PINK1-mediated Drp1 Phosphorylation Modulates Synaptic Development And Plasticity

Background:Parkinson’s disease（PD）is a chronic,progressive,neurodegenerative disease.The post-mortem reports of PD patients and animal models confirmed synaptic abnormalities.Dendritic spines are highly dynamic postsynaptic structures.The morphological change of dendritic spine affects synaptic plasticity and function,which play an essential role in synaptic transmission.These changes require lots of energy,thus,the formation of synapses and neural circuits are closely related to mitochondrial dynamics.Mitochondria are crucial intracellular energy supply organelles.Mitochondrial dynamics is considered as a key mechanism involved in regulating energy supply,calcium homeostasis,and neurotransmitter synthesis.Drp1 is a critical GTPase involved in regulating mitochondrial dynamics.Post-translational modifications,such as phosphorylation and SUMOylation,to Drp1 are important for its fission activity.Disruption of Drp1 leads to impairment of synaptic plasticity and function.PINK1,as one of the pathogenic genes of PD,patients with PINK1 mutations present cognitive and mental abnormalities.Most studies suggest that PINK1 aggregates on damaged mitochondria and induces mitophagy by recruiting Parkin and other autophagy-related proteins.Meanwhile,PINK1 regulates mitochondrial fission by phosphorylation of Drp1 S616.Studies suggested that the loss of PINK1 might affect synaptic plasticity and function.However,the underlying mechanism remains elusive.Objective:To clarify the mechanism of the abnormal synaptic phenotype caused by PINK1 and link mitochondrial dynamics with the development and function of neural circuits,provide new ideas for the detection and treatment of PD non-motor disorders.Methods:1)The morphology and number of dendritic spine/mitochondria of PINK1 knockout（KO）,Drp1 S616A（phosphor-null variant of Drp1）knockin（KI）mutant mice,and primary neurons were detected by immunofluorescence,Western Blot,and transmission electron microscopy,etc.2)The electrophysiological characteristics of cortical pyramidal neurons were recorded by whole-cell patch clamp to detect the effects of PINK1 and phosphorylation of Drp1S616 on the electrophysiological characteristics.3)The effects of PINK1and phosphorylation of Drp1 S616 on long-term potentiation（LTP）of CA3-CA1 synapse was detected by microelectrode array.4)The changes of phosphorylation level and mitochondrial localization of Drp1 S616before/after chemical induction of LTP（c LTP）were detected by immunofluorescences and Western Blot.5)The cognitive function of PINK1^KOand Drp1^{S616A KI} mutant mice were tested by Morris Water Maze（MWM）and Fear-conditioning.Results:We found that:1)PINK1 regulates both the morphology and density of mitochondria and dendritic spines and the formation of excitatory synapses.PINK1^KO mice showed increased mitochondrial length and decreased mitochondrial density in dendrites.As well as the defects of dendritic spines in maturation.PINK1 absence leads to decreased excitatory synapses,PSD95 expression and PSD length;2)Loss of PINK1 impairs synaptic transmission and LTP.Miniature excitatory postsynaptic currents（m EPSCs）decreased significantly in amplitude and frequency of cortical pyramidal neurons in PINK1^KO mice,and LTP induction and maintaenance were also abnormal;3)PINK1regulates synapse maturation through Drp1 S616 phosphorylation.The Drp1 S616 phosphorylation level was decreased in PINK1^KO mice.Drp1^{S616A KI} mice showed a phenotype similar to PINK1^KO mice,such as decreased spine density and abnormal synaptic transmission,etc.The overexpressing of Drp1^WT/Drp1^S616D（phosphor-mimetic）in PINK1^KOneurons restored the abnormal dendritic spines caused by PINK1 deletion;4)PINK1-dependent Drp1 S616 phosphorylation is critical for LTP induction and maintenance.The amplitude and frequency of m EPSC in cortical pyramid neurons of Drp1^{S616A KI} mice were decreased.The induction and maintenance of LTP were abnormal in Drp1^{S616A KI} mice.The phosphorylation of Drp1 S616 increased significantly after c LTP-induction and was localized to mitochondria,and this increase was PINK1-dependent;5)PINK1-mediated Drp1 S616 phosphorylation regulates mitochondrial fission during synapse development.The length of mitochondria in Drp1^{S616A KI} mouse tissues and primary neurons was abnormally elongated.The overexpression of Drp1^WT/Drp1^S616D mutants in PINK1^KO neurons rescued the abnormal mitochondria.The loss of PINK1 caused the decrease of ATP content in dendrites,and piracetam,a drug that can improve mitochondrial function and increase ATP production,can partially restore the changes of dendritic spines caused by PINK1 loss;6)PINK1-mediated Drp1 S616 phosphorylation regulatesmitochondrial localization and invasion to synapses.PINK1 loss leads to decreased mitochondrial localized to the vicinity of dendritic protrusions before and after c LTP-induction.The Drp1^S616D mutant restored the reduction of mitochondria in dendritic protrusions;7)Drp1^{S616A KI} mice exhibited impaired hippocampal-dependent memory.In MWM,Drp1^{S616A KI} mice showed more latency and crossing times compared to the control group.In fear conditioning,Drp1^{S616A KI} mice showed less freezing time than the control group.Conclusions:Our study found that PINK1 absence leads to abnormal synaptic development and plasticity in mice.Provide evidence that Drp1 S616 phosphorylation plays an important role in synaptic development and plasticity.Demostrated that PINK1 regulates mitochondrial dynamics by mediating the phosphorylation of Drp1 at S616,thereby participating in the development and maturation of excitatory circuits and synaptic plasticity in the hippocampus and cortex.