Phosphorylation of 53BP1 by ATM enforce neurodevelopmental programs in cortical organoids.

53BP1 is a well-established DNA damage repair factor recently shown to regulate gene expression and critically influence tumor suppression and neural development. For gene regulation, how 53BP1 is regulated remains unclear. Here, we showed that 53BP1-serine 25 phosphorylation by ATM is required for neural progenitor cell proliferation and neuronal differentiation in cortical organoids. 53BP1-serine 25 phosphorylation dynamics controls 53BP1 target genes for neuronal differentiation and function, cellular response to stress, and apoptosis. Beyond 53BP1, ATM is required for phosphorylation of factors in neuronal differentiation, cytoskeleton, p53 regulation, and ATM, BNDF, and WNT signaling pathways for cortical organoid differentiation. Overall, our data suggest that 53BP1 and ATM control key genetic programs required for human cortical development.

Cardioprotective role of APIP in myocardial infarction through ADORA2B.

In ischemic human hearts, the induction of adenosine receptor A2B (ADORA2B) is associated with cardioprotection against ischemic heart damage, but the mechanism underlying this association remains unclear. Apaf-1-interacting protein (APIP) and ADORA2B transcript levels in human hearts are substantially higher in patients with heart failure than in controls. Interestingly, the APIP and ADORA2B mRNA levels are highly correlated with each other (R = 0.912). APIP expression was significantly increased in primary neonatal cardiomyocytes under hypoxic conditions and this induction reduced myocardial cell death via the activation of the AKT-HIF1α pathway. Accordingly, infarct sizes of APIP transgenic mice after left anterior descending artery ligation were significantly reduced compared to those of wild-type mice. Strikingly, knockdown of APIP expression impaired the cytoprotective effects of ADORA2B during hypoxic damage. Immunoprecipitation and proximity ligation assays revealed that APIP interacts with ADORA2B, leading to the stabilization of both proteins by interfering with lysosomal degradation, and to the activation of the downstream PKA-CREB signaling pathways. ADORA2B levels in the hearts of APIPTg/Tg, APIPTg/+, and Apip+/- mice were proportionally downregulated. In addition, ADORA2B D296G derived from the rs200741295 polymorphism failed to bind to APIP and did not exert cardioprotective activity during hypoxia. Moreover, Adora2b D296G knock-in mice were more vulnerable than control mice to myocardial infarction and intentional increases in APIP levels overcame the defective protection of the ADORA2B SNP against ischemic injury. Collectively, APIP is crucial for cardioprotection against myocardial infarction by virtue of binding to and stabilizing ADORA2B, thereby dampening ischemic heart injury.

Amelioration of amyloid ß-FcγRIIb neurotoxicity and tau pathologies by targeting LYN.

SRC-family kinases (SFKs) have been implicated in Alzheimer's disease (AD), but their mode of action was scarcely understood. Here, we show that LYN plays an essential role in amyloid ß (Aß)-triggered neurotoxicity and tau hyperphosphorylation by phosphorylating Fcγ receptor IIb2 (FcγRIIb2). We found that enzyme activity of LYN was increased in the brain of AD patients and was promoted in neuronal cells exposed to Aß 1-42 (Aß1-42). Knockdown of LYN expression inhibited Aß1-42-induced neuronal cell death. Of note, LYN interacted with FcγRIIb2 upon exposure to Aß1-42 and phosphorylated FcγRIIb2 at Tyr273 within immunoreceptor tyrosine-based inhibitory motif in neuronal cells. With the use of the structure-based drug design, we isolated KICG2576, an ATP-competitive inhibitor of LYN. Determination of cocrystal structure illustrated that KICG2576 bound to the cleft in the LYN kinase domain and inhibited LYN with a half-maximal inhibitory concentration value of 0.15 µM. KICG2576 inhibited Aß- or FcγRIIb2-induced cell death, and this effect was better than pyrazolopyrimidine 1, a widely used inhibitor of SFK. Upon exposure to Aß, KICG2576 blocked the phosphorylation of FcγRIIb2 and translocation of phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 2, a binding protein to the phosphorylated FcγRIIb2, to the plasma membrane, resulting in the inhibition of tau hyperphosphorylation, the downstream event of Aß1-42-FcγRIIb2 binding. Furthermore, intracerebroventricular injection of KICG2576 into mice ameliorated Aß-induced memory impairment. These results suggest that LYN plays a crucial role in Aß1-42-mediated neurotoxicity and tau pathology, providing a therapeutic potential of LYN in AD.-Gwon, Y., Kim, S.-H., Kim, H. T., Kam, T.-I., Park, J., Lim, B., Cha, H., Chang, H.-J., Hong, Y. R., Jung, Y.-K. Amelioration of amyloid ß-FcγRIIb neurotoxicity and tau pathologies by targeting LYN.

TOM1 Regulates Neuronal Accumulation of Amyloid-ß Oligomers by FcγRIIb2 Variant in Alzheimer's Disease.

