Transfusion with Blood Plasma from Young Mice Affects rTg4510 Transgenic Tau Mice Modeling of Alzheimer's Disease.

Alzheimer's disease (AD) is characterized by the buildup of plaques and tangles in the brain. Tangles are formed when the stabilizing protein, tau, becomes hyperphosphorylated and clumps together. There are limited treatments for AD; therefore, the exploration of new treatments is warranted. Previous research showed that plasma transfusion from young donor mice improved spatial memory and increased synaptic proteins in old transgenic APP/PS1 mice, suggesting a remediation of memory and synaptic function. In the current study, plasma was transfused from 2-3-month-old young wildtype mice (WT) to 8-month-old rTg4510 mice expressing human tau (Tau). One week after the transfusions, behavior and tau pathology were examined. We found that Tau mice injected with plasma had lower expression of phosphorylated tau (ptau) in the brain, accompanied by fewer tau tangles in the cortex and CA1 region of the hippocampus and smaller tau tangles in the cortex, when compared to Tau mice injected with saline. Despite no improvement in behavior, the decreased level of ptau and tangles open the door to future studies involving plasma transfusions.

White Button Mushroom (Agaricus bisporus) Supplementation Ameliorates Spatial Memory Deficits and Plaque Formation in an Amyloid Precursor Protein Mouse Model of Alzheimer's Disease.

Alzheimer's Disease (AD) is characterized by cognitive impairment and the presence of amyloid-ß (Aß) plaques and tau tangles. This study was conducted to assess the effects of white button mushroom (WBM) supplementation on spatial memory and plaque formation in mice with mutations in amyloid (Aß). Mice with amyloid precursor protein (hAPP) mutations and their wildtype (WT) littermates were fed a 10% white button mushroom (WBM) feed ad libitum three times per week, in addition to their normal diet. Morris water maze (MWM) was conducted at 14 and 32 weeks of age to assess spatial memory and Aß plaque pathology in the hippocampus was analyzed. Our results showed that hAPP mice on the WBM diet were faster in reaching the platform in the MWM compared to hAPP mice on the control diet at 32 weeks (p < 0.05). Significantly fewer plaque deposits were found in the hippocampi of hAPP mice on the WBM diet compared to those on the control diet at 32 weeks (p < 0.05). Overall, hAPP mice on the WBM diet had improved spatial memory at 32 weeks of age compared to those on the control diet and exhibited fewer amyloid plaques.

Wheel-Running Behavior Is Negatively Impacted by Zinc Administration in a Novel Dual Transgenic Mouse Model of AD.

Alzheimer's disease (AD) is a neurocognitive disorder that impacts both the brain and behavior. Metal ions, including zinc (Zn), have been seen to play an important role in AD-related pathology. In this study, we show alterations in wheel-running behavior both early and late in disease progression in a novel dual Tg mouse model of AD. This mouse includes both amyloid and tau pathology through its cross with the J20 (hAPP) and P301L (Tau) parentage. Animals were given either lab water or water that had been supplemented with 10 ppm Zn. Wheel running was assessed through individually housing mice and measuring wheel-running activity in both the light and dark cycles. Dual Tg mice showed significantly less activity in the first part of the dark cycle than WT mice at both 3.5 and 7 months of age (p < 0.05). Dual Tg mice given Zn water showed less activity compared to dual Tg mice on lab water, tau mice on Zn water, or WT mice given either lab or Zn water (p < 0.05) at 7 months. Female mice in this study consistently showed higher activity compared to male mice in all groups whereas Zn led to reduced activity. Daily activity rhythm was altered in both the tau and dual Tg mice, and Zn impacted this alteration through effects on amyloid, tau, and through circadian pathways.

Nest Building Behavior as an Early Indicator of Behavioral Deficits in Mice.

Nest building is an innate behavior in male and female rodents, even when raised in laboratory settings. As such, many researchers provide rodents synthetic and/or natural materials (such as twine, tissue, cotton, paper, and hay) as a gauge of their overall well-being and as an ancillary assessment to predict the possible decline in cognition. Typically, changes in nesting behaviors, such as failure to create a nest, indicate a change in health or welfare. In addition, nesting behavior is sensitive to many environmental and physiological challenges, as well as many genetic mutations underlying pathological disease states. The following protocol describes a nesting behavior paradigm that explores the usage of four types of nesting material. In addition, the protocol utilizes intraclass correlations to demonstrate that inter-rater reliability is higher when nests are constructed out of shredded paper compared to other common nesting materials such as cotton squares, paper twists, and soft cob bedding. The chosen methodology and statistical considerations (i.e., intraclass correlation) for this assay may be of interest for those conducting experiments assessing the quality of living of mice.

