Carolina Alquézar

Altered cell cycle‐related gene expression in brain and lymphocytes from a transgenic mouse model of Alzheimer’s disease [amyloid precursor protein/presenilin 1 (PS1)]

Cumulative evidence indicates that aberrant re‐expression of many cell cycle‐related proteins and inappropriate neuronal cell cycle control are critical events in Alzheimer’s disease (AD) pathogenesis. Evidence of cell cycle activation in post‐mitotic neurons has also been observed in murine models of AD, despite the fact that most of these mice do not show massive loss of neuronal bodies. Dysfunction of the cell cycle appears to affect cells other than neurons, as peripheral cells, such as lymphocytes and fibroblasts from patients with AD, show an altered response to mitogenic stimulation. We sought to determine whether cell cycle disturbances are present simultaneously in both brain and peripheral cells from the amyloid precursor protein (APP)/presenilin 1 (PS1) mouse model of AD, in order to validate the use of peripheral cells from patients not only to study cell cycle abnormalities as a pathogenic feature of AD, but also as a means to test novel therapeutic approaches. By using cell cycle pathway‐specific RT2Profiler™ PCR Arrays, we detected changes in a number of cell cycle‐related genes in brain as well as in lymphocytes from APP/PS1 mice. Moreover, we found enhanced 5′‐bromo‐2′‐deoxyuridine incorporation into DNA in lymphocytes from APP/PS1 mice, and increased expression of the cell proliferation marker proliferating cell nuclear antigen (PCNA), and the cyclin‐dependent kinase (CDK) inhibitor Cdkn2a, as detected by immunohistochemistry in cortical neurons of the APP/PS1 mice. Taken together, the cell cycle‐related changes in brain and blood cells reported here support the mitosis failure hypothesis in AD and validate the use of peripheral cells as surrogate tissue to study the molecular basis of AD pathogenesis.

Alteration in cell cycle-related proteins in lymphoblasts from carriers of the c.709-1G>A PGRN mutation associated with FTLD-TDP dementia.

Frontotemporal lobar degeneration with neuronal inclusions containing TAR DNA binding protein 43 (TDP-43) is associated in most cases with null-mutations in the progranulin gene (PGRN). While the mechanisms by which PGRN haploinsufficiency leads to neurodegeneration remained speculative, increasing evidence support the hypothesis that cell cycle reentry of postmitotic neurons precedes many instances of neuronal death. Based in the mitogenic and neurotrophic activities of PGRN, we hypothesized that PGRN deficit may induce cell cycle disturbances and alterations in neuronal vulnerability. Because cell cycle dysfunction is not restricted to neurons, we studied the influence of PGRN haploinsufficiency, on cell cycle control in peripheral cells from patients suffering from frontotemporal dementia, bearing the PGRN mutation c.709-1G>A. Here we show that progranulin deficit increased cell cycle activity in immortalized lymphocytes. This effect was associated with increased levels of cyclin-dependent kinase 6 (CDK6) and phosphorylation of retinoblastoma protein (pRb), resulting in a G(1)/S regulatory failure. A loss of function of TDP-43 repressing CDK6 expression may result from altered subcellular TDP-43 distribution. The distinct functional features of lymphoblastoid cells from c.709-1 G>A carriers offer an invaluable, noninvasive tool to investigate the etiopathogenesis of frontotemporal lobar degeneration.

Downregulation of extracellular signal-regulated kinase 1/2 activity by calmodulin KII modulates p21Cip1 levels and survival of immortalized lymphocytes from Alzheimer’s disease patients

