Regulation of human MAPT gene expression

Tau mRNA expression was initially identified in the brain and, more precisely, in
neurons by both northern blot and in situ hybridization analyses; it has since been detected in various tissues, including
the cerebellum, kidney, muscle and testis, using more sensitive methods such as RT-PCR,
microarray and RNA-Seq 57], 114]–116]. However, although Tau protein expression in peripheral tissues has been clearly
observed in rats 116], very little data concerning such expression in human tissues have been reported
117], with the exception of reports of Tau protein expression in pathological peripheral
tissues 37], 41], 118].

Mature Tau transcripts contain up to 16 exons 3], 57]. The different exons have been identified and numbered, corresponding to their locations
within RNA sequences from different animal, primarily human, bovine and murine, models.
The first exon is generally named exon 0 (E0) in the literature, and exon 4A corresponds
to the alternative exon located between exons 4 and 5. The exon numbers provided in
the databases must be carefully considered when determining the functions of analyzed
transcripts because different exons may be assigned the same number depending on the
transcript analyzed.

Three Tau transcripts of 2 kb, 6 kb, and 9 kb in length are encoded by the MAPT gene (reviewed in 57]). These transcripts differ from one another by their splicing patterns, as demonstrated
by the differences between the 6 and 9 kb transcripts 119], and their polyadenylation sites (2 kb versus 6 kb) 120]. The abundance of data in databases largely confirms the observed variations in splicing
patterns and polyadenylation sites but also suggests the existence of several promoters,
as detailed below.

5? UTR/MAPT promoter

MAPT promoter identification in the E0 region

The MAPT promoter has been studied primarily in three different models, namely human, rat
and mouse models. The MAPT promoter is characterized by a high G?+?C content as well as by the absence of TATA
and CAAT boxes 65], 77], 121]. Several regions have been determined to play important roles in Tau transcription
(Fig. 2).

First, the “core” promoter is located immediately upstream of the first exon (exon
E0, previously named exon ?1) (Fig. 2) 64], 65], 77], 101], 121]. More detailed studies of rat and mouse models have enabled differentiation between
the two sub-sequences in this core region 121], 122] (Fig. 1). The sequence located 5? of the core sequence is involved in neuronal-specific expression
in rats and mice but not in humans, whereas expression of the 3? sequence is not tissue
dependent 77], 121]–123]. However, this apparent difference in the neuronal specificity of the core promoter
between mice and humans may be due to differences in the cell type and the length
of the promoter sequence used in constructs, as suggested by Maloney and Lahiri 64]. These authors have reported the influence of E0 on human promoter activity and have
identified a neuronal-specific sequence that includes the last 10 nt of E0 and the
first 200 nt of the intronic sequence located immediately downstream that negatively
regulates promoter activity (Fig. 2).

Other distal sequences (approximately ?7000 to ?1500 nt upstream of exon E0) are involved
in promoter activity 64], 101], 121] (Fig. 2). The 3? end of this sequence (approximately ?3000 to 1500 nt) negatively regulates
Tau expression in rats and humans 64]–66], 101], 121]. The 5? end of this sequence (approximately ?7000 to 3000 nt), which has only been
studied in mice, is also involved in promoter activity 121]. In addition, a putative reverse sequence located immediately downstream of E0 might
be involved in human MAPT regulation (Fig. 2) 64]. However, these authors could not exclude interference from another promoter because
of identification of a hypothetical gene, LOC100128977, immediately upstream of and
in the opposite orientation as the MAPT gene 124].

According to the technique used, several initiation sites for Tau transcription have
been reported in both rats and humans 65], 77]. The human transcript sequences published in databases confirm the diversity of the
transcription start sites. However, some discrepancies exist between the sites described
in the literature and those in databases. In particular, the initiation sites of the
published sequences are located in E0, whereas the Ensembl database shows that various
initiation sites are present all along the gene in addition to those in E0 (Fig. 3, Fig. 4b) 125]. Three of these identified sites are common between the literature and databases.

