William E. Van Nostrand - US grants

The hypotheses that form the basis of this proposal are that protease nexin-2/amyloid beta-protein precursor (PN-2/APP) has a critical role in the physiologic regulation of hemostatic factors XIa and IXa. Human platelets, the major intravascular repository for PN-2/APP, function as the physiologic, intravascular delivery system for this protein. Human brain, enriched with PN-2/APP, utilizes this protein as an important anticoagulant. Abnormal processing an/or expression of PN-2/APP by platelets and cerebral blood vessels contribute to the pathogenesis of Alzheimer's Disease (AD) and Hereditary Cerebral Hemorrhage with Amyloidosis-Dutch Type (HCHWA-D). The objectives of this proposal are to study the effect of PN-2/APP on hemostatic proteins and its expression/processing by human platelets and in cerebral blood vessel tissue. The specific aims are as follows: (1) Studies will be performed to ascertain the interaction of PN-2/APP with factors Ixa and Xia. The kinetics of factor Ixa inhibition by PN-2/APP will be characterized. Pn- 2/APP inactivation rates of factors Xia and Ixa will be determined in the absence or presence of heparin and platelets or cultured endothelial cells. Investigations will determine if, when in excess, factor Ixa and factor Xia proteolyze PN-2/APP. (2) Investigations will be performed to study the processing and expression of human platelet PN-2/APP. Studies will be performed to determine if upon lysis of human platelets, endogenous calpain produces an amyloidogenic fragment in normal and AD platelets. Investigations will determine if thrombin or platelet activating factor can induce calpain processing of PN-2/APP in the absence of cell lysis. Studies will be conducted to characterize the structure of membrane APP remaining with platelets after PN-2/APP secretion. PN-2/APP will be quantitated in whole lysates and releasates from normal and AD platelets. (3) Investigations will determine the expression and processing of PN-2/APP in cerebral blood vessels. Immunohistochemical and in situ hybridization studies on human brain tissue from HCHWA-D patients, AD patients, and normals will determine the synthesis and form of PN-2/APP in cerebral vessel walls. Pn-2/APP inactivation rates of factors Xia and Ixa in the presence of cultured smooth muscle cells will be determined. Cultured smooth muscle cells will be studied to determine how they express and process PN-2/APP. Better understanding of the biochemistry and physiology of PN-2/APP will provide insight into the patho-biochemistry and patho- physiology of AD and HCHWA-D, two disorders intimately involved with PN- 2/APP.

The objectives of the proposed studies are two-fold and reflect research areas in the biochemistry, functional properties, and cell biology of PN- 2/AbetaPP that are currently funded by two research grants. First, the biochemical interactions between PN-2/AbetaPP and coagulation factors will be investigated. Specifically, the present proposal will focus on studies concerning interactions between coagulation factor X1a and PN-2/AbetaPP and the effects of high molecular weight kininogen and the effects of biologic surfaces on these interactions. In addition, characterization of the protease inhibitory and potential anticoagulant properties of a recombinant Kunitz-type protease inhibitor domain of a related AbetaPP- homolog are planned to better understand the functional relatedness of this homolog protein to PN2/AbetaPP. Second, in a somewhat different, but related and important series of studies, PN-2/AbetaPP expression and processing in cerebral blood vessels from postmortem human brain tissue and in cultured cerebrovascular smooth muscle cells will be investigated. These series of investigations will begin to elucidate how PN-2/AbetaPP may normally serve as an anticoagulant in the cerebrovasculature and how it may abnormally be involved with intracerebral hemorrhaging in patients with a rare disorder that is related to Alzheimer's disease known as hereditary cerebral hemorrhage with amyloidosis-Dutch type. Better understanding of the biochemistry and cerebrovascular cell biology of PN- 2/AbetaPP will provide insight into its normal function as well as the patho-biochemistry and pathophysiology of this protein in certain disease states.

The hypotheses that form the basis of this proposal are that the amyloid beta-proteln (Abeta) causes degeneration of vascular smooth muscle cells in cerebral blood vessels which leads to abnormal expression and cellular accumulation of the amylold beta-proteln precursor (AbetaPP) and a concomitant increase in cellular carboxyl terminal AbetaPP fragments and soluble Abeta. Apolipoprotein E (ApoE) can also contribute to increased expression of AbetaPP in cerebrovascular smooth muscle cells. Therefore, Abeta and ApoE can induce subsequent formation of additional Abeta in cerebral blood vessel walls further contributing to the cerebral amylold anglopathy (CAA) In disorders Including Alzheimer's disease (AD) and hereditary cerebral hemorrhage with amyloidosis-Dutch type (HCHWA-D). The proposed studies will provide important information on how Abeta, AbetaPP and cerebrovascular smooth muscle cells potentially interact to play key roles in the initiation and spread of the cerebrovascular pathology associated with CAA in disorders including AD and HCHWA-D. The specific objectives of this proposal are to investigate the effects of Abeta peptides and ApoE on the expression and processing of AbetaPP that accompanies the degeneration of cultured human smooth muscle cells from cerebral blood vessels. Investigations will also be performed to compare the effects of soluble and aggregated forms of Abeta peptides as well as normal and HCHWA-D Abeta peptides on cellular degeneration and expression and processing of AbetaPP in the cultured cerebrovascular smooth muscle cells. We also plan to investigate the mechanisms by which Abeta induces cell death in cerebrovascular smooth muscle cells. Studies will be performed to determine if these combined effects of Abeta peptides are observed in other cultured cerebrovascular and peripheral vascular cells. This may provide insight as to why the cerebrovascular pathology of AD and related disorders is restricted to cerebral blood vessels. Within the framework of the program project, parallel studies on the effects of Abeta peptides on the expression and processing of AbetaPP in cultured neurons will be conducted in collaboration with Dr. Cotman and his colleagues as described in his proposal. These studies will be important to determine if different or similar Abeta-induced pathologic mechanisms occur in cerebrovascular smoo4h muscle cells and neurons. In addition, future investigations will be conducted in collaboration with Dr. Glabe and coworkers concerning the differential uptake and degradation of the different length Abeta peptides by the cultured HLSM cells. These studies coincide with our long term goals to better understand the expression and processing of APP leading to Abeta formation and deposition that contributes to the hallmark cerebrovascular pathology in AD, HCHWA-D and related disorders.

DESCRIPTION (from applicant's abstract) The overall hypothesis that forms the basis of this proposal is that an interaction between the amyloid beta-protein (Abeta) and the amyloid beta-protein precursor (AbetaPP), and the cell surface in human cerebrovascular smooth muscle (HCSM) cells is involved in the initiation and progression of the cerebrovascular smooth muscle cellular pathology of Alzheimer's disease (AD) and related disorders including hereditary cerebral hemorrhage with amyloidosis Dutch-type (HCHWA-D). The applicants' overall research efforts have focused on elucidating the cellular mechanism that underlie the cerebrovascular pathology of AD, HCHWA-D and related disorders. The objectives of the present proposal are to investigate a recently identified interaction between certain forms of Abeta and cellular AbetaPP in cultured HCSM cells that induce several key pathologic responses in these cells. This mechanism may be specific for HSCM and related cells in the cerebral blood vessel wall and, therefore, may help to explain the different etiologies between the parenchymal and cerebrovascular Abeta induced pathologies in AD and relates disorder. They plan to identify the precise domain on AbetaPP where Abeta binds to induce the pathologic responses in HCSM cells. Investigations will be conducted to determine if AbetaPP is implicated in cell-surface Abeta fibril formation and if these fibrils are involved with promoting the pathological responses in HCSM cells. Lastly, they will determine if altering the endogenous level of AbetaPP expression affects the ability of Abeta to induce pathologic responses in HCSM cells. These combined investigations intend to utilize cultured HCSM cells as an in vitro paradigm to identify the Abeta binding site on AbetaPP and characterize the consequences of this interaction on the cells surface that potentially leads to the initiation and progression of the cerebrovascular smooth muscle cellular pathology of AD, HCHWA-D and related disorders. Although early in its discovery, the pathologic interaction between Abeta and AbetaPP in HCSM cells have potential therapeutic implications. Identification of the precise Abeta binding site on AbetaPP and determining the nature of the interactions that are involved with inducing the key pathologic responses may provide an opportunity for the future development of novel intervention strategies to block the initiation and/or halt the further progression of the cerebrovascular pathology of AD, HCHWA-D, and related disorders.

