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Keywords:

  • Alzheimer's;
  • amyloid;
  • immediate-early genes;
  • induction;
  • plasticity;
  • transgenic

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Mice transgenic for mutated forms of the amyloid precursor protein (APP) plus presenilin-1 (PS1) genes (APP + PS1 mice) gradually develop memory deficits which correlate with the extent of amyloid deposition. The expression of several immediate-early genes (IEGs: Arc, Nur77 and Zif268) and several other plasticity-related genes (GluR1, CaMKIIα and Na-K- ATPase αIII) critical for learning and memory was normal in young APP + PS1 mice preceding amyloid deposition, but declined as mice grew older and amyloid deposits accumulated. Gene repression was less in APP + PS1 mouse brain regions that contain less Aβ and in APP mice compared with APP + PS1 mice, further linking the extent of amyloid deposition and the extent of gene repression. Critically, we demonstrated that amyloid deposition led specifically to impaired induction of the IEGs with no effects on basal expression using exposure to a novel environment 30 min prior to being killed to induce IEGs. These data imply that Aβ deposition can selectively reduce expression of multiple genes linked to synaptic plasticity, and provide a molecular basis for memory deficiencies found in transgenic APP mice and, most likely, in early stage Alzheimer's disease (AD). Presumably, pharmacological agents blocking the Aβ-related inhibition of gene expression will have benefit in AD.

Abbreviations used
AD

Alzheimer's disease

APP

amyloid precursor protein

Ct

threshold cycle

Gap43

growth-associated protein 43

IEGs

intermediate-early genes

PS1

presenilin-1

qRT-PCR

quantitative real-time reverse transcriptase-polymerase chain reaction

Rted

reverse transcribed

Mutations in the amyloid precursor protein (APP) and presenilin-1 (PS1) genes accelerate Aβ deposition and lead to an aggressive form of early onset Alzheimer's disease [AD; (Selkoe 2001)]. Mice transgenic for the mutant forms of the human APP (Hsiao et al. 1996) and PS1 (Duff et al. 1996) genes have been bred to generate a viable doubly transgenic mouse presenting accelerated amyloid pathology [APP + PS1 mice; (Holcomb et al. 1998)]. Despite the lack of intracellular fibrillary tangles and neuronal degeneration, these mice do exhibit anterograde amnesia and increased neuro-inflammation with age, providing a good model for the study of amyloid-associated memory loss at a stage of pathology similar to that in early AD (Morgan et al. 2000; Mukaetova-Ladinska et al. 2000; Arendash et al. 2001; Gordon et al. 2002).

Based on work involving DNA microarray comparisons, we previously described the down-regulation of several genes in 18+-month-old APP + PS1 mice using quantitative real-time reverse transcription-polymerase chain reaction [qRT-PCR; (Dickey et al. 2003)]. Of particular interest was a panel of down-regulated immediate-early genes (IEGs) implicated in memory function, such as Zif268 (Miyashita et al. 1998; Hall et al. 2000) and activity-regulated cytoskeletal-associated protein [Arc; (Guzowski et al. 2001)]. We demonstrated that several more synaptic genes were also selectively down-regulated in the amyloid-containing hippocampus of APP + PS1 mice (e.g. the ionotropic glutamate receptor 1 (GluR1) and Na, K ATPase αIII), while other vital synaptic mRNAs remained unchanged [e.g. synaptophysin and growth-associated protein 43 (Gap43)].

Our earlier work found that in most instances, gene expression in APP + PS1 mice was normal in the amyloid-free cerebellum. Here, we describe additional studies to test the linkage between amyloid deposition and suppression of these genes, and address whether the diminished IEG expression is due to reduced basal expression or impaired induction.

Mice were bred in our facility and genotyped using previously described methods (Gordon et al. 2002). For most experiments, each mouse was individually transported out of the vivarium to a holding room 30 min before being killed. Mice were deeply anesthetized with pentobarbitol (100 mg/kg) and perfused transcardially with 0.9% saline. Brains were quickly removed and regions dissected and frozen on dry ice. We have shown previously that the behavior and amyloid burden of 18 month-old APP + PS1 mice were significantly different from the non-transgenic littermates used for these studies (Austin et al. 2003).

