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.

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.

SLC2A8 (GLUT8) is a mammalian trehalose transporter required for trehalose-induced autophagy.

Trehalose is a disaccharide demonstrated to mitigate disease burden in multiple murine neurodegenerative models. We recently revealed that trehalose rapidly induces hepatic autophagy and abrogates hepatic steatosis by inhibiting hexose transport via the SLC2A family of facilitative transporters. Prior studies, however, postulate that intracellular trehalose is sufficient to induce cellular autophagy. The objective of the current study was to identify the means by which trehalose accesses the hepatocyte cytoplasm, and define the distal signaling mechanisms by which trehalose induces autophagy. We provide gas chromatographic/mass spectrometric, fluorescence microscopic and radiolabeled uptake evidence that trehalose traverses the plasma membrane via SLC2A8 (GLUT8), a homolog of the trehalose transporter-1 (Tret1). Moreover, GLUT8-deficient hepatocytes and GLUT8-deficient mice exposed to trehalose resisted trehalose-induced AMP-activated protein kinase (AMPK) phosphorylation and autophagic induction in vitro and in vivo. Although trehalose profoundly attenuated mTORC1 signaling, trehalose-induced mTORC1 suppression was insufficient to activate autophagy in the absence of AMPK or GLUT8. Strikingly, transient, heterologous Tret1 overexpression reconstituted autophagic flux and AMPK signaling defects in GLUT8-deficient hepatocyte cultures. Together, these data suggest that cytoplasmic trehalose access is carrier-mediated, and that GLUT8 is a mammalian trehalose transporter required for hepatocyte trehalose-induced autophagy and signal transduction.

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.

Global biochemical profiling identifies ß-hydroxypyruvate as a potential mediator of type 2 diabetes in mice and humans.

Glucose-dependent insulinotropic polypeptide (GIP) and GLP-1 are incretins secreted by respective K and L enteroendocrine cells after eating and amplify glucose-stimulated insulin secretion (GSIS). This amplification has been termed the "incretin response." To determine the role(s) of K cells for the incretin response and type 2 diabetes mellitus (T2DM), diphtheria toxin-expressing (DT) mice that specifically lack GIP-producing cells were backcrossed five to eight times onto the diabetogenic NONcNZO10/Ltj background. As in humans with T2DM, DT mice lacked an incretin response, although GLP-1 release was maintained. With high-fat (HF) feeding, DT mice remained lean but developed T2DM, whereas wild-type mice developed obesity but not diabetes. Metabolomics identified biochemicals reflecting impaired glucose handling, insulin resistance, and diabetes complications in prediabetic DT/HF mice. ß-Hydroxypyruvate and benzoate levels were increased and decreased, respectively, suggesting ß-hydroxypyruvate production from d-serine. In vitro, ß-hydroxypyruvate altered excitatory properties of myenteric neurons and reduced islet insulin content but not GSIS. ß-Hydroxypyruvate-to-d-serine ratios were lower in humans with impaired glucose tolerance compared with normal glucose tolerance and T2DM. Earlier human studies unmasked a neural relay that amplifies GIP-mediated insulin secretion in a pattern reciprocal to ß-hydroxypyruvate-to-d-serine ratios in all groups. Thus, K cells may maintain long-term function of neurons and ß-cells by regulating ß-hydroxypyruvate levels.

Synthesis and properties of Asante Calcium Red--a novel family of long excitation wavelength calcium indicators.

Although many synthetic calcium indicators are available, a search for compounds with improved characteristics continues. Here, we describe the synthesis and properties of Asante Calcium Red-1 (ACR-1) and its low affinity derivative (ACR-1-LA) created by linking BAPTA to seminaphthofluorescein. The indicators combine a visible light (450-540 nm) excitation with deep-red fluorescence (640 nm). Upon Ca2+ binding, the indicators raise their fluorescence with longer excitation wavelengths producing higher responses. Although the changes occur without any spectral shifts, it is possible to ratio Ca(2+)-dependent (640 nm) and quasi-independent (530 nm) emission when using visible (< 490 nm) or multiphoton (∼780 nm) excitation. Therefore, both probes can be used as single wavelength or, less dynamic, ratiometric indicators. Long indicator emission might allow easy [Ca2+]i measurement in GFP expressing cells. The indicators bind Ca2+ with either high (Kd = 0.49 ± 0.07 µM; ACR-1) or low affinity (Kd = 6.65 ± 0.13 µM; ACR-1-LA). Chelating Zn2+ (Kd = 0.38 ± 0.02 nM) or Mg2+ (Kd∼5mM) slightly raises and binding Co2+ quenches dye fluorescence. New indicators are somewhat pH-sensitive (pKa = 6.31 ± 0.07), but fairly resistant to bleaching. The probes are rather dim, which combined with low AM ester loading efficiency, might complicate in situ imaging. Despite potential drawbacks, ACR-1 and ACR-1-LA are promising new calcium indicators.

