Jorge Ghiso - US grants

Beta-amyloid protein (Abeta), the major constituent of the fibrils composing senile plaques and amyloid deposits in cerebral blood vessels in Alzheimer's disease (AD) and other beta-amyloid related disorders, is a degradation product of a larger precursor APP. The deposition of Abeta in cerebral vessel wall and brain parenchyma may involve a complex interaction of different cell types and various protein factors. The comprehension of the mechanisms by which Abeta is processed from its precursor and the determination of the extent to which early and mature plaque formation contribute to neuronal cell damage and disease progression remain central and important questions in the understanding of AD. Recent findings from different groups including ours, indicate the existence of different APP degradation products, one of them containing only part of the Abeta sequence (nexin II) while another possess the intact Abeta being, therefore, potentially "amyloidogenic". Interestingly, some of these fragments including Abeta and nexin II can mediate cell adhesion. We found that the tetrapeptide RHDS can mimic the behavior of RGDS, a widely distributed cell adhesion recognition signal found in many extracellular matrix proteins. We propose i) to identify the intermediate APP C-terminal fragment(s) containing Abeta by immunochemical analysis and N-terminal sequence; ii) to use this information to engineering a construct and express it in cells in order to obtain enough amounts of fragment(s) to be used in cell adhesion and enzymatic degradation/fibril formation experiments; iii) to determine the biological importance of the sequence RHDS for both nexin II and Abeta.

This proposal is to fund a Procise Protein Sequencing System. The Model 494 is composed of three integrated molecules: I) the Procise Protein Sequencer, ii) the Model 190-00 Procise PTH System, that includes the Model 140C Microgradient Delivery System and the Model 785A UV Detector, and iii) a Macintosh computer equipped with Procise control software and the Model 610A Data Analysis software. The Procise system will replace an obsolete Applied Biosystems 477A that was purchased in 1987 through a DRR-BRS Shared Instrumentation Grant. The new instrument has been designed to run up to four sample cartridge assemblies independently; thus, the run order, sample type and sequencing method may be customized for each sample. It utilizes multiple types of cartridge assemblies, including the Blot and Micro-cartridge, which are customized for diverse samples: liquid, PVDF blot membranes and micro-quantities. It has the versatility needed to develop new methods for particular samples. A new coupling base, N-methylpiperidine, which is more stable then triethylamine has been substituted in the Edman chemistry. This has effected higher repetitive yields and the ability to perform high- sensitivity sequence analysis. The internal volume from the reaction cartridge to the injection loop has been reduced to less than one-third of the volume in previous models resulting in improved precision and efficiency of the sequence chemistry. For better peak detection and integration, the Procise has a build-in analog to digital converter providing four times the resolution of previous systems. The sensitivity, flexibility and advanced technology of the Procise system is eminently suitable for today's research prerequisites. This instrument would be critical to the needs of a group of highly productive and strongly NIH funded scientists who share a common interest in the misfolding of proteins, with specific focus in amyloid formation and the pathogenesis of Alzheimer's disease.

Amyloid beta (Abeta), the major constituent of the amyloid fibrils deposited in cerebral blood vessels and parenchymal plaques Alzheimer s brains, is also normally found as a soluble component (sAbeta) in biological fluids, where it appears to be transported (greater than 90 percent) in association with lipoprotein particles. Although peptides homologous to sAbeta spontaneously polymerize in vitro, the potential contribution of the circulating soluble forms to the deposited Abeta remains unknown. It has been demonstrated that the blood brain barrier has the capability to modulate the uptake of Abeta species and that the interaction of Abeta with carrier apolipoproteins E and J results in either prevention or enhancing of the brain uptake respectively. In addition, an increased amount of sAbeta was found in soluble fractions of Alzheimer s disease and Down s syndrome brain homogenates, and this elevation appears to precede the appearance of Abeta deposits. The overall goal of this proposal is to investigate the contribution of circulating Abeta peptides to the vascular pathology observed in aging, Alzheimer s disease and related disorders. The knowledge of physiologic aspects of sAbeta turn-over, among them the peptide s half-life in circulation, its interaction(s) with circulating transport molecules, the organs involved in its catabolism and/or excretion and the putative cerebrovascular cell receptors responsible for its uptake by the cerebral vessel wall as well as how these physiologic parametes are modulated under pathologic situations will certainly contribute to better understanding of the mechanism(s) leading to the formation of vascular and perhaps parenchymal amyloid deposits. The four specific aims outlined in the project are focused a) to identify cell receptors for free and complexed Abeta species in cerebral endothelial cells (aim 1), b) to investigate the half- life of free and complexed Abeta species in circulation as well as to determine their systemic sites of catabolism and/or excretion (aim 2), c) to ascertain whether oxidation of the carrier lipoparticle may favor complexdisequilibrium resulting either in the release of free sAbeta or in the formation of oxidized Abeta (aim 3), and d) to clarify whether the physiologic variables evaluated in aims 1 and 2 are compromised in aging and Alzheimer s disease (aim 4).

