Transient Activation of GABA_B Receptors Suppresses SK Channel Currents in Substantia Nigra Pars Compacta Dopaminergic Neurons

<div><p>Dopaminergic (DA) neurons in the substantia nigra pars compacta (SNc) are richly innervated by GABAergic neurons. The postsynaptic effects of GABA on SNc DA neurons are mediated by a mixture of GABA_A and GABA_B receptors. Although activation of GABA_A receptors inhibits spike generation, the consequences of GABA_B receptor activation are less well characterized. To help fill this gap, perforated patch recordings were made from young adult mouse SNc DA neurons. Sustained stimulation of GABA_B receptors hyperpolarized SNc DA neurons, as previously described. However, transient stimulation of GABA_B receptors by optical uncaging of GABA did not; rather, it reduced the opening of small-conductance, calcium-activated K⁺ (SK) channels and increased the irregularity of spiking. This modulation was attributable to inhibition of adenylyl cyclase and protein kinase A. Thus, because suppression of SK channel activity increases the probability of burst spiking, transient co-activation of GABA_A and GABA_B receptors could promote a pause-burst pattern of spiking.</p></div

PKA activation prevents GABA_B modulation of SK.

<p>(A) Schematic diagram showing the hypothesized signaling pathway from GABA_B receptor activation to SK channels, and the site of action of 8-bromo-cAMP and H-89 in that pathway. (B) A 50 ms uncaging pulse elicited an immediate and significant change in SOP variance (black, n = 10, Wilcoxon signed rank test, p = 0.002). (C) The same was not seen when cells were incubated in 1 μM 8-bromo-cAMP (orange, n = 4, Wilcoxon signed rank test, p = 0.875). (D) Directly activating PKA with 1 μM 8-bromo-cAMP does not have an effect on SOP variance (n = 4, Wilcoxon signed rank test, p = 0.75). (E) Inhibiting PKA with 10 μM H89 increases SOP variance (n = 6, Wilcoxon signed rank test, p = 0.0938). (F) Summary data for panels A-B. (G) Summary data for panels C-D. (H) Top, inhibiting PKA with 10 μM H89 significantly decreases SK current (n = 6, Wilcoxon signed rank test, p = 0.0313). Bottom, directly activating PKA with 1 μM 8-bromo-cAMP does not have an effect on SK current (n = 5, Wilcoxon signed rank test, p = 1.00). (I) Summary data for PKA modulators from panel H and Rp-8-CPT-cAMPS (n = 3, Wilcoxon signed rank test, p = 0.25).</p

SNc DA neuron physiology.

<p>(A) 2P reconstruction of SNc DA neuron. (B) Left, normal pacemaking of a SNc DA neuron. Middle, TTX (1 μM) application uncovered slow oscillatory potential (SOP). Right, 5 μM baclofen application hyperpolarized the cell. (C) Summary of hyperpolarization due to application of 5 μM baclofen (n = 8, median = -25.24 mV). (D) Sustained uncaging of 5 μM RuBi-GABA in the presence of 25 μM gabazine (to block GABA_A receptors) hyperpolarized cells in a manner similar to that seen following baclofen application. (E) Summary of hyperpolarization due to sustained 5 μM RuBi-GABA uncaging (n = 7, median = -11.71 mV).</p

VGCC contribution to SK.

<p>(A) Application of 10 μM isradipine (orange) to inhibit Ca_V1 channels did not reduce total SK charge (n = 8, Wilcoxon signed rank test, p = 0.3828), while inhibiting Ca_V3 channels with 10 μM mibefradil (blue) inhibited roughly half the charge (n = 8, Wilcoxon signed rank test, p = 0.0078). (B) 5 μM baclofen (green) application did not inhibit T-type calcium current (n = 12, Wilcoxon signed rank test, p = 0.1099).</p

RuBi-GABA uncaging.

