The role of splicing factors and upstream proteins in Tau isoform regulation and it’s

implication for Alzheimer’s disease pathophysiology

SSU 1
Date: 27.11.23 - 15.12.2023
690065413
Total word count:
Abstract
(200 words -> I need to cut out 200 words from my essay first- plz help!)

Introduction

Dementia, affecting 55 million people worldwide, is characterised by memory loss,

impaired thinking and consequently an inability to perform daily tasks. (1)
Alzheimer’s disease (AD) is the most prevalent form of dementia and affects 60-70%
of cases. (1,2)

AD is characterised by two abnormal protein aggregates in the brain: amyloid beta

proteins can cause beta-amyloid plaques and tau can lead to tauopathies,
specifically neurofibrillary tangles. (2)

AD is characterised by two abnormal protein aggregates in the brain. (2,3) Tau, a

microtubule-associated protein, maintains axonal cytoskeletal structure (2,3).
However, in AD, Tau is hyperphosphorylated and leads to tauopathies, specifically
neurofibrillary tangles in AD. The second abnormal protein aggregates cause beta-
amyloid plaques. The build-up of these abnormal protein aggregates can lead to the
abnormal function of neurones and lead to their eventual death. (2)

Tau is transcribed from the Microtubule-Associated Protein Tau (MAPT) gene

contains 16 exons. (4,5) Exons 2,3 and 10 play a role in the alternative splicing of
Tau precursor messenger ribonucleic acid (pre-mRNA) in the brain of an adult
human.

Exons 2 and 3 encode the N-terminal region (NTR), influencing Tau distribution into
axons, signalling, nuclear interaction, and aggregation. (6–9) Exon 10 is part of the

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microtubule-binding domain segment and encodes the second repeat segment (R2
segment). (9–14) The inclusion exon 10, and there the R2 segment, elongates the
microtubule-binding domain segment of Tau, and therefore increases Tau’s
microtubule affinity. (9–14)

Due to alternative splicing, Tau exists as 6 isoforms. This can be divided into 3R-Tau
(3R0N, 3R01, 3R02) and 4R-Tau (4R0N, 4R1N, 4R2N) (see Figure 1). (9) In normal
physiology, 3R:4R expression is 1:1. (9) The expression of different 3R to 4R
isoforms of Tau have been attributed to AD pathophysiology. (9)

Mature mRNA creation is controlled by splicing, which involves several splicing

factors which form the spliceosome. (15) Despite transcription of Tau exons 2,3 and
10, these can be spliced to be included or excluded in the mature mRNA Tau and
therefore Tau protein. (9,15)

Tau hyperphosphorylation is commonly attributed to post-translational modifications,

but splicing may also play a role. (9) Certain Tau isoforms could be more prone to
phosphorylation, and an imbalance in isoform ratios might facilitate
hyperphosphorylation and subsequent Tau aggregate formation. (9)

Splicing, involving the spliceosome machinery, generates tau isoforms. (15–17)

Identifying the splicing factors and understanding their regulation by upstream
proteins is fundamental for uncovering therapeutic targets to prevent aberrant Tau
splicing and hyperphosphorylation.

This review aims to synthesise literature which has investigated splicing factors of
Tau and proteins which affect splicing factors of Tau, and how they presented in AD.

Methods

TRIP Pro and PubMed database was used to conduct a search for relevant
literature. The key terms used were the following: ‘Tau’, ‘splicing’, ‘Alzheimer’s
disease’ and linked with the Boolean term ‘AND’ to conduct the search. The term
‘spliceosome’ was used alongside ‘splicing’ which was linked with the Boolean term
‘OR’ to ensure all relevant papers were found. This search gave 158 results in TRIP
and 258 in PubMed.

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The use of the filter “primary research” on TRIP Pro database was used, narrowing
down the search to 107. The use of the filter “MEDLINE” was used in Pubmed,
creating 224 results.

Abstracts were screened for relevance which left 15 papers in TripPro and 13 in
PubMed.

Further screening of the papers excluded those which did not include all the key
terms, leaving 6 papers in TRIP Pro and 7 in PubMed.

