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Keywords:

  • ageing;
  • hippocampus;
  • immunohistochemistry;
  • messenger RNA;
  • neurogranin;
  • plasticity

Abstract

  1. Top of page
  2. Abstract
  3. Experimental Procedures
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Brain ageing is associated with a dysregulation of intracellular calcium (Ca2+) homeostasis which leads to deficits in Ca2+-dependent signalling pathways and altered neuronal functions. Given the crucial role of neurogranin/RC3 (Ng) in the post-synaptic regulation of Ca2+ and calmodulin levels, age-dependent changes in the levels of Ng mRNA and protein expression were analysed in 3, 12, 24 and 31-month-old mouse brains. Ageing produced significant decreases in Ng mRNA expression in the dorsal hippocampal subfields, retrosplenial and primary motor cortices, whereas no reliable changes were seen in any other cortical regions examined. Western blot indicated that Ng protein expression was also down-regulated in the ageing mouse brain. Analysis of Ng immunoreactivity in both hippocampal CA1 and retrosplenial areas indicated that Ng protein in aged mice decreased predominantly in the dendritic segments of pyramidal neurones. These data suggest that age-related changes of post-synaptic Ng in selected brain areas, and particularly in hippocampus, may contribute to altered Ca2+/calmodulin-signalling pathways and to region-specific impairments of synaptic plasticity and cognitive decline.

Abbreviations used
Ca2+

calcium

CaM

calmodulin

IR

immunoreactivity

LTP

long-term-potentiation

MAP2

microtubule-associated protein

Ng

neurogranin/RC3

PKC

protein kinase C

RA

retinoic acid

T3

thyroid hormone.

There is increasing evidence that disruption of calcium (Ca2+) homeostasis in the ageing brain greatly contributes to altered synaptic functioning and neurodegeneration (Landfield et al. 1992; Hartmann et al. 1994; Disterhoft et al. 1996; Thibault et al. 1998; Verkhratsky and Toescu 1998; Foster 1999). As many of the effects of Ca2+ are mediated via calmodulin (CaM), modifications in Ca2+/CaM homeostatic mechanisms would be expected to underlie marked changes in a number of neuronal processes, including synaptic plasticity (i.e. long-term potentiation (LTP); Malenka et al. 1989), regulation of gene expression (Rasmussen and Means 1989), maintenance of the cytoskeleton (Marcum et al. 1978), neuronal differentiation and synaptogenesis (Alvarez-Bolado et al. 1996), response to stress (Landry et al. 1988), and cell death (Dowd et al. 1991; Dawson et al. 1995). In this context, recent studies have indicated that the normal ageing process is associated with structural modifications of CaM (Gao et al. 1998), reducing its ability to activate target proteins, including the plasma membrane CaM-activated Ca2+-ATPase, kinases, phosphatases, cyclic nucleotide phosphodiesterase, nitric oxide synthase, adenylyl cyclase, ion channels and cytoskeletal proteins (Landry et al. 1988; Taussig and Gilman 1995; Molday 1996; Yao et al. 1996; Beckingham et al. 1998; Lee et al. 1999; Leonard et al. 1999; Prichard et al. 1999).