<p>(A) Top, plot of the normalized firing rate before during and after a 60 s uncaging pulse (blue bar) of 5 μM RuBi-GABA in the presence of 25 μM gabazine (n = 4). Bottom, plot of normalized firing rate (black line) and running standard deviation (grey area) before, during and after a 50 ms uncaging pulse in the presence of 5 μM RuBi-GABA and 25 μM gabazine (n = 12); application of 2 μM CGP 55845 blunted the changes in spiking induced by RuBi-GABA uncaging (orange line, n = 4). Example raster plots are shown at the top of the panel. (B) Left, two different time scales showing action potentials just prior to and after GABA uncaging. Right, overlaid action potentials from just prior to and after GABA uncaging showing a clear reduction in the mAHP. (C-D) As in panel A (bottom) and B, but in the absence of gabazine (n = 9).</p

DAPT raises APP-CTF levels and lowers Aβ levels in brains of 4-month-old Ts65Dn mice.

<p>Four-month-old Ts65Dn mice and wild type colony mate controls were treated with vehicle or DAPT (100 mg/kg/day) for 4 days. (A) Representative western blots of APP, CTFs and β-actin from control (ctrl) and Ts65Dn (Ts) mice. (B) Left panel, quantification of APP (Students t-test, mean±s.e.m.,unpaired, two-tailed, n = 8 per group). Ctrl+Vehicle vs. Ts+Vehicle, p = 0.0003; Ctrl+DAPT vs. Ts+DAPT, p = 0.0002; Ctrl+Vehicle vs. Ts+DAPT, p = 0.0001; Ctrl+DAPT vs. Ts+Vehicle, p = 0.0006. Right panel, combined (C99, C89 and C83) CTFs (all means differ significantly, n = 8, 1-way ANOVA, p = 0.0002; significant differences between individual pairs of mean calculated by Students t-test, mean±s.e.m., unpaired, two-tailed). (C) Aβ40 and Aβ42 quantification from control and Ts65Dn mice. Left panel, Aβ40 (Students t-test, mean±s.e.m., unpaired, two-tailed, n = 6 per group); Ctrl+Vehicle vs. Ts+Vehicle, p = 0.0173; Ctrl+Vehicle vs. Ctrl+DAPT, p = 0.0043; Ctrl+DAPT vs. Ts+Vehicle, p = 0.0079; Ts+Vehicle vs. Ts+DAPT, p = 0.0082. Right panel, Aβ42 (Students t-test, mean±s.e.m., unpaired, two-tailed, n = 6 per group); Ctrl+Vehicle vs. Ts+Vehicle, p = 0.0169; Ctrl+DAPT vs. Ts+Vehicle, p = 0.0003; Ts+Vehicle vs. Ts+DAPT, p = 0.0052.</p

DAPT reverses cognitive deficits in 4-month-old Ts65Dn mice in the Morris water maze.

<p>DAPT was administered to Ts65Dn and control mice (100 mg/kg/day) two days prior to, and throughout, the maze testing. (A) Hidden platform test, latency to reach platform during training. (B) Probe trial on day 12, number of platform crossings. (C) Visible platform test, latency to reach platform. (D) Thigmotaxis. Statistical Analysis: n = 6 for all groups (A–D). (A) 2-way ANOVA with repeated measures revealed a main effect of genotype F1,20 = 11.31, p = 0.003 & Day F10,200 = 4.90, p = 3.00E-06 and an interaction between genotype and DAPT F1,20 = 7.73, p = 0.012. Post-hoc planned comparison test between Ts65Dn+vehicle and all 3 other groups (Ts65Dn+vehicle vs. Ts65Dn+DAPT p = 0.02, Ts65Dn+vehicle vs. control+vehicle p = 0.0003, Ts65Dn+vehicle vs. control+DAPT p = 0.008, n = 6 in all groups for all figures). (B) 2-way ANOVA for number of target platform crossings revealed an interaction between genotype and DAPT F1,20 = 8.46, p = 0.009. Post-hoc planned comparison test revealed a significant difference between Ts65Dn+vehicle vs. Ts65Dn+DAPT p = 0.01 and between Ts65Dn+vehicle vs. control+vehicle p = 0.007. No significant differences were observed for number of crossings of the analogous, virtual opposite platform location (not shown). (C) 2-way ANOVA with repeated measures revealed significant effects of genotype, F1,20 = 9.91, p = 0.005 and day, F10,200 = 21.42, p = 0.001, as well as a significant interaction between genotype and DAPT, F1,20 = 5.43, p = 0.03. Post-hoc planned comparison test revealed significant differences between Ts65Dn+vehicle vs. all 3 other groups (vs. Ts65Dn+DAPT p = 0.04, vs. control+vehicle p = 0.003, and vs. control+DAPT p = 0.005). (D) 2-way ANOVA with repeated measures revealed main effects of genotype, F1,20 = 5.13, p = 0.03 & day F10,200 = 21.94, p<1.00E-06 with an interaction between genotype and DAPT, F1,20 = 5.43, p = 0.03. Post-hoc planned comparison test revealed only a significant difference between Ts65Dn+vehicle vs. control+vehicle p = 0.004.</p

