Could the 14q23.2 microdeletion or AKAP5 haploinsufficiency be a potential cause of intellectual disability?

Intellectual disability is characterized by impairment in at least two of the following areas: social skills, communication skills, self-care tasks, and academic skills. These impairments are evaluated in relation to the expected standards based on the individual's age and cultural levels. Additionally, intellectual disability is typically defined by a measurable level of intellectual functioning, represented by an intelligence quotients core of 70 or below. Autism spectrum disorder is a developmental disability resulting from differences in the brain, often characterized by problems in social communication and interaction, and limited or repetitive behaviors or interests. Hereditary spherocytosis is a disease characterized by anemia, jaundice, and splenomegaly as a result of increased tendency to hemolysis with morphological transformation of erythrocytes from biconcave disc-shaped cells with central pallor to spherocytes lacking central pallor due to hereditary injury of cellular membrane proteins. An 11-year-old female patient was referred to Pediatric Genetics Subdivision due to the presence of growth retardation and a diagnosis of hereditary spherocytosis. Since she also had dysmorphic facial features, such as frontal bossing, broad and prominent forehead, tubular nasal structure, and thin vermillion, genetic tests were performed. Chromosomal microarray analysis revealed a 2.5 Mb deletion in the 14q23.2q23.3 region. Deletion was also identified in the same region in her father, who had the same phenotypic characteristics, including hereditary spherocytosis and learning difficulties. We propose that the PLEKHG3 and AKAP5 genes, which are located in this region, may contribute to the development of intellectual disability.

A mutation in the cardiac KV7.1 channel possibly disrupts interaction with Yotiao protein.

Here, we characterized the p.Arg583His (R583H) Kv7.1 mutation, identified in two unrelated families suffered from LQT syndrome. This mutation is located in the HС-HD linker of the cytoplasmic portion of the Kv7.1 channel. This linker, together with HD helix are responsible for binding the A-kinase anchoring protein 9 (AKAP9), Yotiao. We studied the electrophysiological characteristics of the mutated channel expressed in CHO-K1 along with KCNE1 subunit and Yotiao protein, using the whole-cell patch-clamp technique. We found that R583H mutation, even at the heterozygous state, impedes IKs activation. Molecular modeling showed that HС and HD helixes of the C-terminal part of Kv7.1 channel are swapped along the C-terminus length of the channel and that R583 position is exposed to the outer surface of HC-HD tandem coiled-coil. Interestingly, the adenylate cyclase activator, forskolin had a smaller effect on the mutant channel comparing with the WT protein, suggesting that R583H mutation may disrupt the interaction of the channel with the adaptor protein Yotiao and, therefore, may impair phosphorylation of the KCNQ1 channel.

The scaffolding protein AKAP12 regulates mRNA localization and translation.

Regulation of subcellular messenger (m)RNA localization is a fundamental biological mechanism, which adds a spatial dimension to the diverse layers of post-transcriptional control of gene expression. The cellular compartment in which mRNAs are located may define distinct aspects of the encoded proteins, ranging from production rate and complex formation to localized activity. Despite the detailed roles of localized mRNAs that have emerged over the past decades, the identity of factors anchoring mRNAs to subcellular domains remains ill-defined. Here, we used an unbiased method to profile the RNA-bound proteome in migrating endothelial cells (ECs) and discovered that the plasma membrane (PM)-associated scaffolding protein A-kinase anchor protein (AKAP)12 interacts with various mRNAs, including transcripts encoding kinases with Actin remodeling activity. In particular, AKAP12 targets a transcript coding for the kinase Abelson Tyrosine-Protein Kinase 2 (ABL2), which we found to be necessary for adequate filopodia formation and angiogenic sprouting. Moreover, we demonstrate that AKAP12 is necessary for anchoring ABL2 mRNA to the PM and show that in the absence of AKAP12, the translation efficiency of ABL2 mRNA is reduced. Altogether, our work identified a unique post-transcriptional function for AKAP12 and sheds light into mechanisms of spatial control of gene expression.

The focal adhesion protein talin is a mechanically gated A-kinase anchoring protein.

