Treatment with MDL 72527 Ameliorated Clinical Symptoms, Retinal Ganglion Cell Loss, Optic Nerve Inflammation, and Improved Visual Acuity in an Experimental Model of Multiple Sclerosis

Multiple Sclerosis (MS) is a highly disabling neurological disease characterized by inflammation, neuronal damage, and demyelination. Vision impairment is one of the major clinical features of MS. Previous studies from our lab have shown that MDL 72527, a pharmacological inhibitor of spermine oxidase (SMOX), is protective against neurodegeneration and inflammation in the models of diabetic retinopathy and excitotoxicity. In the present study, utilizing the experimental autoimmune encephalomyelitis (EAE) model of MS, we determined the impact of SMOX blockade on retinal neurodegeneration and optic nerve inflammation. The increased expression of SMOX observed in EAE retinas was associated with a significant loss of retinal ganglion cells, degeneration of synaptic contacts, and reduced visual acuity. MDL 72527-treated mice exhibited markedly reduced motor deficits, improved neuronal survival, the preservation of synapses, and improved visual acuity compared to the vehicle-treated group. The EAE-induced increase in macrophage/microglia was markedly reduced by SMOX inhibition. Upregulated acrolein conjugates in the EAE retina were decreased through MDL 72527 treatment. Mechanistically, the EAE-induced ERK-STAT3 signaling was blunted by SMOX inhibition. In conclusion, our studies demonstrate the potential benefits of targeting SMOX to treat MS-mediated neuroinflammation and vision loss.


Introduction
Multiple Sclerosis (MS), a highly disabling neurological disease affecting more than 2.5 million worldwide [1,2], is characterized by inflammatory responses, neuronal damage, and demyelination [3]. MS has a heterogeneous presentation, and patients suffer from various disabilities, such as vision problems, memory loss, cognitive deficit, and movement disorders [1,4]. In addition to the motor and sensory disturbances, neuropsychiatric symptoms, such as depression and anxiety, are more common in MS than in other chronic neurological conditions [5,6]. While it is evident that neurodegeneration contributes significantly to MS pathology, the mechanisms underlying the progressive neurodegeneration remain unclear. During the last two decades, several disease-modifying therapies, including oral medications and monoclonal antibodies have been approved for treating MS [7][8][9][10][11]. While these medications have helped to manage symptoms and

Immunofluorescence Staining of the Retinal and Optic Nerve Sections and Retinal Flatmounts
Immunostaining was performed on the retinal and optic nerve sections, as described previously [32,43,44]. The eyes were enucleated, fixed in 4% PFA (overnight at 4 • C), washed in PBS, and cryoprotected in 30% sucrose. The cryostat sections (10 µm) were prepared and mounted on glass slides and stored at −80 • C until used. The sections were permeabilized in 0.05% Triton X-100 (10 min) and blocked in 10% normal goat serum for 1 h at room temperature. The sections were then incubated with primary antibodies (Table 1) overnight, followed by 1 h incubation with fluorescein-conjugated secondary antibodies (Invitrogen, Carlsbad, CA, USA; 1:400), as previously described. The sections were washed in PBS and covered with mounting medium (Vector Laboratories Cat. # H-1000, Burlingame, CA, USA). Images were taken using a confocal microscope (LSM 780; Carl Zeiss, Thornwood, NY, USA).
Immunostaining of the retinal flatmounts was performed, according to the standardized methods, in our laboratory [37,49]. The eyeballs were fixed in 4% PFA at 4 • C overnight and the retinal flatmounts were permeabilized and incubated with NeuN antibody (1:200 dilution) for 2 h at 37 • C. Incubation with fluorescein-conjugated secondary antibody (Invitrogen, Carlsbad, CA, USA; 1:400) was performed overnight at 4 • C. Four images per retina were taken in the mid-periphery (500 µm from the optic nerve head) of the ganglion cell layer (GCL) using a confocal microscope (LSM 780; Carl Zeiss, Thornwood, NY, USA). NIH ImageJ software was used for the quantification of NeuN-positive cells.

