Daniela P. Ponce

Phosphorylation of AKT/PKB by CK2 is necessary for the AKT-dependent up-regulation of β-catenin transcriptional activity

β‐Catenin is a key protein in the canonical Wnt signaling pathway and in many cancers alterations in transcriptional activity of its components are observed. This pathway is up‐regulated by the protein kinase CK2, but the underlying mechanism of this change is unknown. It has been demonstrated that CK2 hyperactivates AKT/PKB by phosphorylation at Ser129, and AKT phosphorylates β‐catenin at Ser552, which in turn, promotes its nuclear localization and transcriptional activity. However, the consequences of CK2‐dependent hyperactivation of AKT on β‐catenin activity and cell viability have not been evaluated. We assessed this regulatory process by manipulating the activity of CK2 and AKT through overexpression of wild‐type, constitutively active and dominant negative forms of these proteins as well as analyzing β‐catenin‐dependent transcriptional activity, survivin expression and viability in HEK‐293T cells. We observed that CK2α overexpression up‐regulated the β‐catenin transcriptional activity, which correlated to an increased nuclear localization of β‐catenin as well as survivin expression. Importantly, these effects were strongly reversed when an AKT‐S129A mutant was co‐expressed in the same cells, followed by a significant decrease in cell viability but no changes in β‐catenin stability. Taken together, the data suggest that the CK2α‐dependent up‐regulation of β‐catenin activity requires phosphorylation of AKT in human embryonic kidney cells. J. Cell. Physiol. 226: 1953–1959, 2011.

CK2 functionally interacts with AKT/PKB to promote the β-catenin-dependent expression of survivin and enhance cell survival

Abstractβ-Catenin is crucial in the canonical Wnt signaling pathway. This pathway is up-regulated by CK2 which is associated with an enhanced expression of the antiapoptotic protein survivin, although the underlying molecular mechanism is unknown. AKT/PKB kinase phosphorylates and promotes β-catenin transcriptional activity, whereas CK2 hyperactivates AKT by phosphorylation at Ser129; however, the role of this phosphorylation on β-catenin transcriptional activity and cell survival is unclear. We studied in HEK-293T cells, the effect of CK2-dependent hyperactivation of AKT on cell viability, as well as analyzed β-catenin subcellular localization and transcriptional activity and survivin expression. CK2α overexpression led to an augmented β-catenin-dependent transcription and protein levels of survivin, and consequently an enhanced resistance to apoptosis. However, CK2α-enhancing effects were reversed when an AKT mutant deficient in Ser129 phosphorylation by CK2 was co-expressed. Therefore, our results strongly suggest that CK2α-specific enhancement of β-catenin transcriptional activity as well as cell survival may depend on AKT hyperactivation by CK2.

Inverse Susceptibility to Oxidative Death of Lymphocytes Obtained From Alzheimer's Patients and Skin Cancer Survivors: Increased Apoptosis in Alzheimer's and Reduced Necrosis in Cancer

A paucity of cancer in individuals with Alzheimers disease (AD) and low rates of AD in cancer survivors has been reported in epidemiological studies. Deregulation in opposite directions of biological mechanisms, such as susceptibility to cell death, might be shared in the two disorders. We analyzed lymphocytes from AD and skin cancer patients as well as healthy controls and found significantly increased vulnerability of AD lymphocytes to H(2)O(2)-induced apoptotic death and higher resistance to death of skin cancer lymphocytes, due to reduced necrosis, as compared with healthy controls by pairwise comparisons adjusted for age and sex. H(2)O(2)-induced death in lymphocytes was caspase independent and significantly reduced by PARP-1 inhibition in all three groups. These differences in the susceptibility to cell death observed for lymphocytes from AD and skin cancer patients may be one of the mechanisms that help explain the inverse correlation detected between these diseases in epidemiological studies.