Schematic diagram depicting hypothesized signaling pathways involved in the GABA_B receptor-mediated inhibition of SK channels.

<p>GABA_B receptor inhibition of AC by G_i signaling is hypothesized to be responsible for reduced cAMP levels and PKA signaling. The reduction in PKA activity is hypothesized to reduce SK channel opening through mechanism that are independent of either plasma membrane Ca²⁺ channels or release from intracellular stores.</p

Inhibitor of the Tyrosine Phosphatase STEP Reverses Cognitive Deficits in a Mouse Model of Alzheimer's Disease

<div><p>STEP (STriatal-Enriched protein tyrosine Phosphatase) is a neuron-specific phosphatase that regulates N-methyl-D-aspartate receptor (NMDAR) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) trafficking, as well as ERK1/2, p38, Fyn, and Pyk2 activity. STEP is overactive in several neuropsychiatric and neurodegenerative disorders, including Alzheimer's disease (AD). The increase in STEP activity likely disrupts synaptic function and contributes to the cognitive deficits in AD. AD mice lacking STEP have restored levels of glutamate receptors on synaptosomal membranes and improved cognitive function, results that suggest STEP as a novel therapeutic target for AD. Here we describe the first large-scale effort to identify and characterize small-molecule STEP inhibitors. We identified the benzopentathiepin 8-(trifluoromethyl)-1,2,3,4,5-benzopentathiepin-6-amine hydrochloride (known as TC-2153) as an inhibitor of STEP with an IC₅₀ of 24.6 nM. TC-2153 represents a novel class of PTP inhibitors based upon a cyclic polysulfide pharmacophore that forms a reversible covalent bond with the catalytic cysteine in STEP. In cell-based secondary assays, TC-2153 increased tyrosine phosphorylation of STEP substrates ERK1/2, Pyk2, and GluN2B, and exhibited no toxicity in cortical cultures. Validation and specificity experiments performed in wild-type (WT) and STEP knockout (KO) cortical cells and <i>in vivo</i> in WT and STEP KO mice suggest specificity of inhibitors towards STEP compared to highly homologous tyrosine phosphatases. Furthermore, TC-2153 improved cognitive function in several cognitive tasks in 6- and 12-mo-old triple transgenic AD (3xTg-AD) mice, with no change in beta amyloid and phospho-tau levels.</p></div

Compound 3 fractionation and initial characterization.

<p>(A) Commercially purchased Compound <b>3</b> was dissolved in methanol at 10 mg/mL, and 300 µL portions were injected onto a Zorbax (Agilent) 5 µm 300SB-C18 column (0.94×25 cm, 3 mL/min 75% methanol/25% pH 4.0 0.1 M ammonium acetate). Thirty-five fractions (3 mL each) were collected, evaporated, and reconstituted in 100 µL of DMSO. Fractions were tested with pNPP assays to determine inhibition of STEP activity by using 0.1 µL of each fraction and 100 nM of STEP protein in 96-well plates. DMSO alone was used as a control. Shown in the insert is a representative chromatogram (UV absorbance detection, 350 nm). Peaks A, B, and C indicate early unretained material, Compound 3, and the unknown compound. (B) Structure of S₈, the benzopentathiepin core, and 8-(trifluoromethyl)-1,2,3,4,5-benzopentathiepin-6-amine hydrochloride (known as TC-2153). (C and D) Dose–response curves for S₈ and TC-2153. (C) The IC₅₀ for S₈ was determined to be 17.2±0.4 nM (mean ± s.e.m., <i>n</i> = 4). (D) The IC₅₀ for TC-2153 was determined to be 24.6±0.8 nM (mean ± s.e.m., <i>n</i> = 4).</p