Imaging and Quantification
Images of the retinal sections were captured using a Keyence fluorescence microscope (BZ-X800, Itasca, IL, USA) and/or confocal microscope (LSM 780; Carl Zeiss, Thornwood, NY, USA). A minimum of two sections (20 µm apart) per optic nerve or three sections per retina were utilized per animal for each antibody treatment. As described in our previous publications, [38,39], the images were acquired at 500 µm from the optic nerve head of the retinal sections and used for quantification. A minimum of two non-overlapping fields per optic nerve section were taken, resulting in a minimum of 6 images per mouse per antibody. A minimum of 5 animals per group were included in each study unless otherwise stated.

Hematoxylin and Eosin (H&E) Staining and Analysis of Cellular Infiltration
H&E staining was performed on the optic nerve sections at Augusta University's histology core. Images were taken using Zeiss AxioPlan 2 microscope (Carl Zeiss, Thornwood, NY, USA) and the analysis was performed on three different non-overlapping fields of view from each optic nerve using the "point tool" function in the NIH ImageJ software (National Institutes of Health, Bethesda, MD, USA). A minimum of two sections were used from each optic nerve.

Optokinetic Studies
The visual acuity threshold was measured daily with the optokinetic tracking (OKT) response using Optometry software and apparatus (Cerebral Mechanics Inc., Medicine Hat, AB, Canada) as described [45,46]. Briefly, mice situated on a pedestal within a four-Cells 2022, 11, 4100 5 of 18 sided chamber were presented with vertical sine-wave gratings moving at 12 • /s or gray of the same mean luminance. The grating rotation elicited reflexive tracking, which was scored via live video using a method-of-limits procedure with a yes/no criterion. Spatial resolution was taken as the asymptote of a staircase procedure. The two eyes were tested in an interleaved fashion. In situations where the mice were unsteady due to motor deficits to adequately perform OKT analysis, the measurement was not recorded for that mouse on that day.

Statistical Analysis
Statistical analyses were performed using GraphPad Prism 9 (GraphPad Software Inc., La Jolla, CA, USA). One-way ANOVA followed by the Tukey test was used for multiple comparisons. A p-value < 0.05 was considered statistically significant.

MDL 72527 Treatment Improved EAE-Induced Motor Deficits
In the present study, we utilized an active immunization model to induce chronic EAE in mice using a myelin oligodendrocyte glycoprotein (MOG) peptide, as published previously [49], and EAE progression was assessed up to 36 days following induction. The initial sign of paralysis in the EAE mice, indicated by the loss of tail tonicity, was observed, starting on day nine in the vehicle-treated EAE mice. Other clinical symptoms of EAE increased gradually in the EAE mice treated with vehicle, as measured by the clinical scores. As presented in Figure 1, the clinical scores were markedly reduced in EAE mice treated with MDL 72527 throughout the induction period. Interestingly, in the group of EAE mice treated with MDL, the appearance of the initial signs of paralysis was observed to be delayed (beginning at day 14) and the clinical scores were significantly lower at most of the time points, compared to the vehicle-treated EAE mice. While many EAE mice in the vehicle-treated group showed clinical scores between three and four, the scores stayed below three in most of the mice treated with MDL 72527. Those mice showing a 15% or more reduction in body weight were removed from the study and sacrificed. These results suggest that the motor deficits resulting from EAE were reduced in response to SMOX inhibition.
Cells 2022, 11, 4100 6 of 20 Figure 1. Treatment with MDL 7252 improved EAE-induced motor deficits. Animals were evaluated every day and the clinical scores were recorded according to a 0-5 scale (as described in the methods section). The mice in the vehicle-treated EAE group showed progressively increasing clinical scores starting at day 9 post-induction. EAE mice that received MDL 72527 treatments showed significantly lower clinical scores as compared to the vehicle-EAE group. Mice in both the control groups showed no signs of motor deficits. # p < 0.05; * p < 0.01; n = 9-15 per group. Data are presented as mean ± SEM.

