Willin/FRMD6 is part of a family of proteins with a 4.1 ezrin-radixin-moesin (FERM) domain. It has been identified as an upstream activator of the Hippo pathway and, when aberrant in its expression, is associated with human diseases and disorders. Even though Willin/FRMD6 was originally discovered in the rat sciatic nerve, most studies have focused on its functional roles in cells outside of the nervous system, where Willin/FRMD6 is involved in the formation of apical junctional cell-cell complexes and in regulating cell migration. Here, we investigate the biochemical and biophysical role of Willin/FRMD6 in neuronal cells, employing the commonly used SH-SY5Y neuronal model cell system and combining biochemical measurements with Elastic Resonator Interference Stress Micropscopy (ERISM). We present the first direct evidence that Willin/FRMD6 expression influences both the cell mechanical phenotype and neuronal differentiation. By investigating cells with increased and decreased Willin/FRMD6 expression levels, we show that Willin/FRMD6 not only affects proliferation and migration capacity of cells but also leads to changes in cell morphology and an enhanced formation of neurite-like membrane extensions. These changes were accompanied by alterations of biophysical parameters such as cell force, the organization of actin stress fibers and the formation of focal adhesions. At the biochemical level, changes in Willin/FRMD6 expression inversely affected the activity of the extracellular signal-regulated kinases (ERK) pathway and downstream transcriptional factor NeuroD1, which seems to prime SH-SY5Y cells for retinoic acid (RA)-induced neuronal differentiation.

The FERM domain-containing protein 6 (FRMD6), also known as Willin, is an upstream regulator of Hippo signaling that has recently been shown to modulate actin cytoskeleton dynamics and mechanical phenotype of neuronal cells through ERK signaling. Physiological functions of Willin/FRMD6 in the nervous system include neuronal differentiation, myelination, nerve injury repair, and vesicle exocytosis. The newly established neuronal role of Willin/FRMD6 is of particular interest given the mounting evidence suggesting a role for Willin/FRMD6 in Alzheimer’s disease (AD), including a series of genome wide association studies that position Willin/FRMD6 as a novel AD risk gene. Here we describe recent findings regarding the role of Willin/FRMD6 in the nervous system and its actions in cellular perturbations related to the pathogenesis of AD.

Willin/FRMD6 has been reported as a potential Alzheimer’s disease (AD) risk gene in a series of genome-wide association and neuroimaging studies; however, the mechanisms underlying its potential role in AD pathogenesis remain unknown. Here, we demonstrate the direct effects of Aβ on Willin/FRMD6 expression and position mitochondrial oxidative stress as a novel potential mechanism underlying the role of Willin/FRMD6 in AD pathogenesis. Specifically, using mouse hippocampal HT-22 cells and primary mouse neurons, we show that Aβ induces downregulation of Willin/FRMD6 protein expression. Furthermore, we demonstrate that Willin/FRMD6 knockdown leads to mitochondrial dysfunction and fragmentation, as well as upregulation of ERK1/2 signaling, both of which are reported to be key early features of AD pathogenesis. Importantly, increasing Willin/FRMD6 expression was able to rescue Aβ-induced abnormalities in mitochondrial morphology, function, and energetics. Thus, enhancing Willin/FRMD6 expression holds potential as a therapeutic strategy for protecting against Aβ-induced mitochondrial and neuronal dysfunction.

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