As CaM is a widespread transducer of Ca2+-signalling, it appears probable that the regulation of CaM itself may be controlled by peptides that function as intracellular reservoirs to stabilize the inactive form. There are at least two neurone-specific CaM-binding proteins, including GAP43 (also called neuromodulin; P57; B50) and neurogranin/RC3 (Ng) that function as downstream effectors to target the free Ca2+/CaM complex needed to activate CaM-dependent proteins at synaptic compartments (Baudier et al. 1989, 1991; Liu and Storm 1990; Watson et al. 1990; Coggins and Zwiers 1991; Slemmon and Martzen 1994; Gerendasy and Sutcliffe 1997). The interactions between these two proteins and CaM have been characterized biochemically (Cimler et al. 1985; Alexander et al. 1987; Liu and Storm 1990; Gerendasy et al. 1994; Martzen and Slemmon 1995; Sheu et al. 1996; Gerendasy and Sutcliffe 1997; Prichard et al. 1999; Huang et al. 2000). Both Ng and GAP43 release CaM in response to activation of protein kinase C (PKC) and local increase of free Ca2+ concentration, and it has been proposed that a selective enrichment of Ng within dendritic shafts and spines and GAP43 throughout axons would place them in an ideal position to release CaM in response to a transient Ca2+ signal (Van Lookeren Campagne et al. 1989; Watson et al. 1992; Martzen and Slemmon 1995; Gerendasy and Sutcliffe 1997; Rhoads and Friedberg 1997; Chakravarthy et al. 1999; Prichard et al. 1999). Consistent with a role for CaM-binding proteins in Ca2+/CaM-induced synaptic potentiation, an increase in the amount of phosphorylated form of Ng has been reported during the maintenance phase of LTP (Chen et al. 1997) and it has demonstrated that the post-synaptic injection of monoclonal antibodies against the phosphorylation site domain of Ng in hippocampal CA1 pyramidal cells prevents the induction of LTP (Fedorov et al. 1995). More recently, it has been shown that mice lacking Ng gene exhibited deficits in hippocampal synaptic plasticity and spatial learning impairments (Pak et al. 2000).

These observations are of particular interest as previous studies in rat brain indicated that ageing produces a decrease in the phosphorylated form of GAP43 in the hippocampus (Barnes et al. 1988) as well as a decrease in GAP43 immunoreactivity (IR) in selective regions associated with spatial memory and emotional behaviour (Casoli et al. 1996). Concerning post-synaptic Ng, our previous studies using RT-PCR indicated that ageing is associated with a decrease in Ng gene expression in the whole ageing mouse brain (Enderlin et al. 1997). However, whether normal brain ageing produces regionally selective changes in Ng mRNA and protein expression has yet to be elucidated.

To address this issue, we examined specific age-related changes in Ng mRNA expression in hippocampal and cortical regions of C57BL/6 mice aged 3, 12, 24 and 31 months by using an in situ hybridization study. The age-associated changes of Ng at protein level were investigated by using western blot analysis and immunohistochemistry. The comparative study of the distribution of Ng-IR in hippocampal CA1 subfield and the retrosplenial cortex of young and old mice revealed that age-related reductions of Ng-IR occur predominantly in the dendrites of pyramidal neurones. These data suggest that selective age-related alterations of Ng, at both mRNA and protein levels, may contribute, at least in part, to the decline of post-synaptic Ca2+/CaM-dependent mechanisms and may have significant implications for overall synaptic function and, in particular, for synaptic plasticity which is thought to underline learning and memory processes.

Experimental Procedures

  1. Top of page
  2. Abstract
  3. Experimental Procedures
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Animals

Young adult (3-month-old), mature (12-month-old) and aged (24- and 31-month-old) C57BL/6 male mice were obtained from IFFA-CREDO Laboratories (Lyon, France). They were housed with food and water available ad libitum in a constant temperature air-conditioned room maintained on a 12-h light–dark cycle.

Probe synthesis and in situ hybridization analysis

Mice of different age groups [3-month-old (n = 9); 12-month-old (n = 6); 24-month-old (n = 9) and 31-month-old (n= 3)] were decapitated and their brains were fixed overnight in 1% (w/v) paraformaldehyde in 0.1 m phosphate buffer (pH 7.4), immersed in the same buffer containing 20% sucrose, and then stored at − 80°C. Serial coronal sections (20 µm) were thaw-mounted onto gelatine-coated-slides and stored at − 80°C until processing.

