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.     

Synergetic Effects of K,Ca, Cu and Zn in Human Semen in Relation to Parameters Indicative of Spontaneous Hyperactivation of Spermatozoa

We have observed that sperm quality parameters indicative of spermatozoa hyperactivation such are lower “linearity” and “straightness”, and as showed by this research “elongation”, were more pronounced in patients with normal spermiogram compared to the group of men with reduced sperm motility who were undergoing routine in vitro fertilisation. The research encompassed 97 men diagnosed with normozoospermia (n = 20), asthenozoospermia (n = 54) and oligoasthenozoospermia (n = 23). The findings indicate that sperm quality of patients with normal spermiogram diagnosed according to WHO criteria, may be compromised by showing premature spontaneous hyperactivation which can decrease the chances of natural conception. We assessed synergistic effects of multiple chemical elements in ejaculated semen to find if premature spontaneous hyperactivation of spermatozoa can be a sign of imbalanced semen composition especially of elements K, Ca, Cu and Zn. Human semen samples showing low or high baseline status of chemical elements concentrations were found in samples from all three diagnostic groups. However, correlation of K/Ca and Cu/Zn ratios, taking into account samples from all three groups of men, were negative at statistical significance level p = 0.01. We tested if the negative correlation between K/Ca and Cu/Zn ratio works for greater number of semen samples. We found the negative correlation to be valid for 175 semen samples at statistical significance of p = 0.00002. The ratio of K/Ca and Cu/Zn, i.e. increased concentrations of K and Zn in comparison to concentrations of Ca and Cu, were associated with a decrease of “straightness” in the group of men with normal spermiogram and pronounced spontaneous hyperactivation of spermatozoa, implying that these elements act in synergy and that the balance of elements and not their absolute concentrations plays the major role in premature spermatozoa hyperactivation in ejaculated semen.     

Euclein: A new binaphthaquinone from Euclea pseudebenus

7-MethyljugIone, 8,8′-dihydroxy-4,4′-dimethoxy-6,6′-dimethyl-2,2′-binaphthyl-1,1′-quinone, 2-methylnaphthazarin, mamegakinone and euclein have been isolated from Euclea pseudebenus. Euclein is the 3,6′-dimer of 7-methyljuglone.     

Rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Identification of essential sulfhydryl residues in the primary sequence of the enzyme

The kinase and sugar phosphate exchange reactions of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase were inactivated by treatment with 5'-p-fluorosulfonylbenzoyladenosine or 8-azido-ATP, but activity could be restored by the addition of dithiothreitol. This inactivation was accompanied by incorporation of 5'-p-sulfonylbenzoyl[8-14C]adenosine into the enzyme that was not released by the addition of dithiothreitol. The lack of effect of ATP analogs on the ATP/ADP exchange or on bisphosphatase activity and reversal of their effects on the kinase and sugar phosphate reactions by dithiothreitol suggest that 1) they reacted with sulfhydryl groups important for sugar phosphate binding in the kinase reaction, and 2) the inactivation of the kinase by these analogs involves a specific reaction that is not related to their general mechanism of attacking nucleotide-binding sites. In addition, alkylation of the enzymes' sulfhydryls with iodoacetamide prevented inactivation by 5'-p-fluorosulfonylbenzoyladenosine, suggesting that the same thiols were involved. o-Iodosobenzoate inactivated the kinase and sugar phosphate exchange; the inactivation was reversed by dithiothreitol; but there was no effect on the bisphosphatase or nucleotide exchange, indicating that oxidation occurred at the same sulfhydryl that are associated with sugar phosphate binding. ATP or ADP, but not fructose 6-phosphate, protected these groups from modification by 5'-p-fluorosulfonylbenzoyladenosine, 8-azido-ATP, and o-iodosobenzoate. ATP also induced dramatic changes in the circular dichroism spectrum of the enzyme, suggesting that adenine nucleotide protection of thiol groups resulted from changes in enzyme secondary structure. Analysis of cyanogen bromide fragments of 14C-carboxamidomethylated enzyme showed that all radioactivity was associated with cysteinyl residues in a single cyanogen bromide fragment. Three of these cysteinyl residues are clustered in a 38-residue region, which probably plays a role in maintaining the conformation of the kinase sugar phosphate-binding site.     

Enzymatic Protection Against Peroxidative Damage in Isolated Brain Capillaries

The content of polyunsaturated fatty acids, the activities of superoxide dismutase (SOD), glutathione peroxidase, glutathione reductase, and catalase, and the concentration of reduced glutathione were measured in cerebral microvessels isolated from rat brain. Polyunsaturated fatty acids, mainly arachidonic, linoleic, and docosahexaenoic acids, accounted for 32% of total fatty acids in cerebral microvessels. Whereas total SOD activity in the microvessels was slightly lower than that found in cerebrum and cerebellum, glutathione peroxidase and glutathione reductase activities were twice as high and catalase activity was four times higher. Glutathione peroxidase in microvessels is active on both hydrogen peroxide and cumen hydroperoxide, and it is strongly inhibited by mercaptosuccinate. After several hours of preparation, the concentration of reduced glutathione in isolated microvessels was 0.7 mumol/mg of protein, which corresponds to a concentration of approximately 3.5 mM. Our results indicate that the blood-brain barrier contains large amounts of peroxide-detoxifying enzymes, which may act, in vivo, to protect its highly polyunsaturated membranes against oxidative alterations.