Emerging evidences suggest that intraneuronal Aß correlates with the onset of Alzheimer's disease (AD) and highly contributes to neurodegeneration. However, critical mediator responsible for Aß uptake in AD pathology needs to be clarified. Here, we report that FcγRIIb2, a variant of Fcγ-receptor IIb (FcγRIIb), functions in neuronal uptake of pathogenic Aß. Cellular accumulation of oligomeric Aß1-42, not monomeric Aß1-42 or oligomeric Aß1-40, was blocked by Fcgr2b knock-out in neurons and partially in astrocytes. Aß1-42 internalization was FcγRIIb2 di-leucine motif-dependent and attenuated by TOM1, a FcγRIIb2-binding protein that repressed the receptor recycling. TOM1 expression was downregulated in the hippocampus of male 3xTg-AD mice and AD patients, and regulated by miR-126-3p in neuronal cells after exposure to Aß1-42 In addition, memory impairments in male 3xTg-AD mice were rescued by the lentiviral administration of TOM1 gene. Augmented Aß uptake into lysosome caused its accumulation in cytoplasm and mitochondria. Moreover, neuronal accumulation of Aß in both sexes of 3xTg-AD mice and memory deficits in male 3xTg-AD mice were ameliorated by forebrain-specific expression of Aß-uptake-defective Fcgr2b mutant. Our findings suggest that FcγRIIb2 is essential for neuropathic uptake of Aß in AD.SIGNIFICANCE STATEMENT Accumulating evidences suggest that intraneuronal Aß is found in the early step of AD brain and is implicated in the pathogenesis of AD. However, the critical mediator involved in these processes is uncertain. Here, we describe that the FcγRIIb2 variant is responsible for both neuronal uptake and intraneuronal distribution of pathogenic Aß linked to memory deficits in AD mice, showing a pathologic significance of the internalized Aß. Further, Aß internalization is attenuated by TOM1, a novel FcγRIIb2-binding protein. Together, we provide a molecular mechanism responsible for neuronal uptake of pathogenic Aß found in AD.

APIP, an ERBB3-binding partner, stimulates erbB2-3 heterodimer formation to promote tumorigenesis.

Despite the fact that the epidermal growth factor (EGF) family member ERBB3 (HER3) is deregulated in many cancers, the list of ERBB3-interacting partners remains limited. Here, we report that the Apaf-1-interacting protein (APIP) stimulates heregulin-ß1 (HRG-ß1)/ERBB3-driven cell proliferation and tumorigenesis. APIP levels are frequently increased in human gastric cancers and gastric cancer-derived cells. Cell proliferation and tumor formation are repressed by APIP downregulation and stimulated by its overexpression. APIP's role in the ERBB3 pathway is not associated with its functions within the methionine salvage pathway. In response to HRG-ß1, APIP binds to the ERBB3 receptor, leading to an enhanced binding of ERBB3 and ERBB2 that results in sustained activations of ERK1/2 and AKT protein kinases. Furthermore, HRG-ß1/ERBB3-dependent signaling is gained in APIP transgenic mouse embryonic fibroblasts (MEFs), but not lost in Apip-/- MEFs. Our findings offer compelling evidence that APIP plays an essential role in ERBB3 signaling as a positive regulator for tumorigenesis, warranting future development of therapeutic strategies for ERBB3-driven gastric cancer.

Structural and biochemical basis for the inhibition of cell death by APIP, a methionine salvage enzyme.

APIP, Apaf-1 interacting protein, has been known to inhibit two main types of programmed cell death, apoptosis and pyroptosis, and was recently found to be associated with cancers and inflammatory diseases. Distinct from its inhibitory role in cell death, APIP was also shown to act as a 5-methylthioribulose-1-phosphate dehydratase, or MtnB, in the methionine salvage pathway. Here we report the structural and enzymatic characterization of human APIP as an MtnB enzyme with a Km of 9.32 µM and a Vmax of 1.39 µmol min(-1) mg(-1). The crystal structure was determined at 2.0-Å resolution, revealing an overall fold similar to members of the zinc-dependent class II aldolase family. APIP/MtnB exists as a tetramer in solution and exhibits an assembly with C4 symmetry in the crystal lattice. The pocket-shaped active site is located at the end of a long cleft between two adjacent subunits. We propose an enzymatic reaction mechanism involving Glu139* as a catalytic acid/base, as supported by enzymatic assay, substrate-docking study, and sequence conservation analysis. We explored the relationship between two distinct functions of APIP/MtnB, cell death inhibition, and methionine salvage, by measuring the ability of enzymatic mutants to inhibit cell death, and determined that APIP/MtnB functions as a cell death inhibitor independently of its MtnB enzyme activity for apoptosis induced by either hypoxia or etoposide, but dependently for caspase-1-induced pyroptosis. Our results establish the structural and biochemical groundwork for future mechanistic studies of the role of APIP/MtnB in modulating cell death and inflammation and in the development of related diseases.