Tissue remodeling after interference RNA mediated knockdown of transthyretin in a familial amyloidotic polyneuropathy mouse model

Neurobiology of Aging 47 (2016) 91e101

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Tissue remodeling after interference RNA mediated knockdown of transthyretin in a familial amyloidotic polyneuropathy mouse model Nádia Pereira Gonçalves a, b,1, Paula Gonçalves a, b, Joana Magalhães a, b, 2, Miguel Ventosa c, Ana Varela Coelho c, Maria João Saraiva a, b, * a b c

Instituto de Inovação e Investigação em Saúde (I3S), Universidade do Porto, Porto, Portugal Molecular Neurobiology, Instituto de Biologia Molecular e Celular e IBMC, Porto, Portugal Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal

a r t i c l e i n f o

a b s t r a c t

Article history: Received 30 November 2015 Received in revised form 22 July 2016 Accepted 24 July 2016 Available online 1 August 2016

Transthyretin (TTR) deposition in the peripheral nervous system is the hallmark of familial amyloidotic polyneuropathy (FAP). Currently, liver transplantation is the only available treatment to halt the progression of clinical symptoms; however, due to the limitations of this procedure, development of alternative therapeutic strategies is of utmost importance. In this regard, interference RNA (RNAi) targeting TTR is currently in phase III clinical development. To dissect molecular changes occurring in dorsal root ganglia (DRG) upon RNAi-mediated knockdown of TTR, we treated both chronically and acutely an FAP mouse model, in different stages of disease. Our data show that inhibition of TTR expression by the liver with RNAi reverse TTR deposition in DRG, decrease matrix metalloproteinase-2 (MMP-2) protein levels in plasma, inhibit Mmp-2 gene expression and downregulate MMP-9 activity in DRG, indicating extracellular matrix remodeling. Furthermore, protein levels of MMP-2 were found upregulated in plasma samples from FAP patients indicating that MMP-2 might be a novel potential biomarker for FAP diagnosis. Collectively, our data show that silencing TTR liver synthesis in vivo can modulate TTR-induced pathology in the peripheral nervous system and highlight the potential of MMP-2 as a novel disease biomarker. Ó 2016 Elsevier Inc. All rights reserved.

Keywords: Extracellular matrix remodeling Sensory ganglia MMP-2 Amyloidosis Transthyretin Mitochondrial stress

1. Introduction Familial amyloidotic polyneuropathy (FAP) is a chronic neurodegenerative disorder of the peripheral nervous system (PNS), which is pathologically characterized by the presence of transthyretin (TTR) aggregates or amyloid deposits throughout the sciatic nerve, dorsal root ganglia (DRG) and gastrointestinal tract (Andrade, 1952). Point mutations in the TTR gene lead to cardiomyopathy or neuropathy and are associated with different outcomes for disease (Benson and Kincaid, 2007). A substitution of a methionine for a valine at position 30 (V30M) is the most frequent mutation associated with FAP, with the main focus located in Portugal, Sweden, and Japan (Saraiva et al., 1984). TTR functions as a * Corresponding author at: i3S e Instituto de Investigação e Inovação em Saúde da Universidade do PortoRua Alfredo Allen, 208, 4200 e 135 Porto, Portugal. Tel.: þ351 220 408 800. E-mail address: [email protected] (M.J. Saraiva). 1 Presently at Danish Research Institute of Translational Neuroscience DANDRITE, Nordic EMBL Partnership, Department of Biomedicine, Aarhus University, Denmark. 2 Presently at Department of Clinical Neurosciences, Institute of Neurology, University College London, UK. 0197-4580/$ e see front matter Ó 2016 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.neurobiolaging.2016.07.020

transporter for thyroxin and retinol and is mainly synthesized by the liver and choroid plexus of the brain as a homotetrameric protein (Kanai et al., 1968; Soprano et al., 1985; Woeber and Ingbar, 1968). When mutated, the thermodynamic stability of the tetramer decreases leading to dissociation into non-native monomers of low conformational stability, ending up in amyloid fibril formation (Colon and Kelly, 1992; Quintas et al., 2001). Oligomerization or aggregation of TTR has been shown to play an important role in the disease, especially due to cytotoxicity associated with prefibrillar forms (Koike et al., 2004; Sousa et al., 2001a). One of the early hallmarks of FAP development is extracellular matrix (ECM) remodeling. The correlation of biglycan, neutrophil gelatinaseeassociated lipocalin (NGAL) and matrix metalloproteinase-9 (MMP-9) expression with TTR deposition, in tissues from FAP patients and from an animal model of disease, suggests a specific role for ECM in the pathogenesis of FAP (Cardoso et al., 2008; Sousa et al., 2005). The major proteinases implicated in ECM degradation are considered to be MMPs. Interestingly, several studies demonstrated the involvement of MMPs in the pathologic processes behind diverse neurodegenerative disorders including Parkinson’s disease, Alzheimer’s disease, or multiple sclerosis

