measurement-oxygen-consumption-rate-acute-striatal-slices-from-adult

Measurement of Oxygen Consumption Rate in Acute Striatal Slices from Adult Mice

Mitochondria play an important role in cellular ATP production, reactive oxygen species regulation, and Ca2+ concentration control. Mitochondrial ...

Thomas Jefferson University

The striatum is essential for many aspects of mammalian behavior, including motivation and movement, and is dysfunctional in motor disorders such as Parkinson's 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's 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's disease and related disorders.

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

Inhalational general anesthetics, such as sevoflurane and isoflurane, modulate a subset of brain Kv1 potassium channels. However, the Kv1.2 channel is resistant to propofol, a commonly used intravenous alkylphenol anesthetic. We hypothesize that propofol binds to a presumed pocket involving the channel's S4-S5 linker, but functional transduction is poor and, therefore, propofol efficacy is low. To test this hypothesis, we used a photoactive propofol analog (meta-aziPropofol = AziPm) to directly probe binding and electrophysiological and mutational analyses in Xenopus oocytes to probe function. We find that AziPm photolabels L321 in the S4-S5 linker of both the wild-type Kv1.2 and a mutant Kv1.2 (G329 T) with a novel gating phenotype. Furthermore, whereas propofol does not significantly modulate Kv1.2 WT but robustly potentiates Kv1.2 G329T, AziPm inhibits Kv1.2 WT and also potentiates Kv1.2 G329T. Kv1.2 modulation by AziPm was abolished by two mutations that decreased hydrophobicity at L321 (L321A and L321F), confirming the specific significance of the S4-S5 linker in the mechanism of general anesthetic modulation. Since AziPm binds to Kv1.2 G329T and shares the propofol ability to potentiate this mutant, the parent propofol likely also binds to the Kv1.2 channel. However, binding and alkylphenol-induced transduction are seemingly sensitive to the conformation of the S4-S5 linker site (altered by G329T) and subtle differences in the chemical structures of propofol and AziPm. Overall, the results are consistent with a mechanism of general anesthetic modulation that depends on the complementarity of necessary ligand binding and permissive ion channel conformations that dictate modulation and efficacy.

Sites and Functional Consequence of Alkylphenol Anesthetic Binding to Kv1.2 Channels.

Sevoflurane is a commonly used inhaled general anesthetic. Despite this, its mechanism of action remains largely elusive. Compared to other anesthetics, sevoflurane exhibits distinct functional activity. In particular, sevoflurane is a positive modulator of voltage-gated Shaker-related potassium channels (Kv1.x), which are key regulators of action potentials. Here, we report the synthesis and validation of azisevoflurane, a photoaffinity ligand for the direct identification of sevoflurane binding sites in the Kv1.2 channel. Azisevoflurane retains major sevoflurane protein binding interactions and pharmacological properties within in vivo models. Photoactivation of azisevoflurane induces adduction to amino acid residues that accurately reported sevoflurane protein binding sites in model proteins. Pharmacologically relevant concentrations of azisevoflurane analogously potentiated wild-type Kv1.2 and the established mutant Kv1.2 G329T. In wild-type Kv1.2 channels, azisevoflurane photolabeled Leu317 within the internal S4-S5 linker, a vital helix that couples the voltage sensor to the pore region. A residue lining the same binding cavity was photolabeled by azisevoflurane and protected by sevoflurane in the Kv1.2 G329T. Mutagenesis of Leu317 in WT Kv1.2 abolished sevoflurane voltage-dependent positive modulation. Azisevoflurane additionally photolabeled a second distinct site at Thr384 near the external selectivity filter in the Kv1.2 G329T mutant. The identified sevoflurane binding sites are located in critical regions involved in gating of Kv channels and related ion channels. Azisevoflurane has thus emerged as a new tool to discover inhaled anesthetic targets and binding sites and investigate contributions of these targets to general anesthesia.

Photoaffinity Ligand for the Inhalational Anesthetic Sevoflurane Allows Mechanistic Insight into Potassium Channel Modulation.

Voltage-gated ion channels (VGICs) of excitable tissues are emerging as targets likely involved in both the therapeutic and toxic effects of inhaled and intravenous general anesthetics. Whereas sevoflurane and propofol inhibit voltage-gated Na channels (Navs), sevoflurane potentiates certain voltage-gated K channels (Kvs). The combination of these effects would dampen neural excitability and, therefore, might contribute to the clinical endpoints of general anesthesia. As the body of work regarding the interaction of general anesthetics with VGICs continues to grow, a multidisciplinary approach involving functional, biochemical, structural, and computational techniques, many of which are detailed in other chapters, has increasingly become necessary to solve the molecular mechanism of general anesthetic action on VGICs. Here, we focus on electrophysiological and modeling approaches and methodologies to describe how our work has elucidated the biophysical basis of the inhibition Navs by propofol and the potentiation of Kvs by sevoflurane.