Protein kinase A (PKA) is a ubiquitous, promiscuous kinase whose activity is specified through subcellular localization mediated by A-kinase anchoring proteins (AKAPs). PKA has complex roles as both an effector and a regulator of integrin-mediated cell adhesion to extracellular matrix (ECM). Recent observations demonstrate that PKA is an active component of focal adhesions (FA), suggesting the existence of one or more FA AKAPs. Using a promiscuous biotin ligase fused to PKA type-IIα regulatory (RIIα) subunits and subcellular fractionation, we identify the archetypal FA protein talin1 as an AKAP. Talin is a large, mechanosensitive scaffold that directly links integrins to actin filaments and promotes FA assembly by recruiting additional components in a force-dependent manner. The rod region of talin1 consists of 62 α-helices bundled into 13 rod domains, R1 to R13. Direct binding assays and NMR spectroscopy identify helix41 in the R9 subdomain of talin as the PKA binding site. PKA binding to helix41 requires unfolding of the R9 domain, which requires the linker region between R9 and R10. Experiments with single molecules and in cells manipulated to alter actomyosin contractility demonstrate that the PKA-talin interaction is regulated by mechanical force across the talin molecule. Finally, talin mutations that disrupt PKA binding also decrease levels of total and phosphorylated PKA RII subunits as well as phosphorylation of VASP, a known PKA substrate, within FA. These observations identify a mechanically gated anchoring protein for PKA, a force-dependent binding partner for talin1, and a potential pathway for adhesion-associated mechanotransduction.

AKAP150-anchored PKA regulates synaptic transmission and plasticity, neuronal excitability and CRF neuromodulation in the mouse lateral habenula.

The scaffolding A-kinase anchoring protein 150 (AKAP150) is critically involved in kinase and phosphatase regulation of synaptic transmission/plasticity, and neuronal excitability. Emerging evidence also suggests that AKAP150 signaling may play a key role in brain's processing of rewarding/aversive experiences, however its role in the lateral habenula (LHb, as an important brain reward circuitry) is completely unknown. Using whole cell patch clamp recordings in LHb of male wildtype and ΔPKA knockin mice (with deficiency in AKAP-anchoring of PKA), here we show that the genetic disruption of PKA anchoring to AKAP150 significantly reduces AMPA receptor-mediated glutamatergic transmission and prevents the induction of presynaptic endocannabinoid-mediated long-term depression in LHb neurons. Moreover, ΔPKA mutation potentiates GABAA receptor-mediated inhibitory transmission while increasing LHb intrinsic excitability through suppression of medium afterhyperpolarizations. ΔPKA mutation-induced suppression of medium afterhyperpolarizations also blunts the synaptic and neuroexcitatory actions of the stress neuromodulator, corticotropin releasing factor (CRF), in mouse LHb. Altogether, our data suggest that AKAP150 complex signaling plays a critical role in regulation of AMPA and GABAA receptor synaptic strength, glutamatergic plasticity and CRF neuromodulation possibly through AMPA receptor and potassium channel trafficking and endocannabinoid signaling within the LHb.

AKAP12 Upregulation Associates With PDE8A to Accelerate Cardiac Dysfunction.

BACKGROUND: In heart failure, signaling downstream the ß2-adrenergic receptor is critical. Sympathetic stimulation of ß2-adrenergic receptor alters cAMP (cyclic adenosine 3',5'-monophosphate) and triggers PKA (protein kinase A)-dependent phosphorylation of proteins that regulate cardiac function. cAMP levels are regulated in part by PDEs (phosphodiesterases). Several AKAPs (A kinase anchoring proteins) regulate cardiac function and are proposed as targets for precise pharmacology. AKAP12 is expressed in the heart and has been reported to directly bind ß2-adrenergic receptor, PKA, and PDE4D. However, its roles in cardiac function are unclear. METHODS: cAMP accumulation in real time downstream of the ß2-adrenergic receptor was detected for 60 minutes in live cells using the luciferase-based biosensor (GloSensor) in AC16 human-derived cardiomyocyte cell lines overexpressing AKAP12 versus controls. Cardiomyocyte intracellular calcium and contractility were studied in adult primary cardiomyocytes from male and female mice overexpressing cardiac AKAP12 (AKAP12OX) and wild-type littermates post acute treatment with 100-nM isoproterenol (ISO). Systolic cardiac function was assessed in mice after 14 days of subcutaneous ISO administration (60 mg/kg per day). AKAP12 gene and protein expression levels were evaluated in left ventricular samples from patients with end-stage heart failure. RESULTS: AKAP12 upregulation significantly reduced total intracellular cAMP levels in AC16 cells through PDE8. Adult primary cardiomyocytes from AKAP12OX mice had significantly reduced contractility and impaired calcium handling in response to ISO, which was reversed in the presence of the selective PDE8 inhibitor (PF-04957325). AKAP12OX mice had deteriorated systolic cardiac function and enlarged left ventricles. Patients with end-stage heart failure had upregulated gene and protein levels of AKAP12. CONCLUSIONS: AKAP12 upregulation in cardiac tissue is associated with accelerated cardiac dysfunction through the AKAP12-PDE8 axis.

