Neuronal store-operated calcium entry pathway as a novel therapeutic target for Huntington's disease treatment

Abstract

Huntington's disease (HD) is a neurodegenerative disorder caused by a polyglutamine expansion within Huntingtin (Htt) protein. In the phenotypic screen we identified a class of quinazoline-derived compounds that delayed a progression of a motor phenotype in transgenic Drosophila HD flies. We found that the store-operated calcium (Ca(2+)) entry (SOC) pathway activity is enhanced in neuronal cells expressing mutant Htt and that the identified compounds inhibit SOC pathway in HD neurons. The same compounds exerted neuroprotective effects in glutamate-toxicity assays with YAC128 medium spiny neurons primary cultures. We demonstrated a key role of TRPC1 channels in supporting SOC pathway in HD neurons. We concluded that the TRPC1-mediated neuronal SOC pathway constitutes a novel target for HD treatment and that the identified compounds represent a novel class of therapeutic agents for treatment of HD and possibly other neurodegenerative disorders.

Figure 1. Identification of EVP4593 in climbing assay screen with HD transgenic flies

(A) The climbing speed of HD flies is plotted as a function of time following induction of mHtt-128Q transgene. The results with lacZ –expressing flies are shown as a control (red). A number of small molecule compounds were dissolved in DMSO and included in the fly food in the final concentration as indicated. The results obtained in HD flies for 400 µM EVP4593 (orange), 250 µM TSA (green) and 1% DMSO (blue) are compared. (B) Dose-dependence of EVP4593 in rescuing motor dysfunction of HD flies. An average effect size relative to 1% DMSO control was determined at days 8–10 after mHtt-128Q transgene induction (ΔSpeed), normalized to variability of the climbing speed in DMSO-treated group (σ) and plotted as mean ± S.E. (n = 30 flies) against concentration of EVP4593 in the fly food. (C) Chemical structures of EVP4593 and its analogues used in the study. BMS-345541 is an unrelated compound with potent IKK inhibitory activity. 2-APB is an unrelated compound with ability to block SOC with low affinity in some cell types. (D) The potency of 200 µM of EVP4593 or its analogs in the climbing assay was determined as an average effect size at days 8–10 after mHtt-128Q transgene induction and shown as mean ± S.E. (n = 30 flies) (filled bars). The potency of 1 µM of EVP4593 or its analogs was determined as % inhibition of NF-κB activity and shown as mean ± S.E. (n = 4–6) (open bars). Also shown are results for BMS-345541 and 2-APB (400 µM in climbing assay and 1 µM in NF-κB assay for both compounds) See also Figure S1.

Figure 2. SOC pathway in WT and YAC128 MSNs - Ca²⁺ imaging assay

(A, B) WT (A) and YAC128 (B) MSN cultures at DIV10–14 were loaded with Fura-2 Ca²⁺ imaging dye and incubated in Ca²⁺-free media. The intracellular Ca²⁺ stores were depleted by addition of 1 µM of thapsigargin (Tg) as indicated. Readdition of 2 mM Ca²⁺ to the extracellular media resulted in Ca²⁺ influx via SOC pathway. Fura-2 340/380 ratio traces are shown for individual cells (gray thin lines). For each cell 340/380 ratio trace was offset to 0.0 for the time point of Ca²⁺ re-addition. The average trace is also shown (black thick line). (C,D). SOC Ca²⁺ imaging experiments were performed with WT (C) and YAC128 (D) MSN in the presence of 300 nM EVP4593. (E). An average amplitude of an increase in 340/380 Fura-2 signals in response to Ca²⁺ readdition is shown for YAC128 and WT MSN in the presence of DMSO or 300 nM of test compounds as indicated. The results are shown as mean ± S.E. (number of cells is shown on the top of the bar). *, p < 0.05; ***, p<0.001 when compared to DMSO group.