Hybridization was performed using a 60-mer oligodeoxyribonucleotide probe (Isoprim, France) which is complementary to positions 40–99 of the 5′coding region of transcript 140 (Rhyner et al. 1990). The probe was end-labelled with α[35S]deoxy-ATP (Amersham, Arlington Heights, IL, USA) using terminal deoxynucleotidyl transferase (New England Nuclear, Boston, MA, USA) and hybridizations were carried out with minor modifications according to Laurent-Demir et al. (2000). The slides were hybridized overnight at 42°C in the hybridization solution containing [35S]dATP-labelled probe at a concentration of 5 × 105 cpm/100 µL. The hybridized sections were washed in 1 × sodium saline citrate buffer (SSC; 45 min), two times at 55°C (1 × SSC and 0.5 × SSC; 45 min for each) and once in 0.5 × SSC at room temperature (22°C; 1 h). Then, the slides were quickly dehydrated in ethanol, air-dried, exposed to Hyperfilm β-max autoradiography films (Amersham) for 2 days at room temperature and then developed. All slides were processed at the same time under identical experimental conditions.

Quantification and image analysis

For assessment of relative amounts of Ng mRNA in various regions of mouse brain sections, X-ray autoradiographs were digitized using an image analysis system (Densirag software, Biocom, Les Ulis, France). Separate optical densities (OD) measurements within a particular brain region were made using three consecutive sections per animal which were anatomically identified according to Franklin and Paxinos (1997). Background OD was substracted from each image. The mRNA density within each brain region in 12-, 24- and 31-month-old mice was expressed as a percentage of the mean mRNA density observed in the 3-month-old group within the same brain region. Differences in group-mean densities in each region were analysed by anova followed by Scheffe F-tests for post hoc comparisons. The level of significance was set at p < 0.05.

Western blot analysis

For analysis of Ng protein, 3- and 24-month-old mice (n = 6 mice/group) were decapitated and the brains removed and stored at − 80°C for subsequent analysis. Western blot analysis was performed according to the procedure of Watson et al. (1990) with modifications. Frozen brain tissue from either young or aged mice was homogenized in a solution containing 0.16 m NaCl, 11 mm sodium phosphate (pH = 7.4), and 1 mm phenylmethylsulfonyl fluoride. Proteins were separated electrophoretically by size on 12% denaturing sodium dodecyl sulfate (SDS)-polyacrylamide gel and transferred to nitrocellulose membrane by electroblotting. After blocking in phosphate buffer containing 5% fat-free dry milk, 145 mm NaCl and 1% Tween-20, the nitrocellulose sheets were incubated overnight with either affinity-purified polyclonal rabbit anti-Ng antibodies (1 : 3000; Affiniti Research Ltd, Exeter, UK) or monoclonal mouse anti-β-actin antibodies (1 : 8000; Sigma, St Louis, MO, USA). After several washes, protein bands were visualized by alkaline phosphatase-conjugated anti-rabbit or anti-mouse IgG (Sigma). The staining intensity of protein bands was determined using an image analyser (Vilber Loumat, Bio-1D, Marne-La-Vallée, France). The relative levels in Ng and actin proteins in aged mice were determined as percentage of Ng and actin in young mice. Statistical differences between the relative protein levels of each group were analysed using anova followed by Scheffe F-test for post hoc comparisons. The level of significance was set at p < 0.05.

Immunohistochemistry

Mice of 3 and 26 months (n = 5/group) were anaesthetized with Avertin (1%, i.p.) and perfused transcardially with freshly prepared paraformaldehyde (4%) in phosphate buffer. After post-fixation overnight at 4°C, vibratome coronal brain sections (50 µm) were cut, rinsed in phosphate buffered saline (PBS) and incubated with 0.2% H2O2 to block endogenous peroxidase. After three rinses, sections were incubated in a blocking solution (PBS containing 2% bovine serum albumin/2% normal goat serum/0.1% Triton X-100) for 1 h at room temperature. Sections were then incubated at 4°C for 2 days with affinity-purified rabbit polyclonal anti-Ng antibodies (Affiniti) diluted at 1 : 2000. As a control, sections were also incubated with 1 : 5000 dilution of a mouse monoclonal anti-MAP2 (Sigma). After being rinsed, sections were incubated for 2 h at room temperature in biotinylated goat anti-rabbit or goat anti-mouse IgG, rinsed, and then incubated for 2 h in avidin–biotinylated horseradish peroxidase complex (Vector Laboratories, Burlingame, CA, USA). After several rinses, the peroxidase reaction end-product was developed with 0.025% diaminobenzidine tetrahydrochloride; 0.015% glucose oxidase and 2% nickel ammonium sulfate (Shu et al. 1988).

