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.     

A novel method for the evaluation of proximal tubule epithelial cellular necrosis in the intact rat kidney using ethidium homodimer

Background  

Ethidium homodimer is a cell-membrane impermeant nuclear fluorochrome that has been widely used to identify necrotic cells in culture. Here, we describe a novel technique for evaluating necrosis of epithelial cells in the proximal tubule that involves perfusing ethidium homodimer through the intact rat kidney. As a positive control for inducing necrosis, rats were treated with 3.5, 1.75, 0.87 and 0.43 mg/kg mercuric chloride (Hg²⁺, intraperitoneal), treatments which have previously been shown to rapidly cause dose-dependent necrosis of the proximal tubule. Twenty-four h after the administration of Hg²⁺, ethidium homodimer (5 μM) was perfused through the intact left kidney while the animal was anesthetized. The kidney was then removed, placed in embedding medium, frozen and cryosectioned at a thickness of 5 μm. Sections were permeabilized with -20°C methanol and then stained with 4',6-diamidino-2-phenylindole (DAPI) to label total nuclei. Total cell number was determined from the DAPI staining in random microscopic fields and the number of necrotic cells in the same field was determined by ethidium homodimer labeling.     

Diminished Reovirus Capsid Stability Alters Disease Pathogenesis and Littermate Transmission

Reovirus is a nonenveloped mammalian virus that provides a useful model system for studies of viral infections in the young. Following internalization into host cells, the outermost capsid of reovirus virions is removed by endosomal cathepsin proteases. Determinants of capsid disassembly kinetics reside in the viral σ3 protein. However, the contribution of capsid stability to reovirus-induced disease is unknown. In this study, we found that mice inoculated intramuscularly with a serotype 3 reovirus containing σ3-Y354H, a mutation that reduces viral capsid stability, succumbed at a higher rate than those infected with wild-type virus. At early times after inoculation, σ3-Y354H virus reached higher titers than wild-type virus at several sites within the host. Animals inoculated perorally with a serotype 1 reassortant reovirus containing σ3-Y354H developed exaggerated myocarditis accompanied by elaboration of pro-inflammatory cytokines. Surprisingly, unchallenged littermates of mice infected with σ3-Y354H virus displayed higher titers in the intestine, heart, and brain than littermates of mice inoculated with wild-type virus. Together, these findings suggest that diminished capsid stability enhances reovirus replication, dissemination, lethality, and host-to-host spread, establishing a new virulence determinant for nonenveloped viruses.     

Messy eaters: Swabbing prey DNA from the exterior of inconspicuous predators when foraging cannot be observed

Complex coevolutionary relationships among competitors, predators, and prey have shaped taxa diversity, life history strategies, and even the avian migratory patterns we see today. Consequently, accurate documentation of prey selection is often critical for understanding these ecological and evolutionary processes. Conventional diet study methods lack the ability to document the diet of inconspicuous or difficult‐to‐study predators, such as those with large home ranges and those that move vast distances over short amounts of time, leaving gaps in our knowledge of trophic interactions in many systems. Migratory raptors represent one such group of predators where detailed diet studies have been logistically challenging. To address knowledge gaps in the foraging ecology of migrant raptors and provide a broadly applicable tool for the study of enigmatic predators, we developed a minimally invasive method to collect dietary information by swabbing beaks and talons of raptors to collect trace prey DNA. Using previously published COI primers, we were able to isolate and reference gene sequences in an open‐access barcode database to identify prey to species. This method creates a novel avenue to use trace molecular evidence to study prey selection of migrating raptors and will ultimately lead to a better understanding of raptor migration ecology. In addition, this technique has broad applicability and can be used with any wildlife species where even trace amounts of prey debris remain on the exterior of the predator after feeding.     

