For the last two decades, most efforts on new drug development to treat Alzheimer's disease have been focused to inhibit the synthesis of amyloid beta (Aβ), to prevent Aβ deposition, or to clear up Aβ plaques from the brain of Alzheimer's disease (AD) patients. Other pathogenic mechanisms such as the hyperphosphorylation of the microtubular tau protein (that forms neurofibrillary tangles) have also been addressed as, for instance, with inhibitors of the enzyme glycogen synthase-3 kinase beta (GSK3β). However, in spite of their proven efficacy in animal models of AD, all these compounds have so far failed in clinical trials done in AD patients. It seems therefore desirable to explore new concepts and strategies in the field of drug development for AD. We analyze here our hypothesis that a trifunctional chemical entity acting on the L subtype of voltage-dependent Ca 2+ channels (VDCCs) and on the mitochondrial Na + /Ca 2+ exchanger (MNCX), and having additional antioxidant properties, may efficiently delay or stop the death of vulnerable neurons in the brain of AD patients. In recent years, evidence has accumulated indicating that enhanced neuronal Ca 2+ cycling (NCC) and futile mitochondrial Ca 2+ cycling (MCC) are central stage in activating calpain and calcineurin, as well as the intrinsic mitochondrial pathway for apoptosis, leading to death of vulnerable neurons. An additional contributing factor to neuronal death is the excess free radical production linked to distortion of Ca 2+ homeostasis. We propose that an hybrid compound containing a dihydropyridine moiety (to block L channels and mitigate Ca 2+ entry) and a benzothiazepine moiety (to block the MNCX and slow down the rate of Ca 2+ efflux from the mitochondrial matrix into the cytosol), as well as a polyphenol moiety (to sequester excess free radicals) could break down the pathological enhanced NCC and MCC, thus delaying the initiation of apoptosis and the death of vulnerable neurons. In so doing, such a trifunctional compound could eventually become a neuroprotective medicine capable of delaying disease progression in AD patients.

Chondroitin sulfate (CS) proteoglycans (CSPGs) are the most abundant PGs of the brain extracellular matrix (ECM). Free CS could be released during ECM degradation and exert physiological functions; thus, we aimed to investigate the effects of CS on voltage-and current-clamped rat embryo hippocampal neurons in primary cultures. We found that CS elicited a whole-cell Na + -dependent inward current (I CS ) that produced drastic cell depolarization, and a cytosolic calcium transient ([Ca 2+ ] c ). Those effects were similar to those elicited by a-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) and kainate, were completely blocked by NBQX and CNQX, were partially blocked by GYKI, and were unaffected by MK801 and D-APV. Furthermore, I CS and AMPA currents were similarly potentiated by cyclothiazide, a positive allosteric modulator of AMPA receptors. Because CSPGs have been attributed Ca 2+ -dependent roles, such as neural network development, axon pathfinding, plasticity and regeneration after CNS injury, CS action after ECM degradation could be contributing to the mediation of these effects through its interaction with AMPA and kainate receptors.

Our studies suggest that acute hypoxia suppresses I in rapidly inactivating cell population by a mechanism involving Ca-dependent inactivation, while compromised mitochondrial Ca uptake seems also to contribute to I suppression in slowly inactivating cell population. Proximity of cellular Ca pools to sarcolemmal Ca channels may contribute to the variability of inactivation kinetics of I in the two cell populations, while acidosis suppression of I appears mediated by proton-induced block of the calcium channel.

