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The Chick as a Model for the Study of the Cellular Mechanisms and Potential Therapies for Alzheimer's Disease

The Chick as a Model for the Study of the Cellular Mechanisms and Potential Therapies for... SAGE-Hindawi Access to Research International Journal of Alzheimer’s Disease Volume 2010, Article ID 180734, 10 pages doi:10.4061/2010/180734 Review Article The Chick as a Model for the Study of the Cellular Mechanisms and Potential Therapies for Alzheimer’s Disease Radmila Mileusnic and Steven Rose Department of Life Sciences, Faculty of Sciences, The Open University, Milton Keynes, MK7 6AA, UK Correspondence should be addressed to Radmila Mileusnic, r.mileusnic@open.ac.uk Received 5 May 2010; Accepted 17 June 2010 Academic Editor: Gemma Casadesus Copyright © 2010 R. Mileusnic and S. Rose. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. While animal experiments have contributed much to our understanding of the mechanisms of Alzheimer’s disease (AD), their value in predicting the effectiveness of treatment strategies in clinical trials has remained controversial. The disparity between the results obtained in animal models and clinical trials may in part be explained by limitations of the models and species- specific differences. We propose that one trial passive avoidance in the day-old chick is a useful system to study AD because of the close sequence homologies of chick and human amyloid precursor protein (APP). In the chick, APP is essential for memory consolidation, and disrupting its synthesis or structure results in amnesia. RER, a tripeptide sequence corresponding to part of the growth domain of APP, can restore memory loss and act as a cognitive enhancer. We suggest that RER and its homologues may form the basis for potential pharmacological protection against memory loss in AD. 1. Introduction Alzheimer’s disease (AD) manifests itself in humans in terms of behaviour—initially memory loss and confusion, At first, the day-old chick does not seem a likely model with progressive decline in other faculties. Neurologically, system in which to study the molecular processes involved in there is accumulation of amyloid plaques and tangles, neu- a degenerative disease that primarily affects brain function ronal death, notably of cholinergic cells in the hippocampus, in ageing humans. Before proceeding to argue the case and brain shrinkage [6–9]. However, there is no known for including the chick amongst such models, however, we naturally occurring animal equivalent of these features either should consider more carefully what we mean by, and expect behaviourally or neurologically, apart from some partial from, an “animal model” of a human disease. resemblances such as plaque accumulation in aged captive What does one want from an animal model? By an animal apes. Thus, animal models have been directed towards model we mean a nonhuman organism that displays some mimicking the neurological and/or biochemical features or all of the features of the human condition we wish to of the disease, primarily in rodents, including transgenics, understand. These may include some or all of the genetic, and examining the behavioural consequences in terms of molecular, physiological, anatomical, or behavioural features impaired performance on standard memory tasks [10–12]. of the human condition or acceptable analogues thereof. To be of utility, such an organism must be readily amenable to experimental manipulation in one or more of these biologi- 2. Choice of Task cal/behavioural levels. As the manifestation of the disease or disorder is likely to be species-typical, especially when we are The measure of neurological deficit commonly chosen as dealing with neurological or psychiatric diagnoses, inferences an indicator of an animal model’s relevance for AD is an as to the relevance of any animal analogue are always going to impairment in learning or memory retention in a standard be problematic, as much of the literature on animal models task. Such impairment is taken as analogous to, or better, of depression and schizophrenia has demonstrated [1–5]. homologous with, that in human memory in conditions 2 International Journal of Alzheimer’s Disease such as AD. Standard laboratory tasks may be aversive or as the alpha, beta, and gamma secretases associated with appetitive, single or multiple trial. For rodents, they include the misprocessing of APP leading to accumulation of senile passive avoidance and fear conditioning (both single trial) plaques and methods for clearing or diminishing plaque and versions of the Morris water maze (multiple trial). The load. Animal models for AD such as mice transgenic for merit of one trial tasks is that they are sharply timed; the the mutant forms of human APP are therefore in principle brevity of the training trial allows for a separation of events directed towards any of these processes and events [11]. surrounding the training experience from the processes that However, a major weakness of such studies, although very occur during memory consolidation. However, single trial understandable in the earlier days of AD research, has been learning is not typical of learning in general, because many that the striking appearance of the plaques and the early instances of animal and human learning are based on the death of cholinergic cells has focussed excessive attention acquisition of experience in a number of repeated trials, on these end-products of the biochemical chain of events involving processes such as generalisation, categorisation, leading to the disease, on the assumption that they are and discrimination. Furthermore, the memory expressed in both proximal causes of the condition and likely therapeutic such animal models is procedural rather than declarative, targets. An alternative hypothesis would be that the primary and procedural memory is the last, not the first, to be lost lesion in the disease is the disruption of neural processes during the degeneration typical of the progression of AD. that require the normal functioning of APP and are essential While it is a necessary assumption for such studies that the for cognitive functions such as memory. It is towards this biochemical and pharmacological processes explored in the hypothesis that our studies in the chick have proceeded, context of animal memory have their parallels in the human and which in turn has resulted in uncovering a molecular case, the repeated failure of agents which act as cognitive mechanism that could be of therapeutic significance. enhancers in well-controlled animal experiments to affect human cognition in clinical trials is a salutary warning that 4. Avian APP the assumption remains-just that. Although birds and mammals diverged about 270 million years ago, and consequently are very different in morphology, 3. The APP and AD behaviour, lifespan, and in the age-dependent repression of a The amyloid precursor protein (APP) is a multifunctional broad-spectrum of neuronal genes, the chick may be a better transmembrane glycoprotein involved in diverse and oppos- experimental model to study APP than mice because its APP ing cellular functions such as: synapse formation and main- gene sequence and the enzymatic machinery for processing tenance [13–16], memory formation [17–23], regulation of APP are almost identical to that of humans and closer than transcription, and neurotoxicity [13, 14]. It is extensively those in mice [31–38]. processed posttranslationally by specific proteolytic cleavage Chicken APP expression parallels mammalian APP [13–15]. Like APP, the APP-derived fragments initiate or expression both temporally and topographically. Further- execute a variety of cellular functions. Most of the evidence more, the chick embryo expresses the genes that encode that APP is implicated in memory consolidation is based on the main proteases implicated in the production of APP, the effects of intracerebral or intraventricular injections of including BACE-1, BACE-2, presenilin-1, presenilin-2, and exogenous APP, its proteolytic fragments, or antibodies and nicastrin as well as Neprilysin, the main Aβ-degrading antisense oligodeoxynucleotides. For example, smaller solu- enzyme, and ADAM-17, a protease implicated in the non- ble fragments of β-amyloid (Aβ) and structurally mimetic amyloidogenic processing of APP. Importantly, the level of nonpeptidic substances injected centrally antagonise the the APP gene expression is related to the strength of learning binding of Aβ protein and produce amnesia [24]aswellasa in day-old chicks [19]. That makes the chick a useful natural decrease of K -evoked acetylcholine release from hippocam- model in which to study the cell biology and functions of pus [25, 26]. Centrally administered amyloid β peptides APP and a potential “assay system” for drugs that regulate (Aβ) impair retention in the Y-maze, passive avoidance and APP processing. place-learning in the water maze [26] and cause amnesia for The degree of evolutionary conservation of APP is very footshock active avoidance in mice [24]. Multiple bilateral high. The chick APP gene sequence, similar to that of the injections of Aβ into the dorsal hippocampus produce mouse, has 93% amino acid identity and 96% similarity with 1−40 performance decrements in short-term working memory the human sequence (Figure 1). However, it is important [27]. In contrast to the effects of Aβ, the secreted form to stress that avian Aβ has 100% sequence similarity with of APP (sAPP) is neurotrophic and neuroprotective and the human Aβ sequence in contrast to rodent Aβ which