Cerebrovascular amyloid beta-protein (ABeta) deposition is a hallmark of Alzheimer's disease (AD) and related disorders including hereditary cerebral hemorrhage with amyloidosis Dutch-type (HCHWA-D). Cerebrovascular ABeta deposition in these disorders is prominently associated with smooth muscle cellular pathology in the cerebral vessel wall and can lead to a loss of vessel integrity and hemorrhagic stroke. The reasons for this remain unclear. The overall hypothesis that forms the basis of this competing continuation grant proposal is that ABeta deposition in the cerebral vessel wall can modulate the function of hemostatic enzymes thus creating a microenvironment that is conducive to hemorrhagic stroke. The objectives of this proposal are to characterize the effects of ABeta on the regulation of certain hemostatic enzymes at the biochemical and cellular levels. The specific aims of this proposal are as follows: First, the effect of soluble ABeta, solution-assembled fibrillar ABeta, and cell-surface-assembled, fibrillar ABeta on the inhibition of factor XIa (FXIa) and certain other coagulation enzymes by PN-2/AbetaPP will be characterized. The inactivation rates of FXIa and other coagulation enzymes by PN-2/AbetaPP in the presence of soluble and the different fibrillar ABeta peptides will be determined. The proteolysis of ABeta peptides by coagulation enzymes will be investigated. Second, studies will be conducted to determine the effect of soluble ABeta, solution-assembled fibrillar Abeta, and cell-surface-assembled fibrillar ABeta on the stimulation of tissue plasminogen activator (tPA) activity. The effect of ABeta peptides on cellular tPA levels and plasminogen activation activity will be explored. In addition, proteolysis of ABeta peptides by tPA and plasmin will be investigated. Third, investigations will compare the pathologic effects of intact ABeta and hemostatic enzyme proteolyzed ABeta on cultured human cerebrovascular smooth muscle (HCSM) cells, a cell type intimately associated with the cerebrovascular pathology of AD and HCHWA-D. The proposed studies will provide a better biochemical understanding of the influence that ABeta has on the inhibition of certain coagulation proteases and the stimulation of plasminogen activation, two processes that together can create a milieu which could result in loss of vessel integrity and hemorrhagic stroke.

DESCRIPTION: The hypothesis that forms the basis of this proposal is that expression of human hereditary cerebral hemorrhage with amyloidosis Dutch-type (HCHWA-D) mutant amyloid beta-protein precursor (ABPP) in the smooth muscle cells of cerebral blood vessels of transgenic mice will lead to cerebral amyloid angiopathy (CAA). The applicants plan to create transgenic mice that will express human ABPP carrying the HCHWA-D mutation in vascular smooth muscle cells in order to test the above hypothesis. Once they obtain positive transgenic mice, they will conduct specific immunohistochemical staining and immunoblotting studies on brain and other tissues from the transgenic mice to quantitatively and qualitatively determine the extent and distribution of human wild-type or HCHWA-D mutant ABPP protein expression. In addition, they will perform Northern blot analyses. Lastly, they will examine the transgenic mice for key pathological signs that are characteristic of CAA.

Cerebral amyloid angiopathy (CAA) is an age-associated condition in which amyloid is deposited in the medial layer of primarily small- and medium-sized arteries and arterioles of the cerebral cortex and leptomeninges and is present in most patients with Alzheimer's disease (AD) and certain related disorders including hereditary cerebral hemorrhage with amyloidosis Dutch-type (HCHWA-D). The overall hypothesis of this proposal is that age and other risk factor-related changes in cerebrovascular smooth muscle cells renders them more susceptible to the pathogenic effects of Abeta resulting in CAA-related pathology. We plan to utilize novel and unique cell culture and transgenic model systems to investigate the role of age and other risk factors in the development of the smooth muscle cellular pathology of CAA. The studies outlined in this proposal will shed light on the roles of specific molecules, and age-related changes in these molecules, that could contribute to CAA pathology. The specific aims are as follows: First, we will determine if cultured cerebrovascular smooth muscle (CSM) cells from younger and aged comparisons with cultured CSM cells from young and aged non-human primates that develop pronounced or mild CAA (squirrel monkey or rhesus monkey, respectively). Second, we will determine the pathogenic effects of Abeta in cultured CSM cells obtained from control non-transgenic and wild-type or HCHWA-D mutant human AbetaPP transgenic mice. This will ascertain if over-expression of human Abeta PP affects the pathologic leads to earlier and/or more extensive cerebrovascular Abeta deposition and accompanying CAA-related pathology in our human Abeta PP transgenic mice. Lastly, we will determine if over-expression of RAGE influences the pathogenic effects of Abeta in cultured CSM cells obtained from RAGE transgenic mice and RAGE/human Abeta PP pathologic responsiveness of these cells to Abeta. These combined investigations will provide information about how age and changes in key molecules in CSM cells are involved in the development of CAA-related pathology.

DESCRIPTION (provided by applicant): The overall hypothesis of this proposal is that the human cerebrovascular smooth muscle (HCSM) cell surface promotes the assembly of pathogenic forms of amyloid B-protein (AB) into fibrils and that an interaction between secreted amyloid B-protein precursor (sABPP) and HCSM cell surface fibrillar AB contributes to the downstream pathologic changes that occur in cerebral blood vessels of patients with Alzheimer' s disease (AD) and related disorders including hereditary cerebral hemorrhage with amyloidosis Dutch-type (HCHWA-D). Our overall reseach efforts have focused on elucidating the cellular mechanisms that underlie the cerebrovascular pathology of AD, HCHWA-D, and related disorders. The objectives of the present proposal are to investigate the interaction between fibrillar forms of AB and sABPP that occur on the HCSM cell surface that induces several key pathologic responses in these cells. To accomplish this we propose five specific aims. First, we plan to identify specific factors on HCSM cells that promote pathogenic A13 fibril formation. Second, we wifi determine the specific domain on ABPP that mediates its binding to fibrillar forms of AB. Third, we wifi investigate if altering the level of AI3PP expression affects the ability of pathogenic forms of AB to induce pathologic responses in HCSM cells. Fourth, we will determine the specific domain on sAJ3PP that is involved with inducing downstream pathologic responses in cultured HCSM cells. Last, we will begin to identify pathologic signaling pathways that are activated in response to the fibrillar AB-sABPP interaction. These combined investigations intend to utilize cultured HCSM cells as an in vitro paradigm to characterize the consequences of the interaction between fibrillar AB and sABPP that occurs on the cell surface that potentially leads to the downstream cerebrovascular pathologic changes observed in patients with AD and related disorders. Certain aspects of this pathologic cascade may be specific for HCSM and related cells in the cerebral blood vessel wall and, therefore, may help to explain the different etiologies between the parenchymal and cerebrovascular AB induced pathologies in AD and related disorders. Finally, the pathologic interactions that occur between fibrillar AB and sABPP on the surfaces of HCSM cells may have potential therapeutic implications. Characterization of the molecules and domains involved in these interactions and detennining the consequences of these interactions with respect to inducing key pathologic responses may provide an opportunity for the future development of novel intervention strategies to ameliorate the cerebrovascular pathology of AD, HCHWA-D, and related disorders.

