RIG-1 receptor expression in the pathology of Alzheimer’s disease

The study was approved by the University of Miami Miller School of Medicine institutional review board. Written informed consent for research and brain autopsy was obtained for all subjects in this study.

Neuropathologic specimens (3 millimeters) of fresh-frozen human temporal (BA38) and occipital cortex (BA17) were obtained from the University of Miami Brain Endowment Bank™. The temporopolar cortex (BA38) was sampled from frozen tissue blocks at the level of the fundus of the temporopolar sulcus. The occipital cortex was sampled from the primary visual cortex (BA17). Postmortem specimens were selected from age-matched subjects with no cognitive impairment (NCI), MCI, and from AD patients. The diagnosis of AD was made using standard diagnostic criteria [15]. Subjects with NCI, MCI, and AD were selected based on their antemortem clinical dementia rating (CDR) score one year prior to death and postmortem pathologic evaluation for AD pathology and Braak stage. Neuropathologic diagnosis was based on NIA-Regan criteria recommendations of the Consortium to Establish a Registry for AD (CERAD) [16] and Braak staging of neurofibrillary tangles [17]. The diagnosis of MCI included assessment of normal activities of daily living, normal general cognitive function, abnormal memory for age, and no dementia [17]. MCI patients met neuropathologic criteria for possible to probable AD and Braak stages I to IV [17]. AD cases selected for this study included patients with a diagnosis of clinical dementia and definite AD on postmortem examination (Braak stages V or VI; Table 1).

All plasma and serum samples were obtained from the University of Kentucky Alzheimer’s Disease Center Brain Bank. The samples were obtained from patients diagnosed postmortem as either age-matched controls with no cognitive impairment (NCI; Braak stage (0 to I), MCI (Braak stages II to IV), or AD (Braak stages V to VI). The section of the study included six age-matched controls (NCI; Braak stages 0 to I), seven MCI patients with possible AD, determined by pathological evidence of neurofibrillary tangles and senile plaques (Braak stages II to IV), and ten patients who met clinical diagnostic criteria for definite AD (Braak stages V to VI; Table 2).

To prevent interference of immunoglobulin G (IgG) during immunoblot analysis of plasma and serum, IgG was isolated using a Pierce Albumin/IgG Removal kit (Thermo Scientific Waltham, MA, USA) according to manufacturer’s instructions.

Occipital and temporal cortices were homogenized in lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM pyrophosphate, 1 mM β-glycerophosphate) with protease inhibitor cocktail (Sigma). Twenty five micrograms of protein per sample were resolved in 10 to 20% Tris-HCl Criterion precasted gels (Bio-Rad, Hercules, CA, USA), transferred to polyvinylidene difluoride membranes (Applied Biosystems Waltham, MA, USA) and placed in blocking buffer (PBS, 0.1% Tween-20, 0.4% I-Block (Applied Biosystems Waltham, MA, USA) and then incubated for one hour with an antibody against RIG-1 (Anaspec Fremont, CA, USA) at a dilution of 1:1,000. To authenticate the presumptive bands shown in Figures 1 and 2, a RIG-1 positive control sample (Novus Biologicals Littleton, CO, USA) was used. Immunoabsorption is more appropriate to demonstrate the authenticity of the bands. Membranes were incubated for one hour with primary antibodies followed by appropriate secondary horseradish peroxidase (HRP)-linked antibodies (Cell Signaling Danvers, MA, USA). Visualization of signal was enhanced by chemiluminescence using a Phototope-HRP detection kit (Cell Signaling Danvers, MA, USA). To control for protein loading, immunoblots were stripped with Restore, Western blot stripping buffer (Pierce Rockford, IL, USA) and blotted for β-actin using monoclonal anti-β-actin antibody (1:8,000, Sigma St. Louis, MO, USA). Quantification of band density was performed using the UN-SCAN-IT gel software, and data were normalized to β-actin. For immunoblotting of serum and plasma 5 μg of protein were loaded equally across all samples used to keep data normalized.

Human astrocytes were grown in culture as described in de Rivero Vaccari et al. in 2012 [10]. Primary human astrocytes (Lonza Basel, Switzerland) were grown in culture in complete Astrocyte Growth Medium (Lonza Basel, Switzerland) for seven days. RIG-1 signaling was stimulated with 5′ triphosphate double-stranded RNA (5′ppp dsRNA, Invivogen San Diego, CA, USA) as a specific ligand to stimulate RIG-1 signaling at different concentrations (2 and 4 μg/ml) for 18 hours. After stimulation, cells were harvested and immunoblotted for RIG-1 (Anaspec Fremont, CA, USA), phosphorylated IRF3 (Novus Biologicals Littleton, CO, USA), amyloid precursor protein (Abcam Cambridge, MA, USA) and amyloid-β (Epitomics Burlingame, CA, USA) expression as described.