IEGs were induced by exposure to a novel environment. Fourteen mice (six 18 month-old APP + PS1 and eight age-matched non-transgenic littermates) were handled and weighed for three consecutive days to acclimate them to the holding room and the experimenter. On the fourth day, the mice were placed into a 0.6 m × 0.6 m Plexiglas open field containing objects for them to investigate or crawl through for 5 min. All mice actively explored this environment and no differences in activity between transgenic and non-transgenic mice could be discerned in the notes taken by the experimenter. After 5 min, the mice were placed into a new cage located in a different room from their home cage for 30 min to avoid disturbing other mice. Following this 30 min period, mice were killed as described above. The novel environment was cleaned with 30% ethanol between mice. To ascertain basal expression levels of IEGs, 14 other mice (six 18 month-old APP + PS1 and eight age-matched non-transgenic littermates) from the same cohort were removed from their home cage and killed with no delay.

Total RNA was prepared from dissected brain tissue of APP only, PS1 only and APP + PS1 mice along with non-transgenic littermates as previously described (Dickey et al. 2003). Briefly, total RNA samples were reverse transcribed (RTed) with murine monocyte leukemia virus (MMLV) reverse transcriptase and 1 m betaine. A standard curve was established by reverse transcribing increasing amounts of total RNA (covering 3 logs) from an RNA pool compiled from all samples used in that experiment. Two mass quantities (10 and 2 ng) or, in later experiments, a single mass quantity of 5 ng, of total RNA from each sample were RTed for comparison to the standard curve.

Primer pairs for qRT-PCR were generated from the web-based applications Primer3 and the Oligo Toolkit (Operon). Experimental wells containing 25 µl SYBR Green (ABI, Foster City, CA) PCR reactions were run in triplicate in 96-well plates. Two-step PCR was run on the MJ Research Opticon (Boston, MA, USA) as follows: 1 cycle of 95°C for 15 min, followed by 40 cycles of 95°C for 15 s and 60–65°C for 1 min. All primer pairs amplified a single peak of fluorescence by melt curve analysis. The standard curve was calculated by plotting the threshold cycle (Ct) against the log nanogram (ng) quantity of RNA added to the RT reactions. PCR efficiency varied by less than 5% for all amplicons. A linear regression was performed and then the slope, relating Ct to log ng RNA, was calculated and converted to a mass quantity of standard RNA. These mass values for the genes of interest were then divided by 18S ribosomal RNA mass values of the same RT reaction to determine fold change in expression relative to the standard RNA pool. We chose 18S rRNA as the endogenous control gene based on empirical data of our own and results from others (Schmittgen and Zakrajsek 2000) indicating that other commonly used housekeeping genes such as β-actin and GAPDH have more variable expression in tissue, particularly brain, while 18S remained steady. These fold-change values for samples in the experimental and control groups were analyzed for significance using one-way anova (Dickey et al. 2003). For the mice in the behavioral induction study, a two-way anova was performed with the main variables being genotype and induction state.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Quantitative RT-PCR analysis of hippocampal tissue from APP + PS1 mice at 2, 6 and 18 months of age revealed decreases in specific mRNA transcripts with increased amyloid accumulation. At 2 months of age, prior to development of cerebral amyloid, no loss of mRNA expression was seen for any marker (Figs 1a and b). However, by 6 months of age, the IEGs Nur77 (a nuclear orphan receptor), Arc and Zif268 were significantly under-expressed by 35, 40 and 22%, respectively, compared with non-transgenic littermates (Fig. 1a). By 18 months, in addition to further reduced IEG expression, GluR1, calcium/calmodulin kinase IIα (CaMKIIα) and Na, K ATPase αIII mRNA expression was significantly reduced by 20–30% (Fig. 1b). Synaptophysin and Gap43 remained unchanged throughout the lifespan of the transgenic mice. The findings herein also revealed two distinct patterns in gene regulation. The early decrease in IEG expression at 6 months in the APP + PS1 mice (Fig. 1a) is contrasted by the later reduction in the more constitutively expressed plasticity genes GluR1, CaMKIIα and Na, K ATPase αIII seen at 18 months (Fig. 1b). Figure 1(c) was generated by averaging the individual sample percentage reduction values for each gene, then averaging these according to IEG, plasticity-related or non-changing synaptic category, and performing one-way anova on these values to determine significance. Figure 1(c) demonstrates that a significant reduction at 6 months is only seen with the IEGs when analyzed in this manner, while at 18 months both IEG and plasticity-related gene expression are significantly reduced.