DESCRIPTION (provided by applicant): AB plays a central role in AD pathogenesis, although there is still a great need to fully define on which of the multiple AB assemblies underlay the neurotoxic properties and the amyloid-forming ability. While soluble oligomers more than fibrillar structures have been implicated in the mechanism(s) of neuronal toxicity, few in vivo experiments and our own recent data indicate that seeding of AB amyloidogenesis in APP transgenic mice can be accelerated by intracerebral infusion of soluble AD brain extracts but not to the same extent by homologous synthetic peptides in various states of aggregation, suggesting the existence of still undefined amyloidogenic co-factors. Two early-onset neurodegenerative conditions, familial British and Danish dementias (FBD and FDD) show extensive pre-amyloid and amyloid deposits, congophilic angiopathy and neurofibrillary tangle pathology, closely resembling AD. The deposited proteins (ABri in FBD and ADan in FDD), nevertheless, differ from AB in length and in primary structure and yet, all species share great propensity to oligomerize, assemble as ion-channel like structures in lipid bi-layers and form fibrils, all suggestive of common pathogenic pathways. We hypothesize that unrelated peptides could adopt similar altered amyloidogenic configurations that trigger comparable downstream detrimental effects in neuronal cells, being capable of accelerating amyloid deposition in vivo when in conjunction with additional amyloidogenic co-factors. Accordingly, we propose: Aim 1: to compare oligomeric and fibrillar ABri, ADan and AB assemblies isolated from brains with FBD / FDD / AD as well as from Tg mouse models and determine the structural requirements for synthetic ABri, ADan and AB to form in vitro similar assemblies to those found in vivo;Aim 2: to test the functional effect of the oligomeric and fibrillar ABri and ADan species characterized in aim 1 in comparison with analogous AB structures on their differential neurotoxic potential, assessing the induction of specific cell-death mechanisms (activation of initiator and effector caspases, mitochondrial pathways, Ca2+ dysregulation, etc) in ex vivo and in vitro paradigms, validating the results with tissue fractions enriched in oligomeric assemblies as well as synthetic homologues reconstituted in amyloid-depleted tissue extracts;Aim 3: to (a) analyze the exogenous amyloidogenic capability of oligomeric/fibrillar AB, ABri and ADan species in vivo in APP and ADanPP Tg animals using intra-hippocampal injections of brain extracts compared with synthetic homologues with analogous oligomerization in the presence/absence of amyloid-depleted brain extracts and (b) conduct a comparative proteomic analysis of the amyloid-inducing extracts to identify common post-translational modifications and/or additional co-factor(s) responsible for the amyloid-inducing activity. PUBLIC HEALTH RELEVANCE: There is extensive experimental data strongly suggesting that amyloid beta (AB) plays a central role in Alzheimer's disease (AD) although basic questions in the pathogenetic mechanisms remain unclear, particularly the role of oligomers and the existence of amyloidogenic co-factors of the disease. To clarify these issues we propose to study two early-onset non-AB neurodegenerative conditions that closely resemble AD: familial British and Danish dementias. The use of these alternative models of neurodegeneration in close comparison with AD cases will help elucidate whether different amyloids trigger common pathogenic mechanisms and are potentially amenable to similar therapeutics strategies.

