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  • Research article
  • Open Access

Modulation of GSK-3 provides cellular and functional neuroprotection in the rd10 mouse model of retinitis pigmentosa

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Molecular Neurodegeneration201813:19

https://doi.org/10.1186/s13024-018-0251-y

  • Received: 15 June 2017
  • Accepted: 9 April 2018
  • Published:

Abstract

Background

Retinitis pigmentosa (RP) is a group of hereditary retinal neurodegenerative conditions characterized by primary dysfunction and death of photoreceptor cells, resulting in visual loss and, eventually, blindness. To date, no effective therapies have been transferred to clinic. Given the diverse genetic etiology of RP, targeting common cellular and molecular retinal alterations has emerged as a potential therapeutic strategy.

Methods

Using the Pde6b rd10/rd10 mouse model of RP, we investigated the effects of daily intraperitoneal administration of VP3.15, a small-molecule heterocyclic GSK-3 inhibitor. Gene expression was analyzed by quantitative PCR and protein expression and phosphorylation by Western blot. Photoreceptor preservation was evaluated by histological analysis and visual function was assessed by electroretinography.

Results

In rd10 retinas, increased expression of pro-inflammatory markers and reactive gliosis coincided with the early stages of retinal degeneration. Compared with wild-type controls, GSK-3β expression (mRNA and protein) remained unchanged during the retinal degeneration period. However, levels of GSK-3βSer9 and its regulator AktSer473 were increased in rd10 versus wild-type retinas. In vivo administration of VP3.15 reduced photoreceptor cell loss and preserved visual function. This neuroprotective effect was accompanied by a decrease in the expression of neuroinflammatory markers.

Conclusions

These results provide proof of concept of the therapeutic potential of VP3.15 for the treatment of retinal neurodegenerative conditions in general, and RP in particular.

Keywords

  • GSK-3
  • Retina
  • Retinitis pigmentosa
  • rd10
  • Neurodegeneration
  • Therapy

Background

Inherited retinal dystrophies, which include retinitis pigmentosa (RP), are a group of genetic diseases caused by mutations in over 300 genes and loci (http://www.sph.uth.tmc.edu/Retnet/disease.htm). RP is a retinal neurodegenerative condition characterized by primary dysfunction and death of photoreceptor cells, resulting in vision loss and, ultimately, blindness [1]. Although several experimental therapies have advanced to clinical trials (https://clinicaltrials.gov/ct2/results?term=retinitis+pigmentosa&Search=Search), neither approved treatments nor preventive therapies for RP are currently available. Its status as a rare disease and its diverse genetic etiology emphasize the importance of identifying shared pathological mechanisms independent of the causative mutation. Targeting common cellular and molecular retinal responses to mutations could benefit a significant number of RP patients, as well as those with other retinal dystrophies without an exclusively genetic etiology (e.g., glaucoma, age-related macular degeneration, diabetic retinopathy). Recent studies suggest that retinal neurodegeneration is associated with a broad inflammatory response in the retina. This response appears to be mutation-independent and involves microglial activation, reactive macrogliosis, and the production of pro-inflammatory cytokines [25].

GSK-3 is a serine/threonine kinase that exists as 2 highly homologous isoforms, GSK-3α and GSK-3β, each encoded by a distinct gene. GSK-3β is predominantly expressed in the central nervous system (CNS) [6]. Although initially identified as a glycogen synthesis enzyme (the role for which it was named), GSK-3 is currently considered a multitask enzyme involved in the regulation of diverse cellular functions owing to its broad substrate spectrum [7, 8]. In particular, GSK-3 plays a pivotal role in regulating the balance between pro-inflammatory and anti-inflammatory cellular responses. Consequently, GSK-3 is considered a potential therapeutic target for diseases with an inflammatory component. The therapeutic potential of GSK-3 modulators is currently being studied in a variety of neuroinflammatory diseases. These include psychiatric and neurodegenerative disorders, as well as retinal dystrophies; as part of the CNS, the retina shares many physiological and pathological traits with the brain [9].

