Christine L. Lancashire

Long-term potentiation in the rat dentate gyrus is associated with enhanced Arc/Arg3.1 protein expression in spines, dendrites and glia.

Electron microscopic immunocytochemical methods were used to determine the localization, subcellular distribution and expression of activity‐regulated cytoskeletal protein (Arc/Arg3.1) in dentate gyrus after unilateral induction of long‐term potentiation (LTP) in the perforant pathway of anaesthetized rats. At 2 h post‐induction, immunoreaction product was visible in the dentate gyrus in both the granule cell and molecular layers. Arc expression was higher in the potentiated than the unstimulated contralateral hemisphere. Single‐section electron microscopy analysis in unstimulated tissue and in tissue prepared 2 and 4 h after LTP induction showed Arc immunoreactivity (Arc‐IR) in dendrites, dendritic spines and glia. Arc‐IR was associated with synaptic and non‐synaptic plasma membrane apposed to axon terminals and with cytoplasmic organelles, including the cytoskeleton. Arc‐IR was also present in neuronal perikarya and there was occasional labelling of nuclei and axons. At 2 h post‐LTP induction, there were significant increases in Arc‐IR within the granule cell and molecular layers of the dentate gyrus and particularly within the middle molecular layer relative to the inner and outer molecular layers. This increase in Arc expression 2 h after LTP induction was blocked by the N‐methyl‐d‐aspartate receptor antagonist (RS)‐3‐2‐carboxypiperazin‐4‐yl‐propyl‐1‐phosphonic acid. In animals killed 4 h after LTP induction, Arc expression had declined and differences between the potentiated and unpotentiated hemispheres were no longer significant. Our data provide ultrastructural evidence for a transient LTP‐associated increase in the expression of Arc protein in the middle molecular layer of the dentate gyrus, with preferential targeting to dendrites, dendritic spines and glia.

Chemokine production and chemokine receptor expression by human glioma cells: Role of CXCL10 in tumour cell proliferation

The expression of chemokine receptors and chemokine production by adult human non-transformed astrocytes, grade III astrocytoma and grade IV glioblastoma tumour cell lines were determined. Here, we show an increased expression of CXCR3 and CXCR4, and a decreased expression of CXCR1 and CCR4 by glioma cells compared to adult human astrocytes. Glioma cells showed increased production of CXCL10, whereas production of other chemokines was decreased (CXCL8, CCL2, CCL5, and CCL22). CXCL10 induced an ERK1/2-dependent increase in [(3)H] thymidine uptake. These results suggest that expression of chemokine receptor/ligand pairs such as CXCR3/CXCL10 have an important role in the proliferation of glioma cells.

Corticosterone facilitates long-term memory formation via enhanced glycoprotein synthesis.

Long-term memory formation for a passive avoidance task in one-day-old chicks requires a late phase of synaptic glycoprotein synthesis (including the neural cell adhesion molecule), commencing 5.5 h post-training. This phase occurred in chicks trained with a strong, but not a weak aversant, which only retained this memory for a few hours (< 10). In addition, previous work has shown that a corticosteroid action through central corticosteroid receptors is also required for long-term passive avoidance memory. Here, we tested the hypothesis that the corticosteroid action on memory formation might be exerted via modulation of the late phase of neural glycoprotein synthesis. One-day-old chicks were used as experimental subjects. Incorporation of the radiolabelled glycoprotein precursor [3H]fucose into synaptic membranes of the chick forebrain was used as an index of glycoprotein fucosylation. Bilateral intracerebral injections of a corticosterone dose (0.5 micrograms/hemisphere) that facilitates long-term retention of weak learning were able to induce the late phase of glycoprotein synthesis in undisturbed chicks. A further experiment examined the effect of antibodies against the neural cell adhesion molecule on the facilitatory action of corticosterone on long-term memory for the weak passive avoidance training. Chicks trained on a weak aversant were injected with corticosterone (0.5 micrograms/hemisphere) 30 min post-training and testing occurred 24 h post-training. Administration of the neural cell adhesion molecule antibodies during the late phase (5.5 h post-training) blocked the facilitatory action of corticosterone on long-term memory. These findings further support the view that corticosteroids have a role in memory consolidation. In addition to previously proposed effects on gene transcription, these data suggest a post-translational glycosylation mechanism for the modulatory effect of corticosteroids on long-term memory formation.