[unreadable] DESCRIPTION (provided by applicant): Cerebrovascular deposition of the amyloid [unreadable]-protein (A[unreadable]), a condition known as cerebral amyloid angiopathy (CAA), is a common pathological feature of patients with Alzheimer's disease (AD) and several related hereditary cerebral hemorrhage with amyloidosis (HCHWA) disorders. A[unreadable] is proteolytically derived from its parent molecule the amyloid [unreadable]-protein precursor (A[unreadable]PP). Apolipoprotein E (ApoE) genotype can facilitate both cerebrovascular A[unreadable] deposition and hemorrhagic stroke. It is significant that CAA accounts for up to 20% of cases of spontaneous primary intracerebral hemorrhage. Moreover, CAA is most severe in HCHWA patients often resulting in early recurrent and fatal intracerebral hemorrhages. The reason as to why there is preferential cerebrovascular A[unreadable] deposition in HCHWA disorders leading to hemorrhagic stroke and how ApoE may facilitate these pathological processes remains unresolved. [unreadable] [unreadable] We have shown that certain HCHWA mutant forms of A[unreadable], which exhibit a loss or change in charge at peptide residues 22 or 23, possess enhanced pathogenic properties towards cultured cerebrovascular cells. In addition, ApoE genotype can further influence the pathogenic effects of A[unreadable] in these in vitro paradigms. However, many of these issues can be better studied in valid in vivo models for CAA. Therefore, the overall hypotheses that forms the basis of this proposal is that expression of HCHWA mutant A[unreadable]PP in transgenic mice will lead to the preferential development of CAA and human ApoE genotype can further influence this pathology and promote cerebral hemorrhage. The broad objectives of this proposal are two-fold. First, we will compare the pathological consequences of neuronal over-expression of several human A[unreadable]PP forms yielding either wild-type or CAA mutant A[unreadable] with regards to the development of CAA. The CAA mutant forms A[unreadable]PP will contain either a single Dutch E22Q AB substitution or double Dutch/Iowa E22Q,D23N A[unreadable] substitutions. Second, the influence of human ApoE genotype on A[unreadable] deposition, the development of CAA, and cerebral hemorrhage will be investigated in these in vivo models. These proposed studies stem from our overall focus and continuing work on investigating the role of ABPP and its derived fragment A6 in the development of CAA, loss of vessel wall integrity, and hemorrhagic stroke. Completion of these specific aims will produce valuable models for both the further study of pathogenic mechanisms in CAA and in vivo systems to develop and test therapeutic strategies to mitigate cerebrovascular A[unreadable] deposition and the subsequent pathological consequence of hemorrhagic stroke. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Cerebrovascular deposition of the amyloid beta-protein (AB), a condition known as cerebral amyloid angiopathy (CAA), is a common pathological feature of patients with Alzheimer's disease (AD) and related disorders and is a primary cause of cerebral hemorrhage. The reason as to why cerebrovascular AB deposition leads to loss of vessel wall integrity and hemorrhagic stroke in CAA remains unresolved. AB is proteolytically derived from its parent molecule the amyloid beta-protein precursor (ABPP), which possesses potent anticoagulant properties. ABPP and its derived fragment AB are intimately involved in the pathology of CAA. A major thrust in our laboratory is to understand how ABPP and AB contribute to this vessel wall pathology, including cerebrovascular cell death, loss of vessel wall integrity, and cerebral hemorrhage. In this regard, we have focused on developing invaluable in vitro and in vivo models of CAA involving ABPP and AB. Recent findings from our laboratory have implicated ABPP, urokinase-type plasminogen activator (uPA) and members of the matrix metalloproteinase (MMP) family as potential contributors to these pathologic events in CAA. In light of these recent findings, the hypothesis that forms the basis of this proposal is that cerebrovascular deposition of AB enhances the expression and accumulation of factors that alter the local proteolytic environment of the cerebral vessel wall contributing to cerebrovascular cell degeneration, loss of vessel wall integrity and cerebral hemorrhage. The broad objectives of this proposal are two-fold. First, our major emphasis will be to investigate downstream alterations in proteolytic mechanisms that can be influenced by AB deposition and/or ABPP accumulation that may contribute to the pathology of CAA. Second, we will determine if alterations in these proteolytic systems can influence cerebrovascular AB deposition. The planned experiments are multi-tiered and will take advantage of unique cerebrovascular cell culture systems for in vitro studies and novel transgenic mice for in vivo studies modeling CAA. Specifically, the aims of this proposal are to 1) determine if over expression of ABPP on the surfaces of cultured human cerebrovascular cells influences AB deposition and hemostatic reactions; 2) investigate the expression, activity, and consequences of uPA in in vitro and in vivo models of CAA.and 3) investigate the expression, activity, and consequences of specific matrix metallo-proteinases (MMPs) in in vitro and in vivo models of CAA. Completion of these studies will provide new important mechanistic information about pathological functions of AB and ABPP and how they can modulate local proteolytic mechanisms that occur in the cerebral vessel wall in CAA influencing AB deposition and/or loss of vessel wall integrity. The information generated from our proposed studies may provide new insight into the development of therapeutic strategies to mitigate both cerebrovascular AB deposition and the subsequent pathological consequence of hemorrhagic stroke in CAA. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Secreted Kunitz proteinase inhibitor (KPI) domain-containing forms of the amyloid beta-protein precursor (ABetaPP) are also known as the previously described cell secreted proteinase inhibitor designated protease nexin-2 (PN2). Extensive earlier work from our laboratory has shown that PN2/ABetaPP is a potent inhibitor of several key pro-thrombotic enzymes and can inhibit thrombosis in vitro. These defining biochemical features of PN2/ABetaPP, coupled with its abundance in brain and in circulating blood platelets, have suggested a role for this protein in regulating thrombosis during episodes of cerebral vascular injury. Hemorrhagic and ischemic strokes are major health issues that can lead to severe debilitation and morbidity. Both hemorrhagic and ischemic strokes involve alteration of pro-thrombotic pathways. A precise understanding of the molecules and mechanisms involved in regulating cerebral thrombosis during these deleterious vascular events remains unresolved. The goal of this study is to define the role of the proteinase inhibitory properties of the ABetaPP in regulating cerebral thrombosis during cerebral vascular injury. In this regard, the overall hypothesis that forms the basis for this proposal is that the proteinase inhibitory function of PN2/ABetaPP plays a significant role in regulating cerebral thrombosis during cerebral vascular injury. The three specific aims of this proposal are as follows. First, determine if specific over-expression of platelet PN2/ABetaPP in transgenic mice will decrease thrombus formation, brain lesion, and behavioral deficits associated with cerebral vascular injury. Second, determine if specific over- expression of PN2/ABetaPP in brain in transgenic mice will modulate cerebral thrombosis in models of intracerebral hemorrhage and transient focal ischemia. Third, determine if deletion of the proteinase inhibitory activity of PN2/ABetaPP, amyloid precursor-like protein 2 (APLP2), or both will increase thrombus formation, brain lesion, and behavioral deficits in models of cerebral vascular injury. Together, these proposed translational investigations, which stem from our extensive previous in vitro work on the proteinase inhibitory properties of PN2/ABetaPP, will provide new insight into important physiological functions of this protein that currently remain unknown. This may lead to new avenues for developing strategies to regulate cerebral thrombosis and limit damage to the brain as a consequence of hemorrhagic and ischemic stroke.

