Karen Duff, PhD - US grants

Transgenic mice over-expressing mutant APP K670N,M671L and either mutant PS1M146V or PS1M146L transgenes were generated and examined by immunohistochemistry using antibodies directed against 15 different A( subtypes and glial fibrillary acidic protein (GFAP). A( deposition began in the cingulate cortex of double mutant heterozygous transgenic mice at approximately 10 weeks, in a pattern that correlated well with the expression pattern of the PS1 transgene. By six months of age, the mice had extensive amyloid deposition throughout the hippocampus and cortex and deposition had also spread to other regions of the brain. Immunohistochemistry identified deposits consisting of N-terminal normal and modified forms of A( reminiscent of those found in human AD brain and the deposits were highly congophilic. Both immunohistochemistry and mass spectrometry showed that A(42 forms were under-represented relative to A(40 forms and A(43 was undetectable in the transgenic mouse br ains. In P SAPP mice over 14 months of age, there was both compact and diffuse A( immunoreactivity throughout the cortex and hippocampus. Amyloid deposits were associated with prominent gliosis which increased with age, but in 14 month old PSAPP mice, GFAP immunoreactivity in the vicinity of amyloid deposits was substantially reduced, whereas in the APP age-matched littermates, GFAP immunoreactive astrocytes still clustered tightly around the deposit. In conclusion, these mice develop robust AD type amyloid pathology at a young age that recapitulates the human phenotype and they develop a marked inflammatory response that changes with amyloid exposure time, or load.

ovariectomy; amyloidosis; biomedical resource; Alzheimer's disease; macromolecule; genetic models; clinical research; genetically modified animals; mass spectrometry;

DESCRIPTION (provided by applicant): The project aims to explore why inheritance of the E4 variant of apolipoprotein E (ApoE4) is a significant risk factor for onset and progression of AD. The rationale for this proposal is driven by several observations: 1) that inheritance of the ApoE4 variant is a very strong risk factor for AD relative to inheritance of ApoE3, 2) that manifestations of altered brain morphology and function are apparent in ApoE4 individuals, early in life, decades before plaques and tangles are apparent, and 3) that inheritance of ApoE4 is a risk factor for several diseases suggesting a general deficit in pathways affecting neuroprotection, repair, cell survival etc. We anticipate that altered pathways impacted by ApoE of relevance to AD can be identified by a comparison of gene profiles between vulnerable cell populations of ApoE variants. Our second speculation is that while it is clear that ApoE4 affects the accumulation of amyloid-beta (A2) due to its effects on clearance, there are pathways mediated by ApoE receptor family signaling that could also provide a secondary point of vulnerability. This is demonstrated by the observation that inheritance of ApoE4 is a risk factor for other diseases or events that involve brain injury, and that the altered morphology of neurons of ApoE4 individuals suggests less capacity to respond favorably to injury. The combination of a poor overall response to insult, coupled with selective vulnerability of cell types that rely heavily on ApoE mediated pathways could explain why inheritance of ApoE4 variant is more of a problem for AD than other neurodegenerative diseases. The project aims to identify pathways at risk in both humans and a mouse model with different ApoE variants. We anticipate that these studies will generate new ideas about the etiology of AD in this large population group that might suggest novel preventative or therapeutic measures. PUBLIC HEALTH RELEVANCE: ApoE4 carriers are at significantly heightened risk of AD but clinical trials are becoming increasingly stratified to exclude ApoE4 carriers. As it is possible to test for ApoE genotype very early in life, it is imperative to identify pathways that increase risk as it may be possible to intervene therapeutically from an early age. RNA profiling vulnerable cell populations from pathologically normal ApoE4 carriers is expected to identify novel pathways at risk, allowing for biomarker and therapeutic developments. Performing identical experiments on ApoE targeted mice will provide corrolatory data, as well as generating novel information on this valuable mouse model of an important AD risk factor.

