Different MAPT haplotypes influence expression of total MAPT in postmortem brain tissue

Several prior studies have tried to understand the mechanisms underlying the association of the MAPT H1/H1 haplotype as a major risk factor for several neurodegenerative diseases, including sporadic PD. So far, studies evaluating the MAPT expression in the human brain focused on either healthy controls with regard to MAPT haplotypes [34,35,36,37] or investigated PD and control cases without consideration of haplotypes [14]. To our knowledge, the present study is the first investigation of MAPT regional involvement on gene and protein expression in sporadic PD under the paradigm of different homozygous MAPT haplotypes in human brains. Since sporadic PD is classified as synucleinopathy according to its main pathology, we investigated the mRNA and protein levels of the synucleins in the same context. We used ctx-fg as pathological region and ctx-cbl as reference from control brains and PD. Due to the strict selection process, the study group size was rather small, but the included brains were well qualified by their tissue integrity, the severity of PD pathology, and absence of significant concomitant AD pathology.

Our findings support the concept that α-syn pathology in PD does not show up at the transcriptional level or in the soluble protein compartment but is discernible primarily in the insoluble protein compartment. Similar to the study of Quinn et al. [59] we could demonstrate differences on the protein level despite unchanged α-syn mRNA levels. Levels of insoluble α-syn were increased in PD compared to controls independent of the MAPT haplotype. This result was observed only in the ctx-fg, but not in ctx-cbl which supports the value of the former as a relevant region of interest for PD. Our findings confirm results of prior postmortem brain studies in which higher levels of insoluble α-syn protein fractions were found in PD compared to controls, while soluble levels of α-syn did not differ, as observed in the amygdala and anterior cingulate gyrus [60], putamen and frontal cortex [61] and in striatum and inferior frontal gyrus [18]. It is worth noting that some prior studies reported a decrease of soluble α-syn levels in PD in cerebellum and occipital cortex [62, 63], yet, no prior data exist on the α-syn protein level in the same protein fractions and brain regions as studied here, making a direct comparison difficult.

Similar to α-syn, tau pathology in PD was also not detectable at the transcriptional level or in the soluble proteome. However, in the insoluble protein compartment, 0N3R and 1N4R tau isoforms were significantly increased in the fusiform gyrus of PD cases. These isoform-specific findings need to be independently reproduced and understood pathophysiologically. Despite various functions, interactions and the potentially crucial role in tau pathology, little is known about the effect of each single exon within the N-terminal domain [64,65,66]. While for the number of repeats in the C-terminal domain, it is known that compared to 3R tau, 4R tau regulates microtubule dynamics more efficiently, binds with greater affinity to microtubule [67] and is more prone to aggregation [68].

For insoluble total tau, a tendency towards higher levels in PD and H1/H1 was apparent, which did not reach statistical significance due to the small sample size. In contrast to the study of Lei et al., which reported a 44% decrease of soluble levels of tau (phosphate-buffered-saline-soluble) in the substantia nigra in PD compared to controls [69], we did not find significant differences for soluble tau protein. This difference in result may be due to the fact that the SN in PD is strongly degenerated in contrast to the cortex of gyrus fusiformis. Very few studies exist on tau quantity in postmortem human PD brain, however, differences in methodology might explain variable results.

In our study design which allowed a simultanous mRNA and protein extraction from the very same tissue sample, haplotype-specific differences observed at MAPT mRNA level were not reflected in total tau protein expression. Only 1N4R tau isoform of insoluble tau showed a trend towards increased expression in the H1/H1 haplotype, but this did not reach the level of statistical significance. Modulating factors involved in alternative splicing of MAPT might contribute to the observed discrepancy between MAPT mRNA and corresponding tau protein level. The recent work of Bowles et al. 2022 highlighted the role of splicing regulating factors focusing on N-terminal tau splicing. They described differentially altered expression of certain splicing regulating factors and RNA binding proteins by single nuclei gene expression in disease-relevant brain regions of AD and PSP brains compared to controls [3]. Although there are currently no data in PD, such modulating factors might also contribute to the observed discrepancy between MAPT RNA and tau protein levels. This might explain the brain region-specific differences in the effects of MAPT haplotypes at the protein level and subsequently PD pathogenesis.