Protein kinase CK2 promotes cancer cell viability via up-regulation of cyclooxygenase-2 expression and enhanced prostaglandin E2 production

Augmented expression of protein kinase CK2 is associated with hyperproliferation and resistance to apoptosis in cancer cells. Effects of CK2 are at least partially linked to signaling via the Wnt/β‐catenin pathway, which is dramatically enhanced in colon cancer. Cyclooxygenase‐2 (COX‐2), a Wnt/β‐catenin target gene, has been associated with enhanced cancer progression and metastasis. However, the possibility that a connection may exist between CK2 and COX‐2 has not been explored previously. Here we investigated changes in COX‐2 expression and activity upon CK2 modulation and evaluated how these changes affected cell viability. COX‐2 expression and cell viability decreased upon selective inhibition of COX‐2 with SC‐791 or CK2 with 2‐dimethylamino‐4,5,6,7‐tetrabromo‐1H‐benzimidazole (DMAT), both in human colon (HT29‐ATCC, HT29‐US, DLD‐1) and breast (ZR‐75) cancer cells, as well as in human embryonic kidney (HEK‐293T) cells. On the other hand, ectopic CK2α expression promoted up‐regulation of COX‐2 by activating the Wnt/β‐catenin pathway in HEK‐293T cells. Noteworthy, over‐expression of either CK2α, β‐catenin or COX‐2, as well as supplementation of the medium with prostaglandin E2 (PGE2), all were individually sufficient to overcome limitations in cell viability triggered by CK2 inhibition either upon addition of DMAT or over‐expression of a dominant negative CK2α variant. Altogether, these findings provide new insight to the role of CK2 in cancer by up‐regulating COX‐2 expression and thereby PGE2 production. J. Cell. Biochem. 112: 3167–3175, 2011.

Increased Susceptibility to Oxidative Death of Lymphocytes from Alzheimer Patients Correlates with Dementia Severity

We previously reported on enhanced susceptibility to death of lymphocytes from Alzheimers disease (AD) patients when exposed to hydrogen peroxide (H2O2)-induced oxidative stress and an increased resistance to death in those of patients with a history of skin cancer. This is consistent with our hypothesis proposing that the cellular machinery controlling cell death is deregulated in opposite directions in Alzheimers disease (AD) and cancer, to explain the inverse association observed in epidemiological studies. Here we investigated whether the observed increased susceptibility correlates with the degree of dementia severity. Peripheral lymphocytes from 23 AD patients, classified using the Clinical Dementia Rating (CDR) into severe dementia (CDR 3, n=10) and mild-to-moderate dementia (CDR 1- 2, n=13), and 15 healthy controls (HC) (CDR 0), were exposed to H2O2 for 20 hours. Lymphocyte death was determined by flow cytometry and propidium iodide staining. The greatest susceptibility to H2O2-induced death was observed for lymphocytes from severe dementia patients, whereas those with mild-to-moderate dementia exhibited intermediate values, compared to healthy controls. A significant increase in the apoptosis/necrosis ratio was found in AD patients. Poly (ADP-ribosyl) polymerase-1 (PARP-1) inhibition significantly protected from H2O2-induced death of lymphocytes, whereby a lower degree of protection was observed in severe AD patients. Moreover, inhibition of PARP-1 abolished the differences in apoptosis/necrosis ratios observed between the three groups of patients. These results support the notion that AD is a systemic disorder, whereby enhanced susceptibility to H2O2-induced death in peripheral lymphocytes correlates with dementia severity and enhanced death in AD patients is attributable to a PARP-dependent increase in the apoptosis/necrosis ratio.

