Mesenchymal stem cell‐conditioned medium attenuates the retinal pathology in amyloid‐β‐induced rat model of Alzheimer's disease: Underlying mechanisms

Abstract Amyloid‐beta (Aβ) oligomer is known to contribute to the pathophysiology of age‐related macular degeneration. Herein, we aimed to elucidate the in vivo and in vitro effects of Aβ1‐42 application on retinal morphology in rats. Our in vivo studies revealed that intracerebroventricular administration of Aβ1‐42 oligomer caused dysmorphological changes in both retinal ganglion cells and retinal pigment epithelium. In addition, in vitro studies revealed that ARPE‐19 cells following Aβ1‐42 oligomer application had decreased viability along with apoptosis and decreased expression of the tight junction proteins, increased expression of both phosphor‐AKT and phosphor‐GSK3β and decreased expression of both SIRT1 and β‐catenin. Application of conditioned medium (CM) obtained from mesenchymal stem cells (MSC) protected against Aβ1‐42 oligomer‐induced retinal pathology in both rats and ARPE‐19 cells. In order to explore the potential role of peptides secreted from the MSCs, we applied mass spectrometry to compare the peptidomics profiles of the MSC‐CM. Gene ontology enrichment analysis and String analysis were performed to explore the differentially expressed peptides by predicting the functions of their precursor proteins. Bioinformatics analysis showed that 3‐8 out of 155–163 proteins in the MSC‐CM maybe associated with SIRT1/pAKT/pGSK3β/β‐catenin, tight junction proteins, and apoptosis pathway. In particular, the secretomes information on the MSC‐CM may be helpful for the prevention and treatment of retinal pathology in age‐related macular degeneration.


| INTRODUC TI ON
The retina and optic nerve have similar patterns of vascularization and blood-tissue barrier function with brain (Kusne et al., 2017).
Because of these similarities, the retina has been considered to be a source of biomarkers for Alzheimer's disease (AD) (Colligris et al., 2018). Retinal changes in AD include the followings: (a) reduction in the number of retinal ganglion cells (RGC); (b) decreased thickness in the retinal nerve fiber layer (NFL); (c) decreased choroidal thickness in the foveal area; (d) visual field reduction; and (e) accumulation of tau and amyloid-beta (Aβ) (Abulfadl et al., 2018).
Aβ observed in the brain of Alzheimer's disease (AD) patients F I G U R E 1 Learning and memory performance and motor activity for different groups of rats. (a) Retention time (Latency period, second) and (b) the number of errors on the radial-arm maze, the (c) latency (s) and (d) mean velocity (speed, RPM) on rotarod test, and (e) retention latency on passive avoidance were measured in vehicle-treated sham operation rats (Sham+Veh), vehicle-treated Aβ injection rats (Aβ+Veh), normoxia mesenchymal stem cell-conditioned medium treated Aβ injection rats (Aβ+N-CM), and hypoxia mesenchymal stem cell-conditioned medium treated Aβ injection rats (Aβ+H-CM). Data are present mean ± SD. The number of animals used was n = 6 for each experimental group. *p < 0.05 versus the Sham + Veh group; +p < 0.05 versus the Aβ+Veh group F I G U R E 2 Histology of the rat retina (left or right side) using H & E staining. The micrograph shows representative left and right eye from Sham+Veh group (a-f), Aβ+Veh (g-l), Aβ+N-CM (m-r), and Aβ+H-CM (s-x). The black arrow indicates retinal thickness or NFL plus GCL thickness. Values for retinal thickness (y) and nerve fiber and ganglion cell layer (z) are shown. Values are shown as means ± SD (n = 6 of each group). *p < 0.05, compared with the Sham+Veh group; +p < 0.05, compared with the Aβ+Veh group. NFL, nerve fiber layer; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; IS, inner segments; OS, outer segments; RPE, retinal pigment epithelium. Black star (*) indicates the loss of ganglion cells; red star (**) indicates the loss of RPE cells; and hashtag (#) indicates the loss of IS/OS. Scale bar = 20 μm and 50 μm share also been seen in the retinas of patients with retinal neurodegenerative conditions, such as age-related macular degeneration (AMD) the retina has been considered to be a source of biomarkers for AD diagnosis. Dysfunction of retina pigment epithelial (RPE) cells is a significant risk factor for the development of AMD. Drusen or choroidal neovascularization (CNV) is the typical pathological features of AMD. A further experimental study indicated that SIRT1 levels were reduced in human RPE cells after treatment with Aβ, which is one of the constituents of drusen (Cao et al., 2013). Regarding retinal accumulation of Aβ in AD, the update evidence is equivocal. For example, a single intravitreal injection of the oligomeric of Aβ 1-42 has been shown to be toxic to the neural retina of rats (Walsh et al., 2005). Both in vitro and in vivo studies found that Aβ 1-42 reduced mitochondrial redox potential and increased the production of reactive oxygen species, but did not induce apoptosis in RPE cell cultures (Bruban et al., 2009 (Chang et al., 2016). Next sought to determine whether Aβ application caused ARPE-19 cell degeneration and apoptosis. Our study shows that MSCs conditioned medium treatment of intraventricular Aβ 1-42 -microinjected rats could prevent learning and memory deficits and also reduce retinal pathology, which may via the molecular level of SIRT1/pAKT/GSK-3β/β-catenin signaling, TJPs and apoptosis pathways.
2 | RE SULTS 2.1 | Effect of Aβ-induced spatial memory, motor, and learning memory impairment for different groups of rats As shown in Figure 1a, at day 14 to 35 after Aβ 1-42 injection, the vehicle-treated (Aβ+Veh) group performed poorly cognitive functions as indicated by a longer latency period when compared to the non-Aβ control (or sham+Veh group), which ultimately indicates that Aβ 1-42 disrupts the long-term spatial memory. The significant change in the latency period was observed in both rats treated with the con-  and thinner nerve fiber and ganglion cell layers than did Aβ+Veh rats ( Figure 2y).

