Single nucleotide polymorphisms rs148582811 regulates its host gene ARVCF expression to affect nicotine-associated hippocampus-dependent memory

Summary Although numerous susceptibility loci are nominated for nicotine dependence (ND), no report showed any association of ARVCF with ND. Through genome-wide sequencing analysis, we first identified genetic variants associated nominally with ND and then replicated them in an independent sample. Of the six replicated variants, rs148582811 in ARVCF located in the enhancer-associated marker peak is attractive. The effective-median-based Mendelian randomization analysis indicated that ARVCF is a causal gene for ND. RNA-seq analysis detected decreased ARVCF expression in smokers compared to nonsmokers. Luciferase reporter assays indicated that rs148582811 and its located DNA fragment allele-specifically regulated ARVCF expression. Immunoprecipitation analysis revealed that transcription factor X-ray repair cross-complementing protein 5 (XRCC5) bound to the DNA fragment containing rs148582811 and allele-specifically regulated ARVCF expression at the mRNA and protein levels. With the Arvcf knockout mouse model, we showed that Arvcf deletion not only impairs hippocampus-dependent learning and memory, but also alleviated nicotine-induced memory deficits.


INTRODUCTION
Tobacco use remains a high rate worldwide, notably in China. 1 Despite the known healthy risks and strict legislation against it, the prevalence of smoking in China increased 2-fold in 2016 compared with 1985. 2 With an increased tobacco use in China, it was predicted that the death toll could rise to 3 million per year by 2050. 3 Thus, further research on the pathogenesis of nicotine dependence (ND) will promote the development of effective treatment strategies for the disease.
ND plays a crucial role in maintaining smoking-related behaviors. 4Numerous epidemiologic studies have provided strong evidence that genetics affects greatly on the development of ND, with an average heritability of 56%. 5 Therefore, identifying genetic variants and biological function underlying ND are critical for treating those addicted tobacco smokers.
Although a great number of susceptibility loci for ND have been identified during the past years, 6,7 most of them are synonymous variants and difficulty to explain their involvement in the pathogenesis of ND at the molecular level.Even for a few of those identified nonsynonymous variants, the biological functions and regulatory mechanisms underlying their involvement in the pathogenesis of ND have rarely investigated.5][16] In some cases, their contribution to a disease could be much greater than those observed in common variants. 17Thus, it has been suggested that the whole genome sequencing (WGS) analysis holds great potential for finding rare variants with large biological effects, which would greatly increase tractability for biological experiments and reveals novel susceptibility genes as well as biological pathways underlying ND.
1][22][23] Relative to healthy controls, it was found that smokers have cognitive deficits in auditory-verbal and visuospatial learning, visuospatial memory, cognitive efficiency, executive skills, and processing speed. 20In a population-based study of 2,163 participants, smokers were found to have deficits in attention, working memory, and impulse control functions. 23There is increasing evidence suggesting that nicotine has direct effect in the hippocampus altering learning and memory, and these changes in cognitive function may impact susceptibility to develop and maintain ND. 24,25 Additionally, preclinical studies further indicated that nAChR subunits, especially a7 and b2 subunits, play important roles in the cognitive effects of nicotine. 26,27Although information at different levels of analysis are continuing to emerge, the neurobiological mechanisms underlying nicotine's effects on cognitive function remain to be further elucidated.
Specifically, this study had the following two main objectives: 1) to discover more variants, especially those rare ones, from less studied ethnic populations on smoking such as Chinese smokers through WGS analysis; and 2) to select one representative rare variant and the gene where it locates to demonstrate their biological function and molecular mechanism underlying smoking-associated phenotypes by using both in vivo and in vitro approaches.

