Recombinant Integrin β1 Signal Peptide Blocks Gliosis Induced by Aβ Oligomers

Citation: Ortiz-Sanz, C.; Llavero, F.; Zuazo-Ibarra, J.; Balantzategi, U.; Quintela-López, T.; Wyssenbach, A.; Capetillo-Zarate, E.; Matute, C.; Alberdi, E.; Zugaza, J.L. Recombinant Integrin β1 Signal Peptide Blocks Gliosis Induced by AβOligomers. Int. J. Mol. Sci. 2022,23, 5747. https://doi.org/10.3390/ ijms23105747 Academic Editors: Cristina Cereda, Carlo Morasso and Stella Gagliardi Received: 29 April 2022 Accepted: 19 May 2022 Published: 20 May 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). International Journal of Molecular Sciences Article Recombinant Integrin β1 Signal Peptide Blocks Gliosis Induced by AβOligomers Carolina Ortiz-Sanz 1,2, Francisco Llavero 1, Jone Zuazo-Ibarra 1,2, Uxue Balantzategi 1,2, Tania Quintela-López 1,2, Ane Wyssenbach 1,2, Estibaliz Capetillo-Zarate 1,2,3 , Carlos Matute 1,2 , Elena Alberdi 1,2,* and JoséL. Zugaza 1,3,4,* 1Achucarro Basque Center for Neuroscience, Science Park of the UPV/EHU, Sede Building, 3rd Floor, Barrio de Sarriena s/n, 48940 Leioa, Spain; [email protected] (C.O.-S.); [email protected] (F.L.); [email protected] (J.Z.-I.); [email protected] (U.B.); [email protected] (T.Q.-L.); [email protected] (A.W.); [email protected] (E.C.-Z.); [email protected] (C.M.) 2Department of Neurosciences, Faculty of Medicine and Nursery UPV/EHU and CIBERNED, Barrio de Sarriena s/n, 48940 Leioa, Spain 3IKERBASQUE, Basque Foundation for Science, Plaza Euskadi 5, 48009 Bilbao, Spain 4Department of Genetics, Physical Anthropology and Animal Physiology, Faculty of Science and Technology, UPV/EHU, Barrio de Sarriena s/n, 48940 Leioa, Spain *Correspondence: [email protected] (E.A.); [email protected] (J.L.Z.) Abstract: Glial cells participate actively in the early cognitive decline in Alzheimer’s disease (AD) pathology. In fact, recent studies have found molecular and functional abnormalities in astrocytes and microglia in both animal models and brains of patients suffering from this pathology. In this regard, reactive gliosis intimately associated with amyloid plaques has become a pathological hallmark of AD. A recent study from our laboratory reports that astrocyte reactivity is caused by a direct interaction between amyloid beta (A β ) oligomers and integrin β 1. Here, we have generated four recombinant peptides including the extracellular domain of integrin β 1, and evaluated their capacity both to bind in vitro to A β oligomers and to prevent in vivo A β oligomer-induced gliosis and endoplasmic reticulum stress. We have identified the minimal region of integrin β 1 that binds to A β oligomers. This region is called signal peptide and corresponds to the first 20 amino acids of the integrin β 1 N-terminal domain. This recombinant integrin β 1 signal peptide prevented A β oligomer-induced ROS generation in primary astrocyte cultures. Furthermore, we carried out intrahippocampal injection in adult mice of recombinant integrin β 1 signal peptide combined with or without A β oligomers and we evaluated by immunohistochemistry both astrogliosis and microgliosis as well as endoplasmic reticulum stress. The results show that recombinant integrin β 1 signal peptide precluded both astrogliosis and microgliosis and endoplasmic reticulum stress mediated by A β oligomers in vivo . We have developed a molecular tool that blocks the activation of the molecular cascade that mediates gliosis via Aβoligomer/integrin β1 signaling. Keywords: A β oligomers; integrin β 1; interactive region; astrogliosis; microgliosis; interferent peptides 1. Introduction Alzheimer’s disease (AD) is the most common form of dementia and the most prevalent neurodegenerative disease [ 1 ]. Given that the first description made by Alois Alzheimer about pre-senile dementia refers to the formation of senile amyloid plaques and neurofibrillary tangles (aggregates of hyperphosphorylated tau protein) these elements are key pathological hallmarks of AD [2–7]. The formation of neurofibrillary tangles follows wellestablished patterns, while senile plaques appear