| N-CM or H-CM inhibits the Aβ-induced degeneration and apoptosis of both retinal ganglion cells (RGC) and retinal pigment epithelial (RPE) cells in rats
We

| N-CM or H-CM attenuate Aβ-induced βcatenin downregulation in RGC and RPE cells in rats
Immunofluorescence staining was performed to evaluate β-catenin expression in response to retinal stress caused by Aβ. The data revealed that β-catenin expression in all layers of the retina in the Sham+Veh group (Supplementary Figure S4). The β-catenin and NeuN co-labeling were evident in the RGC of GCL. Aβ-injection in experimental rats, this expression significantly reduced in Aβ+Veh rats. At week five, after N-CM or H-CM administration, β-catenin was densely expressed in the GCL and RPE layer in Aβ injection rats.

| N-CM or H-CM maintain the tight junction protein expression in ARPE-19 cells following Aβ administration
The alteration we observed in the retinal structure disorganization

| N-CM or H-CM alleviate Aβ-induced cell morphology alterations and decreased viability in ARPE-19 cell
Phase-contrast micrographs of ARPE-19 cells following treatment with Aβ showed that the cell morphology became irregular and

| N-CM or H-CM inhibit the Aβ-induced ARPE-19 injury by activating SIRT/β-catenin signaling pathway
Results from Figure 5e,f demonstrated that Aβ+Veh group rats had significantly lower levels of both sirtuin 1 (SIRT1) and βcatenin than did the Sham+Veh group rats. In contrast, compared to the Sham+Veh group rats, the Aβ+Veh group rats had significantly higher phosphorylation levels of both pAKT and pGSK3β  Table S2). Next, cellular components, biological processes, and molecular functions of the corresponding proteins were determined by GO analysis to predict the latent functions of the differentially expressed peptide (Figure 6a,b). STRING found 8 proteins out of the 22 proteins (components of N-CM) in Supplementary Table S1 interacted with SIRT1/ pAKT/ pGSK3β/ β-catenin signaling, TJPs, and apoptosis pathways (p < 0.05).
Interaction network analysis was performed using STRING (search tool for the retrieval of interacting genes/proteins, http://strin g-db.org/, version 11.0). The STRING interaction network model is shown in Figure 6c, with the known functions of the proteins in Supplementary  Figure 6d, with the known functions of the proteins in Supplementary Table S2. injury (Chou et al., 2013). Glycogen synthase kinase 3β (GSK3β) has been shown to phosphorylate Tau in intact cells which is involved in the AD pathogenesis (Kitagishi et al., 2014a). GSK3β