Varied and repeated atropine dosages and exercise-heat stress

Comparisons of physiological responses to 0, 0.5, 1, and 2 mg atropine (IM) were made in seven males (X +/- SD: age, 24 +/- 3 years; ht, 174 +/- 12 cm; wt, 76 +/- 3 kg) while they exercised (approximately 390 W) in a hot-dry (40 degrees C, 20% rh) environment. Responses to 4 mg, as well as repeatability of responses to 2 mg, were studied in two and six of these subjects, respectively. On 8 test days an intramuscular injection of atropine or saline control was administered 20 min before subjects walked on a treadmill for two 50-min bouts. Heart rate (HR) during exercise did not change in the control trial but by min 50 increased during all atropine trials (P less than 0.01). Rectal temperature (Tre) increased (P less than 0.01) in all trials by min 50 and continued increasing (P less than 0.01) in the 2-mg trial during the second exercise bout. For the two subjects tested with all dosages (0.5 - 4 mg atropine), the change in HR and Tre between the atropine and control trials at 50 min of exercise was regressed against the various atropine dosages. The relationship (r = 0.92) for HR was curvilinear while the relationship (r = 0.99) for Tre was linear. Mean weighted skin temperature (Tsk) was relatively constant during exercise and was warmer (P less than 0.05) with increasing atropine dosage. In a repeat 2 mg trial, HR was 6 bt . min-1 lower (P less than 0.05) on the second exposure but Tre was the same (P greater than 0.05) on both days. For subjects walking in the heat, three new observations were: 1) 0.5 mg of atropine resulted in increased HR and Tsk compared to control values; 2) HR was elevated but the magnitude of change decreased with increasing dosage, while the elevation in Tre was consistent with increasing dosage; and 3) rectal temperatures (in trials with and without atropine) were unaffected by previous days of atropine administration.     

Deletion of the Nuclear Localization Sequences and C-Terminus of PTHrP Impairs Embryonic Mammary Development but also Inhibits PTHrP Production

Parathyroid hormone-related protein (PTHrP) can be secreted from cells and interact with its receptor, the Type 1 PTH/PTHrP Receptor (PTHR1) in an autocrine, paracrine or endocrine fashion. PTHrP can also remain inside cells and be transported into the nucleus, where its functions are unclear, although recent experiments suggest that it may broadly regulate cell survival and senescence. Disruption of either the PTHrP or PTHR1 gene results in many abnormalities including a failure of embryonic mammary gland development in mice and in humans. In order to examine the potential functions of nuclear PTHrP in the breast, we examined mammary gland development in PTHrP (1–84) knock-in mice, which express a mutant form of PTHrP that lacks the C-terminus and nuclear localization signals and which can be secreted but cannot enter the nucleus. Interestingly, we found that PTHrP (1–84) knock-in mice had defects in mammary mesenchyme differentiation and mammary duct outgrowth that were nearly identical to those previously described in PTHrP^−/− and PTHR1^−/− mice. However, the mammary buds in PTHrP (1–84) knock-in mice had severe reductions in mutant PTHrP mRNA levels, suggesting that the developmental defects were due to insufficient production of PTHrP by mammary epithelial cells and not loss of PTHrP nuclear function. Examination of the effects of nuclear PTHrP in the mammary gland in vivo will require the development of alternative animal models.     

Alleviation of Liver Dysfunction,Oxidative Stress,and Inflammation Underlines the Protective Effects of Polysaccharides from Cordyceps cicadae on High Sugar/High Fat Diet-Induced Metabolic Syndrome in Rats

This study investigated the protective effects of two polysaccharides (CPA-1 and CPB-2) from Cordyceps cicadae against high fructose/high fat diet (HF/HFD) induced obesity and metabolic disorders in rats. Rats were either fed with normal diet or HF/HFD and treated with CPA-1 and CPB-2 (100 and 300 mg/kg) for 11 weeks. Administration of CPA-1 and CPB-2 significantly and dose dependently reduced body and liver weight, insulin and glucose tolerance, serum insulin and glucose levels. Furthermore, serum and hepatic lipid profiles, liver function enzymes and proinflammatory cytokines (TNF-α, IL-1β and IL-6) were markedly reduced. Additionally, CPA-1 and CPB-2 treatment alleviated hepatic oxidative stress by reducing lipid peroxidation level (MDA) and upregulating glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase (CAT) activities as well as ameliorated histological alterations through the reduction of hepatic lipid accumulation. These results suggested that the polysaccharides from C. cicadae showed protective effects against HF/HFD induced metabolic disturbances and may be considered as a dietary supplement for treating obesity.