and distribute in a random manner. The predictable alteration in the pattern and severity of the pathology permits the distinction of initial, intermediate and advanced stages based on investigations carried out by Braak Int. J. Mol. Sci. 2022,23, 5747. https://doi.org/10.3390/ijms23105747 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2022,23, 5747 2 of 15 and Braak [ 7 ]. In addition to plaque distribution, the detection of amyloid β (A β ) as a main constituent of the plaques [ 8 ] and the identification of gene mutations related to A β synthesis in familial AD have led to formulating the amyloid cascade hypothesis [ 9 , 10 ], which postulates that A β deposition in the extracellular space leads to neurodegeneration and subsequent cognitive impairment [ 11 – 13 ]. This hypothesis is not only supported by autosomal-dominant Alzheimer’s disease (ADAD) but also an increase in the copy number of APP (e.g., triplication) is sufficient to cause AD and other amyloidosis [ 14 ]. However, the early CNS inflammation that aggravates the disease starts decades before the onset of AD, and it is characterized by neuronal and microglia-derived cytokines and chemokines, as well as mobilization of microglia toward Aβ-laden neurons [15]. A β peptide oligomers have been isolated from both animal models of AD [ 16 , 17 ] and cerebrospinal fluid (CSF) or brains from AD patients [ 18 ], in whom the presence of this peptide seems to correlate with the progression of the disease [ 19 ]. At nanomolar concentrations, A β oligomers are able to induce neuronal death in hippocampal organotypic slices [ 20 , 21 ], but also to inhibit long-term potentiation [ 21 , 22 ], and to promote abnormal Ca 2+ fluxes as well as cell membrane disruption [ 20 , 23 ]. The biochemical and structural complexity of A β peptides make them very promiscuous molecules able to transduce signals through a repertoire of several receptors and proteins localized at the plasma membrane level both in neurons and in other cell types including glial cells [ 24 , 25 ]. Within the wide variety of effector molecules that interact with A β peptides, integrins have emerged as key molecules in the development of AD [ 25 ] by regulating synaptic dysfunction, diversity of plasticity and long-term potentiation in the early stages of neurodegenerative diseases [ 26 ]. Alpha v integrins mediate A β -induced inhibition of long-term potentiation [ 26 ]. Integrins are a complex family of glycoprotein receptors expressed ubiquitously [ 27 , 28 ]. In turn, they are a class of cellular adhesion molecules with adhesive and signal transduction functions [ 29 , 30 ] that drive to vital cellular events such as cell adhesion, differentiation or migration [ 31 ]. From a structural point of view, integrins are heterodimers constituted by alpha ( α ) and beta ( β ) subunits and bind non-covalently to mediate cell–cell and cell–extracellular matrix interactions. Each integrin recognizes specific ligands, which are either molecules of the extracellular matrix (ECM) (e.g., laminin and fibronectin) or other cell surface counter-receptors of the immunoglobulin superfamily (e.g., intracellular adhesion molecule-1 (ICAM-1)). However, integrins also have functional relationships with other membrane receptors such as ion channels including NMDA receptors and growth factor receptors [32]. During integrin activation, these glycoproteins change configuration from an inactive into an active form (stable extended high-affinity conformation) [ 33 ]. The active form triggers intracellular signaling cascades that are important for relaying information from the external environment to the inside of the cell. One of them is related to clustering between integrin and focal adhesions, leading to the assembly of numerous integrinassociated molecules such as talin, vinculin, paxillin, focal adhesion kinase (FAK), Src and integrin-linked kinase (ILK), that initiate canonical signaling pathways involving small GTPases of the Ras superfamily, ERK, JNK or AKT [ 34 ]. The heterodimers α 2 β 1, α 5 β 1, ανβ 1 and ανβ 3 can facilitate