biomedicines Article Neuromodulation Induced by Sitagliptin: A New Strategy for Treating Diabetic Retinopathy Hugo Ramos 1,2,† , Patricia Bogdanov 1,2,† , David Sabater 1 , Jordi Huerta 1 , Marta Valeri 3 , Cristina Hernández 1,2, * and Rafael Simó 1,2, * 1 Diabetes and Metabolism Research Unit, Vall d’Hebron Research Institute, Universitat Autònoma de Barcelona, 08035 Barcelona, Spain; hugo.ramos@vhir.org (H.R.); patricia.bogdanov@vhir.org (P.B.); david.sabater@vhir.org (D.S.); jordi.huerta@vhir.org (J.H.) 2 Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Instituto de Salud Carlos III (ICSIII), 28029 Madrid, Spain 3 Unit of High Technology, Vall d’Hebron Research Institute, 08035 Barcelona, Spain; marta.valeri@vhir.org * Correspondence: cristina.hernandez@vhir.org (C.H.); rafael.simo@vhir.org (R.S.) † Hugo Ramos and Patricia Bogdanov contributed equally to this study. Abstract: Diabetic retinopathy (DR) involves progressive neurovascular degeneration of the retina. Reduction in synaptic protein expression has been observed in retinas from several diabetic animal models and human retinas. We previously reported that the topical administration (eye drops) of sitagliptin, a dipeptidyl peptidase-4 (DPP-4) inhibitor, prevented retinal neurodegeneration induced by diabetes in db/db mice. The aim of the present study is to examine whether the modulation of presynaptic proteins is a mechanism involved in the neuroprotective effect of sitagliptin. For  this purpose, 12 db/db mice, aged 12 weeks, received a topical administration of sitagliptin (5 µL;  concentration: 10 mg/mL) twice per day for 2 weeks, while other 12 db/db mice were treated with Citation: Ramos, H.; Bogdanov, P.; vehicle (5 µL). Twelve non-diabetic mice (db/+) were used as a control group. Protein levels were Sabater, D.; Huerta, J.; Valeri, M.; assessed by western blot and immunohistochemistry (IHC), and mRNA levels were evaluated by Hernández, C.; Simó, R. reverse transcription polymerase chain reaction (RT-PCR). Our results revealed a downregulation Neuromodulation Induced by Sitagliptin: A New Strategy for (protein and mRNA levels) of several presynaptic proteins such as synapsin I (Syn1), synaptophysin Treating Diabetic Retinopathy. (Syp), synaptotagmin (Syt1), syntaxin 1A (Stx1a), vesicle-associated membrane protein 2 (Vamp2), Biomedicines 2021, 9, 1772. https:// and synaptosomal-associated protein of 25 kDa (Snap25) in diabetic mice treated with vehicle in doi.org/10.3390/biomedicines9121772 comparison with non-diabetic mice. These proteins are involved in vesicle biogenesis, mobilization and docking, membrane fusion and recycling, and synaptic neurotransmission. Sitagliptin was Academic Editor: Josef Troger able to significantly prevent the downregulation of all these proteins. We conclude that sitagliptin exerts beneficial effects in the retinas of db/db mice by preventing the downregulation of crucial Received: 8 November 2021 presynaptic proteins. These neuroprotective effects open a new avenue for treating DR as well other Accepted: 24 November 2021 retinal diseases in which neurodegeneration/synaptic abnormalities play a relevant role. Published: 26 November 2021 Keywords: diabetic retinopathy; retinal neurodegeneration; dipeptidyl peptidase 4 inhibitors; Publisher’s Note: MDPI stays neutral sitagliptin; presynaptic proteins; db/db mouse model with regard to jurisdictional claims in published maps and institutional affil- iations. 1. Introduction The concept of diabetic retina (DR) as a microvascular disease has evolved into that of a more complex diabetic complication in which neurodegeneration plays a significant Copyright: © 2021 by the authors. role [1,2]. Thus, DR is now defined as a highly tissue-specific neurovascular complica- Licensee MDPI, Basel, Switzerland. This article is an open access article tion [3]. Neurodegeneration and glial activation are primary events in the pathogenesis of distributed under the terms and DR and have been observed to occur before overt microangiopathy in experimental models conditions of the Creative Commons of DR and in the retina of diabetic donors [4–6]. Attribution (CC BY) license (https:// Neuronal damage associated with DR is not limited to cell apoptosis and glial activa- creativecommons.org/licenses/by/ tion but also includes other changes such as a reduction in synaptic protein expression [7,8]. 