Distribution of acetylcholinesterase in the hippocampal formation of the Atlantic white-sided dolphin (Lagenorhynchus acutus).

The cetacean hippocampal formation has been noted to be one of the smallest relative to brain size of all mammals studied. This region, comprised of the dentate gyrus, hippocampus proper, subiculum, presubiculum, parasubiculum and the entorhinal cortex, is important in learning, memory, and navigation. There have been a number of studies detailing the distribution of acetylcholinesterase (AChE) in the hippocampal formation of terrestrial mammals with the goal of gaining a greater understanding of some aspects of the cholinergic innervation to this region, as well as its parcellation. The present study was undertaken to describe the organization, cytoarchitecture, and distribution of AChE in the hippocampal formation of the Atlantic white-sided dolphin (AWSD) with the view to understand similarities and differences between this aquatic mammal and terrestrial mammals. Nissl-staining demonstrated cytoarchitecture of the hippocampal formation in the AWSD comparable to that reported in other cetaceans. In addition, the AWSD had a rich pattern of AChE staining that distinctly varied between regions and laminae. A number of differences in the distribution of AChE staining in areas comparable to those of terrestrial species reported suggested possible alterations in connectivity of this region. Overall, however, AChE-staining suggested that cholinergic innervation, neural pathways and function of the hippocampal formation of the AWSD is conserved, similar to other mammals.

Neurotoxic astrocytes express the d-serine synthesizing enzyme, serine racemase, in Alzheimer's disease.

Although ß-amyloid plaques are a well-recognized hallmark of Alzheimer's disease (AD) neuropathology, no drugs reducing amyloid burden have shown efficacy in clinical trials, suggesting that once AD symptoms emerge, disease progression becomes independent of Aß production. Reactive astrocytes are another neuropathological feature of AD, where there is an emergence of neurotoxic (A1) reactive astrocytes. We find that serine racemase (SR), the neuronal enzyme that produces the N-methyl-d-aspartate receptor (NMDAR) co-agonist d-serine, is robustly expressed in A1-reactive neurotoxic astrocytes in the hippocampus and entorhinal cortex of AD subjects and an AD rat model. Furthermore, we observe intracellular signaling changes consistent with increased extra-synaptic NMDAR activation, excitotoxicity and decreased neuronal survival. Thus, reducing neurotoxic d-serine release from A1 inflammatory astrocytes could have therapeutic benefit for mild to advanced AD, when anti-amyloid strategies are ineffective.

PARP inhibition in vivo blocks alcohol-induced brain neurodegeneration and neuroinflammatory cytosolic phospholipase A2 elevations.

Chronic alcoholism promotes brain damage that impairs memory and cognition. High binge alcohol levels in adult rats also cause substantial neurodamage to memory-linked regions, notably, the hippocampus (HC) and entorhinal cortex (ECX). Concurrent with neurodegeneration, alcohol elevates poly (ADP-ribose) polymerase-1 (PARP-1) and cytosolic phospholipase A2 (cPLA2) levels. PARP-1 triggers necrosis when excessively activated, while cPLA2 liberates neuroinflammatory ω-6 arachidonic acid. Inhibitors of PARP exert in vitro neuroprotection while suppressing cPLA2 elevations in alcohol-treated HC-ECX slice cultures. Here, we examined in vivo neuroprotection and cPLA2 suppression by the PARP inhibitor, veliparib, in a recognized adult rat model of alcohol-binging. Adult male rats received Vanilla Ensure containing alcohol (ethanol, 7.1 ±â€¯0.3 g/kg/day), or control (dextrose) ±â€¯veliparib (25 mg/kg/day), by gavage 3x daily for 4 days. Rats were sacrificed on the morning after the final binge. HC and ECX neurodegeneration was assessed in fixed sections by Fluoro-Jade B (FJB) staining. Dorsal HC, ventral HC, and ECX cPLA2 levels were quantified by immunoblotting. Like other studies using this model, alcohol binges elevated FJB staining in the HC (dentate gyrus) and ECX, indicating neurodegeneration. Veliparib co-treatment significantly reduced dentate gyrus and ECX neurodegeneration by 79% and 66%, respectively. Alcohol binges increased cPLA2 in the ventral HC by 34% and ECX by 72%, which veliparib co-treatment largely prevented. Dorsal HC cPLA2 levels remained unaffected by alcohol binges, consistent with negligible FJB staining in this brain region. These in vivo results support an emerging key role for PARP in binge alcohol-induced neurodegeneration and cPLA2-related neuroinflammation.