Mitochondrial Bioenergetics Is Altered in Fibroblasts from Patients with Sporadic Alzheimer's Disease

The identification of an early biomarker to diagnose Alzheimers disease (AD) remains a challenge. Neuropathological studies in animal and AD patients have shown that mitochondrial dysfunction is a hallmark of the development of the disease. Current studies suggest the use of peripheral tissues, like skin fibroblasts as a possibility to detect the early pathological alterations present in the AD brain. In this context, we studied mitochondrial function properties (bioenergetics and morphology) in cultured fibroblasts obtained from AD, aged-match and young healthy patients. We observed that AD fibroblasts presented a significant reduction in mitochondrial length with important changes in the expression of proteins that control mitochondrial fusion. Moreover, AD fibroblasts showed a distinct alteration in proteolytic processing of OPA1, a master regulator of mitochondrial fusion, compared to control fibroblasts. Complementary to these changes AD fibroblasts showed a dysfunctional mitochondrial bioenergetics profile that differentiates these cells from aged-matched and young patient fibroblasts. Our findings suggest that the human skin fibroblasts obtained from AD patients could replicate mitochondrial impairment observed in the AD brain. These promising observations suggest that the analysis of mitochondrial bioenergetics could represent a promising strategy to develop new diagnostic methods in peripheral tissues of AD patients.

Local Klotho Enhances Neuronal Progenitor Proliferation in the Adult Hippocampus

Klotho is an aging-related protein associated with hippocampal cognitive performance in mammals. Klotho regulates progenitor cell proliferation in non-neuronal tissues, but its role in adult hippocampal neurogenesis (AHN) has not been explored. Klotho expression in the adult mouse hippocampus was examined by immunofluorescence and polymerase chain reaction. AHN was evaluated in the hippocampus of klotho knock-out mice (KO), klotho KO/vitamin D-receptor mutant mice, and in a model of local klotho hippocampal knockdown. The recombinant Klotho effect on proliferation was measured in mouse-derived hippocampal neural progenitor cells. Hippocampal-dependent memory was assessed by a dry-land version of the Morris water maze. Klotho was expressed in the granular cell layer of the adult Dentate Gyrus. AHN was increased in klotho KO mice, but not in klotho KO/vitamin D-receptor mutant mice. Inversely, local downregulation of hippocampal Klotho diminished AHN. Recombinant Klotho increased the proliferation rate of neural progenitors. Downregulation of hippocampal Klotho correlated with a decreased performance in hippocampal-dependent memory. These results suggest that Klotho directly participates in regulating AHN. Our observations indicate that Klotho promotes proliferation, AHN and hippocampal-dependent cognition. Increased neurogenesis in klotho KO mice may be secondary to the activation of other pathways altered in the model, such as vitamin D.

PARP-1 and p53 Regulate the Increased Susceptibility to Oxidative Death of Lymphocytes from MCI and AD Patients

Mild cognitive impairment (MCI) is a clinically detectable initial stage of cognitive deterioration with a high conversion rate to dementia. There is increasing evidence that some of the cerebral alterations present in Alzheimer type dementia can be found in peripheral tissues. We have previously shown that lymphocytes from Alzheimer’s disease (AD) patients have increased susceptibility to hydrogen peroxide (H2O2)-induced death that depends on dementia severity. We here investigated whether lymphocytes from MCI patients show increased vulnerability to death, and explored the involvement of Poly [ADP-ribose] polymerase (PARP-1) and p53 in the regulation of this process. Lymphocytes from 16 MCI and 10 AD patients, and 15 healthy controls (HCs) were submitted to increasing concentrations of H2O2 for 20 h. Cell death was determined by flow cytometry, in the presence or absence of PARP-1 inhibitors (3-aminobenzamide (3-ABA) or Nicotinamide (NAM)), or the p53 inhibitor (nutlin-3) or stabilizer (pifithrin-α). PARP-1 and p53 mRNA levels were determined by quantitative PCR (qPCR). Lymphocytes from MCI patients showed increased susceptibility to death, attaining intermediate values between AD and controls. PARP inhibitors -3-ABA and NAM- markedly protected from H2O2-induced death, making the difference between MCI and controls disappear, but not the difference between AD and controls. PARP-1 mRNA expression was increased in MCI lymphocytes. Modulation of p53 with Nutlin-3 or pifithrin-α did not modify the H2O2-induced death of lymphocytes from MCI or AD patients, but augmented the death in control lymphocytes attaining levels similar to MCI and AD. Accordingly, p53 mRNA expression was increased in AD and MCI lymphocytes compared to controls. In all, these results show that increased oxidative death is present in lymphocytes at the MCI stage. PARP-1 has a preponderant role, with complete death protection achieved with PARP inhibition in MCI lymphocytes, but not in AD, suggesting that PARP-1 might have a protective role. In addition, deregulations of the p53 pathway seem to contribute to the H2O2-induced death in MCI and AD lymphocytes, which show increased p53 expression. The results showing a prominent protective role of PARP inhibitors opens the door to study the use of these agents to prevent oxidative death in MCI patients.

