HPA axis adaptive changes in human AD patients and AD mouse models

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Memory formation, synaptic plasticity and modulation by A

AD is a progressive neurodegenerative disease marked by a constellation of cognitive disturbances. The earliest form of memory affected in AD is episodic memory and is one of the most clinically relevant neurological symptom for AD patients. This form of memory helps us to function smoothly in our basic daily activities and to remember critical events. As the disease progresses the patient develops other cognitive abnormalities such as apraxia (inability to carry voluntary movements), aphasia (loss of ability to speak) and agnosia (inability to recognise objects and their use) eventually leading to global cognitive impairment.

Different types of memory

There are mainly two forms of memory depending on the duration of recall: short and long term memory (STM and LTM) (Figure 6). STM is known to hold a limited amount of information in a very accessible state temporarily (Cowan, 2008). STM mainly relies on cortical neuron activity in the lateral prefrontal cortex (Squire and Wixted, 2011). This structure is specifically known to perform higher executive functions, which include working memory and attention.
The LTM system is divided into two broad classes: explicit (declarative) memory, which requires the conscious recall of data, facts and events, and implicit (non-declarative) memory, which is based on non-conscious memory abilities. Implicit memory includes procedural or skilled-based kinds of learning and perceptual representation system (PRS) (Squire et al., 1987). PRS mainly involves priming effects (where one stimulus influences the response of the other stimulus) and operant or classical conditioning (which involves pairing of two stimuli). Long term memory is stored in the neocortex and distributed in different regions, where the area specific processing occurred at the time of learning.

Declarative long term memory

Declarative memory is subdivided into semantic memory and episodic memory (EM). Semantic memory is related to storage of general facts and knowledge, such as the colour of fruits or the capital of countries. While EM is a system that enables an individual to encode, store, and retrieve information about personal experiences and the temporal and spatial contexts of those experiences (first defined by Tulving in 1972).

Encoding, Consolidation, Storage and Retrieval

Memory formation and utilization can be divided into the following stages: Encoding, consolidation, storage and retrieval. The critical structures involved in these processes of memory involve hippocampus, the amygdala and the adjacent entorhinal, perirhinal and parahippocampal cortices, which make up the medial temporal lobe (MTL) and the prefrontal cortex (Squire and Wixted, 2011).
Encoding is the first stage of memory, and it is crucial for the storage and retrieval of voluntary stored information. Encoding begins with perception of sensory inputs that comes from external stimuli. The MTL structures are involved and responsible for transforming these sensory inputs to a memory representation (Squire and Wixted, 2011). All these neuronal activities converge to the medial temporal lobe and specifically to the hippocampus, where they bind to form episodic memory (Figure 7) (Kessels and Kopelman, 2012). After binding, they are allocated to areas in the cortex for long-term storage as they are well protected from the influence of new incoming memories (Straube, 2012). The type of information processing that occurs during the encoding stage determines the quality of encoding and recovery of that information.
Consolidation: Encoded memory undergoes consolidation, a process by which short term memory trace is transferred to stable long term memory before directing it to neocortical areas for long term storage. There are two models of consolidation: the standard model (SM) and the multiple memory trace (MMT) (Nadel et al., 2007). The SM model, proposes that memory storage initially requires the hippocampus to link the different features of memory which are dispersed in several sites in the neocortex. Over time, however, the requirement of the hippocampus decreases and the representation of the memory is solely in the neocortex (Frankland and Bontempi, 2005) (Figure 8). In contrast, the MMT theory poses that all memory traces are combined into a multiple-trace representation. In this model, both the hippocampus and the neocortex continue to interact with each other and that the hippocampus plays a permanent role for the storage and retrieval of the memory. CORT modulate memory consolidation of emotionally aroused experiences (McIntyre et al., 2012) and sleep also plays an active role in memory consolidation (Straube, 2012).

Episodic memory is affected in AD

Episodic memory refers to the conscious recollection of a unique past experience / event in terms of “what”, “where” and “when” it happened. These three components identify a particular object or person (memory for what happened), the context or environment in which the experience occurred (memory for where it happened) and the time at which the event happened (memory for when it happened) (Nyberg et al., 1996, Clayton and Dickinson, 1998, Tulving and Markowitsch, 1998). It is the first form of memory, which is affected in Alzheimer’s patients (deToledo-Morrell et al., 2007). Recall (free and cued) and recognition are considered to provide two different ways to measure episodic memory. Recall depends on declarative memory and recognition on declarative and non-declarative memory. In a free recall experiment, an individual is given a list of items to study and is subsequently asked to recall the items (Arnold and McDermott, 2013), the results of which are noted. While in cued recall, the same protocol is followed with the help of cues. With this test, one can evaluate hippocampus-dependent memory encoding or consolidation. During a recognition experiment, the first phase, called the study phase, a subject is asked to memorize a series of items. Later, in the test phase, randomly ordered items, old and new, are presented and the individual is scored on his ability to distinguish them. In AD patients, both recall and recognition deficits seem to persist, which indicates inability to retrieve information. This could also be related to inability to encode target information (Gold and Budson, 2008).