A Potential Role for α-Amylase in Amyloid-ß-Induced Astrocytic Glycogenolysis and Activation.

BACKGROUND: Astrocytes produce and store the energy reserve glycogen. However, abnormal large glycogen units accumulate if the production or degradation of glycogen is disturbed, a finding often seen in patients with Alzheimer's disease (AD). We have shown increased activity of glycogen degrading α-amylase in AD patients and α-amylase positive glial cells adjacent to AD characteristic amyloid-ß (Aß) plaques. OBJECTIVES: Investigate the role of α-amylase in astrocytic glycogenolysis in presence of Aß. METHODS: Presence of α-amylase and large glycogen units in postmortem entorhinal cortex from AD patients and non-demented controls were analyzed by immunohistological stainings. Impact of different Aß42 aggregation forms on enzymatic activity (α-amylase, pyruvate kinase, and lactate dehydrogenase), lactate secretion, and accumulation of large glycogen units in cultured astrocytes were analyzed by activity assays, ELISA, and immunocytochemistry, respectively. RESULTS: AD patients showed increased number of α-amylase positive glial cells. The glial cells co-expressed the astrocytic marker glial fibrillary acidic protein, displayed hypertrophic features, and increased amount of large glycogen units. We further found increased load of large glycogen units, α-amylase immunoreactivity and α-amylase activity in cultured astrocytes stimulated with fibril Aß42, with increased pyruvate kinase activity, but unaltered lactate release as downstream events. The fibril Aß42-induced α-amylase activity was attenuated by ß-adrenergic receptor antagonist propranolol. DISCUSSION: We hypothesize that astrocytes respond to fibril Aß42 in Aß plaques by increasing their α-amylase production to either liberate energy or regulate functions needed in reactive processes. These findings indicate α-amylase as an important actor involved in AD associated neuroinflammation.

Glutamine synthetase in astrocytes from entorhinal cortex of the triple transgenic animal model of Alzheimer's disease is not affected by pathological progression.

Astrocytes are fundamental for brain physiology and pathology, including Alzheimer's disease (AD). Among their functions, the maintenance of glutamate balance via the glutamate-glutamine (Glu-Gln) shuttle is critical for both normal cognitive functions and excitotoxicity relevant for AD progression. Astroglial glutamine synthetase (GS), converting glutamate to glutamine, is a key element in the Glu-Gln cycle. The entorhinal cortex (EC) is the brain area earliest affected in human AD. We have recently reported an early astrocytic atrophy in the EC in triple transgenic animal model of AD (3×Tg-AD). Here, we studied and analysed whether the changes in astrocytic morphology coincides with alterations of the Glu-Gln cycle by determining astrocytic GS. We found that the numerical density of GS-immunoreactive (GS-IR) cells as well as GS content (measured by optical density, OD) remained constant between 1 and 12 months of age, independent of the presence of senile plaques. Dual labelling images revealed GS-IR, GFAP-IR, GS/GFAP-IR subsets of astroglia. Despite the evident decrease in GFAP-IR surface and volume, the surface and volume of GS-IR and GS/GFAP-IR cells remained unchanged. Therefore, reduced GFAP presence obvious in the progression of AD from early stages does not impair upon glutamate homeostasis in the EC of 3×Tg-AD mice. Our data also indicate distinct functional populations of astrocytes, which may undergo specific remodelling during AD progression.

Caspase-6 activity predicts lower episodic memory ability in aged individuals.

Caspase-6 (Casp6), a cysteinyl protease that induces axonal degeneration, is activated early in Alzheimer Disease (AD) brains. To determine whether Casp6 activation is responsible for early cognitive impairment, we investigated the abundance of Casp6 activity, paired helical filament-1 (PHF-1) phosphorylated Tau and amyloid beta peptide (Aß) pathology by immunohistochemistry in the hippocampal formation of aged non-cognitively impaired (NCI) individuals. Casp6 activity was restricted to the entorhinal cortex (ERC) and CA1 regions of the hippocampus. Pathology scores were then correlated with cognitive scores obtained within 1 year of death. Regression analyses revealed that ERC and CA1 Casp6 activity were the main contributor to lower episodic memory performance, whereas ERC PHF-1 pathology predicted lower semantic and working memory performance. Aß did not correlate with any of the cognitive tests. Because Casp6 activity and PHF-1 pathology are intimately associated with AD pathology and memory decline is an early event in AD, we conclude that Casp6 activity and PHF-1 immunoreactivity in ERC identifies aged individuals at risk for developing AD.