Fig. 4. Splicing patterns of human Tau transcripts. a Classical splicing pattern described in the literature. The insertion of exons 4A
and 6 rarely occurs in the brain (shaded in pale orange) and varies according to which
3’ splice site is used. E8 insertion (shaded in dark orange) has not been reported
in humans but has been described in different animal models and in the human Ensembl
database. b Hypothetical splicing according to the Ensembl database. The MAPT numbers correspond to those in the Ensembl database. White, non-coding regions; gray,
constitutive coding exons. Yellow, orange, pink, green, purple and blue represent
the alternative exons 2, 3, 4A, 6, 8 and 10, respectively

Trans factors

As demonstrated by electrophoretic mobility shift assay (EMSA), the transcription
factors SP1 and AP2 bind to the Tau core promoter region and are necessary for promoter
activity 121], 122]. Another DNA sequence located between the SP1 and AP2 sites influences promoter activity;
however, the transcription factor involved remains to be identified 122]. EMSA has also revealed that SP1, progesterone receptor (PR) and retinoic acid receptor
(RAR) bind to the human exon E0 sequence, although their functionalities remain to
be demonstrated 64].

The human MAPT promoter region also contains putative binding sites for various other transcription
factors, such as Nrf1, MTF1, MBF1, MepI, and GCF 64], 101]. Some of these sites are shared between the H1 and H2 haplotypes, such as the AP2
and SP1 sites mentioned above. Interestingly, among these common sites, four sites
corresponding to an A?-interacting domain have also been identified in the distal
region of the Tau promoter 64], 126]. A? is a pathological peptide generated via deregulated proteolysis of amyloid precursor
protein (APP). This peptide aggregates during AD and leads to the observed pathology
of this disease according to the amyloid cascade hypothesis. This aggregation/accumulation
of A? is not a feature common to all tauopathies, but it does occur in some, such
as AD and Down syndrome. Several studies have suggested that the pathological effect
of A? is mediated by Tau (reviewed in 127], 128]). Thus, determining whether the A?-interacting domain actually regulates Tau expression
directly and thus participates in the development of Tau pathologies in which both
amyloid and Tau aggregates are present would be of interest.

Does MAPT have only one promoter?

Alternative initiation transcripts largely contribute to transcriptome and proteome
diversity, and more than 40 % of genes have at least two promoters, as demonstrated
in flies (reviewed in 129], 130]). Although the region around E0 contains the only promoter for MAPT that has been described in the literature, Andreadis 57] hypothesized the existence of additional Tau promoters to explain variations observed
in neuronal specificity and NGF susceptibility of Tau expression according to the
different Tau transcripts 77], 119], 131]. Currently, new data are available in databases that reinforce this hypothesis; at
least 6 other exons distal to E0 have been identified as the first exons of various
transcripts.

One of the potential additional promoters may be associated with the initiation of
transcription at exon E1. Indeed, considering the number of identified transcripts,
E1 is the most frequent exon where transcription is initiated after E0 (transcripts
initiated at E0: 9 and E1: 5) for the H1 haplotype, further suggesting the possible
existence of an unidentified promoter. E1 is located 68000 nt distal to E0. Similar
to E0 57], sequence analysis has revealed no TATA or CAAT consensus binding sites in E1; however,
a GC box specific for SP family members is present near this exon 132]. Indeed, one potential SP1 site and five sites for AP-2? binding have been identified
200 nt upstream of E1 using the ALGGEN server 133]. The absence of E0 would result in a change in the 5? UTR length but would not alter
the coding sequence because E0 is a non-coding exon, while E1 is a partially coding
exon (Fig. 1). On the reverse strand (H2 haplotype), transcripts listed in the Ensembl database
begin primarily with E1 125] (Fig. 3).

The other initiating exons near the E2, E3, E4A and E5 regions are located more than
10,000 nt and up to 25,000 nt distal to E1, suggesting that more than one alternative
promoter may exist. Interestingly, the CpG
₂₁
island is located immediately upstream of E4A at the beginning of the short transcript
MAP-010/ENST00000576238, as described above in the CpG island section (Fig. 4b).

Tau splicing

Here, we will not describe the splicing mechanisms in detail because they have been
discussed in previous reviews; however, we will focus on the Tau splicing pattern
because splicing is an indirect mechanism of protein function modulation via sequence
modification 24], 57], 61], 134].

Published data

Tau alternative splicing is dependent on the developmental stage, tissue and species.

Central nervous system (CNS)

The following are generally recognized facts regarding Tau splicing in the human CNS.