Human astrocytes were grown in culture for seven days and stimulated with 3-42 amyloid-β (Anaspec Fremont, CA USA) at a concentration of 0.5, 1 and 3 μM for 18 hours. Then cells were harvested and immunoblotted for expression of caspase-1 (Imgenex San Diego, CA, USA) and RIG-1 (Anaspec Fremont, CA USA) as described.

The primary outcome measures were levels of immune proteins in two brain regions. The demographic, clinical and neuropathological characteristics were used to group assignment. Association between individual protein measures and age, gender or postmortem interval were explored in multivariate analyses to ensure that the results were unchanged. Statistical comparisons between groups were made using one-way ANOVA and one-tailed Student’s t-test. The level of statistical significance was set at * P < 0.05.

The demographic and neuropathology characteristics of the cohort used in this section of the study are summarized in Table 1. The study included 22 age-matched controls (NCI), 20 MCI patients with pathologic evidence of senile plaques and neurofibrillary tangles consistent with possible or probable AD (Braak stages I to IV), and 23 patients who met clinical diagnostic criteria for AD and definite pathologic evidence (Braak V to VI). Immunoblot analysis of temporal cortical samples revealed an increase in RIG-1 expression in the MCI group when compared to the NCI and AD groups (Figure 1B). In contrast, the levels of RIG-1 in the occipital cortex were higher in the AD group than in the NCI and MCI groups (Figure 1C). Thus, these results show for the first time that RIG-1 is increased in the temporopolar cortex of MCI patients.

To determine the levels of RIG-1 in the plasma and serum of patients with MCI associated with AD, immunoglobulin G was isolated from serum and plasma obtained from patients corresponding to the NCI, MCI and AD groups, as described above. Figure 2 shows that RIG-1 was significantly increased in the plasma (Figure 2B) from MCI patients compared to the NCI and AD groups, whereas the levels of RIG-1 in serum (Figure 2C) did not differ among the three groups. Thus, these results show for the first time that RIG-1 is increased in the plasma of MCI patients.

3-42 Aβ species have been shown to be the most prevalent form of Aβ peptides present in early and later stages of human AD amyloid pathology [18]. Since we found that levels of RIG-1 expression are elevated in the temporal cortex from MCI patients when compared to end-stage AD pathology (AD, Figures 1 and 2), we stimulated human cortical astrocytes with 3-42 Aβ for 18 hours at different concentrations (C, 0.5, 1 and 3 μM) to determine if Aβ peptide levels regulate the protein expression levels of RIG-1. Interestingly, there was a concentration dependent effect of 3-42 Aβ on the expression of RIG-1. At 0.5 μM treatment, the RIG-1 levels did not change when compared to the control/untreated group, whereas at 1 μM, the levels of RIG-1 increased, and at 3 μM, the protein levels of RIG-1 returned to basal/control levels (Figure 3). Importantly, no morphological or toxic changes were noticed in the cultured astrocytes at the concentrations of 3-42 Aβ used for 18 hours (data not shown). Thus, it appears that Aβ may be involved in regulating the levels of the RIG-1 protein.

5′ppp dsRNA has been shown to be a specific ligand of RIG-1 signaling activation [19]. To determine whether 5′ppp dsRNA is responsible for the activation of RIG-1 in primary human cortical astrocytes, 5′ppp dsRNA was administered to primary astrocytes in culture for 18 hours at two different concentrations (2 and 4 μg/ml). As shown in Figure 4B and 4C, RIG-1 and phospho-interferon regulatory factor 3 (P-IRF3), respectively, were significantly elevated after the administration of 4 μg/ml of 5′ppp dsRNA, thus indicating RIG-1 signaling activation.

To identify if RIG-1 signaling stimulation is involved in the pathogenesis of AD, astrocytes were stimulated with the RIG-1 signaling agonist 5′ppp dsRNA (4 μg/ml) for 18 hours. Samples were then resolved by immunoblotting using antibodies against two hallmark proteins of AD, APP (Figure 4E) and Aβ (Figure 4F). Stimulation of RIG-1 with 4 μg/ml 5′ppp dsRNA, which activates RIG-1 signaling in astrocytes, resulted in a significant elevation in the expression of APP and Aβ when compared to the control group, suggesting an involvement of RIG-1 signaling in the expression of two hallmark proteins in AD pathology.