G9a/GLP histone lysine dimethyltransferase complex activity in the hippocampus and the entorhinal cortex is required for gene activation and silencing during memory consolidation.

Learning triggers alterations in gene transcription in brain regions such as the hippocampus and the entorhinal cortex (EC) that are necessary for long-term memory (LTM) formation. Here, we identify an essential role for the G9a/G9a-like protein (GLP) lysine dimethyltransferase complex and the histone H3 lysine 9 dimethylation (H3K9me2) marks it catalyzes, in the transcriptional regulation of genes in area CA1 of the rat hippocampus and the EC during memory consolidation. Contextual fear learning increased global levels of H3K9me2 in area CA1 and the EC, with observable changes at the Zif268, DNMT3a, BDNF exon IV, and cFOS gene promoters, which occurred in concert with mRNA expression. Inhibition of G9a/GLP in the EC, but not in the hippocampus, enhanced contextual fear conditioning relative to control animals. The inhibition of G9a/GLP in the EC induced several histone modifications that include not only methylation but also acetylation. Surprisingly, we found that downregulation of G9a/GLP activity in the EC enhanced H3K9me2 in area CA1, resulting in transcriptional silencing of the non-memory permissive gene COMT in the hippocampus. In addition, synaptic plasticity studies at two distinct EC-CA1 cellular pathways revealed that G9a/GLP activity is critical for hippocampus-dependent long-term potentiation initiated in the EC via the perforant pathway, but not the temporoammonic pathway. Together, these data demonstrate that G9a/GLP differentially regulates gene transcription in the hippocampus and the EC during memory consolidation. Furthermore, these findings support the possibility of a role for G9a/GLP in the regulation of cellular and molecular cross talk between these two brain regions during LTM formation.

Developmental iodine deficiency and hypothyroidism reduce phosphorylation of calcium/calmodulin-dependent kinase II in the rat entorhinal cortex.

Iodine is essential for the synthesis of triiodothyronine (T3) and thyroxine (T4). Iodine deficiency leads to inadequate thyroid hormone. Hypothyroidism induced by iodine deficiency during gestation and postnatal period leads to cognitive deficits in learning and memory. However, the mechanism underlying these deficits is unclear. Calcium-dependent calmodulin kinase II (CaMKII) known as a potential memory molecule regulates important neuronal functions including learning and memory. Recent studies have shown that hypothyroidism alters phosphorylation of CaMKII in hippocampus or even in sympathetic ganglia of rats. Though the entorhinal cortex (EC) is an important functional structure within the neuronal network responsible for learning and memory, little is known about the effect of hypothyroidism on phosphorylation of CaMKII in the EC. Here, we report that iodine deficiency and propylthiouracil treatment through gestation and lactation reduce phosphorylation of CaMKII in the EC of pups. The increase of calcineurin, as well as reduction of neurogranin and calmodulin, may account for the reduced phosphorylation of CaMKII induced by developmental iodine deficiency and hypothyroidism. These findings in the EC may contribute to understanding the mechanisms that underlie impairment of learning and memory induced by developmental iodine deficiency and hypothyroidism.

Mitochondrial ATP-synthase in the entorhinal cortex is a target of oxidative stress at stages I/II of Alzheimer's disease pathology.

Oxidative stress has been implicated in the pathogenesis of several neurodegenerative diseases including Alzheimer's disease (AD). Several proteins have been identified as targets of oxidative damage in AD dementia (usually stages V/VI of Braak) and in subjects with mild cognitive impairment associated with middle stages of AD pathology (stage IV of Braak). In this study, we investigate whether brain proteins are locally modified by oxidative stress at the first stages of AD-related pathology when morphological lesions are restricted to the entorhinal and transentorhinal cortices of neurofibrillary pathology (stages I/II of Braak). Using a proteomic approach, we show that the alpha subunit of the mitochondrial adenosine triphosphate (ATP)-synthase is distinctly lipoxidized in the entorhinal cortex at Braak stages I/II compared with age-matched controls. In addition, ATP-synthase activity is significantly lower in Braak stages I/II than age-matched control, while electron transport chain, expressed by the mitochondrial complex I activity, remains not affected. This is the first study showing oxidative damage in the first stage, and clinically silent period, of AD-related pathology characterized by entorhinal and transentorhinal tauopathy.

