Kathrin Schulz

Mitochondrial dysfunction: an early event in Alzheimer pathology accumulates with age in AD transgenic mice.

Recent evidence suggests mitochondrial dysfunction as a common early pathomechanism in Alzheimers disease integrating genetic factors related to enhanced amyloid-beta (Ass) production and tau-hyperphosphorylation with aging, as the most relevant sporadic risk factor. To further clarify the synergistic effects of aging and Ass pathology, we used isolated mitochondria of double Swedish and London mutant APP transgenic mice and of non-tg littermates. Pronounced mitochondrial dysfunction in adult Thy-1 APP mice, such as a drop of mitochondrial membrane potential and reduced ATP-levels already appeared at 3 months when elevated intracellular but not extracellular Ass deposits are present. Mitochondrial dysfunction was associated with higher levels of reactive oxygen species, an altered Bcl-xL/Bax ratio and reduction of COX IV activity. We observed significant decreases in state 3 respiration and FCCP-uncoupled respiration in non-tg mice after treatment with extracellular Ass. Similar deficits were seen only in aged Thy-1 APP mice, probably due to compensation within the respiratory chain in young animals. We conclude that Ass dependent mitochondrial dysfunction starts already at 3 months in this AD model before extracellular deposition of Ass and progression accelerates substantially with aging.

Peripheral Mitochondrial Dysfunction in Alzheimer’s Disease: Focus on Lymphocytes

Alzheimer’s disease (AD) is the most common progressive neurodegenerative disease. Today, AD affects millions of people worldwide and the number of AD cases will increase with increased life expectancy. The AD brain is marked by severe neurodegeneration like the loss of synapses and neurons, atrophy and depletion of neurotransmitter systems in the hippocampus and cerebral cortex. Recent findings suggest that these pathological changes are causally induced by mitochondrial dysfunction and increased oxidative stress. These changes are not only observed in the brain of AD patients but also in the periphery. In this review, we discuss the potential role of elevated apoptosis, increased oxidative stress and especially mitochondrial dysfunction as peripheral markers for the detection of AD in blood cells especially in lymphocytes. We discuss recent not otherwise published findings on the level of complex activities of the respiratory chain comprising mitochondrial respiration and the mitochondrial membrane potential (MMP). We obtained decreased basal MMP levels in lymphocytes from AD patients as well as enhanced sensitivity to different complex inhibitors of the respiratory chain. These changes are in line with mitochondrial defects obtained in AD cell and animal models, and in post-mortem AD tissue. Importantly, these mitochondrial alterations where not only found in AD patients but also in patients with mild cognitive impairment (MCI). These new findings point to a relevance of mitochondrial function as an early peripheral marker for the detection of AD and MCI.

Enhanced apoptosis, oxidative stress and mitochondrial dysfunction in lymphocytes as potential biomarkers for Alzheimer's disease.

Alzheimers disease (AD) is the most common progressive neurodegenerative disease. Today, AD affects millions of people worldwide and the number of AD cases will increase with increased life expectancy. The AD brain is marked by severe neurodegeneration like the loss of synapses and neurons, atrophy and depletion of neurotransmitter systems in the hippocampus and cerebral cortex. Recent findings suggest that these pathological changes are causally induced by mitochondrial dysfunction, increased oxidative stress and elevated apoptosis. Until now, AD cannot be diagnosed by a valid clinical method or a biomarker before the disease has progressed so far that dementia is present. Furthermore, no valid method is available to determine which patient with mild cognitive impairment (MCI) will progress to AD. Therefore, a correct diagnosis in the early stage of AD is not only of importance considering that early drug treatment is more effective but also that the psychological burden of the patients and relatives could be decreased. In this review, we discuss the potential role of elevated apoptosis, increased oxidative stress and mitochondrial dysfunction as biomarker for AD in a peripheral cell model, the lymphocytes.

A new link to mitochondrial impairment in tauopathies.