SMOX Expression Is Upregulated in the EAE Retina
Studies were performed to assess the changes in the SMOX expression in response to EAE induction. A significant increase in the SMOX protein was observed in the retinas of vehicle-treated EAE mice (15 days post-induction) compared to the control group ( Figure   Figure 1. Treatment with MDL 7252 improved EAE-induced motor deficits. Animals were evaluated every day and the clinical scores were recorded according to a 0-5 scale (as described in the methods section). The mice in the vehicle-treated EAE group showed progressively increasing clinical scores starting at day 9 post-induction. EAE mice that received MDL 72527 treatments showed significantly lower clinical scores as compared to the vehicle-EAE group. Mice in both the control groups showed no signs of motor deficits. # p < 0.05; * p < 0.01; n = 9-15 per group. Data are presented as mean ± SEM.

SMOX Expression Is Upregulated in the EAE Retina
Studies were performed to assess the changes in the SMOX expression in response to EAE induction. A significant increase in the SMOX protein was observed in the retinas of vehicle-treated EAE mice (15 days post-induction) compared to the control group (Figure 2A,B). Immunofluorescence studies further confirmed the upregulation of SMOX in the EAE retinas, with higher levels in the inner retina. Localization studies performed using markers for retinal ganglion cells (Brn3a) and amacrine cells (ChAT, Choline acetyltransferase) showed expression in RGCs, colocalized with NeuN and Brn3a. SMOX expression was also observed in the outer plexiform layer (OPL) and to a lesser extent in the inner nuclear layer (INL) ( Figure 2C-F).

SMOX Inhibition Reduced EAE-Induced Neurodegeneration
The loss of RGCs is a major feature of EAE-induced retinal damage. In the current study, using immunofluorescence staining and analysis of the retinal sections, we investigated the EAE-induced RGC loss. Figure 3 shows the representative images of retinal sections immunostained using markers Brn3a ( Figure 3A-D), and NeuN ( Figure 3F-I). A significant reduction in GCL neurons is evident in the vehicle-treated EAE retinas, studied by the markers, and the treatment with SMOX inhibitor, MDL 72527, significantly improved the survival of the GCL neurons. The quantification of Brn3a ( Figure 3E) and NeuN ( Figure 3J) positive cells in the GCL showed significant reductions in EAE retinas compared to the vehicle-treated controls. This EAE-induced RGC loss was significantly reduced in response to MDL 72527, as measured by the markers studied. These results are further confirmed by retinal flatmount analyses using a NeuN antibody ( Figure 3K-O). Compared to the vehicle-treated control group, a marked reduction in NeuN-positive cells was observed in response to EAE induction ( Figure 3K,L). Treatment with MDL72527 improved the survival of NeuN-positive cells in the EAE retina ( Figure 3M). Figure 3O demonstrates the quantification studies performed using NIH ImageJ. While MDL 72527 treatment offered significant RGC survival in EAE retinas, no marked changes were observed in the MDL-treated control group in response to SMOX inhibition ( Figure 3O).

