Emerging evidence indicates that brain expression of some OR and TAS2R genes is modulated in neurological diseases 8–11. In AD, for instance, a small subset of mRNAs was found to be deregulated (either up- or down-regulated) in human entorhinal cortex and frontal cortex area 8, albeit exclusively in limbic and isocortical stages where many confounding factors might be in play. In frontal cortex area 8, specifically, only OR4F4 and OR52L1 were found to be up-regulated, and no alteration was observed in TAS2Rs mRNA expression at any stage 10.
In our study, we sought to explore the possible expression and regulation of selected OR and TASR mRNAs in human OFC (areas 10, 11 and 47), a polymodal region of the frontal cortex with great anatomical connectivity to sensory areas and limbic structures involved in emotion and memory. The human genome contains an extensive number of protein-coding OR and TAS2R genes, thus the present study was not intended to provide a comprehensive list of deregulated mRNAs. We found OFC expression of some OR and TAS2R mRNAs both in male and female samples, most at comparable levels, and expression of a subset of sexually dimorphic OR and TAS2R mRNAs. These findings indicate sex-specific vulnerability of certain OR and TAS2R genes. Although steroid hormones such as androstenone and androstadienone have been identified as ORs ligands 36, it is unlikely that steroid hormones or their derivatives play a major role in modulation of these genes sex-specific vulnerability in this particular setting, considering the frequent age- and AD-related decline in circulating and brain levels of sex steroid hormones 37. Thus, at least a subset of these chemoreceptor genes might be modulated by distinct sexually-dimorphic ligands or signals.
On the other hand, we were not able to detect expression of type 1 taste receptors (TAS1R1, TAS1R2, TAS1R3) mRNAs in OFC specimens by RT-qPCR. Although Tas1r expression was described in rodent brain 14,15, and cDNA microarray and RNA sequencing data supported the idea of TAS1R expression in multiple human brain regions, particularly in limbic system areas 38, no study thus far has validated the expression of TAS1R mRNAs in any region of the human brain, which overall indicates that humans most likely lack TAS1Rs expression in OFC. Notwithstanding, considering their proposed involvement in the maintenance of glucose homeostasis in mouse hypothalamic cells 15, and that impaired cerebral glucose metabolism is an invariant pathological feature of AD, comprehensive assessment of TAS1R expression in human brain would be an important topic for future studies.
Subsequently, we found that ORs and TAS2Rs mRNA expression levels are markedly downregulated in AD specimens, both in male and female samples, particularly in its most incipient stage, Braak stage I, and in most cases also in advanced stages. These findings indicate that transcripts deregulation does not follow a disease progression pattern, but rather points toward a regulation mechanism that may play a direct role in incipient AD. Also, these alterations do not appear to be associated with neuronal loss or to be a mere outcome of OFC neurodegeneration, as the appearance of AD-associated neuropathology in the OFC is preceded by a decline in these transcripts mRNA levels at incipient stages wherein neurofibrillary tangle involvement is still limited to trans-entorhinal and entorhinal cortices. Furthermore, in some cases their expression levels seem to be restored at advanced stages wherein neurofibrillary tangles are at that point extensive in the neocortical regions.
In contrast to previous observations, wherein OR and TAS2R expression was mainly found to be upregulated or unchanged in entorhinal and frontal cortex, and alterations were limited to more advanced stages of AD 10, it is puzzling to remark that in our study, we found downregulation in OFC already at early stages. Although dissimilarities can be attributed in part to different ORs being analyzed, and therefore to receptor-specific vulnerability, two common TAS2Rs (TAS2R5, TAS2R14) were investigated, indicating a possible cerebral cortex region-specific vulnerability to ORs and TAS2Rs transcriptional changes.
Beyond cerebral OR and TA2SR mRNAs expression deregulation (manifested either as up- or down-regulation) documented in neurological disorders 8–11, there is limited knowledge regarding their involvement on physiological or pathological processes in neuronal systems. Nevertheless, the characterization of their physiological roles in other non-chemosensory tissues and their involvement in similar pathological settings suggest that these chemoreceptors might have a unique functionality in these systems.