Acute and chronic changes in glycogen phosphorylase in hippocampus and entorhinal cortex after status epilepticus in the adult male rat.

Glial cells provide energy substrates to neurons, in part from glycogen metabolism, which is influenced by glycogen phosphorylase (GP). To gain insight into the potential subfield and laminar-specific expression of GP, histochemistry can be used to evaluate active GP (GPa) or totalGP (GPa + GPb). Using this approach, we tested the hypothesis that changes in GP would occur under pathological conditions that are associated with increased energy demand, i.e. severe seizures (status epilepticus or 'status'). We also hypothesized that GP histochemistry would provide insight into changes in the days and weeks after status, particularly in the hippocampus and entorhinal cortex, where there are robust changes in structure and function. One hour after the onset of pilocarpine-induced status, GPa staining was reduced in most regions of the hippocampus and entorhinal cortex relative to saline-injected controls. One week after status, there was increased GPa and totalGP, especially in the inner molecular layer, where synaptic reorganization of granule cell mossy fibre axons occurs (mossy fibre sprouting). In addition, patches of dense GP reactivity were evident in many areas. One month after status, levels of GPa and totalGP remained elevated in some areas, suggesting an ongoing role of GP or other aspects of glycogen metabolism, possibly due to the evolution of intermittent, recurrent seizures at approximately 3-4 weeks after status. Taken together, the results suggest that GP is dynamically regulated during and after status in the adult rat, and may have an important role in the pilocarpine model of epilepsy.

Exposure to enriched environment improves spatial learning performances and enhances cell density but not choline acetyltransferase activity in the hippocampus of ventral subicular-lesioned rats.

The authors demonstrated the efficacy of enriched housing conditions in promoting the behavioral recovery and neuronal survival following subicular lesion in rats. Chemical lesioning of the ventral subiculum impaired the spatial learning performances in rats. The lesion also induced a significant degree of neurodegeneration in the CA1 and CA3 areas of the hippocampus and entorhinal cortex. Exposure to enriched housing conditions improved the behavioral performance and partially attenuated the neurodegeneration in the hippocampus. The choline acetyl transferase (ChAT) activity in the hippocampus remained unchanged following ventral subicular lesion and also following exposure to an enriched environment. The study implicates the effectiveness of activity-dependent neuronal plasticity induced by environmental enrichment in adulthood following brain insult.

Activation of caspase-6 in aging and mild cognitive impairment.

Active caspase-6 (Csp6) and Tau cleaved by Csp6 (TauDeltaCsp6) are abundant in neuritic plaques (NPs), neuropil threads (NPTs), and neurofibrillary tangles (NFTs) in end-stage Alzheimer's disease (AD) (Guo H, Albrecht S, Bourdeau M, Petzke T, Bergeron C, LeBlanc AC: Active caspase-6 and caspase-6 cleaved Tau in neuropil threads, neuritic plaques and neurofibrillary tangles of Alzheimer's disease. Am J Pathol 2004, 165:523-531). The goal of this study was to determine whether active Csp6 is present in young and aged noncognitively impaired (NCI); aged mild cognitively impaired (MCI); and aged mild, moderate, severe, and very severe AD individuals. Csp6 activity was assessed with anti-p20Csp6 and TauDeltaCsp6 immunoreactivity. Active Csp6 is present in NFTs, NPTs, and NPs at all stages of AD. Active Csp6 is present in NFTs of all MCI cases and present in NPTs and NPs of some MCI cases. Active Csp6 is present in NFTs and NPTs of all NCI cases but is absent in younger cases. The level of TauDeltaCsp6-positive NFTs and NPTs correlates inversely with global cognitive scores in NCI individuals. Therefore, Csp6 activity can occur with aging in the absence of AD and is always associated with clinical and pathological features of confirmed AD cases. Given the ability of active Csp6 to increase amyloid-beta peptide production and cleave Tau and several synaptic proteins (LeBlanc AC, Liu H, Goodyer C, Bergeron C, Hammond J: Caspase-6 role in apoptosis of human neurons, amyloidogenesis and Alzheimer's disease. J Biol Chem 1999, 274:23426-23436; Petzke TL, Rousselet E, Goodyer C, LeBlanc AC: Substrates of caspase-6 in human primary neurons: a proteomic study. Program No. 80.9. 2005 Abstract Viewer/Itinerary Planner. Washington, DC: Society for Neuroscience. Online), we suggest that active Csp6 could be an early instigator of neuronal dysfunction.