DESCRIPTION (provided by applicant): Secreted Kunitz proteinase inhibitor (KPI) domain-containing forms of the amyloid beta-protein precursor (ABetaPP) are also known as the previously described cell secreted proteinase inhibitor designated protease nexin-2 (PN2). Extensive earlier work from our laboratory has shown that PN2/ABetaPP is a potent inhibitor of several key pro-thrombotic enzymes and can inhibit thrombosis in vitro. These defining biochemical features of PN2/ABetaPP, coupled with its abundance in brain and in circulating blood platelets, have suggested a role for this protein in regulating thrombosis during episodes of cerebral vascular injury. Hemorrhagic and ischemic strokes are major health issues that can lead to severe debilitation and morbidity. Both hemorrhagic and ischemic strokes involve alteration of pro-thrombotic pathways. A precise understanding of the molecules and mechanisms involved in regulating cerebral thrombosis during these deleterious vascular events remains unresolved. The goal of this study is to define the role of the proteinase inhibitory properties of the ABetaPP in regulating cerebral thrombosis during cerebral vascular injury. In this regard, the overall hypothesis that forms the basis for this proposal is that the proteinase inhibitory function of PN2/ABetaPP plays a significant role in regulating cerebral thrombosis during cerebral vascular injury. The three specific aims of this proposal are as follows. First, determine if specific over-expression of platelet PN2/ABetaPP in transgenic mice will decrease thrombus formation, brain lesion, and behavioral deficits associated with cerebral vascular injury. Second, determine if specific over- expression of PN2/ABetaPP in brain in transgenic mice will modulate cerebral thrombosis in models of intracerebral hemorrhage and transient focal ischemia. Third, determine if deletion of the proteinase inhibitory activity of PN2/ABetaPP, amyloid precursor-like protein 2 (APLP2), or both will increase thrombus formation, brain lesion, and behavioral deficits in models of cerebral vascular injury. Together, these proposed translational investigations, which stem from our extensive previous in vitro work on the proteinase inhibitory properties of PN2/ABetaPP, will provide new insight into important physiological functions of this protein that currently remain unknown. This may lead to new avenues for developing strategies to regulate cerebral thrombosis and limit damage to the brain as a consequence of hemorrhagic and ischemic stroke.

[unreadable] DESCRIPTION (provided by applicant): Extracellular deposition of the amyloid [unreadable]-protein (A[unreadable]) in brain is a prominent pathological feature of Alzheimer's disease (AD) and related disorders. Cerebral parenchymal A[unreadable] deposition can occur as diffuse plaques, with little surrounding pathology, or as fibrillar plaques associated with dystrophic neurons and inflammation. Fibrillar A[unreadable] deposition in the cerebral vasculature, a condition known as cerebral amyloid angiopathy (CAA), is also commonly found in Alzheimer's disease. Additionally, several familial monogenic forms of CAA exist that result from mutations that reside within the A[unreadable] peptide sequence of ASPP gene including Dutch- type (E22Q) and Iowa-type (D23N) which cause early and severe cerebral vascular amyloid deposition. Recent studies have implicated cerebral microvascular AS deposition in promoting neuroinflammation and dementia in patients with CAA. Cerebral microvascular, but not parenchymal, amyloid deposition is more often correlated with dementia in individuals afflicted with Alzheimer's disease and CAA. Neuroinflammation remains a viable target for the treatment of amyloid-depositing diseases in the central nervous system, particularly the neuroinflammation associated with cerebral microvascular amyloid. [unreadable] [unreadable] Recently, we generated novel transgenic mice that express human vasculotropic Dutch/Iowa mutant human amyloid B-protein precursor (ASPP) in brain, designated Tg-SwDI, that develop early-onset and robust fibrillar cerebral microvascular A[unreadable] deposition in the absence of parenchymal fibrillar plaque amyloid. More recent work from our laboratory has demonstrated that Tg-SwDI mice exhibit neuroinflammation that is strongly associated with the cerebral microvascular amyloid deposition. Furthermore, Tg-SwDI mice show marked deficits in behavioral performance. In light of these findings, the overall hypothesis that forms the basis for this proposal is that cerebral microvascular fibrillar AB deposition promotes neuroinflammation and behavioral deficits in the absence of fibrillar plaque amyloid. In the present proposal, we plan to thoroughly characterize the microvascular amyloid and the neuroinflammatory response, as well as investigate the effects of anti-inflammatory drug treatment on modulating microvascular amyloid, neuroinflammation, and behavioral decline in Tg-SwDI mice, a novel and unique transgenic model that only develops microvascular fibrillar A[unreadable] deposition. Completion of these studies should provide important insight into microvascular amyloid-mediated neuroinflammation and dementia that is an understudied and likely important, aspect of disorders that involve CAA. Additionally, since CAA pathology is commonly found in Alzheimer's disease this pathologic target may have far reaching implications in combined treatment strategies for this neurodegenerative condition and its related CAA disorders. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Cerebrovascular accumulation of the amyloid [unreadable]-protein (A[unreadable]), a condition known as cerebral amyloid angiopathy (CAA), is a common pathological feature of patients with Alzheimer's disease (AD) and several related familial CAA disorders. There is substantial evidence that neuronally-derived A[unreadable] peptides normally migrate to the cerebral microvasculature where they are cleared from the CNS by transport across the capillary blood-brain barrier into the circulation. Familial forms of CAA involve the early and severe cerebrovascular accumulation of A[unreadable] peptides with specific mutations such as the Dutch mutant E22Q and Iowa mutant D23N. However, in familial forms of CAA generally only one mutant A[unreadable]PP allele is present indicating that the pool A[unreadable] peptides in brain is a mixture of wild-type and CAA mutant forms. How wild-type and CAA mutant A[unreadable] peptides interact in vivo to affect clearance and produce CAA pathology is not understood. Recently, we generated a transgenic mouse model that produces Dutch/Iowa CAA mutant A[unreadable] peptides in brain (Tg-SwDI) and develops cerebral microvascular amyloid with associated neuroinflammation and behavioral deficits. The Tg-SwDI mouse has provided an invaluable model to study the genesis and consequences of familial microvascular CAA. However, the amyloid pathology of Tg-SwDI mice is the result of only human Dutch/Iowa CAA mutant A[unreadable] peptides. The impact of human CAA mutant A[unreadable] peptides on the accumulation and clearance of human wild-type A[unreadable] peptides in this model is unknown. Thus, we hypothesize in this proposal that human CAA mutant A[unreadable] and human wild-type A[unreadable] peptides interact in the CNS to influence clearance and cerebrovascular accumulation of A[unreadable] in transgenic mice. To test our hypothesis we will take the experimental approach of crossing the base Tg-SwDI mice with two different transgenic models that produce either 1) elevated levels of human wild-type A[unreadable] peptides in brain and develop abundant amyloid pathology or 2) low, physiological levels of human wild-type Ass peptides in brain with no amyloid pathology. Then we will evaluate the effects on development of CAA, CNS A[unreadable] peptide efflux into the circulation, and the downstream neuroinflammation and behavioral deficits associated with CAA. Completion of the above goals will provide new and significant information regarding how wild- type and CAA mutant A[unreadable] peptides interact in vivo in the brain. These studies will provide useful insight into understanding mechanisms involved in A[unreadable] clearance and pathological accumulation at the cerebral vasculature, particularly in familial forms of CAA, and may lead to new avenues for approaches to impede the initiation and progression of CAA and/or facilitate A[unreadable] efflux from the CNS. PUBLIC HEALTH RELEVANCE: Accumulation of a protein fragment, known as amyloid [unreadable] -protein (A[unreadable]), in the brain is a key pathological feature of Alzheimer's disease and related disorders. Mutations have been identified in A[unreadable] that cause it to accumulate in brain more severely than normal A[unreadable]. The purpose of this proposal is to investigate how mutant A[unreadable] interacts with normal A[unreadable] to increase its accumulation in brain.