RESULTS
Single nucleotide polymorphisms rs148582811 located in ARVCF was significantly associated with tobacco smoking Our WGS analysis of 1,329 Chinese male subjects consisting of 805 heavy smokers and 524 age-matched non-smokers revealed several novel loci associated significantly with smoking status or CPD at a genome-wide significance (p < 5 3 10 À8 ; Figure S1).A detailed description of these subjects is given in Table 1.For these putative susceptibility loci, 31 SNPs of potential interest based on their predicted biological functions were further genotyped using TaqMan OpenArray in 3,726 independent samples (Figure S2 and Table S3), and six of them were successfully replicated (Table 2).
By annotating potential biological functions for these six significant SNPs using chromatin immunoprecipitation sequencing (ChIP-seq) data from the ENCODE consortium, 28 we found that an intronic SNP rs148582811 in ARVCF was located in a genomic region which was enriched for the enhancer-associated H3K4me1 and H3K27ac histone marks [29][30][31] in both SK-N-SH and K562 cells (Figure 1A), suggesting this genomic region may function as an enhancer.Further, gene-based association test using extended Simes procedure (GATES) indicated that ARVCF was significantly associated with ND (p = 0.00056).We also did gene-based association analysis for ARVCF with the same approach based on the GTEx eQTLs data, which revealed a significant association of ARVCF expression with ND in forebrain region (p = 0.0012).
Next, we compared RNA expression level of ARVCF between smokers and non-smokers in two independent datasets with one from our laboratory (Figure 1B) 32 and the other from the GEO database (GDS1348; Figure 1C) and found that the mRNA expression of ARVCF was significantly lower in the smoker group compared to non-smoker group (p < 0.01).Further, we did the effective-median-based Mendelian randomization for inferring the causal genes (EMIC) approach for ARVCF with the GWAS results from our sample and GTEx expression data in brain, we found that ARVCF is a causal gene for ND in forebrain region (min EMIC p = 0.021), suggesting that ARVCF overexpression might increase the risk of ND (Figure S3).

Both rs148582811 and its containing DNA region allele-specifically regulate ARVCF expression
To examine if the intronic DNA region containing rs148582811 acts as an enhancer, we first cloned the DNA fragment containing rs148582811-C or -T allele into a luciferase vector and then conducted luciferase reporter assay in HEK293T cells.We found that the DNA fragment containing rs148582811-T allele significantly increased the enhancer activity compared with that containing rs148582811-C allele (p < 0.001) (Figure 1D).
To determine whether rs148582811 and its containing DNA region regulate ARVCF expression, we used CRISPR/Cas9 to independently edit SH-SY5Y and HEK293T cells with a 217 bp fragment deletion containing rs148582811 site (Figure S4A), which were confirmed by Sanger sequencing (Figures S4B and S4C).As shown in Figures 2A-2E, significant decreases in ARVCF mRNA and protein expression were detected in both rs148582811-KO SH-SY5Y and HEK293T cells compared with the WT cell lines (p < 0.05).Further, we investigated the role of rs148582811 in regulating ARVCF expression.Both SH-SY5Y and HEK293T cells were transfected with homologous repair template including rs148582811-T allele in addition to Cas9-sgRNA plasmid (Figure S4D).And then, the cells carrying T-allele were generated and verified by Sanger sequencing (Figures S4E and S4F).Subsequently, qRT-PCR and Western blotting analyses demonstrated that ARVCF expression increased significantly in the rs148582811-T SH-SY5Y and HEK239T cells (p < 0.01) (Figures 2F-2J) compared with rs148582811-C cells.These results provided direct evidence that rs148582811 plays a regulatory role on ARVCF expression.X-ray repair cross-complementing protein 5 binds to rs148582811-containing DNA region Next, we wanted to determine if there existed any transcriptional factor (TF) that interacts with the rs148582811-containing DNA region.By conducting EMSA through incubation nuclear extracts from SH-SY5Y cells with double-stranded DNA probes of predicted enhancer element, we found that the nuclear proteins bound to the probes leading to the formation of one predominant complex (Figure 3A).In the competition assay, the unlabeled DNA diminished the shifted band in a dose-dependent manner, but the mutant probe had no obvious effect on the band.To better understand which protein binds specifically to rs148582811-containing DNA region, DNA pull-down assay was conducted to purify the binding TFs with the DNA probes used in the EMSA assay and nuclear extracts from SH-SY5Y cells.The silver staining of proteins recovered from pull-down experiments showed proteins with a molecular weight of around 90 kDa bound to rs148582811-C and -T probes but not to the control probe (Figure 3B).Among these $90 kDa proteins, XRCC5 was firstly identified by mass spectrometry (Figure 3C) and then validated by Western blotting (Figure 3D).To further confirm that XRCC5 indeed binds to rs148582811-containing DNA region, we performed super-shift EMSA by using anti-XRCC5 antibody as well as probes for both C and T alleles of rs148582811.As a result, when the C and T probes were incubated with the anti-XRCC5 antibody, the shifted band was markedly diminished along with the formation of a super-shifted band (Figure 3E, Lanes 1 and 2).Together, these results indicated that the rs148582811-containing DNA region indeed interacts with XRCC5.Rs148582811 allele-specifically modulates ARVCF expression by binding to X-ray repair cross-complementing protein 5 Next, we determined whether XRCC5 executed its regulatory effect on ARVCF expression through binding to the rs148582811-C or -T allele.By the co-transfection of luciferase reporter constructs containing rs148582811-C or -T allele with either XRCC5-siRNA or control-siRNA, we found that the cells transfected with T allele significantly increased luciferase activity compared to the C allele in co-transfected with control-siRNA group (p < 0.01) (Figure 3F, left), which was consistent with the finding when the luciferase construct was transfected alone.However, in co-transfected with XRCC5-siRNA group, the luciferase activity showed no significant difference between rs148582811-C and T alleles (Figure 3F, right).To further investigate whether the regulation of rs148582811 on ARVCF expression was modulated by XRCC5, XRCC5-siRNA was used to knockdown its expression.We found that there were significant differences between rs148582811-C and rs148582811-T cells in control-siRNA group, however, the downregulation of XRCC5 in rs148582811-C and rs148582811-T SH-SY5Y cells did not show allele-specific regulation on ARVCF (Figures 3G-3J).Together, these findings support our hypothesis that XRCC5 plays an important role in the regulatory effect of rs148582811 on ARVCF expression by binding to both alleles of the SNP.