(i) In the fetal brain, only one isoform is predominantly expressed, with insertion
of the constitutive coding exons (E1, E4, E5, E7, E9, E11, E12, and E13/14). E1 is
considered a coding exon because it contains the initiating ATG codon. In addition,
there are no reports in the literature suggesting the presence of non-coding E0 during
the fetal stage 3], 57], 135], 136].

(ii) E2, E3 and E10 are specific to the adult brain (Fig. 4a). Consistent with this statement, 6 protein isoforms have been identified in the
human adult brain that contain sequences encoded by exon 2, exon 3 and/or exon 10.
Generally, these 6 isoforms are named 2N3R, 1N3R, 0N3R, 2N4R, 1N4R, and 0N4R. The
designations 0 N, 1 N and 2 N indicate the exclusion of E2 and E3, the inclusion of
E2 and the inclusion of both E2 and E3, respectively. Notably, the insertion of E3
is dependent on the presence of E2, which corresponds with an atypical feature of
the splicing event. Globally, E3 inclusion can be considered a rare event compared
with E2 insertion or complete Tau transcription, as evidenced by RT-PCR 114], 137]. Conversely, 3R and 4R indicate the exclusion and inclusion of E10 115], 138], respectively, which has been previously reviewed 3], 139].

Peripheral tissues

Considering peripheral tissues, including the peripheral nervous system (PNS), the
alternative exons described above are often included with other exons that are nearly
absent in the CNS. Indeed, exon 4A (E4A) is present specifically in the PNS and retina,
and exon 6 (E6) is found in the spinal cord and skeletal muscle. Interestingly, the
lengths of these latter two exons, E4A and E6, are modulated by the choice of 3? splice
site. Indeed, two possible 3? splice sites exist for E4A (E4A, E4A
_Long
), and there are 3 splice sites for E6 (E6c, E6p, E6d) 41], 117], 137] (Fig. 4a). However, although isoforms containing E4A
_Long
, E6p, and/or E6d have been reported in the literature, they are not present in human
databases (Ensembl, UCSC). These observations bring into question the frequencies
at which these different 3? splice sites are used. Considering the possible co-insertion
of alternative exons, in ENSEMBL/UCSC sequences, E4A is always inserted when E6 or
E6 and E8 are included with E2, E3 and E10. However, an E4A-containing isoform without
E6 but containing E2, E3 and E10 has been cloned 140]. Furthermore, in Ensembl database, the isoforms containing exon 6c also contain exons
2, 3, 4A and 10 with or without exon 8, although studies by Andreadis’s group have
demonstrated that all combinations of E6c, E6p, and E6d with the other alternative
exons are possible 141]. The inclusion of E6p and E6d results in the translation of a truncated protein that
lacks microtubule-binding sites because of a change in the reading frame that introduces
a stop codon. Tau proteins containing E6c- or E6d-encoded sequences have been identified
in several tissues, including brain and muscle tissues, using specific antibodies
142], 143]. These new forms, although minor, could interfere with the role of Tau in axonal
transport 144], 145]. Note also the rare but possible inclusion of E6 in the fetal brain 57].

Suggestions from databases

Recent data from databases suggest that the splicing pattern of MAPT may be more complicated than suggested by the primary characteristics described above,
although particular splicing events are most likely rare. Although the majority of
transcripts described in databases are coding transcripts, some are not (Fig. 4b). Non-coding transcripts have been identified that correspond primarily to isoforms
with the deletion of at least one constitutive exon. One such non-coding transcript
(MAPT-011 ENST00000571311) (Fig. 4b) may be processed by NMD. Notably, none of these variants have been described in
the literature, although they are listed in Ensembl. Their existence and potential
roles (perhaps as lncRNAs) remain to be investigated and confirmed.

Splicing and H1/H2 haplotypes

A comparison of the transcripts from the forward (H1) and reverse (H2) strands that
have been published in databases such as Ensembl has revealed that the haplotype might
affect some splicing events. This situation has already been reported for E10 (see
the sub-chapter “Functional consequence of H1 haplotype on pathology”).