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.

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.

Reprogramming of single-cell-derived mesenchymal stem cells into hair cell-like cells.

HYPOTHESIS: Adult mesenchymal stem cells (MSCs) can be converted into hair cell-like cells by transdetermination. BACKGROUND: Given the fundamental role sensory hair cells play in sound detection and the irreversibility of their loss in mammals, much research has focused on developing methods to generate new hair cells as a means of treating permanent hearing loss. Although MSCs can differentiate into multiple cell lineages, no efficient means of reprogramming them into sensory hair cells exists. Earlier work has shown that the transcription factor Atoh1 is necessary for early development of hair cells, but it is not clear whether Atoh1 can be used to convert MSCs into hair cells. METHODS: Clonal MSC cell lines were established and reprogrammed into hair cell-like cells by a combination of protein transfer, adenoviral based gene transfer, and co-culture with neurons. During transdetermination, inner ear molecular markers were analyzed using reverse transcriptase-polymerase chain reaction, and cell structures were examined using immunocytochemistry. RESULTS: Atoh1 overexpression in MSCs failed to convert MSCs into hair cell-like cells, suggesting that the ability of Atoh1 to induce hair cell differentiation is context dependent. Because Atoh1 overexpression successfully transforms VOT-E36 cells into hair cell-like cells, we modified the cell context of MSCs by performing a total protein transfer from VOT-E36 cells before overexpressing Atoh1. The modified MSCs were transformed into hair cell-like cells and attracted contacts from spiral ganglion neurons in a co-culture model. CONCLUSION: We established a new procedure, consisting of VOT-E36 protein transfer, Atoh1 overexpression, and co-culture with spiral ganglion neurons, which can transform MSCs into hair cell-like cells.

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.

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.

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.

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.

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.

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.

Stem cell therapy for hearing loss: Math1 overexpression in VOT-E36 cells.

HYPOTHESIS: VOT-E36 cells acquire mechanosensitivity after mammalian atonal homolog 1 (Math1) overexpression. BACKGROUND: VOT-E36 cells are derived from a population of epithelial cells in the ventral region of the otocyst at embryonic Day 10.5, before hair cell differentiation. These cells express a number of specific molecular markers for hair cells under both proliferation and differentiation states. Overexpression of Math1 can convert nonsensory epithelial cells into hair cells in the cochlea. Based on this information, we tested whether VOT-E36 cells can be converted into hair cells by Math1 overexpression. METHODS: Using reverse transcriptase-polymerase chain reaction-based analysis, we first compared the expression patterns of various molecular markers for hair cell development in VOT-E36 cells between proliferation and differentiation states, and also before and after overexpression of Math1. Subsequently, with a standard calcium imaging method, we examined whether VOT-E36 cells overexpressing Math1 could detect mechanical vibrations and activate spiral ganglion neurons in a coculture model. In addition, using confocal and scanning electron microscopes, we examined morphologic changes of VOT-E36 cells after Math1 overexpression. RESULTS: Consistent with previous reports, this study has shown that VOT-E36 cells express a number of specific molecular markers for hair cells in both proliferation and differentiation states. Under appropriate culture conditions, Math1 is transiently expressed in this cell line during conditional differentiation. In VOT-E36 cells overexpressing Math1, the normal expression pattern of certain molecular markers for mature hair cells is partially restored. Interestingly, after coculture with spiral ganglion neurons, VOT-E36 cells overexpressing Math1 are able to respond to mechanical vibrations and activate spiral ganglion neurons. Possible molecular mechanisms underlying this novel finding have been explored. CONCLUSION: Math1 overexpression can partially restore presumably downstream signaling cascades for normal hair cell differentiation in VOT-E36 cells, which are able to detect mechanical vibrations after being cocultured with spiral ganglion neurons.