Evidence for proteolytic cleavage of brevican by the ADAMTSs in the dentate gyrus after excitotoxic lesion of the mouse entorhinal cortex.

BACKGROUND: Brevican is a member of the lectican family of aggregating extracellular matrix (ECM) proteoglycans that bear chondroitin sulfate (CS) chains. It is highly expressed in the central nervous system (CNS) and is thought to stabilize synapses and inhibit neural plasticity and as such, neuritic or synaptic remodeling would be less likely to occur in regions with intact and abundant, lectican-containing, ECM complexes. Neural plasticity may occur more readily when these ECM complexes are broken down by endogenous proteases, the ADAMTSs (adisintegrin and metalloproteinase with thrombospondin motifs), that selectively cleave the lecticans. The purpose of these experiments was to determine whether the production of brevican or the ADAMTS-cleaved fragments of brevican were altered after deafferentation and reinnervation of the dentate gyrus via entorhinal cortex lesion (ECL). RESULTS: In the C57Bl6J mouse, synaptic density in the molecular layer of the dentate gyrus, as measured by synaptophysin levels in ELISA, was significantly attenuated 2 days (nearly 50% of contralateral) and 7 days after lesion and returned to levels not different from the contralateral region at 30 days. Immunoreactive brevican in immunoblot was elevated 2 days after lesion, whereas there was a significant increase in the proteolytic product at 7, but not 30 days post-lesion. ADAMTS activity, estimated using the ratio of the specific ADAMTS-derived brevican fragment and intact brevican levels was increased at 7 days, but was not different from the contralateral side at 2 or 30 days after deafferentation. CONCLUSION: These findings indicate that ADAMTS activity in the dentate outer molecular layer (OML) is elevated during the initial synaptic reinnervation period (7 days after lesion). Therefore, proteolytic processing of brevican appears to be a significant extracellular event in the remodeling of the dentate after EC lesion, and may modulate the process of sprouting and/or synaptogenesis.

Protein kinase associated with gating and closing transmission mechanisms in temporoammonic pathway.

The entorhinal cortex (EC) is a major source of afferent input to the hippocampus via the perforant and temporoammonic pathways; however, the detailed transmission mechanism in the temporoammonic pathway remains to be clarified. Thus, we determined interaction among GABA(A), AMPA/glutamate receptors and protein kinases (PKA and PKC) in the exocytosis of GABA and glutamate using multiprobe microdialysis, as well as propagation of neuronal excitability using optical recording in the EC-Hippocampal formation. Multiprobe microdialysis demonstrated that EC-evoked GABA release in ventral CA1 was predominantly regulated by the PKC-related rather than PKA-related exocytosis mechanism and was augmented by the activation of glutamatergic transmission. Contrary to GABA release, EC-evoked glutamate release was predominantly regulated by PKA-related rather than PKC-related mechanisms and was suppressed by activation of GABAergic transmission. Optical recording demonstrated that there are two sub-pathways in the temporoammonic pathway; direct projects from EC layers (II-IV) to dendrites on pyramidal cells and GABAergic interneurons in ventral hippocampal CA1. PKC activation enhanced trisynaptic transmission, whether the GABA(A) receptor was functional or blocked, whereas PKC activation enhanced and inhibited temporoammonic transmission when the GABA(A) receptor was functional and blocked, respectively. Thus, GABAergic inhibition, which is regulated by PKC activity, in the temporoammonic pathway is more significant than that in the trisynaptic pathway.

Nitric oxide synthase and arginase expression changes in the rat perirhinal and entorhinal cortices following unilateral vestibular damage: a link to deficits in object recognition?

