Tdrd3-null mice show post-transcriptional and behavioral impairments associated with neurogenesis and synaptic plasticity

Abstract

The Topoisomerase 3B (Top3b) - Tudor domain containing 3 (Tdrd3) protein complex is the only dual-activity topoisomerase complex that can alter both DNA and RNA topology in animals. TOP3B mutations in humans are associated with schizophrenia, autism and cognitive disorders; and Top3b-null mice exhibit several phenotypes observed in animal models of psychiatric and cognitive disorders, including impaired cognitive and emotional behaviors, aberrant neurogenesis and synaptic plasticity, and transcriptional defects. Similarly, human TDRD3 genomic variants have been associated with schizophrenia, verbal short-term memory and educational attainment. However, the importance of Tdrd3 in normal brain function has not been examined in animal models. Here we generated a Tdrd3-null mouse strain and demonstrate that these mice display both shared and unique defects when compared to Top3b-null mice. Shared defects were observed in cognitive behaviors, synaptic plasticity, adult neurogenesis, newborn neuron morphology, and neuronal activity-dependent transcription; whereas defects unique to Tdrd3-deficient mice include hyperactivity, changes in anxiety-like behaviors, olfaction, increased new neuron complexity, and reduced myelination. Interestingly, multiple genes critical for neurodevelopment and cognitive function exhibit reduced levels in mature but not nascent transcripts. We infer that the entire Top3b-Tdrd3 complex is essential for normal brain function, and that defective post-transcriptional regulation could contribute to cognitive and psychiatric disorders.

Introduction

Topoisomerase 3B (Top3b) is the only dual-activity topoisomerase that can solve topological problems for both DNA and RNA. Increasing evidence shows that Top3b works with Tdrd3 in a conserved complex that can act in biological processes on both DNA and RNA in animals(Ahmad et al., 2017; Ahmad et al., 2016; Siaw et al., 2016; Stoll et al., 2013; Xu et al., 2013). Human genetic studies have indicated that TOP3B deletion or mutations are associated with psychiatric and cognitive disorders, including schizophrenia, autism, epilepsy, and intellectual disability(Daghsni et al., 2018; Garavelli et al., 2011; Kaufman et al., 2016; Riley et al., 2020; Stoll et al., 2013; Xu et al., 2012). This inference is supported by analyses of cultured neurons(Xu et al., 2013) and several animal models, including mouse(Joo et al., 2020; Rahman et al., 2021), zebrafish(Doolittle, 2017; Williams, 2015), and Drosophila(Ahmad et al., 2017; Xu et al., 2013). Specifically, our group has observed that Top3b-null mice(Kwan and Wang, 2001) display behavioral phenotypes related to psychiatric disorders and cognitive impairment, as well as impairments in hippocampal neurogenesis and synaptic plasticity(Joo et al., 2020).

On DNA, Top3b -Tdrd3 has been reported to promote transcription(Joo et al., 2020; Su et al., 2023), R-loop resolution(Saha et al., 2022; Yang et al., 2014b; Yuan et al., 2021), siRNA-guided heterochromatin formation and transposon silencing(Lee et al., 2018b). At the RNA level, Top3b-Tdrd3 has been shown to localize with the mRNA translation machinery(Ahmad et al., 2016; Stoll et al., 2013; Xu et al., 2013), and regulate mRNA translation and turnover(Su et al., 2022b). Tdrd3 has several functions in the Top3b-Tdrd3 complex. First, it can enhance the binding and topoisomerase activity of Top3b on both DNA and RNA(Siaw et al., 2016; Yang et al., 2022). Second, Tdrd3 can anchor Top3b to other proteins functioning on DNA or RNA, including RNA polymerase II (pol II)(Joo et al., 2020; Sims et al., 2011; Su et al., 2023), FMRP (Fragle X mental retardation syndrome protein)(Stoll et al., 2013; Xu et al., 2013), polyribosomes(Stoll et al., 2013; Xu et al., 2013) and other proteins(Kashima et al., 2010; Lee et al., 2018b; Stoll et al., 2013; Xu et al., 2013; Yang et al., 2010). Third, Tdrd3 and Top3b mutually stabilize each other, as depletion of either reduces the level of the other(Joo et al., 2020; Su et al., 2022b; Xu et al., 2013; Yang et al., 2014b). These finding suggest that the two proteins act coordinately in a molecular machine to solve topological problems of DNA or RNA. In support of this notion, we recently found that the two proteins coordinately regulate transcription(Su et al., 2023), translation and turnover of mRNAs(Su et al., 2022b) in human cell lines.

One interacting partner of Top3b-Tdrd3 complex, syndromic FMRP, is silenced in Fragile X Mental retardation syndrome, which is a leading cause of autism(Hagerman et al., 2017). The findings that both interacting partners of TDRD3 (TOP3B and FMRP) are linked to psychiatric and cognitive disorders imply that TDRD3 itself could be associated with these disorders. Consistent with this hypothesis, independent genome-wide association studies (GWAS) have reported association between several single nucleotide variants (SNVs) within or near TDRD3 genomic locus and schizophrenia(Lee et al., 2018a; Trubetskoy et al., 2022); cognitive dysfunction(Lahti et al., 2022a; Ohi et al., 2014), language impairment or delay(Bartlett et al., 2002; Bradford et al., 2001; Lahti et al., 2022b), and education attainment(Lahti et al., 2022a; Lee et al., 2018a; Okbay et al., 2022). Although Tdrd3-null Drosophila and mouse have been reported(Xu et al., 2013; Yang et al., 2014a), neither animal model has been subjected to neurological or behavior studies. In contrast, Top3b and FMRP knockouts, both of which have been extensively analyzed in various animal models, show phenotypes consistent with impaired brain function(Doolittle, 2017; Hagerman et al., 2017; Joo et al., 2020; Rahman et al., 2021; Williams, 2015). Only one study in Drosophila showed that Tdrd3 can genetically interact with FMRP to promote eye formation(Xu et al., 2013), implying that Tdrd3 could function in neurodevelopment.

Here we establish a new Tdrd3-null mouse model to test for its importance in normal brain function. We found that Tdrd3-null mice exhibit behavioral and neurological defects that overlap but are not identical to those of Top3b-null mice. The shared defects include cognitive functions, synaptic plasticity, adult neurogenesis, and neuron morphology, indicating that the entire Top3b-Tdrd3 complex is indeed critical for normal brain function. Specific to Tdrd3-null mice, reduced anxiety and myelination as well as elevated neuron complexity was observed, suggesting that Tdrd3 may also have function(s) independently of Top3b. Interestingly, multiple genes critical for neurodevelopment and mental functions exhibit reduced levels in mature but not nascent transcripts, suggesting that defective post-transcriptional regulation could contribute to cognitive and neurological disorders.

Methods and Materials

Mice

The Tdrd3-null mouse line was generated using the gene-trap (GT) strategy (Taniwaki et al., 2005). Briefly, a Gene-trap vector pU21B (Genbank Accession number: AB255647, 9399 bp) was transfected in mouse ES cells to randomly integrate into the genome and disrupt gene function. This vector has been shown to be highly selective for integrations into the introns adjacent to the exon containing the start codon, and can trap and disrupt the expression of the gene where it is inserted. The transfected ES cells with stably-integrated vectors were selected, and the colonies were picked and grown up in new plates. The genomic location of the inserted vector in each ES clone was then identified by rapid amplification of cDNA 5’-ends (5’-RACE) and long PCR of genomic DNA. The subsequent sequencing of the PCR product allows identification of the vector insertion site for each ES clone. One ES cell clone (Ayu21-B171) was found containing the GT vector inserted in the first intron of Tdrd3 genomic locus (Fig. S1b–d). More information about this clone is available on its website [http://egtc.jp/action/access/clone_detail?id=21-B171]. For this clone, the ES cells are KTPU8. KTPU8 is a feeder-free ES cell line derived from TT2 ES cell line. TT2 ES cell line is widely used in Japan which was established from F1 embryo of C57BL/6N and CBA. This ES clone was subsequently used to generate chimeric mice by aggregation with eight cell embryos of ICR mice. The chimeric mice where then mated with C57BL/6N mice to produce F1 heterozygotes. The male and female heterozygous mice were then mated to produce homozygous Tdrd3-null mice in C57BL/6N background. The sperms of these mice were used to impregnate female C57BL/6J mice. The mice produced were further crossed with C57BL/6J mice to establish the Tdrd3-null mouse line in the same C57BL/6J background as that of Top3b-null mice. RT-qPCR and Western Blot analysis revealed that the levels of Tdrd3 mRNA and protein are undetectable in the brain extracts from the homozygous mice (Fig. S1e), confirming inactivation of this gene.