Previously, we reported a Ca(2+)/calmodulin (CaM)-dependent impairment of apoptosis induced by serum deprivation in Alzheimers disease (AD) lymphoblasts. These cell lines showed downregulation of extracellular signal-regulated kinase (ERK)1/2 activity and elevated content of p21 compared with control cells. The aim of this study was to delineate the molecular mechanism underlying the distinct regulation of p21 content in AD cells. Quantitative reverse transcription polymerase chain reaction analysis demonstrated increased p21 messenger RNA (mRNA) levels in AD cells. The ERK1/2 inhibitor, PD98059, prevented death of control cells and enhanced p21 mRNA and protein levels. The CaM antagonist, calmidazolium, and the CaMKII inhibitor, KN-62, normalized the survival pattern of AD lymphoblasts by augmenting ERK1/2 activation and reducing p21 mRNA and protein levels. Upregulation of p21 transcription in AD cells appears to be the consequence of increased activity of forkhead box O3a (FOXO3a) as the result of diminished ERK1/2-mediated phosphorylation of this transcription factor, which in turn facilitates its nuclear accumulation. Murine double minute 2 (MDM2) protein levels were decreased in AD cells relative to control lymphoblasts, suggesting an impairment of FOXO3a degradation.

Control of cell adhesion and migration by podocalyxin. Implication of Rac1 and Cdc42

Podocalyxin (PODXL) is a type I membrane sialomucin, originally described in the epithelial cells (podocytes) of kidney glomeruli. PODXL is also found in extra-renal tissues and in certain aggressive tumors, but its precise pathophysiological role is unknown. Expression of PODXL in CHO cells enhances their adhesive, migratory and cell-cell interactive properties in a selectin and integrin-dependent manner. We aimed at defining the PODXL domains responsible for those cell responses. For this purpose we have analyzed the cell adhesion/migration responses to deletion mutants of human PODXL, and the correlation with the activities of Rac1 and Cdc42 GTPases. The results obtained indicate that integrity of the PODXL ectodomain is essential for enhancing cell adhesion but not migration, while the integrity of the cytoplasmic domain is required for both adhesion and migration. Deletion of the carboxy-terminal DTHL domain (PODXL-ΔDTHL) limited only cell adhesion. The activities of Rac1 and Cdc42 GTPases parallel the PODXL-induced variations in cell adhesion and migration. Moreover, silencing the rac1 gene virtually abolished the effect of PODXL in enhancing cell adhesion.

Targeting TDP-43 phosphorylation by Casein Kinase-1δ inhibitors: a novel strategy for the treatment of frontotemporal dementia

BackgroundMutations in the progranulin gene (GRN) are the most common cause of frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP). TDP-43 pathology is characterized by the hyperphosphorylation of the protein at Serine 409/410 residues. Casein kinase-1δ (CK-1δ) was reported to phosphorylate TDP-43 directly. Previous works from our laboratory described the presence of CDK6/pRb-dependent cell cycle alterations, and cytosolic accumulation of TDP-43 protein in lymphoblast from FTLD-TDP patients carriers of a loss-of function mutation in GRN gene (c.709-1G > A). In this work, we have investigated the effects of two brain penetrant CK-1δ inhibitors (IGS-2.7 and IGS-3.27) designed and synthetized in our laboratory on cell proliferation, TDP-43 phosphorylation and subcellular localization, as well as their effects on the known nuclear TDP-43 function repressing the expression of CDK6.ResultsWe report here that both CK-1δ inhibitors (IGS-2.7 and IGS-3.27) normalized the proliferative activity of PGRN-deficient lymphoblasts by preventing the phosphorylation of TDP-43 fragments, its nucleo-cytosol translocation and the overactivation of the CDK6/pRb cascade. Moreover, ours results show neuroprotective effects of CK-1δ inhibitors in a neuronal cell model of induced TDP-43 phosphorylation.ConclusionsOur results suggest that modulating CK-1δ activity could be considered a novel therapeutic approach for the treatment of FTLD-TDP and other TDP-43 proteinopathies.

