Nicholas M Kanaan - US grants

DESCRIPTION (provided by applicant): Taupathology is a prominent feature of multiple neurological diseases known collectively as tauopathies. These include Alzheimer's disease (AD), Progressive Supranuclear Palsy (PSP), Cortical Basal Degeneration (CBD), Pick's disease, and Frontotemporal Dementia with Parkinsonism linked to chromosome 17. Some of these diseases are hereditary, associated with mutations in the tau gene, but normal tau may also be pathological. Although each tauopathy has a disease specific phenotype, histological presentation, morphology, and neurological presentation, all of them are associated with misfolded tau and altered phosphorylation of tau. The search for a common pathogenic mechanism has been hindered by this clinical diversity. Two recent findings provide new insight into tau pathology. The first is identification of conformation specific tau antibodies that recognize some, but not all, pathological forms of tau, suggesting conformational diversity within the tauopathies. Second, our recent demonstration of a biologically active motif in the tau amino terminus that activates a signaling pathway involving protein phosphatase 1 (PP1) and glycogen synthase kinase 3b (GSK3b): 17 amino acids comprising a Phosphatase Activation Domain (PAD) provides a molecular basis for altered kinase activities in tauopathies. The central hypothesis of this application is that pathogenic forms of tau represent a misregulation of a normal biological function for tau as a scaffold for localization and regulation of microtubule based kinases and phosphatases. This PAD region is aberrantly displayed in all pathological forms of tau examined to date and is a necessary component of at least two forms of tau toxicity: inhibition of fast axonal transport and cell toxicity in culture. We propose that pathological forms of tau in different tauopathies are structurally distinct with variable degrees f toxicity. Experiments in this application will characterize the conformations of tau from different tauopathies and evaluate their relative toxicity in affecting the PP1/GSK3b pathway and axonal transport using authentic and synthetic aggregates. We further hypothesize that toxicity of different tau conformers may be modulated by disease specific patterns of tau phosphorylation and conformation. Disease specific patterns of these alterations will be determined for AD, PSP and CBD. Normal and pathological functions of tau will be analyzed to test the hypothesis that tau serves as a scaffold for localizing and regulating specific kinases and phosphatases to microtubules. We will focus on the role of tau in the normal regulation of PP1 and GSK3b in microtubule rich domains of the axon and identify interaction domains with tau for these phosphotransferases. The localization of the PP1/GSK3b pathway by tau allows for spatial and temporal control of these activities and we propose that presentation of PAD is restricted to specific subcellular compartments in normal neurons and deregulated in pathological states. Consistent with this model, tau, PP1 and GSK3b have all been implicated in neuronal development. Developmental regulation of tau isoforms, conformation, and phosphorylation may play critical roles in neuronal development. We suggest that the regulated presentation of PAD is important for neurite outgrowth and targeting of axonal proteins during normal neuronal development and function, allowing us to understand the relationship between the toxicity of misfolded tau and normal tau function.

DESCRIPTION (provided by applicant): Alzheimer's disease and other tauopathies are aging-related neurodegenerative diseases that are representative of a significant impending economic and treatment burden for the US healthcare system that will only increase as the population shifts to a more aged demographic. These diseases are characterized by the pathological accumulation of abnormally modified tau proteins, which is closely linked to their observed cognitive deficits. Since the underlying causes of tauopathies remain unknown, it is accordingly difficult to develop effective therapeutic interventions. Some of the earliest pathological changes, especially in AD, follow a dying-back pattern in which axons are the first to exhibit abnormal structural changes. A likely pathogenic factor contributing to axonal degeneration is the protein tau, as it is critical in maintaining axonal function. Indeed, studies using human tissue and animal model systems suggest that tau abnormalities and axonal degeneration are interconnected components of the early degenerative sequelae of AD. Our preliminary data indicate that disease-related modifications of tau that expose the amino terminus of the protein cause axonal dysfunction and degeneration in cultured neurons and in vivo. The primary goal of this proposal is to test whether disease-associated abnormalities in tau can induce axonal degeneration. Three independent specific aims are proposed to take a multifaceted approach aimed at addressing this hypothesis. Aim 1 will establish the relative contribution of tau modifications and the molecular events associated with tau-induced axon degeneration in primary cultured hippocampal neurons as well as a novel, viral vector-based rat model. Aim 2 will define the functional relationship between tau protein and enzymes linked to tau-induced axonal dysfunction (i.e. protein phosphatase 1 and glycogen synthase kinase 3?). Lastly, Aim 3 will define the relationship between abnormal forms of tau protein and axonal degeneration in the progression of human AD using post-mortem tissue from cases ranging between non-demented controls to severely demented AD. If successful, these studies will identify a molecular mechanism for tau-induced axon dysfunction/degeneration that could be targeted for disease-modifying therapeutic interventions in AD patients, as well as those suffering from other tauopathies.