Adult male and female (3–10 month old) mice were used in behavioral tests; and 10-week-old mice were used for all other experiments. All animal procedures were approved by the NIA animal care and use committee (ACUC) and following the NIH animal guidelines.

Behavioral tests

Morris water maze: This test was carried out as previously described(Joo et al., 2020). Mice were trained in the Morris water maze (Morris, 1982) with 4 trials per day for 8 days. Probe tests were carried out at both 4 and 24 hours after training. Three such trials were excluded from analysis due to excessive floating (more than 20 seconds immobile prior to first swimming). To avoid confounding influence from remaining floating in training and probe trials, measures of path length rather than escape latency were used for analysis.

Spontaneous alternation. Mice were allowed to freely explore for 15 minutes in an opaque plexiglass maze containing four arms (5 cm × 30 cm, height 120 cm) radiating from the center at 90° angles. Arm entries were automatically detected by an overhead camera and ANY-Maze software (Stoelting; Wood Dale, IL). Alternations were scored as sequences of entries to four unique arms without repeats (e.g., ABCD or ABDC, but not ABCA). Alternation rate was calculated as number of alternations divided by number of opportunities for alternation. One mouse failed to leave the arm it was initially placed in and was excluded from analysis.

Fear conditioning: Procedures were modified from previous reports(Joo et al., 2020). In brief, mice were trained with three tone-shock pairings, then tested for context at 24 hours and for auditory cue at 48 hours post-training. Freezing was detected by camera and scored automatically with Video Freeze computer software (MED-Associates; St Albans, Vt).

Marble burying: Mice were placed in a 22 × 38 × 18 cm clear plastic chamber containing 5 cm of fresh bedding and 20 regularly spaced 13mm glass marbles. After 30 minutes, mice were gently removed, and the number of marbles covered more than 2/3 in bedding material were counted.

Additional tests: Open field test, zero maze, light-dark box, buried food test, 3-chamber sociability, reciprocal social interaction, acoustic startle, and prepulse inhibition were carried out as previously described(Iba et al., 2022; Joo et al., 2020).

Electrophysiological tests

The procedures followed a previous protocol (Joo et al., 2020). 6–8 slices from 5 WT and 6 Tdrd3-null mice were used.

BrdU labeling

For analysis of the number of proliferating cells in the dentate gyrus of the hippocampus, Bromodeoxyuridine (BrdU, Sigma) was diluted in PBS to make a 10 mg/ml stock solution. The 10-week-old mice were given single dose 200 mg/kg BrdU intraperitoneally (i.p.) and sacrificed after 2 hours(Wang et al., 2019). For analysis of new cell survival, the mice were given 4 consecutive doses (50 mg/kg each time) i.p. with a 12-hour interval and sacrificed after 4 weeks(Wang et al., 2019). The collected brains were tested by immunofluorescence staining as described below. WT and Tdrd3-null mice (n=3–4 per group) were used for each analysis.

Preparation of retrovirus and stereotaxic surgery.

The experiments were performed as previously described (Joo et al., 2020).

Preparation of retroviral vector.

We prepared a replication-defective murine moloney leukemia retroviral vector(Lewis and Emerman, 1994) to label dividing stem/progenitor cells in the adult dentate gyrus of the hippocampus. These cells become new neurons over several weeks (van Praag et al., 2002; Zhao et al., 2006). Concentrated viral solution was prepared using 90% confluent human embryonic kidney (HEK293T) cells as a packaging cell line. Briefly, the HEK293T cells were transfected with 7.5 μg vector DNA (CAG-GFP), and plasmids encoding retroviral structure proteins (5 μg CMV-Gag-Pol and 2.5 μg CMV-VSVG) by Lipofectamine 2000 (InVitrogen). The virus-containing supernatant was collected 36 h later, and then filtered and concentrated by ultracentrifugation (2 × 2h at 19,400 rpm). Virus titer was estimated by plaque assay after serial dilution into HEK293T cells (1 × 10⁸ i.u./ml).

Stereotaxic surgery and viral vector injections.

4–5-week-old WT and Tdrd3-null male mice were used for retrovirus CAG-GFP labeling experiments. Mice were housed individually before stereotaxic surgery and allowed to acclimatize for 3–5 days. The experimental mice were anesthetized with avertin (0.4 mg/g, i.p.) and placed within a stereotactic frame (Stoelting stereotaxic Instruments, USA). 1 μL of retrovirus at 0.1 μL/min was injected into the right dorsal dentate gyrus (DG). The coordinates were as follows: Dorsal DG, anterior-posterior (AP) = −2.10 mm; medial-lateral (ML) = 1.9 mm; dorsal-ventral (DV) = −2.10 mm. One month later, the injected mice were deeply anesthetized with isoflurane and perfused with 0.9% saline followed by 4% paraformaldehyde (PFA). The brains were extracted and post-fixed in 4% PFA for 24 h, followed by cryoprotection in 30% sucrose. Subsequently hippocampal tissue sections were utilized for immunohistochemistry and imaging analysis.

Immunofluorescent staining (IF)

Briefly, after isoflurane anesthesia, mice were perfused by PBS and 4% paraformaldehyde sequentially. Then brains were isolated and incubated overnight at 4 °C in 4% paraformaldehyde. After being immersed and incubated in 30% sucrose for 2 days at 4 °C, brains were embedded in OCT (optimal cutting temperature) medium at −80 °C for at least 30 mins, cryo-sectioned by 30 μm setting, and washed 3 times in PBS.

For BrdU staining, brain slices mounted on glass slides were incubated at 37°C, 20 min, then samples were circled by ImmEdge Pen for another 10 min. The slide was washed with PBS for 3 times, permeabilized by 0.2%Triton X-100 permeabilization buffer (in PBS) and incubated for 15–20 minutes at room temperature. Then the slide was treated by 1N HCl, 30 minutes on ice, followed by 2N HCl 30 minutes at 37 °C, and then was neutralized by 0.1N borate sodium buffer, pH 8.5, 20 minutes at room temperature. Afterwards, the slide was washed with PBS buffer for 3 times.

Tissues were incubated at room temperature for 1 hr in blocking solution (10% normal goat serum and 0.1% Triton X- 100 in PBS). Tissues were then incubated overnight at 4 °C with primary antibodies. The antibodies we used were rabbit anti-GFP conjugated with Alexa Fluor488 (1:200; Life technologies, A-21311), anti-BrdU (1:50, Abcam ab6326), anti-ki67 (1:200, Abcam, ab15580), anti-GFAP (1:250, Abcam, G3893), anti-Sox2 (1:400, Abcam, ab97959), anti-Parvalbumin (1:100, Millipore, MAB1572, anti-retrieval at pH6.0). The samples were washed 3 times with PBS and incubated at room temperature for 2 hr with a secondary antibody: Fluorescence anti-Mouse Alexa Fluor 488 (1:1000, Abcam, ab150113), anti-Rat Alexa Fluor 568 (1:1000, Invitrogen, A-11077), anti-rabbit Alexa Fluor 647 (1:1000, Abcam, ab150079) and DAPI (working concentration 300 nM, Life technologies, D1306). Then the slides were mounted by ProLong^™ Glass Antifade Mountant (Thermofisher, P36982) and imaged by confocal microscope, ZEISS LSM 780 or 880. The cell numbers were counted in 3–4 sections from each mouse. Cell density is calculated by the cell count in DG zone on each slide/brain section thickness (30 μm).

RNAscope

Here we used RNAscope Multiple Fluorescent Assay kit V2 from (ACDBio, REF323110) reported previously(Zhang et al., 2020). The main procedure followed instructions (DOC NO., USM-323100). Briefly, after the 24 h 4% PFA fixation and 48 h 30% sucrose dehydration, the brains were embedded into OCT and then cryo-sectioned at 8 μm. Then samples were dehydrated by 50%, 70%, 100% ethanol and treated by Hydrogen Peroxide. The target retrieval was done by mildly boiled ddH₂O with temperature monitoring. After being treated by Protease III, the samples were hybridized by probe (Mm-Tdrd3-C2, REF1032531-C2; Positive control mixture from mouse genes, REF320881; Negative control mixture from bacteria genes, REF320871). Then the signals were amplified by triple hybridizations in HybEZ^™ Oven. The fluorescent signals were developed by incubated with Opal^™ 570 (Akaya Bioscience Reagent kit, Part #: OP-001003) with 1:1500 dilution. Following that, the slides were counterstained by DAPI and mounted using Prolong Gold Antifade Mountant provided by the kit.

