PKA regulation of neuronal function requires the dissociation of catalytic subunits from regulatory subunits.

Protein kinase A (PKA) plays essential roles in diverse cellular functions. However, the spatiotemporal dynamics of endogenous PKA upon activation remain debated. The classical model predicts that PKA catalytic subunits dissociate from regulatory subunits in the presence of cAMP, whereas a second model proposes that catalytic subunits remain associated with regulatory subunits following physiological activation. Here we report that different PKA subtypes, as defined by the regulatory subunit, exhibit distinct subcellular localization at rest in CA1 neurons of cultured hippocampal slices. Nevertheless, when all tested PKA subtypes are activated by norepinephrine, presumably via the ß-adrenergic receptor, catalytic subunits translocate to dendritic spines but regulatory subunits remain unmoved. These differential spatial dynamics between the subunits indicate that at least a significant fraction of PKA dissociates. Furthermore, PKA-dependent regulation of synaptic plasticity and transmission can be supported only by wildtype, dissociable PKA, but not by inseparable PKA. These results indicate that endogenous PKA regulatory and catalytic subunits dissociate to achieve PKA function in neurons.

The polymorphisms of ANXA6 influence head and neck cancer susceptibility in the Chinese Han population.

Background: Head and neck cancer (HNC) is the sixth most common malignant tumor worldwide and imposes a serious economic burden on society and individuals. Annexin has been implicated in multiple functions which are essential in HNC development, including cell proliferation, apoptosis, metastasis, and invasion. This study focused on the linkage between ANXA6 variants and HNC susceptibility in Chinese people. Methods: Eight SNPs in ANXA6 from 139 HNC patients and 135 healthy controls were genotyped by the Agena MassARRAY platform. The correlation of SNPs with HNC susceptibility was evaluated using odds ratio and 95% confidence interval calculated by logistic regression using PLINK 1.9. Results: Overall analysis results demonstrated that rs4958897 was correlated with an increased HNC risk (allele: OR = 1.41, p = 0.049; dominant: OR = 1.69, p = 0.039), while rs11960458 was correlated with reduced HNC risk (OR = 0.54, p = 0.030). In age ≤ 53, rs4958897 was related to reduce HNC risk. In males, rs11960458 (OR = 0.50, p = 0.040) and rs13185706 (OR = 0.48, p = 0.043) were protective factors for HNC, but rs4346760 was a risk factor for HNC. Moreover, rs4346760, rs4958897, and rs3762993 were also correlated with increased nasopharyngeal carcinoma risk. Conclusions: Our findings suggest that ANXA6 polymorphisms are linked to the susceptibility to HNC in the Chinese Han population, indicating that ANXA6 may serve as a potential biomarker for HNC prognosis and diagnosis.

Myristoylation alone is sufficient for PKA catalytic subunits to associate with the plasma membrane to regulate neuronal functions.

Myristoylation is a posttranslational modification that plays diverse functional roles in many protein species. The myristate moiety is considered insufficient for protein-membrane associations unless additional membrane-affinity motifs, such as a stretch of positively charged residues, are present. Here, we report that the electrically neutral N-terminal fragment of the protein kinase A catalytic subunit (PKA-C), in which myristoylation is the only functional motif, is sufficient for membrane association. This myristoylation can associate a fraction of PKA-C molecules or fluorescent proteins (FPs) to the plasma membrane in neuronal dendrites. The net neutral charge of the PKA-C N terminus is evolutionally conserved, even though its membrane affinity can be readily tuned by changing charges near the myristoylation site. The observed membrane association, while moderate, is sufficient to concentrate PKA activity at the membrane by nearly 20-fold and is required for PKA regulation of AMPA receptors at neuronal synapses. Our results indicate that myristoylation may be sufficient to drive functionally significant membrane association in the absence of canonical assisting motifs. This provides a revised conceptual base for the understanding of how myristoylation regulates protein functions.

