D-F) Teased sciatic nerve fibers were treated with 1% Triton X-100 and antibodies specific for neurofilament (red) (D), and Myo1d (E). in the Purkinje cell layer, granule cell layer, and region of the cerebellar nuclei. Upon the onset of myelination, myosin-1d enrichment expands along axonal tracts, while still present in the Purkinje and granule cell layers. However, myosin-1d was undetectable in Rucaparib (Camsylate) oligodendrocyte progenitor cells at early and late time points. We also show that myosin-1d interacts and is co-expressed with aspartoacylase, an enzyme that plays a key role Rucaparib (Camsylate) in fatty acid synthesis throughout the nervous system. Together, these studies provide a foundation for understanding the role of myosin-1d in neurodevelopment and neurological disorders. development (Hozumi et al., 2006; Speder et al., 2006). Aside from these initial reports, our understanding of Myo1d function in the context of vertebrate physiology remains largely unexplored. Three recent lines of evidence suggest that Myo1d plays an important role in nervous system tissues. First, linkage analysis of autistic individuals revealed a potential association with MYO1D (Stone et al., 2007). Second, mass spectrometry studies have identified Myo1d as an enriched component of the myelin proteome (Ishii et al., 2009; Jahn et al., 2009; Yamaguchi et al., 2008). Third, Myo1d is usually a significantly upregulated transcript during oligodendrocyte maturation, along with other classical myelin-associated components (Cahoy et al., 2008; Nielsen et al., 2006). All of these investigations implicate Myo1d in neurodevelopment and further suggest that this Rucaparib (Camsylate) motor plays a role in the process of myelination. However, there is currently no cell biological data to validate or extend the results derived from these broad screening studies. The goal of this study was to investigate the expression, localization, and function of Myo1d during neurodevelopment. Here, we show that Myo1d is present in both peripheral (PNS) and central nervous systems (CNS). In the CNS, our analysis focused on the cerebellum, where Myo1d expression is limited to neurons, exhibiting a punctate distribution along axons and in cell bodies. This motor was not found in glial cells as expected based on previous studies (Cahoy et al., 2008; Nielsen et al., 2006). We also identified aspartoacylase as a putative binding partner for Myo1d in Purkinje cells. Aspartoacylase functions in fatty acid synthesis and mutations in this protein lead to leukodystrophy (Namboodiri et al., 2006). Together, these findings hold implications for understanding the contribution of Myo1d to neurodevelopment and neurological disorders such as autism or Canavan disease. 2. Results 2.1 Myo1d is present in myelinating and non-myelinating cells of the PNS Myo1d was originally identified in the rat cerebrum, spinal cord (Bahler et al., 1994), and sciatic nerve (Lund et al., 2005). Recently, microarray studies exhibited that Myo1d transcripts are present in oligodendrocytes (Cahoy et al., 2008), and proteomic studies suggest that this motor is also associated with myelin (Ishii et al., 2009; Jahn et al., 2009; Yamaguchi et al., 2008). To further develop our understanding Myo1d function in myelinating cells and neurons, we used high-resolution confocal imaging to characterize the distribution of this motor in the PNS and CNS. To this end, we first dissected mouse sciatic nerve bundles for immuno-fluorescence labeling and confocal imaging. To visualize the distribution of Myo1d in sciatic nerve, the nerve bundle was teased into constituent fibers (a single axon wrapped by Schwann cells), which were then stained with antibodies targeting Myo1d, myelin basic protein (MBP) to label Schwann cells Prkd2 (Mirsky et al., 1980), or neurofilament light chain to label axons (Fabrizi et al., 1997; Sotelo-Silveira et al., 2000). Interestingly, Myo1d exhibited strong co-localization with MBP along the myelin sheath enveloping neurons (Fig. 1ACC). In teased fibers that were exposed to higher levels of Triton X-100 (1%) to increase permeabilization, the motor maintained localization along the myelin sheath, but also co-localized with neurofilament labeling along the length of axons (Fig. 1DCF). These high-resolution confocal images indicate that Myo1d is present in both neurons and myelinating cells in the PNS. These data are also consistent with Western blots of sciatic nerve samples (data not shown), brightfield studies demonstrating Myo1d is in the sciatic nerve.