when administered intracerebroventricularly, shows potent is lacking residues Arg, Tyr, and His in the Aβ domain, memory-enhancing effects [26]; amongst other effects it shown to be crucial for amyloidogenesis. In addition, the prevents the learning deficits induced by scopolamine in rodent 5 upstream regulatory region of the APP gene is an object recognition task and improves spatial recognition only 82% homologous to the corresponding region of the memory [28–30]. human APP gene [39]. These differences may be functionally APP is generally accepted to be directly involved in AD related to the fact that Aβ plaques do not accumulate in aged and consequently has been extensively studied in a number memory impaired rodents. Another important difference of different mammalian and nonmammalian systems [13, between rodents and humans is related to the sequence of 14]. Thus, attention has been focussed on enzymes such the last 101 C-terminal amino acids of the human APP International Journal of Alzheimer’s Disease 3 Mouse M L P S L A L L L L A AWT V R A L E V P T D G N A G L L A E P Q I A M F C G K L N M HM N VQ N G KW E S D P S G T K M L P G L A L L L L A AWT A R A L E V P T D G N A G L L A E P Q I A M F C G R L N M HM N VQ N G KWD S D P S G T K Human 60 M L P H L A L L L L A A G A A R A L E V P A D G N A G L L A E P Q I A M F C G K L N M HM N VQ N G KW E S D P S G T K Chick 60 * * * * * * * * * * * : . * * * * * * : * * * * * * * * * * * * * * * * * : * * * * * * * * * * * * : * * * * * * * Mouse T C I G T K E G I L Q Y C Q E V Y P E L Q I T N V V E A N Q P V T I Q N WC K R G R K Q C K T H T H I V I P Y R C L V G 120 Human T C I D T K E G I L Q Y C Q E V Y P E L Q I T N V V E A N Q P V T I Q N WC K R G R K Q C K T H P H F V I P Y R C L V G 120 Chick T C I D T K E G I L Q Y C Q E V Y P E L Q I T N V V E A N Q P V T I Q N WC K R GWK Q C N G H P H I V V P Y R C L V G 120 * * * . * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * : * . * : * : * * * * * * * Mouse E F V S D A L L V P D K C K F L H Q E R MD V C E T H L H W H T V A K E T C S E K S T N L H D Y GM L L P C G I D K F R 180 E F V S D A L L V P D K C K F L H Q E R MD V C E T H L H W H T V A K E T C S E K S T N L H D Y GM L L P C G I D K F R Human 180 E F V S D A L L V P D K C K L L H Q E R MD V C E T H L H W H T V A K E S C S E K S MN L H D Y GM L L P C G I D K F R 180 Chick * * * * * * * * * * * * * * : * * * * * * * * * * * * * * * * * * * * * : * * * * * * * * * * * * * * * * * * * * * * Mouse G V E F V C C P L A E E S D S V D S A D A E E D D S DV WWG G A D T D Y A D G G E D K V V E V A E E E - - E V A D V E 238 Human G V E F V C C P L A E E S D N V D S A D A E E D D S DV WWG G A D T D Y A D G S E D K V V E V A E E E - - E V A E V E 238 Chick G V E F V C C P L A E E S D N L D S A D A E D D D S DV WWG G A D A D Y A D G S D D K V V E E Q P E E D E E L T V V E 240 * * * * * * * * * * * * * * . : * * * * * * : * * * * * * * * * * * : * * * * * . : * * * * * * * * : : * * Mouse E E E A D D D E D V E D G D E V E E E A E E P Y E E A T E R T T S T A T T T T T T T E S V E E V V R E V C S E Q A E T G 298 Human E E E A D D D E D D E D G D E V E E E A E E P Y E E A T E R T T S I A T T T T T T T E S V E E V V R E V C S E Q A E T G 298 Chick D E D A D D D D D - D D G D E I E E T E E E - Y E E A T E R T T S I A T T T T T T T E S V E E V V R E V C S E Q A E T G 298 : * : * * * * : * : * * * * : * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * Mouse P C R AM I S RWY F D V T E G K C V P F F Y G G C G G N R N N F D T E E Y C MA V C G S V S T Q S L L K T T S E P L P 358 Human P C R AM I S RWY F D V T E G K C A P F F Y G G C G G N R N N F D T E E Y C MA V C G S AM S Q S L L K T T Q E P L A 358 Chick P C R AM I S RWY F D V A E G K C A P F F Y G G C G G N R N N F D S E E Y C MA V C G S V - - - - - - - - - - - - - - 344 * * * * * * * * * * * * * : * * * * . * * * * * * * * * * * * * * * : * * * * * * * * * * . Mouse Q D P D K L P T T A A S T P D A V D K Y L E T P G D E N E H A H F Q K A K E R L E A K HR E R M S Q VM R E W E E A E R 418 Human R D P V K L P T T A A S T P D A V D K Y L E T P G D E N E H A H F Q K A K E R L E A K H R E R M S Q VM R E W E E A E R 418 Chick - - - - - L P T T A A S T P D A V D K Y L E T P G D E N E H A H F Q K A K E R L E A K H R E R M S Q VM R E W E E A E R 399 * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * Mouse Q A K N L P K A D K K A V I Q H F Q E K V E S L E Q E A A N E R Q Q L V E T H M A R V E AM L N D R R R L A L E N Y I T 478 Human Q A K N L P K A D K K A V I Q H F Q E K V E S L E Q E A A N E R Q Q L V E T H M A R V E AM L N D R R R L A L E N Y I T 478 Chick 459 Q A K N L P K A D K K A V I Q H F Q E K V E S L E Q E A A N E R Q Q L V E T H M A R V E AM L N D R R R I A L E N Y I T * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * : * * * * * * * Mouse A L Q A V P P R P H H V F NM L K K Y V R A E Q K D R Q H T L K H F E H V R MV D P K K A A Q I R S Q VM T H L R V I Y 538 A L Q A V P P R P R H V F NM L K K Y V R A E Q K D R Q H T L K H F E H V R MV D P K K A A Q I R S Q VM T H L R V I Y Human 538 A L Q T V P P R P R H V F NM L K K Y V R A E Q K D R Q H T L K H F E H V R MV D P K K A A Q I R S Q VM T H L R V I Y Chick 519 * * * : * * * * * : * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * E R MN Q S L S L L Y N V P A V A E E I Q D E V D E L L Q K E Q N Y S D D V L A N M I S E P R I S Y G N D A L M P S L T 598 Mouse E R MN Q S L S L L Y N V P A V A E E I Q D E V D E L L Q K E Q N Y S D D V L A N M I S E P R I S Y G N D A L M P S L T 598 Human E R MN Q S L S F L Y N V P A V A E E I Q D E V D E L L Q K E Q N Y S D D V L A N M I S E P R I S Y G N D A L M P S L T 579 Chick * * * * * * * * : * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * Mouse E T K T T V E L L P V N G E F S L D D L Q P W H P F G V D S V P A N T E N E V E P V D A R P A A D R G L T T R P G S G L 598 Human E T K T T V E L L P V N G E F S L D D L Q P W H S F G A D S V P A N T E N E V E P V D A R P A A D R G L T T R P G S G L 598 Chick E T K T T V E L L P V D G E F S L D D L Q P W H P F G V D S V P A N T E N E V E P V D A R P A A D R G L T T R P G S G L 579 * * * * * * * * * * * : * * * * * * * * * * * * . * * . * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * Mouse T N I K T E E I S E V KMD A E FGH D S GF E VRH Q K L V F F A E D V G S N K G A I I G L MV G G V V I A T V I V I Human T N I K T E E I S E V KMD A E F R H D S G Y E V H H Q K L V F F A E D V G S N K G A I I G L MV G G V V I A T V I V I Chick T N V K T E E V S E V KMD A E F R H D S G Y E V H H Q K L V F F A E D V G S N K G A I I G L MV G G V V I A T V I V I * * : * * * * : * * * * * * * * * * * * * : * * : * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * Mouse T L VM L K K K Q Y T S I H H G V V E V D A A V T P E E R H L S KMQ Q N G Y E N P T Y K F F E QMQ N 770 Human T L VM L K K K Q Y T S I H H G V V E V D A A V T P E E R H L S KMQ Q N G Y E N P T Y K F F E QMQ N 770 Chick T L VM L K K K Q Y T S I H H G V V E V D A A V T P E E R H L S KMQ Q N G Y E N P T Y K F F E QMQ N 751 * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * Figure 1: Alignment of the amino acid sequences of human, mouse, and chick APP. The numbering refers to the human APP sequence. The RERMS sequence is in gray. Amino acid sequences of Aβ domain are underlined. Residues implicated in amyloidogenesis are indicated in bold. The human (P05067), chicken (Q9DGJ7) and mouse (P12023) APP sequences were obtained from the EMBL database (CLUSTAL 2.0.12 multiple sequence alignment). 4 International Journal of Alzheimer’s Disease sequence (corresponding to the Aβ, transmembrane and small bead coated in the bitter, distasteful, but nontoxic intracytoplasmic domains). In contrast to mouse and rat, methylanthranilate (MeA). The task has the merits of being chick and human sequence are identical. That makes the rapid and sharply timed (chicks peck a bead within 10 chick a useful natural model in which to study regulation of seconds) and as many as 60 chicks can be trained in a APP gene expression and the amyloidogenic characteristics single session. Table 1 compares this chick task with those of Aβ. commonly used in rodents. In the standard version of the In contrast to many transgenic models wild type task in our lab, day-old chicks are held in pairs in small heterozygous chicks do not carry a burden of genetic pens, pretrained by being offered asmall drywhite bead,and background which might be a possible confounding factor those that peck trained with a larger (4 mm dia) chrome or with regard to crucial aspects of AD [40, 41]. Although coloured bead coated with MeA. Chicks that peck such a bead sophisticated and precise molecular genetic tools are applied show a disgust reaction (backing away, shaking their heads to transgenic animals in order to study the pathophysiology and wiping their bills) and will avoid a similar but dry bead of AD [11, 42, 43], animal performance in the behavioural for at least 48 hours subsequently. However, they continue to tests used to assess learning and memory is often affected discriminate, as shown by pecking at control beads of other by variables apparently unrelated to memory function, as colours. Chicks trained on the bitter bead are matched with shown in an extensive study analysing data from 3003 controls which have pecked at a water-coated or dry bead, mice tested in the Morris water maze [44]. This meta- and which peck the dry bead on test. Generally some 80% analysis showed that genetic background and environmental of chicks in any hatch group can be successfully trained and differences between laboratories in rearing and handling tested on this