It has been shown that TAS2Rs act as immune sentinels, mobilizing defense mechanisms against pathogenic aggression. For instance, these receptors are stimulated in the presence of gram-negative quorum-sensing molecules, leading to increased nitric oxide production and mucociliary clearance activation during upper airways inflammation 39. In contrast, recent evidence has shown that downregulation of key components of the taste signaling cascade is associated with increased production of pro-inflammatory mediators and oxidative stress molecules in diabetic nephropathy 40, which initiate inflammatory responses similar to those observed in neurological diseases. In this sense, although there is no evidence so far on the involvement of TAS2Rs in neuroinflammatory responses, it is plausible that the downregulation of cerebral TAS2R mRNAs, previous to the appearance of AD-associated neuropathology in the OFC, could be associated with a possible deficiency on the response to toxigenic substances/neurotoxins and microbial components, which under normal conditions could be necessary to prevent their pathogenic action in the cerebral microenvironment, and consequently necessary to prevent neuroinflammatory reactions. This might represent an important etiopathogenetic mechanism, notwithstanding additional comprehensive studies are required to understand the potential molecular mechanisms connecting TAS2Rs-mediated surveillance of the extracellular milieu and neuroinflammation.
Reports on the potential ORs involvement on neuropathological processes have also started to emerge. Remarkably, it has been shown that the activation by acetophenone of overexpressed human OR4M1 in mouse primary cortico-hippocampal neurons could lead to attenuation of abnormal microtubule-associated tau protein phosphorylation via a JNK signaling pathway 41. These findings suggest that ORs may interfere with aberrant tau hyperphosphorylation, which is one of the pathological hallmarks of AD, and that ligand-induced activation of ORs might result in protection against tau neuropathological features. Therefore, it is plausible that decreased expression of OR transcripts, as documented in our study, might be associated with AD-related tauopathy. Nonetheless, additional studies are required to dissect in detail the involvement of cerebral ORs in the attenuation of tau neuropathology.
On the other hand, the transcriptional control mechanisms responsible for OR and TAS2R genes regulation in humans still remains elusive. In mouse olfactory epithelium, repression of Olfr genes has been associated with H3K9me3 and another inhibitory histone methylation mark, H4K20me3 18. The presence of H3K27me3, another inhibitory histone methylation mark, over gene promoters has also been highly correlated with gene repression. Yet, it has been shown that promoters marked by H3K27me3 remain accessible to binding by general transcription factors and paused RNA polymerase 42,43, while chromatin marked by H3K9me3, instead, occludes transcription factors binding with diverse DNA-binding domains 44.
Despite the assumption that the mechanisms regulating OR expression outside of the olfactory epithelium could be markedly different, it is enigmatic whether this heterochromatic silencing mechanism is implemented also in human tissues in physiological or pathological settings.
Therefore, we sought to investigate the potential alterations on H3K9me3 in human OFC in sporadic AD. Remarkably, we found a significant increase of H3K9me3 global levels at both early and advanced stages of sporadic AD. These findings suggest that a gain of specific histone methyltransferases or loss of specific histone demethylases function might be mediating the upsurge of H3K9me3 global levels observed at early stages of the disease and could be associated with the OR and TAS2Rs genes repression. This epigenetic alteration can also be associated with the repression of a number of AD-related genes, such as neuronal activity-related genes, Aβ clearance or production machinery genes, and tau-related genes, which makes the scenario a subject of profound interest. Thus far, the study of the potential alterations on H3K9 methylation in AD has been inconclusive as only a few studies have addressed this question. Increased global H3K9me2 levels were reported in human occipital and prefrontal (area 10) cortex 45,46 and H3K9me3 in temporal cortex 47, whereas reduced H3K9me2 levels were observed in hippocampal neurons 48,49. Likewise, the potential alterations on H3K9 methylation at gene-specific locus in AD has only been explored in a familial AD mouse model, wherein H3K9me2 enrichment was found at glutamate-receptor genes promoter in prefrontal cortex 46. Interestingly, integrated analysis of genome-wide ChIP- and mRNA-sequencing data showed H3K9me3 promoter occupancy in synaptic function-related genes in human temporal cortex in advanced stages of AD 47.