A Highly Sensitive A-Kinase Activity Reporter for Imaging Neuromodulatory Events in Awake Mice.

Neuromodulation imposes powerful control over brain function, and cAMP-dependent protein kinase (PKA) is a central downstream mediator of multiple neuromodulators. Although genetically encoded PKA sensors have been developed, single-cell imaging of PKA activity in living mice has not been established. Here, we used two-photon fluorescence lifetime imaging microscopy (2pFLIM) to visualize genetically encoded PKA sensors in response to the neuromodulators norepinephrine and dopamine. We screened available PKA sensors for 2pFLIM and further developed a variant (named tAKARα) with increased sensitivity and a broadened dynamic range. This sensor allowed detection of PKA activation by norepinephrine at physiologically relevant concentrations and kinetics, and by optogenetically released dopamine. In vivo longitudinal 2pFLIM imaging of tAKARα tracked bidirectional PKA activities in individual neurons in awake mice and revealed neuromodulatory PKA events that were associated with wakefulness, pharmacological manipulation, and locomotion. This new sensor combined with 2pFLIM will enable interrogation of neuromodulation-induced PKA signaling in awake animals. VIDEO ABSTRACT.

Ultrafast neuronal imaging of dopamine dynamics with designed genetically encoded sensors.

Neuromodulatory systems exert profound influences on brain function. Understanding how these systems modify the operating mode of target circuits requires spatiotemporally precise measurement of neuromodulator release. We developed dLight1, an intensity-based genetically encoded dopamine indicator, to enable optical recording of dopamine dynamics with high spatiotemporal resolution in behaving mice. We demonstrated the utility of dLight1 by imaging dopamine dynamics simultaneously with pharmacological manipulation, electrophysiological or optogenetic stimulation, and calcium imaging of local neuronal activity. dLight1 enabled chronic tracking of learning-induced changes in millisecond dopamine transients in mouse striatum. Further, we used dLight1 to image spatially distinct, functionally heterogeneous dopamine transients relevant to learning and motor control in mouse cortex. We also validated our sensor design platform for developing norepinephrine, serotonin, melatonin, and opioid neuropeptide indicators.

Liberated PKA Catalytic Subunits Associate with the Membrane via Myristoylation to Preferentially Phosphorylate Membrane Substrates.

Protein kinase A (PKA) has diverse functions in neurons. At rest, the subcellular localization of PKA is controlled by A-kinase anchoring proteins (AKAPs). However, the dynamics of PKA upon activation remain poorly understood. Here, we report that elevation of cyclic AMP (cAMP) in neuronal dendrites causes a significant percentage of the PKA catalytic subunit (PKA-C) molecules to be released from the regulatory subunit (PKA-R). Liberated PKA-C becomes associated with the membrane via N-terminal myristoylation. This membrane association does not require the interaction between PKA-R and AKAPs. It slows the mobility of PKA-C and enriches kinase activity on the membrane. Membrane-residing PKA substrates are preferentially phosphorylated compared to cytosolic substrates. Finally, the myristoylation of PKA-C is critical for normal synaptic function and plasticity. We propose that activation-dependent association of PKA-C renders the membrane a unique PKA-signaling compartment. Constrained mobility of PKA-C may synergize with AKAP anchoring to determine specific PKA function in neurons.

The Effect of PKCα on the Light Response of Rod Bipolar Cells in the Mouse Retina.