Localization of Free and Bound Metal Species through X-Ray Synchrotron Fluorescence Microscopy in the Rodent Brain and Their Relation to Behavior.

Biometals in the brain, such as zinc, copper, and iron, are often discussed in cases of neurological disorders; however, these metals also have important regulatory functions and mediate cell signaling and plasticity. With the use of synchrotron X-ray fluorescence, our lab localized total, both bound and free, levels of zinc, copper, and iron in a cross section of one hemisphere of a rat brain, which also showed differing metal distributions in different regions within the hippocampus, the site in the brain known to be crucial for certain types of memory. This review discusses the several roles of these metals in brain regions with an emphasis on hippocampal cell signaling, based on spatial mapping obtained from X-ray fluorescence microscopy. We also discuss the localization of these metals and emphasize different cell types and receptors in regions with metal accumulation, as well as the potential relationship between this physiology and behavior.

A Novel hAPP/htau Mouse Model of Alzheimer's Disease: Inclusion of APP With Tau Exacerbates Behavioral Deficits and Zinc Administration Heightens Tangle Pathology.

The brains of those with Alzheimer's disease have amyloid and tau pathology; thus, mice modeling AD should have both markers. In this study, we characterize offspring from the cross of the J20 (hAPP) and rTg4510 (htau) strains (referred to as dual Tg). Behavior was assessed at both 3.5 and 7 months, and biochemical differences were assessed at 8 months. Additionally, mice were placed on zinc (Zn) water or standard lab water in order to determine the role of this essential biometal. Behavioral measures examined cognition, emotion, and aspects of daily living. Transgenic mice (dual Tg and htau) showed significant deficits in spatial memory in the Barnes Maze at both 3.5 and 7 months compared to controls. At 7 months, dual Tg mice performed significantly worse than htau mice (p < 0.01). Open field and elevated zero maze (EZM) data indicated that dual Tg and htau mice displayed behavioral disinhibition compared to control mice at both 3.5 and 7 months (p < 0.001). Transgenic mice showed significant deficits in activities of daily living, including burrowing and nesting, at both 3.5 and 7 months compared to control mice (p < 0.01). Dual Tg mice built very poor nests, indicating that non-cognitive tasks are also impacted by AD. Overall, dual Tg mice demonstrated behavioral deficits earlier than those shown by the htau mice. In the brain, dual Tg mice had significantly less free Zn compared to control mice in both the dentate gyrus and the CA3 of the hippocampus (p < 0.01). Dual Tg mice had increased tangles and plaques in the hippocampus compared to htau mice and the dual Tg mice given Zn water displayed increased tangle pathology in the hippocampus compared to htau mice on Zn water (p < 0.05). The dual Tg mouse described here displays pathology reminiscent of the human AD condition and is impaired early on in both cognitive and non-cognitive behaviors. This new mouse model allows researchers to assess how both amyloid and tau in combination impact behavior and brain pathology.

Zinc Exacerbates Tau Pathology in a Tau Mouse Model.

Hyperphosphorylated tau protein is a key pathology in Alzheimer's disease (AD), frontotemporal dementia, chronic traumatic encephalopathy, and Parkinson's disease. The essential trace element zinc exacerbates tauopathy in vitro as well as in a Drosophila model of AD. However, the interaction has never been assessed behaviorally or biochemically in mammals. Zinc supplementation is prevalent in society, finding use as a treatment for macular degeneration and cataracts, and is also taken as an immune system booster with high levels appearing in multivitamins marketed toward the elderly. Using a transgenic mouse model that contains the human gene for tau protein (P301L), we assessed the effects of excess chronic zinc supplementation on tau pathology. Behavioral tests included nest building, circadian rhythm, Morris Water Maze, fear conditioning, and open field. Biochemically, total tau and Ser396 phosphorylation were assessed using western blot. Number of tangles were assessed by Thioflavin-S and free zinc levels were assessed by Zinpyr-1. Tau mice demonstrated behavioral deficits compared to control mice. Zinc supplementation exacerbated tauopathic deficits in circadian rhythm, nesting behavior, and Morris Water Maze. Biochemically, zinc-supplemented tau mice showed increased phosphorylation at pSer396. Zinc supplementation in tau mice also increased tangle numbers in the hippocampus while decreasing free-zinc levels, demonstrating that tangles were sequestering zinc. These results show that zinc intensified the deficits in behavior and biochemistry caused by tau.