ARVCF impacts hippocampus-dependent memory in mice
As a member of p120 catenin family linked to the synapse reorganization and memory, we were interested to determine whether ARVCF has any role in learning and memory.By using CRISPR/Cas9 system, we generated the Arvcf-KO mouse model with a 5805bp deletion (Figures S4G and S4H) and used them to test the effects of Arvcf on learning and memory by comparing Arvcf WT/KO and Arvcf KO/KO mice with WT mice.In MWM test, the time to platform was significantly enlonged on day 6 in Arvcf KO/KO mice compared with that in the WT mice (p < 0.01) (Figure 4B).Moreover, the platform crossing times were significantly less in Arvcf KO/KO mice compared with those in the WT mice (p < 0.01) (Figure 4C).We then assessed the contextual and cued fear memory in Arvcf-KO mice (Figure 4D).In the contextual fear memory test, Arvcf KO/KO mice were frozen at significantly lower level than the WT mice (p < 0.01) (Figure 4E).However, the results of cued (tone) fear memory test showed no obvious difference of freezing between the WT and Arvcf KO/KO mice (Figure 4F).No significant change of memory was observed between the WT and Arvcf WT/KO mice in both MWM and fear conditioning experiments.Moreover, the significant change of contextual memory but not cued memory indicated that Arvcf-KO led to the hippocampus-dependent memory deficient.
To further investigate the role of ARVCF on hippocampus-dependent learning and memory, Adeno-associated virus (AAV) system, harboring an Arvcf-shRNA, was used to knockdown Arvcf expression in mouse hippocampal CA3 subregion, an important brain region in  learning and memory. 27Both immunofluorescent staining and Western blotting analysis showed a significant reduction of Arvcf protein expression in mouse hippocampal CA3 (p < 0.001) (Figures 5A-5C).Then, we assessed the changes of object and spatial memory of Arvcf-KD mice by using novel object recognition (Figure 5D) and MWM experiments (Figures 5E-5G).Compared with the control group, Arvcf-KD mice showed reduced preference toward novel object (p < 0.01; Figure 5E).In MWM test, the number of platform crossings was less in Arvcf-KD group compared with that in the control group (p < 0.0001) (Figure 5F), and no difference was detected in the average swimming speed between the two groups (Figure 5G).