Employing a chemical genetic approach, we recently investigated the neuroprotective effects of 3 chemically diverse GSK-3 modulators in photoreceptor cells [10]. In the present study we selected one of these compounds, VP3.15, based on its favorable pharmacokinetic and IC50 properties. This molecule is a 5-imino-thiadiazole that has been described not only as substrate-competitive GSK-3 inhibitor [11], but also as allosteric inhibitor of PDE7 [12].

We sought to validate in vivo the potential of GSK-3 as a therapeutic target for the treatment of RP, and to demonstrate the therapeutic potential of VP3.15 in this neurodegenerative condition. We characterized the expression of GSK-3β and its inactive serine-9-phosphorylated form in the dystrophic retina of the rd10 mouse, a model of RP. Moreover, we demonstrated that chronic systemic in vivo VP3.15 treatment preserved visual function by delaying neurodegeneration. Our results indicate that VP3.15 is an innovative drug candidate for the treatment of RP.

Methods

Animals and drug delivery

The rd10 mouse model of retinal neurodegeneration carries a homozygous phosphodiesterase 6b mutation (Pde6b rd10/rd10 ) on a C57BL/6 J background. Mice were kindly provided by Bo Chang from The Jackson Laboratory (Bar Harbor, ME, USA). Wild type (WT) control mice of the same background were also obtained from The Jackson Laboratory. All animals were housed and handled in accordance with the ARVO statement for the Use of Animals in Ophthalmic and Vision Research, European Union guidelines, and those of the local ethics committees of the CSIC and the Comunidad de Madrid. Mice were bred at the CIB core facilities on a 12/12-h light/dark cycle.

VP3.15 was synthesized in our laboratory as previously described [11]. This small molecule is a member of the iminothiadiazole family, the first group of substrate-competitive GSK-3 inhibitors described ([11]; see Additional file 1: Figure S1 for the structure data). The dose administrated was selected based on pharmacokinetic and IC50 data ([1113] and data not shown). Mice received daily intraperitoneal injections of vehicle (10% DMSO, 0.9% NaCl) or VP3.15 (10 mg/kg) for the indicated period of time.

IP administration was selected based on the ability of VP3.15 to cross the brain blood barrier [13] and the necessity of performing repetitive administration without damaging the eye.

Electroretinography

Mice underwent electroretinographic recordings (ERG) at different time points, using a previously described ERG protocol [14]. ERGs were performed by an observer blind to treatment. ERG signals were amplified and band filtered between 0.3 Hz and 1000 Hz (CP511 Preamplifier, Grass Instruments, Quincy, MA, USA) and digitized at 10 kHz using a PowerLab acquisition data card (AD Instruments Ltd., Oxfordshire, UK). Graphical representations of the signals recorded and the control light stimuli were generated using Scope v6.4 PowerLab software. ERG wave amplitudes were measured off-line and the results averaged.

Histology and immunostaining

Mice were euthanized at the indicated ages and their eyes enucleated. The right eye was processed for histological analyses and the left eye for retinal RNA extraction. For histological analyses, eyes were fixed for 1 h in 4% (w/v) paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4, and then cryoprotected by incubation in increasing concentrations of sucrose (final concentration 50% (w/v) in PB). The eyes were then embedded in Tissue-Tek OCT (Sakura Finetec, Torrance, CA, USA) and frozen on isopropanol/dry ice. Cryostat equatorial sections (12 μm) were mounted on Superfrost® Plus slides (Thermo Scientific, Massachusetts, USA), dried at room temperature, and stored at − 20 °C. Before performing further analyses, sections were fixed in acetone for 10 min at − 20 °C and dried at 65 °C for 10 min. After rinsing in PBS and permeation with 1% (w/v) Triton X-100 in PBS, sections were blocked in BGT (2.5 g/L BSA, 100 mM glycine, 0.25% (w/v) Triton X-100 in PBS) for 1 h and then incubated overnight at 4 °C with primary antibodies (Table 1) diluted in BGT. Sections incubated in the absence of primary antibody were used as specificity controls. After rinsing in PBS and incubation with the appropriate secondary antibodies (Table 1), sections were stained with DAPI (4′,6-diamidino-2-phenylindole; Sigma-Aldrich Corp., St. Louis, MO, USA) and coverslipped with Fluoromont-G.
Table 1