The peptide sequence Arg-Glu-Arg, present in the amyloid precursor protein, protects against memory loss caused by Aβ and acts as a cognitive enhancer

Amino acid sequences containing the palindromic tripeptide RER, matching amino acids 328–330 of the amyloid precursor protein APP, when injected intracerebrally prior to or just after training, protect against memory loss induced by amyloid‐beta (Aβ) in a one‐trial passive avoidance task in the young chick. RER also acts as a cognitive enhancer, strengthening memory for a weak version of the task. N‐terminal acylation of RER protects it against rapid degradation, and AcRER is effective in restoring memory if administered peripherally. Biotinylated RER binds to chick neuronal perikarya in an APP‐displaceable manner via 66 and ≈ 110 kDa neuronal cell membrane proteins. We suggest that RER binding is likely to exert effects on memory retention via receptor‐mediated events that include activation of second messenger pathways. These findings suggest that RER and its derivatives may offer a novel approach to enhancing the neuroprotective effects of APP and alleviating the effects of memory loss in the early stages of Alzheimers disease.

Amyloid precursor protein: from synaptic plasticity to Alzheimer’s disease

Abstract: The amyloid precursor protein (APP) has been shown to be implicated in age‐associated plastic changes at synapses that might contribute to memory loss in Alzheimers disease. As APP has previously been reported to have multiple functions during normal development, and as human and avian APP share 95% homology in amino acid sequence, we have employed a one‐trial passive avoidance task in day‐old chicks to study its role in the process of memory formation. Administration of anti‐APP antibodies, raised against human APP, APP‐antisense, and Aβ during pre‐training, prevented memory formation without effects on general behavior or initial acquisition. Amnesia is apparent by 30 min post‐training and lasts for at least 24 hours. Injection of APP‐derived peptides RERMS (APP328‐332) and RER (APP328‐330) homologous to the short stretches of amino acids in the Kang sequence (APP319‐335), rescue the memory in animals rendered amnestic by previous (anti‐APP antibody, antisense, and Aβ pretreatments. The protected form of RER, with a prolonged half‐life (acetylated RER), proved to be effective when injected intracranially and peripherally. The tripeptide RER exerts its biological activity by binding to two neuronal plasma membrane proteins (60 and 110 kDa). The results obtained in this study suggest that RER alleviates memory deficits via receptor‐mediated events, and that short APP‐derived peptides might represent a novel group of therapeutically active molecules for the alleviation of memory deficits in age‐related dementias.

Passive avoidance training enhances cell proliferation in 1-day-old chicks

One‐day‐old domestic chicks were injected i.p. with bromodeoxyuridine (BrdU) before training on a one‐trial passive avoidance task where the aversive experience was a bead coated with a bitter tasting substance, methyl anthranilate (MeA). Animals were tested 24 h later; those avoiding (if MeA‐trained) or pecking if water (W)‐trained (which they peck appetitively), along with a group of untrained naïve chicks, were used to determine cell proliferation either 24 h or 9 days post BrdU injection. In all three groups, BrdU positive cells were identified sparsely throughout the forebrain but labelling was pronounced around ventricular zone (VZ) surfaces at both 24 h and 9 days post‐BrdU‐injection. Double immunolabelling with neuronal specific antibodies, to either NeuN, or β‐tubulin III, confirmed that most BrdU labelled cells appeared to be neurons. Unbiased stereological analysis of labelled cells in selected forebrain areas 24 h post BrdU injection showed a significant MeA‐training induced increase in labelled cells in both the dorsal VZ surface bordering the intermediate and medial hyperstriatum ventrale (IMHV) and the tuberculum olfactorium (TO). By 9 days post‐BrdU‐injection, there was a significantly greater number of BrdU labelled cells in MeA‐trained birds within the IMHV, lobus parolfactorius (LPO) and TO. These results demonstrate that avoidance training in 1‐day‐old chicks has a marked effect on cell proliferation, in the LPO and IMHV, regions of the chick previously identified as a key loci of memory formation, and in a second region (TO), which has olfactory functions, but has not been previously investigated in relation to avoidance learning.

