Papel de la progranulina en los mecanismos de regulación de superviviencia-muerte celular en la demencia frontotemporal

Abstract

Frontotemporal lobar degeneration (FTLD) is a genetically complex neurodegenerative disorder, accounting for 20% of patients with an onset of dementia before 65 years (Ikeda, et al., 2004). The clinical symptoms associated with FTLD are diverse, including behavior and personality changes, language disorders of expression and comprehension and cognitive impairment with early preservation of memory (Neary, et al., 1998). To date, the clinicopathologic spectrum of FTLD includes not only the frontal and temporal variants of FTD, frontotemporal degeneration with abnormal behavior (bvFTD), primary progressive aphasia (PPA) or semantic dementia (SD), but also corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), progressive subcortical gliosis (PSG) and FTD with motor neuron disease (FTD-EMN) (Van Langenhove, et al., 2012). Under a microscope, common pathologic changes of FTLD are atrophy brain region presenting neuronal loss and gliosis in cortices of atrophied frontal and temporal lobes. Histochemically, FTLD can be categorized according to the major component of the celular inclusions deposited in the brain. In the majority of cases, the pathological protein is either the microtubule-associated protein tau or the transactive response DNA-binding protein TDP-43 (FTLD-tau, FTLD-TDP respectively), a small number of cases present inclusions of fused in sarcoma (FUS) protein (FTLD-FUS) or inclusions of a protein that can not be identified and that can only be unveiled by immunostaining against the proteins of the ubiquitin proteasome system (UPS) (FTLD-UPS) (Cairns, et al., 2007,Mackenzie, et al., 2011). A positive family history of FTLD is present in 25-50% of cases and the transmission is usually autosomal domi nant. A few genes have been associated with familial FTLD including microtubule-associated protein tau (MAPT), progranulin (GRN) (Baker), transactive response (TAR) DNA-binding protein-43 (TARDBP), chromatin-modifying 2B protein (CHMP2B), Valosin-containing protein (VCP), and chromosome 9 open reading frame 72 (C9ORF72) genes (Sieben, et al., 2012). Mutations in the GRN gene are the major cause of familial FTLD-TDP (Baker, et al., 2006,Cruts, et al., 2006). To date, more than 60 mutations have been reported in GRN gene (Gijselinck, et al., 2008). Most of the pathogenic mutations are predicted to cause functional null alleles with premature termination of the GRN coding sequence, resulting in nonsense-mediated decay (NMD) of the mutant messenger RNAs (mRNAs), suggesting that progranulin (PGRN) haploinsufficiency is a major pathogenic mechanism. Little is known about the normal function of PGRN in the Central Nervous Sistem (CNS) and the mechanism by which PGRN haploinsufficency leads to neurodegeneration in FTLD remained speculative. In the last decade, mounting evidences support the hypothesis that cell cycle reentry of postmitotic neurons and neural stem cells precedes many instances of neuronal dead (Herrup, et al., 2004). Based in the mitogenic and neuotrophic activities of PGRN, we hypothesized that PGRN deficit may induce cell cycle disturbances and alterations in neuronal vulnerability.

An alternative strategy to study the pathogenesis of FTLD is the use of non-neuronal cells from patients. Numerous observations indicate that, while the predominant clinical expression arises from brain, the neurodegenerative diseases have systemic expression at the cellular and molecular levels. Considerable precedents exist for studying neurodegenerative diseases with peripheral tissues including lymphocytes, fibroblast and platelets (Bialopiotrowicz, et al., 2012). The use of peripheral tissues could complement studies of autopsy samples and provide a useful tool with which to investigate dynamic processes such as signal transduction mechanisms, oxidative metabolism among others. Aims: The main goal of this work was to study the influence of PGRN haploinsufficiency, caused by a prevalent ancestral mutation in GRN gene related to Basque population, c.709- 1G>A, in cell cycle and survival regulation of peripheral blood cells from carriers of the GRN mutation. The possibility to detect molecular alterations in non-neuronal cells, easily accessible, from control, asymptomatic and patients, could provide a useful tool for the early diagnosis of this complex disorder, and particularly to search for treatment or neuroprotective strategies. To this end, we generated lymphoblastoid cell lines from c.709-1G>A carriers diagnosed of FTLD or asymptomatic and control individuals by infecting with the Epstein Barr virus (EBV). Although FTLD symptoms reflect preferential neuronal loss in specific brain regions, PGRN is expressed in almost all tissues, so abnormalities outside the brain might be expected. Moreover, this experimental model has been previously used to study cell cyclerelated events associated with neurodegeneration in other neurodegenerative diseases, e. g. Alzheimer’s disease. Results: Our results show that PGRN haploinsufficiency increased cell cycle activity in immortalized lymphocytes from FTLD patients. This effect was associated with increased levels of cyclin-dependent kinase 6 (CDK6) and phosphorylation of retinoblastoma protein (pRb), resulting in a G1/S regulatory failure. In addition, the lymphoblast carriers of the c.709- 1G>A mutation display a small but significant decrease in the nuclear content of TDP-43. Taken together, these observations suggest that a loss of nuclear TDP-43 function in repressing CDK6 may be involved in the increased pRb phosphorylation, and enhanced proliferative activity of PGRN deficient cells. Assuming that the cell cycle disturbances reported here could be peripheral signs of the disease, our results suggest that neurons of c.709- Degeneración Lobar Frontotemporal asociada a mutaciones en progranulina 1G>A mutation carriers are at high risk of entering an unscheduled cell cycle that would then drive them to death. It is suggested that therapeutic interventions aimed at ameliorating cell cycle progression activity may have a positive impact in FTLD. Using the same cells of FTLD patients, we performed a comparative study of cell viability after serum withdrawal of established lymphoblastoid cell lines from control and carries of c.709-1G>A GRN mutation. Our results suggest that the CDK6/pRb pathway also is enhanced after the serum deprivation. Apparently, this feature allows PGRN deficient cells to escape from serum withdrawal induced apoptosis by decreasing the activity of executive caspases and lowering the dissipation of mitochondrial membrane potential and the release of cytochrome c from the mitochondria. Inhibitors of CDK6 expression levels like sodium butyrate or the CDK6 activity such as PD332991 were able to restore the normal response of lymphoblasts from FLTD-TDP patients to trophic factor withdrawal or cell proliferation. Our results suggested that CDK6 could be potentially a therapeutic target for the treatment of the FTLD-TDP.

