Scott E. Counts, Ph.D. - US grants

DESCRIPTION (Adapted from applicant's abstract): Mutations in the gene encoding presenilin-1 (PS1) protein are causative in the majority of early-onset familial Alzheimer's disease (FAD). Thus, a central focus of Alzheimer's research is to resolve the physiological role of PS1 in normal and PS1-linked FAD brain. Recent studies show that PS1 is a transmembrane protein confined to intracellular membrane compartments, where it undergoes endoproteolytic cleavage into N- and C-terminal fragments. The principal goal of this research is to utilize the specificity of monoclonal antibodies recognizing N- and C-terminal domains of PS1 and a receptor epitope fused to serial truncations of PS1 to address two fundamental aspects of PS1 biology. First, an epitope protection assay and complementary immunogold-EM study will be adapted to determine the transmembrane topology of wild type PS1. Secondly, immunogold-EM will be employed to define the precise ultrastructural localization of processed PS1 derivatives. Taken together, these studies will be critical to understanding the subcellular interactions of PS1 with other molecules along the functional pathways of PS1 and along the structure of the protein itself. We will also examine the effects of select PS1 mutations on wild type topology and native N- and C-terminal fragment distribution to explore potential sources of mutant PS1 malfunction.

DESCRIPTION (provided by applicant): Dementia research has become increasingly focused on the prodromal stages of Alzheimer's disease (AD). Elucidating the mechanisms of disease pathogenesis and the reliable identification of people in early stages of cognitive decline will be critical to the development of efficacious therapies for AD. Recently, a clinical entity termed mild cognitive impairment (MCI) has been recognized as a transitional state between the cognitive changes seen in normal aging and AD. The prevalence of MCI is more than double that of dementia and in many cases MCI may be prodromal AD. Therefore, MCI is a suitable condition for exploring the neurobiology underlying AD pathogenesis. There is a paucity of data on pathological and biological markers that distinguish individuals with MCI from healthy aged individuals. To investigate pathobiological markers for MCI, we will employ a novel two-tiered, discovery-based proteomics approach. First, we will use two-dimensional gel electrophoresis (2DGE) and liquid chromatography-tandem mass spectrometry (LCMS/ MS) to identify specific alterations in protein levels in the entorhinal cortex (EC) of subjects with no cognitive impairment (NCI), MCI, or early stage/mild AD. The EC is the initial site of degeneration in MCI and is an ideal area to explore MCI pathogenesis. Second, we will use new surface-enhanced laser desorption/ionization-time of flight MS (SELDI-TOF MS) protein chip technology to evaluate the changes in the cerebrospinal fluid (CSF) of these same individuals. Both tissue and fluid samples will be obtained from our ongoing NIA-funded longitudinal study of aging and AD, the Religious Orders Study (ROS). The proposed studies will hopefully lead to the discovery of new proteins involved in the pathogenesis of AD and new biomarkers for the timely diagnosis of persons in the earliest stages of this devastating disease. Both of these factors will be critical for the development of neuroprotective strategies for slowing or preventing cognitive decline in the elderly.

[unreadable] DESCRIPTION (provided by applicant): The higher incidence rate of Alzheimer's disease (AD) in elderly women indicates that gender plays a role in AD pathogenesis. Several lines of evidence from clinical and pharmacologic studies, neuropathological examinations, and models of menopause and sex hormone replacement suggest that the cholinergic basal forebrain (CBF) projection system, which mediates memory and attentional processes and degenerates in AD, may be preferentially vulnerable to degenerative processes in elderly women as compared to men. In support of this hypothesis, we have intriguing pilot data suggesting that the number of CBF nucleus basalis (NB) cortical projection neurons in women with no cognitive impairment (NCI) may be reduced compared to men with NCI. In addition, preliminary gene expression profiling studies of single cholinergic NB neurons indicate that nerve growth factor (NGF) receptor mRNA levels are selectively reduced in female subjects relative to male subjects diagnosed with mild cognitive impairment (MCI), a prodromal stage of AD. As CBF neurons depend on NGF for survival, these data suggest that NB cortical projection neurons are selectively vulnerable in women compared to men in the earliest stages of cognitive decline. To explore the potential involvement of the CBF in gender-specific mechanisms of AD, we will first perform unbiased stereological methods to test that there is greater cell loss, atrophy, and phenotypic alteration