As expected, the MAPT haplotype, located on chromosome 17, did not influence mRNA expression of SNCA, encoded on chromosome 4. The predisposition to PD by the MAPT haplotype did not seem to manifest itself at the transcriptional level of the synuclein genes. The lack of influence of disease on expression levels of synuclein genes SNCA, SNCB and SNCG is in good agreement with other postmortem studies. SNCA mRNA levels were reported to be increased only in substantia nigra in PD, [40, 70, 71], but not differ in cerebellum or temporal cortex [40, 59] in comparison to controls. To our knowledge, the expression of SNCB and SNCG in postmortem tissue had so far not been examined in the context of PD, as to our knowledge.

In line with the literature, we found a statistically significant increase of mRNA expression of total MAPT in donors with the H1/H1 haplotype [72]. Despite the trend towards higher MAPT expression in the cerebellum of H1/H1 MAPT haplotype, statistically significant elevated total MAPT mRNA levels were only detected in the ctx-fg only, a brain region affected by α-syn pathology. Our results are in agreement with previous studies describing the same association between homozygous MAPT haplotypes in frontal cortex and cerebellum of healthy donors [34]. This result was also described in prefrontal cortex of PD cases [73] not stratified for the MAPT haplotype. However, there are also studies which could not detect haplotype-specific differences in total MAPT expression in healthy human brains. These contradictory findings could result from analysis of groups with heterozygous MAPT haplotype status [35] or from different brain regions being examined, since MAPT was found to be differently expressed across brain regions [37].

Some studies suggest that the MAPT haplotype affects more differential splicing and thus the expression of certain MAPT transcripts rather than the overall gene expression [35, 37]. Evidence for this has been provided by postmortem studies of healthy individuals with elevated levels of 4R MAPT [36], but lower levels of 2N MAPT transcripts for H1/H1 compared to H2/H2 [37]. However, we did not observe a haplotype- or disease-specific influence on the expression of MAPT transcripts [35, 74, 75]. To our knowledge, the only study on both mRNA and protein levels in human brain is the large-scale work of Trabzuni et al. which found no clear relationship between MAPT mRNA and tau isoforms in healthy donors [37]. The work of Strauß et al. reported the same observation in induced pluripotent stem cells with homozygous MAPT haplotype [58]. Given that only few studies exist regarding tau protein stability and its half-life in postmortem brain, this remains difficult to interpret at first glance. Another possible explanation could be the lower stability of RNA compared to protein in postmortem tissue [76].

In contrast to the increased total MAPT expression in H1/H1 haplotype, we found MAPT-AS1 mRNA to be markedly decreased in H1/H1 in the ctx-cbl regardless of disease state. This effect was not observed in ctx-fg. The expression of MAPT-AS1 has also been studied in postmortem tissue with similar sample size, but only in a comparison of PD versus control. In these studies, a PD-specific decrease in MAPT-AS1 was found across brain regions with different levels of PD pathology, including SN and cerebellum [40, 41]. Overexpression of the promotor-associated long non-coding RNA MAPT-AS1 was shown to inhibit MAPT promotor activity and MAPT expression in human striatal progenitor cells [41]. These findings suggest that haplotype-specific regulation of MAPT may not be restricted to a specific brain region and could be independent of disease status. Further research is required to gain a deeper understanding of the potential regulatory role of MAPT-AS1 in the context of PD.

The impact of MAPT haplotype on the other genes within the MAPT inversion locus remains enigmatic. For the other investigated genes neither an effect of the MAPT haplotype nor disease was detectable in our study. In contrast to previous studies, in which expression of PLEKHM1 was found increased in the cerebellum of healthy H1/H1 compared to H2/H2 carriers [34] and increased expression of STH was observed in cerebellum of PD patients compared to controls [14]. However, the remaining genes of interest might be addressed in future experiments since differences in mRNA expression play a crucial role in pathologic changes mediated by the MAPT haplotypes.