Project Summary Alzheimer?s disease (AD) and AD-related dementias have a severe impact on those affected, the healthcare system and the US economy, but currently no treatments exist and a critical need for effective biomarkers persists. The development of ultrasensitive assays such as the single molecule array (SIMOA) platform has facilitated a significant biomarker opportunity that may transform the current landscape and have a large impact on our ability to manage these diseases. Measuring CSF tau is the primary staple for tau biomarkers, but the SIMOA technology has facilitated a significant biomarker opportunity because the presence of very low levels of tau, a microtubule-associated protein that composes the hallmark pathologies of tauopathies (including AD and ADRDs), in plasma can be reliably measured. Continued pursuit of CSF biomarkers is critical, but plasma provides an important opportunity to potentially obtain critical biomarker information through routes that are relatively non-invasive, inexpensive and easy to collect serially in large volumes. Unfortunately, only two SIMOA assays available for measuring plasma tau and neither distinguishes between mild cognitive impairment (MCI) from nondemented control (ND) cases. Thus, there is a critical need for developing novel tau-based assays. Our overall goal is to use our large resource of established, well-characterized monoclonal tau antibodies and newly created and characterized tau monoclonal antibodies to develop a set of novel SIMOA assays for assessing normal and pathological tau in plasma and CSF. We will pursue two independent aims to achieve this goal. In specific aim 1, we will develop tau assays that detect total tau by targeting specific epitopes throughout the protein. Our experience indicates that the forms of tau in biological fluids (such as CSF) are detected with differential sensitivity depending on the antibody, and the best tau region to target in plasma is not defined. Using a step-wise assay development framework, we will validate novel assays using a combination of recombinant tau protein and human plasma and CSF reference samples. Rigorous criteria will be required to further develop an assay, at which point we will compare its performance using plasma and CSF samples from ND, MCI and AD cases. In specific aim 2, we will develop novel tau assays that detect specific pathogenic forms of tau using antibodies that specifically detect conformational or aggregated states of tau, as well as cleaved tau. The presence/abundance of these pathogenic forms of tau in the plasma and utility as biomarkers remains unexplored. We will use the same workflow for assay development as in aim 1 and will test validated assays for their performance with ND, MCI and AD samples. Overall, we propose to use a rigorous set of studies to develop completely novel tau-SIMOA assays for the detection of total tau and pathogenic tau conformations/truncations in human plasma and CSF across the spectrum of ND, MCI and AD. Developing such assays is critical to advance the field toward obtaining assays with high specificity, sensitivity, reproducibility and predictability for identifying tauopathies, such as AD and other ADRD tauopathies.