ARVCF plays a vital role in the rewarding effect of nicotine and nicotine-associated memory
To assess the biological role of ARVCF on the rewarding effect of nicotine, we performed conditional place preference (CPP) assay in Arvcf-KO mice and corresponding WT mice.Both WT and Arvcf KO/KO mice were conditioned to nicotine treatment in a 7-day unbiased CPP paradigm (Figure 6A), and were compared with the saline-control mice.On the day 9 of treatment (Figure 6B), CPP test showed a robust preference for the nicotine-paired compartment in WT group (p = 0.00027), whereas Arvcf KO/KO mice did not show an obvious preference for nicotine treatment (p > 0.05).These results demonstrated that Arvcf plays a role in mediating the rewarding effect of nicotine on mice.
As nicotine is known to execute its effects on hippocampus-dependent learning and memory, 27,33 we then determined whether Arvcf was involved in the effects of chronic nicotine treatment on nicotine-associated memory in WT and Arvcf KO/KO mice (Figure 6C).In MWM test, during the training phase on days 8-12 after pump implantation (Figures 6D and 6E), although the time to platform and distance traveled to platform were reduced in all four groups day by day, the Arvcf KO/KO mice spent significantly more time and traveled more distances to reach the platform.During the test phase on day 13, compared with WT mice in the saline group, the WT mice in the nicotine-treated group spent significantly greater time reaching the location of platform (p < 0.05; Figure 6F) and crossed less numbers to the platform (p < 0.05; Figure 6G).However, no significant differences were observed in Arvcf KO/KO mice between the saline and nicotine group.Together, these results indicated that Arvcf knockout attenuated chronic nicotine-induced spatial memory impairment.Further, in the fear conditioning test during the days 13 and 14 of nicotine or saline treatment, we examined whether Arvcf knockout could alter nicotine-associated fear memory.As shown in Figures 6H and 6I, in WT mice, chronic nicotine treatment led to significantly reduction of freezing time during contextual fear response test (p < 0.05), but there was no effect on cued fear response.In contrast, in Arvcf KO/KO mice, chronic nicotine treatment led to no significant change in freezing time during both contextual (Figure 6H, right) and cued fear responses (Figure 6I, right), suggesting that Arvcf deletion significantly attenuated chronic nicotine-induced deficits in hippocampus-dependent fear memory.Together, these findings indicated that Arvcf plays a key role in chronic nicotine-associated hippocampus-dependent memory deficient.

