AKAP3-mediated type I PKA signaling is required for mouse sperm hyperactivation and fertility†.

The protein kinase A (PKA) signaling pathway, which mediates protein phosphorylation, is important for sperm motility and male fertility. This process relies on A-kinase anchoring proteins that organize PKA and its signalosomes within specific subcellular compartments. Previously, it was found that the absence of A-kinase anchoring protein 3 (AKAP3) leads to multiple morphological abnormalities in mouse sperm. But how AKAP3 regulates sperm motility is yet to be elucidated. AKAP3 has two amphipathic domains, here named dual and RI, in its N-terminus. These domains are responsible for binding regulatory subunits I alpha (RIα) and II alpha (RIIα) of PKA and for RIα only, respectively. Here, we generated mutant mice lacking the dual and RI domains of AKAP3. It was found that the deletion of these domains caused male mouse infertile, accompanied by mild defects in the fibrous sheath of sperm tails. Additionally, the levels of serine/threonine phosphorylation of PKA substrates and tyrosine phosphorylation decreased in the mutant sperm, which exhibited a defect in hyperactivation under capacitation conditions. The protein levels of PKA subunits remained unchanged. But, interestingly, the regulatory subunit RIα was mis-localized from principal piece to midpiece of sperm tail, whereas this was not observed for RIIα. Further protein-protein interaction assays revealed a preference for AKAP3 to bind RIα over RIIα. Collectively, our findings suggest that AKAP3 is important for sperm hyperactivity by regulating type-I PKA signaling pathway mediated protein phosphorylation via its dual and RI domains.

Multi-proteomic Analysis Revealed Distinct Protein Profiles in Cerebrospinal Fluid of Patients Between Anti-NMDAR Encephalitis NORSE and Cryptogenic NORSE.

New-onset refractory status epilepticus (NORSE) is rare but intractable. Anti-N-methyl-d-aspartate receptor (NMDAR) encephalitis and cryptogenic etiologies are the two major causes of NORSE with distinct clinical features. To elucidate the underlying mechanisms, 6 patients with anti-NMDAR encephalitis NORSE and 5 with cryptogenic NORSE (C-NORSE) were enrolled. Five patients of cerebrovascular disorders were used as controls. Quantitative proteomic analysis of the cerebrospinal fluid (CSF) samples of the patients revealed 101 and 56 proteins were changed, respectively. The average fold-change of the upregulated proteins, namely up-proteomic score in this study, was positively correlated with the severity and prognosis of the diseases, including ICU stay (r = 0.9308, P = 0.0035 in NMDAR group; r = 0.8977, P = 0.0193 in C-NORSE group), mRS score at discharge (r = 0.9710, P = 0.0111 in NMDAR group; r = 0.7071, P = 0.2000 in C-NORSE group), and time taken for patients awaking from a coma (r = 0.8823, P = 0.0100 in NMDAR group; r = 0.7906, P = 0.2000 in C-NORSE group). Pathways involved in humoral immune response, wound healing, and epigenetic regulation of transcription were upregulated in anti-NMDAR encephalitis NORSE. Pathways of innate and lymphocyte mediated immune response, synaptic functions, ubiquitination, and cell apoptosis were up-regulated in C-NORSE, which was consistent with a mouse model of status epilepticus. Fc receptor and B cell mediated immunity signaling pathways were downregulated in C-NORSE. Immunome microarray analysis demonstrated high autoantibody targeting 48 proteins in CSF samples of anti-NMDAR encephalitis NORSE. While the reaction was kept at a very low level in C-NORSE. There is no significant difference in inflammatory cytokine level between each group. The level of IL-4 (r = 0.7435, P = 0.0451), IL-13 (r = 0.7643, P = 0.0384), IFN-γ (r = 0.7973, P = 0.0287) and TNF-α (r = 0.8598, P = 0.0141) in NMDAR group, and IL-6 (r = 0.8479, P = 0.0348), IL-8 (r = 0.9076, P = 0.0166) in C-NORSE group were positively correlated with the up-proteomic score. The present study suggests that the up-proteomic score of CSF could be a promising indicator for assessment of the severity of anti-NMDAR encephalitis NORSE and C-NORSE. The distinct CSF proteomes imply different pathogenic mechanisms of the two diseases, and immunotherapy strategies as well.

Reduced mitochondrial size in hippocampus and psychiatric behavioral changes in the mutant mice with homologous mutation of Timm8a1-I23fs49X.