Antibodies

Antibody against

Host species

Dilution

Manufacturer

Catalog number

Rhodopsin

Mouse

IH,1:500

Abcam, Cambridge, UK

AB3267

L/M-opsin

Rabbit

IH,1:200

Abcam

AB5405

Mouse-Igs, Rabbit-Igs Alexa 488-546-647 labeled

Goat

IH, 1:200–500

ThermoFisher Scientific, Waltham, MA

A-11001

A-11008

A-11004

A-11011

A-21235

GSK-3Ββ

Mouse

IH,1:100

Cell Signaling Technology, Danvers, Massachusetts, USA

9832S

pGSK-3ΒβS9

Mouse

IH,1:400

Cell Signaling Technology

93,235

Recoverin

Rabbit

WB,1:5000

Millipore, Billerica, Massachusetts, USA

AB5585

GSK-3Ββ

Mouse

WB,1:1000

Cell Signaling Technology

9832S

pGSK-3ΒβS9

Rabbit

WB,1:1000

Cell Signaling Technology

93,235

Akt

Rabbit

WB,1:1000

Cell Signaling Technology

9272

pAktS473

Rabbit

WB,1:1000

Cell Signaling Technology

9271 L

pAktS473

Rabbit

IH,1:100

Cell Signaling Technology

4060

GFAP

Rabbit

IH, 1:500

Dako, Glostrup (Denmark)

Z0334

IH immunohistochemistry, WB Western blot

To assess preservation of the photoreceptor layer, we compared the thickness of the ONL (which primarily contains photoreceptors) with that of the corresponding INL (which contains bipolar, horizontal, and amacrine neurons and Müller glial cell bodies), and quantified the length of rod and cone outer segments (OS). Three sections per eye were analyzed: for each section, one image was captured for each of the 6 retinal zones (T1, T2, T3, T4, T5, and T6; Additional file 2: Figure S2). In each image, 3 measurements were recorded at random positions to obtain an average value per retinal zone per section. Measurements were performed using the “freehand line” and “measure” tools in Fiji software. The ONL thickness was normalized to that of the INL (not affected by the degeneration at this stage) to correct for possible inclinations of the sectioning plane.

Immunoblots

Protein extraction was carried out by sonicating individual retinas in RIPA lysis buffer (containing 2 mM Na3VO4, 10 mM NaF, and 4 mM Na4P2O7) and chilling the resulting solution on ice for 30 min. Protein (30 μg) from each sample was fractionated by electrophoresis on precast 10–12% (w/v) SDS-polyacrylamide gels (Criterion TGX, Bio-Rad, Munich, Germany), after which proteins were transferred to PVDF membranes using the Trans-Blot Turbo system (Bio-Rad). Blots were incubated overnight at 4 °C with primary antibodies (Table 1) diluted in TBS (Tris-buffered saline) containing 1% (w/v) Triton-X100, followed by incubation with the appropriate peroxidase-conjugated secondary antibody (Table 1). Proteins were visualized using the Pierce® ECL Western Blotting Substrate (ThermoFisher Scientific, Waltham, MA, USA), and quantified using ChemiDoc™ Touch Imaging System (Bio-Rad).

RNA isolation and quantitative PCR

Total RNA from individual retinas was extracted using TRIzol Reagent, and 2.5 μg of RNA was typically reverse transcribed using the Superscript III Kit and random primers (all from ThermoFisher Scientific). Quantitative PCR (qPCR) was performed with the ABI Prism 7900HT Sequence Detection System using TaqMan Universal PCR Master Mix, no-AmpErase UNG, and Taqman assays (listed below) for detection (all from Thermo Fisher). The relative change in gene expression was calculated using the 2ΔCt method, normalizing to expression levels of the Tbp (TATA-binding protein) gene. The primer-probe sets used are listed in Table 2.
Table 2