4.0/). Biomedicines 2021, 9, 1772. https://doi.org/10.3390/biomedicines9121772 https://www.mdpi.com/journal/biomedicines Biomedicines 2021, 9, 1772 2 of 16 In this regard, there is evidence that diabetes reduces the retinal content of several presy- naptic proteins critical to the exocytosis of neurotransmitters and synaptic maintenance in murine models [8–10]. In postmortem human retinas, it has been reported that the protein content of presynaptic was depleted compared to non-diabetic donors [11]. Therefore, to prevent or ameliorate the downregulation of presynaptic proteins induced by diabetes would be a desirable effect of any neuroprotective drug. We have previously demonstrated that topical administration of sitagliptin, a DPP-4 inhibitor, is effective in preventing retinal neurodegeneration (glial activation and neuronal apoptosis), as well as vascular leakage in db/db mouse model [12]. On this basis, the aim of the present study is to examine in an experimental model of diabetes (db/db mice) whether the modulation of presynaptic proteins is a mechanism involved in the neuroprotective effect of sitagliptin. 2. Materials and Methods 2.1. Animals A total of 24 diabetic male db/db (BKS.Cg-Dock7m +/+ Leprdb/J) mice and 12 non- diabetic mice (db/+; (BKS.Cg-Dock7m + Leprdb/+) aged 7 weeks were obtained from Charles River Laboratories Inc (Calco Italy for the study). We have characterized the neurodegenerative process that occurs in the retinas of db/db mice, and we found that db/db mouse reproduces the features of the neurodegenerative process that occurs in the human diabetic retina [13]. Animals were bred and maintained at VHIR’s animal facility. Animals had free access to ad libitum food (ENVIGO Global Diet Complete Feed for Rodents, Mucedola, Milan, Italy) and filtered water. In order to minimize variability, animals were randomly housed (block randomization) in groups of 2 mice per cage, in Tecniplast GM-500 cages under standard laboratory conditions at 22 ± 2 ◦ C, with 12 h light/dark cycle and relative humidity of 50–60%. Each cage held absorbent bedding and nesting material (BioFresh Performance Bedding 1/800 Pelleted Cellulose, Absorption Corp., Ferndale, WA, USA). Blood glucose levels were weekly measured through tail vein sampling (glucose assay kit; Abbott, Chicago, IL, USA). All performed experiments with animals were conducted in compliance with Eu- ropean Community (86/609/CEE) and ARVO (Association for Research in Vision and Ophthalmology) tenets for the use of laboratory animals. This study was approved by the Animal Care and Use Committee of Vall d’Hebron Research Institute (VHIR) (Pas- seig de la Vall d’Hebron 119–129, Barcelona, Spain. Approval code 75/15, approval date 2 December 2015). 2.2. Topical Ocular Treatment Sitagliptin [sitagliptin phosphate monohydrate (Y0001812, Merck KGaA, Darmstadt, Germany) eyedrops (10 mg/mL; 5 µL; n = 12) or vehicle [phosphate buffered saline (PBS)] eyedrops (5 µL; n = 12) were randomly administered directly onto the superior corneal surface of each eye using a micropipette in 10-week-old mice. The treatment (sitagliptin or vehicle) was administered twice daily for 15 days. Non-diabetic mice (n = 12) matched by age were used as control group. On day 15, the drop of sitagliptin or vehicle was administered animals 1h before euthanasia. 