the deposition of A β and induce neurotoxicity, which results in neuronal loss [ 35 – 37 ]. However, the molecular mechanisms by which integrins participate in the development of AD are still unknown. Here, we have mapped the extracellular region of integrin β 1 in order to identify which domain binds to A β oligomers. Using an in vitro binding assay, we have revealed that A β oligomers bind to integrin β 1 signal peptide localized at the first 20 amino acids (aa) at the N-terminal (hereinafter referred as R s ). Application of this recombinant peptide in primary cultures of astrocytes inhibits ROS generation by A β oligomers. Moreover, we have analyzed in vivo the effects of the R s peptide in A β oligomer-mediated astrogliosis and microgliosis, and in endoplasmic reticulum stress by performing intrahippocampal injections in mice. The findings reveal that integrin β 1 signal peptide, R s , prevents gliosis and endoplasmic reticulum stress induced by A β oligomers in mouse hippocampus. Together, Int. J. Mol. Sci. 2022,23, 5747 3 of 15 these data show that R s peptide diminish A β oligomer-induced gliosis by interfering with integrin β1 signaling. 2. Results 2.1. Integrin β1 Signal Peptide Specifically Binds to AβOligomers First, we analyzed the amino acid sequence of integrin β 1 and selected four regions from its extracellular domain. The first region was constituted by the first 20 amino acids (aa) and it was identified as R s , the second one, R w , included up to aa 139, the third region covered aa 1 to 378 including the VWA domain (R d ), and the last region included the whole extracellular domain (from aa 1 to 728, R t ) (Figure 1A). Now, to determine what amino acid stretch could represent an effective binding domain for A β oligomers, four recombinant GST fusion proteins were generated (R s , R d , R w , and R t , fused to the GST protein), and their binding capacities to A β oligomers were determined by affinity chromatography as described in the Experimental procedures section. As shown in Figure 1B, the four fused proteins bound not only to the monomeric A β peptide (the most intense band) but also to the oligomeric forms, the strongest interaction being between the oligomeric forms with the GST-R s recombinant fusion protein (Figure 1B, lane 7). On the other hand, in order to verify that GST protein (GST 0 ) was not involved in the interaction between A β and the fused proteins GST-R s , GST-R d , GST-R w , and GST-R t , we examined this possibility by affinity chromatography. As shown in Figure 1B (lane 2), GST 0 had no ability to bind either monomeric or oligomeric Aβ. Figure 1B (lane 1) represents the reconstitution of synthetic A β (as an internal control) in its different forms visualized by Western blotting. Together, these findings identified that the signal peptide (R s ) of the extracellular domain of integrin β1 was responsible for binding to Aβoligomers in vitro. Int. J. Mol. Sci. 2022, 23, 5747 4 of 15 . Figure 1. Rs peptide, carrying the signal peptide of integrin β1, specifically binds to Aβ peptide and blocks Aβ-induced ROS production in primary astrocyte cultures. (A) Schematic representation of the structure of integrin β1with its different extracellular regions. (B) Interaction of synthetic Aβ peptide with the indicated GST fusion proteins, GST0, GST-Rt, GST-Rd, GST-Rw, and GST-Rt. After incubation, glutathione beads were washed and proteins separated by SDS-PAGE under non-reducing conditions and analyzed by Western blot using anti-Aβ1–42 antibody (6E10, from Covance). (C) ROS generation was measured by fluorimetry with 10 µM CM-H2DCFDA. Data are expressed as the relative fluorescence normalized to values of untreated or treated cells (100%). *** p < 0.001 compared to non-treated cells; # p < 0.05 compared to GST0; unpaired one-way ANOVA. Figure 1. Cont. Int. J. Mol. Sci. 2022,23, 5747 4 of 15 Int. J. Mol. Sci. 2022, 23, 5747 4 of 15 . Figure 1. Rs peptide, carrying the signal peptide of integrin β1, specifically binds to Aβ peptide and blocks Aβ-induced ROS production in primary astrocyte