image

Figure 1. Time course of gene expression in the hippocampus of transgenic mice by qRT-PCR. (a) The differential expression [transgenic expression (n = 7–8 APP + PS1) relative to non-transgenic expression (n = 7–8 Non-Tg; set as 100% for each gene)] for three IEGs (Arc, Zif268 and Nur77) that were (i) unaffected at 2 months, prior to amyloid development (ii) significantly down-regulated in transgenic animals by 6 months of age, and (iii) further down-regulated by 18 months. Synaptophysin remained equivalent to non-transgenic animals throughout their lifespan. (b) The differential expression for a set of plasticity-related genes (CaMKIIα, GluR1 and Na, K ATPase αIII) that were unchanged at both the 2 and 6 month time points, with the exception of CaMKIIα at 6 months. However, by 18 months these genes were all significantly down-regulated. Gap43 remained equivalent to non-transgenic animals throughout their lifespan. (c) Summary of the findings in (a) and (b) by averaging the values of the individual genes for each sample and analyzing these for significance between representative groupings by one-way anova. The values represented in this figure are the mean ± SEM. *Indicates significant differences between APP + PS1 mice and non-transgenic littermates (p < 0.05).

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To further understand the relationship between amyloid deposition and mRNA expression, we investigated the gene expression of 18-month-old singly transgenic APP or PS1 mice compared with mice expressing both APP + PS1 transgenes. The amyloid-free PS1 transgenic mice had no changes in expression of any genes (Fig. 2). However, the singly transgenic APP mice exhibited a significant 30–40% loss of the IEGs Nur77, Arc and Zif268, without significant loss of the more constitutively expressed synaptic mRNAs GluR1, CaMKIIα and Na, K ATPase αIII (Fig. 2).

image

Figure 2. Gene expression in hippocampi of APP only, PS1 only and APP + PS1 transgenic mice at 18 months of age by qRT-PCR. This figure compares expression of doubly-transgenic APP + PS1 mice and age-matched single transgenics with non-transgenic littermates. Black bars indicate PS1 only mice, gray bars indicate APP only mice and white bars indicate APP + PS1 mice. The far left panel describes the differential expression [transgenic expression (n = 7–8 APP + PS1) relative to non-transgenic expression (n = 7–8 Non-Tg; set as 100% for each gene)] for Arc, Zif268 and Nur77. These genes were down-regulated in both APP only and APP + PS1 transgenic animals. However, the PS1 only transgenics had no deficits in expression. The center panel reveals that the plasticity-related genes CaMKIIα, GluR1 and Na, K ATPase αIII were down-regulated at 18 months only in the APP + PS1 transgenic mice, while APP only and PS1 only expression remained equivalent to that of non-transgenics. The right panel demonstrates the specificity of this effect by showing that other synaptic genes were not influenced by genotype in any of the three transgenic models. Expression values for the APP only and APP + PS1 transgenics could also be compared statistically to determine whether the differing levels of expression were significant between the two models. The values represented in this figure are the mean ± SEM. *Indicates significant differences between APP + PS1 mice and non-transgenic littermates (p < 0.05), determined by one-way anova; † above the bracket indicates differences (p < 0.05) in expression between APP + PS1 mice and APP only mice, determined by one-way anova.

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A regional analysis was then performed on 18-month-old APP + PS1 mice, comparing gene expression levels in the hippocampus, posterior cortex and caudate nucleus (striatum). In the posterior cortex, APP + PS1 mice had a significant reduction in the IEGs, while the other plasticity-related genes, GluR1 and Na, K ATPase αIII, remained unchanged (Fig. 3). CaMKIIα was also significantly reduced, but not to the same extent as in the hippocampus (Fig. 3). Caudate nucleus analysis revealed a similar pattern of expression: significantly reduced IEGs (25–35%) without reductions in the other plasticity-related genes (Fig. 3). For all genotypes and brain regions, Gap43 and synaptophysin expression was unaltered (Figs 2 and 3).

