PGRN haploinsufficiency increased Wnt5a signaling in peripheral cells from frontotemporal lobar degeneration-progranulin mutation carriers

European Neuropsychopharmacology (]]]]) ], ]]]–]]]

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Increasing progranulin levels and blockade of the ERK1/2 pathway: Upstream and downstream strategies for the treatment of progranulin deficient frontotemporal dementia Carolina Alquezara,f, Noemí Esterasa, Ana de la Encarnacióna, Fermín Morenob,c,e, Adolfo López de Munainb,c,d,e, Ángeles Martín-Requeroa,f,n a

Department of Cellular and Molecular Medicine, Centro de Investigaciones Biológicas (CSIC), Madrid, Spain b Neuroscience Area-Institute Biodonostia, San Sebastián, Spain c Department of Neurology, Hospital Donostia, san sebastian, Spain d Department of Neurosciences, University of Basque Country, San Sebastián, Spain e CIBER de Enfermedades neurodegenerativas (CIBERNED), Madrid, Spain f CIBER de Enfermedades Raras (CIBERER), Madrid, Spain Received 10 December 2013; received in revised form 3 September 2014; accepted 24 December 2014

KEYWORDS

Abstract

FTLD-TDP; Lymphocytes; Cell proliferation; Progranulin; ERK1/2; CDK6

Frontotemporal lobar degeneration (FTLD) is a neurodegenerative disorder marked by mild-life onset and progressive changes in behavior, social cognition, and language. Loss-of-function progranulin gene (GRN) mutations are the major cause of FTLD with TDP-43 protein inclusions (FTLD-TDP). Disease-modifying treatments for FTLD-TDP are not available yet. Mounting evidence indicates that cell cycle dysfunction may play a pathogenic role in neurodegenerative disorders including FTLD. Since cell cycle re-entry of posmitotic neurons seems to precede neuronal death, it was hypothesized that strategies aimed at preventing cell cycle progression would have neuroprotective effects. Recent research in our laboratory revealed cell cycle alterations in lymphoblasts from FTLD-TDP patients carrying a null GRN mutation, and in PGRN deficient SH-SY5Y neuroblastoma cells, involving overactivation of the ERK1/2 signaling pathway. In this work, we have investigated the effects of PGRN enhancers drugs and ERK1/2 inhibitors, in these cellular models of PGRN-deficient FTLD. We report here that both restoring

n Corresponding author at: Centro de Investigaciones Biológicas (CSIC), Ramiro de Maeztu 9, 28040 Madrid, Spain. Tel.: +34 91 837 3112; fax: +34 91 536 0432. E-mail address: [email protected] (Á. Martín-Requero).

http://dx.doi.org/10.1016/j.euroneuro.2014.12.007 0924-977X/& 2015 Elsevier B.V. and ECNP. All rights reserved.

Please cite this article as: Alquezar, C., et al., Increasing progranulin levels and blockade of the ERK1/2 pathway: Upstream and downstream.... European Neuropsychopharmacology (2015), http://dx.doi.org/10.1016/j.euroneuro.2014.12.007

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C. Alquezar et al. the PGRN content, by suberoylanilide hydroxamic acid (SAHA) or chloroquine (CQ), as blocking ERK1/2 activation by selumetinib (AZD6244) or MEK162 (ARRY-162), normalized the CDK6/pRb pathway and the proliferative activity of PGRN deficient cells. Moreover, we found that SAHA and selumetinib prevented the cytosolic TDP-43 accumulation in PGRN-deficient lymphoblasts. Considering that these drugs are able to cross the blood-brain barrier, and assuming that the alterations in cell cycle and signaling observed in lymphoblasts from FTLD patients could be peripheral signs of the disease, our results suggest that these treatments may serve as novel therapeutic drugs for FTLD associated to GRN mutations. & 2015 Elsevier B.V. and ECNP. All rights reserved.

1.

Introduction

The term frontotemporal lobar degeneration (FTLD) refers to a group of progressive brain rare diseases, which involve shrinkage of specific areas of the brain that regulate behavior, personality, and language. The onset of symptoms usually occurs before the age of 60 years, accounting for 5– 10% of dementia patients (Graff-Radford and Woodruff, 2007; Ratnavalli et al., 2002). The course of this disease is progressive with mortality within 6–8 years. FTLD patients can be classified into three clinical syndromes depending on the early and predominant symptoms: a behavioural variant (bvFTD) and two language variants; semantic dementia (SD) and primary progressive non-fluent aphasia (PPNFA) (Neary et al., 1998; Rabinovici and Miller, 2010). Each clinical variant is associated with a distinct regional pattern of brain atrophy and, to a varying degree, a characteristic histopathology. Additionally, other neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), progressive supranuclear palsy (PSP), parkinsonism with frontotemporal dementia (FTDP) or corticobasal degeneration syndrome (CBS) are closely related to FTLD (Burrell et al., 2011; Deng et al., 2011; Lomen-Hoerth, 2004; Van Langenhove et al., 2012). Pathologically, these disorders share deposits of abnormal proteins in neuroectodermic cells, and severe cell loss with atrophy of vulnerable cortical and subcortical structures. 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) (Cairns et al., 2007; Mackenzie et al., 2011a, 2011b). A positive family history of FTLD is present in 25–50% of cases and the transmission is usually autosomal dominant (Goldman et al., 2007; Rademakers et al., 2012; Sieben et al., 2012). A few genes have been associated with familial FTLD including microtubule-associated protein tau (MAPT) (Poorkaj et al., 1998; Spillantini et al., 1998), progranulin (GRN) (Baker et al., 2006; Cruts et al., 2006), transactive response (TAR) DNA-binding protein-43 (TARDBP) (Gitcho et al., 2009), chromatin-modifying 2B protein (CHMP2B) (Holm et al., 2007), valosin-containing protein (VCP) (Watts et al., 2004), and chromosome 9 open reading frame 72 (C9ORF72) (DeJesusHernandez et al., 2011). Recentely, two new rare mutations

associated with FTLD and ALS have been identified in the sequestome1/p62 (SQSTM1) (Le Ber et al., 2013) and Ubiquilin2 (UBQLN2) (Deng et al., 2011; Vengoechea et al., 2013) genes. A large number of FTLD-TDP patients have been identified to harbor loss-of-function mutations (including null mutations) in the gene encoding progranulin (GRN) (Baker et al., 2006; Cruts et al., 2006). Up to now more than 60 different mutations in GRN have been described associated with the etiology of the disease (www.molgen.ua.ac. be/FTDmutations/) (Gijselinck et al., 2008). Most of the pathogenic mutations result in null allele, suggesting that FTLD in these families results from progranulin (PGRN) haploinsufficiency (Cruts et al., 2006; Cruts and Van Broeckhoven, 2008). Current treatment options for FTLD associated to PGRN deficiency remain very limited, mainly involving therapy for the mood and behavioral symptoms (Kirshner, 2010). Identification of molecular targets to slow or hopefully to prevent the neurodegenerative process relies in a better understanding of both the biological functions of PGRN, and the role of protein haploinsufficiency in the development of dementia. For this purpose, familial forms of the disease with known pathogenic mutations provide an opportunity to get inside in FTLD-TDP pathogenesis and for fasttrack development of new therapies for PGRN-deficient FTLD. Cell cycle-related events are now considered as an important pathogenic mechanism for neurodegenerative disorders including Alzheimer disease (AD) (Mosch et al., 2007; Yang et al., 2001), Parkinson disease (PD) (Hoglinger et al., 2007), ALS and FTLD (Husseman et al., 2000). In these studies, it was suggested that cell cycle signaling might affect neuronal death pathway. The cell cycle is associated with the phase specific expression or modification of defined sets of regulatory genes that control proliferation, differentiation or entry into a quiescent state (Ross, 1996). However, re-entry of quiescent, post-mitotic neurons into the cell cycle may result in a mitotic catastrophe and cell death (Copani et al., 2001; Herrup et al., 2004; Zhu et al., 2004). Therefore, understanding the molecular pathways underlying this cell cycle-mediated neurodegeneration may be important to find new therapeutic targets to slow or prevent the onset and progression of FTLD. Interestingly, it seems that dysfunction of the cell cycle in neurodegenerative disorders is a general phenomenon affecting cells other than neurons (Nagy et al., 2002; Stieler et al.,

Please cite this article as: Alquezar, C., et al., Increasing progranulin levels and blockade of the ERK1/2 pathway: Upstream and downstream.... European Neuropsychopharmacology (2015), http://dx.doi.org/10.1016/j.euroneuro.2014.12.007

Targeting PGRN levels and ERK1/2 activity in peripheral cells from FTLD patients 2001; Urcelay et al., 2001). Therefore, peripheral cells from patients, mainly lymphocytes or fibroblasts, have been extensively used as a model to study pathogenic molecular mechanisms and to evaluate therapeutic strategies aimed at blocking cell cycle progression (Bialopiotrowicz et al., 2012; de las Cuevas et al., 2003; Munoz et al., 2008; Nagy et al., 2002; Sala et al., 2008; Zhang et al., 2003). We have previously reported alterations in cell survival/death mechanisms in immortalized lymphocytes from FTLD-TDP patients carriers of a loss-offunction mutation in the GRN gene (c.709-1G4A) (Alquezar et al., 2012a, b). This mutation was previously described in members of a small number of families of Basque descent (Lopez de Munain et al., 2008; Moreno et al., 2009). We found that PGRN deficit increased cell cycle activity in lymphoblasts from FTLD-TDP patients. This effect was associated with enhanced levels of cyclin-dependent kinase 6 (CDK6) and phosphorylation of retinoblastoma protein (pRb), resulting in a G1/S regulatory failure. Moreover, we demonstrated that activation of the CDK6/pRb is the consequence of increased stimulation of the ERK1/2 signaling, induced by PGRN haploinsufficiency (Alquezar et al., 2014). The present work was undertaken to evaluate whether restoring PGRN levels or targeting the ERK1/2 cascade would normalize the cell cycle alterations found in lymphoblasts harboring the c.709-1G4A GRN mutation. We investigated the effects of already approved drugs for use in clinical practice or in on-going clinical trials for unrelated condictions, which should make it easier to move quickly to human FTLD trials. PGRN levels were modulated by suberoylanilide hydroxamic acid (SAHA) that increases GRN mRNA expression levels (Cenik et al., 2011) or by chloroquine (CQ) that prevents proteolytic degradation of the protein (Capell et al., 2011). The blockade of ERK1/2 activation was achived by using Selumetinib (AZD6244) and MEK162 (ARRY-438162). Both compounds are small molecules ATP-uncompetitive highly selective inhibitors of MEK1/2 (Mitogen-activated protein kinase kinase or ERK kinase 1 and 2), the upstream activator of ERK1/2 (Davies et al., 2007; Yeh et al., 2007) We report here that increasing PGRN levels or alternatively using ERK1/2-targeted drugs, restore the normal proliferative activity of c.709-1G4A GRN mutation carriers, by preventing the overactivation of the CDK6/pRb cascade, unraveling molecular targets to design novel therapeutic

Table 1

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approaches in the FTLD linked to PGRN haploinsufficiency.

2. 2.1.

