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. 2023 Apr 21:16:1175364.
doi: 10.3389/fnmol.2023.1175364. eCollection 2023.

Endoplasmic reticulum stress and ubiquitin-proteasome system impairment in natural scrapie

Jenny Lozada Ortiz  1 , Marina Betancor  1 , Sonia Pérez Lázaro  1 , Rosa Bolea  1 , Juan J Badiola  1 , Alicia Otero  1
Affiliations

Endoplasmic reticulum stress and ubiquitin-proteasome system impairment in natural scrapie

Jenny Lozada Ortiz et al. Front Mol Neurosci. .

Abstract

Chronic accumulation of misfolded proteins such as PrPSc can alter the endoplasmic reticulum homeostasis triggering the unfolded protein response (UPR). In this pathogenic event, the molecular chaperones play an important role. Several reports in humans and animals have suggested that neurodegeneration is related to endoplasmic reticulum stress in diseases caused by the accumulation of misfolded proteins. In this study, we investigated the expression of three endoplasmic reticulum stress markers: PERK (protein kinase R-like endoplasmic reticulum kinase), BiP (binding immunoglobulin protein), and PDI (Protein Disulfide Isomerase). In addition, we evaluated the accumulation of ubiquitin as a marker for protein degradation mediated by the proteasome. These proteins were studied in brain tissues of sheep affected by scrapie in clinical and preclinical stages of the disease. Results were compared with those observed in healthy controls. Scrapie-infected sheep showed significant higher levels of PERK, BiP/Grp78 and PDI than healthy animals. As we observed before in models of spontaneous prion disease, PDI was the most altered ER stress marker between scrapie-infected and healthy sheep. Significantly increased intraneuronal and neuropil ubiquitinated deposits were observed in certain brain areas in scrapie-affected animals compared to controls. Our results suggest that the neuropathological and neuroinflammatory phenomena that develop in prion diseases cause endoplasmic reticulum stress in brain cells triggering the UPR. In addition, the significantly higher accumulation of ubiquitin aggregates in scrapie-affected animals suggests an impairment of the ubiquitin-proteasome system in natural scrapie. Therefore, these proteins may contribute as biomarkers and/or therapeutic targets for prion diseases.