PURPOSE: Protein kinase C α (PKCα) is abundantly expressed in rod bipolar cells (RBCs) in the retina, yet the physiological function of PKCα in these cells is not well understood. To elucidate the role of PKCα in visual processing in the eye, we examined the effect of genetic deletion of PKCα on the ERG and on RBC light responses in the mouse. METHODS: Immunofluorescent labeling was performed on wild-type (WT), TRPM1 knockout, and PKCα knockout (PKC-KO) retina. Scotopic and photopic ERGs were recorded from WT and PKC-KO mice. Light responses of RBCs were measured using whole-cell recordings in retinal slices from WT and PKC-KO mice. RESULTS: Protein kinase C alpha expression in RBCs is correlated with the activity state of the cell. Rod bipolar cells dendrites are a major site of PKCα phosphorylation. Electroretinogram recordings indicated that loss of PKCα affects the scotopic b-wave, including a larger peak amplitude, longer implicit time, and broader width of the b-wave. There were no differences in the ERG a- or c-wave between PKCα KO and WT mice, indicating no measurable effect of PKCα in photoreceptors or the RPE. The photopic ERG was unaffected consistent with the lack of detectable PKCα in cone bipolar cells. Whole-cell recordings from RBCs in PKC-KO retinal slices revealed that, compared with WT, RBC light responses in the PKC-KO retina are delayed and of longer duration. CONCLUSIONS: Protein kinase C alpha plays an important modulatory role in RBCs, regulating both the peak amplitude and temporal properties of the RBC light response in the rod visual pathway.

TRPM3 expression in mouse retina.

Transient receptor potential (TRP) channels constitute a large family of cation permeable ion channels that serve crucial functions in sensory systems by transducing environmental changes into cellular voltage and calcium signals. Within the retina, two closely related members of the melastatin TRP family, TRPM1 and TRPM3, are highly expressed. TRPM1 has been shown to be required for the depolarizing response to light of ON-bipolar cells, but the role of TRPM3 in the retina is unknown. Immunohistochemical staining of mouse retina with an antibody directed against the C-terminus of TRPM3 labeled the inner plexiform layer (IPL) and a subset of cells in the ganglion cell layer. Within the IPL, TRPM3 immunofluorescence was markedly stronger in the OFF sublamina than in the ON sublamina. Electroretinogram recordings showed that the scotopic and photopic a- and b-waves of TRPM3(-/-) mice are normal indicating that TRPM3 does not play a major role in visual processing in the outer retina. TRPM3 activity was measured by calcium imaging and patch-clamp recording of immunopurified retinal ganglion cells. Application of the TRPM3 agonist, pregnenolone sulfate (PS), stimulated increases in intracellular calcium in ~40% of cells from wild type and TRPM1(­/­) mice, and the PS-stimulated increases in calcium were blocked by co-application of mefenamic acid, a TRPM3 antagonist. No PS-stimulated changes in fluorescence were observed in ganglion cells from TRPM3(-/-) mice. Similarly, PS-stimulated currents that could be blocked by mefenamic acid were recorded from wild type retinal ganglion cells but were absent in ganglion cells from TRPM3-/- mice.

Voriconazole, an antifungal triazol that causes visual side effects, is an inhibitor of TRPM1 and TRPM3 channels.

PURPOSE: Administration of voriconazole, an antifungal triazole, causes transient visual disturbances in patients and attenuates the b-wave of the ERG. We sought to identify the retinal target of voriconazole underlying the effect on the ERG b-wave. METHODS: Electroretinograms were recorded from mice before and after intraperitoneal injection of voriconazole. The effect of voriconazole on ON-bipolar cells was tested by patch-clamp recordings of ON-bipolar cells in mouse retinal slices. Effects of voriconazole on mGluR6 and TRPM3 were assessed by patch-clamp recordings of Chinese hamster ovary (CHO) and HEK293 cells transfected with either TRPM3 or mGluR6 plus Kir3.1/Kir3.4. RESULTS: Voriconazole attenuated the ERG b-wave in mice, and inhibited ON-bipolar cell responses evoked by application of CPPG, an mGluR6 antagonist, onto the ON-bipolar cell dendrites, indicating that voriconazole blocks a step in the mGluR6-TRPM1 signal transduction pathway. Voriconazole almost completely blocked capsaicin-activated currents in ON-bipolar cells, which have been attributed to direct activation of the TRPM1 cation channel. Furthermore, application of voriconazole to CHO cells expressing TRPM3, a closely related channel to TRPM1, showed that voriconazole reversibly blocked pregnenolone sulfate-stimulated TRPM3 currents in transfected cells. In contrast, voriconazole only slightly inhibited mGluR6-mediated activation of G-protein activated inward rectifier potassium (GIRK) currents in cotransfected cells, suggesting that mGluR6 is not the primary target of voriconazole in ON-bipolar cells. CONCLUSIONS: The visual disturbances associated with voriconazole are likely due to block of TRPM1 channels in retinal ON-bipolar cells. Other neurological effects of voriconazole may be due to block of TRPM3 channels expressed in the brain.