Background: Deafness-dystonia-optic neuronopathy (DDON) syndrome, a condition that predominantly affects males, is caused by mutations in translocase of mitochondrial inner membrane 8A (TIMM8A)/deafness dystonia protein 1 (DDP1) gene and characterized by progressive deafness coupled with other neurological abnormalities. In a previous study, we demonstrated the phenotype of male mice carrying the hemizygous mutation of Timm8a1-I23fs49X. In a follow-up to that study, this study aimed to observe the behavioral changes in the female mutant (MUT) mice with homologous mutation of Timm8a1 and to elucidate the underlying mechanism for the behavioral changes. Materials and methods: Histological analysis, transmission electron microscopy (EM), Western blotting, hearing measurement by auditory brainstem response (ABR), and behavioral observation were compared between the MUT mice and wild-type (WT) littermates. Results: The weight of the female MUT mice was less than that of the WT mice. Among MUT mice, both male and female mice showed hearing impairment, anxiety-like behavior by the elevated plus maze test, and cognitive deficit by the Morris water maze test. Furthermore, the female MUT mice exhibited coordination problems in the balance beam test. Although the general neuronal loss was not found in the hippocampus of the MUT genotype, EM assessment indicated that the mitochondrial size showing as aspect ratio and form factor in the hippocampus of the MUT strain was significantly reduced compared to that in the WT genotype. More importantly, this phenomenon was correlated with the upregulation of translation of mitochondrial fission process protein 1(Mtfp1)/mitochondrial 18 kDa protein (Mtp18), a key fission factor that is a positive regulator of mitochondrial fission and mitochondrial size. Interestingly, significant reductions in the size of the uterus and ovaries were noted in the female MUT mice, which contributed to significantly lower fertility in the MUT mice. Conclusion: Together, a homologous mutation in the Timm8a1 gene caused the hearing impairment and psychiatric behavioral changes in the MUT mice; the latter phenotype might be related to a reduction in mitochondrial size regulated by MTP18.

Identification of Anti-Collapsin Response Mediator Protein 2 Antibodies in Patients With Encephalitis or Encephalomyelitis.

Background and Purpose: An increasing number of autoimmune encephalitis (AE)-associated autoantibodies have been successfully characterized. However, many cases of AE remain unexplained on account of unknown antibodies. The aim of the present study was to identify a novel antibody against collapsin response mediator protein 2 (CRMP2) in suspected AE patients. Methods: A patient's serum and cerebrospinal fluid samples tested negative for known AE antibodies; however, strong immunolabel signals were observed in the neuronal cytoplasm of the cortex, hippocampus, and Purkinje cells on rat brain sections. Immunoprecipitation from the rat brain protein lysate, followed by mass spectrometry analysis, was used to identify the targeting antigen. Western blotting and cell-based assay with antigen-overexpressing HEK293T cells were used for antibody specificity, epitope, IgG subtype determination, and retrospective study. Results: An antibody against CRMP2, a synaptic protein involved in axon guidance, was identified. The immunostains of the patient's samples on rat brain sections were eliminated by pre-absorption with HEK293T cells overexpressing CRMP2. The samples specifically immunoreacted with CRMP2, but not with CRMP1, CRMP3, CRMP4, and CRMP5. The C-terminus of CRMP2 with 536 amino acids contained the epitope for antibody binding. The subtype analysis showed that the anti-CRMP2 antibody was IgG4. Furthermore, a screening of 46 patients with neurological disoders and neuro-cytoplasm immunostainings on rat brain sections resulted in the identification of anti-CRMP2 antibodies in a case of encephalomyelitis. The two patients responded well to immunotherapies. Conclusions: This study discovered that a novel anti-CRMP2 antibody was associated with suspected AE and thus should be included in the testing list for AE.

RNA helicase DDX5 acts as a critical regulator for survival of neonatal mouse gonocytes.