Episodic-like object recognition memory in rodents

It has been challenging to study declarative forms of memory in mice since it is an integrative memory for “what”, “where” and “when” component and also it needs to be expressed non-verbally. Thus, in rodents, the equivalent studies done are hippocampus-dependent spatial and contextual memory tests. The most commonly used tasks to study spatial memory in AD mouse models are the Morris water maze (MWM), the Barnes Maze also called circular platform maze, the continuous and forced-choice spatial alternation task, and the radial arm water maze task (Stewart et al., 2011).
To integrate the component of familiarity of items along with recollection of contextual (spatial and/or temporal) information of items, the object recognition test was developed. The novel object recognition task measures spontaneous behaviour in rodents as they readily approach and explore novel objects (Frick and Gresack, 2003). This widely used task consists of a sample trial wherein two identical objects are explored by the mouse, followed by a delayed test trial wherein one familiar object presented during the sample test is replaced by a novel object (see Figure 9A). Animals with adequate memory spend longer time exploring the novel object. The NOR test could be interpreted as the study of the ‘What’ component of episodic memory. This task has been widely used for evaluation of memory in AD mouse models (Balducci et al., 2010). Two key points confirm that novel object recognition can be dependent on hippocampal activity. Firstly, it was noted that hippocampal lesions result in impaired object recognition and secondly a 24-hour inter-trial interval between the two phases (sample and test) confirms retrieval of long term memory consolidated via hippocampal activity (Reed and Squire, 1997). In NOR memory, particularly the dorsal hippocampus plays an important role, especially when spatial or contextual information is a relevant factor (Goulart et al., 2010).

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Table of contents :