of cholinergic NB neurons in women compared to men during the progression of AD. Tissue sections for this study will be harvested from subjects clinically diagnosed antemortem with NCI, amnestic MCI (a putative preclinical AD syndrome), or mild AD. Secondly, we will perform single cell gene expression analysis of cholinergic NB neurons from these same cases to test whether levels of NGF receptor and other functional classes of mRNAs (e.g., cytoskeletal, synaptic, metabolic) are altered in female subjects during disease progression relative to males. Taken together, these studies will explore cholinergic mechanisms underlying gender differences in the risk for AD and may identify novel targets for gender-specific therapy. PUBLIC HEALTH RELEVANCE: Alzheimer's disease research has become increasingly focused on identifying neurobiologic risk factors to confront the looming crisis as the "baby boomer" generation reaches the ages at greatest risk for the disease. As female gender is a risk factor for AD, new lines of investigation must be undertaken to explore the molecular pathology of gender differences in AD. The current proposal explores whether the brain cholinergic system underlying memory and attention is selectively more vulnerable to AD degeneration in aged women compared to men. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The presence of small non-coding microRNAs (miRNAs) which regulate mRNA stability has become increasingly appreciated as a critical factor in fine-tuning specific neuronal protein levels to mediate diverse brain functions. This widespread influence of miRNA regulation on neuronal physiology suggests that perturbations in miRNA function are involved in the pathogenesis of complex neurodegenerative disorders such as Alzheimer's disease (AD). However, despite initial insights that miRNAs contribute to amyloid pathology during disease progression, the field remains in its infancy and must expand to focus on identifying key multifarious miRNA network changes occurring during the onset of AD which will drive novel therapeutic targets. In particular, whether miRNA networks are dysregulated in the brains of people in the prodromal stages of AD such as amnestic mild cognitive impairment (aMCI) and the extent to which these changes have physiologic consequences for AD progression remain underexplored. To this end, our preliminary microarray and quantitative PCR (qPCR) studies discovered two families of miRNAs, miR-212/132 and miR-23a/b, that were down-regulated in the frontal cortex of aMCI subjects compared to controls. Human miRNA databases revealed that the down-regulation of either miR-212/132 or miR-23a/b was predicted to up-regulate two targets that interact to mediate neuroprotective cell stress responses, the deacetylase sirtuin 1 (sirt1) and the forkhead transcription factor foxo3a; pilot qPCR studies using the same frontal cortex samples revealed that both sirt1 and foxo3a mRNA levels were higher in aMCI compared to controls. Given the relatively delayed involvement of frontal cortex in AD pathogenesis and the ability of this region to respond to the onset of dementia by neuronal reorganization, these data suggest that miRNA-mediated up-regulation of the sirt1/foxo3a pathway represents a compensatory neuroprotective response to mounting disease. In fact, qPCR analysis performed on temporal cortex, an area affected early in the progression of AD, showed no changes in miR-212, miR-23a, sirt1, or foxo3a transcripts in the aMCI subjects. Moreover, pilot in vitro mechanistic studies showed that the coordinated down-regulation of miR-212 and miR-23a increased Sirt1 and Foxo3a protein expression and provided neuroprotection from ¿-amyloid toxicity in human neuronal cells. Hence, our preliminary data suggest that we have uncovered a novel miRNA-mediated neuroprotective pathway activated during prodromal AD. This proposal will test this hypothesis using human tissue molecular, biochemical, and histochemical approaches as well as mechanistic pathway modeling in human neurons. These studies may reveal new insights into gene regulation pathways leading to innovative therapeutic avenues for modifying AD progression. PUBLIC HEALTH RELEVANCE: MicroRNA (miRNA) regulation of neuronal gene networks affects a wide variety of complex cellular processes; hence, multifarious miRNA function could influence the progression of neurodegenerative disorders such as Alzheimer's disease (AD). Based on our exciting pilot studies, this proposal will test the extent to which coordinated down-regulation of two related miRNA families activates a neuroprotective pathway during the prodromal stages of AD. Our findings will validate the novel proposition that innate compensatory miRNA-mediated pathways are activated early in AD progression. A greater understanding of these miRNA pathways during the onset of AD will reveal innovative targets for drug discovery, biomarker development, and disease modification.

