To determine if rG4s form in the human brain, we used the well-characterized single-chain BG4 anti- quadruplex antibody (Sigma-Aldrich MABE917, antibody null controls in Figure 1—figure supplement 2) to stain human hippocampal postmortem tissue of different ages under conditions that strongly favor cytoplasmic rG4 identification using 4% paraformaldehyde fixation in cells (Laguerre et al., 2016; Mestre-Fos et al., 2020). The tissues were obtained in collaboration with the brain bank at the Medical University of South Carolina. The study included 22 cases, male and female, of different ages and with or without a clinical or neuropathological diagnosis of AD (see Figure 1b). All cases had received a neuropathological examination including assessment of Braak and Consortium to Establish a Registry for Alzheimer’s Disease (CERAD) stages according to the updated NIA criteria except in those cases in which no pathology was noted (Braak and Braak, 1995; Jack et al., 2012).

First, the rG4 staining profiles were compared between different ages, with an age range of 30–92 years of age (Figure 1c). We discovered a significant age-related increase in rG4 immunostaining, with the densest staining observed in older individuals (Figure 1c and d). The largest difference in staining between young and older cases was observed in the outer molecular layer (OML) and in the hilar region, CA4 (see Figure 1c, legend arrows). The strongest overall staining for BG4 was found in the dentate gyrus granular cell layer (DG), but with less observable age-related increases. Staining density measurements confirmed these findings and demonstrated a highly significant increase in BG4 staining in the oldest cases studied within the OML (Figure 1d) and the CA4 regions of cornu ammonis (Figure 2—figure supplement 2a), confirming increased intracellular BG4 accumulation as a function of age. Plotting BG4 percent area versus age confirmed the observed findings and showed correlation (Spearman R = 0.49) with high significance (p=0.038 in the OML region (Figure 1d). Positive and significant Pearson but not Spearman correlations were observed between age and BG4 area in the CA4 region, Figure 2—figure supplement 2a), and nonsignificant correlations were observed in the dentate granule cell layer. No significant correlation was found with BG4 stain and postmortem interval (PMI) (Spearman R = –0.32, p=0.2 see Figure 1—figure supplement 3). In total, these data show that rG4s increased as a function of age in the human brain. We observed multiple outliers in both older and younger individuals due to Braak stage. For example, an 85-year-old individual with Braak stage 0 or a 59-year-old individual with Braak stage 5.5 were outliers as a function of age. This led us to analyze whether rG4 formation increased with Braak stage, a measure of AD severity.

Next, we examined whether the formation of rG4s was associated with AD neuropathology. Braak staging is a neuropathological staging method that takes into account the density of NFTs both within each brain region and also from region to region (Trejo-Lopez et al., 2022), with higher numerical values representing greater AD severity (Braak and Braak, 1995). Like in the case of aging, increased BG4 staining was readily apparent in AD cases, especially in those cases associated with a higher Braak stage (Figure 2a). In the OML, plotting BG4 percent area versus Braak stage demonstrated a strong correlation (Spearman R = 0.72) with highly significantly increased BG4 staining with higher Braak stages (p=0.00086) (Figure 2b). Positive significant correlations (R = 0.52, p=0.028) between Braak stage severity and BG4 staining were also observed within the CA4 region (Figure 2—figure supplement 2). Interestingly, the OML region is highly involved in AD pathology and is a hippocampal region where AD pathology is often observed, (Dong et al., 2007), suggesting that the presence of rG4s in this particular brain region might have significance for amyloid as well as NFT formation during the AD disease process. In addition, a prominent loss of synapses in AD is found in the OML, suggesting a potential close connection with layer 2 of the entorhinal cortex (Lassmann et al., 1993).

If rG4 formation is more prevalent in AD than in non-AD cases and directly involved in AD pathology, we would also predict that rG4 formation would be more prevalent in APOE genotypes that are predisposed for higher LOAD risk, that is, patients with one or two copies of the APOE4 genotype. Indeed, there was a significant (t-probability 0.002) increase in BG4 staining levels in the OML in postmortem cases with one or more APOE4 allele compared to those with only APOE2 or APOE3 alleles (Figure 2c). This result, although correlative, is consistent with a previous study performed in vitro in which the RNA sequences found in aggregates in different APOE genotype cell lines were different (Shmookler Reis et al., 2021).