image

Figure 3. Gene expression profile in hippocampus, posterior cortex and caudate nucleus of APP + PS1 transgenic mice at 18 months of age by qRT-PCR. This figure compares gene expression in three different brain regions of doubly-transgenic APP + PS1 mice with non-transgenic littermates. Black bars indicate caudate nucleus, gray bars indicate posterior cortex and white bars indicate hippocampus in APP + PS1 mice. The far left panel describes the differential expression in each brain region [transgenic expression (n = 7–8 APP + PS1) relative to non-transgenic expression (n = 7–8 Non-Tg; set as 100% for each gene)] for Arc, Zif268 and Nur77. These genes were significantly down-regulated in all three regions. The center panel reveals that the plasticity-related genes CaMKIIα, GluR1 and Na, K ATPase αIII were down-regulated at 18 months only in the hippocampus (HC) of APP + PS1 transgenic mice, with the exception of CaMKIIα which is significantly down-regulated in the posterior cortex (CX). The right panel demonstrates the specificity of this effect by showing that other synaptic genes were not influenced in any of the brain regions. Expression values for the HC and CX could also be compared statistically to determine whether the differing levels of expression were significant between the two regions. Caudate nucleus (CN) was not compared, as these values were even closer to non-transgenic than those of CX. The values represented in this figure are the mean ± SEM. *Indicates significant differences between APP + PS1 brain regions and those of non-transgenic littermates (p < 0.05), determined by one-way anova; † above the bracket indicates differences (p < 0.05) in expression between PCX and HPC of APP + PS1 mice, determined by one-way anova.

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To determine whether the reduction of the IEGs in the memory-deficient APP + PS1 mice was a result of lower basal expression level or decreased experience-dependent induction, we exposed 18-month-old APP + PS1 mice and their non-transgenic littermates to a novel environment for 5 min, followed by 30 min in a new cage. This manipulation provided the means to increase IEG expression, resulting in increased representative mRNA levels (see Materials and methods). For the non-induced (basal) control groups, additional 18-month-old APP + PS1 mice and their non-transgenic littermates were taken from their home cage and rapidly killed. Upon analyzing the hippocampi of these mice by qRT-PCR, we found that there was a significant 2.5-fold induction of IEGs in our behavioral paradigm in non-transgenic mice (Fig. 4). This induction was significantly blunted by half for Arc and Nur77 in the APP + PS1 mice compared with induced non-transgenic littermates (Fig. 4). The basal expression of the IEGs was nearly identical in the two genotypes (Fig. 4). Zif268 expression was also induced significantly by the environmental novelty, but suppression of its induction in APP + PS1 mice was not statistically significant. The other plasticity-related genes, GluR1, CaMKIIα and Na, K ATPase αIII, were not induced by exposure to the novel environment. However these genes did have significantly reduced expression when analyzed by genotype, while Gap43 and synaptophysin remained unaffected by genotype or exposure to a novel environment (Table 1).

image

Figure 4. IEG expression measured by qRT-PCR in hippocampi of APP + PS1 transgenic mice and non-transgenic littermates following induction by environmental novelty. This figure compares expression of IEGs between APP + PS1 mice and non-transgenic littermates either following induction by a 5 min exposure to a novel environment with a subsequent 30 min period in a new cage, or immediate killing after removal from their home environment. White bars indicate those mice that were killed quickly to measure basal expression. Black bars indicate those animals that were exposed to environmental novelty to measure induced expression. anova indicated a significant effect of induction for Arc (F = 38.8; p < 0.0001), Nur77 (F = 26.1; p < 0.0001) and Zif268 (F = 27.992; p < 0.0001). There was also a significant interaction between genotype and induction for Arc (F = 4.4; p < 0.05) and Nur77 (F = 4.3; p < 0.05), but not Zif268. The values represented in this figure are the mean ± SEM. *Indicates a significant interaction effect between genotype and treatment (p < 0.05) as determined by two-way anova.

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Table 1.  Expression of non-inducible genes in APP + PS1 mice stimulated by environmental novelty compared to non-transgenic littermates by qRT-PCR
Gene propertiesMarker mRNANon-induced
  1. Values indicate percentage reduction in APP + PS1 mouse hippocampus RNA expression compared with non-transgenic littermates' ± SEM. No induction occurred for these genes, therefore mice were analyzed by genotype only using one-way anova. *Indicates significantly different from non-transgenic mice at p < 0.05.