Simvastatin overcomes the resistance to serum withdrawal-induced apoptosis of lymphocytes from Alzheimer’s disease patients

Statins may exert beneficial effects on Alzheimer’s disease (AD) patients. Based on the antineoplastic and apoptotic effects of statins in a number of cell types, we hypothesized that statins may be able to protect neurons by controlling the regulation of cell cycle and/or apoptosis. A growing body of evidence indicates that neurodegeneration involves the cell-cycle activation in postmitotic neurons. Failure of cell-cycle control is not restricted to neurons in AD patients, but occurs in peripheral cells as well. For these reasons, we studied the role of simvastatin (SIM) on cell survival/death in lymphoblasts from AD patients. We report here that SIM induces apoptosis in AD lymphoblasts deprived of serum. SIM interacts with PI3K/Akt and ERK1/2 signaling pathways thereby decreasing the serum withdrawal-enhanced levels of the CDK inhibitor p21Cip1 (p21) and restoring the vulnerability of AD cells to trophic factor deprivation.

G1/S Cell Cycle Checkpoint Dysfunction in Lymphoblasts from Sporadic Parkinson's Disease Patients.

Parkinson’s disease (PD) is the second most prevalent neurodegenerative disease among aging individuals, affecting greatly the quality of their life. However, the pathogenesis of Parkinson’s disease is still incompletely understood to date. Increasing experimental evidence suggests that cell cycle reentry of postmitotic neurons precedes many instances of neuronal death. Since cell cycle dysfunction is not restricted to neurons, we investigated this issue in peripheral cells from patients suffering from sporadic PD and age-matched control individuals. Here, we describe increased cell cycle activity in immortalized lymphocytes from PD patients that is associated to enhanced activity of the cyclin D3/CDK6 complex, resulting in higher phosphorylation of the pRb family protein and thus, in a G1/S regulatory failure. Decreased degradation of cyclin D3, together with increased p21 degradation, as well as elevated levels of CDK6 mRNA and protein were found in PD lymphoblasts. Inhibitors of cyclin D3/CDK6 activity like sodium butyrate, PD-332991, and rapamycin were able to restore the response of PD cells to serum stimulation. We conclude that lymphoblasts from PD patients are a suitable model to investigate cell biochemical aspects of this disease. It is suggested that cyclin D3/CDK6-associated kinase activity could be potentially a novel therapeutic target for the treatment of PD.

Targeting cyclin D3/CDK6 activity for treatment of Parkinson's disease

At present, treatment for Parkinsons disease (PD) is only symptomatic; therefore, it is important to identify new targets tackling the molecular causes of the disease. We previously found that lymphoblasts from sporadic PD patients display increased activity of the cyclin D3/CDK6/pRb pathway and higher proliferation than control cells. These features were considered systemic manifestations of the disease, as aberrant activation of the cell cycle is involved in neuronal apoptosis. The main goal of this work was to elucidate whether the inhibition of cyclin D3/CDK6‐associated kinase activity could be useful in PD treatment. For this purpose, we investigated the effects of two histone deacetylase (HDAC) inhibitors, suberoylanilide hydroxamic (SAHA) acid and sodium butyrate (NaB), and the m‐TOR inhibitor rapamycin on cell viability and cyclin D3/CDK6 activity. Moreover, the potential neuroprotective action of these drugs was evaluated in 6‐hydroxy‐dopamine (6‐OHDA) treated dopaminergic SH‐SY5Y cells and primary rat mesencephalic cultures. Here, we report that both compounds normalized the proliferative activity of PD lymphoblasts and reduced the 6‐OHDA‐induced cell death in neuronal cells by preventing the over‐activation of the cyclin D3/CDK6/pRb cascade. Considering that these drugs are already used in clinic for treatment of other diseases with good tolerance, it is plausible that they may serve as novel therapeutic drugs for PD. We report here that peripheral cells from Parkinsons disease (PD) patients show an enhanced proliferative activity due to the activation of cyclin D3/CDK6‐mediated phosphorylation of retinoblastoma protein (pRb). Treatment of PD lymphoblasts with inhibitors of histone deacetylases like suberoylanilide hydroxamic acid (SAHA) and sodium butyrate (NaB), or with rapamycin, inhibitor of mechanistic target of rapamycin (mTOR) normalized the proliferation of PD lymphoblasts by preventing the over‐activation of the cyclin D3/CDK6/pRb cascade. These drugs were shown to have neuroprotective effects in both human neuroblastoma SH‐SY5Y cells and primary rat mid‐brain dopaminergic neuronal cultures toxicity induced by 6‐hidroxydopamine. Considering that these drugs are already used in clinic for treatment of other diseases with good tolerance, it seems reasonable to believe that the repositioning of these drugs toward PD holds promise as a novel therapeutic strategy.