Figure 3. SOC pathway in YAC128 MSNs - Mn²⁺ quenching assay

(A–C). SOC pathway in YAC128 MSN is quantified by Mn²⁺ quenching assay. The YAC128 MSNs loaded with Fura-2 were placed in Ca²⁺ -free and Mn²⁺-free media containing 100 µM EGTA. The intracellular Ca²⁺ stores are depleted by 10 min incubation with 30 µM of CPA. Following addition of 150 µM of Mn²⁺ to the extracellular media the Fura-2 quenching is quantified as reduction of the Fura-2 fluorescent signal at isobestic λ_ex = 360 nM (F₃₆₀). The rate of Fura-2 Mn² quenching is calculated as the slope of the curve and determined for each cell before (phase a) and after (phase b) addition of DMSO (A), 300 nM of EVP4593 (B) or 300 nM EVP14808 (C). Fura-2 F₃₆₀ traces are shown for individual cells (gray thin lines). For each cell F₃₆₀ signal was normalized to F₃₆₀ signal at the time point of Mn²⁺ addition. The average trace is also shown (black thick line). (D) The changes in Fura-2 Mn²⁺ quench rates in YAC128 MSNs are plotted as b/a slope ratios for each compound tested at 300 nM concentration. The average b/a ratios for each compound are shown as mean ± S.E. (number of cells is indicated above each bar). ***, p<0.001 when compared to DMSO group.

Figure 4. Recordings of Isoc in SK-N-SH cells transfected with Htt-15Q and Htt-138Q

(A) The amplitude of Isoc currents recorded in whole-cell experiments are shown as a function of time after application of 1 µM Tg (indicated by arrow) to non-transfected SK-N-SH cells (Ctrl, green triangles) and to SK-N-SH cells transiently transfected with Htt-15Q (red squares) or Htt-138Q (black circles). The amplitude of Isoc currents for all groups of cells was measured every 10 seconds at −80 mV test potential. Data from representative experiments are shown. (B) The average current-voltage relationships recorded after full development of Isoc in non-transfected SK-N-SH cells (Ctrl, green trace) and SK-N-SH transiently transfected with Htt-15Q (red trace), or Htt-138Q (black trace). Each trace is an average based on a number of experiments as indicated in panel C. (C) The average Isoc amplitude in non-transfected SK-N-SH cells (Ctrl) and in SK-N-SH transfected with Htt-15Q or Htt-138Q constructs. For all groups of cells Isoc amplitude was determined at −80 mV test potential and plotted as mean ± SE (n = number of experiments). ***, p<0.001 when compared to Htt138Q-transfected cells.. (D) Isoc currents in non-transfected SK-N-SH cells (Ctrl) and SK-N-SH cells transfected with Htt-138Q plasmid are shown as a function of the time after 1 µM Tg application (indicated by arrow). The representative data obtained in the presence of DMSO (blue triangles for non-transfected cells and black circles for transfected cells), 300 nM EVP4593 (red squares for transfected cells and cyan rhombs for non-transfected cells) and 300 nM EVP14808 (green triangles for transfected cells) are shown. The amplitude of Isoc currents for all groups of cells was measured every 10 seconds at −80 mV test potential. The times of EVP4593, EVP14808 and DMSO applications are shown above the Isoc plot. (E) The average current-voltage relationships recorded after full development of Isoc in non-transfected SK-N-SH cells (Ctrl) and in SK-N-SH transfected with Htt-138Q in the presence of DMSO (blue trace for control cells and black trace for transfected cells), 300 nM EVP4593 (cyan trace for control cells and red trace for transfected cells), and 300 nM EVP14808 (green trace). Each trace is an average based on a number of experiments indicated in panel F. (F) The average Isoc amplitude in non-transfected (Ctrl) SK-N-SH cells and in SK-N-SH cells transfected with Htt-138 and recorded in the presence of DMSO, 300 nM EVP4593 or 300 nM EVP14808. For all groups of cells Isoc amplitude was determined at −80 mV test potential and plotted as mean ± SE (n = number of experiments). ***, p<0.001 when compared to Htt138Q-transfected cells in the presence of DMSO.