SMOX Inhibition Reduced EAE-Induced Neurodegeneration
The loss of RGCs is a major feature of EAE-induced retinal damage. In the current study, using immunofluorescence staining and analysis of the retinal sections, we investigated the EAE-induced RGC loss. Figure 3 shows the representative images of retinal sections immunostained using markers Brn3a ( Figure 3A-D), and NeuN ( Figure 3F-I). A significant reduction in GCL neurons is evident in the vehicle-treated EAE retinas, studied by the markers, and the treatment with SMOX inhibitor, MDL 72527, significantly improved the survival of the GCL neurons. The quantification of Brn3a ( Figure 3E) and NeuN ( Figure 3J) positive cells in the GCL showed significant reductions in EAE retinas compared to the vehicle-treated controls. This EAE-induced RGC loss was significantly reduced in response to MDL 72527, as measured by the markers studied. These results are further confirmed by retinal flatmount analyses using a NeuN antibody ( Figure 3K-O). Compared to the vehicle-treated control group, a marked reduction in NeuN-positive cells was observed in response to EAE induction ( Figure 3K,L). Treatment with MDL72527 improved the survival of NeuN-positive cells in the EAE retina ( Figure 3M). Figure 3O demonstrates the quantification studies performed using NIH ImageJ. While MDL 72527 treatment offered significant RGC survival in EAE retinas, no marked changes were observed in the MDL-treated control group in response to SMOX inhibition ( Figure 3O). Further studies using Tuj1 ( Figure 4A-D) and synaptophysin ( Figure 4F-I) antibodies support the EAE-induced degeneration of RGCs. A marked reduction in the Tuj1 immunostaining observed in the GCL and IPL of the vehicle-treated EAE retinas indicated axonal loss in response to EAE treatment. Our results show that MDL 72527 treatment significantly improved the levels of Tuj1 expression in the EAE retinas in comparison with the retinas from the vehicle-treated EAE mice. The quantification of Tuj1 fluorescence intensity ( Figure 4E) using NIH ImageJ analysis confirmed the significantly reduced level of Tuj1 expression in the vehicle-treated EAE retina and its improved level in response to SMOX inhibition. Synaptophysin (a pre-synaptic marker) immunostaining was utilized to examine the loss of synapses due to neurodegeneration in the EAE retina. A significant reduction in synaptophysin expression was observed in the IPL and OPL of vehicletreated EAE mice. However, MDL 72527 treatment significantly improved the levels of synaptophysin in the EAE retinas, suggesting improved synaptic contacts by SMOX inhibition. These observations are confirmed by the quantification of fluorescence intensity of the synaptophysin signal using NIH ImageJ ( Figure 4J). Further studies using Tuj1 ( Figure 4A-D) and synaptophysin ( Figure 4F-I) antibodies support the EAE-induced degeneration of RGCs. A marked reduction in the Tuj1 immunostaining observed in the GCL and IPL of the vehicle-treated EAE retinas indicated axonal loss in response to EAE treatment. Our results show that MDL 72527 treatment significantly improved the levels of Tuj1 expression in the EAE retinas in comparison with the retinas from the vehicle-treated EAE mice. The quantification of Tuj1 fluorescence intensity ( Figure 4E) using NIH ImageJ analysis confirmed the significantly reduced level of Tuj1 expression in the vehicle-treated EAE retina and its improved level in response to SMOX inhibition. Synaptophysin (a pre-synaptic marker) immunostaining was utilized to examine the loss of synapses due to neurodegeneration in the EAE retina. A significant reduction in synaptophysin expression was observed in the IPL and OPL of vehicle-treated EAE mice. However, MDL 72527 treatment significantly improved the levels of synaptophysin in the EAE retinas, suggesting improved synaptic contacts by SMOX inhibition. These observations are confirmed by the quantification of fluorescence intensity of the synaptophysin signal using NIH ImageJ ( Figure 4J).

SMOX Inhibition Improved Visual Acuity in EAE Mice
In the present study, using the OptoMotry system 30 days post-induction, we investigated the impact of MDL 72527 treatment effects on visual acuity in EAE mice. Figure 5 shows the results of the optokinetic experiments utilized to study the EAE effects on visual impairment. Mice in the vehicle-treated control group showed an average response of 0.394 ± 0.02 cycles/degree (c/d). In the vehicle-treated EAE group, the average response was 0.170 ± 0.06 c/d, demonstrating a significant reduction in the OKT threshold compared to the vehicle-treated control group. However, treatment with SMOX inhibitor significantly improved the OKT threshold in the EAE mice, with 0.246 ± 0.05 c/d suggesting the improvement in vision. In comparison to the vehicle-treated control group, there was no difference in the measurements of visual acuity in the control mice treated with MDL 72527.

SMOX Inhibition Improved Visual Acuity in EAE Mice
In the present study, using the OptoMotry system 30 days post-induction, we investigated the impact of MDL 72527 treatment effects on visual acuity in EAE mice. Figure 5 shows the results of the optokinetic experiments utilized to study the EAE effects on visual impairment. Mice in the vehicle-treated control group showed an average response of 0.394 ± 0.02 cycles/degree (c/d). In the vehicle-treated EAE group, the average response was 0.170 ± 0.06 c/d, demonstrating a significant reduction in the OKT threshold compared to the vehicle-treated control group. However, treatment with SMOX inhibitor significantly improved the OKT threshold in the EAE mice, with 0.246 ± 0.05 c/d suggesting the improvement in vision. In comparison to the vehicle-treated control group, there was no difference in the measurements of visual acuity in the control mice treated with MDL 72527.