protocol. Each chick is usually trained and procedures alone can produce sufficient variation to span the tested only once. Because the pecking response requires a range of most, if not all, behavioural variables and can thus positive, accurate act by the bird, it also controls for effects easily mask or fake mutational effects. In addition, disparity on attentional, visual, and motor processes [47, 48]. attributable to evolutionary divergence between humans and The training task has two variants: strong, and weak. In rodents, brings about another type of problem: the strong the strong version, the aversive substance used to coat the but incomplete homology between human and mouse APP beads is 100% MeA and it produces high and persisting levels sequences and the weaker but still considerable homology of avoidance. However, if the MeA is diluted to 10%, the between APP and APP-like protein (APLP2), compromise high level of avoidance for the training bead persists only up accurate measurements of total APP transcript levels in 8 hours; long term memories are not formed. In its strong humanised APP transgenic mice and make assessment of form, the task can be used to identify the molecular cascade the neuropathogenic potential of human APP gene products involved in memory formation and the interventions that rather difficult [42, 45, 46]. impair consolidation; in its weak form the task can be used to explore potential memory enhancing agents. These features make the young chick a highly suitable model for the analysis of the biochemical (and in our hands 5. The Chick as an Animal Model morphological and physiological) correlates of memory for the Study of Memory consolidation. Table 1 compares the passive avoidance task Our route towards research on AD led through our lab’s in the chick with commonly used tasks in the mouse. interest, over many years, in the molecular mechanisms involved in memory storage, on which we have worked extensively with the young chick. The suitability of the chick 6. The Biochemical Cascade of Memory as a model system for such studies is well documented. Consolidation in the Chick Chicks are precocial birds, and need actively to explore and learn about their environment from the moment they hatch. Over the past decades a combination of correlative and Thus, they learn very rapidly to identify their mother on interventive experimental strategies has enabled us to iden- the basis of visual, olfactory and auditory cues (imprinting), tify a biochemical cascade that is associated with memory to distinguish edible from inedible or distasteful food, and consolidation in the minutes to hours following training. to navigate complex routes. Training paradigms that exploit These have been fully reviewed elsewhere [47]soonlya these species-specific tasks work with the grain of the brief summary will be given here. In the minutes following animal’s biology, and because such learning is a significant training on this task, there is upregulation of N-methyl- event in the young chick’s life the experiences involved D-aspartate receptor activity, phosphorylation of the presy- may be expected to result in readily measurable brain naptic membrane protein B50, and genomic activation of changes. Chicks have large and well-developed brains and the immediate early genes c-fos and c-jun. During the next soft unossified skulls, making localised cerebral injections hours after training, increased incorporation of fucose into of drugs easy without the use of implanted cannulae or brain glycoproteins occurs. During this time, memory for anaesthesia. The virtual lack of any blood-brain barrier in the passive avoidance task can be impaired by inhibitors these young animals also ensures rapid entry into the brain of protein and glycoprotein synthesis injected around the of peripherally injected agents (for review see [47]). time of training. Two regions of the chick brain are involved The training task that we employ is one trial passive specifically in the biochemical responses to the learning avoidance, in which chicks learn to avoid pecking at a experience. These are the intermediate medial mesopallium International Journal of Alzheimer’s Disease 5 Table 1: Comparison of training tasks in chicks and mice. Chicks Mice Passive avoidance Training paradigm Passive avoidance Fear conditioning Water maze (multiple trials) Passive avoidance - Brief Timing 10 secends training time Fear conditioning - Brief Water maze - multiple trials Passive avoidance - yes Suitable for biochemical analysis Yes Fear conditioning - yes Water maze - unsuitable Natural distribution of males and females Sex in the hatch. (Sex determined post hoc by Generally males only inspection of gonads) Group size Large (20 chicks/group) Small Intracranial injections Anaesthesia not required Anaesthesia required BBB Not fully developed Fully developed Genome Sequenced in 2004 Sequenced in 2002 Yes, many (for review see Transgenic model No Crews et al., 2010; [11]) (IMMP), an association “cortical” area, and the medial striatum (MS), a basal ganglia homologue. The chick brain is strongly lateralized and many, but not all, of the molecular events we observe are confined to the left IMMP. All these events depend on de novo protein synthesis and insertion of a variety of proteins, especially glycoproteins, into pre- and postsynaptic membranes. Cell adhesion or cell recognition molecules constitute a major group amongst these glycoproteins. They are expressed both pre- and −30 0 +30 1 2 3 4 5 6 7 8 9 10 11 post-synaptically and involved in the process that allows Time of training (hr) information about synaptic activity to be simultaneously ANI communicated to both side of the synapse. Our early work 2-D-d-Gal identified two such adhesion molecule, L1 and NCAM, which are recruited into this cascade of cellular events Figure 2: Two time-windows when protein synthesis is sensitive at different periods posttraining. Injection of inhibitors to inhibitors of protein synthesis, such as anisomycin (ANI) and of protein and glycoprotein synthesis (anisomycin and 2- glycoprotein synthesis, such as 2-d-Galactose (2-d-Gal). deoxy-galactose resp.) at times corresponds to these periods of recruitment (Figure 2) result in amnesia for the task [47]. This and related data on the effects of application of explore the role of APP itself. Chick APP cross-reacts with protein synthesis inhibitors on memory retention led us the mouse monoclonal antibody raised against human APP. to propose that there were two waves of protein synthesis Therefore, we tested the effect of anti-APP antibody on occurring following a learning experience, the first within memory. The residence time for the anti-APP corresponds an hour of the experience and involving the synthesis of to the relatively rapid turnover time for membrane-bound proteins expressed by immediate early genes, and the second, APP. When injected pre-training, anti-APP did not interfere some 4–6 hours later, involving structural proteins such as with the chicks pecking and learning the avoidance task; the adhesion molecules. Both are necessary for consolidation however, it did result in amnesia in birds tested 30 minutes of long-term memory. later. Amnesia persisted for at least the subsequent 24 hours and was not apparent if the antibody was injected just posttraining or 5.5 hours after training [17, 49]. 7. APP and Memory Consolidation in the Chick This finding indicated that APP might be required APP is an important member of the family of cell adhesion at an early phase and not continually during memory molecules, and having identified a role for NCAM and L1 consolidation. Given that blocking APP function by use [51–53] in the consolidation cascade, it seemed logical to of specific antibodies outside of a specific time window Protein synthesis 6 International Journal of Alzheimer’s Disease Table 2: Effect of anti-APP and APP antisense on memory retention. Memory retention Memory retention Time of injection Time of injection (% Avoidance) (% Avoidance) Control (Saline, 30 minutes pre-training 78–95 5.5 hours Posttraining 78–95 non-immune sera) ∗∗ Anti APP 30 minutes pre training 28–35 5.5 hours Posttraining 78–95 Control (SC) 6 hours pre-training 78–95 30 minutes pre-training 78–95 AS 6 hours pre-training 32–37 30 minutes pre-training 7 8–95 ∗ ∗∗ N = 18–25; P <.05; P <.01. Anti APP: monoclonal human antibody mAb22C11 [17]; AS: 16-mer end-protected phosphodiester oligodeoxynucleotide, 5 CXCGAG GACTGA XCCA 3 , designed to correspond to the transcription start sites −146 and AUG1786 of the βAPP mRNA, immediately upstream of a ribozyme binding site [17]; SC: Scrambled AS sequence [17]; For further details see [18, 49, 50]. 