Taqman assays

Gene

Taqman assay

α2m

Mm00558642_m1

Gfap

Mm01253033_m1

Il1β

Mm00434228_m1

Tbp

Mm00446971_m1

Tnfα

Mm00443260_g1

Rho

Mm01184405_m1

Iba1

Mm00479862_g1

Cd68

Mm03047343_m1

Cd11b

Mm00434455_m1

Statistical analysis

Statistical analyses were performed with GraphPad Prism software 6.0 (GraphPad Software Inc., La Jolla, CA, USA). Protein levels were compared using an unpaired t-test. Gene expression levels were compared using either one-way or 2-way ANOVA tests as indicated in the Figure Legend. For ERG data, differences in wave amplitudes over time between vehicle- and VP3.15-treated mice were assessed using a 2-way ANOVA. Histological differences between vehicle- and VP3.15-treated mice were assessed by 2-way ANOVA. In all cases, p-values ≤0.05 were considered statistically significant.

Results

Inflammatory genes are upregulated in rd10 mice during the early stages of retinal neurodegeneration

Pathological neuroinflammation is a hallmark of neurodegeneration in many retinal dystrophies [25]. We analyzed temporal alterations in pro-inflammatory gene expression in the rd10 retina between P14 (before the appearance of morphological signs of retinal degeneration) and P21 (at which stage the degenerative process has become clearly established) to identify the optimal period for therapeutic intervention. Quantitative PCR analysis of WT and rd10 retinas revealed increased expression of the pro-inflammatory markers α2m, Il1β and Tnfα in the degenerating retinas, beginning in the early stages of degeneration (P18) and increasing dramatically (by 25 to 50 fold) by P21 (Fig. 1). Reactive gliosis in response to retinal damage, as measured by upregulated Gfap expression, correlated with the increased expression of inflammatory markers. Likewise, the expression of the microglia genes Cd68 and Cd11b also showed a 4–5 fold increase (Fig. 1).
Fig. 1
Fig. 1

Expression of inflammatory genes in the retina. RT-qPCR of WT and rd10 retinas at the indicated ages. Levels of different transcripts were normalized to those of Tbp RNA and relativized to P14 WT level (= 1). Results represent the mean + SEM. n = 3–4, *p ≤ 0.05, **p ≤ 0. 01, ***p ≤ 0.001, **** p ≤ 0.0001 (2-way ANOVA)

GSK-3β expression in wild type and rd10 retinas

We next determined expression levels of GSK-3β, the most abundant GSK-3 isoform in the CNS [6], during the same degenerative period as above. We measured the expression of total GSK-3β and of its inactive Ser9-phosphorylated form (GSK-3βSer9) in rd10 mouse retinas. No significant differences in GSK-3β RNA levels were observed between rd10 and WT retinas (Fig. 2a). Furthermore, despite the morphological differences caused by the degenerative process, we observed similarly broad distribution patterns for GSK-3β protein in both P21 rd10 and WT retinas (Fig. 2b).
Fig. 2
Fig. 2

GSK-3β expression in the retina. a RT-qPCR of WT and rd10 retinas at the indicated ages. Gsk-3β transcript levels were normalized to Tbp RNA levels and relativized to P14 WT level (= 1). Results represent the mean + SEM. n = 4. b Representative images of P21 retinal sections from WT and rd10 mice, immunostained for GSK-3β (green). Nuclei are stained with DAPI (blue). ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar: 60 μm

GSK-3β is an unusual kinase in that it is constitutively activated but is inhibited upon stimulation of its regulatory signaling pathways (reviewed in [7]). Activation of the PI3K/Akt pathway induces phosphorylation of GSK-3β at Ser9, and subsequent downregulation of GSK-3β activity [15]. During early retinal degeneration (P16 and P18), no differences in Ser473-phosphorylated Akt (pAktSer473) or Ser9-phosphorylated GSK-3β (pGSK-3βSer9) levels were observed between WT and rd10 retinas (data not shown). Conversely, at P19 and P21, at which point the degenerative process is well established, as evidenced by decreased expression of the photoreceptor protein recoverin, pAktSer473 and pGSK-3βSer9 levels were significantly higher in rd10 versus WT retinas (Fig. 3a and b). These observations in retinal extracts were in agreement with the immunostaining pattern observed at P21, which showed an increment in pAktSer473 and pGSK-3βSer9 in the rd10 retina (Fig. 3c). This inhibitory phosphorylation of GSK-3β, which coincided with activation of the pro-survival Akt signaling pathway, may represent an intrinsic neuroprotective response to the genetic damage caused in the retina. To test this hypothesis, we sought to potentiate this intrinsic response by pharmacologically inhibiting GSK-3 activity.
Fig. 3
Fig. 3