Localization of the zinc binding tubulin polymerization promoting protein in the mice and human eye.

Tubulin Polymerization Promoting Protein (TPPP/p25) modulates the dynamics and stability of the microtubule network by its bundling and acetylation enhancing activities that can be modulated by the binding of zinc to TPPP/p25. Its expression is essential for the differentiation of oligodendrocytes, the major constituents of the myelin sheath, and has been associated with neuronal inclusions. In this paper, evidence is provided for the expression and localization of TPPP/p25 in the zinc-rich retina and in the oligodendrocytes in the optic nerve. Localization of TPPP/p25 was established by confocal microscopy using calbindin and synaptophysin as markers of specific striations in the inner plexiform layer (IPL) and presynaptic terminals, respectively. Postsynaptic nerve terminals in striations S1, S3 and S5 in the IPL and a subset of amacrine cells show immunopositivity against TPPP/p25 both in mice and human eyes. The co-localization of TPPP/p25 with acetylated tubulin was detected in amacrine cells, oligodendrocyte cell bodies and in synapses in the IPL. Quantitative Western blot revealed that the TPPP/p25 level in the retina was 0.05-0.13 ng/µg protein, comparable to that in the brain. There was a central (from optic nerve head) to peripheral retinal gradient in TPPP/p25 protein levels. Our in vivo studies revealed that the oral zinc supplementation of mice significantly increased TPPP/p25 as well as acetylated tubulin levels in the IPL. These results suggest that TPPP/p25, a microtubule stabilizer can play a role in the organization and reorganization of synaptic connections and visual integration in the eye.

Circadian wheel running behavior is altered in an APP/E4 mouse model of late onset Alzheimer's disease.

Circadian rhythms are altered in several diseases associated with aging, one of which is Alzheimer's disease (AD). One example of a circadian rhythm is the rest-activity cycle, which can be measured in mice by monitoring their wheel-running. The present study sought to investigate differences in light phase/dark phase activity between a mouse model of late onset AD (APP/E4) and control (C57Bl6J) mice, in both the pre-plaque and post-plaques stages of the disease. To assess activity level, 24-h wheel running behavior was monitored at six months (pre-plaque) and twelve months (post-plaque) for a period of nine days. The following measures were analyzed: counts (wheel rotations) during the dark phase, counts during the light phase, hour of activity onset, and hour of activity offset. Key findings indicate that activity onset is delayed in APP/E4 mice at six and twelve months, and activity profiles for APP/E4 and C57Bl6J mice differ during the light and dark phase in such a way that APP/E4 mice run less in the early hours of the dark phase and more in the later hours of the dark phase compared to C57Bl6J mice. These findings imply that rest-activity cycle is altered in the pre-plaque stages of AD in APP/E4 mice, as they show impairments as early as six months of age.

Both Genetic and Environmental Changes Can Enhance Learning and Memory.

This review discusses two papers from the same lab that directly compared the effects of genetic enhancement with environmental enrichment on learning and memory in mice. In the first study mice were genetically manipulated to have an increased expression of the NR2B component of the NMDA receptor, associated with learning. These transgenic (Tg) mice showed greater current flow, larger EPSPs, and improved learning and memory on a variety of tasks. In the second experiment both the Tg mice and normal wild type (Wt) mice were raised in either a standard environment or given an enriched environment for two weeks. The differences in behavior and in receptor expression were compared among the four groups. The enriched Wt mice performed as well as both Tg groups on measures of fear conditioning. For the more difficult task of novel object recognition the enriched Wt mice performed as well as the Tg raised in a standard environment, but the enriched Tg mice performed significantly better than all other groups. Environmental enrichment caused an increase in receptor expression in both the Wt and Tg groups, but the Tg enriched mice had the highest expression levels. These papers clearly demonstrated that the mice's environmental enrichment caused behavioral differences for both Wt and Tg-enriched mice - with important implications for humans. They also raise questions about how a lab animal's environment might change its brain and/or behavior, with a potential impact on the results of studies using animals raised in impoverished conditions.