Experimental procedures Lymphoblastic cell lines

Blood samples were obtained from 29 individuals: 19 carriers of the c.709-1G4A GRN gene mutation (7 of them patients of FTLD-TDP and 12 asymptomatic) and 10 control individuals without sign of FTLD. Asymptomatic and control individuals were relatives of patients. All patients were diagnosed as FTD in the Donostia Hospital by applying consensus criteria as published elsewhere (McKhann et al., 2001). Demographic information for control and GRN mutation carriers is presented in Table 1. All study protocols were approved by the Donostia Hospital and the Spanish Council of Higher Research Institutional Review Board and are in accordance with National and European Union Guidelines. In all cases, peripheral blood samples were taken after written informed consent of the patients or their relatives to determine the presence of the c.709-1G4A GRN mutation and to establish the lymphoblastoid cell lines. Establishment of lymphoblastic cell lines was performed in our laboratory as previously described (Ibarreta et al., 1998), by infecting peripheral blood lymphocytes with the Epstein Barr virus (EBV). Cells were grown in suspension in T flasks in an upright position, in approximately 8 ml of RPMI-1640 medium (Life technologies, Barcelona, Spain) that contained 2 mM L-glutamine, 100 μg/ml streptomycin/ penicillin and 10% (v/v) fetal bovine serum (FBS) (Life technologies, Barcelona, Spain) and maintained in a humidified 5% CO2 incubator at 37 1C. Fluid was routinely changed every three days by removing the medium above the settled cells and replacing it with an equal volume of fresh medium.

2.2. GRN knockdown neuroblastoma SH-SY5Y cell lines culture Stable GRN knockdown neuroblastoma SH-SY5Y cells (Clone # 207) (gift from Drs. Drs. Joselin and Wu) were grown in high glucose DMEM (Dulbecco’s modified Eagle’s medium) (Life-Technologies, Barcelona, Spain) containing 10% (v/v) heat-inactivated fetal bobine serum (Sigma-Aldrich, Tres Cantos, Spain) and 1% penicillin/streptomycin (Life-technologies, Barcelona, Spain) at 37 1C in 5% CO2. GRN knockdown was achieved by using pSUPERIOR RNAi construct as previously described (Gao et al., 2010). The target sequence of 19 nucleotides targeted against nucleotides 207–226

Characteristics of individuals enrolled in this study. Control n =10

Age (years) Sex, female, % (n) Age at onset Phenotype Family history, % (n) Serum PGRN, pg/ml

51.874.3 50% (5) – Asymptomatic 70% (7) 126.7713

c.709-1G4A mutation carriers Asymptomatic n =12a

FTLD patients n =7

52.874.3 50% (6) – Asymptomatic 54.5%(6) 47.577.3

65.372.3 100% (7) 6170.6 FTD-bv; CBS 57.1% (4) 4475.6

Control: Individuals without sing of neurological degeneration; c.709-1G4A: GRN mutation carriers; n: number of subjects. Values are expressed as mean7SEM. CBS: Cortico basal syndrome; FTD-bv: Frontotemporal dementia (behaviour). Please cite this article as: Alquezar, C., et al., Increasing progranulin levels and blockade of the ERK1/2 pathway: Upstream and downstream.... European Neuropsychopharmacology (2015), http://dx.doi.org/10.1016/j.euroneuro.2014.12.007

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(#207) of the human GRN mRNA was designed. The 64 nt short hairpin RNA sense and antisense primer sequences were: 50 -gatccccggccactcctgcatctttattcaagagataaagatgcaggagtggcctttttggaaa-30 and 50 -agcttttccaaaaaggccactcctgcatctttatctcttgaataaagatgcaggagtggccggg-30 . The sense and antisense primer pairs were annealed and ligated into the pSUPERIOR vector (OligoEngine) according to manufacturer’s instructions. The vector control was also stably introduced into SH-SY5Y cells to generate the control cell line.

2.3.

Drugs and treatments

The PGRN enhancer drugs, suberoylanilide hydroxamic acid; Vorinostat (SAHA) (Cayman Chemical company, Ann Arbor, MI, USA), or chloroquine diphosphate salt (CQ) (Sigma-Aldrich, Tres Cantos, Spain) were used. SAHA was prepared in dimethylsulfoxide (DMSO) and used to a final concentrations between 0.25 and 1 μM. In the other hand, CQ was dissolved in water and used to final concentrations between 2.5 and 20 μM. Progranulin (human) (recombinant) (rhPGRN) (Enzo, Zandhoven, Belgium) was utilized as positive control for its proven efficacy in normalizing PGRN levels and was used in a final concentration of 100 ng/ml. For blocking the ERK1/2 activity, the MEK1/2 inhibitors selumetinib (AZD6244) (Selleck Chemicals, Houston, TX, USA), MEK162 (ARRY438162) (AdooQ Bioscence, Irvine, CA, USA), and PD98059 (Calbiochem, Darmstadt, Germany) were used. All three drugs were prepared in DMSO and were used to a final concentration between 0.25 and 2.5 μM for Selumetinib, between 0.5 and 1 μM for MEK162, and 20 μM for PD98059. Elastase from human leukocytes (Sigma-Aldrich, Tres Cantos, Spain) was diluted in water and used a final concentration of 0.01 U/ml.

2.4.

Determination of cell proliferation

Cell proliferation was determined by total cell counting, using a TC10TM Automated Cell Counter from Bio-Rad Laboratories, S.A. (Madrid, Spain). EBV-immortalized lymphocytes from control and GRN mutation carriers were seeded at an initial cell concentration of 1  106 cells  ml  1. Cells were enumerated everyday thereafter. In some experiments, cell proliferation was assessed by the 5-bromo-20 -deoxyuridine (BrdU) incorporation method using an enzyme-linked immunoassay kit procured from Roche (Madrid, Spain) or by the MTT assay (Mitsiades et al., 2002) obtaining similar results.

2.5.

Cell cycle analysis

Exponentially growing cultures of cell lines were seeded at an initial concentration of 1  106 cells  ml  1. Cell cycle analysis was performed using a standard method (Krishan, 1975). Cells were fixed in 75% ethanol for 1 h at room temperature. Subsequent centrifugation of the samples was followed by incubation of cells in PBS containing 1 mg/ml of RNase at room temperature for 20 min and staining with propidium iodide (PI; 25 μg/ml). Cells were analyzed in an EPICS-XL cytofluorimeter (Coulter Científica, Móstoles, Spain). Estimates of cell cycle phase distributions were obtained by computer analysis of DNA content distributions.

2.6. Preparation of whole-cell extracts and subcellular fractionation To prepare whole-cell extracts, cells were harvested, washed in PBS and then lysed in ice-cold buffer (50 mM Tris pH 7.4, 150 mM NaCl, 50 mM NaF, 1% Nonidet P-40), containing 1 mM sodium orthovanadate, 1 mM PMSF, 1 mM sodium pyrophosphate and protease inhibitor Complete Mini Mixture (Roche, Madrid, Spain).

To separate the cytosolic and nuclear fractions, cells were harvested, washed in PBS and then lysed in ice-cold hypotonic buffer (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM ethylendiaminetetraacetic acid (EDTA), 0.1 mM ethylene glycol tetraacetic acid (EGTA), 1 mM sodium orthovanadate, 1 mM sodium pyrophosphate, 1 mM PMSF and protease inhibitor mixture). After extraction on ice for 15 min, 0.5% Nonidet P-40 was added and the lysed cells were centrifuged at 4000 rpm for 10 min. Supernatants containing cytosolic proteins were separated and after extraction on ice for 30 min, the samples were centrifuged at 15,000 rpm for 15 min at 4 1C. Antibody against αtubulin was used to assess the purity of the cytosolic fraction. The protein content of the extracts was determined by the Pierce BCA Protein Assay kit (Thermo Scientific).

2.7.

Immunoblotting analysis

Cells were harvested, washed in PBS and then lysed in ice-cold lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 50 mM NaF, 1% Nonidet P-40), containing 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonylfluride (PMSF), 1 mM sodium pyrophosphate and protease inhibitor Complete Mini Mixture (Roche, Mannheim, Germany). 50– 100 mg of protein from cell extracts were fractionated on a SDS polyacrylamide gel, and transferred to PVDF membrane. The membranes were then blocked with 1–5% Bovine Serum Albumin (BSA) (Sigma) and incubated, overnight at 4 1C, with the following primary antibodies: CDK6 (1:1000), pRb (1:500), β-actin (1:500) and α-tubulin (1:1000) obtained from Santa Cruz Biotechologies (Santa Cruz, CA, USA). Anti-Progranulin (1:500) from Abcam (Cambridge, UK). Phospho-p44/42 MAPK (Erk1/2) (XP) (1:2000) and p44/42 MAP Kinase (ERK1/2) (1:1000) obtained from Cell Signaling (Danvers, MA, USA) and Anti-TDP-43 (1:500) from Proteintech Group (Manchester, UK). The specificity of the antibodies used in this work was checked by omitting the primary antibody in the incubation medium. Signals from the primary antibodies were amplified using species-specific antisera conjugated with horseradish peroxidase (Bio-Rad) and detected with a chemiluminiscent substrate detection system ECL. Protein band densities were quantified using Image J software (National Institutes of Health, Bethesda, Maryland, USA) after scanning the images with a GS-800 densitometer from Bio-Rad.

2.8.

Quantitative real-time PCR

Total RNA was extracted from cell cultures using Trizol reagent (Invitrogen, Alcobendas, Madrid, Spain). RNA yields were quantified spectrophotometrically and RNA quality was checked by the A260/ A280 ratio and on a 1.2% agarose gel to determine the integrity of 18S and 28S ribosomal RNA. RNA was then treated with DNase I Amplification Grade (Invitrogen, Alcobendas, Madrid, Spain). One microgram was reverse transcribed with the Superscript III Reverse Transcriptase kit (Invitrogen, Alcobendas, Madrid, Spain). Quantitative real-time polymerase chain reaction (PCR) was performed in triplicates using TaqMan Universal PCR MasterMix No Amperase UNG (Applied Biosystems, Alcobendas, Madrid, Spain) reagent according to the manufacturer’s protocol. Primers were designed using the Universal ProbeLibrary for Human (Roche Applied Science, Madrid, Spain) and used at a final concentration of 20 μM. The sequences of the forward and reverse primers used are the following: for GRN, 50 tctgtagtctgagcgctaccc-30 and 50 -agggtccacatggtctgc-30 ; for β-actin, 50 -ccaaccgcgagaagatga-30 and 50 -ccagaggcgtacagggatag-30 . Real time quantitative PCR was performed in the Bio-Rad iQ5 system using a thermal profile of an initial 5- min melting step at 95 1C followed by 40 cycles at 95 1C for 10 s and 60 1C for 60 s. Relative messenger RNA (mRNA) levels of the genes of interest were normalized to β-actin expression using the simplified comparative threshold cycle delta-delta CT method (2-[ΔCT PGRN -ΔCT Actin]).