Cholinergic basal forebrain (CBF) neurons display the classic hallmark lesion of AD, neurofibrillary tangles (NFT) prior to the cognitive decline seen in AD . Although cholinesterase inhibitors remain the mainstay for the management of mild to moderate AD, these drugs produce only moderate benefits to cognition in AD''. Earlier treatment provides a greater response, highlighting the need to define the pathogenic mechanisms underlying CBF neurodegeneration early in the disease. The CBF connectome consists of four cytoarchitectonic subfieids, anterior medial (Ch4am), anterolateral (Ch4al), intermediate dorsal (Ch4id)/ventral (Ch4iv) and posterior (Ch4p) subfieids, which innervate select cortical regions in a semi topographical pattern. The magnitude of cholinergic neuronal loss within the Ch4 subfieids of AD patients displays subregional variations with the greatest loss in Ch4p, which projects to medial temporal cortex followed by Ch4i which projects to the parietal lobule. Ch4am is the least affected subfield; it innervates medial parietal cortex. All three areas are involved in cognitive processing. This suggests a spatiotemporal course of Ch4 cellular dysfunction similar to the selective disconnection of the medial temporal entorhinalhippocampal circuit in AD. During the current grant period we found that an antibody that recognized tau phosphorylated at S422 (pS422) is a very early marker of tau's disease-association, localizing within pretangle Ch4 neurons in prodromal AD. Moreover, localization of this phospho-epitope correlated better with cognitive decline than did frank NFTs that contain tau truncated at D421 cleaved by caspase . This critical observation suggests the, existence of separate toxic pools of tau independent of NFT formation. Herein, we provide novel preliminary evidence for the existence of oligomeric tau species that form from dimers. Recently, the role of protein oligomeric aggregation intermediate has received considerable attention in AD because of their link to toxicity. Importantly; our collaborative PPG group has contributed to the concept of a linear tau epitope model for NFT evolution, which can be tracked by well-characterized antibodies to site-specific tau epitopes marking pretangle, intermediate; and late stages of NFT formation . These antibodies combined with our novel tau oligomer and PAD antibodies will be used to determine the trajectory of the earliest evolution of NFT tau alterations within discrete Ch4 subfieids (see Preliminary data) in preclinical phases of the disease compared to subjects with a diagnosis of MCI. It is now well established that certain forms of tau can have toxic effects on neurons, which underlie the onset of various tauopathies including AD. The development of pathological-tau-containing neurons within the CBF subfieids is a major event in prodromal AD. Since tau has been shown to be the main protein in NFTs, it seemed logical that the mere aggregation of tau into filaments and NFTs was responsible for at least some part of the cellular degenerative cascade underlying cognitive decline. More recently, models of beta-amyloid (A(3) toxicity in culture and in transgenic mice'' were shown to depend on the presence of tau; if the tau gene was absent, AB was not toxic to cultured neurons and amyloid precursor protein (APP) transgenic mice did not display behavioral deficits. It is interesting to note, however, that in neither of these cases did tau form NFTs or any obvious aggregates. Additionally, NFTs are believed to persist in neurons for 20-30 years making them unlikely candidates for catalyzing immediate toxicity, yet stereological studies support significant neuronal loss in both MCI and AD . In this regard, our study on CBF neurons in the NBM demonstrated that pretangle neuron and neuropil thread staining with the early tau marker, pS422, correlated extremely well With cognitive decline long before the emergence of significant frank NFT pathology . Moreover, synaptic loss correlates better with cognitive decline than NFTs, again suggesting the possibility of a pretangle tau toxicity. Until recently, the nature of this pre-tangle tau moiety remained elusive. Perhaps the most widely held view was that aggregates physically occluded neuronal processes leading to neuronal dysfunction and death. The most elegant support for this contention comes from work of the Mandelkow lab in which they overexpress a truncated, proaggregate construct of tau that results in neuronal death in transgenic mice. However, in AD, tau aggregates as a full-length molecule and it is not overexpressed in the disease. Another possibility is that tau aggregates sequester tau from the microtubule, causing it to become unstable. Support for this contention comes from transgenic mouse work in which tauopathy mice are treated with, a microtubule stabilizing compound. More recently, our work has suggested that tau aggregates and even certain conformations of tau monomers can lead to unmasking of the PAD region of tau (amino acids 2-18), resulting in inhibition of PP1, activation of GSK3 and inhibition of anterograde transport . Additionally, the PAD region ends in Y18, the fyn site on human tau; phosphorylation of tau with fyn prevents tau aggregates from inhibiting anterograde FAT. Moreover, others