Abnormal accumulation and deposition of the amyloid ss-protein (Ass), a prominent pathological feature of patients with Alzheimer's disease (AD) and related disorders, can occur from increased production but in most cases is likely due to decreased clearance mechanisms in the CNS. Clearance mechanisms involve factors that can promote Ass efflux from the CNS, enhance Ass degradation, and/or inhibit Ass assembly and deposition. Although much has been learned about specific molecules that can either influence Ass assembly and deposition or enzymes that can proteolytically degrade Ass our understanding of these processes in brain remains incomplete. Recently, we identified myelin basic protein (MBP) as a novel factor in brain that can bind Ass and potently inhibit its assembly into fibrils. Furthermore, through the course of our recent studies we made the exciting new discovery that MBP, with recently reported serine proteinase activity, can also proteolytically degrade Ass peptides. In light of these novel findings the hypothesis that forms the basis of this proposal is that MBP may contribute to the regulation of Ass levels, amyloid formation, and deposition in brain through its respective proteolytic and fibril-inhibiting activities. The broad objective of this proposal is to better understand these novel activities of MBP that involve its interaction with Ass peptides. The specific aims of the proposal are as designed to: 1) determine the respective binding regions on MBP and Ass peptides and investigate the consequences of this interaction, 2) characterize the proteolytic activity of MBP and its degradation of Ass, and 3) determine if MBP can modulate Ass levels and amyloid deposition in cell culture models. In the present proposal we plan to implement a multi-faceted approach to investigate how MBP interacts with Ass peptides to regulate their assembly and degradation. We will utilize a combination of biochemical, molecular, ultrastructural, and cell culture approaches to understand these interactions and the consequences of them.

Accumulation of the amyloid ss-protein (Ass) in brain, either as parenchymal plaques or cerebrovascular deposits, is a key pathological feature of patients with Alzheimer's disease (AD) and several related disorders. The Ass peptides are derived from the amyloid ss-protein precursor (AssPP) by sequential proteolytic cleavages by ss- and ¿-secretase enzymes. The factors that either promote or impede Ass assembly into fibrillar structures that deposit in brain remain largely undefined. Recent in vitro work from our laboratory has shown that myelin basic protein (MBP), a prominent component of myelin in brain, is a potent inhibitor of Ass fibrillar assembly and can protect cultured primary neurons from the toxic effects of Ass. Although the spatial deposition of Ass in brain is consistent with this finding (i.e. brain white matter rich in MBP is largely devoid of fibrillar Ass deposits) and there is a relationship between decreased MBP levels and increased Ass levels it remains unknown if MBP does indeed influence Ass assembly and accumulation in vivo. Several well-characterized human AssPP transgenic mouse models have been generated that develop AD-like fibrillar amyloid deposits. To study the consequences of the absence of MBP on fibrillar amyloid assembly and deposition in these established AssPP transgenic models one could breed them onto an MBP gene knockout background. Such a model, known as the shiverer mouse, exists but comes with the significant shortcomings in that they do not form myelin and die within several months after birth. Unfortunately, human AssPP transgenic mouse models require aging well beyond several months to develop significant pathologic amyloid formation. Instead of knocking out expression of the entire MBP protein a more sophisticated approach that we plan to employ will be to mutate a highly specific domain on the MBP protein to disable a specific function. To this end, in the R21 Phase of this application we propose to generate a novel knock in mouse model where we will introduce alanine mutations into a specific KRG motif in the endogenous mouse Golli-MBP gene. Our recent studies have identified this specific KRG motif as an essential element for binding to Ass peptides and inhibiting their fibrillar assembly. The resulting new knock in model will produce MBP that lacks the ability to bind Ass peptides and inhibit their assembly. These novel MBP- KRG/AAA knock in mice will be generated and initially characterized for viability, growth, behavior, and myelination. After successful completion of the R21 Phase of this application we plan to proceed to the R33 Phase where we propose to cross the MBP-KRG/AAA knock in mice with two different human AssPP transgenic mouse models that develop fibrillar amyloid deposition. The crossed mouse lines will be aged and quantitatively evaluated for the acumulation, asembly, and deposition of Ass peptides and the resulting downstream pathological and behavioral consequences. Completion of these studies will provide new insight into potential physiological mechanisms that govern pathogenic amyloid assembly and may lead to new avenues for intervention into this pathologic process.

DESCRIPTION (provided by applicant): The amyloid ¿-protein is implicated as a key pathogenic molecule in the pathogenesis of Alzheimer's disease (AD) and related disorders. A¿ possesses the strong propensity to self-assemble into soluble, oligomeric species and ultimately into insoluble, fibrillar structures that deposit in the central nervous system. Although soluble A oligomeric assemblies and A¿ deposits exist largely in the extracellular compartment of the CNS growing evidence suggests that initial assembly stages of A¿ and potential sites for its toxic activities occurs within neurons suggesting that this may be an early site for targeting the disruption of this process. However, our present understanding of what regulates these processes in the CNS, in particular within neurons, remains incomplete. Recently, we identified myelin basic protein (MBP) as a novel factor isolated from brain that can strongly bind to A¿ peptides and potently inhibit their assembly into fibrils and its neurotoxicity. Moreover, this particular function of MBP was mapped to N-terminal residues 1-64 (MBP1-64), a region that is contained in most related Golli proteins as well. In addition to its prominent role in myelin sheat formation, Golli-MBP proteins are present in numerous cells, including neurons, and have been proposed as intracellular multifunctional scaffolds that can bind a number of intracellular proteins and small molecule ligands affecting diverse cellular processes. In light of these points the overall hypothesis of this exploratory proposal is that Golli-MBP proteins interact with intracellular A¿ peptides to influence their assembly, accumulation, and toxicity within neurons and extracellularly in the brain. In the present proposal we plan to implement a multi-faceted approach to investigate how a biologically active MBP fragment interacts with A¿ peptides both in vitro and in vivo to regulate their intracellular and extracellular assembly, deposition, and pathological consequences. First, we will utilize cultured cortical neurons prepared from Tg-5xFAD mice that produce high levels of intracellular and extracellular A¿ peptides in vitro. We will express the MBP1-64 fragment in these neurons to investigate how it influences the fate of intracellular A¿ peptide. Second, we will use the well-characterized Tg-5xFAD mice that exhibit intraneuronal A¿ and develop abundant, age-dependent extracellular A¿ plaque deposits with accompanying neuroinflammation and behavioral deficits. The Tg-5xFAD mice will be crossed with newly generated transgenic mice that express the biologically active MBP1-64 fragment in neurons to investigate its interaction with A¿ peptides and how this might alter pathological outcomes. Completion of these studies will provide new insight into the biology of intracellular Golli-MBP interactions with A¿ that may contribute to the spatial and quantitative regulation of A¿ levels, assembly, and deposition in brain as well as the accompanying downstream pathological consequences. PUBLIC HEALTH RELEVANCE: Assembly and deposition of a protein fragment, known as amyloid ¿ -protein (A¿), in the brain is a key pathological feature of Alzheimer's disease and related disorders. Factors that regulate its assembly play an important role in determining if and where A¿ accumulates during disease. The purpose of this proposal is to investigate the activity of a newly identified factor, myelin basic protein (MBP), on A¿ assembly and deposition in cultured neuronal cells and in brain using a mouse model of intraneuronal and extracellular A¿ accumulation.

Abnormal accumulation, assembly and deposition of the amyloid ss-protein (Ass) is a prominent pathological feature of patients with Alzheimer's disease (AD) and related disorders. Ass peptides are derived through sequential proteolytic processing of the Ass precursor protein (AssPP) by ss- and ¿- secretase activities. AssPP is highly expressed in brain although its physiological functions remain poorly understood. Many functional domains have been identified on secreted forms of AssPP proteins that could participate in variety of neuroprotective activities ranging from proteinase inhibition to ligand binding to cytoprotection. For example, during the previous funding period we unequivocally demonstrated that the Kunitz proteinase inhibitory (KPI) activity of sAssPP limits the extent of cerebral thrombosis. Additional protective activities are likely associated with other biologically active domains present on sAssPP proteins in response to cerebral injuries including chronic neurodegenerative disorders such as AD. The abnormal accumulation and deposition of cerebral Ass peptides can occur from increased production but in most cases is likely due to decreased clearance mechanisms in the CNS. Clearance mechanisms involve factors that can promote Ass efflux from the CNS, mediate Ass degradation, and/or inhibit Ass assembly and deposition. Although numerous molecules have been identified that can influence Ass assembly and deposition in vitro our present understanding of these processes in brain remains incomplete. In this regard, the N-terminal region of AssPP (AssPP18-119) is a highly structured region of the protein that binds to Ass peptides and can inhibit their assembly. Thus, the overall hypothesis that forms the basis of this exploratory R21 proposal is that the N-terminal region of secreted AssPP proteins contributes to the regulation of Ass levels, amyloid formation and deposition in brain through its Ass assembly inhibiting activities. In the present proposal we plan to implement studies to investigate how the N-terminal region of AssPP interacts with Ass peptides in vivo to regulate their assembly, deposition and the pathological consequences associated with these processes. For these studies we will utilize two distinct and well- characterized transgenic mouse models of human Ass deposition coupled with approaches to increase AssPP N-terminal fragment levels in them, to understand how this region of sAssPP might alter pathological outcomes. Finally, this newly identified activity of sAssPP, and in particular the N-terminal AssPP18-119 fragment, may lead to new approaches for developing therapeutic agents to combat pathological Ass accumulation, assembly and deposition that occurs in AD and related amyloid depositing diseases.

? DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is a progressive neurodegenerative condition that is the sixth leading cause of death with a prevalence of every one in eight Americans of 65 and older in the United States. Presently, there are no effective pharmacological therapeutic agents to prevent or treat AD. In light of the present shortcomings, there is a growing call that a concerted effort be made to discover modifiable risk factors for dementia and exploit those already identified. To this point, epidemiological studies have shown that demographic and lifestyle factors such as cardiovascular exercise are related to a lower risk of developing cognitive impairments as one ages and in AD. Of the lifestyle factors, cardiovascular exercise is particularly promising since reports suggest that moderate, but positive, benefits are produced when cardiovascular exercise has been introduced as an intervention in clinical trials for older adults in early stages of mild cognitive impairment. Additionally, while many benefits of cardiovascular exercise as an intervention and/or preventative lifestyle factor towards AD pathology and cognitive impairments are possible, the age of onset, duration and intensity of exercise required for improved outcomes is largely unknown. In this case, animal studies designed to answer these key questions comprehensively and in a manner that will inform prospective human clinical trials would be highly advantageous. Indeed, several studies have shown benefits of exercise in mouse models of AD pathology. However, in all of these studies exercise was generally presented as a qualitative treatment solely contrasted with a sedentary control condition rather than as a quantitative, dimensional factor having potential dose-response properties. Thus, the overall hypothesis of this proposal is that specific cardiovascular exercise regimens can provide prevention and/or interventional benefits towards cognitive impairment in the presence of Aß pathologies and in normal aging. While earlier studies in humans and mouse models are encouraging, neither has systematically evaluated the beneficial effects of cardiovascular exercise based on age of onset, duration and intensity. Here, we plan to fill this critical void in the existing knowledge and determine the potential preventative and interventional benefits of varying levels of cardiovascular exercise on cognitive decline in normal aging mice and in two distinct mouse models of amyloid pathologies commonly associated with AD, with corresponding cognitive impairments. Presently, recruitments are underway for an NIH supported clinical trial to evaluate Cognitive Benefits of Aerobic Exercise Across the Age Span ://www.nia.nih.gov/alzheimers/clinical-trials/cognitive-benefits- aerobic-exercise-across-age-span. Therefore, our proposed studies are not only novel, but also very timely, and could provide invaluable translational information to further guide study design in human trials.

? DESCRIPTION (provided by applicant): Cerebrovascular accumulation of the amyloid ß-protein (Aß), a condition known as cerebral amyloid angiopathy (CAA), is an important cause of vascular cognitive impairment (VCI) and a common pathological feature of patients with Alzheimer's disease (AD). In addition, several related familial CAA disorders result from mutations that reside within the Aß peptide sequence of AßPP gene including Dutch-type (E22Q) and Iowa- type (D23N). Evidence continues to accumulate indicating that cerebral microvascular amyloid can promote small vessel pathology, neuroinflammation and cognitive deficits in patients with AD and related CAA disorders. Previously, we generated unique transgenic mice that produce Dutch/Iowa CAA mutant human Aß in brain, designated Tg-SwDI, that develop early-onset and prominent subcortical fibrillar cerebral microvascular Aß deposition. Despite the value and unique insight that Tg-SwDI mice have provided in the study of subcortical small vessel CAA, associated pathologies and cognitive impairment there remains significant shortcomings with the use of this model. For example, in contrast to humans and higher animals, mice possess small brains with little white matter thus limiting neuroimaging capabilities and the study of important vascular mediated changes in these regions. In addition, the study of cognitive abilities in mice are much more restricted compared to higher species. Thus, there is an important need for better models to further our understanding of the impact of small vessel CAA on brain pathology and function. In light of the limitations of current mouse models, advances in the production of transgenic rats provide the opportunity to develop a more appropriate and reproducible species to model small vessel CAA. Thus, the overall hypothesis and aim of this exploratory R21 proposal is that the generation and characterization of novel transgenic rats will provide a superior model to study the impact of subcortical small vessel CAA on brain pathology and cognitive function. To accomplish this goal we propose to generate novel transgenic rats expressing Dutch/Iowa CAA mutant Aß in brain and subsequently 1) conduct temporal biochemical and pathological characterization; 2) determine the consequences of small vessel CAA on cognitive functions; and 3) perform neuroimaging studies to determine the impact of small vessel CAA on brain pathology using microMRI. Here, we take the unorthodox approach of submitting a multi-PI exploratory proposal that will bring together three collaborative investigators with distinct, but highly complimentary, expertise to generate and characterize novel transgenic rats for CAA. The investigators range in expertise from production of numerous transgenic mouse models and biochemical and pathological characterization (Dr. Van Nostrand), advanced behavioral and cognitive characterization of rodents (Dr. Robinson) and high-resolution neuroimaging morphometric analysis of rodent models (Dr. Benveniste). The aim of our group is to generate a superior model for the study of small vessel CAA and provide a much needed, more advanced and invaluable animal model of this condition to the research community for investigating pathogenic mechanisms and to evaluate potential diagnostic and therapeutic interventions with relevant cognitive and neuropathological endpoints.

? DESCRIPTION (provided by applicant): There are two distinct processes involved in the deposition of amyloid in the brain during aging. Accumulation of the amyloid ?-protein (A?) in the brain parenchyma is the hallmark of Alzheimer's disease (AD), while accumulation of the A? protein in the cerebrovascular network is a condition known as cerebral amyloid angiopathy (CAA). There are familial mutations associated with both conditions. For AD, many of the mutations reside in the A? amyloid precursor protein and are responsible for the increased production of A?42 over the more prevalent A? 40 form of the peptide. For CAA, familial CAA disorders result from specific mutations in the A? peptides themselves, including the Dutch-type (E22Q) and Iowa-type (D23N) mutations. Despite the highly fibrillogenic nature of Dutch and Iowa mutant A? peptides, fibrillar A? is restricted to the cerebral vasculature in these familil disorders. Recent evidence suggests the parenchymal plaque amyloid is distinct from cerebral vascular amyloid. However, there is a poor understanding as to why either amyloid forms, and it is not known whether there are unique structural motifs that promote the distinct pathological consequences leading to dementia. The focus of this proposal is to fill this critical void in knowledge. Accordingly, the overall hypothesis of this proposal is that the A? peptides forming parenchymal and vascular amyloid have distinct structures that determine their location and pathology. To address this hypothesis we propose four specific aims. First, we plan to isolate parenchymal plaque amyloid and cerebral vascular amyloid from post mortem brain tissue of AD and familial CAA cases. Parallel studies will be undertaken on unique transgenic mouse models of AD and CAA. The studies on transgenic mice provide a direct method to assess the level of parenchymal and vascular amyloid, and the ability to engineer mutations. The isolated amyloid will serve as seeds to nucleate fibril growth for in vitro studies in Aim 2, which will reval the distinct structural signatures of cerebral vascular and parenchymal plaque amyloid deposits. Next, we will generate a collection (library) of homogeneous fibrils isolated from brain tissue. Fluorescence, infrared and NMR spectroscopy will be used to determine the structural features of these fibrils. Preliminary results show the A? peptides in parenchymal amyloid adopt ? - sheet structure with parallel, in-register ?-strands, whereas the ?-sheets in vascular amyloid have anti-parallel structure. Third, differences in cell toxicity and activation of different fibri (and oligomer) forms will be assessed using neuronal, vascular and microglial cell cultures. Preliminary results reveal profound differences of different A? fibrils on microglial activation. Fourth, we will assess the influence of A? inhibitors on different A? fibril structures. We wil first test the ability of known inhibitors to dissociate the different fibril structures isolated from brin tissue. Next, we will design dual-site inhibitors that combine elements of known inhibitors starting with designed, synthetic inhibitors that target the GxxxG sequences in the hydrophobic C-terminus of A? and inhibitors based on fragments of the myelin basic protein that interact with the N-terminus of A?.

