Novel Vaccine against Pathological Pyroglutamate-Modified Amyloid Beta for Prevention of Alzheimer's Disease.

Post-translationally modified N-terminally truncated amyloid beta peptide with a cyclized form of glutamate at position 3 (pE3Aß) is a highly pathogenic molecule with increased neurotoxicity and propensity for aggregation. In the brains of Alzheimer's Disease (AD) cases, pE3Aß represents a major constituent of the amyloid plaque. The data show that pE3Aß formation is increased at early pre-symptomatic disease stages, while tau phosphorylation and aggregation mostly occur at later stages of the disease. This suggests that pE3Aß accumulation may be an early event in the disease pathogenesis and can be prophylactically targeted to prevent the onset of AD. The vaccine (AV-1986R/A) was generated by chemically conjugating the pE3Aß3-11 fragment to our universal immunogenic vaccine platform MultiTEP, then formulated in AdvaxCpG adjuvant. AV-1986R/A showed high immunogenicity and selectivity, with endpoint titers in the range of 105-106 against pE3Aß and 103-104 against the full-sized peptide in the 5XFAD AD mouse model. The vaccination showed efficient clearance of the pathology, including non-pyroglutamate-modified plaques, from the mice brains. AV-1986R/A is a novel promising candidate for the immunoprevention of AD. It is the first late preclinical candidate which selectively targets a pathology-specific form of amyloid with minimal immunoreactivity against the full-size peptide. Successful translation into clinic may offer a new avenue for the prevention of AD via vaccination of cognitively unimpaired individuals at risk of disease.

BKCa channel regulates calcium oscillations induced by alpha-2-macroglobulin in human myometrial smooth muscle cells.

The large-conductance, voltage-gated, calcium (Ca(2+))-activated potassium channel (BKCa) plays an important role in regulating Ca(2+)signaling and is implicated in the maintenance of uterine quiescence during pregnancy. We used immunopurification and mass spectrometry to identify proteins that interact with BKCain myometrium samples from term pregnant (≥37 wk gestation) women. From this screen, we identified alpha-2-macroglobulin (α2M). We then used immunoprecipitation followed by immunoblot and the proximity ligation assay to confirm the interaction between BKCaand both α2M and its receptor, low-density lipoprotein receptor-related protein 1 (LRP1), in cultured primary human myometrial smooth muscle cells (hMSMCs). Single-channel electrophysiological recordings in the cell-attached configuration demonstrated that activated α2M (α2M*) increased the open probability of BKCain an oscillatory pattern in hMSMCs. Furthermore, α2M* caused intracellular levels of Ca(2+)to oscillate in oxytocin-primed hMSMCs. The initiation of oscillations required an interaction between α2M* and LRP1. By using Ca(2+)-free medium and inhibitors of various Ca(2+)signaling pathways, we demonstrated that the oscillations required entry of extracellular Ca(2+)through store-operated Ca(2+)channels. Finally, we found that the specific BKCablocker paxilline inhibited the oscillations, whereas the channel opener NS11021 increased the rate of these oscillations. These data demonstrate that α2M* and LRP1 modulate the BKCachannel in human myometrium and that BKCaand its immunomodulatory interacting partners regulate Ca(2+)dynamics in hMSMCs during pregnancy.

Domain organization of the ATP-sensitive potassium channel complex examined by fluorescence resonance energy transfer.

K(ATP) channels link cell metabolism to excitability in many cells. They are formed as tetramers of Kir6.2 subunits, each associated with a SUR1 subunit. We used mutant GFP-based FRET to assess domain organization in channel complexes. Full-length Kir6.2 subunits were linked to YFP or cyan fluorescent protein (CFP) at N or C termini, and all such constructs, including double-tagged YFP-Kir6.2-CFP (Y6.2C), formed functional K(ATP) channels. In intact COSm6 cells, background emission of YFP excited by 430-nm light was ∼6%, but the Y6.2C construct expressed alone exhibited an apparent FRET efficiency of ∼25%, confirmed by trypsin digestion, with or without SUR1 co-expression. Similar FRET efficiency was detected in mixtures of CFP- and YFP-tagged full-length Kir6.2 subunits and transmembrane domain only constructs, when tagged at the C termini but not at the N termini. The FRET-reported Kir6.2 tetramer domain organization was qualitatively consistent with Kir channel crystal structures: C termini and M2 domains are centrally located relative to N termini and M1 domains, respectively. Additional FRET analyses were performed on cells in which tagged full-length Kir6.2 and tagged SUR1 constructs were co-expressed. These analyses further revealed that 1) NBD1 of SUR1 is closer to the C terminus of Kir6.2 than to the N terminus; 2) the Kir6.2 cytoplasmic domain is not essential for complexation with SUR1; and 3) the N-terminal half of SUR1 can complex with itself in the absence of either the C-terminal half or Kir6.2.