have presented evidence indicating that defects in axonal transport were likely causing neuronal dysfunction in AD and other tauopathies. Therefore, we seek to determine what tau alterations affect unmasking of the PAD region as assessed by binding of our PAD-specific antibody, TNT1 and what genes are expressed that can be associated with potential tau toxicity within the Ch4 subfieids (Aims 1 and 2). Data from our laboratory indicates that, during aggregation, tau first dimerizes prior to forming oligomers that presumably can transition to filaments . During this process, the PAD region, generally unavailable in monomers, becomes unmasked as oligomers form (see Section 3, below). Although we previously assumed the polymeric filaments were the toxic species of tau, recently, Hsp70, a tau binding protein, was shown to prevent the tau aggregate inhibition of anterograde transport in the squid model by binding to oligomers and not filaments. It now appears that most of the toxic potential in full-length tau is displayed in the oligomer and our monoclonal antibodies selective for the oligomer (T0C1) and specific for the PAD region on tau (TNT1) allow us to ascertain in situ, 1; when tau is likely in an oligomeric state; and, 2. When the toxic PAD domain is displayed (Aim 1). From this we can infer when axonal transport is becoming dysfunctional in the transition from prodromal to authentic AD by following the evolution and spread of the tau staining patterns in the Ch4 subfieids during the progression of AD. Furthermore, using our in vitro aggregation assays and recombinant technology, we can also approach the mechanisms of PAD unmasking and oligomer formation (Aim 3) while determining which oligomers are most toxic (Aim 4).

DESCRIPTION (provided by applicant): This proposal will provide the first cellular and molecular mechanistic profile of noradrenergic locus coeruleus (LC) projection system loss during the preclinical course of AD. LC neurons provide the sole source of norepinephrine (NE) to the hippocampus/medial temporal lobe (MTL) and cortex, where they regulate memory, attention and arousal. Notably, these cells are likely the initial site of neurofibrillary tagle (NFT) formation, suggesting that progressive LC neurodegeneration may help drive NFT formation in its target fields. However, the extent to which LC vulnerability impacts the onse of disease is unclear. Our preliminary studies show that LC neurons from subjects who died with amnestic mild cognitive impairment (aMCI) display significant cell loss compared to control subjects and that noradrenergic fiber density is selectively reduced in the hippocampus compared to cortical target fields during aMCI. These data suggest that selective LC neuronal loss and noradrenergic deafferentation of the hippocampus is a pathogenic preclinical event underlying the transition from normal cognition to prodromal AD. To understand the role of LC system degeneration in AD etiology, we will quantify LC cell loss and fiber density within the MTL and frontal cortex using rarely acquired cases of individuals who displayed no cognitive impairment at the time of death, but who were found to have moderate to high Braak scores that are predictive of AD; these cases are the tissue equivalent of a pre- MCI condition called preclinical AD (PCAD). Whether LC fiber loss directly impacts NFT formation in MTL target fields is unclear. Pilot studies in our laboratory revealed that chemical lesioning of the LC wth the compound DSP4 increased NFT pathology in hippocampal CA1 neurons of the 3xTg-AD mouse, providing novel mechanistic evidence that LC degeneration propagates NFT pathology. To explore this mechanism, we used custom microarrays to analyze gene expression differences in CA1 neurons microdissected from control and DSP4-treated 3xTg-AD mice. Quantitative analysis revealed that noradrenergic deafferentation in DSP4-treated mice resulted in a pronounced 80% down- regulation of the transcription factor nuclear respiratory factor 1 (NRF1) in CA1 neurons. Moreover, pathway analysis unveiled a striking pattern wherein several NRF1 transcriptional targets were also down-regulated, including functional classes of transcripts regulating calcium-mediated neuronal excitabilit (e.g., GluR2 AMPA receptor) and mitochondrial biogenesis (e.g., cytochrome oxidase V). Subsequent pilot studies showed that NRF1 expression is tightly regulated by NE and that NRF1 is selectively reduced in the hippocampus compared to frontal cortex in aMCI subjects, tracking with the pattern of LC deafferentation. Therefore, our hypothesis is that LC projection system degeneration potentiates NFT pathology in MTL neurons during PCAD by disrupting NRF1-mediated calcium and mitochondrial homeostasis. To gain a better understanding of the effects of NE depletion on MTL neurofibrillary degeneration during the preclinical course of AD, we will combine in vivo manipulations of the 3xTg-AD mouse with exploratory microarray and pathway analysis of CA1 neurons. Altogether, this proposal will advance our understanding of fundamental mechanisms underlying multisystem deafferentation within the LC-MTL memory circuit, resulting in new information about disease etiology and new targets for timely diagnostic and therapeutic approaches.