2.3. Electroretinogram (ERG) Full field electroretinography (ERG) recordings were estimated using the Ganzfeld ERG platform (Phoenix Research Laboratories, Pleasanton, CA, USA) following ISCEV (International Society for Clinical Electrophysiology of Vision) recommendations [14]. Animals were dark adapted for at least 8 h prior to ERG recording and then anesthetized with isoflurane. Tropicamide (1%) was utilized to each eye prior to the test. A cutaneous ground electrode was placed near the base of the tail, a needle electrode was placed on the head between the two eyes, and a corneal electrode was placed near each eye. Carboxymethylcellulose (1%) drops were applied to the interior surface of the contact lens Biomedicines 2021, 9, 1772 3 of 16 electrodes prior to their placement on the eyes. Three ERG components were assessed in terms of amplitude and timing. 2.4. Retinal Tissue Processing On day 15, each animal was transcardially perfused with paraformaldehyde 4% (sc-281692, Santa Cruz Biotechnology, Dallas, TX, USA). Previously they received a 200 µL intraperitoneal injection of anesthesia, prepared with a mix containing 1 mL ketamine (GmbH, Hameln, Germany) and 0.3 mL xylazine (Laboratorios Calier S.A., Barcelona, Spain). Eyes were rapidly enucleated and for mRNA and protein evaluations the retinas were separated instantly after enucleation, frozen in liquid nitrogen, and stored at −80 ◦ C. For RNA extraction, the retinas were introduced in individual tubes with 140 µL of TRIzol reagent (15596018, InvitrogenTM , Carlsbad, CA, USA). For Western Blot assays, retinas were directly dispensed in an empty tube until protein extraction. Finally, for immunohis- tochemistry assessment, eyes were not dissected after enucleation and were fixed in 4% paraformaldehyde during 5 h before paraffin embedding. Sections were mounted on slides and stored at 4 ◦ C. 2.4.1. RNA Extraction and Quantitative Reverse Transcription Polymerase Chain Reaction (RT-PCR) Assay Neuroretinas (previously stored at −80 ◦ C in TRIzol) were treated with DNase (18068015, ThermoFisher Scientific, Waltham, MA, USA) to eliminate genomic contamina- tion and were purified on RNeasy MinElute column (74106, Qiagen, Hilden, Germany). After supernatant removal, RNA sediment was obtained and resuspended in 30 µL of RNAse free water (AM9937, ThermoFisher Scientific, Waltham, MA, USA). An Agilent 2100 Bioanalyzer and a Nanodrop spectrophotometer were used for samples integrity and quantity respectively. cDNA reverse transcription was performed in a T100 Thermal Cycler (Bio-rad, Hercules, CA, USA) using High-Capacity cDNA Reverse Transcription Kit (4368814, ThermoFisher Scientific, Waltham, MA, USA) and Oligo(dT)18 Primer (SO131, ThermoFisher Scientific, Waltham, MA, USA). RT-PCR was carried out using SYBR Green PCR Master Mix (4309155, Applied Biosystems, Warrington, UK) and a 7.900 HT Sequence Detection System in 384-well optical plates with specific primers (displayed in Table 1). Relative quantities were calculated using the ABI SDS 2.4 RQ software and presented as a ratio between them and the endogenous controls (B2m and Actin B (Actb)). Table 1. Primers used for RT-PCR. Primers Nucleotide Sequence B2m Forward (50 -30 ) 50 -GTATGCTATCCAGAAAACCC-30 Reverse (50 -30 ) 50 -CTGAAGGACATATCTGACATC-30 Actb Forward (50 -30 ) 50 -CTAAGGCCAACCGTGAAAG -30 Reverse (50 -30 ) 50 -CAGTATGTTCGGCTTCCCATTC-30 Syn1 Forward (50 -30 ) 50 -AATCACAAAGAGATGCTCAG-30 Reverse (50 -30 ) 50 -GGACACGCACATCATATTTAG-30 Syp Forward (50 -30 ) 50 -TGCCAACAAGACGGAGAGTG-30 Reverse (50 -30 ) 50 -TAGTGCCCCCTTTAACGCAG-30 Syt1 Forward (50 -30 ) 50 -ACCCTGGGCTCTGTATCCC-30 Reverse (50 -30 ) 5 -CCCTGACCACTGAGTGCAAA-30 0 Stx1a Forward (50 -30 ) 50 -CGCTGTCCCGAAAGTTTGTG-30 Reverse (50 -30 ) 50 -GTGTCTGGTCTCGATCTCACT-30 Vamp2 Forward (50 -30 ) 50 -ATCATCGTTTACTTCAGCAC-30 Reverse (50 -30 ) 50 -TGAAAGATATGGCTGAGAGG-30 Snap25 Forward (50 -30 ) 50 -CAACTGGAACGCATTGAGGAA-30 Reverse (50 -30 ) 50 -GGCCACTACTCCATCCTGATTAT-30 Biomedicines 2021, 9, 1772 4 of 16 2.4.2. Western Blotting Protein extraction consisted in a sonication of neuroretinas for 10–15 s in 80 µL of lysis buffer [phenylmethanesulfonylfluoride (PMSF), 1mM; NaF, 100mM; Na3VO4 2mM; RIPA buffer (R0278, Sigma-Aldrich, St Louis, MO, USA); 1x protease inhibitor cocktail (P8340, Sigma-Aldrich, St. Louis, MO, USA)]. 