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(Backstrom et al., 1996; Cossins et al., 1997; Lorenzl et al., 2002, 2003, 2004). Therefore, drugs modulating, directly or indirectly, the levels of MMPs might reveal beneficial in pathophysiologic neural processes. Currently, the only therapy available for FAP is the orthotopic liver transplantation. Despite its efficacy, especially when performed at early stage of disease, liver transplant encompasses several limitations: (1) lifelong treatment with immunosuppressors; (2) limited organ availability; (3) it is extremely expensive; (4) both carriers without clinical symptoms and patients in advanced stage of disease cannot undergo liver transplantation; and (5) substantial morbidity and mortality (Ando, 2005). Thus, it is of outstanding importance the development of new therapeutic strategies for this disorder. Recently, 2 TTR stabilizers, Tafamidis and Diflunisal, underwent clinical trials showing some benefit slowing disease progression (Berk et al., 2013; Coelho et al., 2012). However, there is a significant need for novel therapeutics to treat patients with TTR amyloidosis. RNA interference (RNAi) is an endogenous biological process in which small interfering RNAs (siRNAs) mediate the cleavage of target messenger RNA (mRNA) for the control of gene expression (Elbashir et al., 2001). Formulations of lipid nanoparticles (LNPs) have been used for the delivery of siRNAs to hepatocytes resulting in the knockdown of several hepatocyte-related genes (Akinc et al., 2010; Frank-Kamenetsky et al., 2008; Zimmermann et al., 2006). This advancing innovative technology was already tested in normal human volunteers to demonstrate the efficient mechanism of action in the knockdown of serum TTR (Coelho et al., 2013). Since this RNAi has the ability to knock down both wild type and all mutant forms of TTR synthesized by the liver, it might be used for FAP in alternative to liver transplantation. To test this hypothesis, clinical trials are currently being conducted showing a favorable tolerability profile and disease improvement or stabilization (www.alnylam.com). FAP has been modeled in mice expressing human TTR V30M in a heterozygous heat shock factor 1 (Hsf-1) background (Hsf/V30M) (Santos et al., 2010). With this tool, novel drugs or therapeutic strategies can be preclinically tested with dissection of diseaseassociated mechanisms. Likewise, TTR siRNA efficacy was previously experienced in this FAP mouse model, and results denote inhibition of TTR deposition both in gastrointestinal tract and PNS, when compared with vehicle-injected animals (Butler et al., 2016). In the present study, we used Hsf/V30M transgenic mice with different ages, to evaluate the efficacy of RNAi in the pathology profile. Our findings support that inhibition of TTR synthesis by the liver ameliorates TTR deposition in DRG and targets MMPs, which may be meaningful in the treatment of FAP. 2. Materials and methods 2.1. Ethics statement Mice were handled in accordance with the European Community Council Directive (2010/63/EU), and efforts were made to minimize pain and distress. The total number of animals for this research was approved by the institutional ethical committee and by the National General Veterinarian Board. Human plasma (n ¼ 11 FAP V30M patients and n ¼ 9 normal control subjects) was collected after informed consent and approval from the ethics committee of the Hospital Geral de Santo António (Porto, Portugal), following the declaration of Helsinki. 2.2. siRNA nanoparticles formulation Human-specific TTR siRNA was designed and synthesized using 50 -O-(4,4 0 -dimethoxytrityl) 0 3 0 O-(2-cyanoethyl-N,N-diisopropyl)

phosphoramidite monomers of uridine, 4-N-benzyoylcytidine, 6-N-benzoyladenosine, and 2-Nisobutyrlguanosine with 20 -O-tbutyldimethylsilyl-protected phosphoramidites according to standard solid-phase oligonucleotide synthesis protocols. After cleavage and deprotection, RNA oligonucleotides were purified by anion-exchange high-performance liquid chromatography and characterized by mass spectrometry. To generate siRNAs from RNA single strands, equimolar amounts of complementary sense and antisense strands were mixed and annealed, and siRNAs were further characterized by capillary gel electrophoresis. For in vivo TTR-silencing studies, TTR or control siRNA was formulated into a LNP delivery system (Semple et al., 2010). Briefly, LNPs were prepared with the lipid DLin-KC2dimethylaminopropane, disteroylphosphatidyl choline, cholesterol, and polyethyleneglycol dimyristoylglycerol using a spontaneous-formed vesicle formulation procedure as previously described (Semple et al., 2010). The LNPs had a component molar ratio of approximately 50/10/38.5/1.5 (DLin-KC2dimethylaminopropane/disteroylphosphatidyl choline/cholesterol/polyethyleneglycol dimyristoylglycerol). 2.3. Animals and study design Transgenic mice for human TTR V30M, in the 129/Sv and endogenous Ttr null background, heterozygous for the Hsf-1 (here designated as Hsf/V30M) (Santos et al., 2010) were used for the experiments. Animals were maintained in a controlled temperature room (24  1  C) under a 12-hour light/dark cycle and fed regular chow and tap water ad libitum. Five-month-old (n ¼ 9) mice were injected in the tail vein with human TTR siRNA, at a concentration of 1 mg/kg over 4 weeks. One intravenous injection was performed per week, and animals were euthanized 48 hours after the last injection. Untreated age-matched controls received vehicle intravenously only (n ¼ 7). An additional experiment was performed where 6-month-old (n ¼ 5 per group) or 10- to 12-month-old (n ¼ 11 per group) mice were treated with TTR siRNA or vehicle at 1 mg/kg only for 1 week. In this case, 2 injections were performed with 4-day intervals and similarly, animals were sacrificed 48 hours after the last injection. After the treatment period, animals were euthanized with a lethal anesthesia of ketamine and/or medetomidine. Mice tissues in particular stomach, colon, DRG, and sciatic nerve were immediately excised and frozen at 80  C or fixed in 10% neutral-buffered formalin and embedded in paraffin for light microscopy techniques. Blood was also collected for plasma preparation. 2.4. Semiquantitative immunohistochemistry and double immunofluorescence DRG were excised and postfixed in 10% formalin. Threemicrometer paraffin sections were deparaffinized in Histoclear (National Diagnostics, Atlanta, GA, USA) and rehydrated by a sloping concentration of ethanol (100%, 90%, 80%, and 70%). Endogenous peroxidase activity was quenched using 3% H2O2 in methanol for 30 minutes. After 1-hour blocking with 10% fetal bovine serum and 0.5% Triton X-100 in phosphate-buffered saline (PBS), sections were overnight incubated with anti-human TTR (1:600, rabbit polyclonal, DAKO, Glostrup, Denmark) or anti-MMP-2 (1:1200, rabbit polyclonal, Abcam, Cambridge, UK) antibodies, diluted in the previous solution. Next, sections were incubated for 45 minutes with biotinylated antirabbit (Vector, Burlingame, CA, USA) followed by 30-minute incubation with avidin-biotin-peroxidase complex (ABC Elite, Vector). Peroxidase labeling was visualized using 3.30 -diaminobenzidine as a chromogen. Between all incubation steps, sections were extensively washed with PBS. Semiquantitative immunohistochemical analysis