Project Summary Alzheimer's disease (AD) is now feared more than cancer among the elderly, yet we still do not understand the mechanisms of neuronal cell death in AD well enough to know which pathways to target for therapy. Notably, rare cases of familial AD (FAD) cause a more aggressive clinical course than more common cases of sporadic AD (SAD), suggesting that studying FAD cases may inform SAD pathophysiology. However, no one has ever studied this possibility on a molecular level with single neuron resolution. To this end, we used laser capture microdissection to harvest individual pre-tangle bearing frontal cortex layer III neurons from presenilin-1 (PS1)- linked FAD cases and SAD cases, as well as naïve, unlabeled neurons from aged control cases. We then used custom microarrays to compare gene expression profiles within each neuron. The most remarkable observation from this study was that FAD and SAD neurons displayed ~40-70% increases in the expression of several chaperones (e.g., DNAJA3/HSP40) and proteases (e.g., CLPP) that function in the mitochondrial unfolded protein response (mtUPR), a critical pathway for maintaining mitochondrial proteostasis that helps the cell counter mitochondrial stress. Moreover, we found evidence for mtUPR gene activation in pre-tangle bearing layer III neurons harvested from cases of amnestic mild cognitive impairment (aMCI), a putative prodromal stage of AD. These novel pilot data suggested that mitochondrial proteostatic stress is an early event in FAD and SAD and that the mtUPR is activated as a neuroprotective mechanism. However, when we induced mitochondrial proteostatic stress in human hNT neurons, these cells first displayed an up-regulation of mtUPR genes and then, paradoxically, underwent cell death. This response was highly reminiscent of the endoplasmic reticulum UPR, where sustained activation shifts a normally protective pathway to an apoptotic one. Additional in vitro studies showed that mtUPR+ neurons exhibited signs of mitochondrial fission, lysosomal accrual, and mitophagy prior to cell death. Given that one of the prominent theories in the field is that lysosomal/autophagy abnormalities contribute to neurodegeneration in both FAD and SAD, these findings suggest that mtUPR dysfunction in vulnerable neurons acts upstream of lysosomal perturbations to trigger a feed-forward cell death cascade. To test this novel hypothesis, we propose to expand upon our molecular profiling studies to show that neuronal mtUPR activation precedes lysosomal/autophagy activation during the progression of AD. We will then use mitochondrial proteostatic stressors +/- pharmacological interventions in human neuronal cultures to dissect the mechanistic role of mtUPR activation in mitochondrial fission, lysosomal dysfunction and cell death. Altogether, this proposal will establish chronic mtUPR activation as a pivotal early event in familial and sporadic AD and build the knowledge scaffold necessary to model and target this pathway as a disease modifying therapeutic.        

DNA ligases are critical enzymes for virtually all DNA transactions, including DNA replication, repair and recombination. Vertebrates have three DNA ligase genes: Lig1, Lig3 and Lig4; none can be directly deleted from the mouse germline. The Lig3 gene encodes two isoforms: mitochondrial Lig3 (which is cell essential) and the more abundant nuclear Lig3. Recently, we established a mutant mouse strain harboring a knock-in mutation that specifically ablates nuclear Lig3 using a one-step CRISPR/Cas9- mediated genome editing strategy in mouse embryos. Nuclear Lig3 is widely considered to be the primary ligase for DNA single strand break (SSB) repair due to its strong interaction with an essential SSBR factor called X-ray cross complementation factor 1 (XRCC1). Our unique mouse model will allow for the first time, a thorough dissection of the function of Lig3 in nuclear DNA repair in vivo. Experiments proposed in this application will provide the initial characterization of these mice. A comprehensive pathological phenotype analysis will be performed. Because DNA repair defects are often associated with genomic instability and neurodegenerative diseases, additional experiments will focus on the impact of Lig3 deficiency on spontaneous tumor development and neuropathology. Successful completion of the proposed studies will provide unprecedented insight into Lig3's normal physiologic role. Conversely, Lig3 overexpression is found in a number of tumor cell lines and tumor samples from human patients; this impacts DNA double strand break (DSB) repair such that the Lig4-dependent canonical non-homologous end-joining (NHEJ) is, to varying extents, replaced by the Lig3-dependent alternative end-joining (A-EJ), which has been implicated in chromosomal translocations. We will address the impact of nuclear Lig3 deficiency on IgH/c-myc translocation that occurs during immunoglobulin (Ig) heavy (H) chain class switch recombination (CSR). These particular translocations are critical to the development of many human and mouse B cell tumors. Furthermore, characterizing the dependence of these translocations on Lig3 may be relevant to many other tumor types. Successful completion of these studies may elucidate a pathological role of Lig3 in tumorigenesis.