GSK-3β activation in the retina. a Representative Western blots of protein extracts from WT and rd10 retinas (at P19 and P21). b Densitometric analysis of several membranes as those shown in a. Levels of phosphorylated Akt and GSK-3β were normalized to those of total Akt and GSK-3β, respectively. Results represent the mean + SEM. n = 4–5, *p ≤ 0.05 (unpaired 2-tailed Student’s t test). c Representative images of P21 retinal sections from WT and rd10 mice, immunostained for pAktSer473 and pGSK-3βSer9 (green). Nuclei are stained with DAPI (blue). ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar: 60 μm

In vivo VP3.15 treatment decreases inflammatory and degenerative signs in the rd10 mouse retina

In light of our findings concerning proinflammatory gene expression and GSK-3β modulation, we selected the P15–P21 interval to assess the effects of short-term treatment with VP3.15, a GSK-3 inhibitor. VP3.15 inhibitory activity on GSK-3 was confirmed by determining its effect on the β-catenin levels in the microglia cell line N9 (Additional file 3: Figure S3). rd10 littermates received daily intraperitoneal injections of either vehicle or VP3.15 (10 mg/kg). One eye from each animal was analyzed for gene expression and the other for retinal morphology. VP3.15 treatment significantly decreased the expression of the proinflammatory genes Il1β and α2m. A similar trend was observed for Tnfα expression (Fig. 4a), and secretion by rd10 retinal explants (Additional file 7: Figure S7b), although these failed to reach statistical significance. The microglia genes Cd11b and Iba1 were also decreased upon VP3.15 treatment, while the activated microglia marker Cd68 showed a decline trend (Fig. 4b). In parallel, VP3.15 significantly reduced the expression of Gfap, an effect that correlates with a reduced GFAP staining depicted by the radial process of Müller glial cells and by the most inner retinal layer formed by Müller end feet and astrocytes (Fig. 4c and d). Conversely, a trend towards increased gene expression of rod photoreceptor-specific rhodopsin was observed in VP3.15-treated rd10 mice (Fig. 4e). This observation correlated with the maintenance of ONL thickness and OS length, both of which were better preserved in VP3.15-treated than in the vehicle-treated rd10 mice (Fig. 4f). Moreover, while rhodopsin expression was specifically localized in the photoreceptor outer segment (OS) in WT and VP3.15-treated rd10 retinas, vehicle-treated rd10 retinas displayed shortening of the OS and mislocalization of rhodopsin in the ONL (Fig. 4f).
Fig. 4
Fig. 4

In vivo effect of short-term VP3.15 administration. rd10 mice received daily intraperitoneal injections of vehicle or VP3.15 from P15 to P21, and were analyzed one day after the last injection (P22). a-c and e RT-qPCR of WT as well as untreated rd10 retinas, and vehicle- and VP3.15-treated rd10 retinas (all harvested at P22). Levels of all transcripts were normalized to those of Tbp RNA and relativized to WT levels (= 1). Results represent the mean + SEM. n = 2–4, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, **** p ≤ 0.0001 (one-way ANOVA). d and f Representative images of P22 retinal sections from WT mice and from vehicle- and VP3.15-treated rd10 mice, immunostained for GFAP (d, red) and rhodopsin (f, cyan). Nuclei are stained with DAPI. In d arrows point to the radial processes of Müller cells and the arrow head to the most inner retinal layer containing the Müller end feet and astrocytes. Arrows in f point to mislocalized rhodopsin in the ONL. OS, outer segments; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar: 60 μm

