Sandrine Humez

Nuclear Tau, a Key Player in Neuronal DNA Protection

Tau, a neuronal protein involved in neurodegenerative disorders such as Alzheimer disease, which is primarily described as a microtubule-associated protein, has also been observed in the nuclei of neuronal and non-neuronal cells. However, the function of the nuclear form of Tau in neurons has not yet been elucidated. In this work, we demonstrate that acute oxidative stress and mild heat stress (HS) induce the accumulation of dephosphorylated Tau in neuronal nuclei. Using chromatin immunoprecipitation assays, we demonstrate that the capacity of endogenous Tau to interact with neuronal DNA increased following HS. Comet assays performed on both wild-type and Tau-deficient neuronal cultures showed that Tau fully protected neuronal genomic DNA against HS-induced damage. Interestingly, HS-induced DNA damage observed in Tau-deficient cells was completely rescued after the overexpression of human Tau targeted to the nucleus. These results highlight a novel role for nuclear Tau as a key player in early stress response.

Store depletion and store-operated Ca2+ current in human prostate cancer LNCaP cells: involvement in apoptosis

1 In the present study, we investigated the mechanisms involved in the induction of apoptosis by the Ca2+‐ATPase inhibitor thapsigargin (TG), in androgen‐sensitive human prostate cancer LNCaP cells. 2 Exposure of fura‐2‐loaded LNCaP cells to TG in the presence of extracellular calcium produced an increase in intracellular Ca2+, the first phase of which was associated with depletion of intracellular stores and the second one with consecutive extracellular Ca2+ entry through plasma membrane, store‐operated Ca2+ channels (SOCs). 3 For the first time we have identified and characterized the SOC‐mediated membrane current (Istore) in prostate cells using whole‐cell, cell‐attached, and perforated patch‐clamp techniques, combined with fura‐2 microspectrofluorimetric and Ca2+‐imaging measurements. 4 I store in LNCaP cells lacked voltage‐dependent gating and displayed an inwardly rectifying current‐voltage relationship. The unitary conductance of SOCs with 80 mM Ca2+ as a charge carrier was estimated at 3.2 ± 0.4 pS. The channel has a high selectivity for Ca2+ over monovalent cations and is inhibited by Ni2+ (0.5–3 mM) and La3+ (1 μM). 5 Treatment of LNCaP cells with TG (0.1 μM) induced apoptosis as judged from morphological changes. Decreasing extracellular free Ca2+ to 200 nM or adding 0.5 mM Ni2+ enhanced TG‐induced apoptosis. 6 The ability of TG to induce apoptosis was not reduced by loading the cells with intracellular Ca2+ chelator (BAPTA‐AM). 7 These results indicate that in androgen‐sensitive prostate cancer cells the depletion of intracellular Ca2+ stores may trigger apoptosis but that there is no requirement for the activation of store‐activated Ca2+ current and sustained Ca2+ entry in induction and development of programmed cell death.

Role of Cationic Channel TRPV2 in Promoting Prostate Cancer Migration and Progression to Androgen Resistance

Castration resistance in prostate cancer (PCa) constitutes an advanced, aggressive disease with poor prognosis, associated with uncontrolled cell proliferation, resistance to apoptosis, and enhanced invasive potential. The molecular mechanisms involved in the transition of PCa to castration resistance are obscure. Here, we report that the nonselective cationic channel transient receptor potential vanilloid 2 (TRPV2) is a distinctive feature of castration-resistant PCa. TRPV2 transcript levels were higher in patients with metastatic cancer (stage M1) compared with primary solid tumors (stages T2a and T2b). Previous studies of the TRPV2 channel indicated that it is primarily involved in cancer cell migration and not in cell growth. Introducing TRPV2 into androgen-dependent LNCaP cells enhanced cell migration along with expression of invasion markers matrix metalloproteinase (MMP) 9 and cathepsin B. Consistent with the likelihood that TRPV2 may affect cancer cell aggressiveness by influencing basal intracellular calcium levels, small interfering RNA-mediated silencing of TRPV2 reduced the growth and invasive properties of PC3 prostate tumors established in nude mice xenografts, and diminished expression of invasive enzymes MMP2, MMP9, and cathepsin B. Our findings establish a role for TRPV2 in PCa progression to the aggressive castration-resistant stage, prompting evaluation of TRPV2 as a potential prognostic marker and therapeutic target in the setting of advanced PCa.

Lysophospholipids stimulate prostate cancer cell migration via TRPV2 channel activation.