Plasticity-relatedCaMKIIα87.3 ± 5.2*
genesGluR183.8 ± 6.6*
Na, K ATPase αIII83.3 ± 5.9*
Non-changingGap4395.6 ± 4
synaptic genesSynaptophysin95.4 ± 5.1

Glial fibrillary acidic protein (GFAP) mRNA increased in the hippocampi of APP + PS1 mice. At 2 months, GFAP was unchanged, by 6 months it was increased by 80% and at 18 months, levels had increased to over 300% that of non-transgenic littermates (data not shown), demonstrating the potential role for glial cells in the pathogenesis of amyloid-associated dementias and confirming earlier ELISA and histochemical results in APP + PS1 mice (Morgan et al. 1991; Gordon et al. 2002). We have also increased the breadth of our investigations into gene expression by analyzing many more genes, finding that several more remain unaltered in the 18-month-old APP + PS1 hippocampus compared with non-transgenic littermates. These include Ha-Ras, Rheb, Shank3, synapsin, microtubule-associated protein 2 (MAP2), the Na, K ATPase γ subunit and the two metabotropic glutamate receptors, mGluR1 and mGluR5. These findings reiterate the selectivity of gene dysregulation associated with either amyloid deposition itself or its subsequent deleterious effects on the brain.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Post-mortem brain tissue from AD patients is riddled with amyloid plaques comprising Aβ peptide polymers and intracellular neuro-fibrillary tangles of hyperphosphorylated tau filaments. Neuron loss and gliosis are also evident. The cognitive impairments of the disease could be attributed to any combination of these pathologies, and recent evidence supports each of these components. The development and subsequent usage of transgenic animal models expressing genes known to produce AD-like pathology have provided a way to elucidate the contributions of these individual pathological features. Here, we have utilized the memory-deficient APP + PS1 mouse model to analyze expression of genes that appear to be essential for the formation of memories. Using qRT-PCR, we have found that as amyloid becomes reliably detectable in the brains of the APP + PS1 mice (6 months old), IEGs begin to realize decreased expression. As amyloid burden increases, resulting in further gliosis and onset of memory loss, several other more constitutively expressed genes critical for plasticity and memory function are significantly reduced. Perhaps of greatest significance here was the finding that the induction of the IEGs was significantly impaired, indicating that the triggering mechanism for their rapidly increased expression is being inhibited somehow in these mice that develop amyloid. These data provide strong support for the hypothesis that amyloid, itself, impairs the induction and expression of genes thought to be essential for retaining newly acquired information.

Our first indication that amyloid might repress genes essential for memory came via a microarray study (Dickey et al. 2003). Intrigued by the novelty of the idea that amyloid may suppress molecular mechanisms for memory, independent of neuron or synapse loss, we examined other memory-associated genes and as of this writing have found 11 that are down-regulated. For these studies, we chose a representative panel of the down-regulated genes associated with long-term potentiation (LTP) and memory consolidation, three of which are immediate-early (inducible) genes (Arc, Nur77 and Zif268) and three with more constitutive expression (Na, K ATPase αIII, GluR1 and CaMKIIα). Mice receiving antisense oligonucleotides targeting the mRNA of Arc, an IEG that is translated at the synapses (Lyford et al. 1995), have disrupted LTP maintenance and demonstrate impaired long-term memory (Guzowski et al. 2000). Nur77 mRNA, also an IEG product, is the transcript for an orphan thyroid hormone receptor that is dramatically increased in response to several excitotoxic stimuli (Watson and Milbrandt 1989; St Hilaire et al. 2003). Zif268 is a zinc finger transcription factor that is rapidly up-regulated in the hippocampus of rats after exposure to a novel stimulus (Hall et al. 2000) and by water maze training (Jones et al. 2001). Additionally, homozygous Zif268 knockout mice exhibit long-term memory deficits (Jones et al. 2001), underscoring the critical nature of this gene to proper cognitive function. The Na, K ATPase αIII subunit, when inhibited by oubain, is known to disrupt memory consolidation (Mark and Watts 1971; Watts and Mark 1971). Recent reports have implicated the ionotropic glutamate receptor GluR1 (AMPA1) as essential for various forms of synaptic plasticity and memory retention (Lee et al. 2003; Schmitt et al. 2003). CaMKIIα is also thought to be critical for memory retention, as evidenced by mice transgenic for activated forms of the protein having impaired memory retention (Mayford et al. 1995, 1996). Expression of both the growth-associated protein, Gap43, a gene important for growth cone formation as well as memory (Routtenberg et al. 2000), and synaptophysin, a protein widely used as a marker for nerve terminals, remains unchanged in the APP + PS1 mouse brain. Thus, not all genes involved in synaptic function or plasticity are down-regulated by Aβ deposits.

