IB4-binding sensory neurons in the adult rat express a novel 3' UTR-extended isoform of CaMK4 that is associated with its localization to axons.
ABSTRACT: Calcium/calmodulin-dependent protein kinase 4 (gene and transcript: CaMK4; protein: CaMKIV) is the nuclear effector of the Ca(2+) /calmodulin kinase (CaMK) pathway where it coordinates transcriptional responses. However, CaMKIV is present in the cytoplasm and axons of subpopulations of neurons, including some sensory neurons of the dorsal root ganglia (DRG), suggesting an extranuclear role for this protein. We observed that CaMKIV was expressed strongly in the cytoplasm and axons of a subpopulation of small-diameter DRG neurons, most likely cutaneous nociceptors by virtue of their binding the isolectin IB4. In IB4+ spinal nerve axons, 20% of CaMKIV was colocalized with the endocytic marker Rab7 in axons that highly expressed CAM-kinase-kinase (CAMKK), an upstream activator of CaMKIV, suggesting a role for CaMKIV in signaling though signaling endosomes. Using fluorescent in situ hybridization (FISH) with riboprobes, we also observed that small-diameter neurons expressed high levels of a novel 3' untranslated region (UTR) variant of CaMK4 mRNA. Using rapid amplification of cDNA ends (RACE), reverse-transcription polymerase chain reaction (RT-PCR) with gene-specific primers, and cDNA sequencing analyses we determined that the novel transcript contains an additional 10 kb beyond the annotated gene terminus to a highly conserved alternate polyadenylation site. Quantitative PCR (qPCR) analyses of fluorescent-activated cell sorted (FACS) DRG neurons confirmed that this 3'-UTR-extended variant was preferentially expressed in IB4-binding neurons. Computational analyses of the 3'-UTR sequence predict that UTR-extension introduces consensus sites for RNA-binding proteins (RBPs) including the embryonic lethal abnormal vision (ELAV)/Hu family proteins. We consider the possible implications of axonal CaMKIV in the context of the unique properties of IB4-binding DRG neurons.
Project description:The enzyme calcium/calmodulin-dependent protein kinase II (CaMKII) is associated with memory and its alpha isoform is critical for development of activity-induced synaptic changes. Therefore, we hypothesized that CaMKII is involved in altered function of dorsal root ganglion (DRG) neurons after neuronal injury. To test this hypothesis, Sprague-Dawley rats were made hyperalgesic by L5 and L6 spinal nerve ligation (SNL), and changes in total phosphorylated and unphosphorylated CaMKII (tCaMKII) and phosphorylated form of its alpha isoform (pCaMKIIalpha) were analyzed using immunochemistry in different subpopulations of DRG. SNL did not induce any changes in tCaMKII between experimental groups, while the overall percentage of pCaMKIIalpha-positive neurons in injured L5 DRG SNL (24.8%) decreased significantly when compared to control (41.7%). SNL did not change the percentage of pCaMKIIalpha/N52 colabeled neurons but decreased the percentage of N52-negative nonmyelinated neurons that expressed pCaMKIIalpha from 27% in control animals to 11% after axotomy. We also observed a significant decrease in the percentage of small nonpeptidergic neurons labeled with IB4 (37.6% in control vs. 4.0% in L5 SNL DRG), as well as a decrease in the percentage of pCaMKIIalpha/IB4 colabeled neurons in injured L5 DRGs (27% in control vs. 1% in L5 DRG of SNL group). Our results show that reduction in pCaMKIIalpha levels following peripheral injury is due to the loss of IB4-positive neurons. These results indicate that diminished afferent activity after axotomy may lead to decreased phosphorylation of CaMKIIalpha.
Project description:BACKGROUND:The expression of calcium/calmodulin-dependent kinase IV (CaMKIV) was hitherto thought to be confined to the nervous system. However, a recent genome-wide analysis indicated an association between hypertension and a single-nucleotide polymorphism (rs10491334) of the human CaMKIV gene (CaMK4), which suggests a role for this kinase in the regulation of vascular tone. METHODS AND RESULTS:To directly assess the role of CaMKIV in hypertension, we characterized the cardiovascular phenotype of CaMK4(-/-) mice. They displayed a typical hypertensive phenotype, including high blood pressure levels, cardiac hypertrophy, vascular and kidney damage, and reduced tolerance to chronic ischemia and myocardial infarction compared with wild-type littermates. Interestingly, in vitro experiments showed the ability of this kinase to activate endothelial nitric oxide synthase. Eventually, in a population study, we found that the rs10491334 variant associates with a reduction in the expression levels of CaMKIV in lymphocytes from hypertensive patients. CONCLUSIONS:Taken together, our results provide evidence that CaMKIV plays a pivotal role in blood pressure regulation through the control of endothelial nitric oxide synthase activity. (J Am Heart Assoc. 2012;1:e001081 doi: 10.1161/JAHA.112.001081.).