DESCRIPTION (provided by applicant): In the earliest stages of AD, tangle pathology is limited to the hippocampal formation. As the disease progresses however, pathology is seen in cortical areas and these later stages correlate with the onset of overt dementia. Although the progressive spread of pathology has been mapped in humans, most transgenic mouse models of the disease do not model what is seen in humans due to the use of promoters that drive high level expression of AD-related transgenes in inappropriate, or regionally diverse areas of the brain. To model the initial stages of the disease, and to map the spread of pathology out of the hippocampal formation, we have created a novel line of mice with regionally restricted expression of human tau in parahippocampal/hippocampal regions of relevance to the earliest affected regions in the AD brain. A second mouse model will change the regions in which tau is expressed through injection of tau-containing extract into synaptically connected, and unconnected areas of the brain to allow further insight into the significance of network activity in pathology propagation. Three specific aims will address the following issues 1) if the anatomical progression of pathology out of the entorhinal cortex supports the hypothesis that tau pathology spreads transynaptically. 2) the spatio-temporal relationship between basal metabolic function (cerebral blood volume assessed by functional imaging) and pathological progression to test the hypothesis that functional decline is associated with accumulation of pathological tau species in vulnerable brain regions and 3) the spatio-temporal relationship between metabolic function and cognitive impairment, and the relationship with pathological progression to test the hypothesis that cognitive impairment occurs after metabolic dysfunction, when pathology is extensive in extrahippocampal regions. All three measures (neuropathology, metabolic function and cognitive performance) will be assessed relative to each other to provide a spatial and temporal ordering of events. These studies will allow us to not only address a key issue in AD pathobiology - whether transynaptic spread is implicated in propagation of the disease, but mapping the anatomical progression of the disease and correlating it with functional measures of metabolic function (fMRI) and cognitive performance will give insight into spatial and temporal relationships between these measures. These insights could inform on future therapeutic approaches that could prevent the progression of the disease when administered at an early stage. PUBLIC HEALTH RELEVANCE: Alzheimer's Disease is a progressive disease characterized by the accumulation of amyloid/Abeta and tau tangles in defined regions of the brain. In the earliest stages, tangle pathology is limited to the hippocampal formation but as the disease progresses, pathology is seen in cortical areas and these later stages correlate with the onset of overt dementia. Although the progressive spread of pathology has been mapped in humans, most transgenic mouse models of the disease do not model this feature of the disease. To model the initial stages of the disease, and to map the spread of pathology, metabolic dysfunction and cognitive impairment through the brain, we have created a novel line of mice with regionally restricted expression of human tau. Insights from these mice could inform on future diagnostic or therapeutic approaches that could prevent the progression to severe stages.

DESCRIPTION (provided by applicant): Intracellular inclusions of misfolded proteins are the hallmark of many neurodegenerative diseases. Dysfunction of either of the two proteolytic pathways involved in clearing abnormal or obsolete cellular proteins, the ubiquitin-proteasome system (UPS) and the autophagic-lysosomal system (A-LS) may underlie the development of the disease. Macroautophagy (autophagy), a major degradative pathway of the lysosomal system, plays a significant role in the removal of organelles and protein aggregates that are too large, or that cannot be unfolded by chaperone proteins and that are consequently unable to be degraded by the UPS. An equilibrium exists between autophagosome formation and clearance by lysosomes, and uncompromised vesicular trafficking, heterotypic organelle fusion and lysosomal function are critical for the terminal stages of autophagosomal degradation. The A-LS has been shown to play an important role in the clearance of misfolded, aggregate-prone proteins such as ?-synuclein and huntingtin. In general, we hypothesize that the UPS is upregulated to clear misfolded tau species early during the disease, but the system becomes overwhelmed as larger aggregates of tau accumulate. We envisage that the A-LS is then upregulated in an effort to compensate for the lost UPS activity and to clear the aggregates but ultimately both systems fail resulting in accelerated pathology and decline. The relationship between the accumulation of tau, the interplay between the UPS and A-LS, and the effect of relevant pharmacologic manipulations on outcome measures of relevance to human tauopathy will be assessed in three specific aims. Aim 1 will examine the interplay between the UPS, AL-S and tau accumulation in human tissue from patients with 4R tauopathies and will compare to two mouse models of 4R tauopathy that have been modified to express an autophagic marker. The mouse models will allow us to manipulate components of the autophagic pathways to further study the interplay with the UPS, with specific examination of what happens to specific ubiquitinated forms of proteins to confirm the significance of autophagic sufficiency in tauopathy progression in vivo. Aim 2 will use primary neurons from the aforementioned animal models to test the impact of dysfunction in a particular pathway (abnormal transport of autophagic vacuoles leading to failure of autophagic flux) and its impact on tauopathy, and whether compounds that activate A-LS or reduce the levels of hyperphosphorylated tau ameliorate the pathological phenotype. Aim 3 will identify if compounds identified by NCGC/NIH (National Chemical and Genetic Center) using the MLPCN (Molecular Library Probes Center Network) to reduce huntingtin aggregates and cell death are viable autophagic enhancers that can be used to treat tauopathy. Cumulatively, these studies will add insight into the relationship between clearance pathways, how and when they fail, and the impact of drugs that target autophagy as a therapeutic intervention for the tauopathies