Western Blotting

The procedures were performed as described previously (Su et al., 2022a). The primary antibodies used were Top3b (1:2000, Sigma Aldrich, WH0008940M1-100UG), Tdrd3 (1:2000, Cell Signaling Technology, REF5942S), GAPDH (1:2000, Cell Signaling Technology, REF2118s).

TUNEL staining

The procedure followed the In Situ Cell Death Detection Kit, Fluorescein kit (Roche, 11684795910) protocol. Briefly, the sections were air-dried at room temperature overnight, then were fixed with a 4% PFA in PBS for 20 min at RT. After being washed 30 min with PBS, the samples were incubated in 0.1% Triton X-100 in 0.1% sodium citrate for permeabilization about 2 min on ice. For positive control, PBS-rinsed slides were incubated with DNase I recombinant (40 U/ml) in DNase I buffer for 10 min at RT then treated by solution 1 and solution 2 mixture. Negative control was treated the same but no terminal deoxynucleotidyl transferase (solution 2). Samples were then treated by solution 1 and solution 2 mixture. All the treated samples were incubated in a humidified box for 60 min at 37°C in the dark. Later, the slides were rinsed 3 times with PBS. Following that, rinsed slides were incubated with DAPI for 15 mins at RT. The samples were washed for 3 times with PBS and then mounted with Prolong Gold Antifade Mountant. Samples were analyzed under a fluorescence microscope (10 slices for 16.5 um in Z stack, LSM 880).

Black Gold II Myelin staining

The myelin staining followed the instructions of kit (Biosensis, TR-100-BG). Briefly, the sectioned samples were air-dried at 37°C for 2 h on a slide warmer until thoroughly dry. Then they were washed in distilled water for 2 minutes, and incubated in preheated solution A for about ~12 minutes in a clean covered Coplin jar in water bath, 60–65°C. The slides were rinsed in distilled water for 2 minutes and incubated in 1 X Solution B for 3 minutes. Following that, the slides were rinsed in distilled water for three times, each 5 minutes. The samples were then dehydrated via nature air-drying and immersed in xylene for 1–2 minutes to total remove water. Finally, the samples were mounted using a non-aqueous mounting media DPX (Sigma, 06522-100ML) and imaged by a lightscope.

RT-qPCR

Half of a mouse brain was homogenized, and 100 μL was removed for RNA extract by Trizol (InVitrogen, 15596026). The sample was centrifuged for 5 minutes at 12,000 × g at 4°C, then clear supernatant was transferred to a new tube with 0.2 mL of chloroform per 1 mL of TRIzol used for lysis. Samples were spun for 10 minutes at 12,000 × g at 4°C. After that, the upper layer was transferred to a new tube and 600 μL phenol: chloroform : isoamyl acid was added, mixed and incubated at RT for 5 minutes. Samples were spun for 10 minutes at 12,000 × g at 4°C. The upper aqueous phase was transferred into new tube. After addition of 2–3 μL GlycoBlue^™ Coprecipitant (Catalog: AM9515), the RNA was pelleted at 18000 rpm, 15 min, 4°C. The pellet was washed twice with 1 mL pre-chilled 75% ethanol, air-dried and resuspended in 20–50 μL of RNase-free water. RNA (1 μg) was reverse transcribed using Taqman Reverse Transcription Reagents (Applied Biosystems, N8080234). The cDNA was diluted to 1/5 and used as a template to perform qPCR with SYBR Green PCR Master Mix (Applied Biosystems, 4309155). The PCR primer sequences were listed in supplementary table S4. The gene expression relative to Gapdh was calculated by 2^−ΔΔCT method(Livak and Schmittgen, 2001).

RNAseq

The sample preparation and sequencing steps followed published protocols(Joo et al., 2020; Su et al., 2022a).

PROseq

The procedure followed published protocol(Mahat et al., 2016) with small modifications. Briefly, the brain tissues were prepared by permeabilization buffer (10 mM Tris-HCl, pH 7.4, 300 mM sucrose, 10 mM KCl, 5 mM MgCl2, 1 mM EGTA, 0.05% Tween-20, 0.1% NP40 substitute, 0.5 mM DTT, one tablet of protease inhibitors cocktail per 50 ml and 4 units per ml RNase inhibitor) and co-incubated with 2 × nuclear run on buffer for 10 min at 37 °C, 200 rpm. RNA was extracted by Trizol LS (Invitrogen, #10296028) and fragmented by 5 μl of ice-cold 1 N NaOH for 10 min on ice. After being neutralized with 25 μl of 1 M Tris-HCl, pH 6.8, the RNA was precipitated again with isopropanol. Biotin RNA was enriched by prewashed Dynabeads^™ M-280 Streptavidin beads (Invitrogen, #11206D), washed by high salt buffer, binding buffer, low salt buffer separately, and then extracted on beads by Trizol and ZYMO RNA clean & concentrator (Zymoresearch, #R1016). RNA was ligated with 1 μl of 100 μM VRA3 RNA adaptor at 20 °C overnight. Then biotin RNA was enriched and extracted again. RNA was modified in 5’ end by RppH (NEB, #M0356S) and performed hydroxyl repair by T4 PNK (NEB, #M0201L). After being extracted by ZYMO RNA clean & concentrator (Zymoresearch, #R1016), RNA was ligated with 1 μl of 100 μM VRA5 RNA adaptor at 20 °C overnight. Biotin RNA was enriched and extracted for the third time. The extracted RNA was used to perform reverse transcription, PCR amplification and then sent to sequence.

Transmission electron microscopy (TEM)

10 week old male mice were perfused with perfusion/fixation buffer (2% paraformaldehyde, 2% glutaraldehyde, 15% picric acid in 1 × PB). Brains were post-fixed with perfusion/fixation buffer for 2 hours at 4°C, followed by 2% paraformaldehyde in 1 × PB for 16 hours at 4°C. Then brains were sliced coronally using a brain mold, and the sliced blocks containing corpus callosum were cut into small blocks for obtaining the coronal view of axon. Thereafter, blocks were collected in 1 × PB buffer, fixed with 0.5% osmium tetroxide and contrasted in 1% uranyl acetate. After that, samples were dehydrated through a series of graded ethanol and with propylene oxide, and embedded in Durcupan ACM epoxy resin (Electron EMS, Fort Washington, PA). Resin-embedded samples were polymerized at 60°C for 2 days. After resin-embedded, 60 nm thick sections were cut with an ultramicrotome UC7 (Leica Microsystems Inc., Buffalo Grove, IL) using a diamond knife (Diatome, Fort Washington, PA). Ultrathin sections were collected on formvar-coated single slot grids and counterstained with Reynolds lead citrate. The ultrathin sections were examined and imaged using a Transmission Electron Microscope at 80,000 V (6800X and 9300X).

Bioinformatical analysis

RNAseq.

RNAseq raw data were removed adaptor by TrimGalore and checked by FastQC. Trimmed data were mapped using RSEM with the STAR alignments(Li and Dewey, 2011). The bedgraph files were produced by BEDTools. The above programs were run in Biowulf in NIH. Downstream analysis was done by R using Rstudio. The differentially expressing genes were analyzed mainly by DESeq2 package and gene annotations were performed by clusterProfiler, DOSE, ReactomePA and MeSHDbi packages (Foldchage >1.3, p-value < 0.05).

PROseq.

The adaptors were removed and checked as RNAseq. Fastq files were mapped by bowtie2. Mapped data were sorted and indexed by samtools. Bedgraph files were produced by BEDTools. Counts were calculated by featureCounts in subread(Liao et al., 2013).

Imaging and statistical analysis.

The illustration of behaviors in Fig. 1 and model in Fig. S7b were created with BioRender (BioRender.com). Quantification of 50 to 60 min LTP magnitude compared with the 10 min baseline, Data: mean ± SEM. Statistical significance was performed with two-tailed Student’s t test, ANOVA, or mixed effects model (restricted maximum likelihood). Dunnet’s, Tukey’s or Sidak’s multiple comparisons tests were used following significance in ANOVA or mixed effects model tests. Several outlier data points were excluded in behavior data, using outlier criteria of > 3 standard deviations outside of group mean. *p < 0.05, **p < 0.01, ***p < 0.001.The imaged figures were analyzed by ZEN (blue version) from Zeiss and Image J (Fiji) from NIH. Statistical analysis and graph preparation were performed using GraphPad or R. The bedgraph files in sequencing were examined by IGV genome browser. Tracing of dendrites and spines of GFP positive adult-born neurons was performed using Bitplane Imaris software (Oxford instruments) and Image J (FIJI distribution, NIH) with SNT plugin. The traced neurons were compared and draw by Inkscape (https://inkscape.org/).