Confocal microscopy images showed that a large amount of the BG4 immunostaining was localized in the cytoplasm near the nucleus in a punctate pattern, reminiscent of known rG4 formation in vitro and in cell lines in rRNA ribosomal extensions (Mestre-Fos et al., 2020). We therefore also stained with an anti-rRNA antibody to determine whether this was the case in human tissue as well. As expected, the stains overlapped (Figure 3b, Figure 3—figure supplement 1), showing that at least a large percentage of the observed BG4 signal arose from rG4s and not nuclear DNA G4s. This result also confirms that previously observed rG4 formation in ribosomes (Mestre-Fos et al., 2020) also occurs in human brain tissue.

To further explore the formation of rG4s in the human hippocampus, we examined the staining pattern in different cell types based on specific antibody markers. Co-staining with markers for the most common brain cell types, rG4s were clearly abundant in neurons, oligodendrocytes, and astrocytes (Figure 3c). rG4s exhibited no apparent colocalization with microglia (Figure 3c). This pattern is especially noticeable in the double labeling with SOX10 (Figure 3c), which shows the nuclear oligodendrocyte marker SOX10 (Turnescu et al., 2018) staining in proximity to the cytoplasmic BG4 immunostaining.

We co-stained BG4 with different p-Tau epitope antibodies to determine whether pathological aggregation of Tau proteins co-occurred with BG4 immunostaining (Figure 4). Indeed, we found that BG4 immunostaining co-localized with p-Tau immunostaining in neurons located in the CA1–4 regions of the hippocampus (Figure 4). We used two different antibodies directed against the serine 396 (S396) and the threonine 231 (T231) phosphorylation sites to investigate co-localization with different p-Tau epitopes. Quantifying the percentage of cells co-stained with BG4 and p-Tau revealed that 85% of the S396 p-Tau-positive cells also had positive BG4 staining, and 82% of the T231 p-Tau-positive cells also had positive BG4 staining.

This work was approved by both the University of Colorado and Medical University of South Carolina Institutional Review Boards (IRB), and the study was performed in accordance with the ethical standards of the 1964 Declaration of Helsinki and its later amendments. The University of Colorado IRB protocol was approved in January 2022 (#21-4648) and was reviewed by the UCD Panel D COMIRB review committee. The Medical University of South Carolina IRB protocol was approved in August 2019 (#00079529) and was reviewed by the IRB I review committee. Based on these reviews, this project met the Not Human Research criteria set forth by the Code of Federal Regulations (45CFR46). Since the use of postmortem brain tissue for research is exempt according to federal IRB regulations and does not require a full IRB review and approval, each laboratory that participated in the research of human brain tissue had a valid non-human research (NHR) protocol.

The next of kin, in the absence of actual contrary indications by the decedent, gave the decedent’s brain and associated structures for educational and research purposes. The next of kin signed a postmortem consent form and the original of this postmortem consent form is kept in a locked drawer and the identities are never revealed to researchers studying the brain tissue and associated biofluids. All efforts were made to consider diversity, equity, and inclusion in the selection of brain tissues for this study. Figure 1b shows the demographics for the 21 cases used for this study. The tissue was sliced in 1 cm coronal slices and subjected to free-floating fixation in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 72 hours. The fixed tissue was then transferred to a 30% sucrose in phosphate buffer (PB), followed by transfer to a cryoprotection solution in PBS (30% glycerol, 30% ethylene glycol, and 40% PO₄ buffer) and stored at –20°C until dissection. Anatomical blocks from 19 brain regions were dissected from the 1 cm slabs, embedded in paraffin, and cut at a thickness of 5 µm on a Microm Microtome (Thermo Fisher Scientific Inc, Waltham, MA) and stained for routine neuropathological staging according to the NIA-AA Revised Criteria for Diagnosis and Staging of Alzheimer’s Disease (Hyman et al., 2012).