Type I phosphotidylinosotol 4-phosphate 5-kinase γ regulates osteoclasts in a bifunctional manner.

Type 1 phosphotidylinosotol-4 phosphate 5 kinase γ (PIP5KIγ) is central to generation of phosphotidylinosotol (4,5)P(2) (PI(4,5)P(2)). PIP5KIγ also participates in cytoskeletal organization by delivering talin to integrins, thereby enhancing their ligand binding capacity. As the cytoskeleton is pivotal to osteoclast function, we hypothesized that absence of PIP5KIγ would compromise their resorptive capacity. Absence of the kinase diminishes PI(4,5) abundance and desensitizes precursors to RANK ligand-stimulated differentiation. Thus, PIP5KIγ(-/-) osteoclasts are reduced in number in vitro and confirm physiological relevance in vivo. Despite reduced numbers, PIP5KIγ(-/-) osteoclasts surprisingly have normal cytoskeletons and effectively resorb bone. PIP5KIγ overexpression, which increases PI(4,5)P(2), also delays osteoclast differentiation and reduces cell number but in contrast to cells lacking the kinase, its excess disrupts the cytoskeleton. The cytoskeleton-disruptive effects of excess PIP5KIγ reflect its kinase activity and are independent of talin recognition. The combined arrested differentiation and disorganized cytoskeleton of PIP5KIγ-transduced osteoclasts compromises bone resorption. Thus, optimal PIP5KIγ and PI(4,5)P(2) expression, by osteoclasts, are essential for skeletal homeostasis.

Maintaining energy homeostasis is an essential component of Wld(S)-mediated axon protection.

Wld(S) mutation protects axons from degeneration in diverse experimental models of neurological disorders, suggesting that the mutation might act on a key step shared by different axon degeneration pathways. Here we test the hypothesis that Wld(S) protects axons by preventing energy deficiency commonly encountered in many diseases. We subjected compartmentally cultured, mouse cortical axons to energy deprivation with 6mM azide and zero glucose. In wild-type (WT) culture, the treatment, which reduced axon ATP level ([ATP]axon) by 65%, caused immediate axon depolarization followed by gradual free calcium accumulation and subsequent irreversible axon damage. The calcium accumulation resulted from calcium influx partially via L-type voltage-gated calcium channel (L-VGCC). Blocking L-VGCC with nimodipine reduced calcium accumulation and protected axons. Without altering baseline [ATP]axon, the presence of Wld(S) mutation significantly reduced the axon ATP loss and depolarization, restrained the subsequent calcium accumulation, and protected axons against energy deprivation. Wld(S) neurons possessed higher than normal nicotinamide mononucleotide adenylyltransferase (NMNAT) activity. The intrinsic Wld(S) NMNAT activity was required for the Wld(S)-mediated energy preservation and axon protection during but not prior to energy deprivation. NMNAT catalyzes the reversible reaction that produces nicotinamide adenine dinucleotide (NAD) from nicotinamide mononucleotide (NMN). Interestingly, preventing the production of NAD from NMN with FK866 increased [ATP]axon and protected axons from energy deprivation. These results indicate that the Wld(S) mutation depends on its intrinsic Wld(S) NMNAT activity and the subsequent increase in axon ATP but not NAD to protect axons, implicating a novel role of Wld(S) NMNAT in axon bioenergetics and protection.

Apolipoprotein M Attenuates Anthracycline Cardiotoxicity and Lysosomal Injury.