Fig 1. Tdrd3-null mice show impaired spatial memory, anxiety, olfactory sensitivity, and synaptic plasticity.

a-b Tdrd3-null mice show memory impairments in the Morris water maze. a Path length until first crossing of the platform’s former location during probe tests at 4 and 24 hours post-training is longer in Tdrd3-null mice than WT mice. ** p<0.01 main effect of genotype. b During 24-hour probe test, WT (left) but not Tdrd3-null mice (right) preferred the target as measured by % path length (n=26–28 per genotype). Dashed lines indicate random chance. No sex differences were observed; for visual simplicity, water maze data is collapsed across sexes. *** p<0.001 Dunnett’s post-hoc. c Tdrd3-null mice show a reduced tendency to enter least-recently visited arm during free exploration of a 4-arm maze. Dashed line indicates random chance. n=12/genotype. ** p<0.01 main effect of genotype. d Tdrd3-null mice are more active compared to WT mice in the open field (middle). Male but not female Tdrd3-null mice show less avoidance of the center of an open field (right). n=35–36/genotype. * p<0.05 Sidak post-hoc test. ** p<0.01 main effect of genotype. e During fear conditioning, male Tdrd3-null mice do not differ from WT at baseline; but after 3 tone-shock pairings (“recovery”), they show increased freezing when later presented with shock-associated contextual or auditory cues. n=13/genotype. * p<0.05 Sidak post-hoc test. f Male but not female Tdrd3-null mice show less avoidance of the brightly lit compartment in a light-dark box. n=35–36/genotype. *, p<0.05 Sidak post-hoc test. g Tdrd3-null mice show a reduced anxiety-like digging response when placed in a novel environment, as assessed by fewer marbles buried. n=35−26/genotype. ** p<0.01 main effect of genotype. h Tdrd3-null mice take longer to find food when guided by olfactory cues. n=16/genotype. ** p<0.01 main effect of genotype. i-j Tdrd3-null mice show impairments in synaptic plasticity, as being suggested by abnormal excitatory post-synaptic potentials (EPSP) from field recordings of hippocampal slices during induction of (i) long-term potentiation (LTP) or (j) long-term depression (LTD). Data presented as mean + SD (a-h), mean ± SE (i-j). p-values < 0.05, 0.01, 0.001 are marked as: *, **, ***; p-value > 0.5 is marked as no significance (ns). Mixed effects model was performed in (a). One-way ANOVA was performed in (b). 2-way ANOVA with factors sex and genotype was performed in (c,d,f,g,h,i,j). Repeated measures ANOVA was performed in (e).

Results

Tdrd3-null mice displays elevated embryonic lethality

To investigate the function of Tdrd3 in mouse brain, we analyzed its mRNA expression pattern by RNAscope. We found that Tdrd3 mRNA is widely expressed throughout the brain and especially enriched in the hippocampus (Fig. S1a). These patterns are similar to those described in the Allen Brain Atlas (https://mouse.brain-map.org/static/atlas) and the patterns of Top3b(Joo et al., 2020), suggesting that Tdrd3 may function in hippocampus together with Top3b.

We next developed a Tdrd3-null mouse line using gene-trap (GT) strategy indicated in the method (Fig. S1b–d). The mRNA and protein of Tdrd3 are undetectable as shown in (Fig. S1e).

The Tdrd3-null mice developed to maturity at a rate similar to wildtype littermates. However, the observed percentage of Tdrd3^−/− newborn mice in litters from heterozygous male and female mice is about 12% (Fig. S1f), 2-fold fewer than the expected Mendelian ratio (25%). In comparison, the percentage of Top3b^−/− newborn mice was 20%, similar to the expected Mendelian ratio (<1.5-fold difference), suggesting that Tdrd3 but not Top3b is required for normal embryo viability.

Tdrd3-null mice are impaired in learning and memory tasks

We gave Tdrd3-null mice a panel of behavior tests to examine whether they exhibit cognitive and anxiety abnormalities comparable to Top3b-null and other mouse models of psychiatric disorders. In the Morris water maze, WT and Tdrd3-null mice learned the location of a hidden platform at similar rates, with trends for impairment in null mice emerging only in the last two days of invisible platform training (Fig. S2a). We then conducted two probe tests at 4- and 24-hours post-training to assess memory for the platform location. In both trials, null mice showed longer distance traveled until the first visit to the platform location (Fig. 1a; main effect of genotype F_1,98=10.5, p=0.002). Wild-type mice showed a more selective bias for the target quadrant compared to other non-target quadrants (Fig. 1b, left; significantly longer distance traveled; F_1.75,43.75 =23.1, p<0.0001; target vs other quadrants p=0.0008, p<0.0001, p<0.0001), whereas Tdrd3-null mice searched equally in the target and one of the adjacent non-target quadrants (Fig. 1b, right; F_1.56,40.64=18.5, p<0.0001; target vs other quadrants p=0.5 (adjacent, 2^nd column), p=0.004, p<0.0001). There were no sex differences in these measures (Fig. S2b).

We next assessed spatial working memory in a continuous spontaneous alternation task. Tdrd3-null mice had a significantly lower alternation rate (Fig. 1c; main effect of genotype F_1,20=14.8, p=0.001) and did not perform above random chance (Huttenrauch et al., 2016) (t₁₁=0.10, p=0.92). Because both of these tasks are hippocampus dependent (Lalonde, 2002; Vorhees and Williams, 2006), these results suggest defective hippocampal function in Tdrd3-null mice, as in Top3b-null mice (Joo et al., 2020). Tdrd3-null mice were also hyperactive in an open field test (Fig. 1d, middle; main effect of genotype F_1,67=8.2, p=0.006), a hallmark of animals with hippocampal lesions (Dillon et al., 2008).

In a fear conditioning task performed in males, Tdrd3-null mice did not differ from WT controls during the baseline training session but showed heightened conditioned freezing during tests of context and cue at 24 and 48 hours post-training, respectively (Fig, 1e; genotype × stage interaction F_3,72=3.5, p=0.02; context test p=0.02, cue test p=0.03). This pattern of results is again consistent with that seen in Top3b-null mice(Joo et al., 2020), indicating that the Top3b-Tdrd3 complex is required for normal associative fear learning.

Tdrd3-null mice exhibit reduced anxiety-like behaviors

Top3b-null mice display heightened anxiety-like behavior in several behavior tests (Joo et al., 2020), a phenotype prevalent in patients and animal models of schizophrenia and autism (Achim et al., 2011; Kazdoba et al., 2016). In the light-dark box, avoidance of a brightly-lit compartment was affected by Tdrd3-null in a sex-dependent manner, with reduced avoidance seen only in males (Fig. 1f; genotype × sex interaction F_1,67=8.3, p=0.005; male wt vs Tdrd3-null p=0.03, female wt vs Tdrd3-null p=0.2, Fig. S2c). In an open field test, a similar pattern of results was seen in avoidance of the center (Fig. 1d, right; genotype x sex interaction F_1,66=4.2, p=0.04; male wt vs Tdrd3-null p=0.04, female wt vs Tdrd3-null p=0.8). Although increased time in the center of open field could reflect reduced anxiety of Tdrd3-null mice, it is possible that it could also be a simple consequence of increased overall physical activity, as measured by total distance traveled in the open field (Fig. S2d–e).

To corroborate an anxiolytic effect and rule out hyperlocomotion as a confound, we conducted the marble burying test. In this test, anxiolytic drugs produce behavioral changes associated with reduced physical activity (Njung’e and Handley, 1991), whereas these drugs increase activity in other test environments (Crawley and Goodwin, 1980). Accordingly, Tdrd3-null mice buried fewer marbles (Fig. 1g, main effect of genotype F_1,65=10.9, p=0.002). This result confirms an anxiolytic effect of knockout, rather than simple hyperlocomotion. In the elevated zero maze, Tdrd3-null mice showed no difference in time spent in the open quadrants (Fig. S2f). Collectively, these results indicate reduced anxiety in Tdrd3-null mice, a phenotype that is directly opposite to Top3b-null mice, which show increased anxiety in a nearly identical battery of tests.

Tdrd3-null mice show impairments in olfactory function but no deficit in social behaviors

The buried food test requires mice to locate food using only odor cues (Yang and Crawley, 2009). Tdrd3-null mice were delayed in finding food (Fig. 1h, main effect of genotype F_1,28=9.4, p=0.005), suggesting an impairment in olfactory sensitivity. To rule out motivational effects on food retrieval latency, we performed a control experiment with food placed on the surface of the bedding, thus allowing use of visual cues in locating food. In this experiment, Tdrd3-null mice were not impaired in food retrieval latency (Fig. S2g; main effect of genotype: F_1,28=2.124, p=0.15; main effect of sex: F_1,28=2.824, p=0.10). This is consistent with Top3b-null mice, which performed normally in this task (Joo et al., 2020). Conversely, Tdrd3-null mice were normal in two tests of social behaviors in which Top3b-null mice showed abnormalities (Fig. S2h,i). Moreover, Tdrd3-null mice were normal in acoustic startle and prepulse inhibition, identical to behaviors of Top3b-null (Fig. S2j–k). These findings indicate that Top3b-null and Tdrd3-null mice have shared and unique behavior phenotypes.