Based on the increase of H3K9me3 global levels we observed in OFC and the effects that H3K9me3 may entail depending on the specific genomic loci, we interrogated both H3K9me3-mediated transcriptional repression and H3K9me3-mediated alternative splicing at the proximal promoter and the coding region of the candidate OR and TAS2R genes. We focused on possible alterations at early stages in which changes cannot be attributed to AD-derived neuronal loss in comparison with possible changes occurring with disease progression. We found a prominent enrichment of the H3K9me3 repressive mark in the proximal promoter of the target chemoreceptor genes at Braak stage I, which suggests that an H3K9me3- mediated mechanism could be responsible for ORs and TAS2Rs transcriptional control in this brain region. In addition, these findings raise the possibility that this silencing mechanism might also be implemented in other non-chemosensory tissues and organs and in other physiological and pathological contexts. On the other hand, at later stages, we found a pronounced reduction of the H3K9me3 repressive mark in both promoter and coding regions, suggesting that the inhibitory function associated with H3K9me3 is not required at these sites and that an alternative repression mechanism might be involved at advanced stages.
There are, however, some hindrances in understanding the transcriptional control of these genes expression due to the lack of a complete characterization of OR promoters and transcription start sites (TSS). The analysis of each proximal promoter regulatory region was based on a consensus localization of transcription factor binding sites clustered at a distance of 100 to 300 bp from the TSS reported on the upstream regions of a number of human OR genes 50. We cannot exclude that transcription regulation might be controlled by different regulatory sites or the presence of different promoters for the selected OR and TAS2R genes.
Afterwards, we inquired the potential differences in the H3K9me3 interactome in OFC between early and advanced stages by mass spectrometry-based proteomics, and the analysis pinpointed MeCP2 as a candidate interactor of H3K9me3 in Braak stage I. In fact, in addition to methylated DNA, MeCP2 has been shown to interact with specific histone methylation marks. MeCP2 has been shown to bind to H3K9me2 and H3K27me3 nucleosomes in mouse brain nuclear extracts 33, and was found to be associated with H3K9me2 in the IL-6 gene upstream region in pancreatic adenocarcinoma cell lines 34. Recently, it has also been shown that MeCP2 can regulate gene expression through recognition of H3K27me3 and that MeCP2-H3K27me3 interaction is independent of DNA methylation 35.
We then sought to investigate the potential alterations on MeCP2 protein levels in the human OFC and we found a pronounced increase in this protein expression already at early stages of the disease. Alterations in the homeostatic levels of this protein at this early stage could have important functional consequences for AD pathogenesis. Further validation of H3K9me3-MeCP2 interaction at early stages of AD by reciprocal co-IPs puts forwards this epigenetic repressive mechanism as an early event in AD that could be implicated in the transcriptional regulation of OR and TAS2R genes and other AD-related genes.
Validation of H3K9me3-MeCP2 interaction was just the first step in elucidating this mechanism, notwithstanding, tissue amount requirements and differences in antibody efficiency have hindered further analysis on the co-enrichment of these two chromatin-associated proteins on the selected OR and TAS2R proximal promoters by sequential ChIP.
In summary, our findings indicate that the upsurge in H3K9me3 and MeCP2 proteins and epigenetic repression of chemoreceptor genes are early events in AD pathogenesis, as these alterations seem to precede more conventional pathological features such as amyloid plaque formation and tauopathy. It is possible that OR and TAS2R genes and other AD-related genes become inactive through H3K9me3 engagement in early stages of AD, and as the disease progresses, the reversible relaxation of H3K9me3-enriched chromatin may be compromised and lead to the constitutive silencing of these genes. Therefore, it will be necessary to further investigate the mechanisms responsible for H3K9me3 increase in early stages of AD and whether H3K9me3-enriched chromatin and H3K9me3-landscaped genes can be reversibly modulated.