cultures. (A) Schematic representation of the structure of integrin β1with its different extracellular regions. (B) Interaction of synthetic Aβ peptide with the indicated GST fusion proteins, GST0, GST-Rt, GST-Rd, GST-Rw, and GST-Rt. After incubation, glutathione beads were washed and proteins separated by SDS-PAGE under non-reducing conditions and analyzed by Western blot using anti-Aβ1–42 antibody (6E10, from Covance). (C) ROS generation was measured by fluorimetry with 10 µM CM-H2DCFDA. Data are expressed as the relative fluorescence normalized to values of untreated or treated cells (100%). *** p < 0.001 compared to non-treated cells; # p < 0.05 compared to GST0; unpaired one-way ANOVA. Figure 1. R s peptide, carrying the signal peptide of integrin β 1, specifically binds to A β peptide and blocks A β -induced ROS production in primary astrocyte cultures. ( A ) Schematic representation of the structure of integrin β 1with its different extracellular regions. ( B ) Interaction of synthetic A β peptide with the indicated GST fusion proteins, GST 0 , GST-R t , GST-R d , GST-R w , and GST-R t . After incubation, glutathione beads were washed and proteins separated by SDS-PAGE under nonreducing conditions and analyzed by Western blot using anti-A β 1–42 antibody (6E10, from Covance). ( C ) ROS generation was measured by fluorimetry with 10 µ M CM-H2DCFDA. Data are expressed as the relative fluorescence normalized to values of untreated or treated cells (100%). *** p< 0.001 compared to non-treated cells; # p< 0.05 compared to GST0; unpaired one-way ANOVA. 2.2. RsPeptide Blocks AβOligomer-Induced ROS Generation in Cultured Astrocytes Next, we investigated whether GST-R s affected ROS generation mediated by A β oligomers in primary astrocyte cultures, as previously shown [ 38 ]. For that, we treated primary astrocyte cultures with 5 µ M A β oligomers for 60 min alone or together with 5 µ g/ µ L GST 0 (control) or 5 µ g/ µ L GST-R s , and measured ROS levels by fluorimetry using 10 µ M CM-H2DCFDA for 20 min. As expected, A β oligomers induced ROS generation (Figure 1C, empty bar). Regarding GST 0 , this peptide did not interfere in A β oligomermediated ROS generation (Figure 1C, gray bar). Nevertheless, GST-R s totally prevented ROS generation mediated by A β oligomers (Figure 1C, solid bar). Taken together, these results show that integrin β 1 signal peptide (R s ) binds in vitro to A β oligomers, and that it is able to prevent ROS generation induced by A β oligomers in primary astrocyte cultures. 2.3. AβOligomers Trigger Gliosis in Mouse Hippocampus In Vivo A β injection in mouse brain causes reactive astrogliosis in the dentate gyrus (DG) [ 38 ]. However, it is still unclear whether A β injection in mice brain also drives microgliosis. To investigate that possibility, we performed intrahippocampal injections of vehicle (control) or A β oligomers (A β ) and examined astrocyteand microglia-occupied areas by immunohistochemistry with astrocyte (GFAP and S100 β ) and microglia (Iba1) markers in dentate gyrus (DG). As expected, the intrahippocampal administration of A β strongly increased the presence of both the GFAP and S100 β markers compared to control (Figure 2A). In addition, A β also boosted the presence of the Iba1 marker in DG compared to control (Figure 2A). Quantification of the immunohistochemical analysis showed significant increases in the GFAP, S100 β and Iba1 markers in DG values due to A β treatment compared to control (Figure 2B, 1.00 ± 0.04 vs. 1.21 ± 0.04 for GFAP, 1.00 ± 0.04 vs. 1.46 ± 0.08 for S100 β 1.00 ± 0.07 vs. 1.31 ± 0.08 for Iba1). These results confirm that A β induces astrogliosis and show that Aβoligomers also lead to microgliosis in adult mouse DG. Int. J. Mol. Sci. 2022,23, 5747 5 of 15 Int. J. Mol. Sci. 2022, 23, 5747 5 of 15 2.3. Aβ Oligomers Trigger Gliosis in Mouse Hippocampus In Vivo Aβ injection in mouse brain causes reactive astrogliosis in the dentate gyrus (DG) [38]. However, it is still unclear whether Aβ injection in mice brain also drives microgliosis. To investigate that possibility, we performed