Characterization of PIH1D3 Deficiency in Knockout Rats: Insights into Ciliopathy Mechanisms and Phenotypic Manifestations

PIH1 domain-containing protein 3 (PIH1D3) plays a central role in the assembly and function of primary cilia, which are cellular organelles that act as signaling hubs for many pathways. Ciliopathies are a group of disorders caused by defects in the structure or function of cilia, which leads to a wide range of clinical manifestations, including kidney disease, retinal degeneration, brain anomalies, and skeletal malformations. The importance of studying PIH1D3 in ciliopathies is due to its involvement in the regulation of ciliary protein trafficking and ciliogenesis. PIH1D3 is part of the R2TP/Prefoldin-like complex, which assists in the assembly and stability of protein complexes essential for ciliary function, in particular the assembly and function of dynein arms within motile cilia. Dynein arms are essential components of the ciliary axoneme, responsible for the cilia’s motility. The pre-assembly of dynein arms in the cytoplasm and their subsequent loading onto the axoneme are vital for proper ciliary function. Understanding how PIH1D3 contributes to the formation and maintenance of cilia, researchers can discover the mechanisms causing ciliopathies and potentially identify new targets for therapy.

A new study published in Frontiers in Cell & Developmental Biology led by Professor Hongxia Zhou from the Florida International University and conducted by Tingting Zhang, Shiquan Cui, Xinrui Xiong, Ying Liu, Qilin Cao, Xu-Gang Xia, Investigated the role of PIH1 domain-containing protein 3 (PIH1D3) in ciliopathies, the team employed transcription activator-like effector nuclease (TALEN) technology, to target exon 5 of the rat Pih1d3 gene to induce a frameshift mutation that resulted in a lack of PIH1D3 protein expression. The new model was important to mirror the spectrum of ciliopathy symptoms, including situs inversus, impaired mucociliary clearance, hydrocephalus, and male infertility. They selected rats over mice for their investigation because rats have more physiological similarities with humans which is important in the translational value of their findings. They bred heterozygous KO female rats with wild-type male rats across 20 generations to establish a stable lineage of PIH1D3-KO rats. The team monitored the growth, survival rates, and physical characteristics of PIH1D3-KO rats and compared to wild-type counterparts. They showed that KO rats exhibited key symptoms of ciliopathies, including hydrocephalus, male infertility, and respiratory dysfunction, attributed to the defective assembly and function of cilia due to PIH1D3 deficiency. To investigate the impact of PIH1D3 deficiency on cerebrospinal fluid (CSF) circulation, the researchers injected evans blue dye into the lateral ventricle of both KO and wild-type rats and revealed impaired CSF flow in PIH1D3-KO rats, implicating its deficiency in the development of hydrocephalus. This finding is particularly significant, providing a fresh perspective on the mechanisms underlying ciliopathy-associated hydrocephalus and spotlighting potential therapeutic targets.

When they authors conducted histological and immunostaining analyses on various tissues from PIH1D3-KO and wild-type rats and assessed cellular and structural changes, focusing on ciliary composition and function in tissues like the brain, respiratory tract, and testes. Moreover, they also used transmission electron microscopy to examine the ultrastructural integrity of cilia in the respiratory tract to confirm defects in the outer and inner dynein arms in KO rats. The team transfected HEK293 cells with plasmids expressing PIH1D3 and various dynein arm components to investigate PIH1D3’s interactions with proteins involved in ciliary function and found that PIH1D3 deficiency leads to the malformation of both outer and inner dynein arms in cilia, essential for their motility and function. Moreover, PIH1D3 interacts with proteins crucial for the pre-assembly and trafficking of dynein arms, highlighting its central role in maintaining ciliary function. Indeed, the study’s exploration of the molecular interactions between PIH1D3 and proteins involved in the dynein arm assembly and function is another highlight and demonstrate the complex regulatory networks governing ciliary function and the pathogenesis of ciliopathies. The identification of protein partners that interact with PIH1D3 provides a foundation for future studies aimed at unraveling the intricate molecular pathways involved in ciliopathy development. In conclusion, the development of a PIH1D3-KO rat model by Professor Hongxia Zhou and her team has facilitated a deeper understanding of the molecular underpinnings of these disorders, particularly the role of PIH1D3 in dynein arm function and CSF circulation. The newly developed PIH1D3-KO rat model has successfully replicated the full spectrum of ciliopathy features, provided   insights into the disease’s underlying molecular mechanisms and can be invaluable for studying these diseases and developing therapeutic strategies.