Discovery of a Cushing's syndrome protein kinase A mutant that biases signaling through type I AKAPs.

Adrenal Cushing's syndrome is a disease of cortisol hypersecretion often caused by mutations in protein kinase A catalytic subunit (PKAc). Using a personalized medicine screening platform, we discovered a Cushing's driver mutation, PKAc-W196G, in ~20% of patient samples analyzed. Proximity proteomics and photokinetic imaging reveal that PKAcW196G is unexpectedly distinct from other described Cushing's variants, exhibiting retained association with type I regulatory subunits (RI) and their corresponding A kinase anchoring proteins (AKAPs). Molecular dynamics simulations predict that substitution of tryptophan-196 with glycine creates a 653-cubic angstrom cleft between the catalytic core of PKAcW196G and type II regulatory subunits (RII), but only a 395-cubic angstrom cleft with RI. Endocrine measurements show that overexpression of RIα or redistribution of PKAcW196G via AKAP recruitment counteracts stress hormone overproduction. We conclude that a W196G mutation in the kinase catalytic core skews R subunit selectivity and biases AKAP association to drive Cushing's syndrome.

Expression of RASSF1A, DIRAS3, and AKAP9 Genes in Thyroid Lesions: Implications for Differential Diagnosis and Prognosis of Thyroid Carcinomas.

Thyroid carcinoma is the primary endocrine malignancy worldwide. The preoperative examination of thyroid tissue lesion is often unclear. Approximately 25% of thyroid cancers cannot be diagnosed definitively without post-surgery histopathological examination. The assessment of diagnostic and differential markers of thyroid cancers is needed to improve preoperative diagnosis and reduce unnecessary treatments. Here, we assessed the expression of RASSF1A, DIRAS3, and AKAP9 genes, and the presence of BRAF V600E point mutation in benign and malignant thyroid lesions in a Polish cohort (120 patients). We have also performed a comparative analysis of gene expression using data obtained from the Gene Expression Omnibus (GEO) database (307 samples). The expression of RASSF1A and DIRAS3 was decreased, whereas AKAP9's was increased in pathologically changed thyroid compared with normal thyroid tissue, and significantly correlated with e.g., histopathological type of lesion papillary thyroid cancer (PTC) vs follicular thyroid cancer (FTC), patient's age, tumour stage, or its encapsulation. The receiver operating characteristic (ROC) analysis for the more aggressive FTC subtype differential marker suggests value in estimating RASSF1A and AKAP9 expression, with their area under curve (AUC), specificity, and sensitivity at 0.743 (95% CI: 0.548-0.938), 82.2%, and 66.7%; for RASSF1A, and 0.848 (95% CI: 0.698-0.998), 54.8%, and 100%, for AKAP9. Our research gives new insight into the basis of the aggressiveness and progression of thyroid cancers, and provides information on potential differential markers that may improve preoperative diagnosis.

The miR-669a-5p/G3BP/HDAC6/AKAP12 Axis Regulates Primary Cilia Length.

Primary cilia are conserved organelles in most mammalian cells, acting as "antennae" to sense external signals. Maintaining a physiological cilium length is required for cilium function. MicroRNAs (miRNAs) are potent gene expression regulators, and aberrant miRNA expression is closely associated with ciliopathies. However, how miRNAs modulate cilium length remains elusive. Here, using the calcium-shock method and small RNA sequencing, a miRNA is identified, namely, miR-669a-5p, that is highly expressed in the cilia-enriched noncellular fraction. It is shown that miR-669a-5p promotes cilium elongation but not cilium formation in cultured cells. Mechanistically, it is demonstrated that miR-669a-5p represses ras-GTPase-activating protein SH3-domain-binding protein (G3BP) expression to inhibit histone deacetylase 6 (HDAC6) expression, which further upregulates A-kinase anchor protein 12 (AKAP12) expression. This effect ultimately blocks cilia disassembly and leads to greater cilium length, which can be restored to wild-type lengths by either upregulating HDAC6 or downregulating AKAP12. Collectively, these results elucidate a previously unidentified miR-669a-5p/G3BP/HDAC6/AKAP12 signaling pathway that regulates cilium length, providing potential pharmaceutical targets for treating ciliopathies.

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.

A-kinase anchoring protein 79/150 coordinates α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor sensitization in sensory neurons.