Tdrd3-null mice have impaired hippocampal synaptic plasticity

Tdrd3-null and Top3b-null mice both have impaired hippocampus-dependent cognition, therefore, the former may also resemble the latter in defective hippocampal synaptic plasticity (Joo et al., 2020). To investigate this hypothesis, we performed electrophysiological recordings of CA1 neurons in hippocampal slices of Tdrd3-null mice. These include assays of long-term potentiation (LTP) and long-term depression (LTD), both of which are broadly used to study activity-dependent long-lasting changes in synaptic plasticity(Stacho and Manahan-Vaughan, 2022). In the LTP assay, Tdrd3-null mice exhibited a significantly decreased EPSP slope (about 30%) in response to high-frequency stimulation compared to WT mice (Fig. 1i). In LTD, Tdrd3-null mice also displayed reduced depression in response to a low-frequency stimulus (about 50% of that of WT mice) (Fig. 1j). The observed reduction of LTP and LTD in Tdrd3-null cells is weaker than that in Top3b-null mice (80% and 100% reduction, respectively)(Joo et al., 2020). However, the findings that Tdrd3-null and Top3b-null mice both have decreased synaptic plasticity may account for the hippocampus-dependent cognitive dysfunction observed in their behavioral assays.

We also performed a short-term synaptic transmission assay, paired-pulse facilitation (PPF)(Zucker and Regehr, 2002), and observed no significant difference between Tdrd3-null and WT mice (Fig. S2l), similar to our results with Top3b-null mice (Joo et al., 2020).

Tdrd3-null mice exhibit defective adult neurogenesis

We also examined hippocampal adult neurogenesis, which is known to be important for mood and spatial learning and memory(Aimone et al., 2014; Joo et al., 2020; Schoenfeld and Cameron, 2015). We investigated whether proliferation of adult neural stem cells (aNSCs) in the subgranular zone (SGZ) of hippocampus of Tdrd3-null mice is reduced using BrdU-labeling (Wang et al., 2019). We found that the density of BrdU-labeled aNSCs is significantly lower (about 50%) in Tdrd3-null mice (Fig. 2a–b), identical to the phenotype of Top3b-null mice(Joo et al., 2020), indicating that the entire Top3b-Tdrd3 complex is required for proliferation of aNSCs in hippocampus. In support of this inference, we performed double-staining with BrdU and another cell proliferation marker, Ki67, and obtained identical results (Fig.2a, c).

Fig. 2. Adult neurogenesis is decreased in subgranular zone of mouse hippocampus in Tdrd3-null mice, especially for the type II subtype neural stem cells.

a-c Images (a) and their quantifications (b-c) to show that proliferation of aNSCs in Tdrd3-null mice were reduced significantly compared to WT mice. This is indicated by decreased density of BrdU⁺ cells (b) and BrdU⁺Ki67⁺ cells (c) in BrdU pulsed-labeling experiments. Color codes in (a): Red, BrdU; White, Ki67; Blue, DAPI. Mouse numbers: WT=4, Tdrd3-null=4, with 7–8 slices/mouse. d A scheme of lineage-specific markers during hippocampal neural stem cell development. The cells at different developmental stages are shown at the top, whereas their markers are shown at the bottom. e-h Images (e-f) and their quantifications (g-h) to show that the type II but not type I aNSCs in SGZ are compromised in Tdrd3-null mice. This is revealed by decreased BrdU⁺Sox2⁺GFAP⁻ cell density (e, f, h) but unaltered BrdU⁺Sox2⁺GFAP⁺ cell density (g) (Red, BrdU; Green, GFAP; White, Sox2; Blue, DAPI). Several representative Type I and II aNSCs are marked by brown and red arrows, respectively in (e). Number of mice used: WT=4, Tdrd3-null=4, with 4–6 slices/mouse. We used 10 week-old adult mice for this experiment. Data are presented as mean values + SD. Two-tail Student’s t test was performed for (b), (c), (g), (h). p-values < 0.001, and 0.0001 are marked as: ***, and ****; p-value > 0.5 is marked as ns.

To determine which type of aNSC is defective in proliferation (Fig. 2d), we discriminated aNSCs by triple staining cells with BrdU and two unique markers for type I and II aNSCs, GFAP and Sox2(Miller and Gauthier-Fisher, 2009), respectively. We found that the type II aNSCs (marked by Sox2⁺/GFAP⁻) exhibited about 50% reduction, whereas type I cells (marked by Sox2⁺/GFAP⁺) remained unchanged (Fig. 2e–h), indicating that Tdrd3 inactivation specifically disrupts proliferation of type II aNSCs.

We observed that Sox2-labelled cells were more numerous than those labelled by BrdU, and only a small percentage Sox2-positive cells overlapped with BrdU-positive cells (Fig. 2e). Though Sox2 is a common marker for neural stem cells(Ellis et al., 2004), only a subset of Sox2 positive cells proliferate(Suh et al., 2007). It is possible that some Sox2 signals may be derived from antibody cross-reactivity. Alternatively, some Sox2 signals may be due to its expression in some differentiated neurons, as have been reported before (Ferri et al., 2004; Mercurio et al., 2019).

We failed to detect any apoptotic cells by TUNEL staining in either Tdrd3-null or WT mice (Fig. S3a), thus decreasing the possibility of increased apoptosis accounting for the reduction in number of aNSCs.

Tdrd3-null mice have abnormal newborn neurons and dendritic spines

Our findings that Tdrd3-null mice have defective proliferation of aNSCs raised the possibility that they have abnormal newborn neurons(van Praag et al., 2002; Zhao et al., 2006). To assess this possibility, we injected mice with BrdU and euthanized them after a 4 week interval to allow the dividing progenitor cells to develop into neurons. We then examined the newborn neurons in SGZ of hippocampus by double-staining with BrdU and a mature neuron marker, NeuN. We found that the density of double-positive cells (BrdU⁺NeuN⁺) is significantly reduced (about 40%) in Tdrd3-null mice (Fig. 3a), indicating that the number of newborn neurons in null mice is decreased, consistent with the data that the null mice have defective proliferation of aNSC.

Fig. 3. Neuronal complexity and spine morphology are altered in newborn hippocampal neurons of Tdrd3-null mice while only spines are altered in Top3b-null mice.

a Images (left) and their quantifications show that the newborn neuron density is decreased in Tdrd3-null mice. This is demonstrated by lower BrdU⁺NeuN⁺ cell density in Tdrd3-null mice than that in WT mice, 4 weeks after BrdU labeling (Red, BrdU; Green, NeuN; Blue, DAPI). Mouse numbers: WT=4, Tdrd3-null =4, with 3–4 slices/mouse. b-c Sholl complexity analysis indicates that the newborn neurons are more complex in Tdrd3-null than WT mice. This is illustrated by larger numbers of intersections at distances from soma from 90 to 150 μm in Tdrd3-null than WT mice. d-f Graphs from sholl analysis show that the mean intersection numbers (d), branch numbers (e) and total dendrite lengths (f) are all increased in neurons of Tdrd3-null mice vs. WT control mice. g-h The dendrites are smaller and shorter in Tdrd3-null mice, which are indicated by reduced dendrite lengths (g) and dendrite volumes (h) in Tdrd3-null mice. i The dendrites are thinner in both Tdrd3-null and Top3b-null mice, which are demonstrated by decreased dendrite mean diameters. j Representative images show that the spine density and sizes are smaller in Tdrd3-null mice than WT mice. k-l Graphs show that spine numbers in each dendrite (k) and dendrite spine density (l) are decreased in Tdrd3-null mice. m-n Graphs show that both spine lengths and spine max diameters are decreased in Tdrd3-null and Top3b-null mice. o A graph shows that the spine mean diameters are significantly reduced in Top3b-null mice, but remain unchanged in Tdrd3-null mice. Data are presented as mean values + SD (a, g-i, k-o), mean values ± SD (c-f). Two-tail Student’s t test was performed for all comparisons. p-values < 0.05, 0.01, 0.001, and 0.0001 are marked as: *, **, ***, and ****; p-value > 0.5 is marked as ns.