Apolipoprotein M (ApoM) binds sphingosine-1-phosphate (S1P) and is inversely associated with mortality in human heart failure (HF). Here, we show that anthracyclines such as doxorubicin (Dox) reduce circulating ApoM in mice and humans, that ApoM is inversely associated with mortality in patients with anthracycline-induced heart failure, and ApoM heterozygosity in mice increases Dox-induced mortality. In the setting of Dox stress, our studies suggest ApoM can help sustain myocardial autophagic flux in a post-transcriptional manner, attenuate Dox cardiotoxicity, and prevent lysosomal injury.

Calcium-independent inhibitory G-protein signaling induces persistent presynaptic muting of hippocampal synapses.

Adaptive forms of synaptic plasticity that reduce excitatory synaptic transmission in response to prolonged increases in neuronal activity may prevent runaway positive feedback in neuronal circuits. In hippocampal neurons, for example, glutamatergic presynaptic terminals are selectively silenced, creating "mute" synapses, after periods of increased neuronal activity or sustained depolarization. Previous work suggests that cAMP-dependent and proteasome-dependent mechanisms participate in silencing induction by depolarization, but upstream activators are unknown. We, therefore, tested the role of calcium and G-protein signaling in silencing induction in cultured hippocampal neurons. We found that silencing induction by depolarization was not dependent on rises in intracellular calcium, from either extracellular or intracellular sources. Silencing was, however, pertussis toxin sensitive, which suggests that inhibitory G-proteins are recruited. Surprisingly, blocking four common inhibitory G-protein-coupled receptors (GPCRs) (adenosine A(1) receptors, GABA(B) receptors, metabotropic glutamate receptors, and CB(1) cannabinoid receptors) and one ionotropic receptor with metabotropic properties (kainate receptors) failed to prevent depolarization-induced silencing. Activating a subset of these GPCRs (A(1) and GABA(B)) with agonist application induced silencing, however, which supports the hypothesis that G-protein activation is a critical step in silencing. Overall, our results suggest that depolarization activates silencing through an atypical GPCR or through receptor-independent G-protein activation. GPCR agonist-induced silencing exhibited dependence on the ubiquitin-proteasome system, as was shown previously for depolarization-induced silencing, implicating the degradation of vital synaptic proteins in silencing by GPCR activation. These data suggest that presynaptic muting in hippocampal neurons uses a G-protein-dependent but calcium-independent mechanism to depress presynaptic vesicle release.

Cardiac specific ATP-sensitive K+ channel (KATP) overexpression results in embryonic lethality.

Transgenic mice overexpressing SUR1 and gain of function Kir6.2[∆N30, K185Q] K(ATP) channel subunits, under cardiac α-myosin heavy chain (αMHC) promoter control, demonstrate arrhythmia susceptibility and premature death. Pregnant mice, crossed to carry double transgenic progeny, which harbor high levels of both overexpressed subunits, exhibit the most extreme phenotype and do not deliver any double transgenic pups. To explore the fetal lethality and embryonic phenotype that result from K(ATP) overexpression, wild type (WT) and K(ATP) overexpressing embryonic cardiomyocytes were isolated, cultured and voltage-clamped using whole cell and excised patch clamp techniques. Whole mount embryonic imaging, Hematoxylin and Eosin (H&E) and α smooth muscle actin (αSMA) immunostaining were used to assess anatomy, histology and cardiac development in K(ATP) overexpressing and WT embryos. Double transgenic embryos developed in utero heart failure and 100% embryonic lethality by 11.5 days post conception (dpc). K(ATP) currents were detectable in both WT and K(ATP)-overexpressing embryonic cardiomyocytes, starting at early stages of cardiac development (9.5 dpc). In contrast to adult cardiomyocytes, WT and K(ATP)-overexpressing embryonic cardiomyocytes exhibit basal and spontaneous K(ATP) current, implying that these channels may be open and active under physiological conditions. At 9.5 dpc, live double transgenic embryos demonstrated normal looping pattern, although all cardiac structures were collapsed, probably representing failed, non-contractile chambers. In conclusion, K(ATP) channels are present and active in embryonic myocytes, and overexpression causes in utero heart failure and results in embryonic lethality. These results suggest that the K(ATP) channel may have an important physiological role during early cardiac development.