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was performed by quantifying the area occupied by pixels corresponding to the substrate reaction color, normalized relatively to the total image area. Per animal, 5 pictures at 20 magnification were taken from different areas of the organ and quantified using the Image pro-plus 5.1 software (Media Cybernetics, Rockville, MD, USA). Results shown represent mean values with the corresponding standard error of the mean. For colocalization studies, double-immunofluorescence analyses in the DRG were performed using primary antibodies against human TTR (1:100, DAKO) and mouse albumin (1:2000, DAKO), diluted in the previously described blocking solution. Sections were incubated with secondary antibodies: donkey anti-rabbit Alexa Fluor 488 and donkey anti-mouse Alexa Fluor 568 (1:1000, Molecular Probes, Eugene, OR, USA), for 2 hours. After washing, slides were covered with Vectashield containing 40 .6-diamino-2phenylindole (Vector). Fluorescent analysis was performed with a Leica SP5 confocal laser scanning microscope (Leica Microsystem, Wetzlar, Germany). 2.5. Microarrays and data processing Total mRNA from DRG was isolated using Lipid Tissue Mini Kit (Qiagen, Gaithersburg, MD, USA) according to the manufacturer’s instructions. mRNA quantity and quality were assessed using the Nanodrop ND-1000 (Thermo Scientific, Waltham, MA, USA) and Experion RNA StdSens Analysis Kit (Bio-Rad, Hercules, CA, USA), and only samples with RNA quality indicator above 7.5 were considered for further study. In vitro synthesized transcripts (Spike-in kit), in predeterminated ratios, were used to monitor microarray workflow for linearity, sensitivity, and accuracy. RNA from DRG injected with TTR siRNA or vehicle were labeled using the low-input quick amp labeling kit (Agilent Technologies, California, SC, USA) according to the one-color microarray-based gene expression analysis (Agilent), as per the manufacturer’s instructions. Before hybridization, labeled samples were purified using RNeasy mini columns (Qiagen) to remove nonreacted Cyanine 3 dye and the efficiency of cRNA synthesis and dye incorporation was measured by spectrophotometry (NanoDrop). Amplified cRNA samples were hybridized to mouse whole gene expression 4  44K v2 Microarray slides (Agilent) at 65  C for 17 hours. Microarray slides were washed following the manufacturer’s recommendations and immediately scanned using an Agilent G2565AB microarray scanner. The fluorescence intensities were obtained with Agilent Feature Extraction (FE) software using GE1_105_Dec08 feature extraction protocol. The Biometric Research BrancheArrayTools v3.4.0 software was used to center the median of the processed signal of each array taking median array intensity as reference. Differentially expressed transcripts between the 2 conditions were filtered using the Class Comparison tool, by performing a t-test assuming equal variances with a p < 0.05, using TM4 Microarray Software Suite (MeV) version 4.3. Genes with fold changes over and below 1.5 were considered to be upregulated or downregulated. The NCBI web-based functional annotation tool DAVID (Database for Annotation, Visualization, and Integrated Discovery) was used to investigate functional associations of genes. The mRNA microarrays used in this study were performed in the National DNA-Microarray Facility located at the University of Aveiro, Portugal. 2.6. mRNA isolation and real-time quantitative polymerase chain reaction DRG were dissected free from surrounded tissues, and mRNA was isolated by using the mRNA capture kit (RNA tissue lipid minikit, Qiagen) following the manufacturer’s protocol. mRNA from

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livers was isolated by phenol extraction (Invitrogen, Carlsbad, CA, USA). mRNA was reverse transcribed using the SuperScript doublestranded complementary DNA Kit (Invitrogen) and quantitative polymerase chain reaction was performed, in duplicate, using iQ Syber Green Super Mix (Bio-Rad). Reactions were analyzed on BioRad iQ5 software. Primers were developed using the program Beacon designer 8.0 (Premier Biosoft, Palo Alto, CA, USA). The sequences of primers are as follows: human TTR: sense 50 -ATTCTTGGCAGGATGGCTTC-30 , antisense 50 -CAGAGGACACTT GGATTCACC-30 ; mouse Mmp-2: sense 50 -GATTGACGCTGTGTATGAG-30 , antisense 50 -GATGTATGTCTTCTTGTTCTTAC-30 , and glyceraldehyde 3-phosphate dehydrogenase (Gapdh): sense 50 -GCC TTCCGTGTTCCTACC-30 , antisense 50 -AGAGTGGGAGTTGCTGTTG-30 . Relative gene expression levels in relation to the reference Gapdh were calculated using the mathematical model: 2DDCT. 2.7. TTR aggregates extraction TTR aggregates were extracted from DRG of old mice using the Kaplan method (Kaplan et al., 1994). Briefly, samples were homogenized in ice-cold PBS and centrifuged at 19,000 g for 10 minutes. Pellet was washed with PBS twice, rinsed with distillated water, and resuspended in 20% acetonitrile, 0.1% trifluoroacetic acid. After 1-hour incubation at room temperature, a new centrifugation was performed, the supernatant saved, and the extraction repeated twice. The final supernatant was lyophilized, resuspended in Laemmli SDS sample buffer and resolved in 12% sodium dodecyl sulphate and polyacrylamide gel electrophoresis (SDS-PAGE) gel for TTR Western blot analysis. 2.8. Western blotting DRGs were homogenized, at 4  C, in lysis buffer containing 5-mM ethylenediamine tetraacetic acid, 2-mM ethylene glycol tetraacetic acid, 20-mM 3-(N-morpholino) propanesulfonic acid, 0.5% Triton X100, 30-mM sodium fluoride, 40-mM sodium pyrophosphate, 1-mM sodium orthovanadate, 1-mM phenylmethylsulphonyl fluoride, and a protease inhibitor mix (GE Healthcare, Buckinghamshire). Blood collected with EDTA was centrifuged at 14,000 rpm for 10 minutes and plasma recovered. The protein concentration in tissue lysates and plasma was determined using the Bradford protein assay (BioRad). Samples were resolved on 12% polyacrylamide gels (50 mg per lane), in reducing conditions and transferred to a nitrocellulose Whatman membrane (GE Healthcare) using a Mini Trans-Blot Cell system (Bio-Rad). Membranes were blocked with 5% bovine serum albumin in PBS supplemented with 0.05% Tween 20 (PBS-T) and then probed with rabbit polyclonal anti-TTR (1:1000, DAKO) or mouse monoclonal anti-a-tubulin (1:10,000, Sigma-Aldrich, St. Louis, MO, USA). After washing, membranes were incubated with the appropriate secondary antibodies conjugated to horseradish peroxidase (The Binding Site, Birmingham) and proteins were visualized with enhanced chemiluminescence using the Luminata Crescendo (Millipore, Billerica, MA, USA). Protein bands were quantified by densitometry using Quantity One software (Bio-Rad). Density values were normalized to a-tubulin levels. 2.9. Two-dimensional differential gel electrophoresis Differential protein expression between animals injected with TTR siRNA or vehicle was determined in DRG (n ¼ 4 for each group) as previously described (Batista et al., 2014). Shortly, after tissue homogenization, DRG proteins were extracted in lysis buffer. The supernatant obtained after centrifugation was precipitated with 2D Clean-up kit (GE Healthcare). Dried pellets were dissolved in isoelectric focusing differential gel electrophoresis (DIGE) buffer for 2D