Vascular cognitive impairment & dementia (VCID) is defined as a form of dementia that is triggered by damage to cerebral blood vessels or cerebrovascular disease. Cerebral amyloid angiopathy (CAA), which is accumulation of amyloid ß-protein (Aß) within and along primarily small and medium-sized arteries and arterioles of the brain and in the cerebral microvasculature, is a common cerebral vascular condition that can cause VCID in the elderly. Not surprisingly, with the involvement of Aß, CAA is the most common vascular comorbidity found in the brains of Alzheimer's disease (AD) patients. Although there is evidence that both parenchymal plaque amyloid and cerebral microvascular amyloid can contribute to dementia in patients with AD and related disorders, there is growing recognition that the latter is a potent driver of cognitive impairment. Yet, the reasons as to why cerebral vascular amyloid forms and its contribution to downstream pathologies and early cognitive impairment remain unclear. Altered copper homeostasis has been considered an important factor in the neurodegenerative diseases. Earlier findings suggest that copper may play an important role in the formation of amyloid deposits and in subsequent neuronal dysfunction and cognitive impairment. However, relatively little is known about the accumulation of copper in cerebral vascular amyloid deposits, which are associated with early-onset VCID. Thus, the overall hypothesis of our proposal is that copper plays a role in driving fibrillar amyloid assembly in CAA and that the subsequent accumulation of copper in the cerebrovascular amyloid deposits promotes downstream pathologies and early- onset cognitive impairment. In order to test this hypothesis we propose to three specific aims. First, we will determine if vascular amyloid deposits exhibit high levels of copper compared to parenchymal amyloid plaques in post mortem human brain tissue samples of AD, sporadic CAA and familial CAA patients and in transgenic mouse models. Second, we will investigate the effects of copper on Aß fibril assembly. Third, we will determine the effects of increasing or reducing copper levels on the development of CAA, downstream pathologies and cognitive impairment in Tg-SwDI mice. Currently, there are no effective therapies or reliable biomarkers specifically for CAA. These deficiencies are complicated by our lack of understanding of the assembly and unique structural attributes of cerebral vascular amyloid and their distinctive features that lead to CAA formation and subsequent pathologies. The present proposal, focused on the role of copper in these events, will seek to fill this critical void in our knowledge and will advance our understanding of the pathogenesis of CAA and provide insight into the development of novel diagnostic markers and potential therapeutic interventions for CAA and VCID.

We are proposing a novel approach to diagnosing early Alzheimer?s disease (AD) and predicting progression via a robust biomarker that captures ?glymphatic? pathway transport on a systems level. The glymphatic pathway is a brain-wide system, which was recently discovered to function as a clearance pathway for toxic brain waste proteins including soluble amyloid beta (A?) and tau similarly to the classical body-wide lymphatic system. As such, the glymphatic pathway comprises a previously overlooked and unique compartment of the brain vasculature, the peri-vascular space wherein cerebrospinal fluid (CSF) is flowing and streaming into the brain interstitial fluid (ISF) space thereby forcing waste solutes out of the brain. Except for rare familial AD, where excessive A? production and deposition in the brain clearly drives cognitive decline, there is limited evidence in the more common sporadic AD that cerebral A? accumulation is the result of A??overproduction. In fact, emerging evidence suggests that parenchymal A? accumulation in sporadic AD is driven by reduced A? clearance. The glymphatic pathway is thus a prime candidate for linking disruptive clearance of A? to AD, and we will use this opportunity to develop new tools and computational analysis aimed explicitly at capturing global glymphatic pathway function and serve as a novel diagnostic AD biomarker. Currently there is no method available to capture all of the intricate and dynamic components of glymphatic transport, in particular, parenchymal transport and clearance pathways. We propose to integrate imaging techniques and develop novel computational analysis including optimal mass transport to characterize the glymphatic pathway as a brain-wide dynamic ?unit?. The ultimate goal of the proposed investigation is to apply the glymphatic biomarker and track its disruption in progressing vascular and parenchymal amyloid pathologies. The proposed studies are based on novel preliminary findings that 1) glymphatic transport can be visualized as an integrative system through perivascular and interstitial spaces; 2) that state dependent changes induced by specific anesthetic regimens which dramatically affect the glymphatic transport can be captured by optimal mass transport analysis; and 3) a new transgenic rat model of cerebral amyloid angiopathy (rTg-SwDI) which will be used for specific hypothesis testing against the transgenic rat AD model (rTgF344-AD36) in the proposed studies. The specific aims are the following: (1) To develop biomarkers to visualize and functionally quantify macroscopic, glymphatic transport based on computational analysis of MRI and macroscopic optical imaging of CSF tracers in normal young (3 month old) rats and (2) to determine how and when normal aging and specific AD-like cerebral vascular and parenchymal amyloid pathologies influence glymphatic transport in the brain using the computational pipeline developed in SA1. Successful completion of the proposed highly innovative experiments will yield an entirely new and promising biomarker to track reduced Aß clearance via the glymphatic pathway which is key to the propagation of CAA and AD.

? DESCRIPTION (provided by applicant): Cerebrovascular accumulation of the amyloid ß-protein (Aß), a condition known as cerebral amyloid angiopathy (CAA), is an important driver of vascular cognitive impairment and dementia (VCID) and is a common comorbidity of patients with Alzheimer's disease (AD). CAA can promote VCID through a number of mechanisms including chronic inflammation, hypoperfusion and ischemia, loss of vessel wall integrity and hemorrhage. In addition to its prevalence in AD, several related familial CAA disorders result from specific mutations that reside within the Aß peptide sequence of the Aß precursor protein including the Dutch-type (E22Q) and Iowa-type (D23N) mutations. Despite the highly fibrillogenic nature of Dutch mutant and Iowa mutant Aß peptides, fibrillar Aß is restricted to te cerebral vasculature in these familial disorders. Recent evidence suggests the cerebral vascular amyloid is distinct from parenchymal plaque amyloid. However, there is a poor understanding as to why cerebral vascular amyloid forms and its unique structural features that promotes distinct pathological consequences leading to VCID. Thus, the focus of this proposal is to fill this critica void in knowledge. Accordingly, the overall hypothesis of this proposal is that fibrillar amyloid i cerebral blood vessels possesses distinct structural features compared to parenchymal fibrillar amyloid and unique anti-parallel structures, enhanced by CAA mutations, drives the cerebral vascular specific deposition of amyloid in brain. To address this hypothesis we propose three specific aims. First, we will determine the structure, assembly and membrane interactions of wild-type and the Dutch and Iowa CAA mutants of Aß in solution and model membrane systems that drive their compartmental deposition. Second, we will determine how familial CAA variants of Aß chronologically influence the structural features and assembly of wild-type Aß in the brains of transgenic mice. Third, we will isolate parenchymal plaque amyloid and cerebral vascular amyloid from post mortem brain tissue of AD cases, sporadic CAA cases and familial CAA cases and investigate their ability to promote assembly of wild-type and CAA mutant Aß peptides. These important studies will reveal the distinct structural signatures of cerebral vascular and parenchymal plaque amyloid deposits in human disease. Presently, there are no reliable biomarkers or effective therapies specifically for CAA and VCID. These deficiencies are complicated by our lack of understanding of the unique structural attributes of cerebral vascular amyloid and its early-stage oligomeric precursors, and their distinctive features and processes compared to parenchymal plaque amyloid. The present proposal will seek to fill this critical void in our knowledge and will advance our understanding of the pathogenesis of CAA and provide the basis for the future development of novel therapeutic interventions and diagnostic markers for CAA and VCID.