2 μm 5 μm (a) (b) (c) Figure 3: RER binding detected on chick, human and mouse neuronal cells. Specific binding of the biotinylated RER (arrows) to chick (a), human (b) and mouse neuronal (c) cell. Location of the chick neuronal cells is in the IMMP area; Human and mouse neuronal cells are located in the CA1 are in hippocampus. was without effect, we compromised APP gene expression 8. APP-Related Peptides as a Tool to using antisense oligodeoxynucleotides (AS) designed to Study Memory correspond to the −146 and to AUG1786 transcription Studies conducted on the physiological activity of APP [54– start site of APP [17]. The antisense oligodeoxynucleotides 58] resulted in the identification of a small stretch of amino (AS ODNs) were injected intracerebrally at 6 hours or 30 acids containing the RERMS sequence C-terminal to the KPI minutes pre-training and chicks were tested at different times posttraining. Injection at 6 hours pre-training was aimed insertion site of sAPP-695 as the active domain responsible for growth promotion and neurite extension, neuronal sur- to suppress APP synthesis during the first wave of protein vival, and for sAPP’s ability to interfere with the deleterious synthesis while the injection made at 30 minutes pre-training was aimed at the second wave (Table 2). Thus, in both groups effects of Aβ on neurons. A synthetic peptide homologous to the RERMS sequence, APP 328–332, was identified as the the AS was present for 6 hours before training. Controls were shortest active peptide to exhibit trophic activity through treated with scrambled (SC) ODNs or saline and trained and cell-surface binding and induction of inositol polyphosphate tested as the AS ODNs treated groups. accumulation. Such observations suggested that the RERMS The results showed that APP-antisense both decreased peptide might substitute for sAPP during memory formation APP gene expression and affected memory formation. The and thereby reverse or protect against the blockade resulting time-window of onset of amnesia relative to time of injection from antibody or antisense. of ODNs and to time of training confirmed our previous finding that APP exerts its function during an early phase We first assessed the effects of RERMS on memory in chicks rendered amnesic by APP-antisense and APP- of memory formation and appears to be a necessary factor in the biochemical cascade involved in the transition antibody treatment [17, 49]. In the series of experiments between short- and long-term memory. Our findings on the which followed, we studied the time window in which injection of RERMS might affect amnesia and showed that importance of APP in learning were supported by reports [19] that APP gene expression in the young chick is related if injected either before or just after training on the task, the pepide protected against the memory loss. As a control to the strength of memory retention for an imprinting for the behavioural effect of RERMS, we used the reversed task. International Journal of Alzheimer’s Disease 7 RER rer HO O HO O O O O O H H H H N N N N N N NH NH H H 2 2 O O O O rER HO O HN HN HN HN O O NH H N NH NH H N NH H N H N 2 2 2 2 H H NH H 2 HO O HO O HN HN O O O O H H H H NH H N NH H N 2 2 N N N N N N NH NH H 2 H 2 O O O O HN HN HN HN NH H N NH NH H N NH H N H N 2 2 2 2 REr ReR Figure 4: Structure of D/L tripeptides included in the study. peptide sequence SMRER, but to our surprise SMRER was as in animals rendered amnesic by pretreatment with Aβ.We effective in relieving the memory block as RERMS. However, concluded that the RER sequence acts as a core domain of adifferent control peptide, RSAER, was without effect. Anal- the growth promoting region of APP in the chick because it ysis of these experiments led to two principal observations: appears able to substitute for sAPP. The protection against first, that the APP-derived peptide might exert its action by the amnestic effects of Aβ may also result from RER’s ability compensating for the low presence of APP. According to the to initiate receptor-mediated processes. This interpretation is proposed amyloid hypothesis, the faulty processing of APP strengthened by the evidence that RER binds to two neuronal and accumulation of the amyloid fragments might be one cell membrane proteins, of ∼66 and 110 kDa, respectively. In of the factors leading to neurotoxicity. Therefore, we tested experiments aimed at identifying specific neuronal binding whether the RERMS peptide might also have a potential partners, using a combination of biotinylated tripeptide and protective effect against the memory deficit induced by Aβ. cell-specific antibodies, bound RER was localised in chick Amyloid-beta, injected into the IMMP bilaterally at a dose and human brain sections (Figure 3), suggesting that it might of 2 μg/hemisphere, 30 minutes prior to training, resulted in also be active in humans, and could play an important role in amnesia for the task in chicks tested 24 hours subsequently. the memory formation process which is deficient in the early However, administration of 1 μg/hemisphere of RERMS 10 stages of AD. Moreover, the distribution of biotinylated RER minutes after Aβ injection prevented the memory deficit binding suggested membrane binding. In the chick, binding caused by Aβ. Conversely, if the injection is delayed to 5.5 is displaced by longer peptides derived from APP’s external hours posttraining, there is no subsequent amnesia. domain, but not by Aβ, suggesting that RER competes with The second observation came from analysis of the amino sAPPfor aputativereceptor[17, 49]. acid sequence of the peptides used in this study [17, 49]. Both To overcome the problem of the short half-life of RER the forward and reverse sequences contain the tripeptide we protected it by N-acylation, and showed that Ac-RER palindrome RER. The next step was therefore to investigate is as effective as RER in protecting against memory loss. the ability of RER to relieve memory block under the same More importantly, Ac-RER crosses the partially formed conditions used for testing RERMS. The RER tripeptide, blood brain barrier of the one-day old chicks, enabling the when injected around the time of training, showed the same peptide to be injected intraperitoneally [17]. The immediate potential as the RERMS pentapeptide and rescued memory implication of these findings is that it is possible to introduce 8 International Journal of Alzheimer’s Disease Table 3: Summary of peptides and their effects on memory reported in this study. Enhances Crossing Peptide Injection route Effective dose Rescue of amnesia induced with: t1/2 hour weak training BBB Ic ip Ic μg/brain Ip mg/kg bw Anti APP AS Aβ Up to RERMS 2 Y Y 4 20–25 Y Y Y Y Y SMRER 2 Y Y 4 20–25 Y Y Y Y Y RSAER / NN 4 / N N N N Y RER / Y Y 4 20–25 Y Y Y Y Y Ac-RER 6 Y Y 16 20–25 Y / Y Y Y Ac-RRE / NN 16 / / / N N / Ac-rER >12 Y Y 16 20–25 Y / Y Y Y Ac-REr / NN 16 / N / N N / Ac-ReR / NN 16 / N / N N / Ac-rer / Y Y 16 20–25 N / N N / Y: yes, there is an effectonmemory; N: No,there is no effect on memory; Anti-APP: monoclonal antibody, clone mAb22C11 [17]; AS: 16-mer end-protected phosphodiester oligodeoxynucleotide, 5 CXCGAG GACTGA XCCA3 , designed to correspond to the transcription start sites −146 and AUG1786 of the βAPP mRNA, immediately upstream of a ribozyme binding site [17]; Aβ: amyloid-beta. For further details see [18, 49, 50]. abehaviourally effective form of RER peripherally by N- administration. The fact that there was no difference in the terminal acylation, hence protecting the peptide against magnitude of the effect of the L- and L/D tripeptide on rapid degradation. behaviour suggested that they engage the same biochemical processes [50]. These results are summarised in Table 3. If the RER sequence acts as a substitute for sAPP than What is now required is to determine the identity of the the question to ask is whether it might act as a cognitive RER binding proteins, the specific second messenger systems enhancer in the weak version of the passive avoidance task activated and the genes controlled by RER. Our currently discussed above. The weak training protocol is an ideal unpublished experiments go some way towards answering paradigm to test this hypothesis as memory for the task is these questions, which may be central to understanding not retained beyond an early phase, presumably because the the peptide’s effects both in memory enhancement and, mild aversant does not provide a sufficient signal for the potentially, in neuroprotection. release of sAPP. Under these conditions, tripeptide injected peripherally wasaseffective as memory enhancer as when injected intracerebrally, meaning that even in the weak 9. Concluding Remarks training task, in the presence of the tripeptide peptide, Although it remains important to demonstrate that the memory persists for at least 24 hours. peptide or its related structures is effective in other learning All our results point to the short APP-related peptides tasks and in mammals, we propose that the chick is a used in our experiments as both powerful tools in studying useful animal model in which to study AD, and that Ac- the structure and function of APP and as of potential rER is a molecule which might form the basis for a potential therapeutic interest in AD. We have therefore begun to therapeutic agent in the early stages of AD. Even though explore the effectiveness of a number of compounds struc- some specific details of protein-protein interactions can turally related to RER. Of particular interest have been vary between birds and human, the degree of functional the optically isomeric D- or diasteromeric (D/L) forms of conservation seems to be of particular relevance for the the peptide, which are more resistant to proteolysis than AD field. This animal model, like many other natural their L-equivalents. The diasteromeric forms have begun model-systems, often appears to suffer from publication lately to attract increasing interest as potential immunogens, bias towards transgenic animal models, which may account diagnostic and therapeutic agents [59]. for substantial under-representation of avian model system Our results using different D/L forms (shown in in the experimental literature related to neurodegenerative Figure 4) demonstrated that substitution of C-terminal L- diseasessuchasAD. arginine with the D-isomer essentially abolished the memory retention-enhancing effect of the peptide. 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The Chick as a Model for the Study of the Cellular Mechanisms and Potential Therapies for Alzheimer's Disease