Previous studies have shown that peripheral vestibular damage causes long-term neurochemical changes in the hippocampus which may be related to spatial memory deficits. Since recent studies have also demonstrated deficits in non-spatial object recognition memory following vestibular lesions, the aim of the present study was to extend these investigations into the perirhinal cortex (PRC), which is known to be important for object recognition, and the related entorhinal cortex (EC). We examined the effects of unilateral vestibular deafferentation (UVD) on the expression of four enzymes associated with neuronal plasticity, neuronal nitric oxide synthase (nNOS), endothelial nitric oxide synthase (eNOS), arginase I and arginase II (AI and II), in the rat EC and PRC using Western blotting. Tissue was collected at 10 hs, 50 hs and 2 weeks post-UVD. In the EC and PRC, nNOS protein expression decreased on the contralateral side at 2 weeks post-UVD but not before. At the same time, eNOS protein expression increased in both regions on the contralateral side. In the EC, AII protein expression increased on the ipsilateral side at 2 weeks post-UVD. In the PRC, AI increased and decreased on the contralateral and ipsilateral sides (respectively) at 2 weeks post-UVD. AII showed a bilateral increase in the PRC at 2 weeks post-UVD. These results demonstrate changes in NOS and arginase protein expression in the PRC and EC following UVD, which are unlikely to be due to the initial severity of the vestibular syndrome because they develop well after vestibular compensation has taken place. Neurochemical changes in these regions of the medial temporal lobe may be implicated in the development of object recognition deficits that contribute to cognitive dysfunction following peripheral vestibular damage.

Nitric oxide synthase and arginase in the rat hippocampus and the entorhinal, perirhinal, postrhinal, and temporal cortices: regional variations and age-related changes.

Increasing evidence suggests that nitric oxide synthase (NOS)/nitric oxide (NO) contributes to the aging process. By contrast, the role of arginase, which shares a common substrate with NOS, has not been determined. In the present study, regional variations and age-related changes in NOS and arginase in the hippocampus and its neighboring structures were investigated for the first time. In young adult rats, high levels of NOS activity were found in the entorhinal, perirhinal, and postrhinal cortices, whereas low values were located in the hippocampus and the temporal cortex. Interestingly, arginase activity showed an overall inverse pattern with the lowest levels in the entorhinal and perirhinal cortices. When a comparison was carried out between young (4-month-old) and aged (24-month-old) rats, significant increases in total NOS activity were found in the aged entorhinal and temporal cortices, and a significant decrease in arginase activity was observed in the aged postrhinal cortex. Western blotting demonstrated significant decreases in both neuronal and endothelial NOS expression in the aged hippocampus and postrhinal cortex, whereas arginase I and II expression did not show age-related changes in any region examined. Activity and protein expression of inducible NOS were not detected in any tissue from either group. The present findings of region-specific changes in NOS and arginase appear to support the potential involvement of NOS/NO in the aging process and raise the issue of a possible contribution of arginase to aging.

Changes in NOS protein expression and activity in the rat hippocampus, entorhinal and postrhinal cortices after unilateral electrolytic perirhinal cortex lesions.

The integrity of the perirhinal cortex is critical for certain types of learning and memory. One important issue relating to the function of this region is its interaction with other brain areas that play a role in memory processing. This study investigates the time course of changes in activity and protein expression of nitric oxide synthase (NOS), which transforms L-arginine into nitric oxide (NO) and citrulline, in the hippocampus and the entorhinal and postrhinal cortices after unilateral electrolytic lesions of the perirhinal cortex. Electrolytic lesions of the perirhinal cortex resulted in long lasting changes in NOS activity and protein expression in the entorhinal and postrhinal cortices (< or = 2 weeks post-lesion). In contrast, there was a small and transient decrease in nNOS expression (with no change in NOS activity) in the dorsal portion of the hippocampus. iNOS was not expressed in any region examined at any time point. These findings provide the first evidence that electrolytic lesions of the perirhinal cortex can result in long-term neurochemical changes in its anatomically related structures. Given that NO has been implicated in neuroplasticity processes, the interpretation of memory impairments induced by electrolytic lesions of the perirhinal cortex (and possibly, therefore, other brain regions) need to be considered with regard to these findings.