The physiological role, the mechanisms of activation, as well as the endogenous regulators for the non-selective cationic channel TRPV2 are not known so far. In the present work we report that endogenous lysophospholipids such as lysophosphatidylcholine (LPC) and lysophosphatidylinositol (LPI) induce a calcium influx via TRPV2 channel. This activation is dependent on the length of the side-chain and the nature of the lysophospholipid head-group. TRPV2-mediated calcium uptake stimulated by LPC and LPI occurred via Gq/Go-protein and phosphatidylinositol-3,4 kinase (PI3,4K) signalling. We have shown that the mechanism of TRPV2 activation induced by LPC and LPI is due to the TRPV2 channel translocation to the plasma membrane. The activation of TRPV2 channel by LPC and LPI leads to an increase in the cell migration of the prostate cancer cell line PC3. We have demonstrated that TRPV2 is directly involved in both steady-state and lysophospholipid-stimulated cancer cell migration. Thus, for the first time, we have identified one of the natural regulators of TRPV2 channel, one of the mechanisms of TRPV2 activation and regulation, as well as its pathophysiological role in cancer.

Beneficial effects of exercise in a transgenic mouse model of Alzheimer's disease-like Tau pathology.

Tau pathology is encountered in many neurodegenerative disorders known as tauopathies, including Alzheimers disease. Physical activity is a lifestyle factor affecting processes crucial for memory and synaptic plasticity. Whether long-term voluntary exercise has an impact on Tau pathology and its pathophysiological consequences is currently unknown. To address this question, we investigated the effects of long-term voluntary exercise in the THY-Tau22 transgenic model of Alzheimers disease-like Tau pathology, characterized by the progressive development of Tau pathology, cholinergic alterations and subsequent memory impairments. Three-month-old THY-Tau22 mice and wild-type littermates were assigned to standard housing or housing supplemented with a running wheel. After 9 months of exercise, mice were evaluated for memory performance and examined for hippocampal Tau pathology, cholinergic defects, inflammation and genes related to cholesterol metabolism. Exercise prevented memory alterations in THY-Tau22 mice. This was accompanied by a decrease in hippocampal Tau pathology and a prevention of the loss of expression of choline acetyltransferase within the medial septum. Whereas the expression of most cholesterol-related genes remained unchanged in the hippocampus of running THY-Tau22 mice, we observed a significant upregulation in mRNA levels of NPC1 and NPC2, genes involved in cholesterol trafficking from the lysosomes. Our data support the view that long-term voluntary physical exercise is an effective strategy capable of mitigating Tau pathology and its pathophysiological consequences.

A2A adenosine receptor deletion is protective in a mouse model of Tauopathy

Consumption of caffeine, a non-selective adenosine A2A receptor (A2AR) antagonist, reduces the risk of developing Alzheimer’s disease (AD) in humans and mitigates both amyloid and Tau burden in transgenic mouse models. However, the impact of selective A2AR blockade on the progressive development of AD-related lesions and associated memory impairments has not been investigated. In the present study, we removed the gene encoding A2AR from THY-Tau22 mice and analysed the subsequent effects on both pathological (Tau phosphorylation and aggregation, neuro-inflammation) and functional impairments (spatial learning and memory, hippocampal plasticity, neurotransmitter profile). We found that deleting A2ARs protect from Tau pathology-induced deficits in terms of spatial memory and hippocampal long-term depression. These effects were concomitant with a normalization of the hippocampal glutamate/gamma-amino butyric acid ratio, together with a global reduction in neuro-inflammatory markers and a decrease in Tau hyperphosphorylation. Additionally, oral therapy using a specific A2AR antagonist (MSX-3) significantly improved memory and reduced Tau hyperphosphorylation in THY-Tau22 mice. By showing that A2AR genetic or pharmacological blockade improves the pathological phenotype in a Tau transgenic mouse model, the present data highlight A2A receptors as important molecular targets to consider against AD and Tauopathies.

Hippocampal tauopathy in tau transgenic mice coincides with impaired hippocampus-dependent learning and memory, and attenuated late-phase long-term depression of synaptic transmission.

We evaluated various forms of hippocampus-dependent learning and memory, and hippocampal synaptic plasticity in THY-Tau22 transgenic mice, a murine tauopathy model that expresses double-mutated 4-repeat human tau, and shows neuropathological tau hyperphosphorylation and aggregation throughout the brain. Focussing on hippocampus, immunohistochemical studies in aged THY-Tau22 mice revealed prominent hyper- and abnormal phosphorylation of tau in CA1 region, and an increase in glial fibrillary acidic protein (GFAP) in hippocampus, but without signs of neuronal loss. These mice displayed spatial, social, and contextual learning and memory defects that could not be reduced to subtle neuromotor disability. The behavioral defects coincided with changes in hippocampal synaptic functioning and plasticity as measured in paired-pulse and novel long-term depression protocols. These results indicate that hippocampal tauopathy without neuronal cell loss can impair neural and behavioral plasticity, and further show that transgenic mice, such as the THY-Tau22 strain, might be useful for preclinical research on tauopathy pathogenesis and possible treatment.