Aims:The effects of acute (100s) hypoxia and/or acidosis on Ca 2+ signaling parameters of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) are explored here for the first time.Methods and Results: 1) hiPSC-CMs express two cell populations: rapidly-inactivating I Ca myocytes (τ i <40ms, in 4-5 day cultures) and slowly-inactivating I Ca (τ i ≥ 40ms, in 6-8 day cultures). 2) Hypoxia suppressed I Ca by 10-20% in rapidly-and 40-55% in slowly-inactivating I Ca cells. 3) Isoproterenol enhanced I Ca in hiPSC-CMs, but either enhanced or did not alter the hypoxic suppression. 4) Hypoxia had no differential suppressive effects in the two cell-types when Ba 2+ was the charge carrier through the calcium channels, implicating Ca 2+ -dependent inactivation in O 2 sensing. 5) Acidosis suppressed I Ca by ~35% and ~25% in rapidly and slowly inactivating I Ca cells, respectively. 6) Hypoxia and acidosis suppressive effects on Ca-transients depended on whether global or RyR2-microdomain were measured: with acidosis suppression was ~25% in global and ~37% in RyR2 Ca 2+ -microdomains in either cell type, whereas with hypoxia suppression was ~20% and ~25% respectively in global and RyR2-microdomaine in rapidly and ~35% and ~45% respectively in global and RyR2-microdomaine in slowly-inactivating cells. Conclusions:Variability in I Ca inactivation kinetics rather than cellular ancestry seems to underlie the action potential morphology differences generally attributed to mixed atrial and ventricular cell populations in hiPSC-CMs cultures. The differential hypoxic regulation of Ca 2+signaling in the two-cell types arises from differential Ca 2+ -dependent inactivation of the Ca 2+channel caused by proximity of Ca 2+ -release stores to the Ca 2+ channels.

From experiments performed at room temperature, we know that the buffering of Ca2+ by mitochondria contributes to the shaping of the bulk cytosolic calcium transient ([Ca2+]c) and secretion transients of chromaffin cells stimulated with depolarizing pulses. We also know that the mitochondrial Ca2+ transporters and the release of catecholamine are faster at 37°C with respect to room temperature. Therefore, we planned this investigation to gain further insight into the contribution of mitochondrial Ca2+ buffering to the shaping of [Ca2+]c and catecholamine release transients, using some novel experimental conditions that have not been yet explored namely: (1) perifusion of bovine chromaffin cells (BCCs) with saline at 37°C and their repeated challenging with the physiological neurotransmitter acetylcholine (ACh); (2) separate blockade of mitochondrial Ca2+ uniporter (mCUP) with Ru360 or the mitochondrial Na+/Ca2+ exchanger (mNCX) with CGP37157; (3) full blockade of the mitochondrial Ca2+ cycling (mCC) by the simultaneous inhibition of the mCUP and the mNCX. Ru360 caused a pronounced delay of [Ca2+]c clearance and augmented secretion. In contrast, CGP37157 only caused a tiny delay of [Ca2+]c clearance and a mild decrease in secretion. The mCC resulting in continued Ca2+ uptake and its release back into the cytosol was interrupted by combined Ru360 + CGP37157 (Ru/CGP), the protonophore carbonyl cyanide‐p‐trifluoromethoxyphenylhydrazone, or combined oligomycin + rotenone (O/R); these three treatments caused a mild but sustained elevation of basal [Ca2+]c that, however, was not accompanied by a parallel increase in basal secretion. Nevertheless, all treatments caused a pronounced augmentation of ACh‐induced secretion, with minor changes of the ACh‐induced [Ca2+]c transients. Combined Ru/CGP did not alter the resting membrane potential in current‐clamped cells. Additionally, Ru/CGP did not increase basal [Ca2+]c near subplasmalemmal sites and caused a mild decrease in the size of the readily releasable vesicle pool. Our results provide new functional features in support of the view that in BCCs there are two subpopulations of mitochondria, M1 underneath the plasmalemma nearby exocytotic sites and M2 at the core cell nearby vesicle transport sites. While M1 serves to shape the ACh‐elicited exocytotic response through its efficient Ca2+ removal by the mCUP, M2 shapes the lower [Ca2+]c elevations required for new vesicle supply to the exocytotic machinery, from the large reserve vesicle pool at the cell core. The mCUP of the M1 pool seems to play a more prominent role in controlling the ACh responses, in comparison with the mNCX.