intrahippocampal injections of vehicle (control) or Aβ oligomers (Aβ) and examined astrocyteand microglia-occupied areas by immunohistochemistry with astrocyte (GFAP and S100β) and microglia (Iba1) markers in dentate gyrus (DG). As expected, the intrahippocampal administration of Aβ strongly increased the presence of both the GFAP and S100β markers compared to control (Figure 2A). In addition, Aβ also boosted the presence of the Iba1 marker in DG compared to control (Figure 2A). Quantification of the immunohistochemical analysis showed significant increases in the GFAP, S100β and Iba1 markers in DG values due to Aβ treatment compared to control (Figure 2B, 1.00 ± 0.04 vs. 1.21 ± 0.04 for GFAP, 1.00 ± 0.04 vs. 1.46 ± 0.08 for S100β 1.00 ± 0.07 vs. 1.31 ± 0.08 for Iba1). These results confirm that Aβ induces astrogliosis and show that Aβ oligomers also lead to microgliosis in adult mouse DG. Figure 2. Reactive astrocytes and microglia in the dentate gyrus (DG) of Aβ-injected mice. (A) Coronal sections of mouse brains were immunostained by DAB assay 7 days post -injection with Aβ or with vehicle (Ctrl). Photomicrographs show GFAP and S100β immunolabeling in astrocytes and Iba1 immunolabeling in microglia of the dentate gyrus. Scale bar: 100 µm and Scale bar in zoom is 50 µm. It is included in caption.Inset: 50 µm. (B) Box plot graphs show quantitative analysis of laFigure 2. Reactive astrocytes and microglia in the dentate gyrus (DG) of A β -injected mice. ( A ) Coronal sections of mouse brains were immunostained by DAB assay 7 days post -injection with A β or with vehicle (Ctrl). Photomicrographs show GFAP and S100 β immunolabeling in astrocytes and Iba1 immunolabeling in microglia of the dentate gyrus. Scale bar: 100 µ m and Scale bar in zoom is 50 µ m. It is included in caption.Inset: 50 µ m. ( B ) Box plot graphs show quantitative analysis of labelled areas for GFAP, S100 β and Iba1 under A β and control conditions in the DG. Data are presented as the mean ± S.E.M. Fifteen slices from five animals were analyzed per condition. *** p< 0.001, ** p< 0.01, *p< 0.05 compared with Aβ-injected mice; unpaired Student’s test. 2.4. RsPeptide Prevents Glia Reactivity in the DG of AβOligomer-Injected Mice Brain Before examining the functionality of the GST-R s fused protein in vivo , we evaluated whether GST 0 affected astrocyte and microglia reactivity in A β oligomer-injected brain. For that, we performed intrahippocampal injections of A β and A β with GST 0 (A β + GST 0 ) and quantified the changes in astrocyte and microglia morphology as described in the previous section. As shown in Figure 3A, the intrahippocampal administration of the combination of A β + GST 0 did not modify the area occupied by both the GFAP and S100 β markers compared to A β alone. However, the area occupied by Iba1 staining appeared increased in the combination A β + GST 0 when it was compared to A β alone (Figure 3A). Quantification of the immunohistochemical analysis showed that GST 0 in the presence Int. J. Mol. Sci. 2022,23, 5747 6 of 15 of A β did not produce any significant change in GFAP and S100 β staining (Figure 3B; 0.94 ±0.09 vs. 0.64 ±0.03 for GFAP, 0.93 ±0.08 vs. 0.67 ±0.06 for S100β, whereas it caused microgliosis as compared to A β alone (1.00 ± 0.04 vs. 0.77 ± 0.03 for Iba1). These results suggest that the GST 0 protein did not reduce A β -dependent astrogliosis and/or microgliosis. Int. J. Mol. Sci. 2022, 23, 5747 6 of 15 belled areas for GFAP, S100β and Iba1 under Aβ and control conditions in the DG. Data are presented as the mean ± S.E.M. Fifteen slices from five animals were analyzed per condition. *** p < 0.001, **p < 0.01, * p < 0.05 compared with Aβ-injected mice; unpaired Student’s test. 2.4. Rs Peptide Prevents Glia Reactivity in the DG of Aβ Oligomer-Injected Mice Brain Before examining the functionality of the GST-Rs fused protein in vivo, we evaluated whether GST0 affected astrocyte and microglia reactivity in Aβ oligomer-injected brain. For that, we performed intrahippocampal injections of Aβ and Aβ with GST0 (Aβ + GST0) and quantified the