Please cite this article as: Alquezar, C., et al., Increasing progranulin levels and blockade of the ERK1/2 pathway: Upstream and downstream.... European Neuropsychopharmacology (2015), http://dx.doi.org/10.1016/j.euroneuro.2014.12.007

Targeting PGRN levels and ERK1/2 activity in peripheral cells from FTLD patients

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Figure 1 Proliferative activity of lymphoblasts from control and c.709-1G4A GRN mutation carriers individuals. (A) Immortalized lymphocytes from control and c.709-1G4A GRN mutation carriers, asymptomatic or FTLD-TDP patients, were seeded at an initial density of 1  106  ml  1 and were incubated in RPMI medium containing 10% FBS, 24 h later cells were harvested for cell extracts preparation. PGRN levels were determined by Western blotting. Box plots represent the PGRN content in lymphoblasts form all individuals enrolled in this studio (** po0.01, significantly different form control cells). (B) Proliferative response of control and PGRN deficient cells. 100,000 cells/well were seeded in 96-well plates for 24 h and pulsed with 10 μM BrdU for an additional period of 4 h. DNA synthesis was assessed by BrdU incorporation method according to the manufacturer’s instructions. Proliferation was expressed as absorbance of stimulated minus that of non-stimulated cultures. Each bar represents the mean7SEM of seven independent experiments performed in triplicate. (C) Lymphobastoid cell lines were seeded at an initial density of 1  106  ml  1 and were incubated for 72 h with rhPGRN (100 ng/ml) alone or preincubated with elastase (0.1 U/ml) for 3 h. Cell proliferation was determined everyday by counting the cells excluding trypan blue using a TC10TM Automated Cell Counter. Values shown are the mean7SEM of all cell lines used in this work (*po0.05; **po0.01 significantly different from control cells). (D) rhPGRN (100 mg/ml) was incubated in the absence and presence of 0.1 U/ml of elastase for 3 h and the levels of PGRN protein were determined by Western blot. (E) Representative immunoblots showing the effects of rhPGRN on pERK1/2, CDK6 levels and pRb phosphorylation status.

2.9.

Statistical analysis

Statistical analyses were performed on GraphPad Prism 5 for Macintosh (La Jolla, CA, USA). All the statistical data are presented as mean7

standard error of the mean (SEM). Statistical significance was estimated by analysis of variance (ANOVA) followed by the Bonferroni's test for multiple comparisons. Differences were considered significant at a level of po0.05.

Please cite this article as: Alquezar, C., et al., Increasing progranulin levels and blockade of the ERK1/2 pathway: Upstream and downstream.... European Neuropsychopharmacology (2015), http://dx.doi.org/10.1016/j.euroneuro.2014.12.007

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Figure 2 Effects of SAHA and chloroquine on the proliferation of lymphoblasts from control and c.709-1G4A GRN mutation carriers individuals. Immortalized lymphocytes from control and c.709-1G4A GRN mutation carriers, asymptomatic or FTLD-TDP patients, were seeded at an initial density of 1  106  ml  1 and were incubated in medium containing 10% FBS, in the absence or in presence of increasing doses of SAHA (0–2 μM) (A) and CQ (0–20 μM) (B) for 72 h. A total of 100,000 cells per well were seeded in a 96-well plaque for the MTT assay. Results represent the % of cell survival of treated cells referred to untreated ones (†po0.05 and ††po0.01 significantly different from untreated cells). (C, D) Effect of the treatment with SAHA 1 μM (C) and CQ 10 μM (D) on proliferation in control and PGRN deficient lymphoblasts. Aliquots were taken for cell counting 72 h after the drug administration. Data shown are the mean7SEM of ten determinations from SAHA and seven for CQ carried out with different cell lines (**po0.01 significantly different from control cells. ††po0.01 significantly different from untreated cells).

3.

Results

3.1. Proliferative activity of lymphoblasts from control or c.709-1G4A GRN mutation carriers individuals Figure 1 summarizes the distinct response of EBV-immortalized lymphocytes bearing a loss-of-function GRN mutation c.7091G4A to serum stimulation. As expected PGRN content of mutant lymphoblasts, either from asymptomatic or FTLD-TDP patients, was significantly reduced when compared with the protein content of control cells (Figure 1A) (1.5170.18, 0.6070.09, and 0.5370.10 for control individuals, GRN mutation carriers, asymptomatic and FTLD-TDP patient respectively) (F(2,25) =19.13., po0.0001). In agreement with previous reports (Alquezar et al., 2012a, 2014), PGRN deficient lymphoblasts showed higher proliferative activity, determined by the rate of BrdU incorporation into DNA, than control cells (Figure 1B) (F(2,26) =8.83., p=0.0012), or by direct trypan blue excluding cell counting over three days (Figure 1C, left panel) (Findividualsxtime(4,113) =2.818., p=0.0284). Addition of exogenous recombinant human PGRN (rhPGRN) to the culture medium prevented the enhanced stimulation of proliferation of lymphoblasts bearing the GRN mutation (Figure 1C, medium panel)

(Findividualsxtime(4,103)=0.5359., p=0.7097). The effect of rhPGRN disappeared when the exogenous PGRN was previously degraded by elastase (Figure 1C, right panel). It is known that elastase cleaves full-length PGRN to generate individual granulin peptides (Zhu et al., 2002). As shown in Figure 1D incubation of rhPGRN with 0.1 U/ml of elastase for three hours was sufficient to completely degrade PGRN although the anti-PGRN antibody used did not recognize the individual granulin peptides. PGRN and granulins are biologically active (De Muynck and Van Damme, 2011), but since elastase-digested PGRN had no effect in restoring the proliferative activity (Figure 1C, right panel), it is suggested that the anti-proliferative effect of added rhPGRN in lymphoblasts harboring the c.709-1G4A GRN mutation is due to the full-length protein. The effect of rhPGRN normalizing the proliferative activity of PGRN-deficient lymphoblasts was accompanied by the blockade of ERK1/2 activation, and decreased levels of CDK6 and phosphorylation of pRb as observed in Figure 1E

3.2. Effects of SAHA and chloroquine on proliferation of lymphoblasts from control or c.7091G4A GRN mutation carriers individuals Given that PGRN deficit seems to be causally associated with neurodegeneration in FTLD-TDP patients (Baker et al.,

Please cite this article as: Alquezar, C., et al., Increasing progranulin levels and blockade of the ERK1/2 pathway: Upstream and downstream.... European Neuropsychopharmacology (2015), http://dx.doi.org/10.1016/j.euroneuro.2014.12.007

Targeting PGRN levels and ERK1/2 activity in peripheral cells from FTLD patients

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Figure 3 Cell cycle distribution of lymphoblast from control and c.7091-G4A GRN mutation carriers after SAHA or chloroquine treatment. Immortalized lymphocytes from control and GRN mutation carriers were seeded at an initial density of 1  106  ml  1 and cultured in RPMI medium containing 10% FBS in the absence or in the presence of SAHA (1 μM) and CQ (10 μM). 36 h after drugs addition, cells were harvested, fixed, and analyzed by flow cytometry as described under Section 2. The percentage of cells in the different cell cycle phases is indicated for each condition.

2006; Mackenzie et al., 2011a), treatments to increase the PGRN levels were envisioned as promising therapies. For this reason, we considered interesting to evaluate the efficacy of drugs known to increase PGRN content, such as SAHA, a histone deacetylase (HDAC) that increases the GRN expression (Cenik et al., 2011) or chloroquine, an alkalizing reagent able to prevent the proteolytic degradation of PGRN (Capell et al., 2011), to restore the normal response of PGRN deficient peripheral cells from FTLD patients. These drugs are already US Food and Drug Administrationapproved drugs for the treatment of cutatenous T-cell lymphoma and malaria respectively (Duvic and Vu, 2007; Mann et al., 2007; Pullman et al., 1948). To determine the dose-response effects of SAHA and CQ on cell proliferation, lymphoblasts from control and c.709-1G4A GRN mutation carriers, asymptomatic or FTLD-TDP patients were incubated in the absence or in the presence of escalating concentrations of SAHA (0–2 mM) or CQ (0–20 mM) for 72 h and cell proliferation were determined by the MTT assay. As shown in Figure 2A, SAHA suppressed the cell proliferation in a dose-dependent manner (Figures 2A) (F0–2 mM SAHAxindividuals (8,86) = 0.7851., p=0.6170). Similar results were obtained with increasing concentrations of CQ (Figure 2B) (F0–20 mM CQxindividuals (8,60) = 1.173., p= 0.3303). Maximal effects of SAHA or CQ were obtained at 1 mM or 10 mM, respectively. At these doses, both SAHA and CQ were able to abrogate the enhanced proliferative

response of PGRN-deficient lymphoblasts without affecting the proliferation of control cells (F1 mM SAHAxindividuals (2,27) =13.27., po0.0001; F10 mM CQxindividuals (2,29) =3.224., p=0.0497). The effect of these drugs on cell proliferation mimicked the addition of rhPGRN (see Figure 1). Since the change in cell number depends on the balance between cell proliferation and cell death, we tested whether SAHA or CQ treatment induced cell death by necrosis/apoptosis. For this purpose, we analyzed the distribution of cells in the cell cycle phases. We did not observe significant changes in the proportion of sub-G0/G1 hypodiploid cells, characteristic of apoptosis/necrosis, in control and PGRN deficient lymphoblasts after SAHA or CQ treatment (Figure 3). SAHA was more effective in decreasing the percentage of cells in S/G2M phases than CQ. Taken together, these results suggest that the decreased cell number in cultures of GRN mutation c.709-1G4A carriers in the presence of SAHA or CQ truly reflects a decrease in cell proliferation. The analysis of mRNA and protein levels of PGRN in control and c.709-1G4A GRN mutation carriers lymphoblasts revealed that, at the concentrations used, both drugs were effective in increasing the cellular levels of protein (Figure 4A) (F1 mM SAHAxindividuals (2,32) = 6.105., p= 0.0057; F10 mM CQxindividuals (2,32) = 4.637., p= 0.0170) and, in consonance with the distinct mechanism of action of these drugs,

Please cite this article as: Alquezar, C., et al., Increasing progranulin levels and blockade of the ERK1/2 pathway: Upstream and downstream.... European Neuropsychopharmacology (2015), http://dx.doi.org/10.1016/j.euroneuro.2014.12.007

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C. Alquezar et al. enhanced levels of pERK1/2 (Figure 5A). Incubation of cells with either SAHA or CQ prevented the enhanced ERK1/2 stimulation of c.709-1G4A GRN mutation carrier lymphoblasts without affecting the phosphorylation of ERK in control cells (Figure 5A) (F1 mM SAHAxindividuals (2,35) =4.348., p=0.0206; F10 mM CQxindividuals (2,32) =5.551., p=0.0085). We next sought to check whether SAHA and CQ are blocking effectively the levels and enzyme activity of CDK6. To this end, cells were preincubated with the corresponding drug 30 min before serum stimulation and 24 h later, cells were harvested to determine by Western blotting the levels of CDK6 and pRb phosphorylation status. Figure 5B shows how SAHA or CQ treatments were effective in decreasing the content of CDK6 in lymphoblasts carrying the c.709-1G4A GRN mutation up to levels similar to those of control cells (F1 mM SAHAxindividuals (2,72) =14.16., po0.0001; F10 mM CQxindividuals (2,70) =10.95., po0.0001). As expected, both drugs also diminished the hyperphosphorylation of pRb in PGRN deficient lymphoblasts (Figure 5C) (F1 mM SAHAxindividuals (2,66) =4.824., p=0.0047; F10 mM CQxindividuals (2,66) =6.062., p=0.0038).