Alternative 5? UTR/alternative first exon

In the literature, E0 is always described as the first exon in all MAPT transcripts. However, when analyzing different MAPT transcripts in databases such as Ensembl, it is apparent that E0 can be present or
absent in the H1 haplotype; thus, it may be considered an alternative exon due to
alternative promoter usage. Furthermore, E0 inclusion is less frequent in the sequences
of transcripts from the reverse strand (H2 haplotype) (Fig. 3). Several possible initiating transcription sites may exist in E0 as described above
(see the sub-chapter regarding promoters), leading to different possible lengths of
E0 and of the 5? UTR because E0 is a non-coding exon (Fig. 3). According to the Ensembl database, the length of E1 may also vary depending on
the first exon that is included (E0/E1). Indeed, E1, a partially coding exon, is 150
or 133 nt long when initiated from E0 or E1, respectively, on the forward strand.
For H2 (the reverse strand), in the absence of E0, the length of E1 is three nucleotides
longer at its 5? side (153 nt) or 17 nucleotides shorter at the ATG codon (133 nt).
These variations in E1 length do not affect the sequence of the translated protein
because the 5? end is non-coding; therefore, this length variation only results in
modification of the 5? UTR of the transcript (Fig. 2).

Additional exons

The possible insertion of additional exons, although not yet identified in the published
human transcripts, is suggested by data in the Ensembl database. This possibility
is the case for E8, which was first identified in bovines and then in certain species
such as rhesus monkeys 146], 147] but has not been described in humans 148] (Fig. 4a). However, according to the Ensembl database, transcripts containing E8 include all
of the alternative exons except for E0, which can be included or excluded (MAPT-203 ENST00000344290, MAPT-004 ENST00000415613). Studies of different animal species have shown that E8 insertion
is possible in the absence of certain alternative exons. In particular, 4 transcripts
containing E8 have been identified in cows 148]. All of these transcripts also contain E2 and E10 but lack E4A. These transcripts
differ from each other by the presence or absence of E3 or E14. In mice, only one
transcript (MAPT-006/ENSMUST00000106993) has been identified with E8, and this transcript contains
E10 as the only other alternatively spliced exon.

New exons that have never been reported in the literature are present in certain transcripts.
The insertion of most of these new exons results in truncated transcripts at the 5?
and/or 3? ends of Tau mRNA (Fig. 4b). For example, the exon located between E1 and E2, named E1A, is present in the only
transcript that potentially undergoes NMD (MAPT-011, ENST00000571311/ENST00000625688) (Fig. 4b). In addition, we identified exon E4B, which is positioned between E4A and E5 and is
spliced to E5, producing a non-coding but processed transcript composed of two exons
(MAPT-012, ENST00000577017/ENST00000627800) (Fig. 4b).

Other exons generated by the extension of known exons have been found in particular
isoforms in Ensembl database. Exon E4A
_Long
has been described in MAPT transcripts as another spliced form of exon 4A 41] (see above). E4A
_Long
may also be the only exon in a short transcript listed in Ensembl database (MAPT-010 ENST00000576238/ENST00000625417) (Fig. 4b). Another example is extended E3 (E3
_Ext
, 5658 nt versus E3, 87 nt), which may be the first exon in the MAPT-009 transcript (ENST00000576518/ENST00000626880) (Fig. 4b). Exon E3
_Ext
is composed of exons E3 and E4, the 1603 nt upstream of E3 and the entire retained
intron between E3 and E4 (3903 nt) (Fig. 4b). E3
_Ext
may be a partially coding exon because an ATG is located 100 nt before E4 and is spliced
to exon 5, encoding a protein that lacks part of the 5? region that contains the acidic
projection domain (Fig. 4b). Because the alternative exons E4A, E6, and E10 are excluded from this transcript,
it may be translated more precisely in this shortened form during fetal development.
Interestingly, if this transcript exists, then it has a short 3? UTR. Another example
of an extended exon is extended exon E4 (E4
_Ext
), which corresponds to exon E4 in-frame with partial intronic sequences (2883 nt
upstream and 2066 nt downstream) and which may be the only exon in the non-coding
transcript MAPT-014 (ENST00000572440/ENST00000628437) (Fig. 4b). Certainly, the existence of these two latter transcripts must be confirmed under
physiological conditions.

