Post-translationally modified N-terminally truncated amyloid beta peptide with a cyclized form of glutamate at position 3 (pE
Aβ) is a highly pathogenic molecule with increased neurotoxicity and ...propensity for aggregation. In the brains of Alzheimer's Disease (AD) cases, pE
Aβ represents a major constituent of the amyloid plaque. The data show that pE
Aβ 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 pE
Aβ 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 pE
Aβ
fragment to our universal immunogenic vaccine platform MultiTEP, then formulated in Advax
adjuvant. AV-1986R/A showed high immunogenicity and selectivity, with endpoint titers in the range of 10
-10
against pE
Aβ and 10
-10
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.
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.
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.
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 (ATPaxon) 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 ATPaxon, 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 ATPaxon 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.
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.
The large-conductance, voltage-gated, calcium (Ca2+)-activated potassium channel (BKCa) plays an important role in regulating Ca2+ signaling and is implicated in the maintenance of uterine quiescence ...during pregnancy. We used immunopurification and mass spectrometry to identify proteins that interact with BKCa in 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 BKCa and 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 BKCa in an oscillatory pattern in hMSMCs. Furthermore, α₂M* caused intracellular levels of Ca2+ to oscillate in oxytocin-primed hMSMCs. The initiation of oscillations required an interaction between α₂M* and LRP1. By using Ca2+-free medium and inhibitors of various Ca2+ signaling pathways, we demonstrated that the oscillations required entry of extracellular Ca2+ through store-operated Ca2+ channels. Finally, we found that the specific BKCa blocker paxilline inhibited the oscillations, whereas the channel opener NS11021 increased the rate of these oscillations. These data demonstrate that α₂M* and LRP1 modulate the BKCa channel in human myometrium and that BKCa and its immunomodulatory interacting partners regulate Ca2+ dynamics in hMSMCs during pregnancy.
Abstract WldS 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 WldS protects axons by preventing energy deficiency commonly encountered in many diseases. We subjected compartmentally cultured, mouse cortical axons to energy deprivation with 6 mM azide and zero glucose. In wild-type (WT) culture, the treatment, which reduced axon ATP level (ATPaxon ) 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 ATPaxon , the presence of WldS mutation significantly reduced the axon ATP loss and depolarization, restrained the subsequent calcium accumulation, and protected axons against energy deprivation. WldS neurons possessed higher than normal nicotinamide mononucleotide adenylyltransferase (NMNAT) activity. The intrinsic WldS NMNAT activity was required for the WldS -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 ATPaxon and protected axons from energy deprivation. These results indicate that the WldS mutation depends on its intrinsic WldS NMNAT activity and the subsequent increase in axon ATP but not NAD to protect axons, implicating a novel role of WldS NMNAT in axon bioenergetics and protection.
Cholesterol is the major sterol component of all mammalian cell plasma membranes and plays a critical role in cell function and growth. Previous studies have shown that cholesterol inhibits inward ...rectifier K(+) (Kir) channels, but have not distinguished whether this is due directly to protein-sterol interactions or indirectly to changes in the physical properties of the lipid bilayer. Using purified bacterial and eukaryotic Kir channels reconstituted into liposomes of controlled lipid composition, we demonstrate by (86)Rb(+) influx assays that bacterial Kir channels (KirBac1.1 and KirBac3.1) and human Kir2.1 are all inhibited by cholesterol, most likely by locking the channels into prolonged closed states, whereas the enantiomer, ent-cholesterol, does not inhibit these channels. These data indicate that cholesterol regulates Kir channels through direct protein-sterol interactions likely taking advantage of an evolutionarily conserved binding pocket.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
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. β-Hydroxypyruvate and benzoate levels were increased and decreased, respectively, suggesting β-hydroxypyruvate production from d-serine. In vitro, β-hydroxypyruvate altered excitatory properties of myenteric neurons and reduced islet insulin content but not GSIS. β-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 β-hydroxypyruvate-to-d-serine ratios in all groups. Thus, K cells may maintain long-term function of neurons and β-cells by regulating β-hydroxypyruvate levels.
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