NMDA receptor dysfunction contributes to impaired brain-derived neurotrophic factor-induced facilitation of hippocampal synaptic transmission in a Tau transgenic model

While the spatiotemporal development of Tau pathology has been correlated with occurrence of cognitive deficits in Alzheimers patients, mechanisms underlying these deficits remain unclear. Both brain‐derived neurotrophic factor (BDNF) and its tyrosine kinase receptor TrkB play a critical role in hippocampus‐dependent synaptic plasticity and memory. When applied on hippocampal slices, BDNF is able to enhance AMPA receptor‐dependent hippocampal basal synaptic transmission through a mechanism involving TrkB and N‐methyl‐d‐Aspartate receptors (NMDAR). Using THY‐Tau22 transgenic mice, we demonstrated that hippocampal Tau pathology is associated with loss of synaptic enhancement normally induced by exogenous BDNF. This defective response was concomitant to significant memory impairments. We show here that loss of BDNF response was due to impaired NMDAR function. Indeed, we observed a significant reduction of NMDA‐induced field excitatory postsynaptic potential depression in the hippocampus of Tau mice together with a reduced phosphorylation of NR2B at the Y1472, known to be critical for NMDAR function. Interestingly, we found that both NR2B and Src, one of the NR2B main kinases, interact with Tau and are mislocalized to the insoluble protein fraction rich in pathological Tau species. Defective response to BDNF was thus likely related to abnormal interaction of Src and NR2B with Tau in THY‐Tau22 animals. These are the first data demonstrating a relationship between Tau pathology and synaptic effects of BDNF and supporting a contribution of defective BDNF response and impaired NMDAR function to the cognitive deficits associated with Tauopathies.

Role of endoplasmic reticulum calcium content in prostate cancer cell growth regulation by IGF and TNFalpha

Variations in calcium concentration within the endoplasmic reticulum ([Ca2+]ER) may play a role in cell growth. This study evaluates the regulation of calcium pools by growth modulators of prostate cancer (PC) cells, the insulin growth factor (IGF), and the tumor necrosis growth factor‐alpha (TNFalpha) as well as evaluating the possible role of [Ca2+]ER variations as signals for growth modulation. We show that IGF (5 ng/ml), which increases cell growth, induces an increase in [Ca2+]ER whereas TNFalpha (1 ng/ml) which reduces cell proliferation and induces apoptosis, reduces [Ca2+]ER. IGF‐induced [Ca2+]ER increase is correlated to an overexpression of the sarcoendoplasmic calcium‐ATPase 2B (SERCA2b), whereas TNFalpha‐induced [Ca2+]ER decrease is associated to a reduction in SERCA2b expression. Pretreatment with epidermal growth factors (EGF) or IGF does not prevent TNFalpha from affecting the induction of apoptosis, [Ca2+]ER reduction and SERCA2b downregulation. Reduction in [Ca2+]ER induced by thapsigargin (TG) (from 1 pM to 1 μM, 48 h) reduces LNCaP growth in a dose dependent manner and induces apoptosis when cells are treated with 1 μM TG. We also show that a transient TG application (1 pM, 1 nM, 1 μM 15 min) is insufficient to induce a long lasting decrease in [Ca2+]ER, since [Ca2+]ER remains identical to the control for 48 h following TG application. These treatments (1 pM and 1 nM, 15 min) do not modify cell growth. However, TG (1 μM, 15 min) induces apoptosis. We thus identify [Ca2+]ER and SERCA2b as a central targets for causing LNCaP PC cell life or death induced by growth modulators. Furthermore our results indicate that calcium pool contents can regulate cell growth.

Prolactin stimulates cell proliferation through a long form of prolactin receptor and K+ channel activation

PRL (prolactin) has been implicated in the proliferation and differentiation of numerous tissues, including the prostate gland. However, the PRL-R (PRL receptor) signal transduction pathway, leading to the stimulation of cell proliferation, remains unclear and has yet to be mapped. The present study was undertaken to develop a clear understanding of the mechanisms involved in this pathway and, in particular, to determine the role of K(+) channels. We used androgen-sensitive prostate cancer (LNCaP) cells whose proliferation is known to be stimulated by PRL. Reverse transcriptase PCR analysis showed that LNCaP cells express a long form of PRL-R, but do not produce its intermediate isoform. Patch-clamp techniques showed that the application of 5 nM PRL increased both the macroscopic K(+) current amplitude and the single K(+)-channel open probability. This single-channel activity increase was reduced by the tyrosine kinase inhibitors genistein, herbimycin A and lavandustine A, thereby indicating that tyrosine kinase phosphorylation is required in PRL-induced K(+) channel stimulation. PRL enhances p59( fyn ) phosphorylation by a factor of 2 after a 10 min application in culture. In addition, where an antip59( fyn ) antibody is present in the patch pipette, PRL no longer increases K(+) current amplitude. Furthermore, the PRL-stimulated proliferation is inhibited by the K(+) channel inhibitors alpha-dendrotoxin and tetraethylammonium. Thus, as K(+) channels are known to be involved in LNCaP cell proliferation, we suggest that K(+) channel modulation by PRL, via p59( fyn ) pathway, is the primary ionic event in PRL signal transduction, triggering cell proliferation.