changes in astrocyte and microglia morphology as described in the previous section. As shown in Figure 3A, the intrahippocampal administration of the combination of Aβ + GST0 did not modify the area occupied by both the GFAP and S100β markers compared to Aβ alone. However, the area occupied by Iba1 staining appeared increased in the combination Aβ + GST0 when it was compared to Aβ alone (Figure 3A). Quantification of the immunohistochemical analysis showed that GST0 in the presence of Aβ did not produce any significant change in GFAP and S100β staining (Figure 3B; 0.94 ± 0.09 vs. 0.64 ± 0.03 for GFAP, 0.93 ± 0.08 vs. 0.67 ± 0.06 for S100β, whereas it caused microgliosis as compared to Aβ alone (1.00 ± 0.04 vs. 0.77 ± 0.03 for Iba1). These results suggest that the GST0 protein did not reduce Aβ-dependent astrogliosis and/or microgliosis. Figure 3. GST0 polypeptide is ineffective in preventing gliosis in the DG of Aβ-injected mice. (A) Coronal sections of mouse brains were immunostained by DAB assay 7 days post-injection with Aβ and Aβ + GST0. Photomicrographs show GFAP and S100β immunolabeling in astrocytes and Iba1 immunolabeling in microglia of the dentate gyrus. Scale bar: 100 µm and Scale bar in zoom is 50 µm. It is included in caption: 50 µm. (B) Box plot graphs show quantitative analysis of labelled areas Figure 3. GST 0 polypeptide is ineffective in preventing gliosis in the DG of A β -injected mice. ( A ) Coronal sections of mouse brains were immunostained by DAB assay 7 days post-injection with A β and A β + GST 0 . Photomicrographs show GFAP and S100 β immunolabeling in astrocytes and Iba1 immunolabeling in microglia of the dentate gyrus. Scale bar: 100 µ m and Scale bar in zoom is 50 µ m. It is included in caption: 50 µ m. ( B ) Box plot graphs show quantitative analysis of labelled areas for GFAP, S100 β and Iba1 under A β and A β + GST 0 in the DG. Data are presented as the mean ± S.E.M. Fifteen slices from five animals were analyzed per condition. ns: non-significant; ** p< 0.01 compared with Aβ-injected mice; unpaired Student’s test. Based on that, we examined the ability of recombinant GST-R s peptide to prevent Aβ-mediated astrogliosis in brain. For that, we performed intrahippocampal injections of A β , and A β with GST-R s peptide (A β + GST-R s ) and the glial changes were analyzed and quantified. As shown in Figure 4A, the intrahippocampal administration of the combination of Aβ+ GST-Rsstrongly reduced the presence of three—GFAP, S100βand Iba1—markers compared to Aβ. Int. J. Mol. Sci. 2022,23, 5747 7 of 15 Int. J. Mol. Sci. 2022, 23, 5747 7 of 15 for GFAP, S100β and Iba1 under Aβ and Aβ + GST0 in the DG. Data are presented as the mean ± S.E.M. Fifteen slices from five animals were analyzed per condition. ns: non-significant; ** p < 0.01 compared with Aβ-injected mice; unpaired Student’s test. Based on that, we examined the ability of recombinant GST-Rs peptide to prevent Aβmediated astrogliosis in brain. For that, we performed intrahippocampal injections of Aβ, and Aβ with GST-Rs peptide (Aβ + GST-Rs) and the glial changes were analyzed and quantified. As shown in Figure 4A, the intrahippocampal administration of the combination of Aβ + GST-Rs strongly reduced the presence of three—GFAP, S100β and Iba1—markers compared to Aβ. Quantification of the immunohistochemical analysis showed a significant decrease in GFAP, S100β and Iba 1 (Figure 4B) in the presence of Aβ + GST-Rs compared to Aβ (1.05 ± 0.10 vs. 1.30 ± 0.05 for GFAP, 1.03 ± 0.08 vs. 1.484 ± 0.167 for S100β 1.00 ± 0.05 vs. 1.22 ± 0.04 for Iba1). These results point out that integrin β1 signal peptide Rs blocks Aβ-induced not only astrogliosis but also in microgliosis in adult mouse DG. Figure 4. GST-Rs polypeptide prevents gliosis in the DG of Aβ-injected mice. (A) Coronal sections of mouse brains were immunostained by DAB assay 7 days post-injection with Aβ or Aβ + GST-Rs. Photomicrographs show GFAP and S100β immunolabeling in astrocytes and Iba1 immunolabeling in microglia of the dentate gyrus. Scale bar: 100 µm and Scale bar in zoom is 50 µm. It is included in caption. 