OBJECTIVES: Mammalian spermatogenesis is a biological process of male gamete formation. Gonocytes are the only precursors of spermatogonial stem cells (SSCs) which develop into mature spermatozoa. DDX5 is one of DEAD-box RNA helicases and expresses in male germ cells, suggesting that Ddx5 plays important functions during spermatogenesis. Here, we explore the functions of Ddx5 in regulating the specification of gonocytes. MATERIALS AND METHODS: Germ cell-specific Ddx5 knockout (Ddx5-/- ) mice were generated. The morphology of testes and epididymides and fertility in both wild-type and Ddx5-/- mice were analysed. Single-cell RNA sequencing (scRNA-seq) was used to profile the transcriptome in testes from wild-type and Ddx5-/- mice at postnatal day (P) 2. Dysregulated genes were validated by single-cell qRT-PCR and immunofluorescent staining. RESULTS: In male mice, Ddx5 was expressed in germ cells at different stages of development. Germ cell-specific Ddx5 knockout adult male mice were sterile due to completely devoid of germ cells. Male germ cells gradually disappeared in Ddx5-/- mice from E18.5 to P6. Single-cell transcriptome analysis showed that genes involved in cell cycle and glial cell line-derived neurotrophic factor (GDNF) pathway were significantly decreased in Ddx5-deficient gonocytes. Notably, Ddx5 ablation impeded the proliferation of gonocytes. CONCLUSIONS: Our study reveals the critical roles of Ddx5 in fate determination of gonocytes, offering a novel insight into the pathogenesis of male sterility.

Identification of Plasmodium falciparum-specific protein PIESP2 as a novel virulence factor related to cerebral malaria.

Cerebral malaria (CM) is the most severe complication caused by Plasmodium falciparum infection. The pathophysiological changes caused by parasite virulence factors and the human immune response to parasites contribute to CM. To date, very few parasite virulence proteins have been found to participate in CM. Here, we employed comparative genomics analysis and identified parasite-infected erythrocyte specific protein 2 (PIESP2) to be a CM-related protein. We conducted further experimental investigations and found that PIESP2 is an immunogenic protein. PIESP2 expression begins at the early trophozoite stage and progressively increases with parasite development. Although PIESP2 proteins mainly reside within infected red blood cells (IRBCs), some of them are present on the IRBC surface at the pigmented stage. Moreover, blockage of PIESP2 by antiserum apparently inhibited the adhesion of IRBCs to brain microvascular endothelial cells (BMECs). Western blot analysis detected the binding of PIESP2 to BMECs. Transcriptional analysis revealed that the binding of PIESP2 to BMECs can increase the expression of genes involved in the inflammatory response but decrease the expression of genes related to the anchoring junction. Overall, PIESP2 might be associated with CM by mediating the sequestration of IRBCs, inducing the inflammation response, and impairing the integrity of blood-brain barrier.

Lack of AKAP3 disrupts integrity of the subcellular structure and proteome of mouse sperm and causes male sterility.

The development and maintenance of the correct morphology of sperm is important for their functions. Cellular morphogenesis of sperm occurs during the post-meiotic developmental stage; however, little is known about what coordinates this process. In the present study, we investigated the role of A-kinase anchoring protein 3 (AKAP3) during mouse spermiogenesis, using both mouse genetics and proteomics. It was found that AKAP3 is essential for the formation of the specific subcellular structure of the sperm flagellum, motility of sperm and male fertility. Additionally, lack of AKAP3 caused global changes of the sperm proteome and mislocalization of sperm proteins, including accumulation of RNA metabolism and translation factors and displacement of PKA subunits in mature sperm, which may underlie misregulated PKA activity and immotility in sperm. Interestingly, sperm lacking a complete fibrous sheath from both Akap3 and Akap4 null mice accumulated F-actin filaments and morphological defects during post-testicular maturation in the epididymis. These results suggest that the subcellular structures of sperm could be formed via independent pathways, and elucidate the roles of AKAP3 during the coordinated synthesis and organization of the sperm proteome and sperm morphology.

Domain-functional analyses of PIWIL1 and PABPC1 indicate their synergistic roles in protein translation via 3'-UTRs of meiotic mRNAs.

Translational regulation plays a central role during post-meiotic development of male germ cells. Previous studies suggested that P-element induced wimpy testis like 1 (PIWIL1), a PIWI-interacting RNA (piRNA) binding protein that is critical for sperm development, participates in the maintenance and translational regulation of post-meiotic mRNAs in haploid spermatids. However, how PIWIL1 regulates protein translation remains largely unclear. Using biochemical assays, we show here that PIWIL1 utilizes different domains to interact with post-meiotic mRNAs and Poly-A binding protein cytoplasmic 1 (PABPC1), a general protein translation regulator. PIWIL1 binds 3'-untranslated regions (3'-UTRs) of several spermiogenic mRNAs via its N-terminal domain, whereas its interactions with PABPC1 are mediated through its N- and C-terminal domains in an RNA-dependent manner. Using a heterologous cell system, we analyzed its effects on protein translation via luciferase reporter assay and sucrose gradient sedimentation. It was found that PIWIL1 augments protein translation with PABPC1 in the presence of 3'-UTRs of post-meiotic mRNAs. While both the N-terminal RNA recognition motif (RRM) domain and the central linker region of PABPC1 stimulate translation, only the PIWI Argonaute and Zwille (PAZ) domain of PIWIL1 positively affects translation of reporter mRNAs. Interestingly, the PAZ domain was found absent from polysomal fractions, in contrast to the N- and C-terminal domains of PIWIL1. Taken together, the results suggest that PIWIL1 interacts with various partners using different domains and participates in translational regulation partly through 3'-UTRs. It will be of interest to further explore how PIWIL1 elicit its versatile functions, including translational regulation of post-meiotic mRNAs through intrinsic structural changes and extrinsic signals during mouse spermiogenesis under more physiological settings.