Optic Nerve Inflammation in the EAE Mice Is Reduced by SMOX Inhibition
The inflammation of the optic nerve is a major feature in EAE mice [21,50]. In the present study, we investigated the impact of MDL 72527 on EAE-induced optic nerve inflammation. Cellular infiltration was studied by the histological analysis of optic nerve

Optic Nerve Inflammation in the EAE Mice Is Reduced by SMOX Inhibition
The inflammation of the optic nerve is a major feature in EAE mice [21,50]. In the present study, we investigated the impact of MDL 72527 on EAE-induced optic nerve inflammation. Cellular infiltration was studied by the histological analysis of optic nerve sections stained by H&E. As seen in Figure 6, increased cellular infiltration is evident in the EAE optic nerves. The optic nerve sections from the vehicle-treated EAE group showed hypercellularity, compared to vehicle-treated controls ( Figure 6A,B). However, in response to MDL 72527 treatment, the EAE-induced cellular infiltration was markedly reduced in the optic nerve samples ( Figure 6C). The high magnification images ( Figure 6E-H) collected from the optic nerve sections show clusters of infiltrated cells in the vehicle-treated EAE optic nerves compared to the MDL 72527 treated EAE and the vehicle-treated control optic nerve samples. Figure 6I presents the quantification of the infiltrated cells, demonstrating a two-fold increase in the vehicle-treated EAE optic nerve, compared to the vehicle-treated control. The EAE optic nerves from MDL 72527 treated mice showed significantly decreased levels of cellular infiltration, indicating the reduced optic nerve inflammation offered by SMOX inhibition. Figure 5. MDL 72527 improved visual acuity in EAE mice. Optokinetic studies show a reduction in visual acuity in the EAE mice (36 days post-induction) compared to the control. MDL 72527 treatment significantly improved the visual acuity. Data are presented as the average of clockwise and counterclockwise measurements. n = 6-10 per group; * p < 0.01. Data presented as mean ± SD.

Optic Nerve Inflammation in the EAE Mice Is Reduced by SMOX Inhibition
The inflammation of the optic nerve is a major feature in EAE mice [21,50]. In the present study, we investigated the impact of MDL 72527 on EAE-induced optic nerve inflammation. Cellular infiltration was studied by the histological analysis of optic nerve sections stained by H&E. As seen in Figure 6, increased cellular infiltration is evident in the EAE optic nerves. The optic nerve sections from the vehicle-treated EAE group showed hypercellularity, compared to vehicle-treated controls ( Figure 6A,B). However, in response to MDL 72527 treatment, the EAE-induced cellular infiltration was markedly reduced in the optic nerve samples ( Figure 6C). The high magnification images ( Figure  6E-H) collected from the optic nerve sections show clusters of infiltrated cells in the vehicle-treated EAE optic nerves compared to the MDL 72527 treated EAE and the vehicletreated control optic nerve samples. Figure 6I presents the quantification of the infiltrated cells, demonstrating a two-fold increase in the vehicle-treated EAE optic nerve, compared to the vehicle-treated control. The EAE optic nerves from MDL 72527 treated mice showed significantly decreased levels of cellular infiltration, indicating the reduced optic nerve inflammation offered by SMOX inhibition.  We further evaluated the changes in EAE-induced optic nerve inflammation through immunofluorescence staining. Antibodies against Iba1 (Ionized calcium-binding adaptor molecule 1) and F4/80 (EGF-like module-containing mucin-like hormone receptor-like 1) were utilized to study the activation of microglia and macrophages in response to EAE (Figure 7). Resting microglia appear elongated with well-defined processes, and activated microglia show a more compact ameboid appearance in the optic nerve sections [51]. As shown in Figure 7A,B,E,F, the optic nerve sections from the vehicle-treated EAE mice demonstrated upregulation in Iba1 and F4/80 positive cells with an activated morphology compared to the vehicle-treated control group. Treatment with the SMOX inhibitor ameliorated these EAE-induced changes in the optic nerve ( Figure 7C,G). These changes were evaluated by analyzing the fluorescence intensity measurements of Iba1 and F4/80 immunostaining. The quantification of the Iba1 and F4/80 expression levels ( Figure 7I,R) shows a significant increase in the vehicle-treated EAE group compared with the vehicle-treated control mice, and this change was significantly reduced in the EAE group in response to MDL 72527 treatment. the optic nerve sections from the MDL 72527 treated control group did not show any noticeable alterations in the morphology of the Iba1 or F4/80 positive cells compared with the vehicle-treated control nerves. evaluated by analyzing the fluorescence intensity measurements of Iba1 and F4/80 immunostaining. The quantification of the Iba1 and F4/80 expression levels ( Figure 7I,R) shows a significant increase in the vehicle-treated EAE group compared with the vehicletreated control mice, and this change was significantly reduced in the EAE group in response to MDL 72527 treatment. the optic nerve sections from the MDL 72527 treated control group did not show any noticeable alterations in the morphology of the Iba1 or F4/80 positive cells compared with the vehicle-treated control nerves.