3.4. Effects of ERK1/2 cascade inhibitors on proliferation of lymphoblasts from control or c.709-1G4A GRN mutation carriers individuals

Figure 4 Effects of SAHA and chloroquine on GRN mRNA and PGRN protein levels. Immortalized lymphocytes from control and c.709-1G4A GRN mutation carriers, FTLD patients or asymptomatic individuals were seeded at an initial density of 1  106  ml  1 and cultured in RPMI medium containing 10% FBS in the absence or in the presence of SAHA (1 μM) and CQ (10 μM). 24 h later cells were harvested to isolate RNA and to prepare cell lysates. (A) Progranulin (GRN) mRNA expression levels were analyzed by quantitative RT-PCR. Data shown are the mean7SEM of three different observations in all the subjects enrolled in this study. (B) Representative immunoblot showing PGRN protein content after drugs treatment. Densitometric measurements were performed on individual immunoblots and values indicate the mean of protein levels normalized to the corresponding β-actin levels7SEM for experiments carried out with seven different cell lines for each group (*po0.05 significantly different from control cells; †po0.05 and ††po0.01 significantly different from untreated cells).

only the deacetylase inhibitor SAHA increased the mRNA PGRN levels (Figure 4B) (F1 mM SAHAxindividuals (2,29) = 10.89., p= 0.0003; F10 mM CQxindividuals (2,32) = 0.4814., p= 0.6223).

3.3. Effects of SAHA and chloroquine on downstream signaling pathway Since ERK1/2/CDK6/pRb pathway appears to be increased in PGRN deficient lymphoblast (Alquezar et al., 2014), we have evaluated the impact of SAHA and CQ on this cascade. As expected, lymphoblasts from PGRN deficient individuals show

Providing that ERK1/2 activity alterations underlie the enhanced proliferative response of PGRN deficient lymphoblasts, we were interested in testing the effects of selective ERK inhibitors, such as selumetinib (AZD6244) and MEK162 (ARRY-438162) (Davies et al., 2007; Yeh et al., 2007) that eventually could be used as therapeutic drugs in FTLD. These drugs, orally available, are being investigated for the treatment of various types of cancer including lung cancer and gynecologic malignancies (Akinleye et al., 2013; Miller et al., 2014). The sensitivity of lymphoblastoid cell lines from control or carriers of the c.709-1G4A GRN mutation individuals to selumetinib is shown in Figure 6A. Control cells were moderately sensitive to selumetinib treatment (up to 2.5 mM); however this drug inhibited the proliferation of PGRN deficient cells in a dose-dependent manner (F0–2.5 mM Selumxindividuals (10,82) =1.491., p=0.1577). Maximal effects were observed at 1 mM (Figure 6A). At this dose, selumetinib was able to inhibit the phosphorylation of ERK1/2 after serum stimulation (Figure 6B) (F1 mM Selumxindividuals (4,42) =2.995., p=0.0291). For comparison, the effect of the known pharmacological MEK inhibitor PD98059 (20 mM) is also shown. After 72 h of cell treatment with 1 mM selumetinib, it was observed that the drug abrogated the serum-mediated increased proliferation of PGRN deficient cells, without affecting the proliferation of control cells (Figure 6C) (F1 mM Selumxindividuals (4,49) =2.824., p=0.0343). In Figure 7, we summarized the results obtained with the ERK1/2 inhibitor MEK162. Maximal effect of this drug was observed at 0.1 mM concentration (F0–1 mM MEKxindividuals (10,44) = 1.05., p=0.4199) (Figure 7A). As observed in Figure 7B, treatment with 0.1 μM of MEK162 induced a significant decrease in the levels of pERK1/2 in both control and PGRN-deficient lymphoblasts (F0.1 mM MEKxindividuals (2,16) =4.003., p=0.0389). The inhibition of ERK1/2 activity resulted in the blockade of increased proliferation of PGRN mutated carriers, without affecting that of control cells (F0.1 mM MEKxindividuals (2,39) = 3.474., p=0.0409) (Figure 7C).

Please cite this article as: Alquezar, C., et al., Increasing progranulin levels and blockade of the ERK1/2 pathway: Upstream and downstream.... European Neuropsychopharmacology (2015), http://dx.doi.org/10.1016/j.euroneuro.2014.12.007

Targeting PGRN levels and ERK1/2 activity in peripheral cells from FTLD patients

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Figure 5 Effects of SAHA and chloroquine in ERK1/2 activity, CDK6 levels and pRb phosphorylation status. Immortalized lymphocytes from control and c.709-1G4A GRN mutation carriers, were seeded at an initial density of 1  106  ml  1, and were incubated in RPMI medium containing 10% FBS in the absence or in the presence of SAHA (1 μM) or CQ (10 μM). Cell extracts were prepared 6 h after drug administration to determine ERK1/2 activation (Figure 5A) and 24 h after treatment to analyze CDK6 and pRb levels (Figure 5 B and C). Representative immunoblots are shown. The densitometric data represent the mean7SEM of different experiments using all the individuals enrolled in this study. (*po0.05 and **po0.01 significantly different from control cells; †po0.05 and ††po0.01 significantly different from untreated cells).

We next studied the effects of these ERK1/2 activity inhibitors in CDK6 levels and enzyme activity. Figure 8 shows how both treatments were effective in decreasing the content of CDK6 (F1 mM Selumxindividuals (1,72) =3.753., p=0.0282; F0.1 mM MEKxindividuals (2,65) =4.071., p=0.0216) (Figure 8A) and the levels of pRb (F1 mM Selumxindividuals (1,37) =4.548., p=0.0171; F0.1 mM MEKxindividuals (2,34) =3.440., p=0.0436) (Figure 8B), in lymphoblasts from c.709-1G4A GRN mutation carriers up to levels similar to those of control cells.

3.5. SAHA and selumenitib block pathological cytoplasmic accumulation of TDP-43 in PGRNdeficient lymphoblasts Since one of the hallmarks in FTLD associated to GRN mutations is the presence of TDP-43 protein aggregates in the cytosol of cells from PGRN-deficent FTLD patients (Mackenzie, 2007), we sought to evaluate the impact of the PGRN enhancer drug SAHA or the inhibitor of ERK1/2 selumetinib in cytoplasmic accumulation of TDP-43 in PGRN deficient lymphoblasts. To this end,

we carried out fractionation of cell extracts from control and lymphoblasts harboring the c.709-1G4A GRN mutation after the treatment with the corresponding drug, and processed them for Western blot with an anti-TDP-43 antibody. Our results shown that, as expected, PGRN deficient lymphoblasts present increased levels of TDP-43 in the cytosolic fraction compared with lymphoblast form control individuals (Figure 9). Both treatments with SAHA or selumetinib were able to decrease the levels of cytosolic TDP-43 in PGRN deficient cells without affecting TDP-43 levels in control cells. These results suggest that these treatments could ameliorate the formation of cytosolic TDP-43 aggregates in FTLD-TDP patients (Fdrugsxindividuals (4,38) =6.186., p=0.0006).

3.6. Effects of PGRN enhancers and ERK1/2 inhibitors on PGRN deficient neuroblastoma SH-SY5Y cells We considered interesting to validate the above-described results in lympoblastoid cell lines by testing the effects of PGRN levels-

Please cite this article as: Alquezar, C., et al., Increasing progranulin levels and blockade of the ERK1/2 pathway: Upstream and downstream.... European Neuropsychopharmacology (2015), http://dx.doi.org/10.1016/j.euroneuro.2014.12.007

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Figure 6 Effects of selumetinib on proliferation and ERK1/2 activation on lymphoblasts from control and c.709-1G4A GRN mutation carriers individuals. Immortalized lymphocytes from control and c.709-1G4A GRN mutation carriers were seeded at an initial density of 1  106  ml  1 and were incubated in medium containing 10% FBS. (A) For the MTT assays, triplicates with 100,000 cells per well were seeded in absence or presence of increasing doses of selumetinib (0–2.5 μM) for 72 h. Results represent the % of cell proliferation of treated cells referred to untreated ones (†po0.05 and ††po0.01 significantly different from untreated cells). (B) Efficacy of the selumetinib (1 μM) and PD98059 (20 μM) treatment in the inhibition of ERK1/2 activity. The densitometric data represent the mean7SEM of seven independent determinations carried out with different cell lines. (C) Effect of treatment with 1 μM selumetinib and 20 μM PD98059 on the proliferation of control and PGRN deficient lymphoblasts. Aliquots were taken for cell counting 72 h after the drug administration. Data shown are the mean7SEM of seven determinations carried out with different cell lines (*po0.05; **po0.01 significantly different from control cells. †po0.05; ††po0.001 significantly different from untreated cells).

modifying drugs or ERK1/2 inhibitors in a neuronal cell model of PGRN deficiency. To this end, we used GRN knockdown human neuroblastoma SH-SY5Y cell line, generated in Dr. Wu lab with a pSUPERIOR RNAi construct containing the target sequence corresponding to nucleotide sequences 207 to 226 specific for human GRN gene. In agreement with a previous report from this laboratory (Alquezar et al., 2014), the KD SH-SY5Y cells showed enhanced levels of BrdU incorporation into DNA, compared with control cells. Normal rates of BrdU incorporation could be rescued by the addition of the PGRN enhancers SAHA and CQ, mimicking the effect of the addition of exogenous PGRN (rhPGRN) (Fdrugsxindividuals (3,16) =6.981., p=0.0032) (Figure 10A). The efficacy of these drugs altering the PGRN abundance (Fdrugsxindividuals (2,12) =6.215., p=0.0140) and ERK1/2 activity (Fdrugsxindividuals (2,42) =12.79., po0.0001) in KD SH-SY5Y cells is presented in Figure 10B. Figure 11A depicts the effects of the ERK1/2 inhibitors selumenitib and MEK162 preventing the enhanced BrdU incorporation into DNA in GRN KD SH-SY5Ycells (Fdrugsxindividuals (2,12) = 6.215., p=0.0140). In Figure 11B, it is shown that both

selumenitib and MEK162 effectively reduced the levels of pERK1/2 in control and PGRN-deficient cells (Fdrugsxindividuals (2,23) = 6.517., p=0.0057). Taken together, these results obtained in human neuroblastoma cells, indicate that PGRN deficiency induce similar alterations in the ERK1/2 pathway and proliferative activity in neuronal and non-neuronal cells.

4.