Sperm-specific AKAP3 is a dual-specificity anchoring protein that interacts with both protein kinase a regulatory subunits via conserved N-terminal amphipathic peptides.

cAMP-dependent protein kinase A (PKA) plays important regulatory roles during mouse spermatogenesis. PKA-mediated signaling has been shown to regulate gene expression, chromatin condensation, capacitation, and motility during sperm development and behavior, although how PKA is regulated in spatiotemporal manners during spermatogenesis is not fully understood. In the present study, we found that PKA subunit isoforms are expressed and localized differently in meiotic and post-meiotic mouse spermatogenic cells. Regulatory subunit I alpha (RIα) is expressed in spermatocytes and round spermatids, where it is localized diffusely throughout the cytoplasm of cells. During late spermiogenesis, RIα abundance gradually decreases. On the other hand, RIIα is expressed constantly throughout meiotic and post-meiotic stages, and is associated with cytoskeletal structures. Among several A kinase anchoring proteins (AKAPs) expressed in the testis, sperm-specific AKAP3 can be found in the cytoplasm of elongating spermatids and interacts with RIα, as demonstrated by both in vivo and in vitro experiments. In mature sperm, AKAP3 is exclusively found in the principal piece of the flagellum, coincident with only RIIα. Mutagenesis experiments further showed that the preferential interactions of AKAP3 with PKA regulatory subunits are mediated by two highly conserved amphipathic peptides located in the N-terminal region of AKAP3. Thus, AKAP3 is a dual-specificity molecule that modulates PKA isotypes in a spatiotemporal manner during mouse spermatogenesis.

AKAP3 synthesis is mediated by RNA binding proteins and PKA signaling during mouse spermiogenesis.

Mammalian spermatogenesis is regulated by coordinated gene expression in a spatiotemporal manner. The spatiotemporal regulation of major sperm proteins plays important roles during normal development of the male gamete, of which the underlying molecular mechanisms are poorly understood. A-kinase anchoring protein 3 (AKAP3) is one of the major components of the fibrous sheath of the sperm tail that is formed during spermiogenesis. In the present study, we analyzed the expression of sperm-specific Akap3 and the potential regulatory factors of its protein synthesis during mouse spermiogenesis. Results showed that the transcription of Akap3 precedes its protein synthesis by about 2 wk. Nascent AKAP3 was found to form protein complex with PKA and RNA binding proteins (RBPs), including PIWIL1, PABPC1, and NONO, as revealed by coimmunoprecipitation and protein mass spectrometry. RNA electrophoretic gel mobility shift assay showed that these RBPs bind sperm-specific mRNAs, of which proteins are synthesized during the elongating stage of spermiogenesis. Biochemical and cell biological experiments demonstrated that PIWIL1, PABPC1, and NONO interact with each other and colocalize in spermatids' RNA granule, the chromatoid body. In addition, NONO was found in extracytoplasmic granules in round spermatids, whereas PIWIL1 and PABPC1 were diffusely localized in cytoplasm of elongating spermatids, indicating their participation at different steps of mRNA metabolism during spermatogenesis. Interestingly, type I PKA subunits colocalize with PIWIL1 and PABPC1 in the cytoplasm of elongating spermatids and cosediment with the RBPs in polysomal fractions on sucrose gradients. Further biochemical analyses revealed that activation of PKA positively regulates AKAP3 protein synthesis without changing its mRNA level in elongating spermatids. Taken together, these results indicate that PKA signaling directly participates in the regulation of protein translation in postmeiotic male germ cells, underscoring molecular mechanisms that regulate protein synthesis during mouse spermiogenesis.