DISCUSSION
Up to now, the identified susceptible variants for ND by genetic association analysis could only explain a small portion of the estimated heritability for ND.To find more causal variants, we conducted WGS analysis on 1,329 Chinese samples and found multiple SNPs associated significantly with smoking at the genomic level.Of them, six were replicated in 3,726 independent subjects, indicating they were significantly associated with smoking.Since identified SNPs by GWAS are mostly enriched in DNA regulatory elements and can regulate gene expression, 34 we then used ChIP-seq data from ENCODE database to predict the biological function of the identified variants.This revealed that SNP rs148582811 in ARVCF gene overlapped with the enhancer marker signals (H3K4me1 and H3K27ac) in both SK-N-SH and K563 cells, suggesting the potential regulatory function of rs148582811.Further, our gene-based association test demonstrated that ARVCF was significantly associated with ND at both the GWAS results from our sample and the eQTL results in forebrain region of GTEx dataset. 35In addition, by using the RNA-seq data from our laboratory 32 and public GEO dataset, we found that ARVCF mRNA expression level was significantly lower in smokers compared to non-smokers.Finally, our EMIC analysis revealed that ARVCF is a causal gene for smoking addiction where we found an increased ARVCF expression can lead to an increased risk of ND.
During the past years, although a number of susceptibility genes and variants have been identified for ND, whether these genetic findings could be applied to clinic practices depends largely on their biological functions. 36By using luciferase reporter assay to initially explore the biological function of rs148582811, we found that rs148582811 not only acted as an enhancer, but also discovered that rs148582811-C or T allele had different regulatory functions, which formed the major reason for us to focus on the functional study of this rare variant in the present report.
To determine whether the susceptibility variant rs148582811 has any biological function, CRISPR/Cas9 gene editing technique is commonly used, which can accurately edit the target site or region on the genome and provide effective experimental models for studying functional mechanisms for the variants of interest. 37However, due to the low efficiency of single-base editing of CRISPR/Cas9 system, there were very limited reports regarding the application of CRISPR/Cas9 technology in SNP functional research. 38,39Based on the above genetic and functional evidence, we hypothesized that the SNP rs148582811 could regulate ARVCF expression in an allele-specific manner.By applying CRISPR/Cas9 gene editing technique, the locus of rs148582811 and the region where it locates were deleted.In the rs148582811-KO SH-SY5Y and HEK293T cells, both ARVCF mRNA and protein expression were significantly decreased.Furthermore, in the SH-SY5Y and HEK293T cells carrying rs148582811-T allele, both ARVCF mRNA and protein expression levels were significantly higher than those in rs148582811-C cells.These findings demonstrated that the rs148582811 alleles affected the expression of ARVCF by regulating the activity of enhancer element.
Given the potential important biological function of rs148582811, we also explored the mechanism underlying the function of rs148582811.Considering that the nuclear protein generally regulates gene expression through binding to the regulatory region of the genome, 40 we conducted the EMSA, DNA pull-down and protein mass spectrometry experiments, which revealed that nuclear protein XRCC5 bound to the rs148582811containing DNA region.XRCC5, also known as Ku80, is a component of the DNA-dependent protein kinase, existing as a heterodimer with XRCC6. 41,42Because XRCC5 binds to DNA and was reported to function as a transcription factor, [43][44][45] we then performed rs148582811 luciferase report experiment in XRCC5-KD HEK293T cells, and observed no significant difference of enhancer activity between rs148582811-C and -T cells.Further, we found that the significant difference of ARVCF expression between the rs148582811-C and rs148582811-T SH-SY5Y cells were completely abolished after XRCC5 knockdown, indicating that XRCC5 is able to regulate the functional difference between C and T alleles of rs148582811.
The protein encoded by ARVCF belongs to p120ctn catenin family, which plays a significant functional role in various aspects of neuronal morphogenesis, neurodevelopmental and neurological disorders. 46Recent works have confirmed that ARVCF is involved in the modulation of cell-cell adhesion 47,48 and essential to many developmental processes including neuronal rearrangement and migration. 49,503][54] In addition, it has been reported that ARVCF mutation could decrease the migration of SH-SY5Y cells via the downregulating of RHOA and ROC. 49Meanwhile, during fetal development, ARVCF expression is closely associated with the rearrangement and migration of neurons within ganglionic eminence, a telencephalic structure which gives rise to precursor neurons of the striatum, amygdala and basal nucleus of Meynert. 50By using Arvcf-KO mice generated from this work, we found a decreased spatial hippocampus-dependent learning and memory.Furthermore, Arvcf-KO in mouse hippocampal CA3 also presented impaired learning and memory.These findings strongly indicated a specific action of ARVCF on hippocampus-dependent learning and memory in animals.
ARVCF is located on chromosome 22q11.2,one of the most significant regions (22q11.23-12.1)7][58] Although an association of ARVCF with schizophrenia has been previously reported, whether this gene is associated with other addictive or psychiatric disorders has not been reported yet.Thus, this study represents the first one showing the presence of significant association of SNP rs148582811 in ARVCF with ND.
Given its genomic location and biological function in learning and memory, together with the existence of the functional SNP, we further investigated the biological effect of ARVCF on nicotine dependence.We found the wild-type mice presented a robust preference for nicotine in CPP test, but Arvcf knockout mice did not show any obvious preference, indicating that Arvcf plays a key role in mediating the rewarding effect of nicotine.Further, since nicotine is known to has an effect on hippocampus-dependent learning and memory 27,33 except for the well-known rewarding effects, we also examined the function of ARVCF on learning and memory in nicotine chronically treated mice.We found that chronic nicotine administration impaired spatial and emotional memory which were consistent with previous findings. 59,60However, these hippocampus-dependent learning and memory deficits were abolished in Arvcf KO/KO mice.Taken together, these findings strongly indicate that ARVCF plays a critical role in nicotine-associated learning and memory.