Discussion

In the last decade, remarkable progress made in the understanding of FTLD biology has unraveled it as both a set of clinically different syndromes and disorders with unique genetic and neuropathological profiles. FTLD-TDP is one of the most common subtypes of FTLD that in most of cases is associated with mutations in GRN gene. Increasing knowledge of pathogenic molecular mechanisms of FTLD has provided a rationale for designing novel therapeutic strategies. There are not yet Food and Drug Administration-approved treatments for FTLD-TDP, considered a fatal and progressive rare

Please cite this article as: Alquezar, C., et al., Increasing progranulin levels and blockade of the ERK1/2 pathway: Upstream and downstream.... European Neuropsychopharmacology (2015), http://dx.doi.org/10.1016/j.euroneuro.2014.12.007

Targeting PGRN levels and ERK1/2 activity in peripheral cells from FTLD patients

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Figure 7 Effects of MEK162 on proliferation and ERK1/2 activation on lymphoblasts from control and c.709-1G4A GRN mutation carriers individuals. (A) Cells were seeded in absence or presence of increasing doses of MEK162 (0–1 μM) and 72 h later the MTT assay was performed. The experimental conditions were identical to those described in the legend of Figure 6. Results represent the % of cell proliferation of treated cells referred to untreated ones (†po0.05 and ††po0.01 significantly different from untreated cells). (B) Efficacy of the treatment with 0.1 μM of MEK162 in the inhibition of ERK1/2 activity. The densitometric data represent the mean7SEM of seven determinations carried out in cell extracts from different individuals. (C) Proliferative activity of lymphoblast from control and GRN mutation carriers individuals 72 h after the MEK162 (0.1 μM) treatment. Data shown are the mean7SEM of seven determinations carried out with different cell lines, (*po0.05; **po0.01 significantly different from control cells; †po0.05 and ††po0.01 significantly different from untreated cells).

neurodegenerative dementia (OMIM 607485), however the PGRN haploinsufficiency associated with GRN mutations as well as the insights into pathological processing of TDP-43 and in signaling pathways involved in FTLD-TDP open new perspectives for the identification of appropriate targets. Previously we described a cell cycle control failure in lymphoblasts harboring a single pathogenic splicing mutation in GRN gene (c.709-1G4A). It was found that PGRN haploinsuficiency increased cell proliferation by inducing overactivation of 2 the ERK1/2/CDK6/pRb pathway (Alquezar et al., 2012a, 2014). Since cell cycle reactivation in neurons appears to underlie the development of

neurodegenerative disorders including FTLD (Arendt, 2012; Herrup and Yang, 2007; Hoglinger et al., 2007; Ueberham and Arendt, 2005) and cell cycle disturbances are also found in non-neuronal cells, we considered interesting to evaluate the efficacy of PGRN enhancer drugs or EK1/2 inhibitors in the proliferative activity of PGRN deficient lymphoblasts. In addition, the effects of these drugs were evaluated in GRN KD SH-SY5Y neuroblastoma cells. To the best of our knowledge, this is the first attempt to study whether blocking cell cycle progression could restore the aberrant response of peripheral cells from FTLD patients, holding promise for new therapeutic strategies in PGRN-deficient FTLD. We

Please cite this article as: Alquezar, C., et al., Increasing progranulin levels and blockade of the ERK1/2 pathway: Upstream and downstream.... European Neuropsychopharmacology (2015), http://dx.doi.org/10.1016/j.euroneuro.2014.12.007

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Figure 9 Effects of PGRN enhancers and ERK1/2 inhibitors in the subcellular localization of TDP-43 in control and GRN mutation lymphoblasts. Control and GRN mutation carriers lymphoblasts were seeded at an initial density of 1  106 cells  ml  1 and incubated in presence or absence of SAHA (1 μM) or selumetinib (1 μM). 24 h after treatment, lymphoblasts were collected and lysed to obtain the cytosolic fragment that was analyzed by Western blot. α-tubulin antibody was used as loading and purity control of the cytosolic fraction. A representative experiment is shown. Densitometric analyses represent the mean7SEM of different observations carried out in six cell lines from each group (*po0.05; **po0.01 significantly different from control cells. ††po0.01 significantly different from untreated cells).

Figure 8 Effects of selumetinib and MEK162 on CDK6 levels and pRb phosphorylation status of lymphoblasts from control and c.709-1G4A GRN mutation carriers individuals. Immortalized lymphocytes from control and GRN mutation carriers, asymptomatic or FTLD-TDP patients, were seeded at an initial density of 1  106  ml  1 and incubated in RPMI medium in presence or absence of selumetinib (1 μM) and MEK162 (0.1 μM). 24 h after the treatment, cells were collected to prepare cell extracts for Western blotting. Representative immunoblots of CDK6 levels (A) and pRb status (B) are shown. Densitometric analyses represent the mean7SEM of different observations carried out in six cell lines from each group (*po0.05 significantly different from control cells; †po0.05 significantly different from untreated cells).

found that treatment of cells with drugs able to increase PGRN levels such as SAHA and CQ or with the ERK1/2 inhibitors selumetinib and MEK162 inhibited the increased proliferative activity of PGRN-deficient lymphoblast, without affecting normal basal rates of proliferation in control cells. In agreement with previous results (Alquezar et al., 2012a, 2014), untreated lymphoblasts from individuals carrying the c.709-1G4A GRN mutation, asymptomatic or diagnosed of FTLD-TDP, showed enhanced cell proliferation when compared with cells from control subjects. The inhibition of proliferation

induced by SAHA or CQ correlated with increased PGRN content. SAHA, a known histone deacetylase (HDAC) inhibitor was shown to effectively increase PGRN mRNA and protein levels in human lymphoblasts (Cenik et al., 2011) while CQ increased the PGRN content by a posttranslational mechanism (Capell et al., 2011). Both drugs were able to effectively normalize the relative abundance of PGRN in c.709-1G4A GRN mutation carrier lymphoblasts. Moreover the inhibitory effect of SAHA and CQ mimicked the effect of addition of rhPGRN to lymphoblast from GRN mutation carriers. Interestingly, the effect of exogenous PGRN is most likely due to the full-length protein, rather than to granulin peptides. It was previously reported that PGRN haploinsufficient lymphoblasts show enhanced ERK1/2 activation following serum stimulation (Alquezar et al., 2014). Our results suggest that the mechanism underlying the anti-proliferative effects of SAHA and CQ is the capacity of these drugs to abrogate the serum-mediated stimulation of the ERK1/2 activity, which then leads to decreased CDK6 content and pRb phosphorylation, key regulators of the G1-S cell cycle transition. SAHA demonstrated good safety profile and therapeutic potential in other neurodegenerative diseases, such as Rubinstein-Taybi syndrome, Rett syndrome, Friedreich’s ataxia, Huntington’s disease and multiple sclerosis (Kazantsev and Thompson, 2008) as well as in PD and AD (Harrison and Dexter, 2013; Meng et al., 2014). On the other hand, CQ introduced into clinical practice in 1947 for

Please cite this article as: Alquezar, C., et al., Increasing progranulin levels and blockade of the ERK1/2 pathway: Upstream and downstream.... European Neuropsychopharmacology (2015), http://dx.doi.org/10.1016/j.euroneuro.2014.12.007

Targeting PGRN levels and ERK1/2 activity in peripheral cells from FTLD patients

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Figure 10 Effects of PGRN enhancers in PGRN deficient neuroblastoma SH-SY5Y cells. (A) Control and GRN KD SH-SY5Y cells (15,000 cells/well) were incubated in the absence or in the presence of rhPGRN (100 ng/ml) SAHA (1 μM) or CQ (10 μM) for 24 h. DNA synthesis was assessed by BrdU incorporation method according to the manufacturer’s instructions. Proliferation was expressed as absorbance of stimulated minus that of non-stimulated cultures. Data shown are the mean7SEM for three individual experiments. (B) 24 h after drug administration, whole cell extracts of SH-SY5Y clones expressing either the control vector or a target sequence of human GRN mRNA were prepared to analyze by Western blotting the expression of PGRN and pERK1/2. Densitometric analyses show the mean7SEM for four individual experiments (*po0.05; **po0.01 significantly different from control cells. †po0.05; ††po0.01 significantly different from untreated cells).

prophylaxis treatment of malaria (Pullman et al., 1948), has been shown to alleviate the abnormal proteolytic processing of the amyloid precursor protein (APP) in a neuronal cell model of AD (Cagnin et al., 2012). Therefore, these PGRNenhancers drugs may be useful for prevention and treatment of FTLD-TPD associated to PGRN haploinsufficency. Encouragingly, mice overexpressing PGRN are significantly rescued from the behavioral deficits induced by middle cerebral artery occlusion (Egashira et al., 2013). In addition, the fact that SAHA was shown to induce changes in inflammatory markers in a mouse model of septic shock (Finkelstein et al., 2010) suggest that SAHA may have secondary beneficial effects in FTLD-TDP besides normalizing PGRN deficiency ameliorating the chronic inflammation that is a common feature in different neurodegenerative diseases including FTLD. In the other hand, CQ could also have additional beneficial effects in FTLD patients as it can moderate the lysosomal dysfunction seen in affected brain areas (Cagnin et al., 2012; Tanaka et al., 2014). Nevertheless, caution is

needed before the application of PGRN enhancer drugs therapies, since a fine tuning of PGRN levels is mandatory given its potential tumorigenic action in different tissues (Matsumura et al., 2006). The highly selective inhibitors of MEK1/2, selumenitib and MEK162, used in this work are small molecules, orally available, that have shown promising results in clinical trials, including in previously intractable cancer such as melanoma (Ascierto et al., 2013; Bennouna et al., 2011). The safety profile and tolerability of selumetinib has been evaluated in a 2-part, multicenter, ascending dose, phase I clinical study (Adjei et al., 2008). This trial showed the tolerability of selumetinib, with the most common treatment-related toxicities being rash, diarrhea, nausea, and fatigue. MEK162 is also being evaluated in phase I and II clinical trials in patients with advanced solid tumors (Ascierto et al., 2013). Our results demonstrated the efficacy of both selumetinib and MEK162 in restoring rates of proliferation of PGRN-deficient lymphoblasts to values similar to those of control cells. Although the

Please cite this article as: Alquezar, C., et al., Increasing progranulin levels and blockade of the ERK1/2 pathway: Upstream and downstream.... European Neuropsychopharmacology (2015), http://dx.doi.org/10.1016/j.euroneuro.2014.12.007

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Figure 11 Effects of ERK1/2 inhibitors in PGRN deficient neuroblastoma SH-SY5Y cells. The experimental conditions in this figure were identical to those described in the legend of Figure 10. (A) Effect of the treatment with selumetinib (1 μM) and MEK162 (0.1 μM) on the BrdU incorporation into DNA and in the activation of ERK1/2 in control and knockdown cells. Data shown are the mean7SEM for four individual experiments. (B) Representative immunobloting showing pERK1/2 and ERK1/2 after treatment with selumetinib and MEK162. Densitometric analyses show the mean7SEM for four individual experiments (*po0.05; **po0.01 significantly different from control cells. †po0.05; ††po0.01 significantly different from untreated cells).

