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Abstract The transcriptional repressor DREAM (downstream regulatory element antagonist modulator) is a multifunctional neuronal calcium sensor (NCS) that controls Ca2+ and protein homeostasis through gene regulation and protein-protein interactions. Downregulation of DREAM is part of an endogenous neuroprotective mechanism that improves ATF6 (activating transcription factor 6) processing, neuronal survival in the striatum, and motor coordination in R6/2 mice, a model of Huntington's disease (HD). Whether modulation of DREAM activity can also ameliorate cognition deficits in HD mice has not been studied. Moreover, it is not known whether DREAM downregulation in HD is unique, or also occurs for other NCS family members. Using the novel object recognition test, we show that chronic administration of the DREAM-binding molecule repaglinide, or induced DREAM haplodeficiency delays onset of cognitive impairment in R6/1 mice, another HD model. The mechanism involves a notable rise in the levels of transcriptionally active ATF6 protein in the hippocampus after repaglinide administration. In addition, we show that reduction in DREAM protein in the hippocampus of HD patients was not accompanied by downregulation of other NCS family members. Our results indicate that DREAM inhibition markedly improves ATF6 processing in the hippocampus and that it might contribute to a delay in memory decline in HD mice. The mechanism of neuroprotection through DREAM silencing in HD does not apply to other NCS family members. ; This work was funded by the Instituto de Salud Carlos III/CIBERNED (to JRN, BM and AR), the Madrid regional government/Neurodegmodels (to JRN), and SAF2014–53412-R and SAF2017–89554-R (AEI-FEDER, EU) (to JRN).

DREAM, a neuronal calcium sensor protein, has multiple cellular roles including the regulation of Ca2+ and protein homeostasis. We recently showed that reduced DREAM expression or blockade of DREAM activity by repaglinide is neuroprotective in Huntington's disease (HD). Here we used structure-based drug design to guide the identification of IQM-PC330, which was more potent and had longer lasting effects than repaglinide to inhibit DREAM in cellular and in vivo HD models. We disclosed and validated an unexplored ligand binding site, showing Tyr118 and Tyr130 as critical residues for binding and modulation of DREAM activity. IQM-PC330 binding de-repressed c-fos gene expression, silenced the DREAM effect on KV4.3 channel gating and blocked the ATF6/DREAM interaction. Our results validate DREAM as a valuable target and propose more effective molecules for HD treatment ; This work was funded by the Spanish Ministery of Economy, Industry and Competitivity (AEI-FEDER, EU grants): BFU2015-67284-R (to M.G.R., R.H.), SAF2017-89554-R (to J.R.N.), SAF2016-75021-R (to C.V.) and SAF2015-66275-C2-2-R (to M.M.M.); the Instituto de Salud Carlos III CIBERNED and CIBERCV programs (to J.R.N. and to C.V., respectively) and the Madrid regional government/Neurodegmodels (to J.R.N.); CSIC grant PIE201880E109 (to M.G.R., R.H. and M.M.M.) and PIE201820E104 (to C.V.)

DREAM, a neuronal calcium sensor protein, has multiple cellular roles including the regulation of Ca2+ and protein homeostasis. We recently showed that reduced DREAM expression or blockade of DREAM activity by repaglinide is neuroprotective in Huntington's disease (HD). Here we used structure-based drug design to guide the identification of IQM-PC330, which was more potent and had longer lasting effects than repaglinide to inhibit DREAM in cellular and in vivo HD models. We disclosed and validated an unexplored ligand binding site, showing Tyr118 and Tyr130 as critical residues for binding and modulation of DREAM activity. IQM-PC330 binding de-repressed c-fos gene expression, silenced the DREAM effect on KV4.3 channel gating and blocked the ATF6/DREAM interaction. Our results validate DREAM as a valuable target and propose more effective molecules for HD treatment. ; PC was the recipient of a postgraduate FPI fellowship from the Spanish Ministry de Economy, Industry and Competitivity (MINECO). P.C. also thanks the Spanish Society of Therapeutic Chemistry (SEQT) for a Jassen-Cilag Award for young researchers (XVIII Edition, 2016). T.G. holds a Ramón y Cajal contract. D.A.P. holds a CSIC contract. This work was funded by the Spanish Ministery of Economy, Industry and Competitivity (AEI-FEDER, EU grants): BFU2015-67284-R (to M.G.R., R.H.), SAF2017-89554-R (to J.R.N.), SAF2016-75021-R (to C.V.) and SAF2015-66275-C2-2-R (to M.M.M.); the Instituto de Salud Carlos III CIBERNED and CIBERCV programs (to J.R.N. and to C.V., respectively) and the Madrid regional government/Neurodegmodels (to J.R.N.); CSIC grant PIE201880E109 (to M.G.R., R.H. and M.M.M.) and PIE201820E104 (to C.V.). ; Peer reviewed

Downstream Regulatory Element Antagonist Modulator (DREAM)/KChIP3/calsenilin is a neuronal calcium sensor (NCS) with multiple functions, including the regulation of A-type outward potassium currents (I A). This effect is mediated by the interaction between DREAM and KV4 potassium channels and it has been shown that small molecules that bind to DREAM modify channel function. A-type outward potassium current (I A) is responsible of the fast repolarization of neuron action potentials and frequency of firing. Using surface plasmon resonance (SPR) assays and electrophysiological recordings of KV4.3/DREAM channels, we have identified IQM-266 as a DREAM ligand. IQM-266 inhibited the KV4.3/DREAM current in a concentration-, voltage-, and time-dependent-manner. By decreasing the peak current and slowing the inactivation kinetics, IQM-266 led to an increase in the transmembrane charge ( QKV4.3/DREAM ) at a certain range of concentrations. The slowing of the recovery process and the increase of the inactivation from the closed-state inactivation degree are consistent with a preferential binding of IQM-266 to a pre-activated closed state of KV4.3/DREAM channels. Finally, in rat dorsal root ganglion neurons, IQM-266 inhibited the peak amplitude and slowed the inactivation of I A. Overall, the results presented here identify IQM-266 as a new chemical tool that might allow a better understanding of DREAM physiological role as well as modulation of neuronal I A in pathological processes. ; PC was the recipient of a postgraduate FPI fellowship from the Spanish Ministry of Economy, Industry and Competitivity (MINECO). This work was funded by the Spanish Ministry of Economy, Industry and Competitivity (Ministerio de Economía y Competitividad; AEI-FEDER, EU grants): SAF2012-32209 and BFU2015-67284-R (to MG-R), SAF2014-53412-R and SAF2017- 89554-R (to JN), SAF2013-45800-R, SAF2016-75021-R (to CV) and SAF2015-66275-C2-2-R (to MM-M); Universidad Complutense de Madrid (UCM) grant: PR75/18-21593 (to AA); the Instituto de Salud Carlos III CIBERNED and CIBERCV programs (to JN and to CV, respectively) and the Madrid regional government/Neurodegmodels (to JN); Consejo Superior de Investigaciones Científicas (CSIC) grants: PIE 201820E104 (to CV) and 201880E109 (to MG-R and MM-M). ; We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI). ; Peer reviewed