Identification of hydroxyapatite spherules provides new insight into subretinal pigment epithelial deposit formation in the aging eye.

Accumulation of protein- and lipid-containing deposits external to the retinal pigment epithelium (RPE) is common in the aging eye, and has long been viewed as the hallmark of age-related macular degeneration (AMD). The cause for the accumulation and retention of molecules in the sub-RPE space, however, remains an enigma. Here, we present fluorescence microscopy and X-ray diffraction evidence for the formation of small (0.5-20 µm in diameter), hollow, hydroxyapatite (HAP) spherules in Bruch's membrane in human eyes. These spherules are distinct in form, placement, and staining from the well-known calcification of the elastin layer of the aging Bruch's membrane. Secondary ion mass spectrometry (SIMS) imaging confirmed the presence of calcium phosphate in the spherules and identified cholesterol enrichment in their core. Using HAP-selective fluorescent dyes, we show that all types of sub-RPE deposits in the macula, as well as in the periphery, contain numerous HAP spherules. Immunohistochemical labeling for proteins characteristic of sub-RPE deposits, such as complement factor H, vitronectin, and amyloid beta, revealed that HAP spherules were coated with these proteins. HAP spherules were also found outside the sub-RPE deposits, ready to bind proteins at the RPE/choroid interface. Based on these results, we propose a novel mechanism for the growth, and possibly even the formation, of sub-RPE deposits, namely, that the deposit growth and formation begin with the deposition of insoluble HAP shells around naturally occurring, cholesterol-containing extracellular lipid droplets at the RPE/choroid interface; proteins and lipids then attach to these shells, initiating or supporting the growth of sub-RPE deposits.

Human ApoE ε4 alters circadian rhythm activity, IL-1ß, and GFAP in CRND8 mice.

Disruptions to daily living, inflammation, and astrogliosis are characteristics of Alzheimer's disease. Thus, circadian rhythms, nest construction, IL-1ß and TNF-α, and glial fibrillary acidic protein (GFAP) were examined in a mouse model developed to model late-onset Alzheimer's disease-the most common form of the disease. Mice carrying both the mutated human AßPP transgene found in the CRND8 mouse and the human apolipoprotein E ε4 allele (CRND8/E4) were compared with CRND8 mice and wildtype (WT) mice. Circadian rhythms were evaluated by wheel-running behavior. Activity of daily living was measured by nest construction. This study then examined mRNA levels of the inflammatory cytokines IL-1ß and TNF-α as well as protein levels of GFAP. Behavioral outcomes were then correlated with cytokines and GFAP. Compared to WT controls, both CRND8 and CRND8/E4 mice showed significantly more frequent, but shorter, bouts of activity. In the three groups, the CRND8/E4 mice had intermediate disruptions in circadian rhythms. Both CRND8/E4 mice and CRND8 mice showed significant impairments in nesting behavior compared to WTs. While CRND8 mice expressed significantly increased IL-1ß and GFAP expression compared to WT controls, CRND8/E4 mice expressed intermediate IL-1ß and GFAP levels. Significant correlations between IL-1ß, GFAP, and behavior were observed. These data are congruent with other studies showing that human ApoE ε4 is protective early in life in transgenic mice modeling Alzheimer's disease.

Spatial memory deficits in a mouse model of late-onset Alzheimer's disease are caused by zinc supplementation and correlate with amyloid-beta levels.