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Copyright © 2010 Radmila Mileusnic and Steven Rose. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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SAGE-Hindawi Access to Research International Journal of Alzheimer’s Disease Volume 2010, Article ID 180734, 10 pages doi:10.4061/2010/180734 Review Article The Chick as a Model for the Study of the Cellular Mechanisms and Potential Therapies for Alzheimer’s Disease Radmila Mileusnic and Steven Rose Department of Life Sciences, Faculty of Sciences, The Open University, Milton Keynes, MK7 6AA, UK Correspondence should be addressed to Radmila Mileusnic, r.mileusnic@open.ac.uk Received 5 May 2010; Accepted 17 June 2010 Academic Editor: Gemma Casadesus Copyright © 2010 R. Mileusnic and S. Rose. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. While animal experiments have contributed much to our understanding of the mechanisms of Alzheimer’s disease (AD), their value in predicting the effectiveness of treatment strategies in clinical trials has remained controversial. The disparity between the results obtained in animal models and clinical trials may in part be explained by limitations of the models and species- specific differences. We propose that one trial passive avoidance in the day-old chick is a useful system to study AD because of the close sequence homologies of chick and human amyloid precursor protein (APP). In the chick, APP is essential for memory consolidation, and disrupting its synthesis or structure results in amnesia. RER, a tripeptide sequence corresponding to part of the growth domain of APP, can restore memory loss and act as a cognitive enhancer. We suggest that RER and its homologues may form the basis for potential pharmacological protection against memory loss in AD. 1. Introduction Alzheimer’s disease (AD) manifests itself in humans in terms of behaviour—initially memory loss and confusion, At first, the day-old chick does not seem a likely model with progressive decline in other faculties. Neurologically, system in which to study the molecular processes involved in there is accumulation of amyloid plaques and tangles, neu- a degenerative disease that primarily affects brain function ronal death, notably of cholinergic cells in the hippocampus, in ageing humans. Before proceeding to argue the case and brain shrinkage [6–9]. However, there is no known for including the chick amongst such models, however, we naturally occurring animal equivalent of these features either should consider more carefully what we mean by, and expect behaviourally or neurologically, apart from some partial from, an “animal model” of a human disease. resemblances such as plaque accumulation in aged captive What does one want from an animal model? By an animal apes. Thus, animal models have been directed towards model we mean a nonhuman organism that displays some mimicking the neurological and/or biochemical features or all of the features of the human condition we wish to of the disease, primarily in rodents, including transgenics, understand. These may include some or all of the genetic, and examining the behavioural consequences in terms of molecular, physiological, anatomical, or behavioural features impaired performance on standard memory tasks [10–12]. of the human condition or acceptable analogues thereof. To be of utility, such an organism must be readily amenable to experimental manipulation in one or more of these biologi- 2. Choice of Task cal/behavioural levels. As the manifestation of the disease or disorder is likely to be species-typical, especially when we are The measure of neurological deficit commonly chosen as dealing with neurological or psychiatric diagnoses, inferences an indicator of an animal model’s relevance for AD is an as to the relevance of any animal analogue are always going to impairment in learning or memory retention in a standard be problematic, as much of the literature on animal models task. Such impairment is taken as analogous to, or better, of depression and schizophrenia has demonstrated [1–5]. homologous with, that in human memory in conditions 2 International Journal of Alzheimer’s Disease such as AD. Standard laboratory tasks may be aversive or as the alpha, beta, and gamma secretases associated with appetitive, single or multiple trial. For rodents, they include the misprocessing of APP leading to accumulation of senile passive avoidance and fear conditioning (both single trial) plaques and methods for clearing or diminishing plaque and versions of the Morris water maze (multiple trial). The load. Animal models for AD such as mice transgenic for merit of one trial tasks is that they are sharply timed; the the mutant forms of human APP are therefore in principle brevity of the training trial allows for a separation of events directed towards any of these processes and events [11]. surrounding the training experience from the processes that However, a major weakness of such studies, although very occur during memory consolidation. However, single trial understandable in the earlier days of AD research, has been learning is not typical of learning in general, because many that the striking appearance of the plaques and the early instances of animal and human learning are based on the death of cholinergic cells has focussed excessive attention acquisition of experience in a number of repeated trials, on these end-products of the biochemical chain of events involving processes such as generalisation, categorisation, leading to the disease, on the assumption that they are and discrimination. Furthermore, the memory expressed in both proximal causes of the condition and likely therapeutic such animal models is procedural rather than declarative, targets. An alternative hypothesis would be that the primary and procedural memory is the last, not the first, to be lost lesion in the disease is the disruption of neural processes during the degeneration typical of the progression of AD. that require the normal functioning of APP and are essential While it is a necessary assumption for such studies that the for cognitive functions such as memory. It is towards this biochemical and pharmacological processes explored in the hypothesis that our studies in the chick have proceeded, context of animal memory have their parallels in the human and which in turn has resulted in uncovering a molecular case, the repeated failure of agents which act as cognitive mechanism that could be of therapeutic significance. enhancers in well-controlled animal experiments to affect human cognition in clinical trials is a salutary warning that 4. Avian APP the assumption remains-just that. Although birds and mammals diverged about 270 million years ago, and consequently are very different in morphology, 3. The APP and AD behaviour, lifespan, and in the age-dependent repression of a The amyloid precursor protein (APP) is a multifunctional broad-spectrum of neuronal genes, the chick may be a better transmembrane glycoprotein involved in diverse and oppos- experimental model to study APP than mice because its APP ing cellular functions such as: synapse formation and main- gene sequence and the enzymatic machinery for processing tenance [13–16], memory formation [17–23], regulation of APP are almost identical to that of humans and closer than transcription, and neurotoxicity [13, 14]. It is extensively those in mice [31–38]. processed posttranslationally by specific proteolytic cleavage Chicken APP expression parallels mammalian APP [13–15]. Like APP, the APP-derived fragments initiate or expression both temporally and topographically. Further- execute a variety of cellular functions. Most of the evidence more, the chick embryo expresses the genes that encode that APP is implicated in memory consolidation is based on the main proteases implicated in the production of APP, the effects of intracerebral or intraventricular injections of including BACE-1, BACE-2, presenilin-1, presenilin-2, and exogenous APP, its proteolytic fragments, or antibodies and nicastrin as well as Neprilysin, the main Aβ-degrading antisense oligodeoxynucleotides. For example, smaller solu- enzyme, and ADAM-17, a protease implicated in the non- ble fragments of β-amyloid (Aβ) and structurally mimetic amyloidogenic processing of APP. Importantly, the level of nonpeptidic substances injected centrally antagonise the the APP gene expression is related to the strength of learning binding of Aβ protein and produce amnesia [24]aswellasa in day-old chicks [19]. That makes the chick a useful natural decrease of K -evoked acetylcholine release from hippocam- model in which to study the cell biology and functions of pus [25, 26]. Centrally administered amyloid β peptides APP and a potential “assay system” for drugs that regulate (Aβ) impair retention in the Y-maze, passive avoidance and APP processing. place-learning in the water maze [26] and cause amnesia for The degree of evolutionary conservation of APP is very footshock active avoidance in mice [24]. Multiple bilateral high. The chick APP gene sequence, similar to that of the injections of Aβ into the dorsal hippocampus produce mouse, has 93% amino acid identity and 96% similarity with 1−40 performance decrements in short-term working memory the human sequence (Figure 1). However, it is important [27]. In contrast to the effects of Aβ, the secreted form to stress that avian Aβ has 100% sequence similarity with of APP (sAPP) is neurotrophic and neuroprotective and the human Aβ sequence