Xenin-25 increases cytosolic free calcium levels and acetylcholine release from a subset of myenteric neurons.

Xenin-25 (Xen) is a 25 amino acid neurotensin-related peptide reportedly produced with glucose-dependent insulinotropic polypeptide (GIP) by a subset of K cells in the proximal gut. We previously showed exogenously administered Xen, with GIP but not alone, increases insulin secretion in humans and mice. In mice, this effect is indirectly mediated via a central nervous system-independent cholinergic relay in the periphery. Xen also delays gastric emptying, reduces food intake, induces gall bladder contractions, and increases gut motility and secretion from the exocrine pancreas, suggesting that some effects of Xen could be mediated by myenteric neurons (MENs). To determine whether Xen activates these neurons, MENs were isolated from guinea pig proximal small intestines. Cells expressed neuronal markers and exhibited typical neuron-like morphology with extensive outgrowths emanating from cell bodies. Cytosolic free Ca(2+) levels ([Ca(2+)](i)) were measured using Fura-2. ATP/UTP, KCl, and forskolin increased [Ca(2+)](i) in 99.6%, 92%, and 23% of the MENs imaged, respectively, indicating that they are functional and activated by nucleotide receptor signaling, direct depolarization, and cAMP. [Ca(2+)](i) increased in only 12.7% of MENs treated with Xen. This rise was blocked by pretreatment with EGTA, diazoxide, SR48692, and neurotensin. Thus the Xen-mediated increase in [Ca(2+)](i) involves influx of extracellular Ca(2+) and activation of neurotensin receptor-1 (NTSR1). Xen also increased acetylcholine release from MENs. Amylin, produced by ß-and enteroendocrine cells, delays gastric emptying and increased [Ca(2+)](i) almost exclusively in Xen-responsive MENs. Immunohistochemistry demonstrated NTSR1 expression in human duodenal MENs. Thus myenteric rather than central neurons could mediate some effects of Xen and amylin.

Missense mutations in Otopetrin 1 affect subcellular localization and inhibition of purinergic signaling in vestibular supporting cells.

Otopetrin 1 (Otop1) encodes a protein that is essential for the development of otoconia. Otoconia are the extracellular calcium carbonate containing crystals that are important for vestibular mechanosensory transduction of linear motion and gravity. There are two mutant alleles of Otop1 in mice, titled (tlt) and mergulhador (mlh), which result in non-syndromic otoconia agenesis and a consequent balance defect. Biochemically, Otop1 has been shown to modulate purinergic control of intracellular calcium in vestibular supporting cells, which could be one of the mechanisms by which Otop1 participates in the mineralization of otoconia. To understand how tlt and mlh mutations affect the biochemical function of Otop1, we examined the purinergic response of COS7 cells expressing mutant Otop1 proteins, and dissociated sensory epithelial cells from tlt and mlh mice. We also examined the subcellular localization of Otop1 in whole sensory epithelia from tlt and mlh mice. Here we show that tlt and mlh mutations uncouple Otop1 from inhibition of P2Y receptor function. Although the in vitro biochemical function of the Otop1 mutant proteins is normal, in vivo they behave as null alleles. We show that in supporting cells the apical membrane localization of the mutant Otop1 proteins is lost. These data suggest that the tlt and mlh mutations primarily affect the localization of Otop1, which interferes with its ability to interact with other proteins that are important for its cellular and biochemical function.

TRAF2, an Innate Immune Sensor, Reciprocally Regulates Mitophagy and Inflammation to Maintain Cardiac Myocyte Homeostasis.

Mitochondria are essential for cardiac myocyte function, but damaged mitochondria trigger cardiac myocyte death. Although mitophagy, a lysosomal degradative pathway to remove damaged mitochondria, is robustly active in cardiac myocytes in the unstressed heart, its mechanisms and physiological role remain poorly defined. We discovered a critical role for TRAF2, an innate immunity effector protein with E3 ubiquitin ligase activity, in facilitating physiological cardiac myocyte mitophagy in the adult heart, to prevent inflammation and cell death, and maintain myocardial homeostasis.

