A Mechanistic Role Of HnRNP DL For The Regulation Of Brain Aging And Alzheimer’s Disease-related Cognitive Impairment

Brain aging,a precursor of the organism aging,is an irreversible physiological process,accompanied by compromised cognitive function and memory loss.Brain aging is also a major risk factor for Alzheimer’s disease(AD).During the brain aging process,neuronal cells occur complex cellular and functional changes,including the process of gene expression regulation.In eukaryotic cells,RNA alternative splicing is an essential type of post-transcriptional regulation,which extensively regulates genes expression and function in the brain.Mounting evidence has shown a global change in RNA alternative splicing during brain aging and AD.Moreover,aberrant RNA splicing directly or indirectly affects the expression of synaptic proteins,thus contributing to cognitive decline of elderly individuals and AD patients.However,what causes aberrant RNA splicing in aged or AD brain remains unclear.Heterogeneous nuclear ribonucleoproteins(hnRNPs),a classic trans-splicing regulator,are extensively studied in RNA alternative splicing through modulating core splicing machinery and generating various splicing variants.The hnRNP D-like(hnRNP DL)belongs to the hnRNP family proteins and is highly expressed in the brain.Further,studies have shown that hnRNP DL is functionally associated with AD-related proteins,and proteomic data indicate that hnRNP DL is significantly reduced in AD brains.However,the regulatory role of hnRNP DL in the brain is largely unknown.Therefore,we aim to investigate the physiological and pathological functions of hnRNP DL in the brain,and establish a direct role for LDL in brain aging and AD-related synaptic dysfunction and memory decline.By assessing the levels of hnRNP DL in the hippocampus of young and aged mice,we demonstrate a significant reduction of long isoform hnRNP DL(L-DL)in the hippocampus of aged mice.Moreover,downregulation of L-DL impairs cognition in WT mice.We further demonstrate L-DL is preferentially expressed in neurons,and is exclusively localized to nuclear speckles.Depletion of L-DL results in the collapse of nuclear speckles,suggesting that L-DL serves as a structural component to maintain the structure of nuclear speckles.Moreover,we observe that loss of L-DL in cells leads to a significant reduction in splicing activity.Mechanistically,we illustrate that L-DL deficiency significantly reduces the association of U2AF65 with SF3B1 and SF3B3,disrupting the loading of U2 snRNP on pre-mRNAs.This leads to aberrant changes in RNA splicing patterns and subsequent changes in the expression of synaptic genes.In addition,L-DL levels are found significantly decreased in the hippocampus of APP/PS1 mice.Downregulation of L-DL(L-DL KD)in the hippocampus of these mice further deteriorates memory decline,whereas overexpression of L-DL(L-DL OE)significantly improves memory in APP/PS1 mice.Mechanistically,we demonstrate that L-DL significantly reduces intron 4 retention of CamkV,thus increases the production of CAMKV,whereas L-DL KD significantly increases intron 4 retention of CamkV,thus decreases production of CAMKV.In addition to CAMKV,L-DL OE increases levels of synaptic proteins including SNAP25,PSD95,and NMDA receptor NR2B in the hippocampus of APP/PS1 mice.L-DL KD further decreases levels of synaptic proteins including SNAP25,PSD95,and NMDA receptor NR2B in the hippocampus of APP/PS1 mice.These observed changes in synaptic proteins support the cognitive function of APP/PS1 mice.Taken together,our study defines nuclear speckle specific protein hnRNP DL as a critical protein linking synaptic genes splicing and aging or AD associated cognitive decline,shedding light on a potential therapeutic approach for cognitive decline in aged and AD brains via correcting aberrant RNA splicing.