In vivo treatment of rd10 mice with VP3.15 delays the loss of visual function

Based on the promising results obtained following short-term GSK-3 inhibition, we investigated whether a longer treatment period preserved visual function in the rd10 mouse model. rd10 littermates received daily injections of either vehicle or VP3.15 from P15 to P46 inclusive. Visual function was assessed weekly by ERG between P25 and P46. Compared with vehicle-treated counterparts, VP3.15-treated rd10 mice displayed better-defined ERG waves of greater amplitude (Fig. 5 and Additional file 4: Figure S4). The amplitude of ERG waves (a-mixed, b-mixed, b-photopic, and oscillatory potential [OP]) was significantly higher in VP3.15-treated than in vehicle-treated mice, indicating a prolongation of both rod- and cone-mediated vision function by VP3.15 (Fig. 5).
Fig. 5
Fig. 5

In vivo effect of long-term VP3.15 administration on visual function. rd10 mice received daily intraperitoneal injections of vehicle or VP3.15 from P15 to P45. ERG were performed at the indicated ages. Graphs show averaged ERG wave amplitudes, plotted as a function of animal age. Results represent the mean ± SEM. n = 10, **** p ≤ 0.0001 (2-way ANOVA)

Histological evaluation of retinas at the end point of the study (P47, after the last ERG) revealed a greater relative ONL thickness in VP3.15- versus vehicle-treated retinas despite the advanced degeneration stage (Fig. 6a and b). The photoreceptor preservation effect of VP3.15 was more evident at mid-degeneration stages (P33; Additional file 5: Figure S5). Moreover, specific immunostaining showed better preservation of photoreceptor structure in VP3.15-treated retinas (Fig. 6a). Rhodopsin immunostaining in rod outer segments confirmed the partial preservation of rod outer segments in VP3.15-treated retinas. By contrast, rod outer segments were barely present in vehicle-treated retinas. Similarly, M-L opsin immunostaining revealed longer cone outer segments in VP3.15- versus vehicle-treated rd10 retinas (Fig. 6a and b).
Fig. 6
Fig. 6

In vivo effect of long-term VP3.15 administration on photoreceptor cell preservation. Retinal sections from the same animals described in Fig. 5 were immunostained to assess photoreceptor cell structure. a Representative images of P47 retinal sections from vehicle- and VP3.15-treated rd10 mice immunostained for rhodopsin (cyan) and M/L opsin (green). Nuclei are stained with DAPI (blue). OS, outer segments; ONL, outer nuclear layer; INL, inner nuclear layer; GCL, ganglion cell layer. Scale bar: 60 μm. b ONL and INL thickness and the length of rod and cone outer segments were measured in equatorial sections corresponding to 6 regions of the retina, following a nasotemporal sequence (T1–T6; see Methods and Additional file 2: Figure S2). Plots show the mean + SEM. n = 5 mice, 3 sections per retina, 3 measurements per region and section. ON, optic nerve. ***p ≤ 0.001, **** p ≤ 0.0001 (2-way ANOVA)

Moreover, no toxic effect of VP3.15 administration was observed in other neuronal populations of the retina, particularly rod-bipolar and ganglion cells (Additional file 6: Figure S6).

Taken together, these results demonstrate a neuroprotective effect of VP3.15 on neuroinflammation, retinal structure, and visual function. Our findings thus support the potential of VP3.15 as a novel therapy for retinal dystrophies.

Discussion

This study describes GSK-3β expression and activation of the Akt-GSK-3β pathway during the early stages of retinal neurodegeneration in the rd10 mouse model of RP. Our findings support the involvement of GSK-3 in retinal decay and provide proof of concept of the neuroprotective effect of GSK-3 inhibition on photoreceptor cells [10]. In vivo administration of VP3.15, a small-molecule GSK-3 inhibitor, reduced photoreceptor cell loss and extended visual function. This neuroprotective effect was accompanied by a decrease in the expression of neuroinflammatory genes in the retina, indicating the potential of VP3.15 as a candidate RP therapy.