Increased Wnt Signaling and Reduced Viability in a Neuronal Model of Progranulin-Deficient Frontotemporal Lobar Degeneration.

Progranulin (PGRN) deficiency is considered the major cause of frontotemporal lobar degeneration with TDP-43 protein inclusions (FTLD-TDP). Recent work unveiled a relationship between Wnt signaling and PGRN in cellular models of FTLD and cells of patients carrying loss-of-function GRN mutations. This study was undertaken to explore the relationship between PGRN deficit and Wnt signaling in the regulation of survival of GRN knockdown neuroblastoma SH-SY5Y cells (GRN KD). We report here that both canonical and noncanonical Wnt signaling cascades are overactivated in GRN KD cells. We detected increased expression levels of Wnt1 and Wnt5a ligands of the Frizzled receptors, as well as evidence for increased signaling of the Wnt/β-catenin and Wnt/Ca2+ cascades in PGRN deficient cells, such as increased nuclear content of β-catenin and higher levels of cyclin D1, or increased levels of the active form of the NFAT1 transcription factor, respectively. Upregulation of either Wnt/β-catenin or Wnt/Ca2+ signaling in GRN KD cells leads to the stimulation of BrdU incorporation into DNA, hyperphosphorylation of the pRb family of proteins and reduced cell viability over time. Blocking the Wnt cascades by specific canonical or noncanonical inhibitors of Wnt-dependent signaling, normalized the rate of DNA synthesis, and what it is more important restored the viability of GRN KD cells. Our results suggest an important role of Wnt activation inducing cell cycle disturbance-mediated neuronal loss in the pathogenesis of PGRN deficiency-linked FTLD-TDP. Therefore, it is plausible that modulation of Wnt signaling could be a promising strategy for developing of new disease-modifying treatments for FTLD-TDP.

Activation of the Cannabinoid Type 2 Receptor by a Novel Indazole Derivative Normalizes the Survival Pattern of Lymphoblasts from Patients with Late-Onset Alzheimer’s Disease

BackgroundAlzheimer’s disease is a multifactorial disorder for which there is no disease-modifying treatment yet. CB2 receptors have emerged as a promising therapeutic target for Alzheimer’s disease because they are expressed in neuronal and glial cells and their activation has no psychoactive effects.ObjectiveThe aim of this study was to investigate whether activation of the CB2 receptor would restore the aberrant enhanced proliferative activity characteristic of immortalized lymphocytes from patients with late-onset Alzheimer’s disease. It is assumed that cell-cycle dysfunction occurs in both peripheral cells and neurons in patients with Alzheimer’s disease, contributing to the instigation of the disease.MethodsLymphoblastoid cell lines from patients with Alzheimer’s disease and age-matched control individuals were treated with a new, in-house-designed dual drug PGN33, which behaves as a CB2 agonist and butyrylcholinesterase inhibitor. We analyzed the effects of this compound on the rate of cell proliferation and levels of key regulatory proteins. In addition, we investigated the potential neuroprotective action of PGN33 in β-amyloid-treated neuronal cells.ResultsWe report here that PGN33 normalized the increased proliferative activity of Alzheimer’s disease lymphoblasts. The compound blunted the calmodulin-dependent overactivation of the PI3K/Akt pathway, by restoring the cyclin-dependent kinase inhibitor p27 levels, which in turn reduced the activity of the cyclin-dependent kinase/pRb cascade. Moreover, this CB2 agonist prevented β-amyloid-induced cell death in neuronal cells.ConclusionOur results suggest that the activation of CB2 receptors could be considered a useful therapeutic approach for Alzheimer’s disease.