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gel electrophoresis. The samples and the internal standard were labeled with CyDye DIGE Fluor minimal dyes (GE Healthcare). Samples and internal standards were loaded on a previously rehydrated IPG strip (Immobiline Dry Strips, 24 cm, pH 4e7, GE Healthcare). Isoelectric focusing was carried out on an IPGphor III system (GE Healthcare). After IEF, the proteins were reduced, alkylated, and separated on 12.5% acrylamide SDS-PAGE gels. The DIGE images obtained with a laser-based scanner FLA-5100 (FujiFilm Life Science, Düsseldorf, Germany) under Image Reader FLA 5000 version 1.0 (FujiFilm) were analyzed using Progenesis SameSpots analysis software version 4.5 (NonLinear Dynamics, Durham, NC, USA). Only protein spots differentially expressed with an analysis of variance p value < 0.05, an absolute abundance variation of 1.2 or higher, and a power value above 0.7 were considered. Excised gel spots were submitted to tryptic in-gel digestion. The obtained peptide mixtures were desalted and concentrated using homemade microcolumns (GELoader tip, Eppendorf, Hamburg, Germany) with POROS R2 resin (Applied Biosystems). The tryptic peptides were eluted with a acyano-4-hydrocynnamic acid matrix solution. Peptide mass spectra were acquired by Applied Biosystems 4800 Plus matrix-assisted laser desorption/ionization (MALDI) TOF-TOF Analyzer equipment. Data were acquired in positive MS reflector mode. Processing and interpretation of the MS spectra were performed with 4000 Series Explorer Software (Applied Biosystems). The resulting peak lists and fragmentation patterns were used to search for matches in an inhouse compiled database of UniprotKB/Swiss-Prot database release 2014_01 (541954 sequence entries) using MASCOT search engine (version 2.2; Matrix Science). The processing parameters were set as follows: minimum mass accuracy of 50 ppm for the parent ions, an error of 0.3 Da for the fragments, 1 missed cleavage in peptide masses, and cysteine carbamidomethylation, and methionine oxidation as fixed and variable amino acid modifications, respectively. Peptides were only considered if the ion score indicated extensive homology (p < 0.05). 2.10. Gelatin zymography DRG from mice having 12-month old were excised and homogenized in radioimmunoprecipitation assay buffer (RIPA buffer) (Sigma-Aldrich). Samples were centrifuged (14,000 rpm, 20 minutes, 4  C) and protein extracts quantified using the Bio-Rad protein assay (Bio-Rad). Samples were prepared by mixing 50 mg of protein with loading buffer (Bio-Rad) and resolved in 10% SDS polyacrylamide gels containing gelatin (Invitrogen). Gels were incubated 30 minutes in renaturing buffer (Invitrogen) and incubated in MMP substrate buffer for 16e18 hours at 37  C (developing buffer, Invitrogen). Subsequently, gels were rinsed with water, incubated with Coomassie Blue in 40% methanol, 10% acetic acid for 30 minutes, and destained in 40% methanol, 10% acetic acid until clear proteolytic activity bands were seen against the Coomassiestained, gelatin blue background. 2.11. Enzyme-linked immunosorbent assay Human MMP-2 plasma levels were determined by enzymelinked immunosorbent assay (ELISA) following manufacturers’ guidelines (R&D Systems, Minneapolis, MN, USA). Mice MMP-2 plasma levels were determined by ELISA after treatment with TTR siRNA, Anakinra (Gonçalves et al., 2014), or combined Doxycycline/Taurodeoxycholic acid (TUDCA) (Cardoso et al., 2010), according to the manufacturers’ instructions (R&D Systems). For this measurement, plasma samples from Hsf/V30M transgenic animals treated daily with subcutaneous injections of 25 mg/kg Anakinra over 6 weeks were used. Age-matched controls were injected with PBS. A combination strategy with Doxycycline/