Changes in Conjugated Acrolein Levels
Acrolein, a mediator of oxidative damage, is a major downstream effector of SMOX function [52,53]. In the present study, we employed immunofluorescence experiments to investigate the effect of the SMOX inhibitor, MDL 72527, on the changes in levels of conjugated acrolein in the EAE retina. As evident in Figure 8, an elevated level (around 3-fold) of conjugated acrolein was present in the GCL and INL of the EAE retina, while MDL 72527 treatment reduced the EAE-induced upregulation of conjugated acrolein. Quantitative studies on the fluorescence intensity on retinal sections showed a significant upregulation in the retinal samples from the vehicle-treated EAE group, compared to the vehicle-treated controls. This was significantly reduced in response to MDL 72527 treatment in the EAE retinas ( Figure 8I). The presence of nonspecific staining observed in the retinal sections (as shown by arrows) was subtracted during quantification. These results support the involvement of acrolein-induced cellular damage as a potential mechanism of SMOXregulated neurodegeneration in the EAE retina.
tative studies on the fluorescence intensity on retinal sections showed a significant upregulation in the retinal samples from the vehicle-treated EAE group, compared to the vehicle-treated controls. This was significantly reduced in response to MDL 72527 treatment in the EAE retinas ( Figure 8I). The presence of nonspecific staining observed in the retinal sections (as shown by arrows) was subtracted during quantification. These results support the involvement of acrolein-induced cellular damage as a potential mechanism of SMOXregulated neurodegeneration in the EAE retina.

Changes in Signaling Pathways
In the current study, we investigated changes in ERK1/2 and STAT3 signaling pathways in response to EAE induction. A significant increase in p-ERK1/2 was evident in the EAE retina ( Figure 9A). This was accompanied by a significant upregulation of p-STAT3 ( Figure 9B). Treatment with MDL 72527 reduced the level of p-ERK1, while that of p-ERK2 reduction was statistically not significant ( Figure 9C,D). However, the EAE-induced increase in p-STAT3 was significantly reduced by SMOX inhibition ( Figure 9E). No marked changes were observed in the status of p-ERK1/2 and p-STAT3 in the control retinas in response to MDL 72527 treatment.

Changes in Signaling Pathways
In the current study, we investigated changes in ERK1/2 and STAT3 signaling pathways in response to EAE induction. A significant increase in p-ERK1/2 was evident in the EAE retina ( Figure 9A). This was accompanied by a significant upregulation of p-STAT3 ( Figure 9B). Treatment with MDL 72527 reduced the level of p-ERK1, while that of p-ERK2 reduction was statistically not significant ( Figure 9C,D). However, the EAE-induced increase in p-STAT3 was significantly reduced by SMOX inhibition ( Figure 9E). No marked changes were observed in the status of p-ERK1/2 and p-STAT3 in the control retinas in response to MDL 72527 treatment.