GSK-3 is a constitutively activated multitask enzyme, the activity of which is regulated by several inhibitory signaling pathways [7]. Inhibitory phosphorylation of GSK-3 at Ser9 is one of the main regulatory mechanisms. Our analysis of GSK-3β RNA and protein levels during the degenerative period in rd10 retinas revealed no differences with respect to WT counterparts. However, at both P19 and P21, by when photoreceptor degeneration is well established, inhibitory phosphorylation of GSK-3β at Ser9, and phosphorylation of its Akt regulator, were increased in rd10 retinas. This endogenous activation of the prosurvival Akt-GSK-3β pathway in the rd10 retina is in agreement with previous observations in a photoreceptor cell line subjected to a variety of insults, and in the dystrophic retina [16, 17]. We speculate that the activation of endogenous prosurvival pathways in the dystrophic retina constitutes an attempt to control the degenerative process, a response that, however, results insufficient to counteract the permanent genetic damage underlying the degeneration. In our experiments, administration of the GSK-3 inhibitor VP3.15 may have exogenously potentiated the prosurvival pathway, thus delaying photoreceptor cell death and preserving visual function. Indeed, several agents with neuroprotective effects in the retina exert common stimulatory effects on the Akt-GSK-3β pathway [1820]. GSK-3 is a downstream substrate of the insulin receptor [15, 21] and is thus inhibited upon insulin receptor stimulation. We have previously demonstrated that proinsulin, which binds with high affinity to the A isoform of the insulin receptor in the retina and activates Akt [22, 23], similarly prolongs rod survival and preserves visual function [14, 22, 24].

Neuroinflammation is a key component of neurodegenerative processes affecting the brain and retina, and thus constitutes a promising non-cell-autonomous target for treatments of CNS diseases in general, and of the retina in particular [25, 25]. GSK-3 plays a pivotal role in the regulation of peripheral and central pro-inflammatory cytokine production [26, 27], and its inhibition reduces both systemic and brain inflammation [26, 27]. The specific molecular events that occur in the rd10 retina upon GSK-3 inhibition remain to be established. This is a complicated task since GSK-3 is implicated in the regulation of multiple cellular processes including metabolism, cell structure, cell death, proliferation, and gene expression, with over 100 confirmed and 500 predicted substrates [7]. Exploratory studies employing rd10 retinal explants showed that VP3.15 attenuated NF-kB activation (Additional file 7: Figure S7). NF-kB is a key regulator of the inflammatory response by enabling the transcription of the genes encoding many pro-inflammatory cytokines. Indeed, our results suggest that levels of pro-inflammatory cytokines (Il1β and Tnfα) are reduced in VP3.15-treated rd10 retinas. This could explain, at least in part, the attenuation of photoreceptor degeneration and loss, and the preservation of vision. Moreover, the reduced inflammatory response observed in VP3.15-treated retinas was accompanied by decreases in the reactive gliosis marker GFAP as well as in α2M expression, for which a role in retinal neurodegeneration has been recently described [2830].

In addition to its inhibitory effect on GSK-3, VP3.15 also acts as an allosteric inhibitor of PDE7, an effect that contributes to its neuroprotective activity in other CNS pathologies [13]. However, PDE7 inhibition, via increasing cAMP levels and PKA activation, also converges on GSK-3 inhibition [31]. PKA is able to phosphorylate GSK-3β at Ser9 and, subsequently, to inactivate it [20, 31]. Besides, previous findings by our group support that the prosurvival effect of VP3.15 in photoreceptors may be primarily due to GSK-3 inhibition, since the specific GSK-3 inhibitor tideglusib exerted an even more potent neuroprotective effect in photoreceptors [10].

Most forms of RP involve primary death of rod photoreceptors, in which the mutated gene exerts its function, followed by secondary loss of cone photoreceptors. VP3.15 treatment prolonged photoreceptor survival, as evidenced by the preservation of ONL thickness and rod outer segment length. Furthermore, VP3.15 preserved the integrity of the cone outer segment, the first site of morphological alterations during cone degeneration. Human vision primarily relies on cones, which mediate daylight, color, and high-acuity vision. Accordingly, a mutation-independent treatment for RP that specifically prolongs cone survival would be highly beneficial. VP3.15 had a marked effect on cone cytoarchitecture and visual function, as indicated by an increase in the amplitude of the photopic b-wave compared with the untreated rd10 animals. It remains unclear whether VP3.15 exerts a direct effect on cone integrity, or whether this effect is secondary to the prosurvival effect on rods (present study and [10]). Regardless, VP3.15 resulted in a marked attenuation of the loss of both daylight and dim-light vision.

