Studies On The Roles Of CD59 In Synaptic Transmission And Alzheimer’s Disease

The complement system is a complex innate immune surveillance system,which is tightly regulated to ensure defense against pathogens and self-debris whilst avoiding host tissue damage.Despite these well-documented roles,accumulating evidence suggests that complement components such as C1q and C3,could serve as"eat me signals" through tagging weak synapses to be eliminated by microglial phagocytosis,thus modulating synaptic connectivity and cognitive function.This complement-dependent process can be over-activated and consequently contributes to synapse loss underlying cognitive deficits in a number of neurodegenerative diseases,including Alzheimer’s disease(AD)in which it has been shown that decrease of CD59,a ubiquitously expressed cell surface complement inhibitor,correlates with synaptic loss,indicating that CD59 may protect synapses from complement-mediated elimination.However,in the process of complementdependent synaptic elimination,whether CD59 works downstream as a brake to inhibit complement activation,or functions directly as a "do not eat me" signal,thus protecting synapses against microglial phagocytosis,remains elusive.And the role of CD59 in AD is also unknown.In the present study,using a combination of transgenic knockout mice(CD59-/-)as well as CD59 deficient AD mice(5×FAD/CD59-/-)with analyses of animal behavior,patch-clamp recordings and molecular biology techniques,we have made the following key discoveries:1.Loss of CD59 impairs spatial memoryTo address whether CD59 is involved in learning and memory,we performed behavioral analysis in CD59-/-mice using three general behavioral paradigms:an Open Field test for analysis of locomotor activity,Y Maze test and the Barnes Maze test for assessment of hippocampal-dependent learning and memory.The locomotor activity of CD59-/-mice was normal,as shown by similar total distance traveled compared to WT mice in the Open Field test.In the Y Maze test,there were also no differences in the performance between the two groups.In the probe phase of the Barnes Maze task,CD59-/-mice spent significantly longer time to reach the target hole,indicating impaired spatial reference memory.Other behavioral tests were also performed on WT and CD59-/-mice and no differences were observed between genotypes in the Trace Fear Conditioning test,Novel Object Recognition test and Accelerating Rotarod task.While in the Elevated Plus Maze,CD59-/-mice spent much more time in the center area of the maze,indicating impaired approachavoidance conflict decision-making.2.Loss of CD59 specifically impairs GABAergic synaptic transmissionTo investigate the neuronal mechanisms underlying the deficits in spatial memory in CD59-/-mice,we analyzed synaptic function in acute brain slices with the patch clamp technique.Using extracellular recordings of field excitatory postsynaptic potential and comparing high-frequency stimulation(HBS)induced long term potentiation,we found no differences between WT and CD59-/-mice,indicating that loss of CD59 dose not affect long term synaptic plasticity.Next,we performed whole-cell patch-clamp recordings of excitatory and inhibitory postsynaptic currents(EPSCs and IPSCs),and found that loss of CD59 impairs GABAergic but not glutamatergic synaptic transmission.3.Loss of CD59 does not alter microglial engulfment of inhibitory synapsesAlthough the widely reported complement-dependent microglial phagocytosis of synapses has been mainly focused on excitatory synapses,recent reports have also shown that complement could mediate microglial pruning of inhibitory synapses.To investigate whether the deficits in GABAergic synaptic transmission in CD59-/-mice were resulted from increased microglial engulfinent of inhibitory synapses,we first confirmed the presence of CD59 in inhibitory synapse,but immunofluorescence staining of Ibal and Clqa showed no differences between WT and CD59-/-mice.Three-dimensional surface rendering analysis revealed that a similar amount of VGAT puncta in CD68+phagolysosomes from CD59-/-and WT microglia,demonstrating normal microglial engulfment of VGAT in CD59-/-mice,indicating that microglial pruning of inhibitory synapses dose not account for the deficits in GABAergic synaptic transmission in CD59-/-mice.4.Loss of CD59 impairs Ca2+-dependent GABA releasePrevious studies have shown that CD59 could regulate insulin secretion in pancreatic β-cells,while crosslinking of membrane bound CD59 could lead to elevation of intracellular Ca2+ levels in T cells,indicating that CD59 may be able to regulate Ca2+-dependent synaptic vesicle(SV)release.To this end,we manipulated presynaptic Ca2+influx through Voltage-Gated Calcium Channels(VGCCs)by either blocking VGCCs with Cd2+ or increasing their opening probability with higher extracellular K+concentration,and found that both treatments completely abolished the deficits of GABAergic synaptic transmission in CD59-/-mice.Further analysis revealed a direct interaction between CD5 9 and VAMP2——a core soluble N-ethylmaleimide-sensitive factor attachment protein receptor(SNARE)protein residing on SV and CD59 could increase SNARE complex assembly.Taken together,these results indicate that CD59 regulates GABA SV release via interaction with SNARE complexes.5.Loss of CD59 in AD mice exacerbates complement activation and gliosisPrevious findings indicate that inadequate control of complement activation due to diminished CD59 protein level,could be the primary underlying mechanism of complement over-activation in the AD brain.As a preliminary study,we made a CD59 deficient AD mouse model(5×FAD/CD59-/-)by crossing CD59-/-mice with AD mice(5×FAD),and found increased C1q level as well as microglia activation in the brain compaired to normal 5×FAD mice,indicating that loss of CD59 exacerbates complement activation and gliosis.6.Loss of CD59 in AD mice exacerbates cognitive deficitsFinally,using patch-clamp and behavior analysis,we found that loss of CD59 in AD mice led to worsened spatial memory impairments as well as early onset anxiety-related behaviors,although no changes in neither neuronal excitability nor synaptic transmission were detected.Conclusions:We showed compelling evidence that the endogenous complement inhibitor CD59 could play an active role in modulating synaptic function through direct interaction with a core SNARE protein on SV membrane,which is required for Ca2+-triggered neurotransmitter release including GABA.This broadens the established view of complement tagging weak synapses for elimination by microglial phagocytosis.While under pathological conditions such as AD,loss of CD59 could lead to complement overactivation,as well as worsened cognitive performance.Given that disturbance in GABA signaling in addition to complement dysfunction is increasingly implicated in multiple neurologic and psychiatric disorders in addition to AD,our work may provide additional insights to the molecular mechanisms underlying pathogenesis and disease progression,and lead to novel therapeutic approaches.