Changes in sensory afferent activity contribute to the transition from acute to chronic pain. However, it is unlikely that a single sensory receptor is entirely responsible for persistent pain. It is more probable that extended changes to multiple receptor proteins expressed by afferent neurons support persistent pain. A-Kinase Anchoring Protein 79/150 (AKAP) is an intracellular scaffolding protein expressed in sensory neurons that spatially and temporally coordinates signaling events. Since AKAP scaffolds biochemical modifications of multiple TRP receptors linked to pain phenotypes, we probed for other ionotropic receptors that may be mediated by AKAP and contribute to persistent pain. Here, we identify a role for AKAP modulation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid Receptor (AMPA-R) functionality in sensory neurons. Pharmacological manipulation of distinct AMPA-R subunits significantly reduces persistent mechanical hypersensitivity observed during hyperalgesic priming. Stimulation of both protein kinases C and A (PKC, PKA, respectively) modulate AMPA-R subunit GluR1 and GluR2 phosphorylation and surface expression in an AKAP-dependent manner in primary cultures of DRG neurons. Furthermore, AKAP knock out reduces sensitized AMPA-R responsivity in DRG neurons. Collectively, these data indicate that AKAP scaffolds AMPA-R subunit organization in DRG neurons that may contribute to the transition from acute-to-chronic pain.

Methyl protodioscin reduces c-Myc to ameliorate diabetes mellitus erectile dysfunction via downregulation of AKAP12.

BACKGROUND: Diabetes mellitus erectile dysfunction (DMED) is one of common complications of diabetes. We aimed to investigate the potential efficacy of methyl protodioscin (MPD) in DMED and explored the underlying mechanism. METHODS: Diabetic mice were induced by streptozotocin, while vascular endothelial cells (VECs) and vascular smooth muscle cells (VSMCs) were stimulated with high glucose. MPD was administrated in vitro and in vivo to verify its efficacy on DMED. The interaction of c-Myc and AKAP12 was determined by luciferase reporter assay and chromatin immunoprecipitation assay. RESULTS: c-Myc and AKAP12 were upregulated in penile tissues in DMED mice. In high glucose-stimulated VSMCs or VECs, MPD intervention enhanced cell viability, inhibited apoptosis, decreased c-Myc and AKAP12, as well as elevated p-eNOS Ser1177. MPD-induced apoptosis inhibition, AKAP12 reduction and p-eNOSSer1177 elevation were reversed by AKAP12 overexpression. c-Myc functioned as a positive regulator of AKAP12. Overexpression of c-Myc reversed the effects induced by MPD in vitro, which was neutralized by AKAP12 silencing. MPD ameliorated erectile function in diabetic mice via inhibiting AKAP12. CONCLUSIONS: MPD improved erectile dysfunction in streptozotocin-caused diabetic mice by regulating c-Myc/AKAP12 pathway, indicating that MPD could be developed as a promising natural agent for the treatment of DMED.

AKAP12 inhibits esophageal squamous carcinoma cell proliferation, migration, and cell cycle via the PI3K/AKT signaling pathway.

Esophageal squamous cell carcinoma (ESCC) consistently ranks as one of the most challenging variants of squamous cell carcinomas, primarily due to the lack of effective early detection strategies. We herein aimed to elucidate the underlying mechanisms and biological role associated with A-kinase anchoring protein 12 (AKAP12) in the context of ESCC. Bioinformatic analysis had revealed significantly lower expression level of AKAP12 in ESCC tissue samples than in their non-cancerous counterparts. To gain deeper insights into the potential role of AKAP12 in the progression of ESCC, we conducted a single-gene set enrichment analysis of AKAP12 on ESCC datasets. Our findings suggested that AKAP12 exhibits functions inhibiting cell cycle progression, tumor proliferation, and epithelial-mesenchymal transition. To further validate our findings, we subjected ESCC cell lines to AKAP12 overexpression using CRISPR/Cas9-SAM. In vitro analyses demonstrated that increased expression of AKAP12 significantly reduced cell proliferation, migration, and cell cycle progression. Simultaneously, genes associated with this biological role undergo corresponding regulatory shifts. These observations provided valuable insights into the biological role played by AKAP12 in ESCC progression. In summary, AKAP12 shows promise as a new potential biomarker for early ESCC diagnosis, offering potential advantages for subsequent therapeutic intervention and disease management.

Endothelial deletion of EPH receptor A4 alters single-cell profile and Tie2/Akap12 signaling to preserve blood-brain barrier integrity.

