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

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Glutamergic synapse

Glutamate is the major excitatory neurotransmitter in the nervous system and is particularly predominant in the hippocampus. It is involved in fast transmission and found as a part of the tripartite synapse which involves communication between pre-and post-synaptic neurons along with glial cells. The pre-synaptic release of glutamate and the activation of post synaptic glutamate receptors are essential mechanisms involved in LTP at glutamatergic synapses (Figure 11). Upon vesicular release, glutamate can act on ionotropic glutamate receptors, (AMPA, NMDA and kainate receptors), which are ligand-gated ion channels and allow direct ion influx when activated (Dingledine et al., 1999). AMPA stands for (-amino-3-hydroxy-5-methyl-4-isoxazole-propionate) and NMDA for (N-methyl-D-aspartate). Metabotrophic glutamate receptors (mGluR) can also be activated by glutamate which are G-protein-coupled receptors and modulate secondary messenger pathways. In this chapter, focus will be given to AMPA receptors (AMPAR) and NMDA receptors (NMDAR).

Post synaptic density

Post synaptic density (PSD) refers to the electron-dense region in the postsynaptic membranes of excitatory synapses which mainly contain proteins responsible for synaptic activity. The biochemical methodology of PSD purification was established in 1970s. In brief, initially the synaptosomes were isolated, followed by detergent treatment (Triton X) for purification of PSD fractions (Carlin et al., 1980). Due to their resistance to detergents they are isolated as detergent –insoluble fractions. From these biochemical PSD fractions, an array of structural membrane proteins (e.g. PSD-95 as a scaffolding protein), cytoskeletal proteins (e.g. actin and tubulin), receptors (NMDAR, AMPAR), ion channels, signalling molecules like calcium/calmodulin dependent protein kinase II (CAMKII) and other enzymes were identified (Matus et al., 1982, Chen et al., 2000, Valtschanoff and Weinberg, 2001). This receptor/enzymes system plays a major role in synaptic activity and PSDs are thus identified as one of the main machineries for synaptic transmission and plasticity.

AMPAR and NMDAR

Within the PSD, one can find the AMPARs, which performs most of the fast-synaptic transmission in the brain. They are composed of GluA1-4 subunits (also previously called GluR), which combine to form a heterotetramer (composed of 4 subunits, of more than one type). These tetramers can be formed of different subunits conferring different activity and channel permeability properties (Kessels and Malinow, 2009). When activated by glutamate, AMPAR allow Na+ influx.
NMDARs can be formed from various combinations of GluN1 (previously called NR1), GluN2(A-D) (previously called NR2(A-D)), and GluN3(A/B) (previously called NR3(A/B)) subunits. The most common form of NMDAR on hippocampal excitatory neurons is the heterotetramic combination or GluN1 with GluN2 units (Paoletti and Neyton, 2007). Activation of these receptors requires the binding of glutamate to GluN2 and one of the co-agonists glycine or d-Serine to GluN1. NMDAR activation opens a non-selective ion channel through which Ca2+ and Na+ ions can enter the post synaptic neuron. NMDAR activation requires depolarization of the surrounding membrane, which removes Mg2+ ions from its pore, thus allowing for ion flux through its channel. This property makes NMDARs ideal coincidence detectors, coupling pre-synaptic and post-synaptic activation, and these receptors are thus crucial for information flow and memory processing (Nowak et al., 1984). Both AMPAR and NMDAR are involved in synaptic plasticity processes of LTP and LTD.

Modulatory action of A on synapse function and plasticity

As shown in the above pictorial representation (Figure 13) by (Palop and Mucke, 2010), there is evidence that synaptic activity is differentially modulated depending on the APP/A levels. Intermediate levels of A potentiate presynaptic terminals, while low levels reduce presynaptic efficacy and high levels depress postsynaptic transmission. Abnormally low A level in mice deficient in APP, PS1 or BACE1 is associated with synaptic transmission deficits (Seabrook et al., 1999, Saura et al., 2004). Due to the extensive research currently carried on the pathological effects of high concentration of A, we tend to forget that the proteolytic pathway of production of amyloid- is a physiological process. And only when net A levels become excessive can this process be regarded as pathological condition.

Physiological conditions

Normal levels (picomolar range) of A peptide via nicotinic acetylcholine receptors (nAChRs) in CA1 (Puzzo et al., 2008) positively increase presynaptic release at hippocampal synapses and facilitate LTP (see Figure 14, Figure 15). Parallel evidence indicates that A may have a role in controlling synaptic activity. Kamenetz et al. 2003 proved that evoked activity of hippocampal neurons in brain slices increased the A secretion at the cell membrane. In physiological conditions, this production of A seems to work as a negative feedback mechanism to controls synaptic activity. Without such a tight control, synaptic activity could become excessive leading to excitotoxicity. There are two evidences confirming this function: -secretase inhibition would lead to increased excitatory post synaptic current (EPSC) frequency (Kamenetz et al., 2003) and kainate-induced seizures are potentiated in APP knockout mice (Steinbach et al., 1998). Together these studies suggest that APP processing and presence of A are closely associated with synaptic activity. This may serve as a physiological control for guarding against excessive activity and preventing increased glutamate release.