TUDCA was also achieved in Hsf/V30M mice. Animals were treated with 8 mg/kg Doxycycline daily in the drinking water and received intraperitoneal injections of 500 mg/kg TUDCA twice a week for 4 weeks. Controls were injected with intraperitoneal PBS. Finally, plasma from 10- to 12-month-old mice treated with TTR siRNA was also collected and used for MMP-2 analyses. 2.12. Statistical analysis Statistical comparison of data was performed using the Student t test with Graph Pad Prism software. Quantitative data are expressed as mean  standard error of the mean. Statistical significance was established for *p < 0.05, **p < 0.01, ***p < 0.001. 3. Results 3.1. TTR siRNA lowers TTR deposition in DRG in different stages of disease, both in chronic and acute approaches The PNS is the key target system for TTR deposition which consequently might alter cell metabolism. Therefore, in the present study, we aimed at identifying cellular pathways that can be affected by treatment with TTR siRNA in sensory ganglia. We started by treating chronically, for 4 weeks, 5 month-old Hsf/V30M mice, thus before major TTR accumulation in the connective tissue surrounding sensory neurons. This chronic treatment silenced 94% of TTR liver expression (Fig. 1A) resulting in lower TTR plasma circulating levels (Fig. 1B). Additionally, treated animals showed less TTR deposits in DRG, when compared with vehicle-injected mice (Fig. 1C, upper panel). Thus, we next investigated siRNA efficacy when administered acutely, for 1 week, in animals with the same age. We found that 1-week treatment with 2 injections in the tail vein was sufficient for the silence of 98% TTR synthesis by the liver (data not shown) with consequent prevention of TTR deposition around sensory neurons (Fig. 1C, bottom panel). Since acute treatment efficiently reduced TTR deposition in DRG, we assessed in parallel the effect of TTR siRNA in older mice, by treating 10- to 12-month-old Hsf/V30M transgenic animals. The acute treatment in older mice resulted in 96% TTR mRNA downregulation by the liver (Fig. 1D), and histological analysis indicated that control animals presented significantly higher levels of TTR deposition in sensory ganglia as compared with TTR siRNA treated Hsf/V30M mice (Fig. 1E). Based on these results, older mice were chosen for subsequent analysis. 3.2. Differential TTR levels in DRG of older mice: an approach using proteomics We next evaluated pathways that can be changed in DRG by silencing TTR liver expression, using a proteomics approach based on 2D-DIGE analyses. The DRG proteome was compared in 10- to 12-month-old animals injected with TTR siRNA (n ¼ 4) or vehicle (n ¼ 4) for 1 week. Few protein spots showed a statistically significant (p < 0.05) change in abundance between groups (Fig. 2A). These spots were picked from the gels and identified by MALDI-TOF/TOF MS. Interestingly, spot 2515 corresponding to ATP synthase subunit d presented increased expression in mice treated with TTR siRNA (Fig. 2B). Four additional spots were identified (Table 1) being spot 2204 the one with higher statistical significance that based on molecular weight, isoelectric point, and mass spectrometry corresponded to TTR (Fig. 2B). Based on results obtained with 2D-DIGE, we performed Western blot analysis of DRG from older animals (n ¼ 5 controls and n ¼ 6 siRNA treated) to evaluate quantitatively the total levels of TTR protein in this tissue. Our results validated the proteomic data, for

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Fig. 1. Decreased TTR deposition in DRG with different TTR siRNA therapeutic strategies. (A) Gene expression levels of TTR are decreased in liver of 6-month-old Hsf/V30M mice after 1-month treatment with TTR siRNA. Data were normalized to Gapdh mRNA (n ¼ 6 per group). (B) Western blot analysis of TTR shows that 1-month treatment with siRNA in young mice lowers TTR plasma levels (the figure shows Western blot bands from n ¼ 3 control and n ¼ 4 treated representative animals of the n ¼ 9 treated and n ¼ 7 controlanalyzed mice). (C) Representative SQ-IHC for TTR in DRG of animals with 6 months of age treated both chronically (n ¼ 9, upper panel) or acutely (n ¼ 5, bottom panel) with siRNA as compared with mice receiving only the vehicle (n ¼ 7, n ¼ 5, respectively). Scale bar 50 mm. Plots correspond to the semiquantification of TTR staining represented as the ratio of the area occupied in whole tissue. (D) Liver TTR mRNA levels from acutely treated older mice were determined by qPCR and presented as relative expression compared to Gapdh; n ¼ 4 per group. (E) Photomicrographs demonstrate representative pictures from DRG of old mice treated 1 week with siRNA (n ¼ 4) or vehicle (n ¼ 4). Scale bar 50 mm. Quantification of immunohistochemistry using Image Pro-Plus software is demonstrated in histogram. Data are presented as means  SEM (*p < 0.05, **p < 0.01, and ***p < 0.001). Abbreviations: DRG, dorsal root ganglia; Hsf, heat shock factor; mRNA, messenger RNA; qPCR, quantitative polymerase chain reaction; SEM, standard error of the mean; siRNA, small interfering RNA; SQ-IHC, semiquantitative immunohistochemistry; TTR, transthyretin; V30M, methionine for a valine at position 30.

example, TTR levels were found significantly decreased in DRG from siRNA-treated mice (Fig. 2C). 3.3. Silencing liver TTR expression lowers TTR aggregated forms in the PNS Since this type of siRNA suppresses TTR mRNA transcription by the liver, we next addressed whether TTR present in the DRG, before treatment of transgenic mice, could be derived from blood contamination. For this purpose, double-immunofluorescence analysis of TTR and albumin, a main plasma protein, was

performed in DRGs of older mice (n ¼ 4). Since no major colocalization between these 2 proteins was visualized in controls (Fig. 3A), we reasoned that the majority of TTR observed in tissues is consequent to its deposition and not derived from blood contamination. Although albumin levels are equivalent in both groups, in siRNA-treated animals TTR is almost absent around sensory neurons (Fig. 3A). Thus, we questioned whether regression of pathogenic TTR deposits in older animals resulted from an effect on aggregated TTR or decreased levels of soluble TTR. To answer this question, we extracted TTR aggregates from the DRG of transgenic mice (n ¼ 3 per group). When comparing treated with control mice,