Project description:Systemic lupus erythematosus (SLE) is a chronic inflammatory disease associated with aberrant immune cell function. Treatment involves the use of indiscriminate immunosuppression, which results in significant side effects. SLE T cells express high levels of calcium/calmodulin-dependent protein kinase type IV (CaMKIV), which translocates to the nucleus upon engagement of the T cell receptor-CD3 complex and accounts for abnormal T cell function. The purpose of this study was to determine whether inhibition of CaMKIV would improve disease pathology.We treated MRL/lpr mice with KN-93, a CaMKIV inhibitor, starting at week 8 or week 12 of age and continuing through week 16 and evaluated skin lesions, proteinuria, kidney histopathology, proinflammatory cytokine production, and costimulatory molecule expression. We also determined the effect of silencing of CAMK4 on interferon-? (IFN?) expression by human SLE T cells.CaMKIV inhibition in MRL/lpr mice resulted in significant suppression of nephritis and skin disease, decreased expression of the costimulatory molecules CD86 and CD80 on B cells, and suppression of IFN? and tumor necrosis factor ? production. In human SLE T cells, silencing of CAMK4 resulted in suppression of IFN? production.We conclude that suppression of CaMKIV mitigates disease development in lupus-prone mice by suppressing cytokine production and costimulatory molecule expression. Specific silencing of CAMK4 in human T cells results in similar suppression of IFN? production. Our data justify the development of small-molecule CaMKIV inhibitors for the treatment of patients with SLE.
Project description:Calcium/calmodulin-dependent protein kinases (CaMKs) are key mediators of calcium signaling and underpin neuronal health. Although widely studied, the contribution of CaMKs to Mendelian disease is rather enigmatic. Here, we describe an unusual neurodevelopmental phenotype, characterized by milestone delay, intellectual disability, autism, ataxia, and mixed hyperkinetic movement disorder including severe generalized dystonia, in a proband who remained etiologically undiagnosed despite exhaustive testing. We performed trio whole-exome sequencing to identify a de novo essential splice-site variant (c.981+1G>A) in CAMK4, encoding CaMKIV. Through in silico evaluation and cDNA analyses, we demonstrated that c.981+1G>A alters CAMK4 pre-mRNA processing and results in a stable mRNA transcript containing a 77-nt out-of-frame deletion and a premature termination codon within the last exon. The expected protein, p.Lys303Serfs*28, exhibits selective loss of the carboxy-terminal regulatory domain of CaMKIV and bears striking structural resemblance to previously reported synthetic mutants that confer constitutive CaMKIV activity. Biochemical studies in proband-derived cells confirmed an activating effect of c.981+1G>A and indicated that variant-induced excessive CaMKIV signaling is sensitive to pharmacological manipulation. Additionally, we found that variants predicted to cause selective depletion of CaMKIV's regulatory domain are unobserved in diverse catalogs of human variation, thus revealing that c.981+1G>A is a unique molecular event. We propose that our proband's phenotype is explainable by a dominant CAMK4 splice-disrupting mutation that acts through a gain-of-function mechanism. Our findings highlight the importance of CAMK4 in human neurodevelopment, provide a foundation for future clinical research of CAMK4, and suggest the CaMKIV signaling pathway as a potential drug target in neurological disease.
Project description:The pathogenesis of diabetic neuropathic pain is complicated and its underlying mechanisms remain unclear. Calmodulin-dependent protein kinases (CaMKs) IV (CaMKIV), one of CaMKs, regulates several transcription factors in pain mechanisms. High-mobility group box 1 (HMGB1) is a key mediator in diabetic neuropathic pain. This study aims to find the roles and mechanisms of CaMIV in diabetic neuropathic pain.Diabetic animal models were constructed by injecting with streptozotocin (STZ) intraperitoneally. They were randomly divided into seven groups (n = 6 per group): Naive, Normal Saline, STZ, STZ + Sham, STZ + DMSO and STZ + KN93 (an inhibitor of CaMKIV) (50 μg), STZ + KN93 (100 μg), which received KN93 (50 or 100 μg) intrathecally after the administration of STZ. Phospho-CaMKIV (pCaMKIV) and HMGB1 expression in rat dorsal root ganglion (DRG) and RAW264.7 cell line were measured by western blot. Distribution of pCaMKIV immune reactivity in different subpopulations of DRG neurons was measured by double-immunofluorescence staining.The pCaMKIV and HMGB1 in DRG significantly increased after STZ administration, and pCaMKIV can regulate the expression of HMGB1 based on both cellular and animal models. Pretreatment with CaMKIV inhibitor attenuated STZ-induced mechanical allodynia and thermal hyperalgesia, as well as reduced HMGB1 expression in the DRG.This study demonstrates that CaMKIV can relieve STZ-induced diabetic neuropathic pain. The mechanism of this function depended on the process: pCaMKIV localized in the nuclei of DRG neurons and regulated HMGB1 which was an important mediator of neuropathic pain. These findings reported CaMKIV may be a potential target or important node in relieving diabetic neuropathic pain.