6 of the searches were the same in TRIP Pro and PubMed, with 1 was unique to
PubMed. Therefore, 7 papers in total were used for this literature review discussion.
The searches were conducted in December 2023. (See Figure 2)

Discussion

Splicing factors in AD

Currently, two splicing factor function in AD have been researched: Arginine and
serine rich coiled-coil 1 (RSRC1), and splicing factor proline and glutamine rich
(SFPQ). Figure 3 demonstrates a summary of their effects, if any, on Tau in AD.
(18,19)

Bowles et al. identified the presence if RSRC1 splicing factor in human AD brain
slices, whilst Younas et al. identified SFPQ splicing factor in rapidly progressive
human AD brain slices. (18,19)

Bowles et al. conducted iso-sequencing on temporal cortex tissue of human normal

vs AD brain samples to conduct analysis. (18,19) Younas et al. did not specify which
area of the brain was investigated. (18,19) Bowles et al. iso-sequencing gave results
of a not statistically significant increase in 2N:1N Tau ratio and decrease in 1N:0N as
well as 2N: 0N ratio. (18,19) This was speculated to be due to an increase in 0N
isoforms, rather than decrease of 1N or 2N isoforms. (18,19) Perhaps the statistical
insignificance was influenced by the small sample size (n=6) of human brain
samples.

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This means that exon 2 and exon 3 exclusion occurs, which could imply splicing
factors which are responsible for these exon’s exclusions could be up regulated.
However, these splicing factors are unknown, and Bowles et al. did not explore this.
(18)

To further investigate isoform expression, the same brain slices were used for
Western blotting, where a statistically significant accumulation of 0N and 2N isoforms
was found. (18) There was also a significant increase in 4R0N: 3R0N and 4R2N:
3R2N ratio. This was suggested to be due to an increase in 4R0N and 4R2N,
perhaps due to their impaired degradation, rather than splicing effects. (18)
However, as the genetics or splicing factors of these results were not explored or
accounted for, this could be due to splicing factors affecting exon inclusion, and exon
2 and exon 3 inclusion, respectively.

Whilst Bowles et al. investigated the expression of different tau isoforms within AD,
Younas et al. did not, but instead investigated where in the cell SFPQ can be found
in normal vs AD conditions. (18,19)

Younas et al. used immunofluorescence to show that SFPQ is downregulated and

dislocated from the nucleus in AD brain cells, in comparison to the wild-type. (19)
Bowles et al., on the other hand, did not show the distribution of RSRC1 within the
cell which leaves room for future investigations, using this technique. (18)

Bowles et al. used datasets from the Accelerating Medicines Partnership-

Alzheimer's Disease Target Discovery and Preclinical Validation consortium to
identify RSRC1 overexpression in AD. (18)An RNA-pull down experiment was then
conducted using the same set of human AD brain samples to confirm this finding.
(18) Similarly, an RNA pull-down experiment was conducted by Younas et al., to
investigate the presence of SFPQ in the AD brain and in contrast, also identify its
locality within the cell. (19) Both Younas et al. and Bowles et al. used SH-SY5Y cells
which displaced overexpression of RSRC1 and downregulation of SFPQ,
respectively. (18,19) However, Bowles et al. also compared their findings in HeLa
cells, to show that SFPQ dislocation from the nucleus is correlated significantly to
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increased Tau phosphorylation, and that this was not due to the cell line in which the
experiment was conducted. (18)On the other hand, Younas et al. did not compare
their investigations of RSRC1 in another cell line, which could suggest increased
levels of Tau phosphorylation are enhanced in SH-SY5Y cells, rather than because
of RSRC1’s overexpression. (19)

The use of SH-SY5Y is one of the few similarities in experimental set up shown in
the studies, which implies a clearer standardisation for investigating splicing factors
of Tau expression, and therefore it’s phosphorylation and function in AD, is needed
within the scientific community.

Upstream proteins which affect splicing factors

Currently 5 proteins have been investigated which affect splicing factors of Tau and
therefore Tau isoform formation. These proteins include serine-arginine protein
kinase 2 (SRPK2), dual-specificity tyrosine-phosphorylated and regulated kinase 1A
(Dyrk1a), Loss of fused in sarcoma (Fus), which increase exon 10 inclusion.
(20–22)
Also, casein kinase 1 (CK1) and Sirtuins-1 (Sirt1) suppress Tau exon 10 inclusion
and cause exon 10 exclusions, respectively. See Figure 4 for an overview of their
effects on Tau.

Upstream proteins SRPK2, Dyrk1 and Fus enhance exon 10 inclusion by their
action on splicing factors.