Splicing and pathology

Defects in splicing can be pathological events that lead to or have roles in tauopathy-type
neurodegenerative processes. Indeed, missplicing has been clearly associated with
certain familial tauopathies, such as FTDP-17 and DM (reviewed in 24], 134]). These pathologies have been associated with either mutations in MAPT cis-elements (FTDP-17) (review in 61]) or with microsatellite expansion in the non-coding regions of various genes, leading
to variations in the expression of splicing factors belonging to the CELF and MBNL
families (DM, reviewed in 24]). Notably, numerous mutations in MAPT are located in or near E10 (upstream and downstream), favoring E10 inclusion either
by destabilizing a stem–loop structure that encourages access to the 5? splice site
or by modifying splicing factor or U1/E6 snRNP binding sites (reviewed in 61]). For sporadic tauopathies, the MAPT haplotype may influence splicing, as described above. Moderate Tau missplicing events
have also been observed in numerous other tauopathies, such as AD (reviewed in 24]). In AD and PSP, modified miRNA expression may result in alterations in the expression
of certain splicing regulatory factors, which would then influence Tau splicing 149], 150]. As such, the splicing pattern of MAPT transcripts could be determinant in tauopathy-type neurodegenerative processes. Consistent
with this hypothesis, none of the mouse transgenic models have reproduced Tau pathology
in the absence of a Tau mutation in the coding region. However, Tau pathology has
been observed in mouse models when the human Tau gene is expressed in the absence
of endogenous MAPT expression. These observations suggest that the correct ratio between the differentially
expressed isoforms is determinant in the tauopathy process in the absence of Tau coding
mutations. Thus, the difficulty in obtaining animal models that reproduce tauopathies
in the absence of a Tau mutation most likely results from the MAPT splicing pattern, which differs among animal species 151]–155]. For example, exon 10 inclusion is highly increased in mouse and rat brains, whereas
exon 2 inclusion is observed less frequently in mice compared with humans. Similarly,
differences in the 3? ends of the transcripts, as described below (section 3.3), may
stem from splicing differences that potentially interfere with the human specificity
of the tauopathy process. However, the contribution of other undetermined factors
cannot be excluded at this point.

3? UTR/alternative termination sites: Roles of RNA stability and localization

The 2 and 6 kb forms of mature Tau RNA are generated from different 3? UTRs. Indeed,
two alternative polyadenylation sites followed by a short poly(A) sequence located
at a distance of approximately 4000 nt have been found in different animal species
74], 115], 120], 156] (Fig. 3). Thus, at least two transcription termination sites could be used, resulting in
transcripts with short or long 3? UTRs. Interestingly, the first polyadenylation site
is located within an intronic sequence that is deleted in some transcripts from various
animal species. Indeed, in some Ensemble database transcripts from mouse, rat and
cow, E14 is separated from E13 by a short intronic sequence of approximately 900 nt
148], 157]. The splicing of E13 to E14 results in extension of the C-terminus of the protein
and truncation of the 3? UTR. In humans, the short intronic region between E13 and
E14 is always retained, as described in the literature 74] and databases such as Ensembl. Thus, the splicing of E13/E14 may be a feature that
differs between animals and humans.

The 3? UTR may contain cis-elements that decrease Tau RNA expression. Indeed, binding sites for miR-34a and
miR-34c-5p have been reported that lead to decreased Tau RNA and protein expression
45], 156]. Interestingly, increased Tau expression and decreased miR-34C-5p expression have
been correlated with the chemoresistance of gastric cancer to paclitaxel 45]. Recent studies have demonstrated a role of miR-485-5p in axonal development and
Tau expression downregulation during synaptic plasticity in hippocampal rat neurons.
The Tau 3? UTR sequence has two possible miR-485-5p binding sites 158] and contains a target sequence for miR-132-3p, which is strongly downregulated in
the brains of AD patients 149]. However, a mutation in the binding site does not completely abolish miR-132 activity,
suggesting that other indirect mechanisms exist by which miR-132 affects Tau expression.

In rat transcripts, a 240-nt region in the 3? UTR has been implicated in RNA stability
and axonal localization 159]–161]. Several proteins, such as HuD (an Elav protein family member), insulin-like growth
factor mRNA-binding protein IMP-1, Ras-regulatory protein G3BP, interleukin enhancer
binding factor 3 (Ilf3) and NF90, bind to this sequence and are potentially involved
in the axonal localization of these transcripts 162]. Unfortunately, no such data are available in humans, and these data must be generated
to determine whether similar mechanisms protect human RNA and guide transcripts to
their proper locations.