The next step in this work was analyzing potential alterations in signaling pathways induced by PGRN haploinsufficency to explain the altered cellular response to serum stimulation. To this end we use specific inhibitors of main pathways as well as conditioned medium from control and PGRN-deficient lymphoblast or exogenous recombinant human PGRN (rhPGRN). Our results show that the specific signaling cascade implicated in the upregulation of CDK6 activity and cell proliferation of GRN mutation bearing lymphoblasts is Ca2+, protein kinase C (PKC), and pertussis toxin (PTX)-dependent and involves activationof the extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) pathway. Addition of exogenous PGRN or conditioned medium from control cells normalized the response of PGRN-deficient lymphoblasts to serum activation. Our data indicated that non-canonical Wnt5a signaling might be overactivated by PGRN deficiency. We detected increased cellular and secreted levels of Wnt5a in PGRN-deficient lymphoblasts associated with enhanced phosphorylated calmodulin kinase II (pCaMKII). Moreover, treatment of control cells with exogenous Wnt5a activated Ca2+/calmodulin kinase II (CaMKII), increased activity of the ERK1/2 pathway and cell proliferation up to the levels found in c.709-1G>A carrier cells. PGRN knockdown SH-SY5Y neuroblastoma cells also show enhanced Wnt5a content and signaling. Taken together, our results revealed an important role of Wnt signaling in FTLDTDP pathology and suggest a novel target for therapeutic intervention. One of the aims in this thesis was to search for new clinical approximations and therapies for the treatment of FTLD-TDP using lymphoblastoid cell lines form carriers of c.709-1G>A GRN mutation. In this regard, we investigated the effects of drugs able to increase PGRN levels, either activating the GRN gene expression such as SAHA, or at posttranslational level as chloroquine (CQ), as well as specific inhibitors of the ERK1/2 signaling cascade such as Selumentinib or MEK162. Considering the haploinsufficiency mechanism, GRN is a particularly appealing gene for drug targeting. On the other hand, given that PGRN deficit appears to induce alterations in the ERK1/2 cascade, it is possible to envision new avenues for therapeutic intervention in PGRN-deficient FTLD. In this regard, the use of highly selective inhibitors of BRAF (v-raf-1 murine leukemia viral oncogene homolog B1) and MEK1/2 (Mitogen-activated protein kinase kinase or ERK kinase 1 and 2), the upstream activators of ERK1/2, have shown promising results in clinical trials, including in previously intratable diseases such as melanoma. Our results indicate that SAHA and CQ were effective in restoring the PGRN levels of c.709-1G>A carriers, and prevented the enhanced activation of ERK1/2 activity. Selumetinib, and MEK160, inhibitors of the ERK1/2 cascade, normalized the response of PGRN deficient lymphoblasts to serum activation. Both sets of drugs ultimately modulate the CDK6/pRb pathway and therefore regulate cell survival/death mechanisms. 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 FTLD associated to GRN mutations

. Conclusions: 1.- Our results suggest that the CDK6/pRb pathway is enhanced in the c.709-1G>A mutation bearing lymphoblasts and this feature is controlling the mechanism of regulation of proliferation/death in progranulin deficient cells. 2.- Our results established a molecular link between misslocalization of TDP-43 and increased CDK6 levels, pRb phosphorylation, and the control of proliferation/death in lymphoblasts from c.709-1G>A carriers 3.- We provided evidence that PGRN haplinsufficiency aberrantly increased noncanonical Gαi dependent-Wnt signaling leading to activation of ERK1/2-mediated stimulation of proliferation of lymphoblast derived from individuals bearing the FTLD-associated GRN mutation. 4.-Our results indicate that the use of drugs able to increase PGRN levels or blocking the ERK1/2 pathway, restore the normal cell response to serum stimulation, by preventing the overactivation of the CDK6/pRb cascade in c.709-1G>A GRN mutation carriers. 5.- The distinct functional features of lymphoblastoid cell from c.709-1G>A carriers offer an inavaluable, noninvasive tool to investigate the etiopathogenesis of FTLD