Results

  1. Top of page
  2. Abstract
  3. Experimental Procedures
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Effects of ageing on Ng mRNA levels

Figures 1 and 2 show the effects of ageing on Ng mRNA expression in the dorsal part of the hippocampus from young, mature and old mice. Figure 1 illustrates the distribution pattern of Ng mRNA in both the dorsal hippocampus of 3- and 24-month-old mice. The expression of Ng mRNA in the mouse brain, irrespective of age, was similar to previous descriptions (Represa et al. 1990; Neuner-Jehle et al. 1995). In the dorsal hippocampus, Ng mRNA was uniformly expressed across all CA1–CA3 subfields and in the dentate gyrus. Quantitative analysis in all hippocampal subfields revealed that the message levels of Ng in the 24- and 31-month-old groups were significantly reduced with respect of the 3- and 12-month-old groups, which otherwise did not differ from each other (Fig. 2). A two-factor anova conducted on these data revealed a significant main effect of age (F3,23 = 8.47; p < 0.001) and region (F2,46 = 6.4; p < 0.001) on hippocampal Ng mRNA content, but with no significant age X region interaction (F6,46 = 1.98; p < 0.05). Levels of Ng mRNA were maintained over the first 12 months of age and thereafter, the Ng message level exhibited a significant decrease at 24 and 31 months of age. When the effect of ageing on Ng mRNA content in each individual subfield was analysed separately, a significant main effect of age was found in the dentate gyrus (F3,23 = 12.34; p < 0.0001) as well as in the CA1 (F3,23 = 8.2; p < 0.001) and CA3 (F3,23 = 3.63; p < 0.05) subfields. In the dorsal hippocampal subfields of 31-month-old mice, decreased levels of Ng mRNA were especially evident in the dentate gyrus and subfield CA1 (42 ± 3% and 40 ± 2%, respectively) whereas the age-related difference in subfield CA3 was less marked (30 ± 7.2%). The age-related changes were also evaluated in several regions of the cerebral cortex in which Ng mRNA was strongly to moderately expressed. Although there was only a small age-related decrease in Ng mRNA expression across cortical regions examined, the age-related changes of Ng mRNA in 24-month-old mice were statistically significant in the primary motor (20 ± 2.7%; p > 0.05) and retrosplenial cortices (24 ± 4%; p < 0.05) (Fig. 3).

image

Figure 1. Dark-field photomicrographs illustrating the distribution patterns of Ng mRNA expression in the dorsal hippocampus of (a) 3- and (b) 24-month-old mice as visualized by in situ hybridization histochemistry. Note that ageing significantly reduced Ng mRNA levels in dorsal hippocampal subfields. Pyramidal cell layers of CA1 and CA3 fields (CA1 and CA3, respectively), dentate granular cells (DG).

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image

Figure 2. Changes in Ng mRNA expression in the dorsal hippocampal subfields of 3-, 12-, 24- and 31-month-old mice. Histograms represent the optical density values (mean ± SEM) measured in the hippocampal fields for each group normalized to the 3-month-old group. An anova analysis followed by post hoc Scheffe's test showed that 24- and 31-month-old mice displayed significantly lower levels of Ng mRNA expression in all hippocampal subfields, relative to young animals (*p < 0.05, **p < 0.01). Pyramidal cell layers of CA1 and CA3 fields (CA1 and CA3, respectively), granular cells of dentate gyrus (DG).

