TDP-43 facilitates milk lipid secretion by post-transcriptional regulation of *Btn1a1* and *Xdh*

Mammalian milk is crucial for offspring survival, providing both nutrients and immunoregulatory agents. While breastfeeding offers significant benefits to both mother and child, lactation insufficiency affects a substantial portion of women, underscoring the need to understand the molecular mechanisms regulating milk secretion. Lipids, a major energy source in milk, are secreted as lipid droplets (LDs) enclosed in a membrane. Butyrophilin 1a1 (BTN1A1, encoded by *Btn1a1*) and xanthine oxidoreductase (XOR, encoded by *Xdh*) are known to be involved in LD secretion. However, the post-transcriptional regulation of these genes remains poorly understood. RNA-binding proteins (RBPs) play a crucial role in post-transcriptional gene regulation; therefore, identifying RBPs influencing lactation-related genes could provide insights into milk secretion. This study focuses on identifying and characterizing RBPs that may be involved in regulating milk lipid secretion, with a particular focus on TDP-43 due to its positive selection in mammals and its established role in other biological processes.

Previous research has established the importance of breastfeeding for infant health, including reduced risk of infections and improved immune development. Furthermore, studies have highlighted the significant role of milk lipids in providing energy for newborns. BTN1A1 and XOR have been identified as key players in milk lipid secretion, with knockout studies demonstrating their involvement in this process. However, the regulatory mechanisms controlling the expression of these genes, particularly at the post-transcriptional level, remained largely unknown. Existing literature suggests a significant role for post-transcriptional regulation in lactation, highlighting the need for research into RNA-binding proteins (RBPs) that might modulate the stability and expression of essential lactation genes.

This study employed a multi-faceted approach. First, phylogenomic analysis was conducted on 15 vertebrate genomes to identify RBPs under positive selection in the mammalian lineage. This analysis utilized likelihood ratio tests (LRTs) to identify genes with significant positive selection signals. Next, the researchers generated a *Tardbp* knockout (KO) mouse model using a whey acidic protein (WAP)-Cre system to specifically target TDP-43 deletion in mammary epithelial cells during pregnancy and lactation. The survival rate of pups from *Tardbp* KO mothers was compared with control mice. Morphological analyses, including hematoxylin and eosin (H&E) staining, whole-mount staining, and immunofluorescence, were performed to assess mammary gland structure and lipid droplet formation. Milk volume and lipid composition were also analyzed. RNA-sequencing (RNA-seq) was used to identify genes differentially expressed in *Tardbp* KO mammary glands. The interaction between TDP-43 and target mRNA (*Btn1a1* and *Xdh*) was investigated using RNA immunoprecipitation (RIP) and RNA pull-down assays. mRNA stability assays using actinomycin D were performed to assess the effect of TDP-43 on mRNA half-life. Finally, the study included a clinical component, analyzing human milk samples from lactating women to examine the correlation between TDP-43 expression and milk output.

The study's key findings include: 1. TDP-43 exhibited significant positive selection in the mammalian lineage, suggesting its importance in mammalian-specific functions like lactation. 2. *Tardbp* KO mice showed severely impaired milk lipid secretion, leading to smaller milk volumes and significantly lower triacylglycerol (TAG) concentrations in milk compared to controls. Conversely, *Tardbp* KO mice showed a significant accumulation of TAGs in mammary epithelial cells (MECs), indicating a defect in lipid secretion rather than production. 3. Morphological analysis revealed larger lipid droplets (LDs) in the MECs of *Tardbp* KO mice, suggesting a deficiency in LD secretion. 4. RNA-seq analysis revealed that many genes involved in lipid metabolism were differentially expressed in *Tardbp* KO mice. *Btn1a1* and *Xdh*, essential genes for milk lipid secretion, showed decreased expression in *Tardbp* KO mice at both mRNA and protein levels. 5. RIP and RNA pull-down assays demonstrated that TDP-43 directly binds to the 3'-UTRs of *Btn1a1* and *Xdh* mRNA, specifically at TG-enriched sequences. Deletion mutants lacking these binding sites disrupted the interaction. 6. Actinomycin D treatment showed that TDP-43 is necessary for the stability of *Btn1a1* and *Xdh* mRNA. 7. Clinical data from human milk samples indicated a positive correlation between *TARDBP* expression and exclusive breastfeeding, suggesting that low TDP-43 expression is associated with lactation deficiency in humans.

This study provides strong evidence for the crucial role of TDP-43 in milk lipid secretion, a critical process for offspring survival. The findings connect the positive selection of TDP-43 in the mammalian lineage to its direct involvement in regulating the expression of key genes involved in milk lipid secretion. The mechanism involves the post-transcriptional stabilization of *Btn1a1* and *Xdh* mRNA via binding to their 3'-UTRs. The concordance between mouse model data and the clinical findings in humans highlights the translational potential of these findings. The significant reduction in milk lipid secretion and subsequent pup mortality in the *Tardbp* KO mice emphasizes the vital role of TDP-43 in lactation.

This study demonstrates that TDP-43, an RNA-binding protein, plays a crucial role in milk lipid secretion by post-transcriptionally regulating the expression of *Btn1a1* and *Xdh*. The results from both mouse models and human milk samples strongly support this conclusion. Future research could focus on exploring the therapeutic potential of modulating TDP-43 activity for treating lactation insufficiency and investigating the potential involvement of other RBPs in regulating lactation.

The study's limitations include the use of a conditional knockout mouse model that might not fully reflect the complexity of TDP-43's function in a physiological setting. The clinical study, while showing a correlation, does not establish causation between TDP-43 levels and lactation insufficiency. Further research is needed to confirm causality and explore the underlying mechanisms in more detail. Additionally, the study focuses mainly on lipid secretion; the potential role of TDP-43 in other aspects of milk production requires further investigation.