Two common disorders of the elderly are heart failure and Alzheimer disease (AD). Heart failure usually results from dilated cardiomyopathy (DCM). DCM of unknown cause in families has recently been shown to result from genetic disease, highlighting newly discovered disease mechanisms. AD is the most frequent neurodegenerative disease of older Americans. Familial AD is caused most commonly by presenilin 1 (PSEN1) or presenilin 2 (PSEN2) mutations, a discovery that has greatly advanced the field. The presenilins are also expressed in the heart and are critical to cardiac development. We hypothesized that mutations in presenilins may also be associated with DCM and that their discovery could provide new insight into the pathogenesis of DCM and heart failure. A total of 315 index patients with DCM were evaluated for sequence variation in PSEN1 and PSEN2. Families positive for mutations underwent additional clinical, genetic, and functional studies. A novel PSEN1 missense mutation (Asp333Gly) was identified in one family, and a single PSEN2 missense mutation (Ser130Leu) was found in two other families. Both mutations segregated with DCM and heart failure. The PSEN1 mutation was associated with complete penetrance and progressive disease that resulted in the necessity of cardiac transplantation or in death. The PSEN2 mutation showed partial penetrance, milder disease, and a more favorable prognosis. Calcium signaling was altered in cultured skin fibroblasts from PSEN1 and PSEN2 mutation carriers. These data indicate that PSEN1 and PSEN2 mutations are associated with DCM and heart failure and implicate novel mechanisms of myocardial disease.

Intercellular communication between γ-aminobutyric acid (GABA)ergic suprachiasmatic nucleus (SCN) neurons facilitates light-induced phase changes and synchronization of individual neural oscillators within the SCN network. We used ratiometric Ca2+ imaging techniques to record changes in the intracellular calcium concentration ([Ca2+]i) to study the role of GABA in interneuronal communication and the response of the SCN neuronal network to optic nerve stimulations that mimic entraining light signals. Stimulation of the retinohypothalamic tract (RHT) evoked divergent Ca2+ responses in neurons that varied regionally within the SCN with a pattern that correlated with those evoked by pharmacological GABA applications. GABAA and GABAB receptor agonists and antagonists were used to evaluate components of the GABA-induced changes in [Ca2+]i. Application of the GABAA receptor antagonist gabazine induced changes in baseline [Ca2+]i in a direction opposite to that evoked by GABA, and similarly altered the RHT stimulation-induced Ca2+ response. GABA application induced Ca2+ responses varied in time and region within the SCN network. The NKCC1 cotransporter blocker, bumetanide, and L-type calcium channel blocker, nimodipine, attenuated the GABA-induced rise of [Ca2+]i. These results suggest that physiological GABA induces opposing effects on [Ca2+]i based on the chloride equilibrium potential, and may play an important role in neuronal Ca2+ balance, synchronization and modulation of light input signaling in the SCN network.

Circadian oscillations in the suprachiasmatic nucleus (SCN) depend on transcriptional repression by Period (PER)1 and PER2 proteins within single cells and on vasoactive intestinal polypeptide (VIP) signaling between cells. Because VIP is released by SCN neurons in a circadian pattern, and, after photic stimulation, it has been suggested to play a role in the synchronization to environmental light cycles. It is not known, however, if or how VIP entrains circadian gene expression or behavior. Here, we tested candidate signaling pathways required for VIP-mediated entrainment of SCN rhythms. We found that single applications of VIP reset PER2 rhythms in a time- and dose-dependent manner that differed from light. Unlike VIP-mediated signaling in other cell types, simultaneous antagonism of adenylate cyclase and phospholipase C activities was required to block the VIP-induced phase shifts of SCN rhythms. Consistent with this, VIP rapidly increased intracellular cAMP in most SCN neurons. Critically, daily VIP treatment entrained PER2 rhythms to a predicted phase angle within several days, depending on the concentration of VIP and the interval between VIP applications. We conclude that VIP entrains circadian timing among SCN neurons through rapid and parallel changes in adenylate cyclase and phospholipase C activities.

Glutamate released from retinohypothalamic tract (RHT) synapses with suprachiasmatic nucleus (SCN) neurons induces phase changes

The ability of the neuroactive steroid pregnenolone sulfate to alter N-methyl-D-aspartate (NMDA) receptor-mediated elevations in intracellular Ca2+ ([Ca2+]i) was studied in cultured fetal rat hippocampal neurons using microspectrofluorimetry and the Ca2+ sensitive indicator fura-2. Pregnenolone sulfate (5-250 microM) caused a concentration-dependent and reversible potentiation of the rise (up to approximately 800%) in [Ca2+]i induced by NMDA. In contrast, the steroid failed to alter basal (unstimulated) [Ca2+]i or to modify the rise in [Ca2+]i that occurs when hippocampal neurons are depolarized by high K+ in the presence of the NMDA receptor antagonist CPP. These data suggest that the previously reported excitatory properties of pregnenolone sulfate may be due, in part, to an augmentation of the action of glutamic acid at the NMDA receptor.

The ability of NMDA to alter intracellular pH (pHJ was studied in fetal rat hippocampal neurons and glia using the pH-sensitive fluorescent indicator 2',7'-bis-(2-carboxyethyl)-S(and-6)carboxyfluorescein (BCECF). Brief exposure (60 set) of hippocampal neurons to NMDA (2.5-250 PM) results in a rapid, and in most cells reversible, reduction in pHi, with full recovery to baseline pH, values taking several minutes following removal of NMDA. In contrast, little or no change in pH, was observed in glial cells exposed to these same concentrations of NMDA. The NMDA-induced acidification of neurons was concentration and time dependent, with an EC

Hyperexcited states, including depolarization block and depolarized low amplitude membrane oscillations (DLAMOs), have been observed in neurons of the suprachiasmatic nuclei (SCN), the site of the central mammalian circadian (∼24-hour) clock. The causes and consequences of this hyperexcitation have not yet been determined. Here, we explore how individual ionic currents contribute to these hyperexcited states, and how hyperexcitation can then influence molecular circadian timekeeping within SCN neurons. We developed a mathematical model of the electrical activity of SCN neurons, and experimentally verified its prediction that DLAMOs depend on post-synaptic L-type calcium current. The model predicts that hyperexcited states cause high intracellular calcium concentrations, which could trigger transcription of clock genes. The model also predicts that circadian control of certain ionic currents can induce hyperexcited states. Putting it all together into an integrative model, we show how membrane potential and calcium concentration provide a fast feedback that can enhance rhythmicity of the intracellular circadian clock. This work puts forward a novel role for electrical activity in circadian timekeeping, and suggests that hyperexcited states provide a general mechanism for linking membrane electrical dynamics to transcription activation in the nucleus.

Cultured cerebellar granule neurons maintained in depolarizing concentrations of K + (25 mM) and then switched to physiological concentrations of K' (5 mM) undergo apoptosis. We now report that activation of specific G proteins robustly and bidirectionally affects apoptosis of cultured rat cerebellar granule neurons. Stimulation of GS with cholera toxin completely blocks apoptosis induced by nondepolarizing concentrations of K', whereas stimulation of Go/G, with the wasp venom peptide mastoparan induces apoptosis of cerebellar granule neurons even in high (depolarizing) concentrations of K ' . Moreover, pretreatment of cerebellar granule neurons with cholera toxin attenuates neuronal death induced by mastoparan . By contrast, pertussis toxin, cellpermeable analogues of cyclic AMP, and activators of protein kinase A do not affect apoptosis of cultured cerebellar granule neurons. These data suggest that G proteins may function as key switches for controlling the programmed death of mammalian neurons, especially in the developing CNS.