Topoisomerase 3β (Top3β) is unique among animal topoisomerases for its dual activity, modifying both DNA and RNA topology. It's implicated in transcription and translation, forming a complex with TDRD3 and interacting with FMRP to influence mRNA translation and synapse formation. While Top3β's role in transcription remains unclear, its association with FMRP suggests a possible link to mental disorders like Fragile X syndrome. Genetic studies have linked Top3β mutations to schizophrenia, autism, epilepsy, and cognitive impairment. This study investigated the role of Top3β in brain function using a Top3β knockout (KO) mouse model. Unlike other topoisomerases (Top1 and Top2β) whose roles in neuronal function are more established, Top3β's specific contribution to neuronal activity-dependent transcription (NADT) remained unknown. This research hypothesized that Top3β deficiency would cause brain dysfunction and examined the behavioral and transcriptional effects of Top3β knockout in mice.

Previous research has established the dual-activity nature of Top3β and its interactions with TDRD3 and FMRP, suggesting roles in transcription and translation. Studies have linked Top3β mutations to several neurodevelopmental and psychiatric disorders, but the causal mechanisms remained unclear. Other topoisomerases, Top1 and Top2β, have demonstrated roles in transcription regulation and neuronal function, particularly NADT. However, a direct link between Top3β and the transcription of genes crucial for neuronal function and mental health had yet to be established. The existing research highlighted the need for a more comprehensive investigation into the impact of Top3β deficiency on brain function.

The study utilized a Top3β knockout (KO) mouse model to investigate the effects of Top3β deficiency. Behavioral tests, including the elevated plus maze, light-dark box, three-chamber sociability, reciprocal social interaction, fear conditioning, context discrimination, and Morris water maze were conducted to assess anxiety, social interaction, fear, and cognitive function. Electrophysiological recordings from hippocampal slices were used to analyze synaptic transmission and plasticity (LTP and LTD). Immunohistochemistry was performed to examine adult hippocampal neurogenesis (BrdU labeling, neuron morphology). RNA polymerase II (Pol II) ChIP-seq and total RNA-seq were used to analyze global and specific gene transcription in response to fear conditioning, focusing on neuronal activity-regulated (NER) genes. Additional ChIP-seq analyses were done using antibodies against phospho-specific forms of Pol II (Ser-5 and Ser-2), TDRD3, and several chromatin marks (H3K4me1-3, H3K9ac, H3K27Ac, H3K9me3, H3K27me3). MRI was used to assess ventricular enlargement. Statistical analyses included Student’s t-tests, ANOVA tests, and gene set enrichment analysis.

Top3β-KO mice exhibited increased anxiety and abnormal social interactions, enhanced fear memory, and impaired hippocampus-dependent cognitive function in behavioral tests. Electrophysiological analysis revealed reduced basal synaptic transmission and impaired hippocampal LTP and LTD in KO mice. Adult hippocampal neurogenesis was significantly reduced, and the morphology of newly generated neurons was altered in KO mice. Fear conditioning caused a significant increase in Pol II signals at NER genes (Arc, Fos, Egr1, and Npas4) in WT mice, but this increase was absent in KO mice. Global NADT was significantly impaired in Top3β KO mice; this was observed in more than 60% of the NER genes. TDRD3, a known Top3β binding partner, showed altered binding patterns in KO mice. Genes associated with dementia and schizophrenia exhibited reduced transcription in Top3β-KO mice, particularly genes involved in Alzheimer's disease and learning and memory. Synapse and neurogenesis-related genes also showed reduced transcription.

The results demonstrate that Top3β deficiency leads to a wide range of behavioral and physiological abnormalities consistent with features of schizophrenia and autism spectrum disorders. Impaired hippocampal synaptic plasticity and reduced neurogenesis contribute to the observed cognitive deficits. The crucial role of Top3β in NADT, particularly for genes related to mental health, was confirmed. A model was proposed where neuronal activity increases topological stress on the genome, requiring Top3β and TDRD3 to facilitate transcription of relevant genes, potentially cooperating with Top2β. The altered transcription of dementia, schizophrenia, synapse, and neurogenesis related genes helps explain the observed behavioral impairments in Top3β-KO mice.

Top3β plays a critical role in normal brain function, particularly in regulating NADT of genes important for cognitive function and mental health. The Top3β-KO mouse model provides strong evidence linking Top3β deficiency to neurodevelopmental and psychiatric disorders. Future research should focus on further elucidating the precise mechanisms by which Top3β and TDRD3 interact with Pol II and influence transcription, exploring potential therapeutic targets based on these findings.

The study used a mouse model, and the results may not fully translate to humans. The behavioral tests used may not capture the full complexity of human psychiatric disorders. The analysis focused on a specific set of NER genes and did not examine the full scope of Top3β's transcriptional effects. The study used mainly male mice, and there was some variability in behavioural and transcriptional responses, although the underlying causes of this variability were not fully determined.