25 µg of extracted protein were loaded in a 4–20% (vol./vol.) precast gels for SDS-PAGE (4561096, Bio-rad, Hercules, CA, USA), and electrophoresis was carried out at 100 V for 90 min. Proteins were then transferred to a polyvinylidene difluoride (PVDF) membrane (1620177, Bio-Rad Laboratories, Hercules, CA, USA) at 400 mA for 90 min and blocked in 5% powder milk (Central Lechera Asturiana, Spain) in 0.1% TBS-Tween. Primary antibodies (Table 2) were overnight incubated at 4 ◦ C. On the next day, secondary antibodies (goat anti-rabbit and goat anti-mouse (Dako Agilent, Santa Clara, CA, USA)) were applied (1:10,000). Immunoreactive bands were detected using a WesternBright ECL HRP substrate kit (K-12045-D50, Advansta, CA, USA). Anti- vinculin (1:7.000, sc-73614; Santa Cruz, Dallas, TX, USA) was used as housekeeping to normalize protein levels. The densitometric analysis was assessed with Image J software (Version 1.8, National Institutes of Health, Bethesda, MD, USA). Table 2. Primary antibodies used for Western blotting. Primary Antibodies Description Synapsin I Rabbit polyclonal; 1:2000; ab64581; Abcam, Cambridge, UK Synaptophysin Rabbit monoclonal; 1:200,000; ab32127; Abcam, Cambridge, UK Synaptotagmin Mouse monoclonal; 1:1,000; ab13259; Abcam, Cambridge, UK Syntaxin 1A Rabbit polyclonal; 1:100,000; ab41453; Abcam, Cambridge, UK SNAP-25 Rabbit polyclonal; 1:1000; 14903-1-AP; Proteintech, Rosemont, IL, USA Vamp2 Rabbit polyclonal; 1:1000; 10135-1-AP; Proteintech, Rosemont, IL, USA Cyclophilin A 1:10,000; BML-SA296; Enzo, NY, USA Vinculin Mouse monoclonal; 1:7000; sc-73614; Santa Cruz, Dallas, TX, USA 2.4.3. Immunofluorescence Analysis Paraffin-embedded ocular globes were sectioned (3 µm) and mounted on 25.5 × 75.5 × 1.0 mm Poly-L-Lysine positive charged slides (S21.2113.A, Leica Biosystems, Wetzlar, Germany). Samples were deparaffinized in xylene, rehydrated in grade ethanol series, fixed in ice-cold acid methanol (−20 ◦ C), and washed with PBS 0.01M at pH 7.4. Consecutively, eye sections were heated in a pressure cooker at 150 ◦ C for 4 min in 250 mL of 1:10 diluted antigen retrieval with sodium citrate 10 mM, pH 6 (ab973, Abcam, Cambridge, UK). Then, sections were blocked with blocking solution (X0909, Dako Agilent, Santa Clara, CA, USA) for 1h at room temperature and then incubated overnight at 4 ◦ C with specific primary antibodies (Table 3). The next day, after three washes in PBS, sections were incubated for 1 h in darkness with secondary antibodies (Alexa 488 and Alexa 594; 1/600, Molecular Probes). Samples were washed with PBS, counterstained with Hoechst 33342 (bisbenzimide) (14533, ThermoFisher Scientific, Waltham, MA, USA), and mounted with Prolong Mounting Medium Fluorescence (P36930, Prolong, InvitrogenTM , Thermo Fisher Scientific, Eugene, OR, USA) and a coverslip (15747592, ThermoFisher Scientific, Waltham, MA, USA). The images were obtained at a resolution of 1024 x 1024 pixels using laser confocal microscopy (Fluoview FV1000 Laser Scanning Confocal Microscope Olympus, Hamburg, Germany) and immunofluorescences were quantified with ImageJ software (U. S. National Institutes of Health, Bethesda, MD, USA). Biomedicines 2021, 9, 1772 5 of 16 Table 3. Primary and secondary antibodies used for immunofluorescence experiments. Primary Antibodies Description Synapsin I Rabbit polyclonal; 1:100; ab64581; Abcam, Cambridge, UK Synaptophysin Rabbit monoclonal; 1:100; ab32127; Abcam, Cambridge, UK Synaptotagmin Mouse monoclonal; 1:200; ab13259; Abcam, Cambridge, UK Syntaxin 1A Rabbit polyclonal; 1:200; ab41453; Abcam, Cambridge, UK Vamp2 Rabbit polyclonal; 1:100; 10135-1-AP; Proteintech, Rosemont, IL, USA SNAP-25 Rabbit