Characterization and evaluation of two novel fluorescent sigma-2 receptor ligands as proliferation probes.

We synthesized and characterized two novel fluorescent sigma-2 receptor selective ligands, SW120 and SW116, and evaluated these ligands as potential probes for imaging cell proliferation. Both ligands are highly selective for sigma-2 receptors versus sigma-1 receptors. SW120 and SW116 were internalized into MDA-MB-435 cells, and 50% of the maximum fluorescent intensity was reached in 11 and 24 minutes, respectively. In vitro studies showed that 50% of SW120 or SW116 washed out of cells in 1 hour. The internalization of SW120 was reduced ≈30% by phenylarsine oxide, an inhibitor of endocytosis, suggesting that sigma-2 ligands are internalized, in part, by an endocytotic pathway. Subcellular localization studies using confocal and two-photon microscopy showed that SW120 and SW116 partially colocalized with fluorescent markers of mitochondria, endoplasmic reticulum, lysosomes, and the plasma membrane, suggesting that sigma-2 receptors localized to the cytoplasmic organelles and plasma membrane. SW120 did not colocalize with the nuclear dye 4',6-diamidino-2-phenylindole. In vivo studies showed that the uptake of SW120 in solid tumors and peripheral blood mononuclear cells of mice positively correlated with the expression level of the cell proliferation marker Ki-67, suggesting that sigma-2 fluorescent probes may be used to image cell proliferation in mice.

Regulation of cellular calcium in vestibular supporting cells by otopetrin 1.

Otopetrin 1 (OTOP1) is a multitransmembrane domain protein, which is essential for mineralization of otoconia, the calcium carbonate biominerals required for vestibular function, and the normal sensation of gravity. The mechanism driving mineralization of otoconia is poorly understood, but it has been proposed that supporting cells and a mechanism to maintain high concentrations of calcium are critical. Using Otop1 knockout mice and a utricular epithelial organ culture system, we show that OTOP1 is expressed at the apex of supporting cells and functions to increase cytosolic calcium in response to purinergic agonists, such as adenosine 5'-triphosphate (ATP). This is achieved by blocking mobilization of calcium from intracellular stores in an extracellular calcium-dependent manner and by mediating influx of extracellular calcium. These data support a model in which OTOP1 acts as a sensor of the extracellular calcium concentration near supporting cells and responds to ATP in the endolymph to increase intracellular calcium levels during otoconia mineralization.

The glycine transport inhibitor sarcosine is an NMDA receptor co-agonist that differs from glycine.

Sarcosine is an amino acid involved in one-carbon metabolism and a promising therapy for schizophrenia because it enhances NMDA receptor (NMDAR) function by inhibiting glycine uptake. The structural similarity between sarcosine and glycine led us to hypothesize that sarcosine is also an agonist like glycine. We examined this possibility using whole-cell recordings from cultured embryonic mouse hippocampal neurons. We found that sarcosine is an NMDAR co-agonist at the glycine binding site. However, sarcosine differed from glycine because less NMDAR desensitization occurred with sarcosine than with glycine as the co-agonist. This finding led us to examine whether the physiological effects of NMDAR activation with these two co-agonists are the same. The difference in desensitization probably accounts for rises in intracellular Ca(2+), as assessed by the fluorescent indicator fura-FF, being larger when NMDAR activation occurred with sarcosine than with glycine. In addition, Ca(2+)-activated K(+) currents following NMDAR activation were larger with sarcosine than with glycine. Compared to glycine, NMDAR-mediated autaptic currents decayed faster with sarcosine suggesting that NMDAR deactivation also differs with these two co-agonists. Despite these differences, NMDAR-dependent neuronal death as assessed by propidium iodide was similar with both co-agonists. The same was true for neuronal bursting. Thus, sarcosine may enhance NMDAR function by more than one mechanism and may have different effects from other NMDAR co-agonists.

Cellular calcium deficiency plays a role in neuronal death caused by proteasome inhibitors.

