Harvey J. Armbrecht

The senescence accelerated mouse (SAMP8) as a model for oxidative stress and Alzheimer's disease.

The senescence accelerated mouse (SAMP8) is a spontaneous animal model of overproduction of amyloid precursor protein (APP) and oxidative damage. It develops early memory disturbances and changes in the blood-brain barrier resulting in decreased efflux of amyloid-β protein from the brain. It has a marked increase in oxidative stress in the brain. Pharmacological treatments that reduce oxidative stress improve memory. Treatments that reduce amyloid-β (antisense to APP and antibodies to amyloid-β) not only improve memory but reduce oxidative stress. Early changes in lipid peroxidative damage favor mitochondrial dysfunction as being a trigger for amyloid-β overproduction in this genetically susceptible mouse strain. This sets in motion a cycle where the increased amyloid-beta further damages mitochondria. We suggest that this should be termed the Inflammatory-Amyloid Cycle and may well be similar to the mechanisms responsible for the pathophysiology of Alzheimers disease. This article is part of a Special Issue entitled: Antioxidants and Antioxidant Treatment in Disease.

The SAMP8 Mouse: A Model to Develop Therapeutic Interventions for Alzheimers Disease

The senescence accelerate mouse P8 (SAMP8) is an excellent model of early learning and memory problems. A number of studies have shown that it has cholinergic deficits, oxidative damage, alterations in membrane lipids and circadian rhythm disturbances. The brains of the SAMP8 overproduce amyloid precursor protein (APP), amyloid-beta protein and have an increased physphorylation of tau. An antisense to APP has been developed that reverses the cognitive deficits and oxidative damage. This antisense represents a potential treatment for Alzheimers disease.

Testosterone modulates gene expression pathways regulating nutrient accumulation, glucose metabolism and protein turnover in mouse skeletal muscle

Testosterone regulates energy metabolism and skeletal muscle mass in males, but the molecular mechanisms are not fully understood. This study investigated the response of skeletal muscle to castration and testosterone replacement in 8-week-old male mice. Using microarray analyses of mRNA levels in gastrocnemius muscle, 91 genes were found to be negatively regulated by testosterone and 68 genes were positively regulated. The mRNA levels of the insulin signalling suppressor molecule Grb10 and the glycogen synthesis inhibitors, protein phosphatase inhibitor-1 and phosphorylase kinase-γ, were negatively regulated by testosterone. The insulin-sensitive glucose and amino acid transporters, Glut3 and SAT2, the lipodystrophy gene, Lpin1 and protein targeting to glycogen were positively regulated. These changes would be expected to increase nutrient availability and sensing within skeletal muscle, increase metabolic rate and carbohydrate utilization and promote glycogen accumulation. The observed positive regulation of atrogin-1 (Fbxo32) by testosterone could be explained by the phosphorylation of Akt and Foxo3a, as determined by Western blotting. Testosterone prevented the castration-induced increase in interleukin-1α, the decrease in interferon-γ and the atrophy of the levator ani muscle, which were all correlated with testosterone-regulated gene expression. These findings identify specific mechanisms by which testosterone may regulate skeletal muscle glucose and protein metabolism.

Effect of age on calcium-dependent proteins in hippocampus of senescence-accelerated mice

The senescence-accelerated P8 mouse (SAMP8) is a well-characterized model for the age-related decline in acquisition and retention. Calcium-dependent protein kinase C (PKC) and calcium-calmodulin-dependent protein kinase (CAM K) have been implicated in these processes in the hippocampus. Therefore, the expression of hippocampal PKC and CAM K was determined in SAMP8 mice aged 4, 8, and 12 months. As measured by Western blotting, total hippocampal PKC-gamma protein declined linearly with age. In addition, the distribution of the PKC-gamma also changed with age. The amount of PKC in the particulate fraction declined linearly with age relative to the soluble PKC. The decline in total PKC and particulate PKC correlated with the previously reported decline in retention but not with the decline in acquisition. Western blotting revealed no consistent change in CAM KII protein levels. In addition to protein levels, Ca-dependent protein kinase activity may also be affected by changes in intracellular Ca concentration. Therefore, the levels of calbindin and the plasma membrane Ca pump, two proteins involved in maintaining low levels of intracellular Ca, were measured in the hippocampus. Calbindin protein declined progressively with age, but there was no significant change in total plasma membrane Ca pump expression. These studies demonstrate a decrease in the amount and distribution of hippocampal PKC-gamma in the SAMP8 between 4 and 12 months that is associated with decreased retention.