Figure 5. TRPC1 supports Isoc currents in SK-N-SH cells transfected with Htt-138Q

(A) The amplitude of Isoc currents recorded in whole-cell experiments is shown as a function of time after application of 1 µM Tg (indicated by arrow) to SK-N-SH cells transiently transfected with scrambled siRNA (Ctrl+ctrl siRNA, black circles), to SK-N-SH cells transiently transfected with TRPC1 plasmid (Ctrl TRPC1+, cyan rhombs), to SK-N-SH cells transiently transfected with TRPC1-RNAi (Ctrl TRPC1-, green pentagons), to SK-N-SH cells transiently co-transfected with Htt-138Q and scrambled siRNA (yellow squares) or to SK-N-SH cells co-transfected with Htt-138Q and TRPC1-RNAi (dark yellow triangles). (B) The amplitude of Isoc currents recorded in whole-cell experiments are shown as a function of time after application of 1 µM Tg (indicated by arrow) to non-transfected SK-N-SH cells treated with DMSO (Ctrl, red circles), to SK-N-SH cells transiently transfected with TRPC1 and treated with 300 nM EVP4593 (Ctrl TRPC1+, pink rhombs), to SK-N-SH cells transiently transfected with TRPC1-RNAi and treated with 300 nM EVP4593 (Ctrl TRPC1-, blue pentagons), to SK-N-SH cells co-transfected with Htt-138Q and TRPC1-RNAi and treated with DMSO (navy triangles) or to SK-N-SH cells co-transfected with Htt-138Q and TRPC1-RNAi and treated with 300 nM EVP4593 (purple squares). The times of EVP4593 and DMSO applications are shown above the Isoc plot. On panels A and B the amplitude of the Isoc currents for all groups of cells was measured every 10 seconds at −80 mV test potential. Data from representative experiments are shown for each group of cells. (C) The average current-voltage relationships recorded after full development of Isoc in non-transfected SK-N-SH cells (Ctrl, red trace), in SK-N-SH cells transfected with TRPC1-RNAi and treated with DMSO (yellow trace) or 300 nM EVP4593 (blue trace), in SK-N-SH cells transfected with Htt-138Q and treated with DMSO (black trace), ), in SK-N-SH cells transfected with TRPC1 and treated with DMSO (gray trace), in SK-N-SH cells co-transfected with Htt-138Q and TRPC1-RNAi and treated with DMSO (navy trace), and in SK-N-SH cells co-transfected with Htt-138Q and TRPC1-RNAi and treated with 300 nM EVP4593 (purple trace). Each trace is an average based on a number of experiments indicated in panel D. (D) The average Isoc amplitude in SK-N-SH cells transfected with scrambled siRNA, in non-transfected SK-N-SH cells treated with DMSO, in SK-N-SH cells transfected with TRPC1-RNAi with and without treatment with 300 nM EVP4593, in SK-N-SH cells transfected with TRPC1-cDNA with and without treatment with 300 nM EVP4593, in SK-N-SH cells co-transfected with Htt-138Q and scrambled siRNA, and in SK-N-SH cells co-transfected with Htt-138Q and TRPC1-RNAi untreated, treated with DMSO or treated with 300 nM EVP4593. For all groups of cells Isoc amplitude was determined at −80 mV test potential and plotted as mean ± SE (n = number of experiments). See also Figure S2.

Figure 6. Compounds from EVP4593 series or knockdown of TRPC1 protect YAC128 MSNs from glutamate-induced apoptosis

The fraction of TUNEL-positive nuclei is plotted against glutamate concentration for MSN from WT (open circle) and YAC128 (YAC, filled circles) mice. The results in the absence (black symbols) and presence (red symbols) of compounds or treatments are compared. The data are shown for (A) 0.03 µM of EVP4593; (B) 0.3 µM of EVP4593; (C) 3 µM of EVP14808; (D) 3 µM of EVP14809; (E) treatment with non-targeting RNAi reagent; (F) treatment with TRPC1 RNAi. In experiments shown on panels A-D the compounds were added 30 minutes prior to the application of glutamate. In experiments shown on panels E-F neurons were treated at DIV2–4. For each data point the fraction of TUNEL-positive nuclei is shown as mean ± SE (n = 6–8 microscopic fields, 100–300 MSN per field).