Cytosolic Ca(2+) concentration ([Ca(2+)](i)) is reduced in cultured neurons undergoing neuronal death caused by inhibitors of the ubiquitin proteasome system. Activation of calcium entry via voltage-gated Ca(2+) channels restores cytosolic Ca(2+) levels and reduces this neuronal death (Snider et al. 2002). We now show that this reduction in [Ca(2+)](i) is transient and occurs early in the cell death process, before activation of caspase 3. Agents that increase Ca(2+) influx such as activation of voltage-gated Ca(2+) channels or stimulation of Ca(2+) entry via the plasma membrane Na-Ca exchanger attenuate neuronal death only if applied early in the cell death process. Cultures treated with proteasome inhibitors had reduced current density for voltage-gated Ca(2+) channels and a less robust increase in [Ca(2+)](i) after depolarization. Levels of endoplasmic reticulum Ca(2+) were reduced and capacitative Ca(2+) entry was impaired early in the cell death process. Mitochondrial Ca(2+) was slightly increased. Preventing the transfer of Ca(2+) from mitochondria to cytosol increased neuronal vulnerability to this death while blockade of mitochondrial Ca(2+) uptake via the uniporter had no effect. Programmed cell death induced by proteasome inhibition may be caused in part by an early reduction in cytosolic and endoplasmic reticulum Ca(2+,) possibly mediated by dysfunction of voltage-gated Ca(2+) channels. These findings may have implications for the treatment of disorders associated with protein misfolding in which proteasome impairment and programmed cell death may occur.

Beta-cell excitability and excitability-driven diabetes in adult Zebrafish islets.

Islet ß-cell membrane excitability is a well-established regulator of mammalian insulin secretion, and defects in ß-cell excitability are linked to multiple forms of diabetes. Evolutionary conservation of islet excitability in lower organisms is largely unexplored. Here we show that adult zebrafish islet calcium levels rise in response to elevated extracellular [glucose], with similar concentration-response relationship to mammalian ß-cells. However, zebrafish islet calcium transients are nor well coupled, with a shallower glucose-dependence of cytoplasmic calcium concentration. We have also generated transgenic zebrafish that conditionally express gain-of-function mutations in ATP-sensitive K+ channels (KATP -GOF) in ß-cells. Following induction, these fish become profoundly diabetic, paralleling features of mammalian diabetes resulting from equivalent mutations. KATP -GOF fish become severely hyperglycemic, with slowed growth, and their islets lose glucose-induced calcium responses. These results indicate that, although lacking tight cell-cell coupling of intracellular Ca2+ , adult zebrafish islets recapitulate similar excitability-driven ß-cell glucose responsiveness to those in mammals, and exhibit profound susceptibility to diabetes as a result of inexcitability. While illustrating evolutionary conservation of islet excitability in lower vertebrates, these results also provide important validation of zebrafish as a suitable animal model in which to identify modulators of islet excitability and diabetes.

Regulation of Insulin Receptor Pathway and Glucose Metabolism by CD36 Signaling.

During reduced energy intake, skeletal muscle maintains homeostasis by rapidly suppressing insulin-stimulated glucose utilization. Loss of this adaptation is observed with deficiency of the fatty acid transporter CD36. A similar loss is also characteristic of the insulin-resistant state where CD36 is dysfunctional. To elucidate what links CD36 to muscle glucose utilization, we examined whether CD36 signaling might influence insulin action. First, we show that CD36 deletion specific to skeletal muscle reduces expression of insulin signaling and glucose metabolism genes. It decreases muscle ceramides but impairs glucose disposal during a meal. Second, depletion of CD36 suppresses insulin signaling in primary-derived human myotubes, and the mechanism is shown to involve functional CD36 interaction with the insulin receptor (IR). CD36 promotes tyrosine phosphorylation of IR by the Fyn kinase and enhances IR recruitment of P85 and downstream signaling. Third, pretreatment for 15 min with saturated fatty acids suppresses CD36-Fyn enhancement of IR phosphorylation, whereas unsaturated fatty acids are neutral or stimulatory. These findings define mechanisms important for muscle glucose metabolism and optimal insulin responsiveness. Potential human relevance is suggested by genome-wide analysis and RNA sequencing data that associate genetically determined low muscle CD36 expression to incidence of type 2 diabetes.

Determining calcium concentration in heterogeneous model systems using multiple indicators.