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Fig. 2. 2D-DIGE differential protein expression. (A) Representative overlaid image of a 2D-DIGE gel with DRG protein extracts from older animals injected with vehicle (n ¼ 4) or siRNA (n ¼ 4). Spots indicated showed a statistically significant variation of volume with 95% confidence level (*p < 0.05). (B) Zoomed representative images from the 2D-DIGE gel showing spots 2515 and 2204, corresponding to ATP synthase d and TTR, respectively. (C) Western blot against TTR and a-tubulin in DRG of older transgenic mice either untreated (n ¼ 5) or treated with the siRNA (n ¼ 6). Bar graph represents the quantification of TTR densitometry measurements, normalized for a-tubulin. Data are expressed as mean  SEM (***p < 0.001). Abbreviations: 2D-DIGE, 2-dimensional differential gel electrophoresis; DRG, dorsal root ganglia; SEM, standard error of the mean; siRNA, small interfering RNA; TTR, transthyretin.

we found that in siRNA-treated animals no TTR aggregates were identified by Western blot (Fig. 3B).

3.4. Mmp-2 was found downregulated in DRG from animals treated with TTR siRNA Whole genome microarrays and gene expression comparisons between old animals injected with TTR siRNA or vehicle (n ¼ 2 per group) were performed to search for new pathways involved in the protection-induced by RNAi. The result of DRG transcriptome analysis was a list of genes with differences in expression levels, either increased or decreased. Functional classification shows that most genes found downregulated are involved in the ECM-receptor interaction, focal adhesion, immune system, and in integrin signaling pathway. Since ECM remodeling plays an important role for the progression of FAP, our selection criteria relied on genes differentially expressed coding ECM-related proteins. From the 9 ECM identified genes, downregulated with treatment (Table 2),

only Mmp-2 was validated by quantitative polymerase chain reaction (Fig. 4A). Decrease of MMP-2 protein levels in DRG from old treated animals was further demonstrated by semiquantitative immunohistochemistry (Fig. 4B). Thus, DRG lysates were used to analyze MMP activity. Results illustrated in Fig. 4C show the active form of the enzymes MMP-9 and MMP-2. Under the influence of siRNA, a small trend for decrease was noticed regarding active MMP-2 while a significant difference was found for active MMP-9 (Fig. 4C). We further assessed levels of MMP-2 in younger animals treated acutely (1 week) or chronically (1 month) with TTR siRNA. In both cases, total levels of Mmp-2 mRNA and protein levels were found downregulated with treatment (Fig. 4D and E).

3.5. MMP-2 as a novel potential FAP biomarker Mean plasma MMP-2 levels of old Hsf/V30M mice treated with Anakinra, Doxyclycline/TUDCA, or TTR siRNA are shown in Fig. 5A.

N.P. Gonçalves et al. / Neurobiology of Aging 47 (2016) 91e101 Table 1 The code assignment between spots and differentially expressed proteins identified by MALDI-TOF/TOF MS Protein ID on gel

Transtyretin (TTR) ATP synthase subunit d Ubiquitinconjugating enzyme E2 N Putative hydroxypyruvate isomerase Myelin protein P0

ANOVA (p)

Fold Protein Number of change identification peptides matched

Sequence coverage (%)

7.09e-006 6.9 0.006 1.3

2204 2515

6 5

68.7 41.6

0.022

1.4

2192

7

33.6

0.019

1.3

1670

3

11.2

0.017

1.2

2477

4

20.2

Fold change represents the change in abundance in TTR siRNA-treated mice (10 months of age) over control animals injected with vehicle (n ¼ 4 per group). Key: ANOVA, analysis of variance; siRNA, small interfering RNA; TTR, transthyretin.

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Table 2 ECM-related genes downregulated in DRG of 10-month-old TTR siRNA-treated animals (n ¼ 2) as compared to age-matched controls injected only with vehicle (n ¼ 2) Symbol

Gene name

Fold change

p value

MMP-2 Col1a1 Col14a1 Timp1 Col23a1 Col4a1 Col6a2 Col18a1 Dag1

Matrix metalloproteinase 2 Collagen type I, alpha 1 Collagen type XIV, alpha 1 Tissue inhibitor of metalloproteinase 1 Collagen type XXIII, alpha 1 Collagen type IV Collagen type VI, alpha 2 Collagen type XVIII Dystroglycan 1

2.24 2.19 1.96 1.82 1.62 1.56 1.51 1.51 1.50

0.012 0.001 0.048 0.039 0.008 0.040 0.016 0.013 0.014

Genes were considered downregulated when fold change was over or equal to 1.5 after class comparison assuming significances p < 0.05. Key: DRG, dorsal root ganglia; ECM, extracellular matrix; siRNA, small interfering RNA; TTR, transthyretin.

4. Discussion The only statistically significant difference in mean plasma MMP-2 levels was found between controls and TTR siRNA-treated older mice, suggesting that ECM remodeling with decreased MMP-2 plasma levels may be a specific feature of treatment with RNAi. In younger animals, no differences were detected (data not shown). Importantly, MMP-2 determination by ELISA highlights a significant increase in this enzyme protein levels in plasma from FAP patients carrying the V30M mutation as compared with normal control subjects (Fig. 5B), highlighting the importance of this molecule as a new potential biomarker for the diagnosis of this neurodegenerative disorder. Further studies using patients in different stages of disease or subjects treated with the TTR siRNA should be conducted to deeply confirm this hypothesis.