To understand the contribution of amyloid to the dysregulation of gene expression in the APP + PS1 mice we analyzed hippocampal tissue from 2-month-old mice, an age prior to amyloid deposition (Gordon et al. 2002), 6-month-old mice, when amyloid deposits are just beginning to form, and 18-month-old mice, when there is considerable amyloid in the forebrain (Gordon et al. 2002). Critically, the stable expression of all genes analyzed at 2 months (Fig. 1), an age when APP and PS1 over-expression is present but amyloid deposits have not yet appeared, argues that transgene expression per se is not the cause of the reduced expression. It is the accumulation of amyloid that appears to be regulating expression in such a way that some genes, predominately the IEGs, are diminished when amyloid deposits are still low (6 months old) while others (e.g. GluR1 and Na, K ATPase αIII) are only affected when amyloid deposition is substantial (18 months old). CaMKIIα expression is significantly decreased at 6 months although not to the same degree as the IEGs. This could possibly indicate that CaMKIIα is one of the first genes being affected by the presence of excess amyloid in the brain, which is significant due to its involvement in the calcium signaling cascade thought to mediate receptor activated transcription (Blanquet et al. 2003). As summarized in Fig. 1(c), these findings provide evidence that slightly reduced IEG expression is not sufficient to cause memory impairments but, assuming that protein expression of these genes follows that of their mRNA, the additional down-regulation of these more constitutively expressed plasticity-related genes, as detected at 18 months, may be necessary to disrupt memory. This emphasizes the robustness of mnemonic processes (or the lack of sensitivity of behavioral tests), as only when partial inhibition of the expression of multiple genes is present in aggregate are memory deficits readily apparent.

The APP only mice have roughly one third of the Aβ deposition as APP + PS1 mice (Jaffar et al. 2001), while the PS1 mice resemble non-transgenic mouse brain when analyzed for amyloid burden and amyloid-associated pathogenesis. Our findings in Fig. 2 reveal that the singly transgenic APP mice also have reduced IEG expression without reductions in the other plasticity-related genes, while the PS1 singly transgenic mice do not have reductions in either set of genes. This is consistent with the selective impairment of IEG expression found in 6 month old mice.

We had previously shown that gene expression in the cerebella of APP + PS1 mice, a region without detectable amyloid levels, remained unchanged when compared with non-transgenic littermates (Dickey et al. 2003). Subsequently, we analyzed two additional regions of the forebrain in 18-month-old APP + PS1 mice: posterior cortex, a region with fewer amyloid plaques than the hippocampus, and the caudate nucleus, a brain region that does not accumulate fibrillar amyloid plaques, yet has abundant diffuse Aβ (Gordon et al. 2002). Interestingly, upon analysis of both of these regions, we found that like the APP singly transgenic mice and the 6-month-old APP + PS1 mice, only the IEGs were significantly under-expressed. Similar to the 6-month-old APP + PS1 mice, CaMKIIα was slightly down-regulated in the posterior cortex but not as dramatically as in the hippocampus. Again, this is consistent with the relative amyloid burden associated with these two conditions. Perhaps most significant is the finding that the caudate nucleus, which only contains diffuse Aβ, still has reduced IEG expression. This further implicates Aβ itself, rather than a mediator of neuro-inflammation, as the precipitator of this mRNA reduction. These findings again argue that the IEGs are very sensitive to the presence of amyloid, possibly indicating a direct interaction of the amyloid peptide with some mechanism regulating the expression of these genes.

One final critical issue in this investigation was the nature of the reductions seen in IEG expression. These genes are expressed at low levels when mice are in a resting state. However, upon induction by some stimulus, e.g. maximal electroconvulsive shock [MECS; (Cole et al. 1990)] or a novel environment (Guzowski et al. 2001), there is a rapid and robust increase in their expression, at which point they have dramatic effects on synapse structure and properties. This increase is transient so that within minutes to hours, some IEGs return to their basal expression level. By using a novel environment in which mice were allowed to investigate several new objects for 5 min, we were able to demonstrate a significant induction of the IEGs Arc, Nur77 and Zif268. We selected a 30 min duration as the time point because previous studies have shown that Arc and Nur77 reach a maximal level of expression by 30 min and begin to return to basal levels after 1 h (Lyford et al. 1995; Tang et al. 1997). We felt that genes that were induced maximally would provide the greatest opportunity to see any significant reductions in their expression. Previous studies have indicated that zif268 mRNA has a slightly greater half-life than other IEG RNAs (Guzowski et al. 2000, 2001), which would effectively increase basal levels, making it more difficult to detect differences due to a single behavioral event, possibly explaining the reason we were unable to see significant reductions in its expression. We had suspected that our standard transport and handling procedures prior to killing the mice were sufficient to induce the IEGs. Anticipating this, our standard protocol was to retrieve each mouse from the vivarium 30 min before pentobarbitol injection. The finding that only the induction of these genes was impaired in APP + PS1 mice confirms our suspicions. These observations emphasize the need to control the conditions leading up to killing in studies of gene expression in brain.

