Gene Expression Profile of THP-1 Monocytes Following Knockdown of DAP12, A Causative Gene for Nasu-Hakola Disease

Abstract

Nasu-Hakola disease (NHD), also designated polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy, is a rare autosomal recessive disorder characterized by progressive presenile dementia and formation of multifocal bone cysts, caused by a loss-of-function mutation of DAP12 or TREM2. TREM2 and DAP12 constitute a receptor/adaptor complex expressed on osteoclasts, dendritic cells, macrophages, monocytes, and microglia. At present, the precise molecular mechanisms underlying development of leukoencephalopathy and bone cysts in NHD remain largely unknown. We established THP-1 human monocyte clones that stably express small interfering RNA targeting DAP12 for serving as a cellular model of NHD. Genome-wide transcriptome analysis identified a set of 22 genes consistently downregulated in DAP12 knockdown cells. They constituted the molecular network closely related to the network defined by cell-to-cell signaling and interaction, hematological system development and function, and inflammatory response, where NF-κB acts as a central regulator. These results suggest that a molecular defect of DAP12 in human monocytes deregulates the gene network pivotal for maintenance of myeloid cell function in NHD.

TYROBP in Alzheimer’s Disease

“…The alteration in TYROBP expression under pathological conditions would lead to significant changes in levels of its downstream genes. As demonstrated by a recent study from Satoh et al, in TYROBP knockdown monocytes, a set of 22 genes was identified to be differentially expressed when compared with normal monocytes [27]. More importantly, Zhang and colleagues recently found that overexpression of TYROBP in microglia resulted in dramatic alterations in numerous downstream genes involved in RNA metabolism, cell cycle mitosis, vacuole/autophagy, mitochondrial dysfunction, and histone assembly [7], most of which were closely associated with known molecular pathologies in LOAD including abnormalities of cell cycle as well as dysfunction of autophagy and mitochondrion [47,48].…”

“…Besides, TYROBP was also reported to suppress inflammation response which involves the repression of cytokine production and secretion [26]. Furthermore, recent findings have suggested that TYROBP-mediated signaling also participated in regulating the expression of multiple genes within the brain [7,27].…”

“…However, in some families, no mutation was found in TYROBP suggesting that the disease is characterized by genetic heterogeneity [50]. Although a recent study by Satoh and colleagues reported that a molecular defect of TYROBP in human monocytes deregulates the gene network pivotal for maintenance of myeloid cell function in PLOSL [27], to date, the mechanisms by which loss of function mutations in TYROBP induce cognitive disorder remained to be solved. As TYROBP is known to express on microglia in the brain, some researchers suggested that the loss of function of TYROBP may lead to the microglia dysfunction, which subsequently caused cognitive deficits in these disease [51].…”

“…Early studies have suggested that a molecular defect of TYROBP in human monocytes deregulates the gene network pivotal for maintenance of myeloid cell function in PLOSL [27]. Recently, Zhang and colleagues [7] applied an integrative network-based approach for rank-ordered network structures for relevance to LOAD pathology, highlighting the immune/microglia module as the molecular system most strongly associated with the pathophysiology of LOAD and identified the key network regulators in the pathogen phagocytosis pathways, including TYROBP as the highest ranking in terms of regulatory strength and differential expression in LOAD brains.…”

Deficits in the miRNA-34a-regulated endogenous TREM2 phagocytosis sensor-receptor in Alzheimer's disease (AD); an update

“…Briefly, Aβ42 peptides were initially solubilized in hexafluoroisopropanol (HFIP; Fluka Chemical, cat# 52512; Sigma-Aldrich, St. Louis MO), aliquoted, and stored at −20°C as an HFIP film. After vacuum evaporation of HFIP, aliquoted peptide was re-suspended with DMSO to 5 mM and diluted to 5 μM into the cell culture media; cells were subsequently stained using a murine amyloid beta MABN10 (red fluorescence λ max ~650 nm; anti-Aβ antibody, clone W0-2; Millipore, Bellerica MA), a TREM-2 antibody (M-227): sc-48765 (green fluorescence; λ max ~510 nm; Santa Cruz, Santa Cruz CA) or DAPI nuclear stain; magnification 60×; note self-aggregation of Aβ42 peptide after 24 h and Aβ42 peptide affinity for TREM2 containing cells (leftmost panels) and internalization (rightmost panel; yellow merge; λ max ~580 nm); additional relevant methods have been described (Griciuc et al, 2013; Zhao and Lukiw, 2013); (B) highly schematicized depiction of the possible actions of an NF-kB-regulated, miRNA-34a-mediated TREM2 sensor-phagocytosis protein down-regulated in AD brain; the triggering receptor for myeloid/microglial cells (TREM2) is a variably glycosylated transmembrane receptor known to be enriched in the microglial cell plasma membrane; signaling via the tyrosine kinase-binding protein (DNAX activation protein 12) [TYROBP (DAP12)] accessory receptor results in phagocytosis and ultimately, clearance of Aβ42 peptides (red ovals) from the extracellular space (Satoh et al, 2011; Benitez et al, 2013; Forabosco et al, 2013; Hickman and Khoury, 2014); interestingly, TREM2 knockout/knockdown mice have attenuated immunological and inflammatory responses and/or increases in age-related neuroinflammatory markers and cognitive deficiency (Jiang et al, 2013; Sieber et al, 2013); TYROBP knockout mice exhibit immune system deficits and an impairment in microglial cell differentiation (Nataf et al, 2005; Sieber et al, 2013); it is not clear what, if any, contribution TREM2 makes directly to phagocytosis and Aβ42 clearance (question mark) without TYROBP (DAP12); while no deficits in TYROBP (DAP12) have been observed in sporadic AD brain, insufficient TREM2 may be in part responsible for the inability to adequately phagocytose Aβ42 peptides, resulting in their buildup and self-aggregation in the extracellular space. Inset: miRNA-34a is found to be significantly increased in AD hippocampal CA1 and superior temporal lobe and in stressed microglial cells; miRNA-34a targeting of the TREM2 mRNA 3′-UTR appears to be in part responsible for this (see text); because miRNA-34a is encoded on an NF-kB-sensitive transcript, both anti-NF-kB and/or anti-miRNA strategies may be clinically useful in the restoration of homeostatic phagocytosis in the brain and CNS.…”