polyclonal; 1:100; 14903-1-AP; Proteintech, Rosemont, IL, USA Secondary Antibodies Description Alexa Fluor 488 Goat anti-mouse Goat polyclonal; 1:600; ab150113; Abcam, Cambridge, UK Alexa Fluor 488 Goat anti-rabbit Goat polyclonal; 1:600; ab150081; Abcam, Cambridge, UK Alexa Fluor 594 Goat anti-mouse Goat polyclonal; 1:600; A-11032; ThermoFisher Scientific, Waltham, MA, USA Alexa Fluor 594 Goat anti-rabbit Goat polyclonal; 1:600; A-11012; ThermoFisher Scientific, Waltham, MA, USA 2.4.4. Statistical Analysis Data are presented as mean ± SEM. Statistical comparisons were performed with Student unpaired test. When multiple comparisons were performed, one-way ANOVA followed by the Bonferroni test was used. Statistical significance was set at p < 0.05. 3. Results We found a downregulation (protein and mRNA levels) of several presynaptic pro- teins such as synapsin I (Figure 1), synaptophysin (Figure 2), synaptotagmin (Figure 3), syntaxin 1A (Figure 4), vesicle-associated membrane protein 2 (VAMP2) (Figure 5), and synaptosomal-associated protein of 25 kDa (SNAP25) (Figure 6) in db/db mice treated with vehicle in comparison with non-diabetic mice. All these presynaptic proteins are involved in vesicle biogenesis, mobilization and docking, membrane fusion and recycling, and synaptic neurotransmission [15]. In addition, we found that topical administration of sitagliptin was able to prevent the downregulation of all these synaptic proteins (Figures 1–6) in diabetic mice. These effects were associated with an improvement of functional abnormalities induced by diabetes assessed by full-field ERG (Figure 7). The amplitude of a wave (predominantly produced by photoreceptors cells) was significantly lower in diabetic mice treated with the vehicle than in non-diabetic mice (Figure 7A). An example of an electroretinogram in response to low and high stimulus intensities in a representative non-diabetic mouse, a db/db mouse treated with vehicle, and a db/db mouse treated with saxagliptin or sitagliptin is shown in Figure 7B. Biomedicines 2021, Biomedicines 9, 9, 2021, x FOR 1772 PEER REVIEW 6 of 16 6 of 16 Figure 1. (A) Real-time quantitative RT-PCR analysis of synapsin I gene (Syn1) in db/db mice treated with vehicle (black Figure bars),1.sitagliptin (A) Real-time quantitative eye drops RT-PCR (grey bars), analysis of synapsin and in non-diabetic mice (white I gene bars).(Syn1) in db/db mice treated (B) Immunofluorescence with vehicle of synapsin I (red) (black bars), sitagliptin among eye drops representative (greyofbars), samples andretinas diabetic in non-diabetic mice groups. of experimental (white bars). Hoechst(B)staining Immunofluorescence (blue) was usedoffor synapsin nuclei I (red) among representative labeling. samples ONL: outer nuclear of diabetic layer; OPL: outerretinas of experimental plexifom groups. layer; INL: inner nuclearHoechst staining layer; IPL: (blue) was inner plexiform used layer; GCL:for nuclei labeling. ganglion ONL: outer Scale cell layer. nuclear layer; bars, OPL: 20 µm. (C) outer plexifomoflayer; Quantification INL: synapsin inner nuclear layer; I immunofluoresence in IPL: inner diabetic plexiform mice layer; GCL: treated with ganglion vehicle cell layer. (black eye drops Scale bars), bars, sitagliptin 20 μm. (C) eyeQuantification of synapsin drops (grey bars), I immunofluoresence and in non-diabetic retinas (white in diabetic bars). mice treated (D) Western blot with vehicle bandseye drops (black of synapsin I andbars), sitagliptin eye (E) Densitometric drops analysis in (grey db/dbbars), and in with mice treated non-diabetic retinas vehicle eye drops(white bars).sitagliptin (black bars), (D) Western blot bands of synapsin eye drops I andand (grey bars), (E)inDensitometric analysis non-diabetic mice retinasin(white db/dbbars). mice Protein treatedlevels with were vehicle eye drops normalized (black with bars), sitagliptin cyclophilin A. eye* drops p < 0.05,(grey ** p