Cell-specific changes in metabolism and transmission as an initial cause of Alzheimer’s disease
Our hypothesis is that the initial expression of soluble forms of one protein, characteristic for AD, Amyloid-Beta (A-B), in identified entorhinal cortex layer II neurons eventually doom the network and subsequently downstream brain areas. The project aims to establish the early effects of increased levels of A-B on entorhinal functions.Alzheimer’s disease (AD) is the most common neurodegenerative disorder. Despite great effort, the cause(s) of AD remain(s) unclear. The establishment and progression of AD relates to increased intracellular expression of soluble amyloid-beta (A-B). A-B is derived from the cleavage of the amyloid precursor protein (APP), which can be cleaved into non-amyloidogenic and amyloidogenic species. Amyloidogenic species have a strong propensity to self-aggregate into oligomers. Their clearance depends on autophagy, a process essential for neuronal maintenance. Dysfunction of autophagy is directly linked to a growing number of neurodegenerative disorders, including AD and depends on various adaptor proteins, such as the protein p62. Neurons differ in terms of their vulnerability to AD and reelin-expressing neurons in entorhinal cortex (EC) layer II, projecting to the hippocampus are among the first to die. This essentially disrupts an important pathways in our main memory system, likely causing the early stage disruption of episodic memory. The project aims to assess changes in i) the electrical and chemical communication of entorhinal layer II neurons (synaptic activity), and ii) metabolic pathways that are relevant for keeping neurons healthy (autophagy pathways). We tested the effects of changes in intracellular amyloid-beta expression on synaptic efficacy using an in-vitro slice preparation of a transgenic rat model for AD, in which we can easily measure changes in synaptic connectivity. We submitted a manuscript, in which we report that stimulation of the EC pathway elicited similar activity patterns in slices from transgenic and control rats aged 3 and 9 months. At 12 months, when we noticed the first deposits of insoluble A-B (plaques), this pattern was subtly altered in the transgenic group, without any change in overall excitability. Most neurons in layer II of EC, fan cells and stellate cells, expressed intracellular Aβ. The electrophysiological properties of fan cells were unaltered, whereas stellate cells were slightly more excitable in transgenic than in control rats. We are now in the final stage of a first analysis of potential changes in the role of autophagy in EC layer II neurons in the transgenic rat model. We collected samples from 15 wild type and 15 homozygous transgenic rats from three different age groups, both females and males. We analyzed expression levels of the proteins p62 and intracellular levels of the marker protein reelin, a marker for the vulnerable neurons in EC layer II. The protein reelin is a potent suppressor of some features of AD, protecting against A-B toxicity. Reelin modulates neuronal function and synaptic plasticity and regulates axonal growth and dendritic spine morphology. Expression levels of reelin is therefore chosen as a proxy for changes in layer II neurons. Our results indicate no changes in levels of reelin related to age, and between transgenic and non-transgenic littermates, except for the measure at 6 months of age, where the transgenic animals have an increase in levels of reelin. The functional relevance of this finding is as yet unclear. In contrast, levels of P62 decline with increasing age, but this decline is not different between the transgenics and controls.