50 µm (B) Box plot graphs show quantitative analysis of labelled areas for GFAP, S100β and Iba1 under Aβ and Aβ + GST-Rs in the DG. Data are presented as the mean ± S.E.M. Fifteen slices from five animals were analyzed per condition. ** p < 0.01 compared with Aβ-injected mice; unpaired Student’s test. Figure 4. GST-R s polypeptide prevents gliosis in the DG of A β -injected mice. ( A ) Coronal sections of mouse brains were immunostained by DAB assay 7 days post-injection with A β or A β + GST-Rs. Photomicrographs show GFAP and S100 β immunolabeling in astrocytes and Iba1 immunolabeling in microglia of the dentate gyrus. Scale bar: 100 µ m and Scale bar in zoom is 50 µ m. It is included in caption. 50 µ m ( B ) Box plot graphs show quantitative analysis of labelled areas for GFAP, S100 β and Iba1 under A β and A β + GST-R s in the DG. Data are presented as the mean ± S.E.M. Fifteen slices from five animals were analyzed per condition. ** p< 0.01 compared with A β -injected mice; unpaired Student’s test. Quantification of the immunohistochemical analysis showed a significant decrease in GFAP, S100 β and Iba 1 (Figure 4B) in the presence of A β + GST-R s compared to A β (1.05 ± 0.10 vs. 1.30 ± 0.05 for GFAP, 1.03 ± 0.08 vs. 1.484 ± 0.167 for S100 β 1.00 ±0.05 vs. 1.22 ±0.04 for Iba1). These results point out that integrin β 1 signal peptide Rsblocks Aβ-induced not only astrogliosis but also in microgliosis in adult mouse DG. 2.5. RsPeptide Reduces Endoplasmic Reticulum Stress in Astrocytes in DG of Aβ Oligomer-Injected Mice Brain Acute injection of A β oligomers in mouse brain induces GRP78 chaperone protein overexpression particularly in astrocytes [ 39 ], being used as an endoplasmic reticulum stress marker. Therefore, we investigated whether recombinant R s fused protein to GST (GST-R s ) could also prevent endoplasmic reticulum stress in astrocytes after intrahippocampal A β injection. Accordingly, we carried out a double immunostaining assay for S100 β and GRP78 of brain tissues previously injected with A β , A β + GST-R s and A β + GST 0 . Intrahippocampal administration of the combination of recombinant GST-R s peptide and A β oligomers strongly reduced GRP78 expression in S100 β -positive astrocytes compared Int. J. Mol. Sci. 2022,23, 5747 8 of 15 to A β oligomers alone (Figure 5A,B). Furthermore, the combination of GST 0 and A β oligomers did not alter the effect induced by A β alone (Figure 5B). Quantification of immunofluorescence staining showed a significant decrease in GRP78 in S100 β values in DG from brains injected with GST-R s fusion protein compared to control (A β -injected mice) (26.95 ±1.01 vs. 30.64 ±1.24) (Figure 5A). In contrast, GST 0 protein did not produce any effect in A β -induced endoplasmic reticulum stress in S100 β (Figure 5B) values compared to A β alone (21.42 ±2.48 vs. 22.07 ±1.36) . These findings suggest that R s also prevents endoplasmic reticulum stress induced by Aβoligomers. Int. J. Mol. Sci. 2022, 23, 5747 8 of 15 2.5. Rs Peptide Reduces Endoplasmic Reticulum Stress in Astrocytes in DG of Aβ OligomerInjected Mice Brain Acute injection of Aβ oligomers in mouse brain induces GRP78 chaperone protein overexpression particularly in astrocytes [39], being used as an endoplasmic reticulum stress marker. Therefore, we investigated whether recombinant Rs fused protein to GST (GST-Rs) could also prevent endoplasmic reticulum stress in astrocytes after intrahippocampal Aβ injection. Accordingly, we carried out a double immunostaining assay for S100β and GRP78 of brain tissues previously injected with Aβ, Aβ + GST-Rs and Aβ + GST0. Intrahippocampal administration of the combination of recombinant GST-Rs peptide and Aβ oligomers strongly reduced GRP78 expression in S100β-positive astrocytes compared to Aβ oligomers alone (Figure 5A,B). Furthermore, the combination of GST0 and Aβ oligomers did not alter the effect induced by Aβ alone (Figure 5B). Quantification of immunofluorescence staining showed a significant decrease in GRP78 in S100β values in DG from brains injected with GST-Rs