Paraffin-embedded sections were deparaffinized in xylene (RPI, Cat#111056) and rehydrated using wash steps at 100%, 95%, 70%, and 50% ethanol followed by distilled water. Heat-induced antigen retrieval was performed using 0.05% citraconic acid at pH 7.2 for 20 minutes in a steamer. Slides were then washed with a saline solution buffered with tris(hydroxymethyl) aminomethane (Tris, TBS) and blocked with 0.25% triton-X and 5% normal serum for 1 hour. After blocking, slides were rinsed again with TBS and incubated with 1X True Black Lipofuscin Autofluorescence Quencher (Biotium Cat#23007) diluted in 70% ethanol. Slides were washed three times with TBS then incubated with primary antibodies listed in Supplementary file 1 overnight, followed by goat anti-mouse AlexaFluor 555 (Thermo Scientific #A32727) or Donkey anti-rabbit Alexafluor 488 (Invitrogen #A-21206) at 1:500 dilution for 1 hour. Slides were then washed three times with TBS and cover-slipped using mounting media containing DAPI (Thermo Scientific, Cat#P36931). Staining controls included omission of primary or secondary antibodies (Figure 1—figure supplement 2). In Figure 3b, we performed sequential double labeling, first with BG4, and then with rRNA antibody at 1:250 (Abcam #ab17119).

In addition, we examined whether Braak stage correlated with aging itself (Figure 2—figure supplement 1). Although not significant at the 0.05 level, there was a weak correlation between Braak and Age. Braak staging is a paradigm used to describe density and regional spread of NFTs in the Alzheimer brain and includes levels 0-VI. CERAD is a staging paradigm for AD that takes into account amyloid plaque formation (Trejo-Lopez et al., 2022).

Slides were imaged at high magnification using an Olympus confocal laser scanning microscope equipped with FlowView FV3000 software (Olympus, Tokyo, Japan) with 16-bit grayscale with 65,000 shades of grey at ×10, ×20, and ×60 magnification at wavelengths 355, 488, and 555 m. Images used for quantification were taken at constant microscope and intensity settings. All image quantification was performed using Fiji ImageJ. Quantification was performed by a researcher who was blinded to the neuropathological diagnosis of individual cases and with no knowledge of Braak stage or age for the case being quantified. Supplementary file 2 presents side-by-side comparisons of quantification from two researchers who were both blinded to group diagnosis and individual cases. The data used in the paper are highlighted. One data point with too much variation between quantifications was removed to increase rigor. Background was subtracted using a rolling ball radius. Images were then divided into three regions of interests (ROIs); OML, DG, and CA4 (Figure 1—figure supplement 3). Images were submitted to an autothreshold and particles analyzed to get a percent area measurement of BG4 staining. These were graphed and linearly fit using Kaleidagraph using Pearson fit. Spearman and Pearson R and p-values are reported for 18 cases for age correlation and 18 cases for Braak correlation. An unpaired t-test with unequal variance was performed for the two groups E2/E3 and E3/E3 versus E3/E4 and E4/E4. When a single outlier based on the singular African American case was removed from the APOE quantification, the t-probability remained highly significant at 0.0045 versus 0.002. All quantification was performed on ×10 images obtained from an Olympus microscope.

CD spectra were obtained using a Jasco J-1100 circular dichroism at 25°C. Sequence was resuspended in 10 mM potassium phosphate pH 7.5 buffer and diluted to 12.5 µM (per strand) RNA. The CD measurement was taken from 300 nm to 210 nm at 1 nm intervals using a 50 nm/min scanning speed. The sequence used was 5'-CGGGCGGCGGGGGGGCCCGGGCGGCGGGGGGGCCCGGGCG-3' (Zwierzchowski-Zarate et al., 2022). Parallel structure was determined using previously described quantification methods for quadruplex topology (Cheng et al., 2018).