The establishment and progression of AD relates to intracellular expression and subsequent extracellular deposition of amyloid-ß (Aß). We tested the hypothesis that intracellular Aß expression can alter synaptic efficacy and that autophagy plays a role in the progression of AD in the rat model. The first conformative results were obtained.Alzheimer’s disease (AD) is the most common neurodegenerative disorder. AD manifests during aging, which is one of the most relevant risk factors, by a progressive decline in memory, thinking, and learning capacity. Despite great effort, the pathogenesis of AD remains unclear. The establishment and progression of AD is associated with high levels of amyloid-ß (Aß), which alter synapse function, local dendritic spine number and plasticity, and neuronal loss. Aß is derived from the cleavage of the amyloid precursor protein (APP), which is one of the most extensively studied proteins in AD. APP can be cleaved into non-amyloidogenic and amyloidogenic species. Amyloidogenic species have a strong propensity to self-aggregate into oligomers. Their clearance depends on autophagy and endosomal-lysosomal pathways. These two processes complement each other in mediating protein turnover. Autophagy is essential for neuronal homeostasis, and its dysfunction is directly linked to a growing number of neurodegenerative disorders. In particular, substrate-specific autophagy is important for removing protein aggregates. Specificity is mediated by adaptor proteins, notably the multifunctional protein p62. Neurons differ in terms of their vulnerability to AD and those projecting from the entorhinal cortex (EC) to the hippocampus are among the most vulnerable. This fact reflects the usual course of the disease, where episodic memory is affected in the early stages of AD. The protein reelin, selectively expressed in the early implicated EC layer II neurons, is shown to be a potent suppressor of some features of the disease. Very recently, this protein was shown to protect against Aß toxicity. Reelin modulates neuronal function and synaptic plasticity and regulates axonal growth and dendritic spine morphology. The depletion and cleavage of reelin was shown to occur in EC of AD animal models. We tested the hypothesis that intracellular amyloid-beta expression can alter synaptic efficacy. We focused on the reelin cells in EC layer II, which project to several regions in the hippocampus, and in the transgenic animals, these cells have high levels of intracellular amyloid-ß. In our previous work, we saw subtle changes in the network activity in the dentate gyrus in the transgenic rats, and therefore we have now started with field potential recordings in this region. We induced long-term potentiation (LTP) by stimulating fibers from EC to dentate gyrus, as changes in LTP can indicate alterations in synaptic transmission. We have collected data from control animals, and will continue with transgenic animals, to assess whether the transgenic animals display changes in synaptic plasticity. We continued to address the question if autophagy plays a role in the progression of AD in the rat model. With a focus on EC layer II reelin positive neurons, we collected samples from 15 wild type and 15 homozygous transgenic rats from three different age groups, both females and males. We analyzed protein expression of several markers related to autophagy signaling, with a focus on protein p62/Sqstm, which is a selective substrate of autophagy that targets ubiquitin-conjugated proteins for degradation. There was a significant difference in p62 protein levels in wild type compared with homozygous transgenic rats across age. We now analyze data for other protein markers and we are still collecting samples from hemizygous animals in order to complete our study.
In this project we explore the concept that in Alzheimer’s disease, the initial pathology in identified neurons in the brain triggers a series of changes that eventually doom the local network and subsequently downstream brain areas. We hypothesize that key players in this process are two proteins expressed in neurons, reelin and amyloid-beta.Findings in the brains of Alzheimer patients convincingly point to a specific part of the cortex in the temporal lobe, the so-called entorhinal cortex (EC), as a key inflicted structure in the initial stages of Alzheimer’s disease (AD). Early pathology is mainly confined to brain cells or neurons in a particular layer (II) of EC. These cells express the protein reelin which seem to co-occur with amyloid-beta. Our main hypothesis is that the initial expression of amyloid-beta in these EC-layer II reelin-expressing neurons triggers a series of changes that eventually doom the brain. The aim of this project is to investigate the mechanisms that lead to this devastating neuron death, focusing on four potential mechanisms. The outcome of the proposed research will contribute to a causal description of the devastating series of events following an initial altered amyloid-beta expression in identified neurons. These results potentially lead to the identification of early and specific biomarkers for specific and timely diagnosis as well as to the development of tailored treatment strategies. In this first year, we started to work on two of the initially proposed four projects. First, we tested the hypothesis that increased amyloid-beta expression inside EC layer II reelin-neurons results in increased activity. We used two different approaches allowing us to measure electrical activity in these neurons and their related networks in transgenic rats and compared this to similar experiments in wildtype control animals. The results point to the termination zone of the EC layer II reelin-neurons in their downstream target, the so-called dentate gyrus, as being affected by amyloid-beta pathology, possibly related to synaptic structure and function of the contacts between the reelin-neurons and their targets in the dentate gyrus. These contacts, or synapses, may show altered efficacy of transmitting information from one neuron to the other. We are preparing a manuscript on these data. Second, we tested the hypothesis that a mechanism for handling waste in neurons, the autophagy signaling pathway, is modified as an early event in the progression of AD. Clearance of protein deposits in diseased neurons depends among others on ubiquination of protein aggregates and subsequent clearance through selective autophagy mediated by a protein called p62. We thus investigated the expression levels and distribution of these two relevant proteins, p62 and ubiquitin, in reelin-positive EC layer II neurons from wild type and transgenic animals. We are now analyzing the first data in order to assess differences between wild type and transgenic animals.