Serum TRPM1 autoantibodies from melanoma associated retinopathy patients enter retinal on-bipolar cells and attenuate the electroretinogram in mice.

Melanoma-associated retinopathy (MAR) is a paraneoplastic syndrome associated with cutaneous malignant melanoma and the presence of autoantibodies that label neurons in the inner retina. The visual symptoms and electroretinogram (ERG) phenotype characteristic of MAR resemble the congenital visual disease caused by mutations in TRPM1, a cation channel expressed by both melanocytes and retinal bipolar cells. Four serum samples from MAR patients were identified as TRPM1 immunoreactive by 1. Labeling of ON-bipolar cells in TRPM1+/+ but not TRPM1-/- mouse retina, 2. Labeling of TRPM1-transfected CHO cells; and 3. Attenuation of the ERG b-wave following intravitreal injection of TRPM1-positive MAR IgG into wild-type mouse eyes, and the appearance of the IgG in the retinal bipolar cells at the conclusion of the experiment. Furthermore, the epitope targeted by the MAR autoantibodies was localized within the amino-terminal cytoplasmic domain of TRPM1. Incubation of live retinal neurons with TRPM1-positive MAR serum resulted in the selective accumulation of IgG in ON-bipolar cells from TRPM1+/+ mice, but not TRPM1-/- mice, suggesting that the visual deficits in MAR are caused by the uptake of TRPM1 autoantibodies into ON-bipolar cells, where they bind to an intracellular epitope of the channel and reduce the ON-bipolar cell response to light.

Development and evaluation of a traditional Chinese medicine syndrome questionnaire for measuring sub-optimal health status in China.

OBJECTIVE: Sub-optimal health status (SHS), in which a person's mind and body exists in a low-quality state of being between disease and health, has become a public health problem that cannot be ignored in China. SHS measurement presents a challenge to the academic fields. We developed and evaluated a questionnaire from the perspective of traditional Chinese medicine (TCM) that embodies the features of TCM syndrome diagnosis for measuring SHS in China. METHODS: The construction of the theoretical framework of the questionnaire was based on a literature review, an expert questionnaire survey and group interviews. The subscales and questionnaire items were screened through a pilot study using statistical means and qualitative analysis. Reliability tests that were used included test-retest reliability, Cronbach's a coefficient, split-half reliability; validity tests included content validity, criterion validity, discrimination validity and construct validity. RESULTS: The final questionnaire, the SHSQ-50, included 50 five-class quantifiable items that encompassed nine subscales: liver stagnation syndrome, liver-Qi deficiency syndrome, spleen-Qi deficiency syndrome, liver-fire syndrome, heart-fire syndrome, stomach-fire syndrome, heart-Qi deficiency syndrome, lung-Qi deficiency syndrome and dampness syndrome. Questionnaires were completed by 268 of the 288 SHS subjects (93.0%) and by 86 of the 94 healthy subjects (91.5%). The Cronbach a coefficients, split-half coefficients and stability coefficients ranged from 0.70 to 0.95, 0.67 to 0.87 and 0.88 to 0.98, respectively, for the overall scores and subscales. The Wilcoxon rank test showed statistically significant differences in the subscales and overall scores between the SHS group and the healthy group (P < 0.01). Twelve factors with an eigenvalue greater than one were extracted by factor analysis and merged into nine factors, for which the cumulative contribution rate was 63.63%. The nine factors were corresponded to the overall structure of the questionnaire. CONCLUSION: The SHSQ-50 is a reliable and valid instrument for measuring TCM syndrome diagnosis of SHS in China.