Much of the research in Alzheimer's disease (AD) that uses mouse models focuses on the early-onset form of the disease, which accounts for less than 5% of cases. In contrast, this study used a late-onset AD model to examine the interaction between increased dietary zinc (Zn) and the apolipoprotein E (ApoE) gene. ApoE ε4 is overrepresented in late-onset AD and enhances Zn binding to amyloid-ß (Aß). This study sought to determine if elevated dietary Zn would impair spatial memory in CRND8 mice (CRND8), as well as mice who carry both the mutated human amyloid precursor protein (APP) and ApoE ε4 genes (CRND8/E4). Mice were provided with either lab tap water or water enhanced with 10 ppm Zn (ZnCO3) for 4 months. At 6 months of age, spatial memory was measured by the Barnes maze. CRND8 mice exhibited significant memory deficits compared to WT mice, as shown by an increased latency to reach the escape box. For the CRND8/E4, but not the CRND8 mice, those given Zn water made significantly more errors than those on lab water. During the probe trial for the WT group, those on Zn water spent significantly less time in the target quadrant than those on lab water. These data suggest that increased dietary Zn can significantly impair spatial memory in CRND8/E4. WT mice given Zn water were also impaired on the 24-h probe trial when compared to lab water WTs. Within the CRND8/E4 group only, levels of soluble Aß were significantly correlated with average primary latencies. Within the Zn-treated CRND8/E4 group, there was a significant correlation between insoluble Aß and average primary errors. Levels of the zinc transporter 3, ZnT3, were negatively correlated with soluble Aß (p < 0.01). These findings are particularly relevant because increased intake of dietary supplements, such as Zn, are common in the elderly-a population already at risk for AD. Given the effects observed in the CRND8/E4 mice, ApoE status should be taken into consideration when evaluating the efficacy of therapies targeting metals.

Correlations in distribution and concentration of calcium, copper and iron with zinc in isolated extracellular deposits associated with age-related macular degeneration.

Zinc (Zn) is abundantly enriched in sub-retinal pigment epithelial (RPE) deposits, the hallmarks of age-related macular degeneration (AMD), and is thought to play a role in the formation of these deposits. However, it is not known whether Zn is the only metal relevant for sub-RPE deposit formation. Because of their involvement in the pathogenesis of AMD, we determined the concentration and distribution of calcium (Ca), iron (Fe) and copper (Cu) and compared these with Zn in isolated and sectioned macular (MSD), equatorial (PHD) and far peripheral (FPD) sub-RPE deposits from an 86 year old donor eye with post mortem diagnosis of early AMD. The sections were mounted on Zn free microscopy slides and analyzed by microprobe synchrotron X-ray fluorescence (µSXRF). Metal concentrations were determined using spiked sectioned sheep brain matrix standards, prepared the same way as the samples. The heterogeneity of metal distributions was examined using pixel by pixel comparison. The orders of metal concentrations were Ca ⋙ Zn > Fe in all three types of deposits but Cu levels were not distinguishable from background values. Zinc and Ca were consistently present in all deposits but reached highest concentration in MSD. Iron was present in some but not all deposits and was especially enriched in FPD. Correlation analysis indicated considerable variation in metal distribution within and between sub-RPE deposits. The results suggest that Zn and Ca are the most likely contributors to deposit formation especially in MSD, the characteristic risk factor for the development of AMD in the human eye.

Indications of reduced prefrontal cortical function in chronically homeless adults.

This study investigated why some homeless individuals seem unable to transition towards self-reliance, following traditional supportive services. It was hypothesized that this may be due to some cognitive dysfunction. Chronically homeless adults were compared to controls on three tests of prefrontal competency: the Iowa Gambling Task, Word Fluency (FAS), and the Burglar's Story; they performed significantly worse than controls on all three tests. These results indicate a relationship between chronic homelessness and possible pre-frontal deficits. This may explain why some long-term homeless fail to learn from the consequences of unproductive behavior and to develop more constructive behaviors needed to attain stability.

The effect of metals on spatial memory in a transgenic mouse model of Alzheimer's disease.