We subsequently investigated the morphology of newborn granule neurons in dentate gyrus by GFP-retroviral labeling(Joo et al., 2020; van Praag et al., 2002). Via neurite tracing and complexity analysis, we observed several abnormal features in Tdrd3-null mice. First, the null mice displayed increased intersections in the middle (90–150 mm away from soma) (Fig. 3b–c, S3b), and means of total intersections (Fig. 3d), as well as the number of branches (Fig.3e), indicating increased neuron complexity. Second, the average total length of neurites in each neuron were significantly increased (Fig. 3f), whereas the average lengths of single neurite were decreased (Fig.3g). Third, the volumes, lengths, and thickness of dendrites were all decreased in null mice (Fig. 3h–i). Collectively, these results demonstrate that Tdrd3-null mice have abnormal numbers and morphology in newborn neurons.

Dendritic spines from adult newborn neurons are crucial for the synaptic plasticity underlying learning and memory(Bailey et al., 2015; Mahmmoud et al., 2015), and spine abnormalities have been observed in patients and animal models of neurological diseases(Ammassari-Teule, 2020), including Top3b-null mice(Joo et al., 2020). We found that spines of Tdrd3-null mice resemble Top3b-null in several abnormal features, including reduced spine numbers(Joo et al., 2020) (Fig. 3k); lower spine density(Joo et al., 2020) (Fig. 3l); shorter lengths (Fig. 3m); and smaller maximum diameter (Fig. 3n); except the mean diameter of spines(Joo et al., 2020) (Fig. 3o). The observed abnormality in spines of hippocampal neurons of both Tdrd3 and Top3b-null mice could contribute to their defective synaptic plasticity and cognitive function.

We reanalyzed our previous data(Joo et al., 2020) using the more sensitive neurite tracing and quantitative method, and found that Top3b-null mice exhibited no significant difference in complexity (Fig. S3c–e) and dendrite length (Fig. S3f), This conclusion differs from that for Tdrd3-null mice. Nevertheless, the newborn neurons of Tdrd3-null mice share several features with Top3b-null mice including reduced dendrite mean diameter (Fig. 3i), smaller dendrite length and volume(Joo et al., 2020) (Fig. 3g, 3h) and lower spine density (Fig. 3l). The data reinforce the notion that Tdrd3 and Top3b -null mice have both shared and unique phenotypes.

Tdrd3-null mice exhibit reduced axon myelination

The reduced anxiety and impaired cognitive behaviors observed in Tdrd3-null mice resemble effects seen in rodents following demyelination of the corpus callosum (CC)(Makinodan et al., 2009; Valeiras et al., 2014). In addition, reduced myelination has been reported in patients and animal models of autism and schizophrenia(Galvez-Contreras et al., 2020; Phan et al., 2020; Valeiras et al., 2014). We therefore examined myelination in Tdrd3-null mice by Black Gold II myelin staining, and observed significantly reduced CC thickness in both males (Fig. 4a) and females (Fig. S4a) vs. that of WT mice, suggesting that TDRD3 is required for normal myelination in both sexes. The thickness of CC between male and female Tdrd3-null or WT mice shows no significant difference (Fig. S4a), suggesting that the role of TDRD3 in myelination is equally important between the two sexes. This reduction of CC thickness could be due to decreased myelination, and/or increased density of axons. To distinguish between these alternatives, we further examined CC by transmission electron microscopy (TEM) and observed an increase of axon density in the null mice (Fig. 4b). Notably, the thickness of the myelin sheath was significantly decreased in Tdrd3-null mice (Fig. 4c–e). Consistently, the inner axon diameter was significantly increased while the outer axon diameter unchanged (Fig. 4f). This was also reflected by a significant increase of the G-ratio (Fig. 4g), which is calculated as the ratio between the inner and outer axon diameters(Cercignani et al., 2017), and has been extensively utilized as a structural and functional index of optimal axonal myelination(Chomiak and Hu, 2009). Together, these data suggest that Tdrd3-null mice have reduced myelination and increased axon density in CC, both of which may contribute to the reduced anxiety and impaired cognitive behaviors.

Fig. 4. Tdrd3-null mice show reduced corpus callosum thickness and increased axon density in the brain.

a Images (middle) and their quantifications (right) from Black Gold II staining show that the thickness of corpus callosum (marked by the arrow) is significantly reduced in Tdrd3-null mice. A picture showing the locus of corpus callosum is shown on the left. b Electron micrographs (left) and their quantifications show that axon density in corpus callosum (CC) is increased in Tdrd3-null mice. c-d Enlarged electron micrographs (c) show that single axons from Tdrd3-null mice have reduced thickness in myelin sheath. Illustration of myelinated axons in CC was shown in (d). (e-g) Quantification of electron micrographs show differences between Tdrd3-null and WT mice in sheath thickness (e), inner and outer diameters of axons (f), and G-ratios. e The left graph shows that the average sheath thickness is decreased in Tdrd3-null mice. The right scatter plot shows that the sheath thickness has modest positive correlation with the outer axon diameters. The correlation coefficient (R) and p-values are listed in the graphs. The datapoints and the trendline from Tdrd3 null mice are largely lower than those of WT mice, indicating reduced sheath thickness in the former animals. f A graph shows that the Inner axon diameter is longer, whereas the outer axon diameter is not significantly different, in Tdrd3-null mice when compared to those of WT mice. g The left graph shows that the G-ratios are significantly higher in Tdrd3-null mice than WT. The right scatter plot shows that the G-ratio has weak positive correlation with outer axon diameters. The datapoints and trendline from Tdrd3-null mice are largely higher than those of WT mice, indicating that axons from the former mice have larger G-ratios (meaning reduced sheath thickness). Data are presented as mean values + SD. Two-tail Student’s t test was performed for all comparisons. Linear model fit was used for fitted curves in e (right) and g (right). p-values < 0.001, and 0.0001 are marked as: ***, and ****; p-value > 0.5 is marked as ns.

Tdrd3-null mice display impaired neuronal activity- dependent transcription

We next explored whether the Tdrd3-null mice exhibit defects in neuronal activity-dependent transcription (NADT) of immediate early genes (IEGs) in response to fear conditioning stress as do Top3b-null mice(Joo et al., 2020). We analyzed Tdrd3-null mice treated with the same stimulus and observed significantly reduced induction of several IEG mRNAs in amygdala and hippocampus (Fig. S4b–c), two brain regions critical for fear memory(Kim and Cho, 2020; Tovote et al., 2015). More IEG mRNAs showed significant reduction in amygdala than hippocampus (7 vs. 3 among 9 genes tested) by RT-qPCR, suggesting that Top3b-Tdrd3 may be more important in the former than the latter regions for NADT.

Tdrd3-null mice exhibit abnormal post-transcriptional regulation

Top3b-Tdrd3 has been reported to participate directly in both transcriptional(Joo et al., 2020; Su et al., 2023; Yang et al., 2014b) and post-transcriptional regulation(Su et al., 2022b). The evidence for the latter includes defective mRNA translation and turnover(Su et al., 2022b) in HCT116 null cells inactivated of either protein. To determine whether Tdrd3-null mouse brain exhibit abnormal mRNA turnover, we compared the levels of nascent RNA determined by PROseq versus those of mature RNA by RNAseq(Blumberg et al., 2021; Su et al., 2023). The former is a readout of genome-wide transcription, whereas the latter is the result of both transcription and mRNA turnover; and differences between the two should be due to mRNA turnover (Fig. 5a).