Tau is a microtubule-associated protein that is abnormally phosphorylated and aggregates in Alzheimer?s disease (AD) and AD-related dementias, leading to its impaired functionality. No disease modifying treatment exists for these diseases, making them a significant healthcare priority. Though we have known for decades that several modifications are intimately associated with sporadic disease and mutations in tau directly cause inherited degenerative tauopathies, the precise molecular pathways engaged by pathological tau proteins to actively cause neurodegeneration are unknown. This knowledge gap remains a significant and critical problem that this proposal aims to address. Current thinking in the field suggests that disease-related tau modifications exert toxicity by disruption of microtubules and/or by enhancing tau aggregation. However, evidence from our group and others supports an alternative explanation. That is, tau acts to regulate signaling pathways and tau toxicity is due to an aberration of this function. Using the isolated axoplasm from squid giant axons, we discovered a functional signaling motif in the N-terminus of tau called the phosphatase-activating domain (PAD) that activates a protein phosphatase-1 (PP1)-dependent signaling cascade. We also identified that known pathological changes in tau (e.g. phosphorylation and oligomerization) alter tau?s structure leading to aberrant PAD-dependent activation of this pathway and axonal toxicity. We propose to focus on determining whether tau normally regulates PP1-dependent functions in neurons and on PP1-dependent pathways of tau toxicity. Our central hypothesis is that, through a PP1-dependent mechanism, tau normally regulates neuronal function and health, but in disease tau causes axonal, synaptic and/or neuronal toxicity through aberrations of this mechanism. In Aim 1, we will test the hypothesis that specific motifs within tau and PP1 are critical for the interaction with and activation of PP1 by tau proteins. We will use a combination of point mutations/deletions to map the specific domains in tau and PP1 isoforms that underlie tau?s ability to bind and activate PP1. In Aim 2, we will test the hypothesis that tau localizes with PP1 in specific subcellular compartments where it regulates PP1-dependent pathways. Here, we will use novel human tau-KI mouse primary neuron cultures and a combination of super-resolution microscopy, subcellular fraction and other protein-protein interaction assays to determine where in neurons tau and PP1 interact. Also, we will knockdown tau to determine its functional relationship to multiple PP1-dependent pathways in neurons. In Aim 3, we will test the hypothesis that disease forms of tau drive toxicity via PP1-dependent mechanisms. We will use a novel tau pre-formed fibril seeding model in the hTau-KI mice as well as the PS19 mutant tau mouse line to provide the critical in vivo translational insights for the tau-PP1 relationship and how disease forms of tau lead to PP1-dependent toxicity. Together, the proposed studies will fill critical gaps in our knowledge and will produce a significant impact by identifying important physiological and pathological roles for tau in regulating PP1 in neurons.

ABSTRACT ? CORE F: BIOMARKER CORE Biomarkers are becoming instrumental components of effective clinical management and trial coordination for Alzheimer?s disease and related dementias (ADRD), and will be required to enable precision medicine approaches to diagnosis and treatment. The primary functions of the newly added Biomarker Core (BC) of the Michigan Alzheimer?s Disease Research Center (MADRC) will be to facilitate innovative research into established and novel blood biomarkers, and provide expertise and access to biomarker assay technologies for investigators at the University of Michigan, Michigan State University, Wayne State University, as well as other ADRCs. The overall goal of the BC is to use cutting-edge ultrasensitive ELISA and quantitative mass spectrometry (MS) approaches to measure biomarkers in blood, identify and build novel blood-based biomarker assays, and integrate biomarkers with clinical measures of disease. We will leverage MADRC resources from ongoing and future efforts to achieve this goal and promote studies that align with the MADRC?s central theme to identify, understand and modulate the many factors beyond ?-amyloid contributing to ADRD. The diverse patient population in the MADRC?s longitudinal cohort and other ongoing clinical research efforts, coupled with the ability to develop novel biomarker assays, will significantly enhance the ability of the BC to focus on unique questions regarding the extent to which current and novel biomarker targets enhance diagnostic accuracy, and track disease progression and predictability over time in multiple patient populations, including underrepresented racial/ethnic groups. The BC will pursue the following five aims: 1) provide measurements of established blood biomarkers, 2) develop and implement novel biomarker assays, 3) facilitate integration of blood biomarkers with established clinical practices to advance translational research, 4) contribute blood biomarker data to the National Alzheimer?s Coordinating Center, and 5) educate and train health care professionals, scientists and students in the use of biomarker assays. The BC goals and aims will be achieved by working closely with the MADRC Clinical Core, Data Management and Statistical Core, Neuroimaging Core, Neuropathology Core, and Research Education Component. Through these efforts, the BC will markedly enhance research supported by the MADRC, leading to a sustained and significant impact for the field as we build a comprehensive assessment of the diagnostic and the predictive utility of blood-based biomarker profiles. We will also interface with other ADRCs and investigators to help shape an evolving clinical landscape for dementia evaluation and treatment.