Conclusions
Our GWAS analysis discovered that SNP rs14858281 in ARVCF was significantly associated with ND and such an association was further validated not only in an independent sample from our sample collection at the SNP level, but also in the GTEx dataset at the gene level.Moreover, our RNA-seq analysis of mRNA expression in both our own sample and public GEO dataset revealed that ARVCF is lowly expressed in smokers and EMIC analysis of the ARVCF expression data in the forebrain region of GTEx dataset revealed that ARVCF is a causal gene of ND and ARVCF overexpression might increase the risk of ND.Next, our in vitro reporter assay indicated that the SNP rs14858281 regulates ARVCF expression in an allele-specific manner, which is realized by binding to the transcriptional factor XRCC5.Finally, by generating Arvcf-KO mice, we demonstrated that Arvcf deletion impaired hippocampus-dependent learning and memory and alleviated nicotine-induced memory deficits.Taken together, this study demonstrated for the first time that SNP rs14858281 is significantly associated with ND and ARVCF plays a key role in nicotine-associated hippocampus-dependent memory.

Limitation of the study
There were several limitations of this study that should be noted.Firstly, we did not perform the validation of our genetic findings in other ethnic populations, in which the genetics of smoking behaviors differ from each other. 61,62Secondly, as our genetic and functional data have confirmed the involvement of rs148582811 and the DNA region around it in learning, memory and ND as well, more effort is needed to find causal variant(s) in this region and ARVCF gene through deep sequencing analysis.Thirdly, although ARVCF plays an important role in memory formation and nicotine-induced memory deficits, there was a lack of evidence to clarify the

Figure 1 .
Figure 1.The predicted enhancer role of rs148582811 in ARVCF (A) Prediction of potential regulatory function of rs148582811 by using ENCODE ChIP-seq data.(B) Comparison of ARVCF mRNA expression between 38 smokers and 36 non-smokers.(C) ARVCF mRNA expression levels in normal bronchial epithelial cells exposed to cigarette smoke compared with controls (downloaded from GEO microarray dataset GDS1348).(D) Comparison of relative luciferase activity in Controls, rs148582811-C or -T allele in HEK293T cells (***p < 0.001).

Figure 2 .
Figure 2. Regulatory effect of rs148582811 and its containing DNA genomic region on the expression of ARVCF (A and F) Representative pictures of Western blotting of ARVCF in rs148582811-KO and rs148582811-T SH-SY5Y or HEK293T cells.Regulated ARVCF mRNA and protein levels by the genomic region containing rs148582811 were examined in rs148582811-KO SH-SY5Y (B and C) and rs148582811-KO HEK293T (D and E) cells.Regulated ARVCF mRNA and protein expression level by rs148582811 were determined in rs148582811-T SH-SY5Y (G and H) and rs148582811-T HEK293T (I and J) cells.Values are shown as Mean G SEM of at least three independent experiments.*p < 0.05; **p < 0.01; ****p < 0.0001.