FAP is a life-threatening disorder and few treatment options are available yet. Recent progress has modified prognosis and management of TTR amyloidosis by targeting different disease aspects, such as protein synthesis, stabilization, inhibition of TTR aggregation, and fibril disruption; nevertheless liver transplantation is so far the best therapeutic strategy for FAP (Adams et al., 2014). Liver transplant removes the main source of mutated TTR, slowing disease progression; however, it does not prevent development of arrhythmia and, in some cases, neuropathy might progress even after surgery, suggesting that preformed amyloid deposits might act as a nidus enhancing further TTR polymerization (Ueda and Ando, 2014). A promising alternative to liver transplant has emerged through gene therapy and RNAi. It is

Fig. 3. Treatment with siRNA decreases soluble and aggregated forms of TTR deposition in DRG. (A) Coimmunostaining for human TTR (green) and albumin (red) in DRG of the Hsf/ V30M mice. Superposition of the labels is shown in merged panels (yellow/orange; n ¼ 4 per group). Nuclei stains blue with DAPI. Scale bars: 15 mm. (B) Representative images of Western blot analysis for TTR aggregates in DRG of vehicle (n ¼ 3) or siRNA-treated mice (n ¼ 3). Treatment with siRNA in a late stage of disease inhibits TTR aggregates’ deposition in DRG. Abbreviations: DAPI, 40 ,6-diamidino-2-phenylindole, dihydrochloride; DRG, dorsal root ganglia; Hsf, heat shock factor; siRNA, small interfering RNA; TTR, transthyretin; V30M, methionine for a valine at position 30. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

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Fig. 4. ECM remodeling in animals treated with siRNA. (A) Mmp-2 gene expression measured by qPCR in DRG from Hsf/V30M mice treated for 1 week in the late stage. Mmp-2 mRNA levels were normalized to Gapdh (n ¼ 4 in each group). (B) MMP-2 immunohistochemical staining of DRG in old animals injected with siRNA (n ¼ 4) or vehicle (n ¼ 4) for 1 week. Bar graph illustrates semiquantification of total MMP-2 staining (expressed as % of area occupied) in both groups. (C) DRG protein content was quantified and then resolved by gelatin zymography. Proteolytic white bands were revealed on a Coomassie Blueestained background. One-week treatment with siRNA decreased MMPs activity in old mice. Chart is represented in arbitrary units (n ¼ 6 per group). (D) Histogram represents Mmp-2 expression in DRG of young Hsf/V30M mice treated acutely or chronically with siRNA (n ¼ 5 per group). qPCR was calculated in relation to Gapdh gene levels. (E) Representative pictures of MMP-2 in DRG of 6-month-old Hsf/V30M mice treated for 1 month (n ¼ 7 controls and n ¼ 9 treated) or 1 week (n ¼ 5 in each group) with siRNA (right panels) or vehicle (age-matched controls, left panels). Scale bar: 50 mm. Charts represent the quantification of immunohistochemical images. Data represent mean  SEM (*p < 0.05 and ***p < 0.001). Abbreviations: DRG, dorsal root ganglia; ECM, extracellular matrix; Hsf, heat shock factor; MMP-2, matrix metalloproteinase-2; mRNA, messenger RNA; qPCR, quantitative polymerase chain reaction; SEM, standard error of the mean; siRNA, small interfering RNA; V30M, methionine for a valine at position 30.

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Fig. 5. MMP-2 as a novel potential biomarker for FAP. (A) Decreased systemic levels of MMP-2 in older animals treated with TTR siRNA. Plasma samples were collected after the different treatment outlines and the amount of MMP-2 measured by ELISA in old Hsf/V30M mice (***p < 0.001; n ¼ 6 in each group). (B) MMP-2 protein levels in plasma from FAP patients carrying the TTR V30M mutation (n ¼ 11) as compared with control subjects (n ¼ 9; *p < 0.05). Abbreviations: ELISA, enzyme-linked immunosorbent assay; FAP, familial amyloidotic polyneuropathy; Hsf, heat shock factor; MMP-2, metalloproteinase-2; siRNA, small interfering RNA; TTR, transthyretin; V30M, methionine for a valine at position 30.

a new noninvasive method that by preventing the transfer of genetic information from DNA to protein, silence TTR production by the liver at the translational level, thus reducing plasma TTR levels in a sustained fashion. LNP formulations were developed by Alnylam Pharmaceuticals targeting a conserved sequence in mutant and nonmutant TTR mRNA, expressed specifically by the liver. In this way, the nanoparticle efficiently delivers the siRNA to hepatocytes where they are taken up by low density lipoprotein receptors and released into the cytoplasm (Hanna, 2014). Animal models, healthy volunteers, and FAP patients were already treated with ALN-TTR02 which was generally safe and well tolerated. In addition, sustained suppression of TTR plasma levels was observed, establishing proof of concept for an RNAi therapeutic tool; phase III clinical trials are currently ongoing (Adams et al., 2014; Butler et al., 2016; Coelho et al., 2013). The hypothesis is that blocking TTR synthesis by the liver will lead to less TTR deposition, halting disease progression which in fact was demonstrated in the Hsf/V30M FAP mouse model with significantly reduced TTR deposits in both GI tract and PNS after treatment with TTR siRNA (Butler et al., 2016). The present work reinforces the action of TTR siRNA over TTR deposition in DRG and shows that acute therapeutics remodels the ECM and enhances clearance of TTR nonfibrillar deposits, suggesting that ECM turnover and TTR clearance might be closely associated and that acting on both mechanisms together may potentiate treatment. Excessive ECM dynamics is a feature of different life-threatening pathological conditions, such as Alzheimer’s disease, cancer, or FAP (Cardoso et al., 2015; Mizoguchi et al., 2011; Sousa et al., 2005). In the later case, microarray analysis of salivary glands from FAP patients at different stages of disease showed increased expression of ECM-related genes, namely biglycan, NGAL, and MMP-9. Later on, these results were observed also in an FAP mouse model; thus biglycan overexpression was already present in tissues with TTR nonfibrillar deposition while NGAL, MMP-9, tissue inhibitor of metalloproteinase 1 and also chondroitin sulfate, increased only in mouse TTR amyloid laden tissues (Cardoso et al., 2008). In other studies, histological examination of human tissues also revealed increased expression of laminin, fibronectin, collagen type IV, and heparan sulfate in close association with TTR amyloid fibrils deposition (Misumi et al., 2009; Noborn et al.,