fusion protein compared to control (Aβ-injected mice) (26.95 ± 1.01 vs. 30.64 ± 1.24) (Figure 5A). In contrast, GST0 protein did not produce any effect in Aβ-induced endoplasmic reticulum stress in S100β (Figure 5B) values compared to Aβ alone (21.42 ± 2.48 vs. 22.07 ± 1.36). These findings suggest that Rs also prevents endoplasmic reticulum stress induced by Aβ oligomers. Figure 5. GST-R s polypeptide reduces GRP78 expression in S100 β -positive astrocytes of A β -injected mouse brains. Photomicrographs of double immunofluorescence staining for S100 β (red) and GRP78 (green) on DG of animals injected with different: A β and A β + GST-R s ( A ) or A β and A β + GST 0 ( B ). Quantitative analysis of fluorescence intensity was performed for GRP78 levels in S100 β -positive astrocytes in dentate gyrus after A β and A β + GST-Rs ( A ) or A β and A β + GST 0 ( B ). Scale bar in zoom area: 20 µ m. Data are presented as the mean ± SEM. Fifteen slices from five animals were analyzed per condition. ns: non-significant; * p< 0.05 compared with A β -injected mouse; unpaired Student’s test. Int. J. Mol. Sci. 2022,23, 5747 9 of 15 3. Discussion Our study identifies the integrin β 1 minimal region that binds to A β oligomers. This region spans from aa 1 to aa 20 and corresponds to integrin β 1 signal peptide (R s peptide). From a functional point of view, this peptide is a very useful tool to block A β oligomer-induced ROS generation in primary astrocyte cultures and also in vivo when R s peptide in combination with A β oligomers is directly injected into the mice hippocampus. In this scenario, astroglial stress, astrogliosis and even microgliosis induced by Aβoligomers are efficiently prevented. Several investigations postulate that there are many potential receptors localized at neuronal synapses with both high affinity for A β peptide and the ability to intracellularly transduce the toxic instructions emanating from A β oligomers [ 40 ]. These include NMDA receptors that are directly activated by A β oligomers, altering its physiological function [ 41 ], although those that seem to be acquiring increasing relevance are integrins. In fact, the interaction between integrins and A β oligomers promotes neurotoxicity, inhibition of LTP and an increase in spine density [ 26 , 42 ]. In this regard, synthetic A β monomer binds through its amino acid sequence RHDS to the α 2b β 3 integrin, being directly related to cerebral amyloid angiopathy, which contributes to dementia and AD [43]. Integrins control important cellular responses including proliferation, survival and cell migration [ 44 ]. All of them require the active participation of transducing molecules such as tyrosine kinases FAK, ILK and Src or small GTPases of the Rho family [ 44 ]. In addition, PKCs may also be involved in integrin-mediated signaling [ 45 ]. We have previously observed that A β oligomer-induced PKC phosphorylation is mediated by integrin β 1 in astrocytes and in neurons [ 38 ]. Further, A β oligomers lead to NR2B subunit upregulation on neuronal membranes through the PKC signaling pathway [ 46 ]. Under these circumstances, integrin β 1 transduces the message that A β oligomers brings, generating a cellular response which manifests itself in a higher permeability for calcium ions to alter cellular homeostasis [ 46 ]. Hence, depending on the stimulus or ligands, the same receptor along with its intracellular signaling molecules can switch on/off different pathways that lead to antagonistic cellular responses. Currently, in addition to pharmacotherapy, gene therapeutic approaches for AD have entered phase I/II clinical trials [ 47 ]. The results of this preliminary study obtained with recombinant R s allow us to postulate a new pharmacological therapeutic alternative in AD. This recombinant peptide neutralizes A β oligomer activity from outside the cell (Figure 6panel B compared to panel A). In addition, Rs recombinant peptide is a useful tool that will aid understanding the molecular mechanisms of the deleterious actions initiated by Aβoligomers both in vitro and in vivo.