Fig. 5. Several mRNAs associated with GABAergic interneurons are downregulated by post-transcriptional mechanisms in Tdrd3-null mice.

a A schematic diagram of experimental design depicts that nascent and mature mRNAs are profiled by PROseq and RNAseq, respectively. The former method detects mRNA altered at the transcriptional step, whereas the latter measures mRNA changes caused by both transcriptional and post-transcriptional mechanisms. The up or down arrows indicate differentially expressed genes (DEGs) that are increased or decreased in null mice, respectively, whereas the horizonal arrows mark mRNAs that remain unchanged. The mRNAs that do not show the same direction of alteration in both methods should be regulated by post-transcriptional mechanism (such as turnover). b Volcano plots show differentially expressed genes (DEGs) identified by PROseq (left) and RNAseq (right) in Tdrd3 null mouse brains (green, downregulated; red, upregulated, log2FoldChange > 1.3 and p-value < 0.05). c HEATmap analysis to compare DEGs obtained by PROseq vs. those by RNAseq. The left panel illustrates how the up or down-regulated DEGs from PROseq are altered in RNAseq. The right panel shows the reciprocal comparison: how the up and down-regulated DEGs from RNAseq are altered in PROseq. The data lines marked in the same color indicate DEGs that are altered in the same direction by both assays. The table below the HEATmaps indicate that the percentages of DEGs that are altered in the same or opposite directions, as marked by the directions of arrows. d Gene ontology analysis of downregulated DEGs from RNAseq using molecular function category indicates that the expression of genes associated with chloride channels in GABAergic interneurons (marked by red boxes) is disturbed in Tdrd3-null mice. e Relationship between several enriched GO terms and their associated genes from (d). f Graphs from USCS genome browser show that several representative genes associated with chloride channels exhibit reduced signals by RNAseq but unchanged signals by PROseq (Gabra2, Gabra6, Pltp) in Tdrd3-null mice. The down arrows mark the RNAseq signals thar are reduced in the null mice, whereas the horizontal arrows mark the PROseq signals that are unchanged. Gapdh and Tdrd3 genes are included as negative and positive controls, respectively. g RT-qPCR results of nascent RNA (left) and mature RNA (right) expression of Gabra2, Gabra6 and Pltp in brains of Tdrd3-null mice and Top3b-null mice. h Immunofluorescent images (left) and their quantification (right) show that the density of parvalbumin (PV)-positive cells is significantly reduced in Tdrd3-null mice. The arrows mark representative PV-positive cells in CA areas. Data are presented as mean values + SD. Two-tailed Student’s t test was performed for the comparisons. p-values < 0.05, 0.01 are marked as: *, **; p-value > 0.5 is marked as ns.

Our PROseq and RNAseq from whole brains of Tdrd3-null and WT mice identified 300 and 227 differentially-expressed genes (DEGs) (p<0.05, fold change 1.3), respectively (Fig. 5b; Table S1), suggesting that Tdrd3 regulates only a small fraction of genes at transcription and/or post-transcriptional steps(Su et al., 2022b). In both sequencing analyses, the DEGs that show increase of their levels in null mice (229 and 147, respectively) are about 2–3 fold greater than those that show decrease (71 and 80, respectively), suggesting that Tdrd3 can either positively or negatively regulate gene expression. These data are largely consistent with our previous findings in human KO cell lines and Top3b-null mice(Joo et al., 2020; Su et al., 2023; Su et al., 2022b).

Comparison of DEGs between PROseq and RNAseq by heatmaps revealed substantial differences (Fig. 5c; Table S2,S3). For example, for DEGs that show either an increase or decrease by PROseq in Tdrd3-null mice, the percentages of them showing the same direction of alteration by RNAseq are fewer than 10%, whereas the majority remain unchanged (~80%)(Fig. 5c, left; Table S2, S3). Similarly, For DEGs showing an increase or decrease by RNAseq, the percentages of them showing the same direction of alteration by PROseq are fewer than 20%, whereas the majority (>65%) remained unchanged (Fig. 5c, right; Table S2, S3). The findings that the majority of DEGs altered by one assay are not altered in the same direction by the other suggest that Tdrd3 inactivation in mouse brains has a major effect on mRNA turnover, with only a small effect on transcription.

We also analyzed the RNAseq data from Top3b-null mouse brains(Joo et al., 2020), and identified 156 increased and 69 decreased DEGs vs. WT control mice (Fig. S5a,b). Unexpectedly, the percentage of overlapped DEGs between Top3b-null and Tdrd3-null mice are low (<13% of total DEGs) (Fig. S5c–d). This difference between adult brains of the two null mice may be caused by different functions of each protein and also possible difference in earlier development. More work is needed to determine possible causes.

Tdrd3-inactivation alters the turnover rates of mRNAs encoding each of several GABA receptors

To determine which mRNAs altered in Tdrd3-null mice may account for the observed behavioral and neurological abnormality, we performed Gene Ontology (GO) analysis of DEGs from RNAseq. Molecular functional analysis of DEGs with reduced expression in Tdrd3-null mice identified several enriched GO terms that are associated with GABA-gated chloride channels or ceramide binding (Fig. 5d). We examined DEGs involved in these GO terms and selected three genes: Gabra2, Gabra6, and Pltp (Fig. 5e), for further analysis. UCSC genome browser analysis revealed that RNAseq signals for these genes were reduced (about 47%, 35.4% and 29.6%, respectively) in Tdrd3-null mouse brains (based on average TPM), whereas their PROseq signals remain either unchanged (<1.2 fold difference) or increased (Gabra6, 2.7 fold) (Fig. 5f). As negative controls, the signals of both RNAseq and PROseq for Gapdh were unchanged. As a positive control, the RNAseq signal for Tdrd3 was significantly reduced (reduced 10.4 fold) in Tdrd3-null mice, consistent with RT-qPCR data (Fig. S1e). RT-qPCR analysis confirmed that mature mRNA levels for Gabra2, Gabra6, and Pltp were reduced by about 30% (p<0.05) (Fig. 5g, right), whereas their precursor mRNA levels remained unchanged (Fig.5g, left), in Tdrd3-null mice. Apparently, mature mRNA levels of these gene are reduced in Tdrd3-null mice; and this reduction is not due to reduced transcription, but rather accelerated mRNA turnover. Our data that Gabra2 and Gabra6 are regulated post-transcriptionally by Tdrd3 are reminiscent of earlier findings that GABA receptor mRNAs are subject to post-transcriptional regulation by multiple RBPs, including FMRP(Schieweck and Kiebler, 2019).

Gabra2 and Gabra6 are distinct subunits of the ligand-gated chloride channel receptor for the major inhibitory neurotransmitter GABA. These subunits are vital for the formation of inhibitory GABAergic synapses(Brown et al., 2016). The reduced expression of Gabra2 and Gabra6 led us to investigate whether parvalbumin (PV)-expressing GABAergic interneurons are impaired in Tdrd3-null mice. Immunostaining showed a marked decrease in the density of PV-positive interneurons (p<0.05) in the hippocampus of Tdrd3-null mice (Fig. 5h). These data are consistent with our RNAseq data showing reduced expression of GABAergic-associated genes (Gabra2 and Gabra6) in Tdrd3-null mice.

Tdrd3-null mice exhibit reduced expression of several genes downstream of GABA receptors

The findings that mRNA levels of Gabra2 and Gabra6 are reduced in Tdrd3-null mice raised the possibility that other genes acting in the GABAergic pathway might show similarly reduced expression. We investigated this hypothesis and found that two genes acting in this pathway, Neurod1 and Neurod2(Roybon et al., 2010; Tozuka et al., 2005), also display reduced mRNA levels by RNAseq(Fig. 6a) and RT-qPCR (Fig. 6c) in Tdrd3-null mouse brains. Thus, the signaling cascade between GABA receptors and Neurod molecules could be impaired. Analysis of PROseq and RT-qPCR data showed that nascent RNA levels of Neurod1 and Neurod2 are not decreased in Tdrd3-null mice (Fig. 6a, S6a), so that their reduced mRNA levels are likely due to accelerated turnover.

Fig. 6. Several Gabra2 downstream genes and myelination associated genes are downregulated by post-transcriptional mechanism in Tdrd3-null mice.

a Bedgraphs from UCSC genome browser analysis show that two genes downstream of Gabra2, Neurod1 and Neurod2, exhibit reduced RNAseq signals but unchanged PROseq signals in Tdrd3-null mice. b Bedgraphs from UCSC genome browser analysis show that three genes that are known to be bound and enhanced expression by Neurod1 exhibit reduced signals of both RNAseq and PROseq in Tdrd3-null mice. The down arrows mark reduced signals, whereas horizontal arrows mark genes that are unchanged in the null mice. c RT-qPCR results show that mature RNA levels of Neurod1 and Neurod2 are significantly decreased in Tdrd3-null mice (p<0.05), and both genes also show a strong trend of reduction in Top3b-null mice (for Neurod2, p=0.06; which does not reach statistical significance). d RT-qPCR analyses show that mature RNA levels of Eml1 and Pou3f2, but not Hes6, are significantly reduced in Tdrd3-null mice. e. Gene ontology enrichment analysis of downregulated genes in Tdrd3-null mice using Biological Process category shows that several top enriched GO terms are related to myelination (marked in red boxes). f Relationship between enriched GO items and their associated genes reveals that these terms are closely associated with each other and share several common genes (marked as red rectangles). g Bedgraphs from USSC genome browser analysis of RNAseq and PROseq show that mature but not nascent RNA levels for several myelination-associated genes are reduced in Tdrd3-null mice (Mag, Trf, Itgb4 and Fa2h). The down arrows mark reduced signals, whereas horizontal arrows mark genes that are unchanged in the null mice. h RT-qPCR results show that mature RNA levels of several myelination associated genes are decreased in Tdrd3-null, but not Top3b-null mice. Data are presented as mean values + SD. Two-tailed Student’s t test was performed for the comparisons. p-values < 0.05, 0.01, 0.001, and 0.0001 are marked as: *, **, ***, and ****; p-value > 0.5 is marked as ns.