Keywords: endoplasmic reticulum stress; prion; prion diseases; scrapie; ubiquitin-proteasome system.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
PRK-like Endoplasmic Reticulum Kinase (PERK) expression in clinical and preclinical scrapie-infected sheep and healthy sheep. (A) Immunohistochemical detection of PERK in thalamus. PERK-positive immunostaining was detected in cellular nuclei from clinical, preclinical and control sheep. A higher number of immunopositive cells is observed in the clinical group. (B) Expression levels of protein PERK in eight clinical, five preclinical and eight control animals evaluated using the following semi-quantitative scoring system a rating of 0 (lack of immunostaining) to 5 (very intense immunostaining) in nine brain areas: [spinal cord (Sc), medulla oblongata (Mo), cerebellum (Cbl), hypothalamus (Ht), thalamus (Th), parietal cortex (Pc), basal ganglia (BG), cortex at the level of basal ganglia (BGc), hippocampus (Hc) and frontal cortex (Fc)]. Clinical sheep showed the highest scores in the hippocampus, thalamus, hypothalamus and the cortex at the level of the thalamus followed by preclinical animals with similar scores in the same areas. Comparison of means was analyzed using the nonparametric Mann–Whitney U test (*p < 0.05, **p < 0.01, Mann–Whitney U test) (Figure 1B). (C) Gene expression of EIF2AK3 in frontal cortex, thalamus, hippocampus and medulla oblongata of clinical, preclinical, and control sheep. SDHA and GAPDH were used as housekeeping genes. Results are expressed as the mean ± standard deviation. The expression values were determined using the 2−∆∆Ct method, and differences between experimental groups were analyzed using the one-way ANOVA test, followed by the Bonferroni post hoc test. No significant differences were found.
Figure 2
Figure 2
Binding immunoglobulin protein (BiP) expression in clinical and preclinical scrapie-infected sheep and control sheep. (A) Immunohistochemical detection of BiP in clinical, preclinical and control groups. Representative images correspond to the thalamus. Intense deposits of BiP protein were observed in the cytoplasm of neurons in clinical and preclinical sheep. (B) Expression levels of BiP in eight clinical, five preclinical and eight control animals were evaluated using the following semi-quantitative scoring system a rating of 0 (lack of immunostaining) to 5 (very intense immunostaining) in nine brain areas: spinal cord (Sc), medulla oblongata (Mo), cerebellum (Cbl), hypothalamus (Ht), thalamus (T), parietal cortex (Pc), basal ganglia (BG), cortex at the level of basal ganglia (BGc), hippocampus (Hc) and frontal cortex (Fc). Clinical sheep showed significantly higher deposition of BiP when compared with preclinical and control animals (*p < 0.05, **p < 0.01, Mann–Whitney U test). (C) mRNA expression profiles of the HSPA5 gene in frontal cortex, thalamus, hippocampus and medulla oblongata of clinical, preclinical, and control sheep. Relative expression levels are expressed as the mean ± standard deviation. The results were normalized using the expression of SDHA and GAPDH housekeeping genes. The expression values were determined using the 2−∆∆Ct method, and differences between experimental groups were assessed using the one-way ANOVA test followed by the Bonferroni post hoc test (*p < 0.05).
Figure 3
Figure 3
Protein disulfide isomerase (PDI) expression in clinical and preclinical scrapie-infected sheep and control sheep. (A) A strong intraneuronal labeling of PDI was observed in the medulla oblongata of clinical animals [pictures show the dorsal motor nucleus of the vagus nerve (DMNX)]. (B) PDI protein expression levels are more intense in clinical sheep when compared to controls in most brain areas. Expression levels of PDI protein in eight clinical, five preclinical and eight control animals were semi quantitatively evaluated using a scale of 0 (lack of immunostaining) to 5 (very intense immunostaining) in nine brain areas: [spinal cord (Sc), medulla oblongata (Mo), cerebellum (Cbl), hypothalamus (Ht), thalamus (Th), parietal cortex (Pc), basal ganglia (BG), cortex at the level of basal ganglia (BGc), hippocampus (Hc) and frontal cortex (Fc)]. Clinical sheep showed the highest levels of PDI in almost every brain area evaluated (*p < 0.05, **p < 0.01, Mann–Whitney U test). (C) Gene expression of P4HB in frontal cortex, thalamus, hippocampus and medulla oblongata of clinical, preclinical, and control sheep. The results were normalized using the expression of SDHA and GAPDH housekeeping genes. The expression values were determined using the 2−∆∆Ct method. Mean scores between experimental groups were assessed using the one-way ANOVA test followed by the Bonferroni post hoc test (*p < 0.05).
Figure 4
Figure 4
Ubiquitin accumulation in clinical and preclinical scrapie-infected sheep and healthy sheep. (A) Ubiquitin-protein intraneuronal aggregates are observed in the hippocampus. The CA1-CA2 regions of the hippocampus showed the strongest immunostaining for ubiquitin in clinical and preclinical sheep. (B) Expression levels of ubiquitin protein in eight clinical, five preclinical and eight control animals were semiquantitatively evaluated using a scale of 0 (lack of immunostaining) to 5 (very intense immunostaining) in nine brain areas: [spinal cord (Sc), medulla oblongata (Mo), cerebellum (Cbl), hypothalamus (Ht), thalamus (Th), parietal cortex (Pc), basal ganglia (BG), cortex at the level of basal ganglia (BGc), hippocampus (Hc) and frontal cortex (Fc)]. Comparison of Ubiquitin immunolabeling revealed significant differences between clinical and control sheep in several brain areas. Preclinical animals showed higher levels of ubiquitin accumulation in the frontal cortex, thalamus (**p < 0.01) medulla oblongata and hippocampus (*p < 0.05) when compared to controls. Clinical and preclinical animals showed significant differences in the basal ganglia area. Comparison of means was analyzed using the nonparametric Mann–Whitney U test.
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