Wang et al. used human brain tissue samples to identify SRPK2 as an upstream
protein which has been shown to affect the following splicing factors in Tau:
alternative splicing factor (ASF), splicing factor 2 (AS2), Serine/arginine-rich splicing
factor (SC35) through phosphorylation. (20)Yin et al. used Down syndrome mice, to
show that Dyrk1 phosphorylates ASF, serine/arginine protein 55 (Srp55), SC35 and
9G8 splicing factors in Tau. (21) Whilst Yin et al. did not use AD brain tissue
samples, it is a useful insight in how Tau splicing works. (21) Both studies showed
that exon 10 was increased in inclusion and therefore regulating an up-regulation of
4R-Tau in the samples. (20,21)

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Furthermore, Wang et al. showed that SRPK2 itself is cleaved by delta-secretase
(AEP), which upregulates SRPK2 action and promotes exon 10 inclusion further, as
well as 4R:3R Tau isoform. (20) Immunoblotting of the hippocampus of human-tau
(htau) transgenic mouse was used to draw this conclusion. (20)

Yin et al. also found an increase in the 4R:3R tau ratio, however they used
differentiated-human neuronal progenitor cells and neonatal rat brains. (21)Down-
syndrome increasing the risk of developing AD early in humans. (21) Using neo-natal
samples could mean not all the isoforms have yet been developed, and therefore a
variety of rat age samples could be tested for the isoform Tau ratio’s using brain
slices.

Yin et al. mentioned implications of the findings on AD due to their contribution of

Tau’s splicing mechanism but did not conduct experiments on AD mice. (21)Future
research should use MAPT transgenic mice to knockout the Dyrk1 gene and confirm
the greater 4R:3R tau ratio.

Orozco et al. identified FUS as an upstream protein which leads to an increase in the
2N and 4R Tau isoforms. (20,22) This means exon 2, 3, and exon 10 inclusion is
promoted due to FUS. (22) Orozoco et al. conducted their experiments in
hippocampal neurones. (22) Similarly, Wang et al. completed immunofluorescence
and immunoblotting in the hippocampus of htau mice to examine the 3R:4R
expression. (20)

CK1 and Sirt1 suppress Tau exon 10 inclusion and cause exon 10 exclusion,
respectively.

Chen et al. showed that CK1 protein affects splicing factors by suppressing Tau
exon 10 inclusion, whilst Qian et al. showed that Sirt1 causes exon 10 exclusion.
(23,24) Both findings showed an increase in the expression of 3R Tau, in comparison
to 4R-tau. (23,24) CK1 was shown to also have a significantly negative correlation
with 4-Tau expression in the frontal cortex of AD human brain. (23) Whilst this
supports the conclusion that exon 10 inclusion is suppressed, (23) it also shows that
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exon 10 inclusion is not eliminated, which suggests that could still play a role in exon
10 inclusion, or, perhaps other splicing factors and upstream proteins are active
instead.

Sirt1, unlike CK1, was shown to affect SC35 and 9G8 splicing factor’s specifically
through deacetylation. (24) Both Dyrk1 and Sirt1 therefore act on 9G8, but Dyrk1
phosphorylates it. (21,24) When SC35 and 9G8 were investigated separately in the
context of Sirt1, it was shown that 9G8, when deacetylated suppressed 3R-tau
expression. (24)This suggests that exon 10 exclusion was increased. However,
when Sirt1 deacetylates SC35, this inhibited 4R-tau expression. (24) This implies that
one upstream protein can affect more than one splicing factor at a time. Whilst this
could be directly due to the upstream protein’s affects, it could also be due to one
protein affecting the structure of another, especially if they are interacting with one
another in a specific way as part of the spliceosome.

Conclusion

Limited research exists about Tau splicing factors and the upstream proteins which
influence Tau splicing. Future research should develop standardisation across
sample type, size, and laboratory techniques for investigating Tau splicing.

The splicing factors RSRC1 and SFPQ act as splicing factors in Tau. SRPK2, Dyrk1,
Fus, CK1 and Sirt1 play a role in modulating Tau splicing factors. The ratio of 3R-
Tau and 4R-Tau is influenced by splicing, and how this ratio changes should be
investigated further as it can lead to potential therapeutic targets of Tau and AD.

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Appendix

Figure 1 -> splicing and isoforms

Figure 2 -> Methods
Figure 3 -> splicing factors, tau and AD
Figure 4 -> upstream proteins affecting splicing factors in Tau and AD