DESCRIPTION (provided by applicant): Exfoliation syndrome (XFS), an age-related disorder which constitutes the most common identifiable cause of open-angle and angle-closure glaucoma, is characterized by the accumulation of an abnormal fibrillar material (XFM) in many intraocular tissues, particularly those structures that line the aqueous-bathed surfaces of the anterior segment. Deposits of XFM are composed of a complex mixture of extracellular matrix glycoproteins and proteoglycans assembled in a supramolecular fibrillar structure. Due to the highly diverse biochemical composition it is still not clear which are the primary elements involved in the formation of XF fibers and whether some of the deposited components or their immediate precursors are recruited to the lesions from the surrounding fluids (e.g. aqueous). Thus, despite all available information, there is still no data pointing out to specific candidate genes to support the generation of animal models. An understudied alternative approach is the development of a culture model that produces XF-like fibers in vitro, a potentially valuable paradigm that would allow the search for the pathogenetic pathway(s) leading to disease, conduct time-course kinetics of fibril assembly, supply fibrils for detailed biochemical analysis and be eventually amenable for pharmacologic interventions. In this sense, and as extensively illustrated in the Preliminary Studies section, we have obtained encouraging data from culturing Tenon's capsule fibroblasts from XFS patients in tri-dimensional systems, documenting the production of XF fibrils ultrastructurally similar, if not identical, to those observed in vivo. Based on our results we hypothesize that the use of selected XFS ocular cells in culture will provide a much needed model to study XF fibril assembly. To this end, we will take advantage of cutting-edge proteomic approaches, the availability of surgical specimens to establish a diversity of primary cultures from individuals with and without XFS, the accessibility to aqueous humors from the same cases, the newly developed Tenon's fibroblasts tri-dimensional cultures as well as previous technical experience acquired by the research group in culturing iris pigmented epithelial (IPE) cells to: (a) conduct proteomic analysis of XF-like fibrils produced in vitro by Tenon's fibroblasts from XFS cases;(b) verify whether cells obtained form IPE of XFS patients, cultured under various conditions, produce XF-like fibrils with the same EM characteristics and biochemical composition than Tenon's fibroblasts and/or XF fibrils produced in vivo;(c) identify common proteomic differences in the composition of the various culture supernatants of XFS vs. non-XFS controls and validate, through the parallel analysis of aqueous fluids from the same XFS and non- XFS controls, whether the observed differences might serve as prospective signature markers of the disease. The data generated by the proposed experiments will constitute the basis of a future R01 application. PUBLIC HEALTH RELEVANCE: Exfoliation (XF) syndrome is an age-related disorder which constitutes the most common identifiable cause of glaucoma worldwide. The extreme diversity of the molecules composing the XF lesions has so far precluded the creation of a much needed animal model of the disease. We propose to biochemically and ultrastructurally characterize a promising cell culture paradigm that produces XF-like fibers in vitro, which may prove to be a useful alternative approach to study the molecular basis of the disease and explore novel pharmacologic interventions.

Reprogramming of primary dermal fibroblasts into induced pluripotent stem cells (iPSCs) has recently proven to be instrumental for the generation of viable neurons derived from patients with neurodegenerative disorders. This technology holds tremendous promise for the creation of in vitro models to study disease pathophysiology in relevant human cell types that would otherwise be impossible to obtain. Familial British and Danish dementias (FBD and FDD, respectively) are autosomal dominant conditions that closely resemble many clinical and neuropathological features of Alzheimer's disease (AD) including parenchymal amyloid and pre- amyloid lesions, widespread cerebral amyloid angiopathy and neurofibrillary tangles morphologically and immunochemically indistinguishable from those in AD. Notably, the amyloid subunits isolated from FBD deposits -ABri- and FDD lesions -ADan- are structurally unrelated to the Alzheimer's A¿, a clear indication that different amyloid peptides could trigger similar neuropathological changes leading to the same scenario: CAA-related microvascular dysfunction, neuronal loss and dementia. Thus, these familial disorders constitute promising alternative paradigms to better understand the role of amyloid in the complex mechanisms of disease pathogenesis. In view of the many clinical and neuropathological similarities between AD, FBD and FDD, we are proposing i) to generate and characterize iPSC lines from dermal fibroblasts obtained from a cohort of FBD and FDD patients as well as from non-carrier siblings of both disorders using repetitive mRNA transfections, a safer non- DNA-integrating technology successfully used by the research team; and ii) to further differentiate the newly generated iPSCs into viable and functional neurons and endothelial cells characterized through well- established morphological, molecular and biological criteria. We anticipate that these iPSC-derived mature cells will constitute excellent candidates to study specific molecular and temporal aspects linked to FBD and FDD disease phenotypes. Moreover, they will have a broader impact in the field of neurodegenerative disorders, extending beyond these rare diseases into the field of AD, providing invaluable options for a better understanding of the mechanisms that modulate APP processing, A¿ homeostasis and the process of tau hyperphosphorylation, serving as alternative paradigms for high throughput drug screening platforms, and assisting with the identification of cross-talk pathways connecting CAA-associated blood brain barrier dysfunction and development of microhemorrhages with changes in the neurovascular unit and cognitive impairment. This proposal represents a collaborative effort from investigators of New York University School of Medicine and the University of California, Irvine and builds on the complementary expertise of the participating researchers and their long-standing interest in the molecular pathogenesis of cerebral amyloid disorders.

