br Material and methods br Results br Discussion The
Material and methods
Discussion The results show for the first time that DDRs may be involved in the pathogenic mechanisms of neurodegenerative diseases. We found that DDR1 and DDR2 levels are up-regulated in post-mortem AD and PD brains, suggesting that these receptors play a role in neurodegenerative pathologies. It appears that both isoforms of DDR are involved in neurodegenerative pathologies. DDR1 activation upregulates matrix metalloproteinase (MMP)-9 expression (Roig et al., 2010), which can impair the blood AY 9944 dihydrochloride mg barrier (BBB) (Gao et al., 2016). DDR1 is also activated after middle cerebral artery occlusion, but DDR1 and DDR2 knockdown reduces BBB permeability via MMP-9 inhibition (Zhu et al., 2015). MMP-9 is also upregulated in the ischemic brain (Wu et al., 2012) and may contribute to damaging the BBB (Lakhan et al., 2013). On the other hand, DDR1 inhibition attenuates microglial activity and reduces inflammation (Seo et al., 2008). Taken together, previous findings and our data indicating reduction of microglia and TREM2 in the brain after DDR1 and DDR2 knockdown suggest that DDR1 and DDR2 knockdown may prevent degradation of the BBB and regulate brain immunity. Using RNA-seq Roig et al. (2010) found that DDRs are predominantly expressed on oligodendrocytes. Our data from cell culture, post-mortem brains and mouse brains show that DDR1 and 2 are widely expressed in the brain. It appears that DDR expression is predominantly increased in neurons compared to glial cells in the post-mortem AD and PD brains, but more studies are needed to determine the localization of DDRs to different cell types. This is the aim of our future investigation. It is important to determine the role of DDRs in neurons and oligodendroglia separately to better understand the mechanisms by which these cells may reduce neurotoxic protein levels. α-synuclein and tau accumulate in oligodendrocytes in multiple system atrophy (MSA) and progressive supranuclear palsy (PSP), respectively. Oligodendrocytes may also affect the brain immune response as is seen in multiple sclerosis. Therefore, the role of oligodendrocytes and mechanisms of protein clearance should be carefully examined in future studies. Although, autophagy, BBB and immune regulation are likely responsible for neurotoxic protein clearance after DDRs knockdown, it is also possible that DDRs directly interact with neurotoxic proteins and this is another area for future investigations. TREM2 is found in various tissue macrophages, including CNS microglia and monocyte derived dendritic cells (Paloneva et al., 2002, Colonna and Wang, 2016). We found that both TREM2 and its adaptor protein DAP12 (Peng et al., 2010) are reduced after DDR1 and DDR2 knockdown, suggesting attenuation in TREM2 signaling. TREM2 signaling may promote phagocytosis of neurotoxic proteins (Takahashi et al., 2005) and neuronal survival (Wang et al., 2015). However, TREM2 can have both activating and inhibitory functions (Colonna and Wang, 2016). In vitro studies showed that TREM2 overexpression on microglia improves phagocytosis, while silencing TREM2 impairs microglia function (Takahashi et al., 2005). Further studies showed that TREM2 overexpression on microglia promotes Aβ phagocytosis (Jiang et al., 2014, Melchior et al., 2010). TREM2 may also reduce secretion of pro-inflammatory cytokines by myeloid cells and attenuate macrophage activation (Turnbull et al., 2006, Hamerman et al., 2006, Takahashi et al., 2005). In vivo studies show that Aβ deposition leads to microgliosis in normal TREM2 expressing mice, but microglial accumulation around plaques is reduced in TREM2-deficient mice (Jay et al., 2015, Wang et al., 2015). Our findings show that DDR1 and DDR2 knockdown reduces TREM2 and microglial number, perhaps to attenuate the inflammatory response. TREM2 variants have been found in several neurodegenerative diseases including AD and PD (Lill et al., 2015), suggesting that TREM2 deficiency and microglial function is a possible risk factor in these diseases. However, the precise effect of TREM2 in these pathologies is unclear and data about TREM2 indicate conflicting results (Colonna and Wang, 2016, Lill et al., 2015). The current data suggest that DDR1 and DDR2 are a possible upstream effector of TREM2 signaling, which may be upregulated in response to DDR activation as a possible common pathway in AD and PD. DDR1 and DDR2 knockdown protects against cell death in vivo and in vitro and reduces the levels of several cytokines that regulate adaptive immunity. The levels of IL-2 and IL-3 are reduced after DDR1 and DDR2 knockdown suggesting alteration of adaptive immunity (Ihle et al., 1981). IL-3 promotes proliferation of monocytes and dendritic cells (Ihle et al., 1981), which express TREM2 (Paloneva et al., 2002, Colonna and Wang, 2016). CCL5 recruits T cells into inflammatory sites, and promotes natural-killer cell proliferation (Maghazachi et al., 1996, Vangelista et al., 2010). Therefore, activation of T cell receptors stimulates the secretion of IL-2 and survival of antigen-specific CD4+ and CD8+ T cells (Beadling et al., 1993, Beadling and Smith, 2002, Stern and Smith, 1986) leading to formation of the immunological memory and a long term adaptive immune response. The attenuation of the immunological memory is associated with clearance of α-synuclein and p-tau in A53T mice and reduction of the anti-inflammatory cytokine IL-10, which indicates diminution of CNS inflammation.