Protection against A??-induced memory loss by tripeptide D-Arg-L-Glu-L-Arg

The molecular and cellular mechanisms underlying the memory deficits in Alzheimers disease are increasingly thought to be associated with faulty processing of amyloid precursor protein. Following our earlier findings that it is possible to use the tripeptide RER (NH2-D-Arg-L-Glu-L-Arg-COOH, derived from the external domain of amyloid precursor protein) to rescue memory in animal models, we report here that the diasteromeric (D/L) form of the acetylated tripeptide RER protects against A&bgr;-induced memory loss for a passive avoidance task in young chicks and enhances retention for a weak version of the task when injected peripherally up to 12 h before training. The tripeptide readily crosses the blood–brain barrier, binds to membrane receptor sites in the brain and is without adverse effects on general behaviour. We discuss this finding in the context of other studies of the importance of peptides containing D-amino acids, and conclude that these RER-related peptides may form the basis for a potential therapeutic agent in the early stages of Alzheimers disease.

O3-05-02

of several neurotransmitters in the brain and enhance cognition in rodent models. Several H3 antagonists are being developed as symptomatic agents for the treatment of cognitive deficits in Alzheimer’s Disease and other dementias. Objective: To investigate the in vitro and in vivo properties of GSK189254, a novel non-imidazole histamine H3 receptor antagonist. Methods: GSK189254 was profiled in standard in vitro binding and functional assays. In vivo oral activity of GSK189254 was assessed in several assays including ex vivo binding, cognition models and EEG. Results: GSK189254 had high affinity for native H3 receptors expressed in human and rat cerebral cortex (pKi 9.59 and 8.58 respectively) and for human and rat recombinant H3 receptors (pKi 9.90 and 9.17 respectively). GSK189254 was highly selective for H3 receptors ( 10000-fold vs other receptors, ion channels and transporters) and exhibited high functional antagonist potency at the human recombinant H3 receptor in vitro (pA2 9.1 vs imetit-induced inhibition of forskolin-stimulated cAMP accumulation). In vitro autoradiography studies with [3H]-GSK189254 in rat brain slices demonstrated a specific binding pattern consistent with H3 receptor distribution in cortex, hippocampus, striatum and hypothalamus. Specific [3H]-GSK189254 labelling was also observed in post-mortem cortex and hippocampus from human control and Alzheimer’s disease brains. Following oral administration in rats, GSK189254 exhibited high H3 receptor occupancy in the cerebral cortex (ED50 0.17mg/kg from ex vivo binding studies) indicative of good brain penetration, and potently reversed drinking induced by the H3 selective agonist (R)-alpha-methylhistamine (ID50 0.05mg/kg), supporting functional blockade of H3 receptors in vivo. GSK189254 (0.1-3mg/kg) exhibited efficacy in a number of rodent models of cognitive function including object recognition (48h delay), passive avoidance (scopolamine-induced amnesia) and water maze (aged animals). In addition, GSK189254 induced a transient increase in wakefulness in the rat during the light phase, and a corresponding decrease in slow wave sleep 2. Conclusion: Given these effects on cognition and alertness in preclinical species, the H3 antagonist GSK189254 has the potential to be a new therapeutic agent for the symptomatic treatment of Alzheimer’s Disease and other dementias.

AbstractOral session: TherapeuticO3-05-02: Peptidomimetics derived from the amyloid precursor protein (APP) as potential therapeutic agents in Alzheimer’s disease