2011). Thus, the excessive or uncontrolled remodeling of the ECM observed in FAP disease, might probably contribute to neurodegeneration. No difference regarding MMP-2 levels or activity was previously noticed in FAP; however, the present study denotes that this metalloprotease may also be important for TTR pathogenesis in the PNS. MMP-2 is widely expressed during development and has an important role for successful peripheral nerve regeneration, being normally upregulated at sites of tissue damage or inflammation (Hsu et al., 2006). Accumulating evidence indicates that MMPs are involved in the pathogenesis of various central nervous system disorders, such as stroke (Clark et al., 1997), multiple sclerosis (Maeda and Sobel, 1996), Alzheimer’s disease (Miyazaki et al., 1993), Parkinson’s disease (Lorenzl et al., 2002), or amyotrophic lateral sclerosis (Beuche et al., 2000; Lim et al., 1996). Thus, reduced MMP-2 levels in substantia nigra of postmortem brain tissue from Parkinson’s disease patients or motor cortex and spinal cord from ALS cases were noted (Lim et al., 1996; Lorenzl et al., 2002). In contrast, in both Alzheimer’s and amyotrophic lateral sclerosis patients, an increase in MMP-9 activity was observed (Asahina et al., 2001; Lim et al., 1996); altogether, the available data imply a role for MMP-2 and MMP-9 in remodeling of neuronal circuits in response to neural activity and brain damage. Likewise, differences in MMPs expression have also been reported in peripheral neuropathies. A number of studies with diabetes have described an increase in systemic MMPs (Jacqueminet et al., 2006) and altered expression of these zinc endopeptidases in secondary nephropathy (Thrailkill et al., 2009) and peripheral neuropathy (Ali et al., 2014). In line with this, active MMP-2 was found downregulated in type 1 diabetic neuropathy accompanying a reduced ability for neuronal elongation, indicating that active MMP-2 might potentiate neurite outgrowth from sensory neurons (Ali et al., 2014); a phenomena that could be ascribed to the MMP-2 ability to degrade regenerating inhibitory ECM components such as chondroitin sulfate proteoglycans (Zuo et al., 1998). However, although MMPs may be important for regeneration of neuronal circuits after injury, their overexpression can result in neuronal damage by alteration of the ECM microenvironment and interruption of cell-cell and cellmatrix homeostasis. In fact, upregulation of MMP-9 and MMP-2 has been linked to the development and maintenance of

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neuropathic pain (Ji et al., 2009), and both enzymes were shown significantly increased in heritable peripheral neuropathies with alteration of nerve ECM (Misko et al., 2002; Sousa et al., 2005). So, depending on the threshold of remodeling and on whether it might be functional or dysfunctional, the result may be either neuritic growth or induction of neuronal cell death. MMP-9 is actively inducible, and the present study shows for the first time activation of this enzyme in FAP sensory neurons, independently of amyloid fibril deposition. Inducers of MMPs expression and activity, such as cytokines, mitogen-activated protein kinases, or nitric oxide, are implicated in the pathophysiology of FAP (Monteiro et al., 2006; Sousa et al., 2001b); therefore, it is tempting to speculate that preventing or reducing transgenic TTR deposition in the PNS with RNAi will consequently affect activation of toxic-associated cascades with decreased expression of MMP-2 and MMP-9 activity. Moreover, studies performed with TTR siRNA-treated wild-type mice where no changes in MMP-2 were observed (data not shown), corroborate that the reduced MMP-2 plasma levels found in V30M animals are not an off-target effect linked to the siRNA itself but is instead directly linked to the downregulated transgenic TTR levels. Using 2D-DIGE analysis of DRG, our major findings were the decreased levels of TTR in animals treated with TTR siRNA, together with an increase in ATP synthase subunit d. No evidence for an important role of mitochondria in FAP pathophysiology was reported since both Bcl-2 (B-cell lymphoma 2) and Apaf-1 (apoptotic protease activating factor 1) were not altered in tissues with TTR deposition (Macedo et al., 2007). However, recently, mitochondrial proteins involved in cell energetics were found differentially represented in abdominal white adipose tissue from patients with TTR amyloidosis. Thus, by proteomic analysis, Brambilla et al., 2013 described a reduction in the levels of ATP synthase subunits a and b, which lead authors to hypothesize that impairment in mitochondrial function and energy production, might play a key role in TTR-triggered cardiomyopathy and neuropathy. Based on this observation and in our result, we believe that the role of the ATP synthase in FAP pathogenesis should further be investigated. Based on FAP multifactorial signaling activation and the wide spectrum of activated microenvironmental cues such as mitochondrial dysfunction, endoplasmatic reticulum stress, calcium dysregulation, inflammation, cell death, oxidative stress, and ECM remodeling, we believe that future therapeutical strategies will encompass a combinatorial approach. In this way, TTR siRNA seems a promising alternative to liver transplantation and might be used in combination with drugs acting on inflammatory pathways, such as Anakinra (Gonçalves et al., 2014) or with small molecules stabilizing the TTR tetramer, such as Tafamidis or Diflunisal (Coelho et al., 2012; Takahashi et al., 2014). In addition, inhibition of MMP-9 and MMP-2 by TTR siRNA may provide a new benefit for the prevention and treatment of neuropathic pain often observed in FAP patients. Disclosure statement The authors have no conflicts of interest. Acknowledgements This work was supported by Alnylam Pharmaceuticals, FEDER funds through the Operational Competitiveness Programd COMPETE and by national funding from Portuguese Foundation for Science and Technology (FCT) under the project PTDC/BIM-MEC/ 0282/2012 and fellowship to NPG (SFRH/BD/74304/2010). Neuromed SOE4/p1/E83I finance SUDOE Program, the UE/EU/ FedereERDF also supported the work, granting a fellowship to Paula Gonçalves. The authors would thank Maria Oliveira and Laura

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