Farnesylation of retinal transducin underlies its translocation during light adaptation.

G proteins are posttranslationally modified by isoprenylation: either farnesylation or geranylgeranylation. The gamma subunit of retinal transducin (Talpha/Tbetagamma) is selectively farnesylated, and the farnesylation is required for light signaling mediated by transducin in rod cells. However, whether and how this selective isoprenylation regulates cellular functions remain poorly understood. Here we report that knockin mice expressing geranylgeranylated Tgamma showed normal rod responses to dim flashes under dark-adapted conditions but exhibited impaired properties in light adaptation. Of note, geranylgeranylation of Tgamma suppressed light-induced transition of Tbetagamma from membrane to cytosol, and also attenuated its light-dependent translocation from the outer segment to the inner region, an event contributing to retinal light adaptation. These results indicate that, while the farnesylation of transducin is interchangeable with the geranylgeranylation in terms of the light signaling, the selective farnesylation is important for visual sensitivity regulation by providing sufficient but not excessive membrane anchoring of Tbetagamma.

PHR1, a PH domain-containing protein expressed in primary sensory neurons.

Previously, we identified PHR1 as an abundantly expressed gene in photoreceptors and showed that it encodes four isoforms, each with N-terminal pleckstrin homology (PH) and C-terminal transmembrane domains. To better understand PHR1 function and expression, we made a Phr1 null mouse by inserting a beta-galactosidase/neor cassette into exon 3. In addition to photoreceptors, we found abundant expression of specific Phr1 splice forms in olfactory receptor neurons and vestibular and cochlear hair cells. We also found Phr1 expression in cells with a possible sensory function, including peripheral retinal ganglion cells, cochlear interdental cells, and neurons of the circumventricular organ. Despite this discrete expression in known and putative sensory neurons, mice lacking PHR1 do not have overt sensory deficits.

Rod sensitivity during Xenopus development.

We have measured the sensitivity of rod photoreceptors from overnight-dark-adapted Xenopus laevis through developmental stages 46-66 into adulthood by using suction-pipette recording. The dark current increased gradually from approximately 5 pA at stage 46 to approximately 20 pA at stage 57, compared with an adult (metamorphosed) current of approximately 35 pA. This increase in dark current largely paralleled the progressive increase in length and diameter of the rod outer segment (ROS). Throughout stages 46-66, the dark current increased approximately linearly with ROS surface area. At stage 53, there was a steep (approximately 10-fold) increase in the rod flash sensitivity, accompanied by a steep increase in the time-to-peak of the half-saturated flash response. This covariance of sensitivity and time-to-peak suggested a change in the state of adaptation of rods at stage 53 and thereafter. When the isolated retina was preincubated with 11-cis-retinal, the flash sensitivity and the response time-to-peak of rods before stage 53 became similar to those at or after stage 53, suggesting that the presence of free opsin (i.e., visual pigment without chromophore) in rods before stage 53 was responsible for the adapted state (low sensitivity and short time-to-peak). By comparing the response sensitivity before stage 53 to the sensitivity at/after stage 53 measured from rods that had been subjected to various known bleaches, we estimated that 22-28% of rod opsin in stage 50-52 tadpoles (i.e., before stage 53) was devoid of chromophore despite overnight dark-adaptation. When continuously dark adapted for 7 d or longer, however, even tadpoles before stage 53 yielded rods with similar flash sensitivity and response time-to-peak as those of later-stage animals. In conclusion, it appears that chromophore regeneration is very slow in tadpoles before stage 53, but this regeneration becomes much more efficient at stage 53. A similar delay in the maturity of chromophore regeneration may partially underlie the low sensitivity of rods observed in newborn mammals, including human infants.