Stabilizing ER Ca2+ Channel Function as an Early Preventative Strategy for Alzheimer’s Disease

Alzheimer’s disease (AD) is a devastating neurodegenerative condition with no known cure. While current therapies target late-stage amyloid formation and cholinergic tone, to date, these strategies have proven ineffective at preventing disease progression. The reasons for this may be varied, and could reflect late intervention, or, that earlier pathogenic mechanisms have been overlooked and permitted to accelerate the disease process. One such example would include synaptic pathology, the disease component strongly associated with cognitive impairment. Dysregulated Ca²⁺ homeostasis may be one of the critical factors driving synaptic dysfunction. One of the earliest pathophysiological indicators in mutant presenilin (PS) AD mice is increased intracellular Ca²⁺ signaling, predominantly through the ER-localized inositol triphosphate (IP₃) and ryanodine receptors (RyR). In particular, the RyR-mediated Ca²⁺ upregulation within synaptic compartments is associated with altered synaptic homeostasis and network depression at early (presymptomatic) AD stages. Here, we offer an alternative approach to AD therapeutics by stabilizing early pathogenic mechanisms associated with synaptic abnormalities. We targeted the RyR as a means to prevent disease progression, and sub-chronically treated AD mouse models (4-weeks) with a novel formulation of the RyR inhibitor, dantrolene. Using 2-photon Ca²⁺ imaging and patch clamp recordings, we demonstrate that dantrolene treatment fully normalizes ER Ca²⁺ signaling within somatic and dendritic compartments in early and later-stage AD mice in hippocampal slices. Additionally, the elevated RyR2 levels in AD mice are restored to control levels with dantrolene treatment, as are synaptic transmission and synaptic plasticity. Aβ deposition within the cortex and hippocampus is also reduced in dantrolene-treated AD mice. In this study, we highlight the pivotal role of Ca²⁺ aberrations in AD, and propose a novel strategy to preserve synaptic function, and thereby cognitive function, in early AD patients.     

Doxepin, a tricyclic antidepressant, binds to normal, intact astroglial cells in cultures and inhibits the isoproterenol-induced increase in cyclic AMP production

Doxepin binds to intact astrocytes in primary cultures. The binding is competitively displaceable by excess cold doxepin and it is pronounced (Bmax = 27 nmol/mg protein) but the affinity is rather low (KD = 30 microM). The binding is inhibited by other antidepressants (amitriptyline, desipramine, tranylcypromine, iprindole) and propranolol but not by isoproterenol. Nevertheless, doxepin counteracts effectively the increase in the production of cyclic AMP evoked by isoproterenol.     

Detection of Aldehydes and Their Conjugated Hydroperox Ydiene Precursors in Thermally-Stressed Culinary Oils and Fats: Investigations Using High Resolution Proton Nmr Spectroscopy

High field (400 and 600 MHz) proton NMR spectroscopy has been employed to investigate the thermally-induced autoxidation of glycerol-bound polyunsaturated fatty acids present in intact culinary frying oils and fats. Heating of these materials at 180°C for periods of 30, 60 and 90 min. generated a variety of peroxidation products, notably aldehydes (alkanals, trans-2-alkenals and alka-2, 4-dienals) and their conjugated hydroperoxydiene precursors. Since such aldehydes appear to be absorbed into the systemic circulation from the gut in vivo, the toxicological significance of their production during standard frying practices is discussed.     

MEARA sequence repeat of human CstF-64 polyadenylation factor is helical in solution. A spectroscopic and calorimetric study.

The primary structure of the human CstF-64 polyadenylation factor contains 12 nearly identical repeats of a consensus motif of five amino acid residues with the sequence MEAR(A/G). No known function has yet been ascribed to this motif; however, according to secondary structure prediction algorithms, it should form a helical structure in solution. To validate this theoretical prediction, we synthesized a 31 amino acid residue peptide (MEARA(6)) containing six repeats of the MEARA sequence and characterized its structure and stability by circular dichroism (CD) spectroscopy and differential scanning calorimetry (DSC). No effects of concentration on the CD or DSC properties of MEARA(6) were observed, indicating that the peptide is monomeric in solution at concentrations up to 2 mM. The far UV-CD spectra of MEARA(6) indicates that at a low temperature (1 degrees C) the MEARA(6) peptide has a relatively high helical content (76% at pH 2.0 and 65% at pH 7.0). The effects of pH and ionic strength on the CD spectrum of MEARA(6) suggest that a number of electrostatic interactions (e.g., i, i + 3 Arg/Glu ion pair, charge-dipole interactions) contribute to the stability of the helical structure in this peptide. DSC profiles show that the melting of MEARA(6) helix is accompanied by positive change in the enthalpy. To determine thermodynamic parameters of helix-coil transition from DSC profiles for this peptide, we developed a new, semiempirical procedure based on the calculated function for the heat capacity of the coiled state for a broad temperature range. The application of this approach to the partial molar heat capacity function for MEARA(6) provides the enthalpy change for helix formation calculated per amino acid residue as 3.5 kJ/mol.     

Synthesis and evaluation of 3″- and 4″-deoxy and -fluoro analogs of the immunostimulatory glycolipid,KRN7000

Four 3″- and 4″-deoxy and -fluorogalactosyl ceramides were synthesized, and their ability to stimulate iNKT cells, based on levels of IL-2 production, was assessed in three NKT cell receptor hybridomas. In two of the hybridomas, 1.2 and 2H4, all of the analogs were immunostimulatory, while in the 1.4 hybridoma only the 4″-fluoro analog led to the production of significant levels of IL-2.     

Effects of fasting and insulin on hepatic phosphofructokinase (PFK)

Fasting of susceptible mice for 24 hr led to a sharp decrease of apparent hepatic PFK activity which was increased to near fed levels by incubation of cytosolic fractions with GSH. Incubation of the cytosols with GSSG reduced the PFK to a lower level-(GSSG insensitive activity). The action of insulin was to increase total PFK with a marked increase of the GSSG insensitive activity.