DESCRIPTION (provided by applicant): In AD, neurofibrillary pathology (NFTs) starts in the trans/entorhinal cortex (EC) area and spreads to neuroanatomically connected areas of the brain. The ''Braak stages of tau pathology go from stages I to VI (Braak, H. and Braak, E. (1991). Stages I and II correlate with preclinical AD and alterations that are largely confined to the upper layers of the transentorhinal cortex (transentorhinal stages). Stages III and IV correlate with mild cognitive impairment and are characterized by robust involvement of the transentorhinal and entorhinal regions, with a less severe involvement of the hippocampus and several subcortical nuclei (limbic stages). Stages V-VI are characterized by extensive neurofibrillary pathology in neocortical association areas (isocortical stages) and a further increase in pathology in the brain regions affected during stages I-IV. As tangle pathology correlates well with cognitive impairment, targeting tau may be a good therapeutic strategy. To explore why tauopathy maps the way it does in the brain we created a mouse model of the earliest Braak stages of AD (Liu et. al. 2012). Our new transgenic mouse model (line EC-tau) has predominant EC expression of pathological tau, and it replicates the spatio-temporal aspects of tauopathy in the AD brain. Of significant interest was the observation that human tau could cross a synapse into monosynaptically connected cells (downstream or secondary circuits), which explains how pathology may propagate through the brain, and why it follows a trans-synaptic route. To begin to understand how tauopathy propagates, we need to understand new aspects of cellular biology with respect to tau. We propose that as tauopathy worsens, tau is released into the extracellular space from whence it could be taken up by adjacent cells. Once inside, templating to endogenous tau is likely to occur allowing the process to perpetuate. In aim 1 we will perform a careful timecourse quantifying pre and post synaptic markers with pathological tau distribution to assess the order of events in primary and secondary circuits. In aim 2, to understand the functional consequences of worsening tauopathy, especially on secondary circuits, we will monitor the cellular behavior readout molecule, Arc, and synaptic function, assessed by electrophysiology. In aim 3 we will develop a polarized cell culture model to study how the accumulation of tau conformers impacts pathology propagation from the somatodendritic compartments (transneuronal propagation) or the axonal compartment (trans-synaptic propagation). In aim 4.1 we will test whether a therapeutic approach, immunotherapy using the anti-tau antibody MC1 can prevent cell to cell propagation of tauopathy in the EC-tau mouse line, and in aim 4.2, we will assess whether attenuation of tauopathy correlates with improved structural and functional outcomes.

DESCRIPTION (provided by applicant): Carriers of the apolipoprotein E (APOE) ¿4 gene are at significantly increased risk for developing Alzheimer's disease (AD). Although numerous theories have been proposed, the cause of this association remains unclear. The most widely accepted view is that the accelerated AD pathology observed in APOE ¿4 carriers is due to a decreased ability of the apoE4 protein to clear A¿ from the brain. However, possession of the APOE ¿4 gene also results in a number of other neurological deficits unrelated to A¿ clearance, including alterations in neuronal structure, thinner entorhinal cortex (EC) layers and poorer outcomes after stroke, suggesting that there may be other mechanisms involved in this process. In order to gain a more comprehensive understanding of the how the expression of different apoE isoforms affects the brain and how this may impact the risk of developing AD and other age-related neurological illnesses, we propose to use mass spectrometry to identify lipids and small-molecules whose levels are affected by changes in apoE isoform expression. To accomplish this, we will first extract lipids, small-molecules and proteins from pathology-free EC and primary visual cortex (PVC) tissues obtained from 14-month old mice and postmortem 19-55 year old individuals expressing differing apoE isoforms. The lipid and small-molecule fractions from these extracts will then be used to perform targeted lipidomics and untargeted metabolomics, followed by bioinformatic analysis and a variety of validation experiments, in order to determine the specific lipids, small-molecules and metabolic pathways that are affected by alternative apoE isoform expression in the brain. Thus far, preliminary studies have uncovered significant changes in energy metabolism pathways and several important lipid subclasses, demonstrating the power of these techniques for discovering previously unknown isoform-specific effects of apoE in the brain. We expect that the full study proposed herein will uncover further apoE isoform-specific changes in lipid and small-molecule levels and will lead to a greater understanding of how apoE4 influences AD pathology, potentially leading to new therapeutic strategies for AD and other neurological illnesses influenced by apoE4 expression.