Discussion
Vision impairment is one of the earliest clinical presentations of MS [15,54,55]. MSassociated ON is characterized by neurodegeneration and inflammation of the optic nerve and retina [50,56]. Current medications that are available for MS or ON target inflamma-

Discussion
Vision impairment is one of the earliest clinical presentations of MS [15,54,55]. MSassociated ON is characterized by neurodegeneration and inflammation of the optic nerve and retina [50,56]. Current medications that are available for MS or ON target inflammation and are only partially effective [57], providing symptomatic benefits but have not been evaluated for potential long-term effects of the disease. Employing the well-established EAE model of MS, we investigated the impact of MDL 72527, a pharmacological inhibitor of the SMOX pathway, on EAE-induced neurodegeneration and optic nerve inflammation. Our results demonstrate that MDL 72527 treatment protected against EAE-induced RGC loss, the degeneration of synapses, the inflammation of the optic nerve, and visual acuity reduction. To the best of our knowledge, this is the first report demonstrating the impact of SMOX inhibition on EAE-induced retinal neurodegeneration and inflammation, suggesting the potential benefits of SMOX inhibition to treat vision loss in MS patients.
Polyamines are involved in various cellular functions and the regulation of inflammatory responses. Alterations in polyamine metabolism are shown to be involved in many neurological diseases, including MS [58,59]. Several studies have shown that the inhibition of polyamine oxidation is protective against neurodegenerative and inflammatory diseases [46,48,[60][61][62]. MDL 72527 is a common inhibitor of the major polyamine oxidases (SMOX and acetyl polyamine oxidase, APAO) [63]. While APAO is constitutively expressed, SMOX is an inducible enzyme. Recent studies from our laboratory have shown the protective effects of MDL 72527 in the models of DR [38], retinal excitotoxicity [37,39], and oxygen-induced retinopathy [36]. The MOG-induced chronic EAE model has been extensively used by several laboratories to study MS and ON-associated pathologies and mechanisms [23,26,64,65]. Similar to other studies, the EAE-induced motor deficits were evident in our experimental model [23,26,66]. Further, our results demonstrated that SMOX inhibition using MDL 72527 significantly improved the clinical symptoms in EAE mice throughout the disease, with a delayed onset. These results agree with recent studies in which the supplementation of polyamines, spermine, and spermidine significantly reduced EAE-induced motor deficits [58,59]. MDL 72527 is a competitive inhibitor of polyamine oxidases, including SMOX; hence, its treatment normalizes spermine and spermidine in the pathological states. The upregulation of SMOX expression observed in the EAE retina is consistent with our findings in other retinal disease models [35,37,38]. The localization showing an expression of SMOX in the GCL, INL, and OPL in the EAE retinas is similar to what we have previously reported in OIR, diabetic, and excitotoxicity models [35,37,38]. However, in the EAE retina, RGCs showed higher expression of SMOX compared to other retinal neurons.
The loss of RGCs is a major characteristic feature of MS and EAE [20,21,67,68] and is recognized as a major cause of visual dysfunction in MS patients [69,70]. Similar to the findings from other laboratories, a significant reduction in RGCs was observed in the EAE retina in our studies. Interestingly, the EAE-induced RGC loss was reduced by MDL 72527 treatment. The quantification of RGC loss using markers such as NeuN, and Brn3a showed comparable results, which is consistent with the previously published literature on the EAE model [21,71,72]. Our results are also in support of the neuroprotection offered by MDL 72527, similar to other CNS injury models [46][47][48]61]. This was further evidenced by improved synaptic contacts and preservation of axons studied by Tuj1 and Synaptophysin immunostaining and analysis.
Several studies have indicated reduced visual acuity in the rodent EAE model [71,[73][74][75]. Visual acuity changes and RGC dysfunction have also been reported in MS patients [17,76]. Improved visual acuity in EAE mice treated with MDL 72527 is an important sign of the neuroprotective efficacy of SMOX inhibition. It has been reported that the combination of retinal ganglion cell layer plus inner plexiform layer thinning is significantly correlated with both visual function and vision-specific quality of life in MS and may serve as a marker of disease activity [17]. Thus, any influence on the synaptic connections in the IPL, such as the degeneration of RGCs could be functionally important. Inflammation of the optic nerve is a major feature of MS and EAE [21,50,77], and is presented by microglia, the resident macrophages in the CNS, and other