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image

Figure 3. Age-related changes in Ng mRNA levels within various cortical regions. Histograms represent the optical density values (mean ± SEM) measured in different cortical regions for either 3-, 12-, 24- or 31-month-old groups normalized to 3-month-old group. Note that age-related changes in Ng mRNA levels were statistically indistinguishable in all of the cortical regions examined, except in the primary motor (M1) and retrosplenial (RSA) cortices. Cingulate (Cg), primary somatosensory (S1), posterior parietal (PPta) and auditory (Au) cortices. (*p < 0.05)

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Effects of ageing on Ng protein level

Western blot analysis

In order to extend the results from in situ hybridization, tissue homogenates derived from mouse brains at 3 and 24 months of age were subjected to western blot analysis with polyclonal anti-Ng antibodies to determine whether decrease in Ng mRNA expression is associated with reduced level of Ng protein in the aged brain. Figure 4 shows the effects of age on Ng-IR in homogenized brains from young or aged mice. Densitometric analyses of Ng-IR revealed that Ng-IR (detected as a single band migrating at 15 kDa) was significantly reduced in 24-month-old mice, as compared with young mice (64.3 ± 2% vs. 100 ± 5%; p < 0.0001). In contrast, immunoblots of young and aged homogenized brains with β-actin antibodies revealed no age-related differences between groups in the intensity of β-actin (detected as a single band migrating at 42 kDa).

image

Figure 4. Effects of ageing on Ng and β-actin immunoreactivities in whole homogenized brains from 3- and 24-month-old mice. (a) An aliquot of either Ng or β-actin (15 µg of protein/lane) was run on a 12% polyacrylamide gel, the protein transferred and IR for either Ng or β-actin was probed as described under Experimental Procedures. Lanes (1–2) and (3–4) represent the β-actin and Ng samples, respectively. Immunoreactive bands for β-actin and Ng are apparent at 42 kDa and 15 kDa, respectively. Mol. Wt. (kDa) markers are indicated at left margin of the figure. (b) Histograms represent the relative intensities of the Ng band on immunoblots from young and aged mice. Data represent means ± SEM for six animals at each age. The band for the 3-month group was considered to be 100% and the relative intensity of this band for the old group was calculated. An anova analysis followed by post hoc Scheffe's test showed that 24-month-old mice displayed significantly reduced levels of Ng, relative to the young animals (***p < 0.0001).

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Immunocytochemical analysis

To evaluate age-related changes in Ng protein at the cellular level, immunocytochemical experiments were performed in both the CA1 subfield and retrosplenial cortex of 3- and 26-month-old mice. Irrespective of age, the overall pattern of Ng-IR in the CA1 subfield was similar to the one previously described (Represa et al. 1990; Watson et al. 1992; Neuner-Jehle et al. 1995). Both the somata and the long apical dendrites of CA1 pyramidal cells within stratum pyramidale and stratum radiatum displayed strong Ng-IR (Fig. 5a). In comparison to young mice, Fig. 5(b) indicated that Ng-IR was also present in the somata of aged CA1 pyramidal neurones, but a loss of Ng-IR with ageing occured in the proximal and distal parts of their corresponding dendrites. An examination of the retrosplenial cortex of young and aged mice revealed that cortical pyramidal neurones displayed similar age-related differences in the intensity of Ng immunolabelling (Figs 5c and d). In aged mice, Ng-IR was intense in pyramidal cell bodies, whereas a markedly lower intensity of Ng-IR was found within the dendritic segments (Fig. 5d), compared with young animals (Fig. 5c).

image

Figure 5. Distribution of Ng immunoreactivity in pyramidal neurones of hippocampal CA1 subfield (a,b) and retrosplenial cortex (c,d). Panels (a and c) and (b and d) are from coronal sections of 3- and 26-month-old brains, respectively. In young mice, Ng exhibited prominent staining in perikarya and dendritic segments (arrows) of pyramidal neurones, whereas in old animals, ageing resulted in a marked decline of Ng staining in both proximal and distal dendritic segments. Bars = 20 µm (a,b) and 50 µm (c,d).

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Additional analysis using MAP2 as a dendritic structural marker was investigated in order to determine whether the observed changes in Ng-IR were associated with generalized dendritic alterations in the aged animal. Analyses of adult and aged hippocampal sections revealed no significant difference in the intensity of MAP2-IR in perikarya and dendritic segments of CA1 pyramidal neurones (Figs 6a and b) as well as in any other brain region examined.

image

Figure 6. Distribution of MAP2 immunoreactivity in coronal sections from (a) 3- and (b) 26-month-old mice. In hippocampal CA1 subfield, no significant difference was observed in either perikarya or the dendritic segments of pyramidal neurones between young and aged mice. Bar: 20 µm.