potential neuroprotective effects of these ERK1/2 inhibitors have not been explored, our results showing the blockage of increase BdU incorporation into DNA in selumenitib and MEK162-treated GRN KD neuroblastoma cells suggest that both drugs may have beneficial effects in neurons. In this sense, it would be desirable to test the effects of these drugs in animal models of PGRN deficiency. As mentioned, these drugs have side effects that may compromise their use for FTLD patients, however, given the grave nature of FTLD, and the devastating speed at which FTLD progresses, we believe that selumetinib and MEK162 should be considered for possible treatment for PGRN-deficient FTLD-TDP. It is worth to highlight that both the enhancer of PGRN, SAHA, and the ERK1/2 inhibitor selumetinib, were able to normalize the cytosolic levels of TDP-43 in PGRN deficient lymphoblasts. It is thought that changes in the localization of TDP-43 protein from its preferential nuclear localization to the cytosol, is responsible for increased TDP-43 hyperphosphorylation, ubiquitination, protein fragmentation, and aggregates formation in FTLD-TDP (Arai et al., 2010; Brady et al., 2011; Neumann et al., 2006). Therefore, the efficacy of SAHA and selumetinib in preventing one of the pathological hallmarks of FTLD-TDP, as it is the cytosolic accumulation of TDP-43, reinforces the use of lymphoblasts from FTLD-TDP patients to test disease-modifying drugs. In agreement with previous work, our results show no differences in the proliferative activity and cellular content of CDK6 and ERK1/2 activity among lymphoblasts derived from GRN c.709-1G4A mutation carriers whether asymptomatic or with a clinical diagnosis of FTLD (Alquezar et al., 2012a, 2014). Neither we found differences in the effects of PGRN enhancers or ERK1/2 inhibitors (this work). Because most of the asymptomatic carriers are younger than the patients it is suggested that these features are probably early etiologically relevant events during FTLD-development. Although considered asymptomatic, the PGRN mutation carriers show poorer neuropsychological performance, and reduced thickness of the cortex (Barandiaran et al., 2012),

reflecting a prodromal phase of the disease. Thus, it is possible to consider that these drugs may have beneficial effects in slowing disease progression at its early stages. Although FTLD-TDP-associated changes detected in peripheral cells might not fully reflect those in FTLD-TDP brain, it is evident that in addition to neuronal damage, there are also peripheral aspects of the disease. Peripheral cells from patients mirror some features of brain pathology. In this regard, attention has been drawn to signaling through the ERK1/2 pathway as a system linking stress granules, containing phosphorylated TDP-43, and neuronal loss in FTLD and ALS (Ayala et al., 2011; Parker et al., 2012). Moreover, increased levels of ERK1/2 and TDP-43 can be detected in cerebrospinal fluid samples from FTLD patients (Steinacker et al., 2008) suggesting that both proteins may be released in parallel in this neurodegenerative conditions. Thus our findings showing that TDP-43 pathological changes are also associated with ERK1/2 dysfunction in PGRN-deficient lymphocytes may be another systemic manifestation of the disease. Assuming that the PGRN deficiency-induced overactivation of ERK1/2 signaling, leading to the cycle disturbances, reported here could be peripheral signs of the disease, our results suggest that neurons of c.709-1G4A mutation carriers are at high risk of entering an unscheduled cell cycle that would then drive them to death. The above considerations support the rationale of using peripheral cells from FTLD patients for preclinical studies and testing therapeutic strategies. Our results show that enhancers of PGRN content and blockers of ERK1/2 signaling prevent the cell cycle failure in PGRN-deficient lymphobasts and neuroblastoma cells. This fact, together with that the SAHA, CQ, selumenitib and MEK162 were shown to cross the blood–brain barrier (Ascierto et al., 2013; Hirata et al., 2011; Matsuoka and Yang, 2012; Palmieri et al., 2009), suggest these drugs can be considered promising candidates for novel treatments for FTLD associated to GRN mutations.

Please cite this article as: Alquezar, C., et al., Increasing progranulin levels and blockade of the ERK1/2 pathway: Upstream and downstream.... European Neuropsychopharmacology (2015), http://dx.doi.org/10.1016/j.euroneuro.2014.12.007

Targeting PGRN levels and ERK1/2 activity in peripheral cells from FTLD patients

Role of funding source This work has been supported by grants from Ministerio de Economía y Competitividad (SAF2011-28603) and Fundación Ramón Areces to AM-R. Dr. López de Munain received research support from the Basque Government, Spain (SAIO11.PR11BN002). We thank Dr. C. Belda-Iniesta for critically reading the manuscript. The authors declare that they have no conflict of interest.

Contributors Conceived and designed the experiments: CA, AMR. Performed the experiments: CA NE AE. Analyzed the data CA, NE, AE, AMR, FM, ALM. Wrote the paper: AMR, CA, FM, ALM. Recruited and diagnosed FTLD patients: FM ALM. Read and approved the final manuscript: CA, NE, AE, FM, ALM and AMR.

Conflict of interest The authors declare that they have no conflict of interest.

Acknowledgement We thank Dr. C. Belda-Iniesta for critically reading the manuscript.

References Adjei, A.A., Cohen, R.B., Franklin, W., Morris, C., Wilson, D., Molina, J.R., Hanson, L.J., Gore, L., Chow, L., Leong, S., Maloney, L., Gordon, G., Simmons, H., Marlow, A., Litwiler, K., Brown, S., Poch, G., Kane, K., Haney, J., Eckhardt, S.G., 2008. Phase I pharmacokinetic and pharmacodynamic study of the oral, small-molecule mitogen-activated protein kinase kinase 1/2 inhibitor AZD6244 (ARRY-142886) in patients with advanced cancers. J. Clin. Oncol. Official Journal of the American Society of Clinical Oncology 26, 2139–2146. Akinleye, A., Furqan, M., Mukhi, N., Ravella, P., Liu, D., 2013. MEK and the inhibitors: from bench to bedside. J. Hematol. Oncol. 6, 27. Alquezar, C., Esteras, N., Alzualde, A., Moreno, F., Ayuso, M.S., Lopez de Munain, A., Martin-Requero, A., 2012a. Inactivation of CDK/pRb pathway normalizes survival pattern of lymphoblasts expressing the FTLD-progranulin mutation c.709-1G4A. PLoS One 7, e37057. Alquezar, C., Esteras, N., Bartolome, F., Merino, J.J., Alzualde, A., Munain, A.L., Martin-Requero, A., 2011b. Alteration in cell cycle-related proteins in lymphoblasts from carriers of the c.709-1G4A PGRN mutation associated with FTLD-TDP dementia. Neurobiol. Aging 33, 429.e7–429.e20. Alquezar, C., Esteras, N., De la Encarnacion, A., Alzualde, A., Moreno, F., Ayuso, M.S., Lopez de Munain, A., Martin-Requero, A., 2014. PGRN haploinsufficiency increased Wnt5a signaling in peripheral cells from FTLD-progranulin mutation carriers. Neurobiol. Aging 35, 886–898. Arai, T., Hasegawa, M., Nonoka, T., Kametani, F., Yamashita, M., Hosokawa, M., Niizato, K., Tsuchiya, K., Kobayashi, Z., Ikeda, K., Yoshida, M., Onaya, M., Fujishiro, H., Akiyama, H., 2010. Phosphorylated and cleaved TDP-43 in ALS, FTLD and other neurodegenerative disorders and in cellular models of TDP-43 proteinopathy. NeuropathologyOfficial Journal of the Japanese Society of Neuropathology 30, 170–181. Arendt, T., 2012. Cell cycle activation and aneuploid neurons in Alzheimer’s disease. Mol. Neurobiol. 46, 125–135.

15

Ascierto, P.A., Schadendorf, D., Berking, C., Agarwala, S.S., van Herpen, C.M., Queirolo, P., Blank, C.U., Hauschild, A., Beck, J.T., St-Pierre, A., Niazi, F., Wandel, S., Peters, M., Zubel, A., Dummer, R., 2013. MEK162 for patients with advanced melanoma harbouring NRAS or Val600 BRAF mutations: a non-randomised, open-label phase 2 study. Lancet Oncol. 14, 249–256. Ayala, V., Granado-Serrano, A.B., Cacabelos, D., Naudi, A., Ilieva, E.V., Boada, J., Caraballo-Miralles, V., Llado, J., Ferrer, I., Pamplona, R., Portero-Otin, M., 2011. Cell stress induces TDP-43 pathological changes associated with ERK1/2 dysfunction: implications in ALS. Acta Neuropathol. 122, 259–270. Baker, M., Mackenzie, I.R., Pickering-Brown, S.M., Gass, J., Rademakers, R., Lindholm, C., Snowden, J., Adamson, J., Sadovnick, A. D., Rollinson, S., Cannon, A., Dwosh, E., Neary, D., Melquist, S., Richardson, A., Dickson, D., Berger, Z., Eriksen, J., Robinson, T., Zehr, C., Dickey, C.A., Crook, R., McGowan, E., Mann, D., Boeve, B., Feldman, H., Hutton, M., 2006. Mutations in progranulin cause tau-negative frontotemporal dementia linked to chromosome 17. Nature 442, 916–919. Barandiaran, M., Estanga, A., Moreno, F., Indakoetxea, B., Alzualde, A., Balluerka, N., Marti Masso, J.F., de Munain, A.L., 2012. Neuropsychological features of asymptomatic c.7091G4A progranulin mutation carriers. J. Int. Neuropsychol. Soc. JINS 18, 1086–1090. Bennouna, J., Lang, I., Valladares-Ayerbes, M., Boer, K., Adenis, A., Escudero, P., Kim, T.Y., Pover, G.M., Morris, C.D., Douillard, J.Y., 2011. A Phase II, open-label, randomised study to assess the efficacy and safety of the MEK1/2 inhibitor AZD6244 (ARRY142886) versus capecitabine monotherapy in patients with colorectal cancer who have failed one or two prior chemotherapeutic regimens. Invest. New Drugs 29, 1021–1028. Bialopiotrowicz, E., Szybinska, A., Kuzniewska, B., Buizza, L., Uberti, D., Kuznicki, J., Wojda, U., 2012. Highly pathogenic Alzheimer’s disease presenilin 1 P117R mutation causes a specific increase in p53 and p21 protein levels and cell cycle dysregulation in human lymphocytes. J. Alzheimer’s Dis. JAD 32, 397–415. Brady, O.A., Meng, P., Zheng, Y., Mao, Y., Hu, F., 2011. Regulation of TDP-43 aggregation by phosphorylation and p62/SQSTM1. J. Neurochem. 116, 248–259. Burrell, J.R., Kiernan, M.C., Vucic, S., Hodges, J.R., 2011. Motor neuron dysfunction in frontotemporal dementia. Brain J. Neurol. 134, 2582–2594. Cagnin, M., Ozzano, M., Bellio, N., Fiorentino, I., Follo, C., Isidoro, C., 2012. Dopamine induces apoptosis in APPswe-expressing Neuro2A cells following Pepstatin-sensitive proteolysis of APP in acid compartments. Brain Res. 1471, 102–117. Cairns, N.J., Bigio, E.H., Mackenzie, I.R., Neumann, M., Lee, V.M., Hatanpaa, K.J., White 3rd, C.L., Schneider, J.A., Grinberg, L.T., Halliday, G., Duyckaerts, C., Lowe, J.S., Holm, I.E., Tolnay, M., Okamoto, K., Yokoo, H., Murayama, S., Woulfe, J., Munoz, D.G., Dickson, D.W., Ince, P.G., Trojanowski, J.Q., Mann, D.M., 2007. Neuropathologic diagnostic and nosologic criteria for frontotemporal lobar degeneration: consensus of the Consortium for Frontotemporal Lobar Degeneration. Acta Neuropathol 114, 5–22. Capell, A., Liebscher, S., Fellerer, K., Brouwers, N., Willem, M., Lammich, S., Gijselinck, I., Bittner, T., Carlson, A.M., Sasse, F., Kunze, B., Steinmetz, H., Jansen, R., Dormann, D., Sleegers, K., Cruts, M., Herms, J., Van Broeckhoven, C., Haass, C., 2011. Rescue of progranulin deficiency associated with frontotemporal lobar degeneration by alkalizing reagents and inhibition of vacuolar ATPase. J. Neurosci. The Official Journal of the Society for Neuroscience 31, 1885–1894. Cenik, B., Sephton, C.F., Dewey, C.M., Xian, X., Wei, S., Yu, K., Niu, W., Coppola, G., Coughlin, S.E., Lee, S.E., Dries, D.R., Almeida, S., Geschwind, D.H., Gao, F.B., Miller, B.L., Farese Jr., R.V., Posner, B.A., Yu, G., Herz, J., 2011. Suberoylanilide hydroxamic acid (vorinostat) up-regulates progranulin transcription: rational

