Studies on the effects of FcγRIIb variant 2 and Lyn on amyloid-beta neurotoxicity in Alzheimers disease

Abstract

Amyloid beta (Aβ) is the major component of the extracellular senile plaque, a pathological hallmark seen in the brain of Alzheimers disease (AD) patients, and gives a toxic edge to neuron in several ways. Some membrane proteins have been suggested to be Aβ receptors sine they bind to soluble Aβ and trigger neurotoxic pathway. Fc-receptor IIb (FcγRIIb) serves as a receptor for soluble Aβ oligomer. Genetic deletion of FcγRIIb ameliorates neuronal death and LTP impairment induced by Aβ oligomer and rescues memory deficits seen in PDAPP AD mouse model. Recently, it is reported that alteration of phosphoinositide metabolism elicited by Aβ-FcγRIIb interaction is responsible for the hyperphosphorylation of tau and memory impairment in 3x Tg-AD mouse model. Here, I demonstrate another mechanistic study supporting the pathogenic role of FcγRIIb in AD through neuronal internalization of Aβ. In addition, I identify the role of Lyn in mediating phosphorylation of FcγRIIb under Aβ exposure and propose the protective effect of a putative Lyn inhibitor isolated by virtual screening. Intraneuronal Aβ is characterized in the brain of early-phase AD patients. Some AD mouse models with intensive intraneuronal Aβ even manifest memory loss without extracellular amyloid deposits. Further, the propagation of pathogenic Aβ between neurons is linked to accumulation of Aβ in neuron. However, molecular mediator to be in charge of neuronal Aβ accumulation and its propagation to adjacent neuron is unclear. In chapter I, I demonstrate the role of FcγRIIb2 variant in neuronal uptake and interneuronal spreading of pathogenic Aβ. Neuronal uptake of oligomeric Aβ1-42 was reduced in Fcgr2b knockout neurons compared to WT neuron. In 3x Tg-AD mouse model, both intraneuronal and soluble Aβ1-42 were diminished by loss-of-function of FcγRIIb. Moreover, memory deficits were alleviated by forebrain-specific expression of Aβ-uptake-defective Fcgr2b mutant in 3xTg-AD mice. I also found that enriched expression of transcription variant 2 of FcγRIIb (FcγRIIb2) in neuron is associated with elevated uptake of Aβ. Target of Myb1 (TOM1) isolated from functional screening bind to FcγRIIb2 and suppress FcγRIIb2-mediated Aβ internalization. Accelerated uptake of Aβ via FcγRIIb2 leads to cytoplasmic and mitochondrial accumulation of Aβ over endocytic compartments and causes neurotoxicity. Finally, blockade of propagation of intraneuronal Aβ to connected neuron by Fcgr2b KO is analyzed by using microfluidic devices. To sum up, I demonstrate a novel function of FcγRIIb2 variant in the pathogenesis of AD by regulating intraneuronal Aβ. Lyn is originally considered to be a member of Src-family tyrosine kinase and take a role in inhibiting immune cells to phosphorylate immune complex binding proteins, including FcγRIIb. However, its association to Aβ-FcγRIIb signaling in neuron is uncertain. In Chapter II, I demonstrate that Lyn is critical for Aβ1-42-mediated neurotoxicity by phosphorylating FcγRIIb. Aβ1-42 oligomer activated Lyn and further induced the tyrosine phosphorylation of FcγRIIb in neuronal cells. Interestingly, both Aβ1-42-initiated FcγRIIb phosphorylation and cell death were reduced by knockdown of Lyn in neuronal cells. A small molecule KICG2576, a putative ATP-competitive inhibitor of Lyn was isolated from virtual screening. Binding of KICG2576 to the cleft between the two lobes of Lyn kinase domain was verified by co-crystal structure. Whereas Aβ-induced FcγRIIb phosphorylation was attenuated by KICG2576, Aβ- or FcγRIIb-induced neuronal cell death were also suppressed. Aβ1-42-induced memory impairment determined by Y-maze and novel object recognition tests were also diminished by intracerebroventricularly injected KICG2675. Finally, I confirmed the activation of Lyn in the brain of AD patients compared to non-AD controls. Taken together, Lyn plays an essential role in Aβ–mediated neurotoxicity, providing detailed understanding of Aβ- FcγRIIb neurotoxic pathway in AD pathogenesis.