Project Summary The main hypothesis of this proposal is that degeneration of the locus coeruleus (LC) noradrenergic projection system promotes cognitive impairment in Alzheimer?s disease (AD) by driving forebrain cerebrovascular dysfunction. We recently demonstrated that LC neuron loss occurs in amnestic mild cognitive impairment (aMCI) and correlates with poorer cognitive function. Hence, understanding how LC degeneration impairs cognition may open up new therapeutic avenues. To date, most studies have focused on the role of the LC in regulating AD neuropathology, yet virtually no attention has been paid to the role of the LC in regulating cerebrovascular permeability and perfusion in AD. To gauge the scientific premise for pursuing this question, we administered a dopamine-?-hydroxylase IgG-saporin (DBH-SAP) immunotoxin into the prefrontal cortex (PFC) of Tg344-19 AD rats, which mimicked LC neuron and fiber loss and resulted in memory impairments. Strikingly, postmortem analysis revealed evidence of widespread blood-brain barrier leakage and increased cerebral amyloid angiopathy in DBH-SAP-lesioned AD rats compared to control IgG-saporin (CTL-SAP) rats. Moreover, pressure myography studies showed blunted vasoreactivity of PFC parenchymal arterioles following DBH-SAP lesions. To begin to understand the mechanisms for these LC-mediated pathologies, RNA sequencing was performed on laser-captured PFC vessels from Rush Religious Orders Study (RROS) subjects. These data revealed a dysregulation of genes mediating vessel permeability and calcium signaling in aMCI and AD compared to controls. Many of these genes were also dysregulated in vessels harvested from DBH-SAP-lesioned AD rats, suggesting specific pathomechanic pathways linking LC degeneration with forebrain vascular dysfunction. Therefore, we designed our Specific Aims to test the extent to which LC degeneration drives cerebrovascular pathology in target fields (Aim 1) and the potential mechanisms underlying this phenomenon (Aim 2). In Aim 1, Tg344-19 AD rats will be administered DBH-SAP or CTL-SAP in the PFC in the presence or absence of the norepinephrine pro-drug L-DOPS or the reuptake inhibitor atomoxetine. Rats will be assessed for memory function and for cortical perfusion by MRI. Pressure myography studies will analyze vessel physiology and postmortem analysis will quantify vascular and AD-like pathology. Spontaneously Hypertensive Stroke Prone rats will also be included to test whether LC cell loss exacerbates vascular risk factors. In Aim 2, PFC parenchymal vessels from RROS subjects will be analyzed by RNA-Seq. Target genes that are also dysregulated in PFC vessels from DBH-SAP rats will be validated and tested for their role in vascular dysfunction using in vitro mechanistic assays. If successful, this proposal will show that 1) LC degeneration is a nexus lesion that impacts both vascular and neuropathology during the earliest stages of AD, and that 2) targeting noradrenergic mechanistic pathways in small vessels may allow for more comprehensive disease modification in AD by reducing vascular contributions to cognitive impairment.

