Phosphorylation of a K channel by PKC regulates the excitability of primary sensory neurons and pain signaling

Introduction: Voltage-gated potassium channels (Kvs) play an important role in the termination of neuronal action potentials. Kv3.4 is one of many types of Kv channels, found throughout the human body – including in the axon terminals of dorsal root ganglia neurons. Kv3.4 channels are categorized as A-type currents and have prominent fast-inactivation that has been shown to be phosphorylation dependent. It is hypothesized that following spinal cord injury (SCI), Kv3.4 channels become hyperphosphorylated and their expression reduced – leading to an increase in nociceptive signaling. Methods: We used embryonic DRG neurons, transfected with one of four types of viral AAV6 constructs, to induce expression of Kv3.4 channels. Constructs included EGFP, WT Kv3.4, Kv3.4 A (phosphonull), Kv3.4 D (phosphomimetic), and Kv3.4 dominant negative (DN). Results: Kv3.4 constructs were differentially trafficked across embryonic DRG neurons, following AAV1 transfection, as seen through immunofluorescence. Peak currents were increased for WT, A, and D mutants, relative to GFP and DN. The sustained current for phosphomimetic was significantly higher than for GFP and phosphonull (p=0.031). G/Gmax for all traces showed similar activation kinetics. V1/2 was relatively unchanged across constructs. The slow inactivating current was measured at t=50 sec and AP duration for the phosphomimetic Kv3.4 channels was shortened compared with GFP (p=0.018). The rate of repolarization was increased in phosphomimetic constructs, compared with GFP (p=0.044). Discussion: Phosphorylation of Kv3.4 appears to modulate channel properties and may also play a role in SCI-induced neuropathic pain. Future in vivo experiments can assess pain behavior in animals expressing different Kv3.4 constructs

Tunable Action Potential Repolarization Governed by Kv3.4 Channels in Dorsal Root Ganglion Neurons.

The Kv3.4 channel regulates action potential (AP) repolarization in nociceptors and excitatory synaptic transmission in the spinal cord. We hypothesize that this is a tunable role governed by protein kinase-C-dependent phosphorylation of the Kv3.4 cytoplasmic N-terminal inactivation domain (NTID) at four nonequivalent sites. However, there is a paucity of causation evidence linking the phosphorylation status of Kv3.4 to the properties of the AP. To establish this link, we used adeno-associated viral vectors to specifically manipulate the expression and the effective phosphorylation status of Kv3.4 in cultured dorsal root ganglion (DRG) neurons from mixed-sex rat embryos at embryonic day 18. These vectors encoded GFP (background control), wild-type (WT) Kv3.4, phosphonull (PN) Kv3.4 mutant (PN = S[8,9,15,21]A), phosphomimic (PM) Kv3.4 mutant (PM = S[8,9,15,21]D), and a Kv3.4 nonconducting dominant-negative (DN) pore mutant (DN = W429F). Following viral infection of the DRG neurons, we evaluated transduction efficiency and Kv3.4 expression and function via fluorescence microscopy and patch clamping. All functional Kv3.4 constructs induced current overexpression with similar voltage dependence of activation. However, whereas Kv3.4-WT and Kv3.4-PN induced fast transient currents, the Kv3.4-PM induced currents exhibiting impaired inactivation. In contrast, the Kv3.4-DN abolished the endogenous Kv3.4 current. Consequently, Kv3.4-DN and Kv3.4-PM produced APs with the longest and shortest durations, respectively, whereas Kv3.4-WT and Kv3.4-PN produced intermediate results. Moreover, the AP widths and maximum rates of AP repolarization from these groups are negatively correlated. We conclude that the expression and effective phosphorylation status of the Kv3.4 NTID confer a tunable mechanism of AP repolarization, which may provide exquisite regulation of pain signaling in DRG neurons

Loss of VGLUT3 Produces Circadian-Dependent Hyperdopaminergia and Ameliorates Motor Dysfunction and l-Dopa-Mediated Dyskinesias in a Model of Parkinson\u27s Disease.