| DISCUSS ION
is ubiquitously active and is a critical effector of PI3 K/AKT cellular signaling which involved in the cellular process such as cell metabolism, cell death, and tauopathy for AD (Kitagishi et al., 2014a). In epithelial cells, the SIRT1 deacetylation of β-catenin causes the release of β-catenin from the nucleus (Firestein et al., 2008).
In the present results, exposure to Aβ caused a decrease cyto- However, other studies have failed to find differences in RGC density (Curcio & Drucker, 1993) and myelinated axon number (Davies et al., 1995) between AD patients and matched controls.
Aβ deposits similar to those in the brain are not identified in the eyes of AD patients (Ho et al., 2014). A well-validated mouse model of AD revealed that robust expression of the human amyloid precursor protein (APP) transgene in the retina of transgenic mice, but a lack of identified retinal pathology during the period when Aβ deposits were dramatically escalating in the brain (Chidlow et al., 2017). Wistar rats (Dreixler et al., 2014). By spectral counting, compared to unconditioned medium, 19 proteins that met stringent identification criteria were in the conditioned medium obtained from bone marrow stem cells. The majority of those proteins were involved in cell growth and adhesion in an interactional network.
Previous studies have shown that intravitreal delivery of neurotrophic factors slow down photoreceptor degeneration in rodent glaucoma and optic tract trauma model, but the effect was temporary (Abulfadl et al., 2018). In our present study, slow-release neurotroph-  Table   S1 and Supplementary Table S2). GO analysis and STRING analysis found that 8 proteins out of 22 proteins form N-CM and 3 proteins out of 30 proteins from H-CM interacted with SIRT1/pAKT/pGSK3β/ β-catenin signaling, tight junction proteins, and apoptosis pathways.
Bioinformatics analysis showed that these differentially expressed peptides might be associated with the beneficial effects exerted by MSC-CM in treating retinal pathology.
Our present results are consistent with many previous experiments. For example, the key to successful retina regeneration is Müller glia (MG), the primary glial cell type in the retina (Bernardos et al., 2007). In mammals, β-catenin has been associated with MG proliferation (Osakada et al., 2007;Osakada & Takahashi, 2009).
Nuclear factor-kappa B (NF-κB), a key regulator of the inflammatory response is modulated by reversible acetylation of the NF-κB RelA/p65 subunit (Chen et al., 2001). Full transcription activity of RelA/p65 requires acetylation of Lys310, which can be deacetylated by sirtuin (SIRT1) (Yeung et al., 2004). An activation of SIRT1 (e.g., Resveratol) inhibits NF-κB signaling by promoting the deacetylation of Lys310 of RelA/p65 (Chen et al., 2001). Thus, SIRT1 can be a key negative regulator of inflammation cells via inhibition of NF-κB activation. In our present study, Aβ might induce pro-inflammatory cytokine production and blood-retinal barrier disruption in human adult retina pigment epithelium cells by inhibiting SIRT1. Retinal injury, growth factors, and cytokines converge on β-catenin and pStat3 signaling to stimulate retina regeneration (Wan et al., 2014). The phosphoinositide 3-kinase (PI3K)/AKT/ GSK3β pathway has been shown to play a pivotal role in neuroprotection, enhancing cell survival by stimulating cell proliferation and inhibiting apoptosis. This pathway promotes protein hyperphosphorylation in Tau protein (Kitagishi et al., 2014b;Morroni et al., 2016), which is one of AD pathological features. Donepezil a therapeutic acetylcholinesterase inhibitor being used for the treatment of AD. It has been proposed that donepezil prevents glutamate neurotoxicity through the PI3K/AKT/GSK3β signaling (Haraguchi et al., 2017;Kitagishi et al., 2014b). collection, and analysis, the decision to publish, or preparation of the manuscript.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The authors confirm that the data supporting the findings of this study are available within the article and supplementary materials.