The amyloid-ß protein (Aß) is a metalloprotein with affinity for the metal ions zinc (Zn), copper (Cu), and iron (Fe), which are found in high concentrations in the plaques of Alzheimer's disease (AD). Increasing attention is focused on the role of these metals in AD, and much of the evidence suggests a dyshomeostasis between these metal ions may significantly affect Aß aggregation and deposition in the brain. While the effect of these metals on Aß has been shown in vitro, there is less behavioral data supporting a direct role in cognitive impairment. In order to investigate the cognitive consequences of metal dyshomeostasis, we sought to directly increase metal levels in the brain by dietary means in a transgenic mouse model (Tg2576). We have now examined the effect of increased Zn (10 ppm) and Fe (10 ppm) levels in the drinking water in the Tg2576 mouse. Since increased dietary Zn can lead to Cu deficiency, a Zn group supplemented with copper was also examined (Zn (10 ppm)+Cu (0.025 ppm)). Significant increases in latency and fewer platform crossings on probe trials, which are considered measures of spatial memory impairment, were seen in both Fe and Zn supplemented transgenic mice, compared to those raised on lab water. No significant differences were seen between the Zn + Cu group and in transgenic mice raised on lab water. These data suggest that the negative consequences of Zn may be due to a reduction in copper levels and, therefore, an imbalance between these metal ions rather than a direct effect of increased Zn.

Alterations in fear response and spatial memory in pre- and post-natal zinc supplemented rats: remediation by copper.

The role of zinc in the nervous system is receiving increased attention. At a time when dietary fortification and supplementation have increased the amount of zinc being consumed, little work has been done on the effects of enhanced zinc on behavior. Both zinc and copper are essential trace minerals that are acquired from the diet; under normal conditions the body protects against zinc overload, but at excessive dosages, copper deficiency has been seen. In order to examine the effect of enhanced metal administration on learning and memory, Sprague Dawley rats were given water supplemented with 10ppm Zn, 10ppm Zn+0.25ppm Cu, or normal lab water, during pre- and post-natal development. Fear conditioning tests at 4months showed significantly higher freezing rates during contextual retention and extinction and cued extinction for rats drinking water supplemented with zinc, suggesting increased anxiety compared to controls raised on lab water. During the MWM task at 9months, zinc-enhanced rats had significantly longer latencies to reach the platform compared to controls. The addition of copper to the zinc supplemented water brought freezing and latency levels closer to that of controls. These data demonstrate the importance of maintaining appropriate intake of both metals simultaneously, and show that long-term supplementation with zinc may cause alterations in memory.

The effects of enhanced zinc on spatial memory and plaque formation in transgenic mice.

There is considerable evidence suggesting that metals play a central role in the pathogenesis of Alzheimer's disease. Reports suggest that elevated dietary metals may both precipitate and potentiate an Alzheimer's disease phenotype. Despite this, there remain few studies that have examined the behavioral consequences of elevated dietary metals in wild type and Alzheimer's disease animals. To further investigate this in the current study, two separate transgenic models of AD (Tg2576 and TgCRND8), together with wild type littermates were administered 10 ppm (0.153 mM) Zn. Tg2576 animals were maintained on a zinc-enriched diet both pre- and postnatally until 11 months of age, while TgCRND8 animals were treated for five months following weaning. Behavioral testing, consisting of "Atlantis" and "moving" platform versions of the Morris water maze, were conducted at the end of the study, and tissues were collected for immunohistochemical analysis of amyloid-beta burden. Our data demonstrate that the provision of a zinc-enriched diet potentiated Alzheimer-like spatial memory impairments in the transgenic animals and was associated with reduced hippocampal amyloid-beta plaque deposits. Zinc-related behavioral deficits were also demonstrated in wild type mice, which were sometimes as great as those present in the transgenic animals. However, zinc-related cognitive impairments in transgenic mice were greater than the summation of zinc effects in the wild type mice and the transgene effects.

Evidence that the ZNT3 protein controls the total amount of elemental zinc in synaptic vesicles.

The ZNT3 protein decorates the presynaptic vesicles of central neurons harboring vesicular zinc, and deletion of this protein removes staining for zinc. However, it has been unclear whether only histochemically reactive zinc is lacking or if, indeed, total elemental zinc is missing from neurons lacking the Slc30a3 gene, which encodes the ZNT3 protein. The limitations of conventional histochemical procedures have contributed to this enigma. However, a novel technique, microprobe synchrotron X-ray fluorescence, reveals that the normal 2- to 3-fold elevation of zinc concentration normally present in the hippocampal mossy fibers is absent in Slc30a3 knockout (ZNT3) mice. Thus, the ZNT3 protein evidently controls not only the "stainability" but also the actual mass of zinc in mossy-fiber synaptic vesicles. This work thus confirms the metal-transporting role of the ZNT3 protein in the brain.