UNLABELLED: The striatum is essential for many aspects of mammalian behavior, including motivation and movement, and is dysfunctional in motor disorders such as Parkinson\u27s disease. The vesicular glutamate transporter 3 (VGLUT3) is expressed by striatal cholinergic interneurons (CINs) and is thus well positioned to regulate dopamine (DA) signaling and locomotor activity, a canonical measure of basal ganglia output. We now report that VGLUT3 knock-out (KO) mice show circadian-dependent hyperlocomotor activity that is restricted to the waking cycle and is due to an increase in striatal DA synthesis, packaging, and release. Using a conditional VGLUT3 KO mouse, we show that deletion of the transporter from CINs, surprisingly, does not alter evoked DA release in the dorsal striatum or baseline locomotor activity. The mice do, however, display changes in rearing behavior and sensorimotor gating. Elevation of DA release in the global KO raised the possibility that motor deficits in a Parkinson\u27s disease model would be reduced. Remarkably, after a partial 6-hydroxydopamine (6-OHDA)-mediated DA depletion (∼70% in dorsal striatum), KO mice, in contrast to WT mice, showed normal motor behavior across the entire circadian cycle. l-3,4-dihydroxyphenylalanine-mediated dyskinesias were also significantly attenuated. These findings thus point to new mechanisms to regulate basal ganglia function and potentially treat Parkinson\u27s disease and related disorders. SIGNIFICANCE STATEMENT: Dopaminergic signaling is critical for both motor and cognitive functions in the mammalian nervous system. Impairments, such as those found in Parkinson\u27s disease patients, can lead to severe motor deficits. Vesicular glutamate transporter 3 (VGLUT3) loads glutamate into secretory vesicles for neurotransmission and is expressed by discrete neuron populations throughout the nervous system. Here, we report that the absence of VGLUT3 in mice leads to an upregulation of the midbrain dopamine system. Remarkably, in a Parkinson\u27s disease model, the mice show normal motor behavior. They also show fewer abnormal motor behaviors (dyskinesias) in response to l-3,4-dihydroxyphenylalanine, the principal treatment for Parkinson\u27s disease. The work thus suggests new avenues for the development of novel treatment strategies for Parkinson\u27s disease and potentially other basal-ganglia-related disorders

The Binding and Mechanism of a Positive Allosteric Modulator of Kv3 Channels

Small-molecule modulators of diverse voltage-gated K+ (Kv) channels may help treat a wide range of neurological disorders. However, developing effective modulators requires understanding of their mechanism of action. We apply an orthogonal approach to elucidate the mechanism of action of an imidazolidinedione derivative (AUT5), a highly selective positive allosteric modulator of Kv3.1 and Kv3.2 channels. AUT5 modulation involves positive cooperativity and preferential stabilization of the open state. The cryo-EM structure of the Kv3.1/AUT5 complex at a resolution of 2.5 Å reveals four equivalent AUT5 binding sites at the extracellular inter-subunit interface between the voltage-sensing and pore domains of the channel’s tetrameric assembly. Furthermore, we show that the unique extracellular turret regions of Kv3.1 and Kv3.2 essentially govern the selective positive modulation by AUT5. High-resolution apo and bound structures of Kv3.1 demonstrate how AUT5 binding promotes turret rearrangements and interactions with the voltage-sensing domain to favor the open conformation

Vitamin B12 modulates Parkinson’s disease LRRK2 kinase activity through allosteric regulation and confers neuroprotection

Missense mutations in Leucine-Rich Repeat Kinase 2 (LRRK2) cause the majority of familial and some sporadic forms of Parkinson’s disease (PD). The hyperactivity of LRRK2 kinase induced by the pathogenic mutations underlies neurotoxicity, promoting the development of LRRK2 kinase inhibitors as therapeutics. Many potent and specific small molecule LRRK2 inhibitors have been reported with promise. However, nearly all inhibitors are ATP competitive – some with unwanted side effects and unclear clinical outcome - alternative types of LRRK2 inhibitors are lacking. Herein we find 5’-deoxyadenosylcobalamin (AdoCbl), a physiological form of the essential micronutrient vitamin B12 as a mixed-type allosteric inhibitor of LRRK2 kinase activity. Multiple assays show that AdoCbl directly binds LRRK2, leading to the alterations of protein conformation and ATP binding in LRRK2. STD-NMR analysis of a LRRK2 homologous kinase reveals the contact sites in AdoCbl that interface with the kinase domain. Furthermore, we provide evidence that AdoCbl modulates LRRK2 activity through disruption of LRRK2 dimerization. Treatment with AdoCbl inhibits LRRK2 kinase activity in cultured cells and brain tissue, and importantly prevents neurotoxicity in primary rodent cultures as well as in transgenic C. elegans and D. melanogaster expressing LRRK2 disease variants. Finally, AdoCbl alleviates deficits in dopamine release sustainability caused by LRRK2 disease variants in mouse models. Our study uncovers vitamin B12 as a novel class of LRRK2 kinase modulator with a distinct mechanism, which can be harnessed to develop new LRRK2-based PD therapeutics in the futur

Increased Mitochondrial Calcium Sensitivity and Abnormal Expression of Innate Immunity Genes Precede Dopaminergic Defects in Pink1-Deficient Mice