Project Summary The main hypothesis of this proposal is that degeneration of the locus coeruleus (LC) noradrenergic projection system promotes cognitive impairment in Alzheimer?s disease (AD) by driving forebrain cerebrovascular dysfunction. We recently demonstrated that LC neuron loss occurs in amnestic mild cognitive impairment (aMCI) and correlates with poorer cognitive function. Hence, understanding how LC degeneration impairs cognition may open up new therapeutic avenues. To date, most studies have focused on the role of the LC in regulating AD neuropathology, yet virtually no attention has been paid to the role of the LC in regulating cerebrovascular permeability and perfusion in AD. To gauge the scientific premise for pursuing this question, we administered a dopamine-?-hydroxylase IgG-saporin (DBH-SAP) immunotoxin into the prefrontal cortex (PFC) of Tg344-19 AD rats, which mimicked LC neuron and fiber loss and resulted in memory impairments. Strikingly, postmortem analysis revealed evidence of widespread blood-brain barrier leakage and increased cerebral amyloid angiopathy in DBH-SAP-lesioned AD rats compared to control IgG-saporin (CTL-SAP) rats. Moreover, pressure myography studies showed blunted vasoreactivity of PFC parenchymal arterioles following DBH-SAP lesions. To begin to understand the mechanisms for these LC-mediated pathologies, RNA sequencing was performed on laser-captured PFC vessels from Rush Religious Orders Study (RROS) subjects. These data revealed a dysregulation of genes mediating vessel permeability and calcium signaling in aMCI and AD compared to controls. Many of these genes were also dysregulated in vessels harvested from DBH-SAP-lesioned AD rats, suggesting specific pathomechanic pathways linking LC degeneration with forebrain vascular dysfunction. Therefore, we designed our Specific Aims to test the extent to which LC degeneration drives cerebrovascular pathology in target fields (Aim 1) and the potential mechanisms underlying this phenomenon (Aim 2). In Aim 1, Tg344-19 AD rats will be administered DBH-SAP or CTL-SAP in the PFC in the presence or absence of the norepinephrine pro-drug L-DOPS or the reuptake inhibitor atomoxetine. Rats will be assessed for memory function and for cortical perfusion by MRI. Pressure myography studies will analyze vessel physiology and postmortem analysis will quantify vascular and AD-like pathology. Spontaneously Hypertensive Stroke Prone rats will also be included to test whether LC cell loss exacerbates vascular risk factors. In Aim 2, PFC parenchymal vessels from RROS subjects will be analyzed by RNA-Seq. Target genes that are also dysregulated in PFC vessels from DBH-SAP rats will be validated and tested for their role in vascular dysfunction using in vitro mechanistic assays. If successful, this proposal will show that 1) LC degeneration is a nexus lesion that impacts both vascular and neuropathology during the earliest stages of AD, and that 2) targeting noradrenergic mechanistic pathways in small vessels may allow for more comprehensive disease modification in AD by reducing vascular contributions to cognitive impairment.

Project Summary The objective of this R21 proposal is to give proof that magnetic susceptibility and volume of cerebral microbleeds quantified by an innovative MRI technique, CISSCO, and validated by pathological examination can differentiate postmortem brain samples from control and demented subjects. Microbleeds appear in varying numbers in images, but they are strongly associated with vascular cognitive impairment (VCI), small vessel diseases (SVD), and Alzheimer's disease (AD). They also appear in healthy older people at a lower prevalence. Current clinical diagnosis only counts the number of microbleeds and their mimics from images. However, the number of these ?apparent? micro-objects is subject to imaging parameters (including the MRI field strength) and does not correlate well with the progression of cognitive decline. As microbleeds with hemorrhagic components show magnetic susceptibility effects in MRI, these investigators hypothesize that magnetic properties of microbleeds may help to differentiate certain clinical dementia subtypes. These novel quantitative parameters may be better markers for incipient dementia. They will advance our current understanding of the contribution of microbleeds to dementia beyond postmortem analysis and toward living patients. To design clinical imaging protocols and parameters, susceptibility values of microbleeds used to distinguish between control and demented subjects must be known first. The proposed aims are to image and pathologically examine micro-objects in postmortem samples obtained from the Michigan Brain Bank (MBB), which will blind these investigators to the clinical and neuropathological diagnosis of each subject until the late stage of this project. Formalin fixed, paraffin-embedded blocks where microvascular lesions are suspected will be obtained from subjects who died with no cognitive impairment or mixed dementia (including cases with high- and intermediate- likelihood of AD pathology). The CISSCO method will be applied to MR images of postmortem samples for accurate quantification of the magnetic susceptibility and volume of each micro-object. These micro-objects observed in MRI will then be co-registered and identified histologically in the same samples. These micro-objects, which are microbleeds and their mimics, will be categorized based on pathological results and quantified susceptibility values from MRI. They will also be compared between the diagnostic groups after the investigators are un-blinded. Cox hazard ratios will be calculated for different categorized results. If different magnetic properties can differentiate the clinical groups, then this outcome would indicate that magnetic properties of microbleeds is a potential advance in imaging biomarkers for dementia. With proof from this R21, future clinical testing plans will be proposed to NIH.