Cerebrovascular accumulation of the amyloid b-protein (Ab), a condition known as cerebral amyloid angiopathy (CAA), is a common small vessel disease in the elderly, an important driver of vascular cognitive impairment and dementia (VCID) and a prominent comorbidity of patients with Alzheimer?s disease (AD). Despite the growing recognition of the contribution of CAA to VCID, early and accurate diagnosis of this condition has remained elusive and largely relies on neuroimaging modalities that are only effective in late stages of the disease. The current ?Boston MRI criteria? for CAA are based on the presence of multiple lobar microbleeds in the brain. However, the neuroimaging approaches are limited in that neuropathological findings demonstrate that abundant CAA is prevalent at early stages of disease without the presence of microbleeds, particularly in patients with AD. Thus, there is a need for biomarkers for early stages of disease prior to the presence of microbleeds detected by neuroimaging. The purpose of the is project is to fill in this void by developing and validating robust biological fluid markers for CAA. Recent work from our laboratories has identified novel candidate biomarkers that appear specific for CAA and mechanistically can be linked to the disease process and can be measured in biological fluids. These candidates were derived from a combination of biochemical and immunochemical approaches using potent and specific human cerebral vascular cell cultures and rodent models for CAA, and their presence has been confirmed in human CAA tissues. The overall hypothesis of this proposal is that these novel candidate biomarkers are unique and specific for CAA and will facilitate in an early and accurate diagnosis of CAA- related small vessel disease. There are two specific aims of this project. First, we will study the trajectory of CAA biomarkers in a transgenic rat model for CAA from the presymptomatic phase (prior to microbleeds) to the symptomatic phase (prominent microbleeds). This model provides the powerful and unique prospect to investigate the longitudinal expression of CSF and serum biomarkers in relation to the progression of disease severity, particularly in prodromal states, an opportunity that is not available in humans. Further, our CAA rat model will be used to identify additional candidate biomarkers using complementary proteomic approaches. Lastly, comparative studies will be performed using rat models of parenchymal plaque amyloid pathology or hypertension/stroke, another common cerebral small vessel disease, to further establish the specificity of CAA biomarkers. Second, we will further characterize, develop and validate assays for candidate protein biomarkers for the diagnosis of CAA including: intact and derivatives of Ab40 peptide, the chief component of cerebral vascular amyloid accumulation, heat shock protein B2 (HSPB2), and urokinase-type plasminogen activator (uPA). A priority of our plan is to share our data, provide developed assays, key reagents, patient samples and rat models to other groups and consortiums to advance small vessel disease biomarker development.

Alzheimer's disease is associated with the deposition of amyloid in the brain during aging. Since the correlation between amyloid formation and AD was originally made, it has been recognized that there are many subtypes and forms that the disease can take. For example, accumulation of the amyloid ?-protein (A?) in the brain parenchyma is the hallmark of Alzheimer's disease (AD). Nevertheless, there is a poor understanding as to why amyloid forms, and it is not known whether there are unique structural motifs that promote the distinct pathological consequences leading to dementia. The focus of this proposal is to fill this critical void in knowledge. Accordingly, the overall hypothesis of this proposal is that the A? peptides forming amyloid with distinct subtypes have distinct structures that determine their location and pathology. To address this hypothesis we propose two specific aims. First, we plan to isolate amyloid from different subtypes of post mortem brain tissue of late-onset AD and early- onset familial AD. We plan to compare five different amyloid plaque subtypes: typical AD, atypical AD, cotton wool, early-onset AD (EOAD) and very early-onset AD (VEOAD). The last two subtypes are associated with familial AD mutations. Clinical information is available concerning age and gender, age of onset and duration of disease, course and symptoms of the disease, medication and ApoE genotype. In the EOAD and VEOAD cases genetic testing was performed for APP, PSEN 1, PSEN2 and tau. For most cases, biomarkers in CSF and neuroimaging results are available. The isolated amyloid will serve as seeds to nucleate fibril growth for in vitro studies. The structure and polymorphism of the fibrils will be assessed by complementary structural approaches including solid-state NMR spectroscopy, Fourier transform infrared spectroscopy, transmission electron microscopy, and atomic force microscopy. Using the amyloid isolated from the five different subtypes of AD, we will assess the biofunctional consequences of the different strains using three approaches. First, we will assess the differences in the inflammatory response and cell toxicity due to different fibril forms using microglial cell cultures. Second, we will determine the influence of amyloid strains on promoting neuroinflammation. Third, we will determine the influence of amyloid strains on assembly and propagation in rat brain. The overall objective is to correlate pathologies (biofunctional consequences) of different amyloid subtypes between cell culture, rat brain and human brain, and to relate these pathologies with specific structural characteristics of the A? fibrils that are associated with the isolated amyloid from each subtype.

Cerebrovascular accumulation of the amyloid b-protein (Ab), a condition known as cerebral amyloid angiopathy (CAA), is a common small vessel disease that is prevalent in the elderly, a prominent comorbidity of patients with Alzheimer?s disease (AD) and an important driver of vascular cognitive impairment and dementia (VCID). Despite the growing recognition of the contribution of CAA to dementia in AD and in VCID, there currently exists no effective therapeutic interventions for this condition. CAA can uniquely contribute to the cognitive decline in VCID and AD in several manners. For example, in response to deposited fibrillar Ab in CAA cognitive impairment is worsened by a chronic state of perivascular neuroinflammation and activation that is characterized by reactive astrocytes and activated microglia and endothelial cells that can produce thrombin, pro-inflammatory cytokines and chemokines, and reactive oxygen and nitrogen species. Also, the increase in perivascular expression and activation of certain proteolytic enzymes, including thrombin, can contribute to microinfarcts, disruption of vessel wall integrity and cerebral hemorrhage, all highly deleterious manifestations of the disease. Thus, perivascular neuroinflammation, vascular activation, thrombosis and hemorrhage associated with cerebral vascular amyloid can all be linked to thrombin, which represents a potential therapeutic target to treat CAA/VCID. Based on our preclinical work in AD mouse models we now propose to test the hypothesis that thrombin-mediated vascular activation, neuroinflammation and thrombosis is a central mechanism contributing to small vessel CAA pathology in the brain and that inhibiting thrombin will improve clinically relevant CAA pathological and VCID-associated endpoints. To test our hypothesis, we first propose to test if early and long-term administration of dabigatran is effective as a preventative treatment to reduce CAA pathology and associated VCID. Specifically, we will test if long term treatment with dabigatran reduces CAA, perivascular neuroinflammation, small vessel occlusions, cerebral microbleeds and improves cognitive performance in a novel and unique transgenic rat model of small vessel CAA. Second, we propose to test if administration of dabigatran during later stage disease is effective as an intervention to reduce CAA pathology and associated VCID. Specifically, we will test if shorter term treatment with dabigatran during later stages in the disease can lower CAA-related perivascular neuroinflammation, small vessel occlusions, cerebral microbleeds and improve cognitive performance in our transgenic rat model of small vessel CAA. Successful outcomes in these exploratory studies will pave the way for future studies to understand the precise beneficial mechanisms of dabigatran. This will complement our ongoing clinical studies of dabigatran treatment in early stage AD and could provide a needed therapeutic agent for CAA related vasculopathy and VCID.