in contrast to rodent Aβ which when administered intracerebroventricularly, shows potent is lacking residues Arg, Tyr, and His in the Aβ domain, memory-enhancing effects [26]; amongst other effects it shown to be crucial for amyloidogenesis. In addition, the prevents the learning deficits induced by scopolamine in rodent 5 upstream regulatory region of the APP gene is an object recognition task and improves spatial recognition only 82% homologous to the corresponding region of the memory [28–30]. human APP gene [39]. These differences may be functionally APP is generally accepted to be directly involved in AD related to the fact that Aβ plaques do not accumulate in aged and consequently has been extensively studied in a number memory impaired rodents. Another important difference of different mammalian and nonmammalian systems [13, between rodents and humans is related to the sequence of 14]. Thus, attention has been focussed on enzymes such the last 101 C-terminal amino acids of the human APP International Journal of Alzheimer’s Disease 3 Mouse M L P S L A L L L L A AWT V R A L E V P T D G N A G L L A E P Q I A M F C G K L N M HM N VQ N G KW E S D P S G T K M L P G L A L L L L A AWT A R A L E V P T D G N A G L L A E P Q I A M F C G R L N M HM N VQ N G KWD S D P S G T K Human 60 M L P H L A L L L L A A G A A R A L E V P A D G N A G L L A E P Q I A M F C G K L N M HM N VQ N G KW E S D P S G T K Chick 60 * * * * * * * * * * * : . * * * * * * : * * * * * * * * * * * * * * * * * : * * * * * * * * * * * * : * * * * * * * Mouse T C I G T K E G I L Q Y C Q E V Y P E L Q I T N V V E A N Q P V T I Q N WC K R G R K Q C K T H T H I V I P Y R C L V G 120 Human T C I D T K E G I L Q Y C Q E V Y P E L Q I T N V V E A N Q P V T I Q N WC K R G R K Q C K T H P H F V I P Y R C L V G 120 Chick T C I D T K E G I L Q Y C Q E V Y P E L Q I T N V V E A N Q P V T I Q N WC K R GWK Q C N G H P H I V V P Y R C L V G 120 * * * . * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * : * . * : * : * * * * * * * Mouse E F V S D A L L V P D K C K F L H Q E R MD V C E T H L H W H T V A K E T C S E K S T N L H D Y GM L L P C G I D K F R 180 E F V S D A L L V P D K C K F L H Q E R MD V C E T H L H W H T V A K E T C S E K S T N L H D Y GM L L P C G I D K F R Human 180 E F V S D A L L V P D K C K L L H Q E R MD V C E T H L H W H T V A K E S C S E K S MN L H D Y GM L L P C G I D K F R 180 Chick * * * * * * * * * * * * * * : * * * * * * * * * * * * * * * * * * * * * : * * * * * * * * * * * * * * * * * * * * * * Mouse G V E F V C C P L A E E S D S V D S A D A E E D D S DV WWG G A D T D Y A D G G E D K V V E V A E E E - - E V A D V E 238 Human G V E F V C C P L A E E S D N V D S A D A E E D D S DV WWG G A D T D Y A D G S E D K V V E V A E E E - - E V A E V E 238 Chick G V E F V C C P L A E E S D N L D S A D A E D D D S DV WWG G A D A D Y A D G S D D K V V E E Q P E E D E E L T V V E 240 * * * * * * * * * * * * * * . : * * * * * * : * * * * * * * * * * * : * * * * * . : * * * * * * * * : : * * Mouse E E E A D D D E D V E D G D E V E E E A E E P Y E E A T E R T T S T A T T T T T T T E S V E E V V R E V C S E Q A E T G 298 Human E E E A D D D E D D E D G D E V E E E A E E P Y E E A T E R T T S I A T T T T T T T E S V E E V V R E V C S E Q A E T G 298 Chick D E D A D D D D D - D D G D E I E E T E E E - Y E E A T E R T T S I A T T T T T T T E S V E E V V R E V C S E Q A E T G 298 : * : * * * * : * : * * * * : * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * Mouse P C R AM I S RWY F D V T E G K C V P F F Y G G C G G N R N N F D T E E Y C MA V C G S V S T Q S L L K T T S E P L P 358 Human P C R AM I S RWY F D V T E G K C A P F F Y G G C G G N R N N F D T E E Y C MA V C G S AM S Q S L L K T T Q E P L A 358 Chick P C R AM I S RWY F D V A E G K C A P F F Y G G C G G N R N N F D S E E Y C MA V C G S V - - - - - - - - - - - - - - 344 * * * * * * * * * * * * * : * * * * . * * * * * * * * * * * * * * * : * * * * * * * * * * . Mouse Q D P D K L P T T A A S T P D A V D K Y L E T P G D E N E H A H F Q K A K E R L E A K HR E R M S Q VM R E W E E A E R 418 Human R D P V K L P T T A A S T P D A V D K Y L E T P G D E N E H A H F Q K A K E R L E A K H R E R M S Q VM R E W E E A E R 418 Chick - - - - - L P T T A A S T P D A V D K Y L E T P G D E N E H A H F Q K A K E R L E A K H R E R M S Q VM R E W E E A E R 399 * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * Mouse Q A K N L P K A D K K A V I Q H F Q E K V E S L E Q E A A N E R Q Q L V E T H M A R V E AM L N D R R R L A L E N Y I T 478 Human Q A K N L P K A D K K A V I Q H F Q E K V E S L E Q E A A N E R Q Q L V E T H M A R V E AM L N D R R R L A L E N Y I T 478 Chick 459 Q A K N L P K A D K K A V I Q H F Q E K V E S L E Q E A A N E R Q Q L V E T H M A R V E AM L N D R R R I A L E N Y I T * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * : * * * * * * * Mouse A L Q A V P P R P H H V F NM L K K Y V R A E Q K D R Q H T L K H F E H V R MV D P K K A A Q I R S Q VM T H L R V I Y 538 A L Q A V P P R P R H V F NM L K K Y V R A E Q K D R Q H T L K H F E H V R MV D P K K A A Q I R S Q VM T H L R V I Y Human 538 A L Q T V P P R P R H V F NM L K K Y V R A E Q K D R Q H T L K H F E H V R MV D P K K A A Q I R S Q VM T H L R V I Y Chick 519 * * * : * * * * * : * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * E R MN Q S L S L L Y N V P A V A E E I Q D E V D E L L Q K E Q N Y S D D V L A N M I S E P R I S Y G N D A L M P S L T 598 Mouse E R MN Q S L S L L Y N V P A V A E E I Q D E V D E L L Q K E Q N Y S D D V L A N M I S E P R I S Y G N D A L M P S L T 598 Human E R MN Q S L S F L Y N V P A V A E E I Q D E V D E L L Q K E Q N Y S D D V L A N M I S E P R I S Y G N D A L M P S L T 579 Chick * * * * * * * * : * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * Mouse E T K T T V E L L P V N G E F S L D D L Q P W H P F G V D S V P A N T E N E V E P V D A R P A A D R G L T T R P G S G L 598 Human E T K T T V E L L P V N G E F S L D D L Q P W H S F G A D S V P A N T E N E V E P V D A R P A A D R G L T T R P G S G L 598 Chick E T K T T V E L L P V D G E F S L D D L Q P W H P F G V D S V P A N T E N E V E P V D A R P A A D R G L T T R P G S G L 579 * * * * * * * * * * * : * * * * * * * * * * * * . * * . * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * Mouse T N I K T E E I S E V KMD A E FGH D S GF E VRH Q K L V F F A E D V G S N K G A I I G L MV G G V V I A T V I V I Human T N I K T E E I S E V KMD A E F R H D S G Y E V H H Q K L V F F A E D V G S N K G A I I G L MV G G V V I A T V I V I Chick T N V K T E E V S E V KMD A E F R H D S G Y E V H H Q K L V F F A E D V G S N K G A I I G L MV G G V V I A T V I V I * * : * * * * : * * * * * * * * * * * * * : * * : * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * Mouse T L VM L K K K Q Y T S I H H G V V E V D A A V T P E E R H L S KMQ Q N G Y E N P T Y K F F E QMQ N 770 Human T L VM L K K K Q Y T S I H H G V V E V D A A V T P E E R H L S KMQ Q N G Y E N P T Y K F F E QMQ N 770 Chick T L VM L K K K Q Y T S I H H G V V E V D A A V T P E E R H L S KMQ Q N G Y E N P T Y K F F E QMQ N 751 * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * Figure 1: Alignment of the amino acid sequences of human, mouse, and chick APP. The numbering refers to the human APP sequence. The RERMS sequence is in gray. Amino acid sequences of Aβ domain are underlined. Residues implicated in amyloidogenesis are indicated in bold. The human (P05067), chicken (Q9DGJ7) and mouse (P12023) APP sequences were obtained from the EMBL database (CLUSTAL 2.0.12 multiple sequence alignment). 4 International Journal of Alzheimer’s Disease sequence (corresponding to the Aβ, transmembrane and small bead coated in the bitter, distasteful, but nontoxic intracytoplasmic domains). In contrast to mouse and rat, methylanthranilate (MeA). The task has the merits of being chick and human sequence are identical. That makes the rapid and sharply timed (chicks peck a bead within 10 chick a useful natural model in which to study regulation of seconds) and as many as 60 chicks can be trained in a APP gene expression and the amyloidogenic characteristics single session. Table 1 compares this chick task with those of Aβ. commonly used in rodents. In the standard version of the In contrast to many transgenic models wild type task in our lab, day-old chicks are held in pairs in small heterozygous chicks do not carry a burden of genetic pens, pretrained by being offered asmall drywhite bead,and background which might be a possible confounding factor those that peck trained with a larger (4 mm dia) chrome or with regard to crucial aspects of AD [40, 41]. Although coloured bead coated with MeA. Chicks that peck such a bead sophisticated and precise molecular genetic tools are applied show a disgust reaction (backing away, shaking their heads to transgenic animals in order to study the pathophysiology and wiping their bills) and will avoid a similar but dry bead of AD [11, 42, 43], animal performance in the behavioural for at least 48 hours subsequently. However, they continue to tests used to assess learning and memory is often affected discriminate, as shown by pecking at control beads of other by variables apparently unrelated to memory function, as colours. Chicks trained on the bitter bead are matched with shown in an extensive study analysing data from 3003 controls which have pecked at a water-coated or dry bead, mice tested in the Morris water maze [44]. This meta- and which peck the dry bead on test. Generally some 80% analysis showed that genetic background and environmental of chicks in any hatch group can be successfully trained and differences between laboratories in rearing