Please cite this article as: Alquezar, C., et al., Increasing progranulin levels and blockade of the ERK1/2 pathway: Upstream and downstream.... European Neuropsychopharmacology (2015), http://dx.doi.org/10.1016/j.euroneuro.2014.12.007

16 therapeutic approach to frontotemporal dementia. J. Biol. Chem. 286, 16101–16108. Copani, A., Uberti, D., Sortino, M.A., Bruno, V., Nicoletti, F., Memo, M., 2001. Activation of cell-cycle-associated proteins in neuronal death: a mandatory or dispensable path? Trends Neurosci. 24, 25–31. Cruts, M., Gijselinck, I., van der Zee, J., Engelborghs, S., Wils, H., Pirici, D., Rademakers, R., Vandenberghe, R., Dermaut, B., Martin, J.J., van Duijn, C., Peeters, K., Sciot, R., Santens, P., De Pooter, T., Mattheijssens, M., Van den Broeck, M., Cuijt, I., Vennekens, K., De Deyn, P.P., Kumar-Singh, S., Van Broeckhoven, C., 2006. Null mutations in progranulin cause ubiquitin-positive frontotemporal dementia linked to chromosome 17q21. Nature 442, 920–924. Cruts, M., Van Broeckhoven, C., 2008. Loss of progranulin function in frontotemporal lobar degeneration. Trends Genet. TIG 24, 186–194. Davies, B.R., Logie, A., McKay, J.S., Martin, P., Steele, S., Jenkins, R., Cockerill, M., Cartlidge, S., Smith, P.D., 2007. AZD6244 (ARRY-142886), a potent inhibitor of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1/2 kinases: mechanism of action in vivo, pharmacokinetic/pharmacodynamic relationship, and potential for combination in preclinical models. Mol. Cancer Ther 6, 2209–2219. de las Cuevas, N., Urcelay, E., Hermida, O.G., Saiz-Diaz, R.A., Bermejo, F., Ayuso, M.S., Martin-Requero, A., 2003. Ca2+/calmodulin-dependent modulation of cell cycle elements pRb and p27kip1 involved in the enhanced proliferation of lymphoblasts from patients with Alzheimer dementia. Neurobiol. Dis. 13, 254–263. De Muynck, L., Van Damme, P., 2011. Cellular effects of progranulin in health and disease. J. Mol. Neurosci. MN 45, 549–560. DeJesus-Hernandez, M., Mackenzie, I.R., Boeve, B.F., Boxer, A.L., Baker, M., Rutherford, N.J., Nicholson, A.M., Finch, N.A., Flynn, H., Adamson, J., Kouri, N., Wojtas, A., Sengdy, P., Hsiung, G.Y., Karydas, A., Seeley, W.W., Josephs, K.A., Coppola, G., Geschwind, D.H., Wszolek, Z.K., Feldman, H., Knopman, D.S., Petersen, R.C., Miller, B.L., Dickson, D.W., Boylan, K.B., GraffRadford, N.R., Rademakers, R., 2011. Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS. Neuron 72, 245–256. Deng, H.X., Chen, W., Hong, S.T., Boycott, K.M., Gorrie, G.H., Siddique, N., Yang, Y., Fecto, F., Shi, Y., Zhai, H., Jiang, H., Hirano, M., Rampersaud, E., Jansen, G.H., Donkervoort, S., Bigio, E.H., Brooks, B.R., Ajroud, K., Sufit, R.L., Haines, J.L., Mugnaini, E., Pericak-Vance, M.A., Siddique, T., 2011. Mutations in UBQLN2 cause dominant X-linked juvenile and adult-onset ALS and ALS/ dementia. Nature 477, 211–215. Duvic, M., Vu, J., 2007. Vorinostat in cutaneous T-cell lymphoma. Drugs Today (Barcelona, Spain: 1998) 43, 585–599. Egashira, Y., Suzuki, Y., Azuma, Y., Takagi, T., Mishiro, K., Sugitani, S., Tsuruma, K., Shimazawa, M., Yoshimura, S., Kashimata, M., Iwama, T., Hara, H., 2013. The growth factor progranulin attenuates neuronal injury induced by cerebral ischemiareperfusion through the suppression of neutrophil recruitment. J. Neuroinflamm. 10, 105. Finkelstein, R.A., Li, Y., Liu, B., Shuja, F., Fukudome, E., Velmahos, G.C., deMoya, M., Alam, H.B., 2010. Treatment with histone deacetylase inhibitor attenuates MAP kinase mediated liver injury in a lethal model of septic shock. J. Surg. Res. 163, 146–154. Gao, X., Joselin, A.P., Wang, L., Kar, A., Ray, P., Bateman, A., Goate, A.M., Wu, J.Y., 2010. Progranulin promotes neurite outgrowth and neuronal differentiation by regulating GSK3beta. Protein Cell 1, 552–562. Gijselinck, I., Van Broeckhoven, C., Cruts, M., 2008. Granulin mutations associated with frontotemporal lobar degeneration and related disorders: an update. Hum. Mutat. 29, 1373–1386.

C. Alquezar et al. Gitcho, M.A., Bigio, E.H., Mishra, M., Johnson, N., Weintraub, S., Mesulam, M., Rademakers, R., Chakraverty, S., Cruchaga, C., Morris, J.C., Goate, A.M., Cairns, N.J., 2009. TARDBP 30 -UTR variant in autopsy-confirmed frontotemporal lobar degeneration with TDP-43 proteinopathy. Acta Neuropathol. 118, 633–645. Goldman, J.S., Adamson, J., Karydas, A., Miller, B.L., Hutton, M., 2007. New genes, new dilemmas: FTLD genetics and its implications for families. Am. J. Alzheimer’s Disease Dementias 22, 507–515. Graff-Radford, N.R., Woodruff, B.K., 2007. Frontotemporal dementia. Sem. Neurol. 27, 48–57. Harrison, I.F., Dexter, D.T., 2013. Epigenetic targeting of histone deacetylase: therapeutic potential in Parkinson’s disease? Pharmacol. Ther. 140, 34–52. Herrup, K., Neve, R., Ackerman, S.L., Copani, A., 2004. Divide and die: cell cycle events as triggers of nerve cell death. J. Neurosci. The Official Journal of the Society for Neuroscience 24, 9232–9239. Herrup, K., Yang, Y., 2007. Cell cycle regulation in the postmitotic neuron: oxymoron or new biology? Nature reviews. Neuroscience 8, 368–378. Hirata, Y., Yamamoto, H., Atta, M.S., Mahmoud, S., Oh-hashi, K., Kiuchi, K., 2011. Chloroquine inhibits glutamate-induced death of a neuronal cell line by reducing reactive oxygen species through sigma-1 receptor. J. Neurochem. 119, 839–847. Hoglinger, G.U., Breunig, J.J., Depboylu, C., Rouaux, C., Michel, P. P., Alvarez-Fischer, D., Boutillier, A.L., Degregori, J., Oertel, W. H., Rakic, P., Hirsch, E.C., Hunot, S., 2007. The pRb/E2F cellcycle pathway mediates cell death in Parkinson’s disease. Proc. Natl. Acad. Sci. U.S.A. 104, 3585–3590. Holm, I.E., Englund, E., Mackenzie, I.R., Johannsen, P., Isaacs, A. M., 2007. A reassessment of the neuropathology of frontotemporal dementia linked to chromosome 3. J. Neuropathol. Exp. Neurol. 66, 884–891. Husseman, J.W., Nochlin, D., Vincent, I., 2000. Mitotic activation: a convergent mechanism for a cohort of neurodegenerative diseases. Neurobiol. Aging 21, 815–828. Ibarreta, D., Urcelay, E., Parrilla, R., Ayuso, M.S., 1998. Distinct pH homeostatic features in lymphoblasts from Alzheimer’s disease patients. Ann. Neurol. 44, 216–222. Kazantsev, A.G., Thompson, L.M., 2008. Therapeutic application of histone deacetylase inhibitors for central nervous system disorders. Nature reviews. Drug Discovery 7, 854–868. Kirshner, H.S., 2010. Frontotemporal dementia and primary progressive aphasia: an update. Curr. Neurol. Neurosci. Reports 10, 504–511. Krishan, A., 1975. Rapid flow cytofluorometric analysis of mammalian cell cycle by propidium iodide staining. J. Cell Biol. 66, 188–193. Le Ber, I., Camuzat, A., Guerreiro, R., Bouya-Ahmed, K., Bras, J., Nicolas, G., Gabelle, A., Didic, M., De Septenville, A., Millecamps, S., Lenglet, T., Latouche, M., Kabashi, E., Campion, D., Hannequin, D., Hardy, J., Brice, A., 2013. SQSTM1 mutations in French patients with frontotemporal dementia or frontotemporal dementia with amyotrophic lateral sclerosis. JAMA Neurol. Lomen-Hoerth, C., 2004. Characterization of amyotrophic lateral sclerosis and frontotemporal dementia. Dementia Geriatric Cogn. Disorders 17, 337–341. Lopez de Munain, A., Alzualde, A., Gorostidi, A., Otaegui, D., RuizMartinez, J., Indakoetxea, B., Ferrer, I., Perez-Tur, J., Saenz, A., Bergareche, A., Barandiaran, M., Poza, J.J., Zabalza, R., Ruiz, I., Urtasun, M., Fernandez-Manchola, I., Olasagasti, B., Espinal, J.B., Olaskoaga, J., Ruibal, M., Moreno, F., Carrera, N., Masso, J.F., 2008. Mutations in progranulin gene: clinical, pathological, and ribonucleic acid expression findings. Biol. Psychiatry 63, 946–952. Mackenzie, I.R., 2007. The neuropathology and clinical phenotype of FTD with progranulin mutations. Acta Neuropathol. 114, 49–54.