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Discussion

  1. Top of page
  2. Abstract
  3. Experimental Procedures
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The present study demonstrates that both the message and protein levels of the CaM-binding protein Ng were reduced during ageing in the mouse brain. Our present results indicate that (1) ageing is associated with region-specific alterations of Ng mRNA expression; (2) decreased levels in Ng mRNA were associated with reduced amount of Ng-protein in whole brains of aged mice; (3) at the cellular level, age-related changes in Ng-IR occurred primarily within dendritic processes in the pyramidal neurones of both the CA1 subfield and the retrosplenial cortex. We suggest that age-related alterations of Ng in specific brain regions have the potential to be an important contributing factor in the decline of Ca2+/CaM homeostasis and may provide some insight into the pathophysiological changes that occur in the brains of aged animals.

Molecular mechanisms involved in the age-related decrease of Ng expression

These results complement and further extend our earlier findings using PCR analysis revealing reduced Ng mRNA expression homogenates of whole aged mouse brain (and hippocampi), an effect which was associated with decreased mRNA expression for retinoid (RA) and thyroid hormone (3,5,3′-triiodothyronine, T3) receptors (Enderlin et al. 1997; Etchamendy et al. 2001). As responsive elements for T3 (T3RE) and RA (RARE) are present in the promoter region of the human and rat Ng genes (Iniguez et al. 1994; Martinez de Arrieta et al. 1999; Morte et al. 1999), the down-regulation of RA (RAR; RXR) or T3 (TR) receptors during the ageing process may have deleterious effects on the regulation of Ng expression. This hypothesis is supported by previous findings suggesting that: (1) Ng mRNA expression is affected by T3 status in developing and adult brains and (2) administration of T3 in hypothyroid rats produces a significant transcriptional increase of Ng mRNA (Iniguez et al. 1992; 1996; Guadano-Ferraz et al. 1997; Morte et al. 1997). Furthermore, we have recently demonstrated that acute administration of RA in old mice reverses RAR, RXR and TR hypoexpressions in parallel with restoring Ng mRNA levels (Enderlin et al. 1997; Etchamendy et al. 2001). Immunohistochemical studies of the neostriatum (Watson et al. 1992), cerebral cortex and hippocampus (Neuner-Jehle et al. 1995) of adult rats revealed a preferential distribution of Ng protein in spines and dendrites. As dendritic spine density in hippocampal neurones are highly sensitive to T3 dysregulation (Gould et al. 1990), we suggest that alteration in T3- or RA-mediated signalling pathway in aged mice may have deleterious consequences on Ng translocation to dendrites and, thus, may partly explain the age-related decrease of Ng-protein in dendritic segments of CA1 pyramidal neurones.

There is also evidence that PKC isoforms regulate the activity of the Ng gene promoter (Sato et al. 1995). Furthermore, it has been shown previously that the maintenance phase of LTP in hippocampal area CA1 is associated with an increase in PKC-mediated phosphorylation of Ng (Fedorov et al. 1995; Chen et al. 1997; Ramakers et al. 2000). These findings, along with other reports of dysregulation of PKC translocation in aged rodents (Martini et al. 1994; Battaini et al. 1995; Undie et al. 1995; Colombo et al. 1997), strongly suggest that alterations in the PKC-signalling cascade with increasing age might also contribute to the age-related declines in Ng mRNA and protein. In this context, age-related deficit in PKC activity is associated with decreased phosphorylation state of downstream targets, such as GAP43 (Barnes et al. 1988; Gianotti et al. 1993) and synapsin (Eckles et al. 1997). Because Ng is selectively translocated to dendrites where it is translated locally in response to synaptic activity (Chang et al. 1997), we postulate therefore that there might also be age-related deficits in phosphorylated Ng. Further work will be necessary to determine whether differential localizations and dynamics of phosphorylated/unphosphorylated Ng in aged brain may influence age-related deficits in CaM-regulated cellular functions.