Intestinal plasma membrane calcium pump protein and its induction by 1,25(OH)2D3 decrease with age

The plasma membrane Ca pump of intestinal absorptive cells has been proposed as a component in the vitamin D-dependent active transport of Ca. Because intestinal Ca transport declines with age, the purpose of this study was to determine if changes in Ca pump expression parallel this decline. Intestinal levels of the plasma membrane Ca pump protein were measured by Western blotting in Fischer 344 rats that were 2, 12, and 24 mo of age. Ca pump protein levels declined by 90% in the duodenum and 65% in the ileum between 2 and 12 mo of age, the time during which active Ca transport declines markedly. The effect of age on the induction of the Ca pump by 1,25-dihydroxyvitamin D3[1,25(OH)2D3], the active metabolite of vitamin D, was determined. Rats were made deficient in 1,25(OH)2D3 by feeding a high-strontium diet, and they were then dosed with 1,25(OH)2D3 or vehicle at 48, 24, and 6 h. In 12-mo-old rats 1,25(OH)2D3 induced duodenal Ca pump protein to only 39% and active Ca transport to 33% of that seen in 2-mo-old animals. These studies demonstrate that decreased expression of the plasma membrane Ca pump protein, along with calbindin protein, parallels the decline in intestinal Ca transport and its response to 1,25(OH)2D3 with age.

Expression of plasma membrane calcium pump mRNA in rat intestine: effect of age and 1,25-dihydroxyvitamin D

The capacity of the small intestine to actively transport Ca declines markedly with increasing age in the rat. The basal-lateral plasma membrane Ca pump is thought to be an important component of the active transport mechanism. Therefore, the purpose of this study was to determine if there are changes in the expression of the intestinal Ca pump with age, mRNA levels were quantitated by Northern and dot blot analysis using a cDNA probe based on the sequence of the plasma membrane Ca pump expressed in the rat intestine (PMCA1). In the duodenum, Ca pump mRNA levels were 3-4 times higher in young (2 months) rats compared to adult (12 months) and old (27 months) rats. In the ileum, Ca pump mRNA levels were one third those of the duodenum, and ileal levels were higher in young rats compared to adult rats. These changes in mRNA levels with age and segment were significantly correlated with Ca pump activity as measured in basal-lateral membrane vesicles in vitro. To determine intestinal responsiveness to 1,25(OH)2D, rats were fed a strontium diet to induce vitamin D deficiency. In young animals, 1,25(OH)2D significantly increased Ca pump mRNA levels 4-fold in the duodenum. 1,25(OH)2D had a similar effect in the adult duodenum. These studies demonstrate that there are changes in Ca pump mRNA levels with age and intestinal segment. Since there was no change in the capacity of 1,25(OH)2D to increase Ca pump mRNA levels, the decline in Ca pump expression may be due to the age-related decrease in serum 1,25(OH)2D rather than to decrease responsiveness to 1,25(OH)2D.

SAMP8 mice have altered hippocampal gene expression in long term potentiation, phosphatidylinositol signaling, and endocytosis pathways

The senescence-accelerated mouse (SAMP8) strain exhibits decreased learning and memory and increased amyloid beta (Aβ) peptide accumulation at 12 months. To detect differences in gene expression in SAMP8 mice, we used a control mouse that was a 50% cross between SAMP8 and CD-1 mice and which showed no memory deficits (50% SAMs). We then compared gene expression in the hippocampus of 4- and 12-month-old SAMP8 and control mice using Affymetrix gene arrays. At 12 months, but not at 4 months, pathway analysis revealed significant differences in the long term potentiation (6 genes), phosphatidylinositol signaling (6 genes), and endocytosis (10 genes) pathways. The changes in long term potentiation included mitogen-activated protein kinase (MAPK) signaling (N-ras, cAMP responsive element binding protein [CREB], protein phosphatase inhibitor 1) and Ca-dependent signaling (inositol triphosphate [ITP] receptors 1 and 2 and phospholipase C). Changes in phosphatidylinositol signaling genes suggested altered signaling through phosphatidylinositol-3-kinase, and Western blotting revealed phosphorylation changes in serine/threonine protein kinase AKT and 70S6K. Changes in the endocytosis pathway involved genes related to clathrin-mediated endocytosis (dynamin and clathrin). Endocytosis is required for receptor recycling, is involved in Aβ metabolism, and is regulated by phosphatidylinositol signaling. In summary, these studies demonstrate altered gene expression in 3 SAMP8 hippocampal pathways associated with memory formation and consolidation. These pathways might provide new therapeutic targets in addition to targeting Aβ metabolism itself.