immune cells crossing the blood-brain barrier [22]. In the present study, optic nerve inflammation was characterized by increased cellular infiltration and elevated microglia/macrophages in the optic nerve, while SMOX inhibition reduced the EAE-induced optic nerve inflammation. This inflammation may lead to the demyelination of the axons. Damage to RGCs in optic neuritis has been shown to largely be a consequence of optic nerve inflammation and demyelination, leading to axonal destruction and RGC apoptosis [56,78]. Studies from MS patients and EAE models have shown that neuronal death may occur before, and independent of, immune cell infiltration of the optic nerve [79][80][81][82][83]. Various theories have also been suggested in the field of MS concerning inflammation and neuronal damage in progressive MS, such as that MS-associated tissue damage is driven by inflammatory changes, inflammation in MS is secondary to neurodegeneration, and neurodegeneration and inflammation are independent events in MS pathology [84]. Further investigations are needed in this direction and learning the sequence of events will significantly contribute to treatments with more efficacy.
Several mechanisms are involved in the interaction between neurons and immune cells, leading to the disease pathology of MS. In our study, elevated levels of conjugated acrolein, a downstream effector of SMOX signaling were observed. Acrolein is capable of increasing oxidative modifications and elevating the levels of reactive oxygen species [85]. Oxidative stress-induced ROS formation has been recognized as a key component in MS pathology [86][87][88]. Recent studies have documented the role of acrolein in elevating oxidative stress in MS models [89]. Elevated levels of acrolein metabolites were observed in the urine and spinal cord samples of EAE mice [90]. These studies emphasize the crucial involvement of acrolein-mediated oxidative damage in MS pathology. However, the exact mechanisms by which acrolein is elevated in MS tissues are unknown. A recent study from our lab has shown that acrolein treatment induces the activation of microglia/macrophages in vitro [39], a major source of ROS formation in pathological conditions. In the present study, MDL 72527 treatment reduced both conjugated acrolein levels and optic nerve inflammation. Our results show that SMOX and acrolein upregulation primarily occur in retinal neurons. This suggests that the upregulation of SMOX signaling could be at least partially contributing to the inflammation, supporting the role of neurodegeneration in disease pathology. However, further studies are needed to understand how neuronal damage and inflammation are regulated. In this study, we have not investigated the status of inflammatory cells in the retina, myelination, or axonal damage. According to our previous studies, these changes are more evident during the later stages of EAE [49,50]. In our previous studies, using the excitotoxicity and DR models, the increase in acroleinconjugated proteins was reduced by MDL 72527 treatment [38,39].
We and others have demonstrated the activation of ERK signaling and its role in neuroglial damage in the EAE model [49,59,91,92]. In the present study, EAE-mediated ERK activation is downregulated by SMOX inhibition. Further, the activation of STAT3 signaling, a downstream effector of the ERK pathway, is also regulated by MDL 72527 treatment. STAT3 signaling is demonstrated to be a major player in EAE-induced [79] tissue damage [93][94][95][96][97]. The impact of MDL 72527 indicated in the present study supports the potential of SMOX inhibition in reducing inflammation, in addition to neuroprotection. While several studies have documented the neuroprotective effects of SMOX inhibition using MDL 72527, not much information is available regarding its anti-inflammatory effects. Earlier studies from our laboratory have shown that MDL 72527 treatment reduced inflammation in models of ischemic retinopathy and excitotoxicity [36,39]. In the present study, the results show that MDL72527 does not only curb optic nerve inflammation, but in addition, it offers neuroprotection to RGCs as well. A previous study showed a reduction in H. pylori-induced inflammation in response to MDL 72527 treatment [62]. Chronic inflammation induced by Enterotoxigenic Bacteroides fragilis in C57BL/6 mice was also shown to be reduced by MDL 72527 treatment [60]. However, no studies have investigated the impact of SMOX inhibition on inflammation and neuroprotection associated with retinal diseases.
In summary, the present study highlights the impact of the SMOX blockade in reducing clinical symptoms, as well as neurodegeneration, attenuating optic nerve inflammation, and improving visual acuity, in an experimental model of MS. Therefore, targeting SMOX signaling may provide a viable new option for reducing long-term disabilities in MS patients.