Neurobiological consequences of traumatic brain injury (TBI) result from a complex interplay of secondary injury responses and sequela that mediates chronic disability. Endothelial cells are important regulators of the cerebrovascular response to TBI. Our work demonstrates that genetic deletion of endothelial cell (EC)-specific EPH receptor A4 (EphA4) using conditional EphA4f/f/Tie2-Cre and EphA4f/f/VE-Cadherin-CreERT2 knockout (KO) mice promotes blood-brain barrier (BBB) integrity and tissue protection, which correlates with improved motor function and cerebral blood flow recovery following controlled cortical impact (CCI) injury. scRNAseq of capillary-derived KO ECs showed increased differential gene expression of BBB-related junctional and actin cytoskeletal regulators, namely, A-kinase anchor protein 12, Akap12, whose presence at Tie2 clustering domains is enhanced in KO microvessels. Transcript and protein analysis of CCI-injured whole cortical tissue or cortical-derived ECs suggests that EphA4 limits the expression of Cldn5, Akt, and Akap12 and promotes Ang2. Blocking Tie2 using sTie2-Fc attenuated protection and reversed Akap12 mRNA and protein levels cortical-derived ECs. Direct stimulation of Tie2 using Vasculotide, angiopoietin-1 memetic peptide, phenocopied the neuroprotection. Finally, we report a noteworthy rise in soluble Ang2 in the sera of individuals with acute TBI, highlighting its promising role as a vascular biomarker for early detection of BBB disruption. These findings describe a contribution of the axon guidance molecule, EphA4, in mediating TBI microvascular dysfunction through negative regulation of Tie2/Akap12 signaling.

Fisetin induces the upregulation of AKAP12 mRNA and anti-angiogenesis in a patient-derived organoid xenograft model.

Colorectal cancer (CRC) is associated with high incidence and mortality rates. Targeted therapies for CRC cause various adverse effects, necessitating the development of novel approaches to control CRC progression. In this milieu, we investigated the anti-CRC effects of fisetin, a natural plant flavonoid. Cytotoxicity was performed in CRC patient-derived organoids (30 T and 33 T). Fisetin-induced tumor growth was evaluated in a CRC patient-derived organoid xenograft (PDOX) model. RNA sequencing, immunohistochemistry, and western blotting were performed subsequently. Fisetin significantly decreased organoid viability in a dose-dependent manner. In the PDOX model, fisetin significantly delayed tumor growth, showing a decrease in Ki-67 expression and the induction of apoptosis. In tumor tissues, four genes were identified as differentially expressed between the control and fisetin-treated groups. Among these, A-kinase anchoring protein 12 (AKAP12) level was significantly increased by fisetin treatment (fold change > 2, p < 0.05). Notably, fisetin significantly inhibited vascular endothelial growth factor (VEGF) and epithelial cell adhesion molecule (EpCAM) via upregulation of AKAP12. Our results demonstrate the upregulation of AKAP12 mRNA and inhibition of angiogenesis by fisetin as a therapeutic strategy against CRC.

Integrative analysis identifies AKAP8L as an immunological and prognostic biomarker of pan-cancer.

A-kinase anchoring protein 8L (AKAP8L) belong to the A-kinase anchoring protein (AKAP) family. Recent studies have proved that AKAP8L is associated with the progression of various tumors. To establish a more complete understanding of the significance of AKAP8L across various types of cancers, we conducted a detailed analysis of multiple histological datasets, including the level of gene expression in pancancer, biological function, molecular characteristics, as well as the diagnostic and prognostic value of AKAP8L in pancancer. Furthermore, we focused on renal clear cell carcinoma (KIRC), and of explored the correlation of AKAP8L with clinical characteristics, prognosis of distinct patient subsets, co-expression genes and differentially expressed genes (DEG). We also performed the immunohistochemical staining and semi-quantitative verification of the monoclonal antibody established by AKAP8L. Our findings indicate that AKAP8L expression varied significantly not only across most cancer types, but also across different cancer molecules and immune subtypes. In addition, the robust ability to accurately predict cancer and its strong correlation with the prognosis of cancer strongly suggest that AKAP8L may be a potential biomarker for cancer diagnosis and prognosis. Furthermore, the high expression levels of AKAP8L were related to the worse overall survival (OS), disease-specific survival (DSS) as well as progression-free interval (PFI) of KIRC with statistical significance, especially among distinct clinical subgroups of KIRC. To sum up, AKAP8L has the potential to serve as a critical molecular biomarker for the diagnosis and prognosis of pancancer, an independent prognostic risk factor of KIRC, and a novel molecular target for cancer therapies.