Tauopathies like the “frontotemporal dementia with Parkinsonism linked to chromosome 17” (FTDP-17) are characterized by an aberrant accumulation of intracellular neurofibrillary tangles composed of hyperphosphorylated tau. For FTDP-17, a pathogenic tau mutation P301L was identified. Impaired mitochondrial function including disturbed dynamics such as fission and fusion are most likely major pathomechanisms of most neurodegenerative diseases. However, very little is known if tau itself affects mitochondrial function and dynamics. We addressed this question using SY5Y cells stably overexpressing wild-type (wt) and P301L mutant tau. P301L overexpression resulted in a substantial complex I deficit accompanied by decreased ATP levels and increased susceptibility to oxidative stress. This was paralleled by pronounced changes in mitochondrial morphology, decreased fusion and fission rates accompanied by reduced expression of several fission and fusion factors like OPA-1 or DRP-1. In contrast, overexpression of wt tau exhibits protective effects on mitochondrial function and dynamics including enhanced complex I activity. Our findings clearly link tau bidirectional to mitochondrial function and dynamics, identifying a novel aspect of the physiological role of tau and the pathomechanism of tauopathies.

Inflammation-induced loss of Pdcd4 is mediated by phosphorylation-dependent degradation

The tumor suppressor programmed cell death 4 (Pdcd4) is lost in various tumor tissues. Loss of Pdcd4 has been associated with increased tumorigenic potential and tumor progression. While various mechanisms of Pdcd4 regulation have been described, the effect of an inflammatory tumor microenvironment on Pdcd4 protein expression has not been characterized so far. In the present study, we aimed to elucidate the molecular mechanisms of Pdcd4 protein regulation in tumor cells under inflammatory conditions. 12-O-tetradecanoylphorbol 13-acetate-induced differentiation of human U937 monocytes increased the expression and secretion of inflammatory cytokines such as tumor necrosis factor α, interleukin (IL)-6 and IL-8. Exposure to conditioned medium (CM) of these activated macrophages markedly decreased Pdcd4 protein expression in various tumor cells. Similarly, indirect coculture with such activated U937 monocyte-derived macrophages resulted in the loss of Pdcd4 protein in tumor cells. Decreased Pdcd4 protein levels were attributable to enhanced proteasomal degradation, diminishing Pdcd4 protein half-life. Proteasomal degradation required activation of phosphatidylinositol-3-kinase (PI3K)-mammalian target of rapamycin (mTOR) signaling. Since macrophage-CM sufficed to induce Pdcd4 degradation, Pdcd4 downregulation was determined to be an indirect unidirectional effect of the macrophages on the tumor cells. Pdcd4 protein expression was also attenuated in vivo in mouse colon tissue in response to dextran sodium sulfate-induced colitis. In summary, we characterized PI3K-mTOR-dependent proteasome-mediated Pdcd4 degradation in tumor cells in the inflammatory tumor microenvironment. Consequently, stabilization of Pdcd4 protein could provide a promising novel avenue for therapeutics targeting inflammation-associated tumors.

Erioflorin Stabilizes the Tumor Suppressor Pdcd4 by Inhibiting Its Interaction with the E3-ligase β-TrCP1