Conclusion

Our in vivo findings, together with the results of a recently published in vitro study [10], support the therapeutic potential of GSK-3 modulation for the treatment of retinal neurodegeneration, as described for other neurodegenerative diseases [32], and underscore the potential of GSK-3 inhibitors, in particular VP3.15, as pharmacological therapies for RP.

Abbreviations

CD11B: 

Cluster of differentiation molecule 11B

CD68: 

Cluster of differentiation 68

CNS: 

Central nervous system

ERG: 

Electroretinographic recording

GCL: 

Ganglion cell layer

GFAP: 

Glial fibrillary acidic protein

GSK: 

Glycogen synthase kinase

IBA1: 

Ionized calcium-binding adapter molecule 1

IC50

Half maximal inhibitory concentration

IL1-β: 

Interleukin-1β

INL: 

Inner nuclear layer

NF-κB: 

Nuclear factor kappa-light-chain-enhancer of activated B cells

ON: 

Optic nerve

ONL: 

Outer nuclear layer

OP: 

Oscillatory potential

OS: 

Outer segment

PB: 

Phosphate buffer

PDE: 

Phosphodiesterase

PKA: 

Protein kinase A

PKCα: 

Protein kinase C alpha

qPCR: 

Quantitative polymerase chain reaction

RBPMS: 

RNA binding protein with multiple splicing

RP: 

Retinitis pigmentosa

RT: 

Reverse transcription

TBP: 

TATA-binding protein

TNFα: 

Tumor necrosis factorα

WT: 

Wild-type

α2M: 

α-2-macroglobulin

Declarations

Acknowledgements

We thank Cayetana Murillo, Laura Ramírez and the staff of the CIB animal house and microscopy facilities for technical support. We thank Violeta Gómez-Vicente for advice on GSK-3 immunostaining. We thank Dr. García-Pardo and Dr. Labandeira-García for providing the pNF-κBser536 antibody and the N9 cell line, respectively. ASC and JZD are recipients of UCM and MECD (FPU13-00362) predoctoral fellowships, respectively.

Funding

This work was supported by grants from the Spanish MINECO (SAF2012-37979-C03-01 and SAF2016-76639-R to AM, SAF2013-41059-R and SAF2016-75681-R to EJdlR, and PI13-02098 to PdlV) and Instituto de Salud Carlos III and cofinanced by FEDER (PI17/01601 and RETICS RD16/0019/0009 to IL).

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors’ contributions

ASC, BVZ and MM and JZD performed experimental procedures. VP synthesized VP3.15. CG and AM supervised the medicinal chemistry work. PdlV designed and analyzed the functional studies. EJdlR and CHS designed and supervised the biological experiments. AM, EJdlR and CHS, analyzed and discussed the results. ASC, VP, IL, PdlV, AM, EJdlR and CHS contributed to the writing and edition of the manuscript. All authors read and approved the final manuscript.

Ethics approval

Animal handling and experimental procedures followed the 3Rs principles, and were revised by the Animal Welfare Committee of the Centro de Investigaciones Biológicas (CIB) and the Bioethics Committee of the Consejo Superior de Investigaciones Científicas (CSIC) and authorized by the Comunidad de Madrid (Ref: PROEX 157/14), in accordance with the Spanish legislation and the European Union (EU) Directive 2010/63/EU for animal research.

Consent for publication

All authors consent for the publication of this study.

Competing interests

CSIC has filed a patent for the VP3.15 (AM, VP and CG inventors).

Publisher’s Note

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Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Departments of Molecular Biomedicine (3D Lab) and Structural and Chemical Biology (IPSBB Unit), Centro de Investigaciones Biológicas-CSIC, C/ Ramiro de Maeztu 9, E-28040 Madrid, Spain
(2)
Neurovascular Research Unit, Department of Pharmacology, Facultad de Medicina, Universidad Complutense de Madrid, Madrid, Spain
(3)
Department of Systems Biology, Facultad de Medicina, Universidad de Alcalá, Alcalá de Henares, Spain

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