? DESCRIPTION (provided by applicant): Spatial memory impairment and disorientation are a common problem associated with aging and they are often one of the first symptoms of mild cognitive impairment and Alzheimer's disease (AD). Understanding the properties of cells involved in the formation of spatial memory in a mouse model with early AD pathology will enhance our understanding of the earliest forms of cognitive decline in AD. The cells known to be important in spatial memory are place cells of the hippocampus (HPC) and grid and head direction cells of the entorhinal cortex (EC). We will use a novel approach to simultaneously record the electrophysiological properties of grid and place cells using 128-channel electrode recordings from 3 regions of the entorhinal cortex-hippocampal (EC-HPC) circuit in AD mice. We will then analyze the large-scale electrophysiological data and measure synaptic plasticity using a spike-timing dependent plasticity (STDP) model. Predictions from this model will be used as a guide to adjust spike timing in neurons, either enhancing or suppressing the synaptic strength of cell populations in affected regions of the EC-HPC, using optogenetic modulation. We anticipate that this will allow us to correct the spatial impairment deficits. To recapitulate te spatial orientation impairments seen in early-stage AD patients, behaviorally equivalent tasks in mice such as morphing open fields, spatial novel object recognition task and T-maze alternation tasks will be applied. These tasks have been chosen specifically to study the functioning of EC-HPC circuit neurons (CA1, CA3, dentate gyrus, lateral and medial entorhinal cortex) that get activated in relevant behavioral modes. The proposal brings together diverse fields (electrophysiology, molecular neuroscience and computational neuroscience) applying large-scale recording techniques simultaneously across multiple brain regions to develop analytical and predictive computational tests to interrogate and restore function in an important circuit that is dysfunctional in Alzheimer's disease.

SUMMARY Tauopathy i n A D a n d F T D is associated with diverse disease syndromes, and e s p e c i a l l y f o r F T D , there is variability in phenotype even w i t h i n f a m i l i e s , a n d between patients with the same mutation. Currently, little is known about why abnormal tau can give rise to such varied phenotypes, but recent data have suggested that subtypes of tau (strains) might underlie this diversity. Our working hypothesis is that p h e n o t y p i c d i v e r s i t y i n t h e t a u o p a t h i e s r e f l e c t s t h e c o n t r i b u t i o n o f differentstrainsthatimpactdifferentbrainregions.Ourproposalaimstousebiochemicaland structural analysis techniques to define strains and sophisticated longitudinal imaging to study how and where in the cell strain variants of tau are propagated, and through the use of biosensors, study the effect of different tau strains on cellular processes. Strains will then be inoculated into mice to study pathology morphology and distribution, as well as functional impact. Together, the aims of this proposal will benefit patients w i t h A D a n d F T D by fostering a better understanding of tauopathy, and providing valid cell and animal models for the testing of therapeutics.

Project Summary Alzheimer?s Disease (AD) and Frontotemporal Lobe Degeneration spectrum diseases caused by tau (FTD-tau) are two neurodegenerative diseases that are characterized by accumulation of abnormal tau. It has been known for many years that tau does not accumulate in all cells in the brain despite the widespread expression of the tau gene. Some regions of the brain (and specific cell populations within them) are differentially vulnerable to accumulating pathological forms of tau. The reasons for this are unknown, and addressing this question is critical for AD and FTD, and also other neurodegenerative diseases showing selective vulnerability. We have observed that excitatory neurons (compared to inhibitory neurons) are especially vulnerable to tauopathy and, using a systems biology approach, have identified deficient tau homeostasis (proteostasis) as a likely mechanism. We now wish to extend these studies to a study on the impact of aging on tau homeostasis pathways in excitatory compared to inhibitory neurons, in human and mouse brain, and in a novel human-derived neuron model, testing one pathway (BAG3) that was implicated from the transcriptomics. Additionally, we will examine the selective vulnerability of neurons in patients with primary tauopathies associated with FTD. Lastly we will work with RNA datasets generated by Allen Institute to identify key pathway differences between excitatory and inhibitory neurons from the entorhinal cortex to begin to identify why excitatory and inhibitory cells might differ in their proteostasis capacity. These studies will explore the basis of selective vulnerability to tauopathy, generate well-characterized resources and potentially identify new disease causing pathways.