Taken together, our results show that the MAPT haplotype influences overall MAPT expression and the PD status leads to increased insoluble tau protein in parallel with insoluble α-syn aggregates. This may constitute a risk factor for PD at the biochemical level according to the principle of mutual catalysis of aggregation of tau and α-syn, demonstrated in vitro [19, 20] and in vivo [77]. In all performed analyses, there was no interaction between disease status and MAPT haplotype on MAPT and SNCA gene expression and protein level. All the above observations were detected only in the vulnerable brain region ctx-fg, but not in the control region ctx-cbl, which supports the preferential vulnerability of certain brain regions in PD.

Besides mRNA expression and protein levels itself, the proportion of MAPT transcripts to total MAPT, respectively tau isoforms to total tau, might also be of relevance. For mRNA, this has already been studied in a few cases in adult frontal cortex only (AD n = 3, Control n = 3). There the ratio of 3R to 4R MAPT was about one, while 1N, 0N, and 2N MAPT accounted for 54%, 37% and 9% of total MAPT [68, 78]. Despite the lack of significant differences, we observed an increased ratio of 4R to 3R MAPT in ctx-fg across all groups. A similar finding was observed in the study of Tobin et al. 2012 in cerebellum in PD compared to healthy donors [68, 78]. No MAPT haplotyping was performed in this research publication. With no further reports on splice variants in postmortem human brain, and specifically in ctx-fg, our data contribute to the understanding of MAPT splicing, and the distribution of tau isoforms respectively, in the human postmortem brain. Studies with a larger sample size could further investigate and verify the trends observed here.

In addition, we present further data on the regional distribution of tau and α-syn protein across the human brain. In comparison, tau protein levels of both protein fractions were visibly overall higher in ctx-fg compared to ctx-cbl, which has also been demonstrated by other studies [37]. In line with the main analysis, this regional difference was significantly altered in PD cases for insoluble tau isoforms 0N3R and 1N4R tau. Above that, the H1/H1 haplotype was associated with increased insoluble 1N4R tau in ctx-fg. This finding might be linked to the association of MAPT haplotype H1/H1 with increased risk of sporadic PD which primarly affects regions besides cerebellum. Still, it is difficult to explain these differences to their full extent, since other insoluble and soluble tau isoforms also showed differences between brain regions but without reaching level of significance. A disease-specific increase was also found for insoluble α-syn levels highlighting the relevance of ctx-fg as a target of study in PD.

The present study is subject to several limitations related to the use of postmortem tissue. First, the limited availability of high-quality brain tissue prevented us from working with larger sample sizes. In addition, it turned out to be challenging to find a sufficient number of donors with H2/H2 MAPT haplotype which is less frequent in Europe (5–37.5%) [22, 79]. Secondly, ante- (agonal phase) and postmortem factors (PMI) are suspected to influence postmortem brain tissue quality. For this reason, we selected donors with the shortest PMI and tissue samples with the highest RIN. It is important to mention that the detrimental influence of PMI on mRNA was estimated to be low [80] and the value of RIN as tissue quality marker has been very controversial [81,82,83,84,85,86]. Third, the ctx-cbl was chosen as reference region in accordance with former post mortem brain studies [34, 87, 88]. Even though LB pathology is typically not present in ctx-cbl, PD related changes have been reported previously indicating a possible involvement of the cerebellum in PD [89]. In turn, the disease-specific findings regarding α-syn in the target brain region confirmed the selection of brain tissue as a validated source for the analysis of MAPT haplotype-specific expression differences and the questionable association to PD. With the stringent tissue quality process, we made the best efforts to identify a well-balanced study population in terms of demographic, clinical and neuropathologic parameters. It is important to note that methodological differences (e.g. sample size, brain regions, types of pathologies, or method of quantification) across postmortem brain studies limit the possibility of comparisons among them and with the presented study.

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