RAVER1 Attenuates Hippocampal Neuronal Loss In TDP-43 Conditional Knockout Mice Via Regulating RNA Splicing

BackgroundNeurodegeneration,which is defined as the progressive loss of structure and function of neurons,is a prominent feature in neurodegenerative diseases such as amyotrophic lateral sclerosis(ALS),frontotemporal lobar degeneration(FTLD),Alzheimer’s disease(AD),Parkinson’ s disease(PD),et al.Over the past decade,TAR DNA binding protein 43(TDP-43)proteinopathy has been regarded as a pathological hallmark of some neurodegenerative diseases such as ALS and FTLD.Beyond this,TDP-43 positive inclusions have also been demonstrated to be related to various other neurological diseases including Alzheimer’s disease,hippocampal sclerosis(HS),dementia with Lewy bodies(DLB).Moreover,missense mutations in TDP-43 are also linked to familial ALS,strongly supporting the idea that TDP-43 pathology is central to the pathogenesis of sporadic disease.While gain-and loss-of-function studies over the past decade have been performed in effort to clarify the mechanism of TDP-43 pathology,its precise pathophysiology in neurodegenerative disease remains unclear.Previously,we showed that TDP-43 is a member of RNA binding protein that repress cryptic exons to maintain a normal transcriptome.Importantly,incorporation of aberrant cryptic exons are detected in ALS,FTD and AD brains,raising the possibility that compromised TDP-43 mediated splicing repression may underlie neurodegeneration.Accompanied by TDP-43 nuclear clearance,these non-conserved cryptic exons were spliced into messenger RNAs,and often led to nonsense-mediated decay,which might incur dysfunction on some important signaling pathways such as autophagy and protein nuclear import,which are central in cell physiology.Our previous in vitro study has proved that these cryptic exons are crucial to the cell death.Because several putative roles have been attributed to TDP-43,it will be critical to establish that this splicing function is an important role of TDP-43 in central neurons,cells that are impacted in ALS,FTD and AD.In this experiment,in order to verify whether the splicing repression of TDP-43 is the main role of TDP-43 in hippocampal neurons,we injected the fusion protein CTR composed of the N-terminal fragments of human TDP-43 and Ribonucleoprotein,PTB-binding 1(RAVER1),packaged with Adeno-associated 9(AAV9)virus into the lateral ventricle of newborn pups which TDP-43 were conditinally knockout(ECFF).We hope that by restoring the splicing repression function of TDP-43,the loss of neurons caused by TDP-43 can be attenuated,thus providing potential therapeutic targets for ALS and other neurodegenerative diseases.Methods and MaterialsWe took advantage the Cre-loxP recombinase system to generate the TDP-43 knockout mice in forebrain by crossbreeding CamK2a-CreER;TardbpF/F(ECFF)and TardbpF/F(FF),followed by Tamoxifen feeding at 6-weeks old.Either CTR or GFP packaged in AAV9 virus were injected to the mice at p0.After genotyping,mice brains were collected at 4-month and CTR were detected using immunohistochemistry and immunofluorescence.RT-PCR and RNA in situ hybridization were applied to detect abarent splicing events.Hippocampal neurons were counted at 6-month using CV staining.ResultsOur fusion protein CTR expressed in 39%hippocampal CA neuronal cells at 4-month old.Moreover,CA3/2 neurons in 6-month-old knockout mice treated with AAV9-GFP decreased significantly(FF-GFP vs.ECFF-GFP;n=9 vs.n=9;univariate analysis of variance,p<0.001),while in AAV9-ctr knockout mice,the would be attenuated(ECFF-GFP vs.ECFF-CTR,n=9 vs.n=12;univariate analysis of variance,Bonferroni’ test,p<0.05),indicating that CTR could restore some of the neuronal function.We further described the possible underlying mechanism--the splicing repression function of TDP-43.With the treatment of CTR,the aberrant splicing of 3 mRNA target due to loss of nuclear TDP-43 could be partially restored(n=3,p<0.01).DiscussionThus,our data support the idea that TDP-43 mediated splicing repression is a major function of TDP-43 in hippocampal neurons and identify a novel mechanism-based therapeutic target for neurodegenerative disease exhibiting TDP-43 pathology.