Mutations in Neurod2 have been associated with autism(Bordey, 2022; Runge et al., 2021), similar to mutations in Top3b. This prompted us to examine the expression of Neurod1 and Neurod2 in Top3b-null mouse brains. We observed significant reduction of Neurod1 mRNA levels by RT-qPCR as in Tdrd3-null mice, and a strong decreased trend in Neurod2 mRNA levels (Fig. 6c) (p = 0.06). Interestingly, we did observe statistically significant reduction of Neurod1 nascent RNA (Fig. S6a), consistent impairment of transcription of this gene in Top3b-null mice.

Neurod1 and Neurod2 are pioneer transcriptional factors that establish transcriptional and epigenetic profiles in the neuronal lineage(Hahn et al., 2019; Matsuda et al., 2019; Pataskar et al., 2016). We found that only a small percentage (<0.5%) of these Neurod1-bound genes(Pataskar et al., 2016) exhibited reduced PROseq or RNAseq signals in Tdrd3-null mice (Fig. S6b). UCSC genome browser analysis revealed reduced RNAseq signals for three representative Neurod1-bound genes (Hes6, Eml1 and Pou3f2) (Fig. 6b). RT-qPCR confirmed statistically significant reduction of mature mRNA levels for two genes (Eml1 and Pou3f2) in Tdrd3-null (Fig. 6d) but not Top3b-null mice. We did not observe the significant reduction in nascent RNA of these three genes in both Tdrd3 and Top3b deficient mice (Fig. S6c). Collectively, the discrepant change of nascent and mature mRNA levels of these three genes imply that these genes are also regulated by turnover.

Tdrd3-null mouse brains display reduced expression of several myelination genes.

To investigate how Tdrd3 inactivation leads to demyelination of axons in CC, we performed GO analysis of our RNAseq data using Biological Process Category and observed “myelination” in several top 10 enriched GO terms (Fig. 6e). Examination of these terms revealed several myelination-associated genes (Mag, Trf, Itgb4 and Fa2h)(Fig. 6f) that showed reduced levels of RNAseq, but unchanged levels of PROseq(Fig. 6g), indicating that the reduced expression of these genes in Tdrd3-null mice is due to accelerated turnover. RT-qPCR analysis confirmed this result (Fig. 6h, S6e). As a comparison, no change was found in their mature or pre-mRNA levels in Top3b-null mouse brains (Fig. 6h, S6e). Because inactivation of these myelination-associated genes (Itgb4, Fa2h and Trf) can cause demyelination(de los Monteros et al., 1999; Potter et al., 2011; Van der Zee et al., 2008), their reduced expression can explain the demyelination phenotype observed in Tdrd3-null mice.

Discussion

Tdrd3-null mice display neurological and behavior problems similar to Top3b-null mice.

A major objective of this study is to establish a mouse model to examine a hypothesis that Tdrd3 may resemble its two partners (Top3b and FMRP) in playing roles in psychiatric and cognitive disorders. Our findings that Tdrd3-null mice resemble Top3b-null mice in exhibiting abnormalities in cognitive and anxiety behaviors, synaptic plasticity, adult neurogenesis, and neuron morphology indicate that Tdrd3 could play important roles in psychiatric and cognitive disorders as do its interacting partners Top3b and FMRP.

Though Tdrd3-null and Top3b-null mice share many phenotypes (Fig. S7a), differences between the two mice are also evident. For example, Tdrd3-null mice exhibit reduced anxiety, which is opposite to Top3b-null mice, which show increased anxiety. In addition, Tdrd3-null mice show normal social interactions, unlike the deficits observed in Top3b-null mice. Conversely, mice null for Tdrd3 but not for Top3b have impaired olfactory function. Moreover, Tdrd3-null mice show increased complexity of newborn neurons, whereas Top3b-null mice do not. These differences in behavior and neuron morphology likely result from the differences in gene expression profiles in two knockout strains (Fig. S6d). The differences observed between gene expression profiles are supported by RT-qPCR analysis showing that multiple genes important for neural development and function are altered in Tdrd3-null but not Top3b-null mice (Graba2, Gabra6, etc; Fig. 5–6). The data suggest that although Top3b and Tdrd3 are components of one complex, each subunit may have some functions independent of its partner.

Tdrd3 regulates gene expression at both transcriptional and post-transcriptional steps

Earlier work based on Top3b-null mice indicates that Top3b-TDRD3 complex directly enhances NADT of neuronal early response (NER) genes(Joo et al., 2020). Our current findings that several NER genes show reduced NADT in tissues from Tdrd3-null mouse brains further support this action (Fig. S4).

Recent work using Top3b and Tdrd3-null cell lines demonstrate that Top3b-Tdrd3 also binds to mRNAs and regulates their translation and turnover(Su et al., 2022b). However, whether the complex has the same function in animals remains unclear. Here we performed gene expression profiling of both nascent and mature mRNAs in Tdrd3-null mouse brains, and observed that many genes important for neuronal function exhibit reduced levels in mature mRNAs but are unchanged in nascent RNA (vice versa). This indicates the turnover rates are altered in absence of Tdrd3. We noticed that some mRNAs (Neurod1, Neurod2) showed significant reduction in Tdrd3-null mice (p<0.05) and also displayed a strong trend of reduction in Top3b-null mice (Fig. 6c), suggesting that their turnover rate may be affected by the entire Top3b-Tdrd3 complex.

Tdrd3-null mice have a defective GABA-Neurod signaling cascade

GABAergic signaling plays an indispensable role in neural development by regulating adult neurogenesis, and its disruption has been linked to autism and schizophrenia(de Jonge et al., 2017; Earnheart et al., 2007; Fatemi et al., 2009), disorders associated with Top3b or Tdrd3 mutations. Our finding of defective PV-expression GABAergic interneurons in Tdrd3-null mice suggests that GABAergic pathways are likely compromised, leading impaired adult neurogenesis and behavioral defects. Neurod transcription factors are downstream effectors of GABA cascade(Gonzalez-Nunez, 2015; Tozuka et al., 2005), and mutation of Neurod2 has been associated with autism(Runge et al., 2021). Notably, we found that multiple mRNAs important in the GABA-Neurod signaling cascade show accelerated turnover in Tdrd3-null mouse brains (Fig. 5 and 6; summarized in Fig. S6d). These include mRNAs of Gabra2, Gabra6, Neurod1 and Neurod2; as well as some targets of Neurod1: Pou3f2, and Eml1. It has been shown that GABA Receptor mRNAs are subject to post-transcriptional regulation by various RBPs, including FMRP (which directly interacts with Tdrd3)(Schieweck and Kiebler, 2019). Fmr1-KO mice do share common phenotypes with Tdrd3-null and Top3b-null mice, including impaired adult neurogenesis, and hippocampus-dependent learning(Guo et al., 2011; Luo et al., 2010). Thus, Tdrd3 and Top3b may work with FMRP and other RBPs to regulate mRNAs important for GABAergic signaling by post-transcriptional mechanisms (Fig. S7b). Mutations in any of the three proteins may impair such mechanisms, leading psychiatric and cognitive disorders.

Conclusion

Tdrd3-null mice display neurological and behavioral defects similar to those of Top3b-null mice, including defective cognitive function, synaptic plasticity, and adult neurogenesis. These defects could be due to impaired transcriptional and post-transcriptional regulation of gene expression. The impaired post-transcriptional regulation may account for the defective GABA-Neurod signaling cascade in Tdrd3-null mice. We infer that the entire Top3b-Tdrd3 complex is essential for normal brain function, and that defective post-transcriptional regulation could contribute to cognitive impairment and psychiatric disorders.

Highlights.

• Tdrd3-null mice display phenotypes observed in psychiatric and cognitive disorders

• The behavioral phenotypes include memory deficits and reduced anxiety

• Neurological defects are observed in synaptic plasticity, adult neurogenesis, and myelination

• Both transcriptional and posttranscriptional regulation are impaired in Tdrd3-null mice.

• Impaired post-transcriptional regulation may account for the observed neurological defects.

Abbreviations:

LTP

Long-term Potentiation

LTD

Long-term Depression

NADT

Neuronal Activity-Dependent Transcription

NER

Neuronal Early Response.

IEGs

Immediate Early Genes.

aNSCs

adult neural stem cells

SGZ

subgranular zone

Availability of Data and Materials

All relevant next generation sequencing data are deposited at GEO database (Accession number GSE223568).