Activation of mitogen-activated protein kinase/extracellular signal-regulated kinase in hippocampal circuitry is required for consolidation and reconsolidation of recognition memory.

Consolidation and reconsolidation of long-term memory have been shown to be dependent on the synthesis of new proteins, but the specific molecular mechanisms underlying these events remain to be elucidated. The mitogen-activated protein kinase (MAPK) pathway can trigger genomic responses in neurons, leading to changes in protein synthesis, and several studies have identified its pivotal role in synaptic plasticity and long-term memory formation. In this study, we analyze the involvement of this pathway in the consolidation and reconsolidation of long-term recognition memory, using an object recognition task. We show that inhibition of the MAPK pathway by intracerebroventricular injection of the MEK [MAPK/extracellular signal-regulated kinase (ERK)] inhibitor UO126 blocks consolidation of object recognition memory but does not affect short-term memory. Brain regions of the entorhinal cortex-hippocampal circuitry were analyzed for ERK activation, and it was shown that consolidation of recognition memory was associated with increased phosphorylation of ERK in the dentate gyrus and entorhinal cortex, although total expression of ERK was unchanged. We also report that inhibition of the MAPK pathway blocks reconsolidation of recognition memory, and this was shown to be dependent on reactivation of the memory trace by brief reexposure to the objects. In addition, reconsolidation of memory was associated with an increase in the phosphorylation of ERK in entorhinal cortex and CA1. In summary, our data show that the MAPK kinase pathway is required for both consolidation and reconsolidation of long-term recognition memory, and that this is associated with hyperphosphorylation of ERK in different subregions of the entorhinal cortex-hippocampal circuitry.

Caspase-9 activation and caspase cleavage of tau in the Alzheimer's disease brain.

Accumulating evidence supports a role for the activation of proteolytic enzymes, caspases, in the Alzheimer's disease (AD) brain. Neurons committed to apoptosis may do so through a mitochondrial pathway employing caspase-9 or through an alternative, receptor-mediated pathway involving caspase-8. Considering the role of mitochondrial dysfunction in AD, we examined the possible activation of caspase-9 in the AD brain using an antibody that recognizes the active fragments of caspase-9, but not the full-length proform of the enzyme. In vivo immunohistochemical analysis demonstrated little caspase-9 activation in the majority of hippocampal brain sections from control brains. However, labeling of neurons as well as dystrophic neurites within plaque regions was observed in all AD hippocampal brain sections examined. In addition, active caspase-9 was colocalized with active caspase-8 and the accumulation of caspase-3-cleavage products of fodrin. The activation of caspase-9 was also observed in neurons positive for oxidative damage to DNA/RNA. A quantitative analysis indicates that as the number of neurons containing neurofibrillary tangles (NFTs) increases, the extent of caspase-9 activation decreases, supporting the idea that caspase-9 activation may precede NFT formation. In addition, a site-directed caspase-cleavage antibody was designed to the amino-terminal caspase-3 consensus cleavage site located in tau, and shown to be an effective marker for caspase-cleaved fragments of tau in vitro. Analysis with this antibody using age-matched control or AD brain sections demonstrated no staining in control brains while widespread labeling of NFTs, neuropil threads, and dystrophic neurites was observed in AD sections. Taken together, these results demonstrate the activation of caspases and cleavage of tau in the AD brain, events which may precede and lead to the formation of NFTs.

Beta-amyloid deposition and neurofibrillary tangle association with caspase activation in Down syndrome.

Individuals with Down syndrome (DS) and Alzheimer's disease (AD) develop senile plaques, neurofibrillary tangles (NFT), and neuron loss. Recent studies demonstrate the activation of apoptotic pathways in AD; less data is available in DS. The DS brain was examined using immunocytochemistry and antibodies against the active fragment of caspase-8 (AC, 8) and to caspase-3 cleavage products of fodrin (CCP), a neuronal cytoskeleton protein. The hippocampus demonstrated widespread accumulation of fodrin CCP and AC8 in NFTs and dystrophic neurites. Individual neurons contained intracellular beta-amyloid (Abeta) and fodrin CCP providing evidence that caspase activation can occur with both NFT and Abeta. Abeta within or around neurons in addition to contributing to NFT formation may also trigger apoptotic pathways. Caspase activation may lead to the cleavage of critical cellular proteins and neuronal cell death associated with DS.