Figure 3 .
Figure 3. Rs148582811 allele-specifically regulates ARVCF expression through binding to XRCC5 (A) Electromobility shift assay (EMSA) was performed by using biotinylated double-stranded probes containing the rs148582811 incubated in nuclear extracts from SH-SY5Y with or without competitors.(B) DNA pull-down assay was conducted by separating the bounded proteins to rs148582811-containing DNA probe with 8% acrylamide SDS gel and then the gel was stained with silver.(C) The binding proteins with the size of about 90 kDa were identified by Mass Spectrometry.(D) The XRCC5 protein level identified from mass spectrometry was examined by Western blotting.(E) Super-shift EMSA was performed by incubating biotinylated double-stranded probes in nuclear extract from SH-SY5Y with or without competitors and antibody.Lane 1: probe with rs148582811-C allele, Lane 2: probe with rs148582811-T allele.(F) The luciferase reporter assay was conducted in HEK293T cells by co-transfecting with the pGL4.26vector harboring renilla luciferase, either rs148582811-C or T allele, and either XRCC5-siRNA or control-siRNA.

Figure 3 .
Figure 3. Continued (G and J) Rs148582811-C and -T SH-SY5Y cells were transfected with negative control-siRNA (NC-siRNA) or XRCC5-siRNA, and then ARVCF mRNA (G) and protein (J) levels were determined at 48 h after transfection.(H and I) The representative pictures of Western blotting of ARVCF in rs148582811-C and rs148582811-T SH-SY5Y cells transfected with NC-siRNA (H) or XRCC5-siRNA (I).Values are shown as Mean G SEM of at least three independent experiments.*p < 0.05; **p < 0.01; ns = no significance.

Figure 4 .
Figure 4. Determining the effect of ARVCF on hippocampal-dependent learning and memory in Arvcf KO mice by using Morris water maze (MWM) test and fear conditioning test (A) The reduced time to platform during a 5-day training phase.(B) The time to platform and (C) number of platform crossings during probe test on the day 6 in wild-type (WT), Arvcf WT/KO , and Arvcf KO/KO groups.** p < 0.01.(D) Schematic of fear conditioning test.On the day 1, which is a conditioning test, three pairs of tone (30 s, 5 kHz, 85 dB) and foot shock (0.8 mA, 2 s) were presented.On the day 2, which is context test followed by tone test after 2 h, no tone and foot shock were presented in the context test, and altered apparatus but only tone (30 s, 5 kHz, 85 dB) presented in the tone test.(E and F) The percentage of freezing time during context test (E) and tone test (F) in WT, Arvcf WT/KO , and Arvcf KO/KO groups.** p < 0.01.

Figure 5 .
Figure 5. Determining the effect of ARVCF on hippocampal-dependent learning and memory in mouse hippocampal CA3 region by using Adenoassociated virus system Arvcf was knocked down (KD) by shRNA-AAV in the CA3 brain region of mouse hippocampus, then fluorescence immunostaining (A) and Western blotting (B and C) were used to detect the expression of ARVCF.(D) Schematic of novel object recognition (NOR) test.During the training phase, each mouse was subjected to two identical objects for 8 min, and 24-h later, one of the objects was replaced with a novel object and all mice explored for 5 min.(E) The discrimination ratio of novel object preference in control and Arvcf-KD groups.(F) The number of platform crossings and (G) average speed changes in MWM test after Arvcf knockdown.N = 10-14 per experimental group for Arvcf-KO study, and n = 8-12 per experimental group for Arvcf-KD study.Values are shown as Mean G SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns = no significance.

Figure 6 .
Figure 6.Determining the involvement of ARVCF in chronic nicotine-induced impairment of memory (A) Experimental design of conditioned place preference (CPP) test.(B) Comparison of rewarding effects of nicotine in wild-type (WT) and Arvcf KO/KO mice with the CPP paradigm.(C) Schematic of chronic nicotine treatment with mini-osmotic pump on WT and Arvcf KO/KO mice followed with MWM test and fear conditioning test.(D) The reduced time to platform and (E) distance traveled to platform during a 5-day training phase.(F) The time to platform and (G) number of platform crossings during probe test on day 13 after chronic nicotine treatment.The percentage of freezing time during (H) context test and (I) tone test in WT and Arvcf KO/KO mice after chronic nicotine treatment.Values are shown as Mean G SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ns = no significance.

Table 1 .
Characteristics of clinical samples used in the study