and handling tested on this protocol. Each chick is usually trained and procedures alone can produce sufficient variation to span the tested only once. Because the pecking response requires a range of most, if not all, behavioural variables and can thus positive, accurate act by the bird, it also controls for effects easily mask or fake mutational effects. In addition, disparity on attentional, visual, and motor processes [47, 48]. attributable to evolutionary divergence between humans and The training task has two variants: strong, and weak. In rodents, brings about another type of problem: the strong the strong version, the aversive substance used to coat the but incomplete homology between human and mouse APP beads is 100% MeA and it produces high and persisting levels sequences and the weaker but still considerable homology of avoidance. However, if the MeA is diluted to 10%, the between APP and APP-like protein (APLP2), compromise high level of avoidance for the training bead persists only up accurate measurements of total APP transcript levels in 8 hours; long term memories are not formed. In its strong humanised APP transgenic mice and make assessment of form, the task can be used to identify the molecular cascade the neuropathogenic potential of human APP gene products involved in memory formation and the interventions that rather difficult [42, 45, 46]. impair consolidation; in its weak form the task can be used to explore potential memory enhancing agents. These features make the young chick a highly suitable model for the analysis of the biochemical (and in our hands 5. The Chick as an Animal Model morphological and physiological) correlates of memory for the Study of Memory consolidation. Table 1 compares the passive avoidance task Our route towards research on AD led through our lab’s in the chick with commonly used tasks in the mouse. interest, over many years, in the molecular mechanisms involved in memory storage, on which we have worked extensively with the young chick. The suitability of the chick 6. The Biochemical Cascade of Memory as a model system for such studies is well documented. Consolidation in the Chick Chicks are precocial birds, and need actively to explore and learn about their environment from the moment they hatch. Over the past decades a combination of correlative and Thus, they learn very rapidly to identify their mother on interventive experimental strategies has enabled us to iden- the basis of visual, olfactory and auditory cues (imprinting), tify a biochemical cascade that is associated with memory to distinguish edible from inedible or distasteful food, and consolidation in the minutes to hours following training. to navigate complex routes. Training paradigms that exploit These have been fully reviewed elsewhere [47]soonlya these species-specific tasks work with the grain of the brief summary will be given here. In the minutes following animal’s biology, and because such learning is a significant training on this task, there is upregulation of N-methyl- event in the young chick’s life the experiences involved D-aspartate receptor activity, phosphorylation of the presy- may be expected to result in readily measurable brain naptic membrane protein B50, and genomic activation of changes. Chicks have large and well-developed brains and the immediate early genes c-fos and c-jun. During the next soft unossified skulls, making localised cerebral injections hours after training, increased incorporation of fucose into of drugs easy without the use of implanted cannulae or brain glycoproteins occurs. During this time, memory for anaesthesia. The virtual lack of any blood-brain barrier in the passive avoidance task can be impaired by inhibitors these young animals also ensures rapid entry into the brain of protein and glycoprotein synthesis injected around the of peripherally injected agents (for review see [47]). time of training. Two regions of the chick brain are involved The training task that we employ is one trial passive specifically in the biochemical responses to the learning avoidance, in which chicks learn to avoid pecking at a experience. These are the intermediate medial mesopallium International Journal of Alzheimer’s Disease 5 Table 1: Comparison of training tasks in chicks and mice. Chicks Mice Passive avoidance Training paradigm Passive avoidance Fear conditioning Water maze (multiple trials) Passive avoidance - Brief Timing 10 secends training time Fear conditioning - Brief Water maze - multiple trials Passive avoidance - yes Suitable for biochemical analysis Yes Fear conditioning - yes Water maze - unsuitable Natural distribution of males and females Sex in the hatch. (Sex determined post hoc by Generally males only inspection of gonads) Group size Large (20 chicks/group) Small Intracranial injections Anaesthesia not required Anaesthesia required BBB Not fully developed Fully developed Genome Sequenced in 2004 Sequenced in 2002 Yes, many (for review see Transgenic model No Crews et al., 2010; [11]) (IMMP), an association “cortical” area, and the medial striatum (MS), a basal ganglia homologue. The chick brain is strongly lateralized and many, but not all, of the molecular events we observe are confined to the left IMMP. All these events depend on de novo protein synthesis and insertion of a variety of proteins, especially glycoproteins, into pre- and postsynaptic membranes. Cell adhesion or cell recognition molecules constitute a major group amongst these glycoproteins. They are expressed both pre- and −30 0 +30 1 2 3 4 5 6 7 8 9 10 11 post-synaptically and involved in the process that allows Time of training (hr) information about synaptic activity to be simultaneously ANI communicated to both side of the synapse. Our early work 2-D-d-Gal identified two such adhesion molecule, L1 and NCAM, which are recruited into this cascade of cellular events Figure 2: Two time-windows when protein synthesis is sensitive at different periods posttraining. Injection of inhibitors to inhibitors of protein synthesis, such as anisomycin (ANI) and of protein and glycoprotein synthesis (anisomycin and 2- glycoprotein synthesis, such as 2-d-Galactose (2-d-Gal). deoxy-galactose resp.) at times corresponds to these periods of recruitment (Figure 2) result in amnesia for the task [47]. This and related data on the effects of application of explore the role of APP itself. Chick APP cross-reacts with protein synthesis inhibitors on memory retention led us the mouse monoclonal antibody raised against human APP. to propose that there were two waves of protein synthesis Therefore, we tested the effect of anti-APP antibody on occurring following a learning experience, the first within memory. The residence time for the anti-APP corresponds an hour of the experience and involving the synthesis of to the relatively rapid turnover time for membrane-bound proteins expressed by immediate early genes, and the second, APP. When injected pre-training, anti-APP did not interfere some 4–6 hours later, involving structural proteins such as with the chicks pecking and learning the avoidance task; the adhesion molecules. Both are necessary for consolidation however, it did result in amnesia in birds tested 30 minutes of long-term memory. later. Amnesia persisted for at least the subsequent 24 hours and was not apparent if the antibody was injected just posttraining or 5.5 hours after training [17, 49]. 7. APP and Memory Consolidation in the Chick This finding indicated that APP might be required APP is an important member of the family of cell adhesion at an early phase and not continually during memory molecules, and having identified a role for NCAM and L1 consolidation. Given that blocking APP function by use [51–53] in the consolidation cascade, it seemed logical to of specific antibodies outside of a specific time window Protein synthesis 6 International Journal of Alzheimer’s Disease Table 2: Effect of anti-APP and APP antisense on memory retention. Memory retention Memory retention Time of injection Time of injection (% Avoidance) (% Avoidance) Control (Saline, 30 minutes pre-training 78–95 5.5 hours Posttraining 78–95 non-immune sera) ∗∗ Anti APP 30 minutes pre training 28–35 5.5 hours Posttraining 78–95 Control (SC) 6 hours pre-training 78–95 30 minutes pre-training 78–95 AS 6 hours pre-training 32–37 30 minutes pre-training 7 8–95 ∗ ∗∗ N = 18–25; P <.05; P <.01. Anti APP: monoclonal human antibody mAb22C11 [17]; AS: 16-mer end-protected phosphodiester oligodeoxynucleotide, 5 CXCGAG GACTGA XCCA 3 , designed to correspond to the transcription start sites −146 and AUG1786 of the βAPP mRNA, immediately upstream of a ribozyme binding site [17]; SC: Scrambled AS sequence [17]; For further details see [18, 49, 50]. 