Project description:Kidney podocytes and their slit diaphragms prevent urinary protein loss. T cells from patients with systemic lupus erythematosus display increased expression of calcium/calmodulin-dependent protein kinase IV (CaMKIV). The present study was undertaken to investigate the role of CaMKIV in podocyte function in lupus nephritis (LN).We treated kidney podocytes with IgG derived from healthy individuals or patients with LN and then analyzed gene expression using a DNA microarray. The localization of IgG in podocytes was analyzed by immunofluorescence staining, with or without silencing of neonatal Fc receptor (FcRn). In addition, we silenced CAMK4 in podocytes and analyzed the expression of selected genes. We also examined the expression of CD86 in kidney podocytes from MRL/lpr, MRL/lpr.camkiv(-/-), and MRL/MPJ mice by in situ hybridization.We found that exposure of podocytes to IgG resulted in entry of IgG into the cytoplasm. IgG entered podocytes via the FcRn because less IgG was found in the cytoplasm of podocytes treated with FcRn small interfering RNA. DNA microarray studies of podocytes exposed to LN-derived IgG revealed up-regulation of genes related to the activation of immune cells or podocyte damage. Interestingly, CD86 expression decreased after silencing CAMK4 in podocytes. Also, in situ hybridization experiments showed that the expression of CD86 was reduced in podocytes from MRL/lpr.camkiv(-/-) mice.LN-derived IgG enters podocytes and up-regulates CAMK4, which is followed by increased expression of genes known to be linked to podocyte damage and T cell activation. Targeted inhibition of CAMK4 in podocytes may prove to be clinically useful in patients with LN.
Project description:Muscle lim protein (MLP) has long been regarded as a cytosolic and nuclear muscular protein. Here, we show that MLP is also expressed in a subpopulation of adult rat dorsal root ganglia (DRG) neurons in response to axonal injury, while the protein was not detectable in naïve cells. Detailed immunohistochemical analysis of L4/L5 DRG revealed ~3% of MLP-positive neurons 2 days after complete sciatic nerve crush and maximum ~10% after 4-14 days. Similarly, in mixed cultures from cervical, thoracic, lumbar and sacral DRG ~6% of neurons were MLP-positive after 2 days and maximal 17% after 3 days. In both, histological sections and cell cultures, the protein was detected in the cytosol and axons of small diameter cells, while the nucleus remained devoid. Moreover, the vast majority could not be assigned to any of the well characterized canonical DRG subpopulations at 7 days after nerve injury. However, further analysis in cell culture revealed that the largest population of MLP expressing cells originated from non-peptidergic IB4-positive nociceptive neurons, which lose their ability to bind the lectin upon axotomy. Thus, MLP is mostly expressed in a subset of axotomized nociceptive neurons and can be used as a novel marker for this population of cells.
Project description:Injury and inflammation cause tissue acidosis, which is a common feature of various painful conditions. Acid-Sensing Ion channels (ASICs) are amongst the main excitatory channels activated by extracellular protons and expressed in the nervous system. Six transcripts of ASIC subunits including ASIC1a, ASIC1b, ASIC2a, ASIC2b, ASIC3, and ASIC4 are encoded by four genes (Asic1-4) and have been identified in rodents. Most ASIC subunits are present at substantial levels in primary sensory neurons of dorsal root ganglia (DRG) except for ASIC4. However, their expression pattern in DRG neurons remains largely unclear, mainly due to the lack of antibodies with appropriate specificity. In this study, we examined in detail the expression pattern of ASIC1-3 subunits, including splice variants, in different populations of DRG neurons in adult mice using an in situ hybridization technique (RNAscope) with high sensitivity and specificity. We found that in naïve condition, all five subunits examined were expressed in the majority of myelinated, NF200-immunoreactive, DRG neurons (NF200+). However, ASIC subunits showed a very different expression pattern among non-myelinated DRG neuronal subpopulations: ASIC1 and ASIC3 were only expressed in CGRP-immunoreactive neurons (CGRP+), ASIC2a was mostly expressed in the majority of IB4-binding neurons (IB4+), while ASIC2b was expressed in almost all non-myelinated DRG neurons. We also found that at least half of sensory neurons expressed multiple types of ASIC subunits, indicating prevalence of heteromeric channels. In mice with peripheral nerve injury, the expression level of ASIC1a and ASIC1b in L4 DRG and ASIC3 in L5 DRG were altered in CGRP+ neurons, but not in IB4+ neurons. Furthermore, the pattern of change varied among DRGs depending on their segmental level, which pointed to differential regulatory mechanisms between afferent types and anatomical location. The distinct expression pattern of ASIC transcripts in naïve condition, and the differential regulation of ASIC subunits after peripheral nerve injury, suggest that ASIC subunits are involved in separate sensory modalities.