of several neurotransmitters in the brain and enhance cognition in rodent models. Several H3 antagonists are being developed as symptomatic agents for the treatment of cognitive deficits in Alzheimer’s Disease and other dementias. Objective: To investigate the in vitro and in vivo properties of GSK189254, a novel non-imidazole histamine H3 receptor antagonist. Methods: GSK189254 was profiled in standard in vitro binding and functional assays. In vivo oral activity of GSK189254 was assessed in several assays including ex vivo binding, cognition models and EEG. Results: GSK189254 had high affinity for native H3 receptors expressed in human and rat cerebral cortex (pKi 9.59 and 8.58 respectively) and for human and rat recombinant H3 receptors (pKi 9.90 and 9.17 respectively). GSK189254 was highly selective for H3 receptors ( 10000-fold vs other receptors, ion channels and transporters) and exhibited high functional antagonist potency at the human recombinant H3 receptor in vitro (pA2 9.1 vs imetit-induced inhibition of forskolin-stimulated cAMP accumulation). In vitro autoradiography studies with [3H]-GSK189254 in rat brain slices demonstrated a specific binding pattern consistent with H3 receptor distribution in cortex, hippocampus, striatum and hypothalamus. Specific [3H]-GSK189254 labelling was also observed in post-mortem cortex and hippocampus from human control and Alzheimer’s disease brains. Following oral administration in rats, GSK189254 exhibited high H3 receptor occupancy in the cerebral cortex (ED50 0.17mg/kg from ex vivo binding studies) indicative of good brain penetration, and potently reversed drinking induced by the H3 selective agonist (R)-alpha-methylhistamine (ID50 0.05mg/kg), supporting functional blockade of H3 receptors in vivo. GSK189254 (0.1-3mg/kg) exhibited efficacy in a number of rodent models of cognitive function including object recognition (48h delay), passive avoidance (scopolamine-induced amnesia) and water maze (aged animals). In addition, GSK189254 induced a transient increase in wakefulness in the rat during the light phase, and a corresponding decrease in slow wave sleep 2. Conclusion: Given these effects on cognition and alertness in preclinical species, the H3 antagonist GSK189254 has the potential to be a new therapeutic agent for the symptomatic treatment of Alzheimer’s Disease and other dementias.

O3-05-02: Peptidomimetics derived from the amyloid precursor protein (APP) as potential therapeutic agents in Alzheimer’s disease

of several neurotransmitters in the brain and enhance cognition in rodent models. Several H3 antagonists are being developed as symptomatic agents for the treatment of cognitive deficits in Alzheimer’s Disease and other dementias. Objective: To investigate the in vitro and in vivo properties of GSK189254, a novel non-imidazole histamine H3 receptor antagonist. Methods: GSK189254 was profiled in standard in vitro binding and functional assays. In vivo oral activity of GSK189254 was assessed in several assays including ex vivo binding, cognition models and EEG. Results: GSK189254 had high affinity for native H3 receptors expressed in human and rat cerebral cortex (pKi 9.59 and 8.58 respectively) and for human and rat recombinant H3 receptors (pKi 9.90 and 9.17 respectively). GSK189254 was highly selective for H3 receptors ( 10000-fold vs other receptors, ion channels and transporters) and exhibited high functional antagonist potency at the human recombinant H3 receptor in vitro (pA2 9.1 vs imetit-induced inhibition of forskolin-stimulated cAMP accumulation). In vitro autoradiography studies with [3H]-GSK189254 in rat brain slices demonstrated a specific binding pattern consistent with H3 receptor distribution in cortex, hippocampus, striatum and hypothalamus. Specific [3H]-GSK189254 labelling was also observed in post-mortem cortex and hippocampus from human control and Alzheimer’s disease brains. Following oral administration in rats, GSK189254 exhibited high H3 receptor occupancy in the cerebral cortex (ED50 0.17mg/kg from ex vivo binding studies) indicative of good brain penetration, and potently reversed drinking induced by the H3 selective agonist (R)-alpha-methylhistamine (ID50 0.05mg/kg), supporting functional blockade of H3 receptors in vivo. GSK189254 (0.1-3mg/kg) exhibited efficacy in a number of rodent models of cognitive function including object recognition (48h delay), passive avoidance (scopolamine-induced amnesia) and water maze (aged animals). In addition, GSK189254 induced a transient increase in wakefulness in the rat during the light phase, and a corresponding decrease in slow wave sleep 2. Conclusion: Given these effects on cognition and alertness in preclinical species, the H3 antagonist GSK189254 has the potential to be a new therapeutic agent for the symptomatic treatment of Alzheimer’s Disease and other dementias.