Public transcriptomic studies have shown that several genes display pronounced gender differences in their expression in the human brain, which may influence the manifestations and risk for neuronal disorders. Here, we apply a transcriptome-wide analysis to discover genes with gender-specific expression and significant alterations in public postmortem brain tissue from Alzheimer’s disease (AD) patients compared to controls. We identify the sex-linked ubiquitin-specific peptidase 9 (USP9) as an outstanding candidate gene with highly significant expression differences between the genders and male-specific underexpression in AD. Since previous studies have shown that USP9 can modulate the phosphorylation of the AD-associated protein MAPT, we investigate functional associations between USP9 and MAPT in further detail. After observing a high positive correlation between the expression of USP9 and MAPT in the public transcriptomics data, we show that USP9 knockdown results in significantly decreased MAPT expression in a DU145 cell culture model and a concentration-dependent decrease for the MAPT orthologs mapta and maptb in a zebrafish model. From the analysis of microarray and qRT-PCR experiments for the knockdown in DU145 cells and prior knowledge from the literature, we derive a data-congruent model for a USP9-dependent regulatory mechanism modulating MAPT expression via BACH1 and SMAD4. Overall, the analyses suggest USP9 may contribute to molecular gender differences observed in tauopathies and provide a new target for intervention strategies to modulate MAPT expression. See associated publication: http://link.springer.com/article/10.1007%2Fs12035-016-0299-z

1. Modelling gender-specific regulation of tau in
Alzheimer’s disease

2. Motivation: Age-dependence of gender-differences in AD
Sex-specific cumulative risk for a 65-year-old to develop AD by 95 years of age.
Adapted from Andersen K et al. Neurology 1999;53:1992-1992
 significant difference also after adjusting for differential survival

3. 2
Candidate gene USP9 – Gene expression data analysis
Left: USP9Y is one of the genes with the largest gender differences in gene expression
across multiple brain regions (USP9Y difference is not compensated by USP9X, dataset
by Kang et al., 2011).
Right: USP9Y down-regulation in human post-mortem hippocampus samples for
incipient, moderate and severe cases of Alzheimer’s disease as compared to non-
demented controls (dataset by Blalock et al., 2011)
USP9Y expression
female male
Alzheimer
USP9Yexpression

4. 3
Analysis in zebrafish embryo model (USP9 Knockdown)
A: Zebrafish prior to injection of morpholino oligos (MO) for usp9 knockdown
B: 8 ng control injection
C: 4.8 ng MO injection
D: 8 ng MO injection

5. 4
Analysis of usp9/tau associations in zebrafish
 Zebrafish have 2 MAPT-
paralogs, mapta & maptb,
which resemble the two
tau isoforms in humans 
Analyze usp9 knockdown
effect on mapta & maptb
 The morpholino knockdown
of usp9 results in a
concentration-dependent
decrease of mapta and
maptb gene expression

6. 5
Analysis of USP9/tau associations in a cell culture model
 Knockdown of USP9X/Y
and USP9X results in
significantly reduced
MAPT concentration levels
 The effect is strongest for
the combined knockdown
of USP9X and USP9Y
• High correlation of gene
expression fold changes in
USP9X/Y und USP9X
knockdowns (r = 0.965, p <
2.2E-16 for the top 1000
most significantly altered
genes)  suggests similar
functions of the two USP9
variants

7. 6
Model for the role of USP9 in MAPT Regulation
a) USP9 can modulate MAPT phosphorylation via SNCA and MARK4
b) USP9 can modulate MAPT gene expression via SMAD4 and BACH1

8. 7
Summary
Overview of findings
 Significant alterations observed in USP9 and MAPT gene expression in
Alzheimer patients (post mortem brain samples) as compared to unaffected
controls
 Association between USP9 knockdown und alterations in MAPT gene expression
in both zebrafish and cell culture model experiments (+ significant correlation in
human post mortem brain samples)
 New USP9/MAPT regulation model is consistent with measured experimental
data and regulatory information from the literature

9. 8
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