Modulatory action of A on memory

A levels modulate memory processes in a way similar to how they regulate synaptic activity. At intermediate levels, they have a positive effect on memory formation, while as the concentration of A increases it can cause memory impairment. There have been multiple studies using AD transgenic mice (like Tg2576 mice and other APP mouse models) to study memory impairment in the context of abnormal APP processing and elevated A levels. Results from these mouse studies demonstrate that a chronic increase of A levels, but also possibly an increase of other APP fragments, correlate with spatial and contextual memory impairment (Hsiao et al., 1996, Higgins et al., 1994, Sandhu et al., 1991). These results indicate the role of APP/ APP processing/ A accumulation on memory processes. On the other hand, non-transgenic models of AD (mice/rats) have been used to study acute effect of in vivo application of A peptides or oligomers on memory processes. In vivo local injections have been mostly carried out using synthetic A25-35 and A1-42 in the intracerebral ventricle (icv) or intra-hippocampus. Using this acute model of AD, results have shown that, at intermediate concentrations (picomolar), A can enhance learning and memory retention (Morley et al., 2010). This has been confirmed by (Puzzo et al., 2008) where infusion of pM concentration of A1-42 improved reference and contextual memory. However, a single icv injection of nanomolar concentration of A1-42 impairs memory consolidation within 24 hours, suggesting that A can also rapidly interfere with synaptic activity necessary for stabilization of new memories (Balducci et al., 2010). Whereas, when injected after memory consolidation, no such effects were seen indicating that A oligomers affect memory consolidation rather than retrieval (Balducci and Forloni, 2014). These results hence confirm the bell-shaped relationship between extracellular A and its effect on memory processes.

Putative mechanisms of A actions at synapses

Elevated extracellular A levels particularly in the form of soluble oligomers can alter synaptic transmission and memory processing. However, the mechanism of action of these oligomers are currently nuclear. Diverse lines of evidence show that extracellular oligomers can bind to pre-and post-synaptic elements on cultured neurons and in the cortex of AD patients. Cellular and animal studies have attempted to identify the molecular targets of the oligomers and have yielded an array of candidates. A has been reported to interact functionally and also sometimes structurally with several distinct types of plasma membrane –anchored receptors, including 7 nicotinic acetylcholine receptors (Snyder et al., 2005), NMDA and AMPA receptors (Lacor et al., 2007), insulin receptors, RAGE (the receptor for advanced glycation end products) (Deane et al., 2003), the prion protein PrPc (Um and Strittmatter, 2013), and the Ephrin-type B2 receptor (EphB2) (Lacor et al., 2007).
The extracellular nature of A assumes that the toxic effects are mediated via membrane-bound substrate and/or by internalization of A by affected neurons. Examples of such plasma membrane substrates are the metabotrophic glutamate receptors (mGluR) and PrPc which interact with A at the synapse and these interactions are known to catalyse synaptic dysfunction and eventually cell death. Reports of capability of A to make holes in the lipid bilayers of membranes could also serve as sites for aberrant entry of Ca2+ into cells (Capone et al., 2009).
It is important to reinforce here that when studying the different receptor /transporters, which are putative substrates for A, consideration must be given to the form of A used, its concentration and its relevance to physiology. Physiological concentrations of A peptides in human brain, and cerebrospinal fluid are in low nanomolar range or below (Schmidt et al., 2005, Giedraitis et al., 2007). A concentrations in the most pathobiologically relevant sites of the AD brain, i.e. within and around the synaptic clefts are unknown and might be higher. Also, the binding capacity of monomers to certain receptors will not be the same as binding of oligomers. Hence since these questions are unanswered it is currently hard to conclude on the molecular mediators of A actions.

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Hypothalamus-Pituitary-Adrenal (HPA) axis

The HPA axis activation occurs in response to circadian signalling pathways (Schibler and Sassone-Corsi, 2002) and in presence of a stressor, hence taking the name ‘stress axis’. HPA axis is mainly under the excitatory control of the amygdala and inhibitory control of the hippocampus. Once the hypothalamus receives the signal of the stimulus, the parvocellular neurons within the paraventricular nucleus (PVN) of the hypothalamus release corticotrophin-releasing hormone / factor (CRH/CRF) and vasopressin (AVP) into the portal vessels. Through these vessels, CRH and vasopressin reach the anterior pituitary. This signal further produces adrenocorticotropic hormone (ACTH) which regulates the adrenal cortex to release CORT (Figure 16).

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
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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