SUMMARY
1 ABBREVIATIONS
2 INTRODUCTION
2.1 Alzheimer’s Disease
2.1.1 Aging, Dementia and AD
2.1.2 AD and its discovery
2.1.3 AD neuropathology:
2.1.3.1 Senile plaques
2.1.3.2 Tau neurofibrillary tangles
2.1.4 Two types of AD:
2.1.4.1 Familial AD
2.1.4.2 Sporadic AD
2.1.5 Environmental risk factors:
2.1.6 APP and its processing:
2.1.6.1 Function of APP:
2.1.6.2 APP processing:
2.1.6.3 Amyloid cascade hypothesis
2.1.6.4 Criticism and modifications to the amyloid hypothesis
2.1.7 Evolution of AD in the brain
2.1.8 Treatments
2.2 Memory formation, synaptic plasticity and modulation by A
2.2.1 Different types of memory
2.2.1.1 Declarative long term memory
2.2.2 Memory formation and its stages:
2.2.2.1 Encoding, Consolidation, Storage and Retrieval
2.2.3 Episodic memory is affected in AD
2.2.3.1 Episodic-like object recognition memory in rodents
2.2.4 Hippocampus organization and pathways for memory formation
2.2.5 Basal synaptic transmission and synaptic plasticity
2.2.5.1 Glutamergic synapse
2.2.5.2 Post synaptic density
2.2.5.3 AMPAR and NMDAR
2.2.5.4 Different forms of synaptic plasticity
2.2.5.4.1 LTP
2.2.5.4.2 LTD
2.2.6 Modulatory action of A on synapse function and plasticity
2.2.6.1 Physiological conditions
2.2.6.2 Pathological conditions
2.2.7 Modulatory action of A on memory
2.2.8 Putative mechanisms of A actions at synapses
2.2.9 Mouse models of AD
2.2.9.1 Different mouse models
2.2.9.2 Tg2576
2.2.9.2.1 A accumulation
2.2.9.2.2 Memory deficits
2.2.9.3 A local injections
2.3 Stress axis
2.3.1 Stress
2.3.1.1 Definition
2.3.1.2 Acute and Chronic stress
2.3.2 Hypothalamus-Pituitary-Adrenal (HPA) axis
2.3.2.1 CORT release
2.3.2.2 Effect of CORT on thymus
2.3.2.3 ACTH release and its effect on adrenal glands
2.3.2.4 Negative feedback control of CORT
2.3.3 GRs and MRs
2.3.3.1 Gene sequence and structure of the receptors:
2.3.3.2 Functional role of the receptors:
2.3.3.2.1 Membrane CORT receptors and their functions
2.3.3.2.2 Genomic action of receptors
2.3.4 Techniques to study receptor function
2.3.4.1 Different GR agonist and antagonists and their drawbacks
2.3.4.2 Genetic manipulation studies
2.3.5 Role of the hippocampus in stress/HPA axis
2.3.5.1 Effect of stress on hippocampus:
2.3.5.2 Effect of stress/CORT on synaptic plasticity
2.3.5.2.1 At intermediate CORT level/stress:
2.3.5.2.2 High CORT level/stress
2.3.5.3 Effect of CORT/ stress on memory
2.3.6 GR modulators and their use as therapeutic in AD
2.4 Link between AD and stress
2.4.1 Stress is a major environmental risk factor for AD
2.4.2 HPA axis adaptive changes in human AD patients and AD mouse models
2.4.2.1 CORT levels
2.4.2.2 ACTH levels
2.4.2.3 Stress related disorders in AD patients
2.4.3 Tau and HPA axis
2.4.4 Role of stress/CORT administration on A pathology
2.4.5 Role of GRs in AD
2.4.6 Relationship between A oligomers and GRs
2.4.7 Common link between A and CORT on the glutamatergic system
3 OBJECTIVES
4 MATERIALS AND METHODS:
4.1 Animal Breeding
4.2 Genotyping
4.3 Dissection of hippocampus, thymus and adrenal glands
4.4 Biochemical Techniques
4.4.1 Estimation by ELISA
4.4.1.1 Plasma corticosterone
4.4.1.2 ACTH estimation
4.4.2 Extraction of total proteins from hippocampus
4.4.3 Immunoblotting
4.4.4 Aβ oligomer (oAβ) preparation
4.5 Local in vivo ablation of GR in GRlox/lox mice
4.5.1 Stereotaxic injections of AAV
4.5.2 Immunofluorescence staining of GR
4.5.3 Microscopy and estimation of GR intensity
4.6 Electrophysiology
4.6.1 Slice preparation
4.6.2 Field recordings by electrophysiology
4.6.2.1 To measure basal synaptic plasticity
4.6.2.2 For pharmacological studies
4.6.3 Analysis of fEPSP response:
4.7 Behaviour
4.7.1 Episodic-like object recognition memory
4.7.2 Novel Object Recognition (NOR) after local in vivo injections
4.8 Statistical analysis
5 RESULTS
5.1 Chapter 1
5.1.1 Aim: Study of HPA axis dysregulation in Tg2576 (Tg+) AD mouse model
5.1.1.1 Comparison of CORT levels in WT and Tg+ male mice at 3 and 6-month of age
5.1.1.2 Comparison of plasma ACTH levels in WT and Tg+ male mice at 4 and 6 months
5.1.1.3 Comparison of body, thymus and adrenal gland weights between WT and Tg+ male mice at 4 and 6 month of age
5.1.1.4 Quantification of GR by immunoblotting from hippocampal total protein extract in 4 months male Tg+ and WT mice.
5.1.1.5 Rescue of episodic memory deficits in 4 month Tg+ male mice with GR antagonist RU486 treatment.
5.2 Chapter 2
5.2.1 Aim 2: To check the specific role of GRs in AD like phenotypes in GRlox/lox Tg+ mice.
5.2.1.1 Generation of the GRlox/lox Tg+ mice
5.2.1.2 Basic characterization of the GRlox/lox Tg + mice
5.2.1.3 Verification of AD like phenotypes in GRlox/lox Tg+ as seen in Tg+ mice.
5.2.1.4 Increased CORT levels
5.2.1.5 Exacerbated LTD phenotype
5.2.1.6 Stereotaxic injections with Cre-GFP in GRlox/lox Tg+ mice to ablate GR gene in CA1 neurons in vivo 109
5.3 Chapter 3
5.3.1 Aim 3: To investigate if Aß oligomers act via GRs to promote their acute synaptic effects at hippocampal synapses.
5.3.1.1 Effect of oAß on levels of GR in PSD
5.3.1.2 Effect of GR Antagonist compound 13 (C13) on synaptic transmission and LTP
5.3.1.3 Effect of C13 on LTP impairment caused by oAß
5.3.1.4 Quantification of GR reduction in the CA1 of the GRlox/lox Tg- mice upon in vivo Cre-GFP transduction
5.3.1.5 Effect of GR reduction in CA1 on LTP
5.3.1.6 Effect of GR reduction in CA1 neurons on the LTP impairment caused by oA
5.3.1.7 NOR test after oAß local injections
5.4 Chapter 4
5.4.1 Discovery of -secretase APP processing pathway
5.4.2 Aim 4: Effect of CHO derived Aη- and Aη-ß peptides on LTP
6 DISCUSSION AND PERSPECTIVES
7 CONCLUSION
8 PERSONAL ACCOMPLISHMENTS
9 ANNEXE
10 REFERENCES

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