Antisense against Amyloid-β Protein Precursor Reverses Memory Deficits and Alters Gene Expression in Neurotropic and Insulin-Signaling Pathways in SAMP8 Mice

The senescence-accelerated mouse (SAMP8) strain exhibits an age-related decrease in memory accompanied by an increase in hippocampal amyloid-β protein precursor (AβPP) and amyloid-β peptide (Aβ). We have shown that administration of an antisense oligonucleotide against the Aβ region of AβPP (AβPP antisense) reverses the memory deficits. The purpose of this study was to determine the effect of peripheral (IV) administration of AβPP antisense on hippocampal gene expression. The AβPP antisense reversed the memory deficits and altered expression of 944 hippocampal genes. Pathway analysis showed significant gene expression changes in nine pathways. These include the MAPK signaling pathway (p = 0.0078) and the phosphatidylinositol signaling pathway (p = 0.043), which we have previously shown to be altered in SAMP8 mice. The changes in these pathways contributed to significant changes in the neurotropin (p = 0.0083) and insulin signaling (p = 0.015) pathways, which are known to be important in learning and memory. Changes in these pathways were accompanied by phosphorylation changes in the downstream target proteins p70S6K, GSK3β, ERK, and CREB. These changes in hippocampal gene expression and protein phosphorylation may suggest specific new targets for antisense therapy aimed at improving memory.

Antisense directed at the Abeta region of APP reverses the differences in the phosphatidylinositol signaling pathway seen in aged, senescence accelerated mice

peripheral mononuclear phagocytes promotes brain trafficking and infiltration of these cells in AD model mice. Methods: We have developed a peripheral mononuclear phagocyte model system using thioglycollate-elicited peritoneal macrophages. This system relies on Ab pulse-chase assays and in-gel kinase analyses to evaluate the relative contribution(s) of transforming growth factor-b (TGF-b)-Smad 2/3 and bone morphogenic protein (BMP)Smad 1/5/8 signaling pathways to macrophage Ab uptake. Results: We show that blood-born professional phagocytes demonstrate the capacity to engulf cerebral b-amyloid deposits without inducing deleterious bystander inflammation. Interestingly, following inhibition of classical TGF-b-Smad 2/3 signaling, peripheral macrophages show concomitant hyper-activation of alternative BMP-Smad 1/5/8 signaling and increased uptake and clearance of Ab. We also show that direct stimulation with either BMP-2 or BMP-4 results in elevated Smad 1/5/8 signaling and PAK2 phosphorylation. This shift in favor of BMP-Smad signaling seems to initiate cytoskeletal remodeling required for enhanced Ab uptake and clearance in this scenario. Conclusions: Taken together, our results suggest that shifting from TGF-b-Smad 2/3 to BMP-Smad 1/5/8 signaling is essential for efficient macrophage Ab phagocytosis. Pharmacologic strategies targeting these pathways in peripheral mononuclear phagocytes would, in principle, have significant potential for treatment of AD.

Hippocampal gene expression profiling reveals changes in the phosphatidylinositol signaling and long-term potentiation pathways of aged senescence accelerated mice

Yunwu Zhang, Xiumei Huang, Yaomin Chen, Francesca-Fang Liao, Limin Li, Huaxi Xu, Burnham Institute for Medical Research, La Jolla, CA, USA; Xiamen University, Xiamen, China; University of Tennessee Health Science Center College of Medicine, Memphis, TN, USA; Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China. Contact e-mail: yunzhang@ burnham.org