin-fixed human lung biopsy sections by immunohistochemistry from patients with IPF. In addition, primary human lung fibroblasts were used to study TG2 function in vitro. MEASUREMENTS AND MAIN RESULTS: TG2 knockout mice developed significantly reduced fibrosis compared with wild-type mice as determined by hydroxyproline content and histologic fibrosis score (P < 0.05). TG2 expression and activity are increased in lung biopsy sections in humans with IPF compared with normal control subjects. In vitro overexpression of TG2 led to increased fibronectin deposition, whereas transglutaminase knockdown led to defects in contraction and adhesion. The profibrotic cytokine transforming growth factor-ß causes an increase in membrane-localized TG2, increasing its enzymatic activity. CONCLUSIONS: TG2 is involved in pulmonary fibrosis in a mouse model and in human disease and is important in normal fibroblast function. With continued research on TG2, it may offer a new therapeutic target.

Increased expression of Bim contributes to the potentiation of serum deprivation-induced apoptotic cell death in Huntington's disease knock-in striatal cell line.

Given that mutant huntingtin may cause dysregulation of gene expression in striatal neurons leading to the neuronal death, we examined the expression level of Bcl-2 interacting mediator of cell death (Bim) in immortalized wild type STHdh(Q7) and knock-in mutant STHdh(Q111) striatal cell lines to understand the underlying mechanism by which mutant huntingtin causes selective death of striatal neurons. Mutant STHdh(Q111) exhibited significantly increased expression level of Bim compared to STHdh(Q7). Serum deprivation resulted in potentiated apoptotic death in STHdh(Q111) compared to STHdh(Q7). However, the expression level of Bim was not changed with serum deprivation in both cell lines. Activation of pro-survival pathway with IGF-1 significantly attenuated serum deprivation-induced neuronal death in both cell lines and attenuated mutant huntingtin-mediated potentiated apoptotic death in STHdh(Q111). The level of active Akt was significantly elevated in STHdh(Q111) compared to STHdh(Q7) resulting in the phosphorylation of a FKHRL1, a forkhead transcription factor regulating Bim expression in neuronal cells. These data suggest that the presence of mutant huntingtin causes transcriptional dysregulation favoring apoptosis and that Akt pro-survival pathway in STHdh(Q111) is not compromised due to the presence of mutant huntingtin. Therefore, activation of this pathway may contribute to the protection of striatal neurons in Huntington's disease.

Regulated proteolytic processing of LRP6 results in release of its intracellular domain.

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 gamma-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 gamma-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 gamma-secretase involved proteolytic releasing its intracellular domain (ICD) which then binds to GSK3 inhibiting its activity and thus activates the canonical Wnt signaling pathway.

Mitochondrial-targeted active Akt protects SH-SY5Y neuroblastoma cells from staurosporine-induced apoptotic cell death.

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.