BACKGROUND: PTEN-induced kinase 1 (PINK1) is linked to recessive Parkinsonism (EOPD). Pink1 deletion results in impaired dopamine (DA) release and decreased mitochondrial respiration in the striatum of mice. To reveal additional mechanisms of Pink1-related dopaminergic dysfunction, we studied Ca²+ vulnerability of purified brain mitochondria, DA levels and metabolism and whether signaling pathways implicated in Parkinson\u27s disease (PD) display altered activity in the nigrostriatal system of Pink1⁻/⁻ mice. METHODS AND FINDINGS: Purified brain mitochondria of Pink1⁻/⁻ mice showed impaired Ca²+ storage capacity, resulting in increased Ca²+ induced mitochondrial permeability transition (mPT) that was rescued by cyclosporine A. A subpopulation of neurons in the substantia nigra of Pink1⁻/⁻ mice accumulated phospho-c-Jun, showing that Jun N-terminal kinase (JNK) activity is increased. Pink1⁻/⁻ mice 6 months and older displayed reduced DA levels associated with increased DA turnover. Moreover, Pink1⁻/⁻ mice had increased levels of IL-1β, IL-12 and IL-10 in the striatum after peripheral challenge with lipopolysaccharide (LPS), and Pink1⁻/⁻ embryonic fibroblasts showed decreased basal and inflammatory cytokine-induced nuclear factor kappa-β (NF-κB) activity. Quantitative transcriptional profiling in the striatum revealed that Pink1⁻/⁻ mice differentially express genes that (i) are upregulated in animals with experimentally induced dopaminergic lesions, (ii) regulate innate immune responses and/or apoptosis and (iii) promote axonal regeneration and sprouting. CONCLUSIONS: Increased mitochondrial Ca²+ sensitivity and JNK activity are early defects in Pink1⁻/⁻ mice that precede reduced DA levels and abnormal DA homeostasis and may contribute to neuronal dysfunction in familial PD. Differential gene expression in the nigrostriatal system of Pink1⁻/⁻ mice supports early dopaminergic dysfunction and shows that Pink1 deletion causes aberrant expression of genes that regulate innate immune responses. While some differentially expressed genes may mitigate neurodegeneration, increased LPS-induced brain cytokine expression and impaired cytokine-induced NF-κB activation may predispose neurons of Pink1⁻/⁻ mice to inflammation and injury-induced cell death

Cytokine expression in the striatum and isolated microglial cultures.

<p>(A) Wildtype and <i>Pink1^−/−</i> mice (n = 4 mice/genotype) were injected ip with 0.33 µg LPS/g body weight. Cytokines in striatal homogenates (corresponding to 100 µg total protein) were measured eight hours later by ELISA as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016038#s4" target="_blank">Materials and Methods</a>. * <i>P</i><0.05, compared to wildtype mice. Basal cytokine levels, measured in a separate experiment, were not statistically different between wildtype and <i>Pink1^−/−</i> mice. (B) Microglial cultures derived from the forebrain of neonatal wildtype and <i>Pink1^−/−</i> mice were incubated with 100 ng/ml LPS for 24 hours in 24-well plates and the cytokines were measured with an ELISA as described in the Methods (n = 8 wells per condition). (C) Strong induction of IL-6, TNF-α and G-CSF in wildtype and <i>Pink1^−/−</i> microglia cells demonstrates that the cells were capable of responding to the LPS stimulus. In panels B and C, background-corrected absorbance is plotted (OD450 minus OD570). (D) Real-time PCR expression analysis of CD3 mRNA (specific T cell marker) in the striatum of control and LPS-treated wildtype and <i>Pink1^−/−</i> mice, showing that the T cell marker is barely detectable (Ct values of 39.67 and 38.46) and not increased by LPS treatment.</p

Southern blot analysis of ES cell colonies and F2 generation mice.

<p>(A) Genomic DNA from PCR-positive ES cell clones was double-digested with NcoI/SacI or NdeI/XbaI and hybridized with the indicated probes. The location of the two probes and expected sizes of the various DNA fragments for wildtype (WT) and <i>Pink1^+/−</i> ES cells are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016038#pone-0016038-g001" target="_blank">Figure 1</a>. (B) Tail DNA from offspring of <i>Pink1^+/−</i> breeder pairs was digested with NcoI and SacI and analyzed by Southern blot using “outside exon 8” probe. The 4513 bp band indicates the wildtype <i>Pink1</i> allele and the 4152 bp band is diagnostic for the mutated <i>Pink1</i> allele. Location of the probe and restriction enzyme cleavage sites are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016038#pone-0016038-g001" target="_blank">Figure 1</a>.</p

Dopamine levels, dopamine turnover and dopamine neuron counts.

<p>(A) Decreased DA levels in the striatum of <i>Pink1^−/−</i> mice aged 6 months and older. (B) Normal counts of dopaminergic neurons in the substantia nigra pars compacta (SNc) of 1-year old <i>Pink1^−/−</i> mice. (C) Increased DA turnover in <i>Pink1^−/−</i> mice. Eight mice per genotype were used for catecholamine analysis (A and C). Five wildtype and six <i>Pink1^−/−</i> mice were used to determine nigral DA neuron numbers by unbiased stereology (B). * <i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i><0.001.</p