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  • Collectively the results presented here provide

    2021-07-22

    Collectively, the results presented here provide new insights into ligand binding, clustering, spatial distribution, and phosphorylation of DDR1b and DDR2 in response to soluble collagen I. As depicted in the cartoon of Scheme 1, we postulate a model in which the spatial distribution and assembly of DDRs is dependent on the morphological state of collagen and precedes receptor phosphorylation. In this model, we propose that DDR1b cluster formation is promoted by the presence of non-fibrillar collagen present during the early stages of collagen fibrillogenesis. These DDR1b clusters undergo Tipifarnib to early endosomes, within a few minutes of collagen stimulation, as shown in our earlier studies [16]. At later time points, a fraction of DDR1b receptor clusters may recycle back to the plasma membrane with their cargo. During this process of recycling, a sub-population of DDR1b clusters is enriched with phosphorylated receptor species at Y513. Whether these phosphorylated DDR1b clusters localize in the endosome or the cell-membrane cannot be presently deciphered. Further studies are required to dissect the molecular composition and sub-cellular location of DDR1b clusters, which may be responsible for specific cell-signaling pathways, as has been defined for other members of the receptor tyrosine kinase family [37], [38]. In this regard, it is interesting to note that DDR1 has also been reported to co-internalize with and phosphorylate upon stimulation of insulin-like-growth factor I (IGF-IR) receptor, and the collagen-dependent phosphorylation of DDR1 was impaired in the absence of IGF-IR [39]. Finally, it is important to emphasize the limitations and sensitivity of the techniques and reagents used (e.g., a limited number of anti-phosphotyrosine antibodies) here to follow the spatial–temporal profile of receptor phosphorylation. Another caveat in our studies was that only partial and/or intermittent co-localization of immuno-stained collagen was observed with the clusters and filamentous structures formed by DDRs. We postulate that this could be due to masking of antibody-recognizing epitopes on collagen as a result of DDR binding. Future studies using additional antibodies, fluorescently labeled collagen, submicroscopic high-resolution imaging of molecular complexes, laser capture-microdissection [40] and identification of phosphorylated receptor species by phospho-proteomics approaches at various time points are warranted to elucidate the state of collagen and of these unique collagen receptors upon ligand interactions.
    Acknowledgment This work was supported by NSF CMMI award 1201111 to G.A., AHA predoctoral award 16PRE31160013 to D.Y., and grants from the NIH-NCI (CA1986), Department of Defense (W81XWH-15-1-0226), and the Sky Foundation to R.F. We acknowledge Nabanita Chatterjee at OSU for her assistance with cell-culture experiments.
    Introduction Discoidin domain receptors (DDRs), including DDR1 and DDR2, are members of the receptor tyrosine kinase (RTK) family and they share 89% homology in their tyrosine kinase domain (Vogel et al., 2006). DDRs bind to native forms of various collagens (Shrivastava et al., 1997, Vogel et al., 1997). Peripherally, DDR1 promotes the accumulation of macrophages in atherosclerotic sites (Franco et al., 2009), and DDR1 null mice exhibit reduction of macrophage accumulation, attenuation of the inflammatory response, and suppression of fibrosis in mouse models of atherosclerosis (Franco et al., 2008), renal disorder (Flamant et al., 2006), and lung fibrosis (Avivi-Green et al., 2006). DDR2 activation in fibroblasts promotes cell proliferation and remodeling of the extracellular matrix (ECM) (Olaso et al., 2002). DDRs mediate cell division and differentiation, and may regulate myeloid-derived glial cells (Kamohara et al., 2001, Roig et al., 2010, Seo et al., 2008), providing potential protective mechanisms in neurodegeneration. Nilotinib is a potent small-molecule inhibitor against DDRs (Bantscheff et al., 2007, Rix et al., 2007), and we previously demonstrated that nilotinib promotes autophagic clearance of neurotoxic proteins and improves motor and cognitive symptoms in several models of neurodegeneration without evidence of increased inflammation (Hebron et al., 2013, Hebron et al., 2014a, Hebron et al., 2014b, Lonskaya et al., 2013a, Lonskaya et al., 2014b, Lonskaya et al., 2014a, Lonskaya et al., 2015, Moussa, 2015). In this study, we show that DDR1 and DDR2 levels are increased in post-mortem Alzheimer's (AD) and Parkinson's disease (PD) brains. To determine whether DDRs affect the clearance of neurotoxic proteins and can regulate myeloid-derived glial cells to modulate inflammation, we used lentiviral gene transfer to specifically knockdown DDR1 or DDR2 and measured α-synuclein, tau and β-amyloid (Aβ) levels, cell death, and inflammatory markers in vitro and in vivo. We found that knockdown of DDR1 and DDR2 leads to neurotoxic protein clearance, attenuation of cell death and reduction of the number of microglia, and triggering receptors expressed on myeloid cells (TREM)-2. These findings indicate possible regulation of myeloid-derived microglia and protection against neurotoxic proteins.