Intracellular free calcium concentrations ([Ca2+]i) are assessed by measuring indicator fluorescence in entire cells or subcellular regions using fluorescence microscopy. [Ca2+]i is calculated using equations which link fluorescence intensities (or intensity ratios) to calcium concentrations [G. Grynkiewicz, M. Poenie, R.Y. Tsien, A new generation of Ca2+ indicators with greatly improved fluorescence properties, J. Biol. Chem. 260 (1985) 3440-3450]. However, if calcium ions are heterogeneously distributed within a region of interest, then the observed average fluorescence intensity may not reflect average [Ca2+]i. We assessed potential calcium determination errors in mathematical and experimental models consisting of 'low' and 'high' calcium compartments, using indicators with different affinity for calcium. [Ca2+] calculated using average fluorescence intensity was lower than the actual mean concentrations. Low affinity indicators reported higher (more accurate) values than their high affinity counterparts. To estimate compartment dimensions and respective [Ca2+], we extended the standard approach by using different indicator responses to the same [Ca2+]. While two indicators were sufficient to provide a partial characterization of two-compartment model systems, the use of three or more indicators offered full description of the model provided compartmental [Ca2+] were within the indicator sensitivity ranges. These results show that uneven calcium distribution causes underestimation of actual [Ca2+], and offers novel approaches to estimating calcium heterogeneity.

Optical suppression of seizure-like activity with an LED.

The therapy of focal epilepsy remains unsatisfactory for as many as 25% of patients. We tested the hypothesis that an efficient, ultraviolet light emitting diode (UV LED), coupled with a newly developed "caged" gamma-aminobutyric acid (GABA), might be capable of terminating "ictal-like" events in cultured murine neurons. GABA was released from BC204, a recently described caged GABA, using a small, ultraviolet (UV) LED. Ictal-like events were provoked by removal of extracellular magnesium. In preliminary control experiments, the concentration of GABA released from our caged compound was dependent upon the strength and duration of the illumination, and readily achieved micromolar (microM) levels that are known to activate tonic, extrasynaptic GABA(A) receptors. Ultraviolet illumination had no effect when BC204 was not present in the perfusate and the currents produced by BC204 were eliminated by picrotoxin. Within a few seconds of UV illumination, BC204 rapidly terminated ictal-like events at low microM concentration. Uncaging of BC204 also blocked the elevation of intracellular calcium induced by seizure-like discharges in our cultures. While much more technical development is clearly required to extend our observations to a more intact preparation, these results suggest the intriguing possibility of constructing an implantable device to "optically suppress" focal human seizures under closed loop control.

Intermittent fasting preserves beta-cell mass in obesity-induced diabetes via the autophagy-lysosome pathway.

Obesity-induced diabetes is characterized by hyperglycemia, insulin resistance, and progressive beta cell failure. In islets of mice with obesity-induced diabetes, we observe increased beta cell death and impaired autophagic flux. We hypothesized that intermittent fasting, a clinically sustainable therapeutic strategy, stimulates autophagic flux to ameliorate obesity-induced diabetes. Our data show that despite continued high-fat intake, intermittent fasting restores autophagic flux in islets and improves glucose tolerance by enhancing glucose-stimulated insulin secretion, beta cell survival, and nuclear expression of NEUROG3, a marker of pancreatic regeneration. In contrast, intermittent fasting does not rescue beta-cell death or induce NEUROG3 expression in obese mice with lysosomal dysfunction secondary to deficiency of the lysosomal membrane protein, LAMP2 or haplo-insufficiency of BECN1/Beclin 1, a protein critical for autophagosome formation. Moreover, intermittent fasting is sufficient to provoke beta cell death in nonobese lamp2 null mice, attesting to a critical role for lysosome function in beta cell homeostasis under fasting conditions. Beta cells in intermittently-fasted LAMP2- or BECN1-deficient mice exhibit markers of autophagic failure with accumulation of damaged mitochondria and upregulation of oxidative stress. Thus, intermittent fasting preserves organelle quality via the autophagy-lysosome pathway to enhance beta cell survival and stimulates markers of regeneration in obesity-induced diabetes.