Loss of the tumor suppressor Pdcd4 was reported for various tumor entities and proposed as a prognostic marker in tumorigenesis. We previously characterized decreased Pdcd4 protein stability in response to mitogenic stimuli, which resulted from p70S6K1-dependent protein phosphorylation, β-TrCP1-mediated ubiquitination, and proteasomal destruction. Following high-throughput screening of natural product extract libraries using a luciferase-based reporter assay to monitor phosphorylation-dependent proteasomal degradation of the tumor suppressor Pdcd4, we succeeded in showing that a crude extract from Eriophyllum lanatum stabilized Pdcd4 from TPA-induced degradation. Erioflorin was identified as the active component and inhibited not only degradation of the Pdcd4-luciferase-based reporter but also of endogenous Pdcd4 at low micromolar concentrations. Mechanistically, erioflorin interfered with the interaction between the E3-ubiquitin ligase β-TrCP1 and Pdcd4 in cell culture and in in vitro binding assays, consequently decreasing ubiquitination and degradation of Pdcd4. Interestingly, while erioflorin stabilized additional β-TrCP-targets (such as IκBα and β-catenin), it did not prevent the degradation of targets of other E3-ubiquitin ligases such as p21 (a Skp2-target) and HIF-1α (a pVHL-target), implying selectivity for β-TrCP. Moreover, erioflorin inhibited the tumor-associated activity of known Pdcd4- and IκBα-regulated αtranscription factors, that is, AP-1 and NF-κB, altered cell cycle progression and suppressed proliferation of various cancer cell lines. Our studies succeeded in identifying erioflorin as a novel Pdcd4 stabilizer that inhibits the interaction of Pdcd4 with the E3-ubiquitin ligase β-TrCP1. Inhibition of E3-ligase/target-protein interactions may offer the possibility to target degradation of specific proteins only as compared to general proteasome inhibition.

HIF‐1α protein is upregulated in HIF‐2α depleted cells via enhanced translation

The hypoxia‐inducible factors HIF‐1 and HIF‐2 are primarily regulated via stabilization of their respective α‐subunits under hypoxic conditions. Previously, compensatory upregulation of one HIF‐α‐subunit upon depletion of the other α‐subunit was described, yet the underlying mechanism remained elusive. Here we provide evidence that enhanced HIF‐1α protein expression in HIF‐2α knockdown (k/d) cells neither results from elevated HIF‐1α mRNA expression, nor from increased HIF‐1α protein stability. Instead, we identify enhanced HIF‐1α translation as molecular mechanism. Moreover, we found elevated levels of the RNA‐binding protein HuR and provide evidence that HuR is critical for the compensatory HIF‐1α regulation in HIF‐2α k/d cells.

Abstract A15: Inflammation-dependent deregulation of the tumor suppressor Pdcd4

Tumor suppressor Pdcd4 was shown to inhibit the helicase activity of eIF4A and consequently to suppress the translation of mRNAs with structured 5′UTRs. As a result, Pdcd4 inhibits transformation, migration, and invasion in vitro and tumorigenesis in vivo. Recent reports indicate that Pdcd4 protein levels are attenuated in various human tumor types. Loss of Pdcd4 was not attributable to mutational inactivation, instead miR-21-dependent translational repression and p70 S6K1 -mediated, phosphorylation-dependent attenuation of Pdcd4 protein stability emerged as important mechanisms of Pdcd4 regulation. In this study, we characterize the mechanism of Pdcd4 regulation under inflammatory, tumor-promoting conditions. Our data indicate that Pdcd4 protein is lost in human tumor cells exposed to an inflammatory environment generated by activated macrophages. Downregulation of Pdcd4 required activation of PI3K-mTOR-p70 S6K1 -signaling and was attributable to a markedly reduced Pdcd4 protein half-life resulting from increased proteasomal degradation. Functionally, Pdcd4 protein levels inversely correlated with AP-1 transactivation, migration, and invasion. Based on the proposed mechanism of Pdcd4 regulation, we further developed an assay to identify compounds interfering with tumor-promoter induced degradation of Pdcd4. Identified hit compounds will provide a source for novel, translation-oriented approaches for tumor therapies. In summary, we suggest that p70 S6K1 -dependent phosphorylation and subsequent proteasomal degradation of Pdcd4 protein is a key mechanism in the regulation of the tumor suppressor Pdcd4 under inflammation-associated, tumor-promoting conditions. Consequently, we put forward stabilization of Pdcd4 as a novel tumor therapeutic approach and identify novel compounds disrupting Pdcd49s phosphorylation-dependent destabilization during tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr A15.