Project description:Advillin is a sensory neuron-specific actin-binding protein expressed at high levels in all types of somatosensory neurons in early development. However, the precise role of advillin in adulthood is largely unknown. Here we reveal advillin expression restricted to isolectin B4-positive (IB4+) neurons in the adult dorsal root ganglia (DRG). Advillin knockout (KO) specifically impaired axonal regeneration in adult IB4+ DRG neurons. During axon regeneration, advillin was expressed at the very tips of filopodia and modulated growth cone formation by interacting with and regulating focal-adhesion-related proteins. The advillin-containing focal-adhesion protein complex was shed from neurite tips during neurite retraction and was detectable in cerebrospinal fluid in experimental autoimmune encephalomyelitis, oxaliplatin-induced peripheral neuropathy, and chronic constriction injury of the sciatic nerve. In addition, advillin KO disturbed experimental autoimmune encephalomyelitis-induced neural plasticity in the spinal-cord dorsal horn and aggravated neuropathic pain. Our study highlights a role for advillin in growth cone formation, axon regeneration, and neuropathic pain associated with IB4+ DRG neurons in adulthood.
Project description:In our previous investigations of the role of the extracellular matrix (ECM) in promoting neurite growth we have observed that a permissive laminin (LN) substrate stimulates differential growth responses in subpopulations of mature dorsal root ganglion (DRG) neurons. DRG neurons expressing Trk and p75 receptors grow neurites on a LN substrate in the absence of neurotrophins, while isolectin B4-binding neurons (IB4+) do not display significant growth under the same conditions. We set out to determine whether there was an expression signature of the LN-induced neurite growth phenotype. Using a lectin binding protocol IB4+ neurons were isolated from dissociated DRG neurons, creating two groups - IB4+ and IB4-. A small-scale microarray approach was employed to screen the expression of a panel of ECM-associated genes following dissociation (t=0) and after 24 hr culture on LN (t=24LN). This was followed by qRT-PCR and immunocytochemistry of selected genes.The microarray screen showed that 36 of the 144 genes on the arrays were consistently expressed by the neurons. The array analyses showed that six genes had lower expression in the IB4+ neurons compared to the IB4- cells at t=0 (CTSH, Icam1, Itg?1, Lamb1, Plat, Spp1), and one gene was expressed at higher levels in the IB4+ cells (Plaur). qRT-PCR was carried out as an independent assessment of the array results. There were discrepancies between the two methods, with qRT-PCR confirming the differences in Lamb1, Plat and Plaur, and showing decreased expression of AdamTs1, FN, and Icam in the IB4+ cells at t=0. After 24 hr culture on LN, there were no significant differences detected by qRT-PCR between the IB4+ and IB4- cells. However, both groups showed upregulation of Itg?1 and Plaur after 24 hr on LN, the IB4+ group also had increased Plat, and the IB4- cells showed decreased Lamb1, Icam1 and AdamTs1. Further, the array screen also detected a number of genes (not subjected to qRT-PCR) expressed similarly by both populations in relatively high levels but not detectably influenced by time in culture (Bsg, Cst3, Ctsb, Ctsd, Ctsl, Mmp14, Mmp19, Sparc. We carried out immunohistochemistry to confirm expression of proteins encoded by a number of these genes.Our results show that 1B4+ and IB4- neurons differ in the expression of several genes that are associated with responsiveness to the ECM prior to culturing (AdamTs1, FN, Icam1, Lamb1, Plat, Plaur). The data suggest that the genes expressed at higher levels in the IB4- neurons could contribute to the initial growth response of these cells in a permissive environment and could also represent a common injury response that subsequently promotes axon regeneration. The differential expression of several extracellular matrix molecules (FN, Lamb1, Icam) may suggest that the IB4- neurons are capable of maintaining /secreting their local extracellular environment which could aid in the regenerative process. Overall, these data provide new information on potential targets that could be manipulated to enhance axonal regeneration in the mature nervous system.