2 μm 5 μm (a) (b) (c) Figure 3: RER binding detected on chick, human and mouse neuronal cells. Specific binding of the biotinylated RER (arrows) to chick (a), human (b) and mouse neuronal (c) cell. Location of the chick neuronal cells is in the IMMP area; Human and mouse neuronal cells are located in the CA1 are in hippocampus. was without effect, we compromised APP gene expression 8. APP-Related Peptides as a Tool to using antisense oligodeoxynucleotides (AS) designed to Study Memory correspond to the −146 and to AUG1786 transcription Studies conducted on the physiological activity of APP [54– start site of APP [17]. The antisense oligodeoxynucleotides 58] resulted in the identification of a small stretch of amino (AS ODNs) were injected intracerebrally at 6 hours or 30 acids containing the RERMS sequence C-terminal to the KPI minutes pre-training and chicks were tested at different times posttraining. Injection at 6 hours pre-training was aimed insertion site of sAPP-695 as the active domain responsible for growth promotion and neurite extension, neuronal sur- to suppress APP synthesis during the first wave of protein vival, and for sAPP’s ability to interfere with the deleterious synthesis while the injection made at 30 minutes pre-training was aimed at the second wave (Table 2). Thus, in both groups effects of Aβ on neurons. A synthetic peptide homologous to the RERMS sequence, APP 328–332, was identified as the the AS was present for 6 hours before training. Controls were shortest active peptide to exhibit trophic activity through treated with scrambled (SC) ODNs or saline and trained and cell-surface binding and induction of inositol polyphosphate tested as the AS ODNs treated groups. accumulation. Such observations suggested that the RERMS The results showed that APP-antisense both decreased peptide might substitute for sAPP during memory formation APP gene expression and affected memory formation. The and thereby reverse or protect against the blockade resulting time-window of onset of amnesia relative to time of injection from antibody or antisense. of ODNs and to time of training confirmed our previous finding that APP exerts its function during an early phase We first assessed the effects of RERMS on memory in chicks rendered amnesic by APP-antisense and APP- of memory formation and appears to be a necessary factor in the biochemical cascade involved in the transition antibody treatment [17, 49]. In the series of experiments between short- and long-term memory. Our findings on the which followed, we studied the time window in which injection of RERMS might affect amnesia and showed that importance of APP in learning were supported by reports [19] that APP gene expression in the young chick is related if injected either before or just after training on the task, the pepide protected against the memory loss. As a control to the strength of memory retention for an imprinting for the behavioural effect of RERMS, we used the reversed task. International Journal of Alzheimer’s Disease 7 RER rer HO O HO O O O O O H H H H N N N N N N NH NH H H 2 2 O O O O rER HO O HN HN HN HN O O NH H N NH NH H N NH H N H N 2 2 2 2 H H NH H 2 HO O HO O HN HN O O O O H H H H NH H N NH H N 2 2 N N N N N N NH NH H 2 H 2 O O O O HN HN HN HN NH H N NH NH H N NH H N H N 2 2 2 2 REr ReR Figure 4: Structure of D/L tripeptides included in the study. peptide sequence SMRER, but to our surprise SMRER was as in animals rendered amnesic by pretreatment with Aβ.We effective in relieving the memory block as RERMS. However, concluded that the RER sequence acts as a core domain of adifferent control peptide, RSAER, was without effect. Anal- the growth promoting region of APP in the chick because it ysis of these experiments led to two principal observations: appears able to substitute for sAPP. The protection against first, that the APP-derived peptide might exert its action by the amnestic effects of Aβ may also result from RER’s ability compensating for the low presence of APP. According to the to initiate receptor-mediated processes. This interpretation is proposed amyloid hypothesis, the faulty processing of APP strengthened by the evidence that RER binds to two neuronal and accumulation of the amyloid fragments might be one cell membrane proteins, of ∼66 and 110 kDa, respectively. In of the factors leading to neurotoxicity. Therefore, we tested experiments aimed at identifying specific neuronal binding whether the RERMS peptide might also have a potential partners, using a combination of biotinylated tripeptide and protective effect against the memory deficit induced by Aβ. cell-specific antibodies, bound RER was localised in chick Amyloid-beta, injected into the IMMP bilaterally at a dose and human brain sections (Figure 3), suggesting that it might of 2 μg/hemisphere, 30 minutes prior to training, resulted in also be active in humans, and could play an important role in amnesia for the task in chicks tested 24 hours subsequently. the memory formation process which is deficient in the early However, administration of 1 μg/hemisphere of RERMS 10 stages of AD. Moreover, the distribution of biotinylated RER minutes after Aβ injection prevented the memory deficit binding suggested membrane binding. In the chick, binding caused by Aβ. Conversely, if the injection is delayed to 5.5 is displaced by longer peptides derived from APP’s external hours posttraining, there is no subsequent amnesia. domain, but not by Aβ, suggesting that RER competes with The second observation came from analysis of the amino sAPPfor aputativereceptor[17, 49]. acid sequence of the peptides used in this study [17, 49]. Both To overcome the problem of the short half-life of RER the forward and reverse sequences contain the tripeptide we protected it by N-acylation, and showed that Ac-RER palindrome RER. The next step was therefore to investigate is as effective as RER in protecting against memory loss. the ability of RER to relieve memory block under the same More importantly, Ac-RER crosses the partially formed conditions used for testing RERMS. The RER tripeptide, blood brain barrier of the one-day old chicks, enabling the when injected around the time of training, showed the same peptide to be injected intraperitoneally [17]. The immediate potential as the RERMS pentapeptide and rescued memory implication of these findings is that it is possible to introduce 8 International Journal of Alzheimer’s Disease Table 3: Summary of peptides and their effects on memory reported in this study. Enhances Crossing Peptide Injection route Effective dose Rescue of amnesia induced with: t1/2 hour weak training BBB Ic ip Ic μg/brain Ip mg/kg bw Anti APP AS Aβ Up to RERMS 2 Y Y 4 20–25 Y Y Y Y Y SMRER 2 Y Y 4 20–25 Y Y Y Y Y RSAER / NN 4 / N N N N Y RER / Y Y 4 20–25 Y Y Y Y Y Ac-RER 6 Y Y 16 20–25 Y / Y Y Y Ac-RRE / NN 16 / / / N N / Ac-rER >12 Y Y 16 20–25 Y / Y Y Y Ac-REr / NN 16 / N / N N / Ac-ReR / NN 16 / N / N N / Ac-rer / Y Y 16 20–25 N / N N / Y: yes, there is an effectonmemory; N: No,there is no effect on memory; Anti-APP: monoclonal antibody, clone mAb22C11 [17]; AS: 16-mer end-protected phosphodiester oligodeoxynucleotide, 5 CXCGAG GACTGA XCCA3 , designed to correspond to the transcription start sites −146 and AUG1786 of the βAPP mRNA, immediately upstream of a ribozyme binding site [17]; Aβ: amyloid-beta. For further details see [18, 49, 50]. abehaviourally effective form of RER peripherally by N- administration. The fact that there was no difference in the terminal acylation, hence protecting the peptide against magnitude of the effect of the L- and L/D tripeptide on rapid degradation. behaviour suggested that they engage the same biochemical processes [50]. These results are summarised in Table 3. If the RER sequence acts as a substitute for sAPP than What is now required is to determine the identity of the the question to ask is whether it might act as a cognitive RER binding proteins, the specific second messenger systems enhancer in the weak version of the passive avoidance task activated and the genes controlled by RER. Our currently discussed above. The weak training protocol is an ideal unpublished experiments go some way towards answering paradigm to test this hypothesis as memory for the task is these questions, which may be central to understanding not retained beyond an early phase, presumably because the the peptide’s effects both in memory enhancement and, mild aversant does not provide a sufficient signal for the potentially, in neuroprotection. release of sAPP. Under these conditions, tripeptide injected peripherally wasaseffective as memory enhancer as when injected intracerebrally, meaning that even in the weak 9. Concluding Remarks training task, in the presence of the tripeptide peptide, Although it remains important to demonstrate that the memory persists for at least 24 hours. peptide or its related structures is effective in other learning All our results point to the short APP-related peptides tasks and in mammals, we propose that the chick is a used in our experiments as both powerful tools in studying useful animal model in which to study AD, and that Ac- the structure and function of APP and as of potential rER is a molecule which might form the basis for a potential therapeutic interest in AD. We have therefore begun to therapeutic agent in the early stages of AD. Even though explore the effectiveness of a number of compounds struc- some specific details of protein-protein interactions can turally related to RER. Of particular interest have been vary between birds and human, the degree of functional the optically isomeric D- or diasteromeric (D/L) forms of conservation seems to be of particular relevance for the the peptide, which are more resistant to proteolysis than AD field. This animal model, like many other natural their L-equivalents. The diasteromeric forms have begun model-systems, often appears to suffer from publication lately to attract increasing interest as potential immunogens, bias towards transgenic animal models, which may account diagnostic and therapeutic agents [59]. for substantial under-representation of avian model system Our results using different D/L forms (shown in in the experimental literature related to neurodegenerative Figure 4) demonstrated that substitution of C-terminal L- diseasessuchasAD. arginine with the D-isomer essentially abolished the memory retention-enhancing effect of the peptide. 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