Vitamin D Increases Aβ140 Plasma Levels and Protects Lymphocytes from Oxidative Death in Mild Cognitive Impairment Patients

BACKGROUND Mild cognitive impairment (MCI) has an increased rate of progression to dementia. Alterations of some metabolic factors, such as deficiency of vitamin D, are a risk factor for cognitive deterioration. Vitamin D is involved in the clearance of β-amyloid (Aβ) from the brain. We have reported that lymphocytes from Alzheimers disease (AD) patients have an increased susceptibility to oxidative death by H2O2 exposure, but currently it is unknown if this characteristic is modifiable in vivo. OBJECTIVE To determine if correction of low vitamin D levels protects lymphocytes from oxidative death and increases Aβ1-40 plasma levels in MCI and very early AD (VEAD) patients. METHOD Sixteen MCI, 11 VEAD and 25 healthy control (HC) voluntaries were evaluated with the Clinical Dementia Rating (CDR), Montreal Cognitive assessment (MoCA), and Memory Index score (MIS). Lymphocyte death was measured by flow cytometry after 20h exposure to H2O2. In patients with low levels of vitamin D -11 MCI, 9 VEAD and 20 HC- lymphocyte H2O2-death, plasma Aβ1-40 levels and cognitive status were evaluated pre- and post-vitamin D supplementation for 6 months. RESULTS Lymphocytes from MCI and VEAD patients showed increased susceptibility to oxidative death at study entry. In MCI, but not VEAD patients, lymphocyte susceptibility to death and Aβ1-40 levels plasma levels improved after 6 months of vitamin D supplementation. In addition, cognitive status on follow-up (18 months) improved in MCI patients after vitamin D supplementation. CONCLUSION Vitamin D supplementation may be beneficial in MCI. The lack of effect in VEAD may be due to a more advanced stage or different characteristics of the neurodegenerative process.

Mitochondrial permeability transition pore contributes to mitochondrial dysfunction in fibroblasts of patients with sporadic Alzheimer's disease

In the last few decades, many reports have suggested that mitochondrial function impairment is a hallmark of Alzheimers disease (AD). Although AD is a neurodegenerative disorder, mitochondrial damage is also present in patients’ peripheral tissues, suggesting a target to develop new biomarkers. Our previous findings indicate that AD fibroblasts show specific defects in mitochondrial dynamics and bioenergetics, which affects the generation of adenosine triphosphate (ATP). Therefore, we explored the possible mechanisms involved in this mitochondrial failure. We found that compared with normal fibroblasts, AD fibroblasts had mitochondrial calcium dysregulation. Further, AD fibroblasts showed a persistent activation of the non-specific mitochondrial calcium channel, the mitochondrial permeability transition pore (mPTP). Moreover, the pharmacological blockage of mPTP with Cyclosporine A (CsA) prevented the increase of mitochondrial superoxide levels, and significantly improved mitochondrial and cytosolic calcium dysregulation in AD fibroblasts. Finally, despite the failure of CsA to improve ATP levels, the inhibition of mitochondrial calcium uptake by the mitochondrial calcium uniporter increased ATP production in AD fibroblasts, indicating that these two mechanisms may contribute to mitochondrial failure in AD fibroblasts. These findings suggest that peripheral cells present similar signs of mitochondrial dysfunction observed in the brain of AD patients. Therefore, our work creates possibilities of new targets to study for early diagnosis of the AD.