Electrophysiological Analysis of Voltage-Gated Ion Channel Modulation by General Anesthetics.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene account for most common causes of familial and sporadic Parkinson's disease (PD) and are one of the strongest genetic risk factors in sporadic PD. Pathways implicated in LRRK2-dependent neurodegeneration include cytoskeletal dynamics, vesicular trafficking, autophagy, mitochondria, and calcium homeostasis. However, the exact molecular mechanisms still need to be elucidated. Both genetic and environmental causes of PD have highlighted the importance of mitochondrial dysfunction in the pathogenesis of PD. Mitochondrial impairment has been observed in fibroblasts and iPSC-derived neural cells from PD patients with LRRK2 mutations, and LRRK2 has been shown to localize to mitochondria and to regulate its function. In this review we discuss recent discoveries relating to LRRK2 mutations and mitochondrial dysfunction.

LRRK2 and mitochondria: Recent advances and current views.

Mutations and deletions in PTEN-induced kinase 1 (PINK1) cause autosomal recessive Parkinson's disease (PD), the second most common neurodegenerative disorder. PINK1 is a nuclear-genome encoded Ser/Thr kinase in mitochondria. PINK1 deletion was reported to affect dopamine (DA) levels in the striatum and mitochondrial functions but with conflicting results. The role of PINK1 in mitochondrial function and in PD pathogenesis remains to be elucidated thoroughly. In this study, we measured DA release using fast-scan cyclic voltammetry in acute striatal slices from both PINK1 knockout (KO) and wild-type (WT) mice at different ages. We found that single pulse-evoked DA release in the dorsal striatum of PINK1 KO mice was decreased in an age-dependent manner. Furthermore, the decrease was because of less DA release instead of an alteration of DA transporter function or DA terminal degeneration. We also found that PINK1 KO striatal slices had significantly lower basal mitochondria respiration compared with that of WT controls, and this impairment was also age-dependent. These results suggest that the impaired DA release is most likely because of mitochondrial dysfunction and lower ATP production.

Loss of PINK1 causes age-dependent decrease of dopamine release and mitochondrial dysfunction.

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 identify 5'-deoxyadenosylcobalamin (AdoCbl), a physiological form of the essential micronutrient vitamin B 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 disrupting LRRK2 dimerization. Treatment with AdoCbl inhibits LRRK2 kinase activity in cultured cells and brain tissue, and prevents neurotoxicity in cultured primary rodent neurons 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 B 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 future.

Vitamin B modulates Parkinson's disease LRRK2 kinase activity through allosteric regulation and confers neuroprotection.

The voltage-gated potassium channel Kv3.4 is a crucial regulator of nociceptive signaling in the dorsal root ganglion (DRG) and the dorsal horn of the spinal cord. Moreover, Kv3.4 dysfunction has been linked to neuropathic pain. Although kinases and phosphatases can directly modulate Kv3.4 gating, the signaling mechanisms regulating the expression and stability of the Kv3.4 protein are generally unknown. We explored a potential role of PKCε and found an unexpected interaction that has a positive effect on Kv3.4 expression. Co-immunoprecipitation studies revealed a physical association between PKCε and Kv3.4 in both heterologous cells and rat DRG neurons. Furthermore, in contrast to the wild-type and constitutively active forms of PKCε, expression of a catalytically inactive form of the enzyme inhibits Kv3.4 expression and membrane localization through a dominant negative effect. Co-expression of Kv3.4 with the wild-type, constitutively active, or catalytically inactive forms of PKCε had no significant effects on Kv3.4 gating. These results suggest that a novel physical interaction of the Kv3.4 channel with functional PKCε primarily determines its stability and localization in DRG neurons. This interaction is akin to those of previously identified accessory ion channel proteins, which could be significant in neural tissues where Kv3.4 regulates electrical signaling.

Article	Year
Loss of VGLUT3 Produces Circadian-Dependent Hyperdopaminergia and Ameliorates Motor Dysfunction and l-Dopa-Mediated Dyskinesias in a Model of Parkinson's Disease. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 26558771	2015
Sites and Functional Consequence of Alkylphenol Anesthetic Binding to Kv1.2 Channels. Molecular neurobiology\| PubMed ID: 28204960	2018
Photoaffinity Ligand for the Inhalational Anesthetic Sevoflurane Allows Mechanistic Insight into Potassium Channel Modulation. ACS chemical biology\| PubMed ID: 28333442	2017
Electrophysiological Analysis of Voltage-Gated Ion Channel Modulation by General Anesthetics. Methods in enzymology\| PubMed ID: 29588038	2018
LRRK2 and mitochondria: Recent advances and current views. Brain research\| PubMed ID: 29894679	2019
Loss of PINK1 causes age-dependent decrease of dopamine release and mitochondrial dysfunction. Neurobiology of aging\| PubMed ID: 30504091	2019
Vitamin B modulates Parkinson's disease LRRK2 kinase activity through allosteric regulation and confers neuroprotection. Cell research\| PubMed ID: 30858560	2019
PKCε associates with the Kv3.4 channel to promote its expression in a kinase activity-dependent manner. FASEB journal : official publication of the Federation of American Societies for Experimental Biology\| PubMed ID: 33368632	2021

Lianteng Zhi

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