Please cite this article as: Alquezar, C., et al., Increasing progranulin levels and blockade of the ERK1/2 pathway: Upstream and downstream.... European Neuropsychopharmacology (2015), http://dx.doi.org/10.1016/j.euroneuro.2014.12.007

Targeting PGRN levels and ERK1/2 activity in peripheral cells from FTLD patients Mackenzie, I.R., Neumann, M., Baborie, A., Sampathu, D.M., Du Plessis, D., Jaros, E., Perry, R.H., Trojanowski, J.Q., Mann, D. M., Lee, V.M., 2011a. A harmonized classification system for FTLD-TDP pathology. Acta Neuropathol. 122, 111–113. Mackenzie, I.R., Neumann, M., Cairns, N.J., Munoz, D.G., Isaacs, A. M., 2011b. Novel types of frontotemporal lobar degeneration: beyond tau and TDP-43. J Mol Neurosci 45, 402–408. Mann, B.S., Johnson, J.R., Cohen, M.H., Justice, R., Pazdur, R., 2007. FDA approval summary: vorinostat for treatment of advanced primary cutaneous T-cell lymphoma. Oncologist 12, 1247–1252. Matsumura, N., Mandai, M., Miyanishi, M., Fukuhara, K., Baba, T., Higuchi, T., Kariya, M., Takakura, K., Fujii, S., 2006. Oncogenic property of acrogranin in human uterine leiomyosarcoma: direct evidence of genetic contribution in in vivo tumorigenesis. Clinical Cancer Research: An Official Journal of the American Association for Cancer Research 12, 1402–1411. Matsuoka, Y., Yang, J., 2012. Selective inhibition of extracellular signal-regulated kinases 1/2 blocks nerve growth factor to brainderived neurotrophic factor signaling and suppresses the development of and reverses already established pain behavior in rats. Neuroscience 206, 224–236. McKhann, G.M., Albert, M.S., Grossman, M., Miller, B., Dickson, D., Trojanowski, J.Q., 2001. Clinical and pathological diagnosis of frontotemporal dementia: report of the Work Group on Frontotemporal Dementia and Pick’s Disease. Arch. Neurol. 58, 1803–1809. Meng, J., Li, Y., Camarillo, C., Yao, Y., Zhang, Y., Xu, C., Jiang, L., 2014. The anti-tumor histone deacetylase inhibitor SAHA and the natural flavonoid curcumin exhibit synergistic neuroprotection against amyloid-beta toxicity. PLoS One 9, e85570. Miller, C.R., Oliver, K.E., Farley, J.H., 2014. MEK1/2 inhibitors in the treatment of gynecologic malignancies. Gynecol. Oncol 133, 128–137. Mitsiades, N., Mitsiades, C.S., Poulaki, V., Chauhan, D., Richardson, P.G., Hideshima, T., Munshi, N., Treon, S.P., Anderson, K.C., 2002. Biologic sequelae of nuclear factor-kappaB blockade in multiple myeloma: therapeutic applications. Blood 99, 4079–4086. Moreno, F., Indakoetxea, B., Barandiaran, M., Alzualde, A., Gabilondo, A., Estanga, A., Ruiz, J., Ruibal, M., Bergareche, A., Marti-Masso, J.F., Lopez de Munain, A., 2009. “Frontotemporoparietal” dementia: clinical phenotype associated with the c.709-1G4A PGRN mutation. Neurology 73, 1367–1374. Mosch, B., Morawski, M., Mittag, A., Lenz, D., Tarnok, A., Arendt, T., 2007. Aneuploidy and DNA replication in the normal human brain and Alzheimer’s disease. J. Neurosci. The Official Journal of the Society for Neuroscience 27, 6859–6867. Munoz, U., Bartolome, F., Bermejo, F., Martin-Requero, A., 2008. Enhanced proteasome-dependent degradation of the CDK inhibitor p27(kip1) in immortalized lymphocytes from Alzheimer’s dementia patients. Neurobiol. Aging 29, 1474–1484. Nagy, Z., Combrinck, M., Budge, M., McShane, R., 2002. Cell cycle kinesis in lymphocytes in the diagnosis of Alzheimer’s disease. Neurosci. Lett. 317, 81–84. Neary, D., Snowden, J.S., Gustafson, L., Passant, U., Stuss, D., Black, S., Freedman, M., Kertesz, A., Robert, P.H., Albert, M., Boone, K., Miller, B.L., Cummings, J., Benson, D.F., 1998. Frontotemporal lobar degeneration: a consensus on clinical diagnostic criteria. Neurology 51, 1546–1554. Neumann, M., Sampathu, D.M., Kwong, L.K., Truax, A.C., Micsenyi, M.C., Chou, T.T., Bruce, J., Schuck, T., Grossman, M., Clark, C. M., McCluskey, L.F., Miller, B.L., Masliah, E., Mackenzie, I.R., Feldman, H., Feiden, W., Kretzschmar, H.A., Trojanowski, J.Q., Lee, V.M., 2006. Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Science (New York, N.Y.) 314, 130–133.

17

Palmieri, D., Lockman, P.R., Thomas, F.C., Hua, E., Herring, J., Hargrave, E., Johnson, M., Flores, N., Qian, Y., Vega-Valle, E., Taskar, K.S., Rudraraju, V., Mittapalli, R.K., Gaasch, J.A., Bohn, K.A., Thorsheim, H.R., Liewehr, D.J., Davis, S., Reilly, J.F., Walker, R., Bronder, J.L., Feigenbaum, L., Steinberg, S.M., Camphausen, K., Meltzer, P.S., Richon, V.M., Smith, Q.R., Steeg, P.S., 2009. Vorinostat inhibits brain metastatic colonization in a model of triple-negative breast cancer and induces DNA doublestrand breaks. Clinical Cancer Res. An Official Journal of the American Association for Cancer Research 15, 6148–6157. Parker, S.J., Meyerowitz, J., James, J.L., Liddell, J.R., Crouch, P. J., Kanninen, K.M., White, A.R., 2012. Endogenous TDP-43 localized to stress granules can subsequently form protein aggregates. Neurochem. Int. 60, 415–424. Poorkaj, P., Bird, T.D., Wijsman, E., Nemens, E., Garruto, R.M., Anderson, L., Andreadis, A., Wiederholt, W.C., Raskind, M., Schellenberg, G.D., 1998. Tau is a candidate gene for chromosome 17 frontotemporal dementia. Ann. Neurol. 43, 815–825. Pullman, T.N., Craige Jr., B., et al., 1948. Comparison of chloroquine, quinacrine (atabrine) and quinine in the treatment of acute attacks of sporozoite-induced vivax malaria, Chesson strain. J. Clin. Invest. 27, 46–50. Rabinovici, G.D., Miller, B.L., 2010. Frontotemporal lobar degeneration: epidemiology, pathophysiology, diagnosis and management. CNS Drugs 24, 375–398. Rademakers, R., Neumann, M., Mackenzie, I.R., 2012. Advances in understanding the molecular basis of frontotemporal dementia. Nature reviews. Neurology 8, 423–434. Ratnavalli, E., Brayne, C., Dawson, K., Hodges, J.R., 2002. The prevalence of frontotemporal dementia. Neurology 58, 1615–1621. Ross, M.E., 1996. Cell division and the nervous system: regulating the cycle from neural differentiation to death. Trends Neurosci. 19, 62–68. Sala, S.G., Munoz, U., Bartolome, F., Bermejo, F., Martin-Requero, A., 2008. HMG-CoA reductase inhibitor simvastatin inhibits cell cycle progression at the G1/S checkpoint in immortalized lymphocytes from Alzheimer’s disease patients independently of cholesterollowering effects. J. Pharmacol. Exp. Ther. 324, 352–359. Sieben, A., Van Langenhove, T., Engelborghs, S., Martin, J.J., Boon, P., Cras, P., De Deyn, P.P., Santens, P., Van Broeckhoven, C., Cruts, M., 2012. The genetics and neuropathology of frontotemporal lobar degeneration. Acta Neuropathol. 124, 353–372. Spillantini, M.G., Murrell, J.R., Goedert, M., Farlow, M.R., Klug, A., Ghetti, B., 1998. Mutation in the tau gene in familial multiple system tauopathy with presenile dementia. Proc. Natl. Acad. Sci. U.S.A. 95, 7737–7741. Steinacker, P., Hendrich, C., Sperfeld, A.D., Jesse, S., von Arnim, C. A., Lehnert, S., Pabst, A., Uttner, I., Tumani, H., Lee, V.M., Trojanowski, J.Q., Kretzschmar, H.A., Ludolph, A., Neumann, M., Otto, M., 2008. TDP-43 in cerebrospinal fluid of patients with frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Arch. Neurol. 65, 1481–1487. Stieler, J.T., Lederer, C., Bruckner, M.K., Wolf, H., Holzer, M., Gertz, H.J., Arendt, T., 2001. Impairment of mitogenic activation of peripheral blood lymphocytes in Alzheimer’s disease. NeuroReport 12, 3969–3972. Tanaka, Y., Chambers, J.K., Matsuwaki, T., Yamanouchi, K., Nishihara, M., 2014. Possible involvement of lysosomal dysfunction in pathological changes of the brain in aged progranulin-deficient mice. Acta Neuropathol. Commun. 2, 78. Ueberham, U., Arendt, T., 2005. The expression of cell cycle proteins in neurons and its relevance for Alzheimer’s disease. Curr. Drug Targets. CNS Neurol. Disorders 4, 293–306. Urcelay, E., Ibarreta, D., Parrilla, R., Ayuso, M.S., Martin-Requero, A., 2001. Enhanced proliferation of lymphoblasts from patients with Alzheimer dementia associated with calmodulin-dependent

Please cite this article as: Alquezar, C., et al., Increasing progranulin levels and blockade of the ERK1/2 pathway: Upstream and downstream.... European Neuropsychopharmacology (2015), http://dx.doi.org/10.1016/j.euroneuro.2014.12.007

18 activation of the na+/H + exchanger. Neurobiology of disease 8, 289–298. Van Langenhove, T., van der Zee, J., Van Broeckhoven, C., 2012. The molecular basis of the frontotemporal lobar degenerationamyotrophic lateral sclerosis spectrum. Ann. Med. 44, 817–828. Vengoechea, J., David, M.P., Yaghi, S.R., Carpenter, L., Rudnicki, S. A., 2013. Clinical variability and female penetrance in X-linked familial FTD/ALS caused by a P506S mutation in UBQLN2. Amyotrophic Lateral Sclerosis Frontotemporal Degeneration. Watts, G.D., Wymer, J., Kovach, M.J., Mehta, S.G., Mumm, S., Darvish, D., Pestronk, A., Whyte, M.P., Kimonis, V.E., 2004. Inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia is caused by mutant valosincontaining protein. Nat. Genet. 36, 377–381. Yang, Y., Geldmacher, D.S., Herrup, K., 2001. DNA replication precedes neuronal cell death in Alzheimer’s disease. J. Neurosci. The Official Journal of the Society for Neuroscience 21, 2661–2668.

C. Alquezar et al. Yeh, T.C., Marsh, V., Bernat, B.A., Ballard, J., Colwell, H., Evans, R. J., Parry, J., Smith, D., Brandhuber, B.J., Gross, S., Marlow, A., Hurley, B., Lyssikatos, J., Lee, P.A., Winkler, J.D., Koch, K., Wallace, E., 2007. Biological characterization of ARRY-142886 (AZD6244), a potent, highly selective mitogen-activated protein kinase kinase 1/2 inhibitor. Clinical Cancer Res. An Official Journal of the American Association for Cancer Research 13, 1576–1583. Zhang, J., Kong, Q., Zhang, Z., Ge, P., Ba, D., He, W., 2003. Telomere dysfunction of lymphocytes in patients with Alzheimer disease. Cogn. Behav. Neurol. Official Journal of the Society for Behavioral and Cognitive Neurology 16, 170–176. Zhu, J., Nathan, C., Jin, W., Sim, D., Ashcroft, G.S., Wahl, S.M., Lacomis, L., Erdjument-Bromage, H., Tempst, P., Wright, C.D., Ding, A., 2002. Conversion of proepithelin to epithelins: roles of SLPI and elastase in host defense and wound repair. Cell 111, 867–878. Zhu, X., Raina, A.K., Perry, G., Smith, M.A., 2004. Alzheimer’s disease: the two-hit hypothesis. Lancet Neurol. 3, 219–226.

Please cite this article as: Alquezar, C., et al., Increasing progranulin levels and blockade of the ERK1/2 pathway: Upstream and downstream.... European Neuropsychopharmacology (2015), http://dx.doi.org/10.1016/j.euroneuro.2014.12.007