Neurodegenerative Disease Associated Pathways in the Brains of Triple Transgenic Alzheimer's Model Mice Are Reversed Following Two Weeks of Peripheral Administration of Fasudil

Abstract

The pan Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor fasudil acts as a vasodilator and has been used as a medication for post-cerebral stroke for the past 29 years in Japan and China. More recently, based on the involvement of ROCK inhibition in synaptic function, neuronal survival, and processes associated with neuroinflammation, it has been suggested that the drug may be repurposed for neurodegenerative diseases. Indeed, fasudil has demonstrated preclinical efficacy in many neurodegenerative disease models. To facilitate an understanding of the wider biological processes at play due to ROCK inhibition in the context of neurodegeneration, we performed a global gene expression analysis on the brains of Alzheimer's disease model mice treated with fasudil via peripheral IP injection. We then performed a comparative analysis of the fasudil-driven transcriptional profile with profiles generated from a meta-analysis of multiple neurodegenerative diseases. Our results show that fasudil tends to drive gene expression in a reverse sense to that seen in brains with post-mortem neurodegenerative disease. The results are most striking in terms of pathway enrichment analysis, where pathways perturbed in Alzheimer's and Parkinson's diseases are overwhelmingly driven in the opposite direction by fasudil treatment. Thus, our results bolster the repurposing potential of fasudil by demonstrating an anti-neurodegenerative phenotype in a disease context and highlight the potential of in vivo transcriptional profiling of drug activity.

Keywords: Alzheimer’s disease; Parkinson’s disease; fasudil; transcriptional profiling.

Figure 1

Self-organising maps of the transcriptional data reveal the global effects of fasudil treatment. In the top left, the SOM weights are regressed against treatment status, and the corresponding Z scores are shown, coloured to represent the scores. It is clear that there are islands of positive and negative correlation corresponding to genes that are up- and down-regulated, respectively. On the top right, the weights are correlated with the mouse sex and show a significant sex effect that is, however, muted relative to that of fasudil. The expression data with the variance explained by sex subtracted out leaves a residual expression profile for which the SOM is shown below. As expected, sex no longer shows any significant correlation, bottom right, with the weights, and interestingly, the fasudil correlations are stronger, bottom left.

Figure 2

Fasudil-driven gene expression changes tend to be the reverse of those in multiple neurodegenerative conditions. The significant gene expression Z scores ($\left|Z\right|>2$) are shown for AD, PD, and HD together with those for the fasudil treatment; non-significant values are left blank, and entries are coloured according to the scores. The most down-regulated genes in AD are shown on the left, and the most up-regulated on the right. As expected, there is a high degree of consistency across the other neurodegenerative conditions, PD and HD. Fasudil shows an up-regulation of all but four genes of those most down-regulated in AD. The reversal is less significant for those genes up-regulated in AD, with only 14 out of 25 driven down with fasudil.

Figure 3

Fasudil regulates pathways in an opposite sense to that seen in multiple neurodegenerative conditions. The significant pathway enrichment Z scores ($\left|Z\right|>2$) are shown for AD, PD, and HD together with those for the fasudil treatment; non-significant enrichment scores are left blank with entries coloured according to the scores. The most down-regulated pathways in AD are shown on the left, and the most up-regulated on the right. As expected, there is a high degree of consistency across the other neurodegenerative conditions, PD and HD. Fasudil shows an up-regulation of all the pathways most down-regulated in AD and a down-regulation of 76% of the most up-regulated pathways in AD.

Figure 4

Pathway and gene-based analyses of the CMAP drug-driven transcription profiles in cancer cell lines in relation to target profiles from AD, PD, and HD. The drugs and target profiles are shown as points on a plane with distances between drugs given by $\frac{1}{2}\left(1-c\right)$, where the correlation is $c=\frac{UU+DD-UD-DU}{UU+DD+UD+DU}$, with $U$ and $D$ the number of shared pathways/genes regulated upwards and downwards, respectively. The distance to the target profile is $\frac{1}{2}\left(1+c\right)$, such that proximity now indicates anti-correlation. Points are mapped to the plane with the target at the centre, in black, and distances to the target are preserved. Angular separations of the compounds are optimised through steepest descent iteration. The compounds are scattered in a more diffuse pattern for the pathway-based analysis. Interestingly, fasudil, shown in violet, emerges as a repurposing candidate only in the pathway-based analysis. In particular, the contingency table for AD is $\left[\begin{array}{cc}12& 20\\ 41& 11\end{array}\right]$ $p<0.00017$, that for PD is $\left[\begin{array}{cc}17& 13\\ 24& 6\end{array}\right]$ $p<0.047$, and that for HD is $\left[\begin{array}{cc}20& 12\\ 36& 6\end{array}\right]$ $p<0.016$.