Functional implications of changes in Ng production with ageing

Our immunohistochemical data revealed that age-related decreases in Ng-IR occur predominantly within the proximal and distal segments of the pyramidal dendrites in both the hippocampal CA1 and retrosplenial regions. The reduced Ng-IR cannot be attributed to dendritic structural alterations as the soma/dendritic-specific cytoskeleton protein MAP2 did not exhibit any significant age-related difference in staining intensity. In view of the functional role of Ng, accumulating evidence suggests that dendritic pool of Ng functions as a calmodulin ‘sponge’ releasing or binding CaM for localized responses on Ca2+ influx triggered by glutamate receptors, voltage-sensitive Ca2+ channels and electrical stimulation (Baudier et al. 1991; Ramakers et al. 1995; Chen et al. 1997; Rodriguez-Sanchez et al. 1997; Li et al. 1999). Consistent with our data, previous findings indicated that the membrane-bound pool of CaM also declined with age (Zaidi et al. 1998). A sequel to the age-related alterations in both Ng and CaM is that activation of downstream Ca2+/CaM-regulated targets, such as adenylyl cyclases (Taussig and Gilman 1995; Smit and Iyengar 1998), kinase (Braun and Schulman 1995; Klee et al. 1998), nitric oxide synthase (Bredt and Snyder 1994; Lee and Stull 1998) and NMDA receptors (Ehlers et al. 1996; Hisatsune et al. 1997; Zhang and Huganir 1999), may not occur in a normal fashion. In this context, it has been reported that (i) the activity of Ca2+/CaM-dependent kinase II is significantly reduced in aged rat hippocampus (Mullany et al. 1996) and (ii) there is a progressive decline in the ability of CaM isolated from aged rat brain or aged erythrocytes to activate plasma membrane Ca2+-ATPase (Clark and Shohet 1985; Gao et al. 1998), suggesting that reductions of Ng in aged rodent brain may influence the activity of CaM and CaM-dependent targets.

Ng has been implicated in molecular mechanisms underlying synaptic plasticity associated with information processing in the CA1 subfield (Fedorov et al. 1995; Ramakers et al. 2000). In addition, knock-out studies have shown that mice lacking the Ng gene exhibit severe deficits in hippocampus-dependent spatial memory and hippocampal LTP (Pak et al. 2000). Thus, we suggest that the observed age-related alterations in the intradendritic pool of Ng may have major implications in the ability of neuronal networks to encode information. Overall, these data, along with other studies showing that advanced age is associated with a decline in spatial/relational memory capacities dependent on hippocampal processing (Barnes 1979, 1988; De Toledo-Morrell et al. 1988; Rapp and Amaral 1992; Gallagher et al. 1993; Gabrieli 1996; Marighetto et al. 1999), suggest that altered Ng may contribute substantially to age-associated memory disorders related to hippocampal dysfunction. This hypothesis is supported by recent data in our laboratory showing that restoration of the age-related hypoexpression of Ng and retinoid receptors by RA treatment eliminates the declarative/relational memory impairment seen in aged mice and also alleviates the age-related impairment in hippocampal CA1 LTP (Etchamendy et al. 2001).

In conclusion, our findings indicate that Ng at both mRNA and protein levels is subject to a regionally selective dysregulation in aged mouse brain. As Ng plays a crucial role in regulating the dendritic membrane-bound CaM pool, age-related changes in Ng expression may, at least in part, account for alterations of the neuronal Ca2+/CaM regulation and may have significant implications for age-related disturbances in the processes underlying Ca2+-dependent post-synaptic functions.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Experimental Procedures
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This research was supported by the CNRS and ARMA. The authors wish to thank Dr T. Durkin for english revision, Dr S. Alfos for technical assistance and M. Chaigniau for iconography.

References

  1. Top of page
  2. Abstract
  3. Experimental Procedures
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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