These data, when taken together with the findings that Aβ is precipitating reductions in gene expression, indicate that in amyloid-depositing APP + PS1 mice, the ability to induce IEG expression is suppressed. This suppression may be a direct effect of Aβ on a receptor altering signal transduction cascades [possibly the Ras/MAPK pathway or the Ca2+ signaling cascade (Dineley et al. 2001)]. Alternatively, Aβ deposition may impair increases in general synaptic activity presumed to result from exposure to environmental novelty, leading to less induction of these activity-dependent IEGs. The diminution of Na, K ATPase activity may permit normal resting activity in neurons, but may lead to failure when demands for ion pumping increase with elevated synaptic activity. Another possibility remains that these events are secondary to increased inflammation associated with glial activation, particularly given the considerable effects that cytokines can have on gene expression. However, at least for the IEGs, this seems unlikely as microglial activation and cytokine production are low in 6 month APP + PS1 mice, when the IEGs are already realizing significant decreases [Figs 1a and c; (Benzing et al. 1999; Mehlhorn et al. 2000; Gordon et al. 2001)]. Ultimately, the data presented here point to Aβ, or some direct effect of its presence in the brain, as the causal agent for suppressed induction of IEG expression.

The failure to identify reduced expression of rheb in APP + PS1 mouse hippocampus provides an example of one IEG that does not seem to be influenced by the presence of Aβ. Shank3, involved in formation of the post-synaptic density (Boeckers et al. 2002) along with calsyntenin-1 and synaptopodin (Dickey et al. 2003), also remains unchanged in the APP + PS1 mouse, eliminating the possibility of a generalized but exclusive loss of post-synaptic mRNAs, with axon terminals remaining intact and functional. Also, the two dendritic metabotropic glutamate receptors, mGluR1 and mGluR5, that have been linked to memory formation and LTP maintenance (Riedel et al. 2000), do not exhibit significant decreases (Petersen et al. 2002). Other genes with unchanged expression are MAP-2, thought to propagate growth cone formation of the axon (Gordon-Weeks 1991), and the Na, K ATPase γ subunit, which modulates the activity of the ATPase α subunit (Crambert and Geering 2003), demonstrating a specific reduction in the catalytic α subunit expression level. The stability of these other markers emphasizes the significance of those genes that do suffer from amyloid-related repression.

The recent finding by Hock et al. that vaccination with the amyloid peptide stabilized and, in some cases, improved cognitive ability in humans suffering from AD (Hock et al. 2003), argues that Aβ itself is likely involved in the development of memory dysfunction in AD beyond the effects on neuronal and synapse loss. The data presented here provide new evidence to support the idea that Aβ impedes the induction of genes critical for synaptic plasticity. The APP + PS1 transgenic mouse model therefore provides a means by which to understand how amyloid diminishes the functioning of essential memory-related molecular systems within the neuron. There is a dynamic nature of specific neuronal transcripts that lend to the ability of the brain to retain information, and it seems that amyloid disrupts this system in multiple ways. It is possible that as the IEG induction decreases, it reaches a point at which the strength of the subsequent memory trace is inadequate to modify the mouse's behavior. It has already been shown in multiple mouse models that amyloid deposition is associated with the decline of cognitive function (Chen et al. 2000; Gordon et al. 2001; Westerman et al. 2002; Heikkinen et al. 2002). These data provide the first hints of a molecular basis for this problem. The results herein suggest potential targets for therapy and prophylaxis of amyloid-associated dementias and, possibly, other forms of cognitive impairments that are to this point poorly understood at the level of gene expression.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This work was supported by AG15490 and AG18478 from NIH.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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