? DESCRIPTION (provided by applicant): Synaptic pathology- one of the strongest correlates to cognitive impairment- is related to the progressive accumulation of oligomeric forms of neurotoxic Aß. The assembly of Aß monomers into oligomers is a concentration-dependent process and as such, it is dependent on adverse changes that alter homeostatic mechanisms regulating Aß physiologic levels in the interstitial fluid (ISF), such us impaired normal brain removal and/or deficient catabolism. Surprisingly, very little is known about the brain clearance and local catabolism of Aß oligomers, particularly during the aging process. Based on a wealth of preliminary/feasibility data we propose to start filling this gap in knowledge by bridging together different understudied aspects of these processes: i) the poorly recognized high heterogeneity of the brain Aß species, with multiple N- and C- terminally truncated derivatives co-existing with the classic full-length duo Aß40/Aß42; ii) the ability of local brain resident enzymes to efficiently generate these truncated fragments; iii) the remarkable dissimilarities of these species in solubility and oligomerization propensity, suggesting engagement in opposite mechanisms either amyloidogenesis or clearance; and iv) the negative impact that aging imposes to anatomical and functional components of the clearance pathway e.g. compromised vascular integrity, lower density of cellular Aß transporters, substandard performance of the local proteolytic machinery all likely affecting the brain efflux efficiency of the perivascular drainage, as well as through the blood-brain and brain-CSF barriers. The presence of already established amyloid deposits a seldom considered complication further obscures the clearance scenario, not only through the potential recruitment of soluble Aß species to the lesions but by additionally restricting vessel functionality, contributing to the self-perpetuation of the amyloidogenic loop. We hypothesize that the process of amyloidogenesis goes beyond the simplistic dichotomy Aß40/Aß42, involving locally generated pro-oligomeric truncated fragments and differential in vivo brain clearance for monomeric and oligomeric Aß species likely mediated by the efflux transporters LRP-1 and P-gp, and postulate that these mechanisms are negatively modulated by aging and by the presence of pre- existing amyloid deposits. Assembled in two specific aims, we propose to compare the physiologic in vivo brain clearance of monomeric and oligomeric forms of intact and truncated Aß species, evaluate their local catabolism and relevance of Aß-efflux transporters while assessing the differential effect that normal aging and the presence of already established amyloid deposits exert in the brain removal mechanisms. Through the use of radiolabeled and isotopically-labeled Aß homologues, stereotaxic intra-hippocampal injections in wild-type mice and APPswePS1dE9 transgenics, novel specific antibodies, targeted proteomic/mass spectrometry approaches in mouse CSF, and in vitro cell culture paradigms, the project will provide a better understanding of brain Aß catabolism and clearance in health and disease.

Impaired brain clearance and abnormal A? degradation are considered key events for the formation and progressive accumulation of soluble neurotoxic oligomers and the development synaptic pathology, one of the strongest correlates to cognitive impairment. Clearance studies mostly centered in monomeric A?40 have provided a basic assessment of the participating mechanisms but the complex molecular and structural heterogeneity of the brain A? peptidome has been largely overlooked. The most obvious heterogeneity resides in the multiple N- and C-terminal truncated fragments that populate the A? peptidome; surprisingly, little is known about their homeostasis or their potential contribution to disease pathogenesis. Our studies indicate that while C-terminal truncations increase solubility and abrogate oligomerization/fibrillogenesis, properties associated with clearance mechanisms, N-terminal truncations, particularly A?4-x, exhibit enhanced pro-amyloidogenic and neurotoxic characteristics in vitro and in vivo, being particularly abundant in parenchymal deposits in AD and multiple APP Tg models and requiring detergents/formic acid for tissue extraction. Proteomic studies of tissue deposits and novel antibodies specific for the free N-terminus of A?4-x showed their topographic association with fibrillar plaque cores. Notably, these antibodies prevented A?4-x oligomerization, abrogated toxicity, and rescued behavioral deficits in a passive immunization pilot study. We hypothesize that N-terminal truncation of A? at position 4 generates pro-amyloidogenic fragments that progressively accumulate in brain deposits as a result of their high oligomerization propensity, poor brain removal, and resistance to proteolytic degradation, perpetuating the amyloidogenic loop, and postulate that passive immunization constitute a feasible therapeutic approach to reduce formation of A?4-x toxic oligomers and fibrillar deposits, restoring synaptic function and improving cognitive parameters. We propose to compare A?4-x clearance mechanisms with those of full-length A?40/A?42 in WT mice, assessing involvement of A? efflux transporters LRP-1 and P-gp and how brain efflux is influenced by oligomerization, local proteolytic degradation, and aging (Aim 1). The temporal accruement of A?4-x species as the disease establishes and progresses will be evaluated by proteomic analysis of the brain A? peptidome in APPswe/PS1?E9 and 3xTg of different age as well as in human specimens from MCI, mild, and severe AD. Intracerebral inoculation of AD-isolated A?4-x species will be used to evaluate seeding capabilities in young Tgs (Aim 2). Preventive and therapeutic benefits of MAb anti-A?4-x will be tested in passive immunization protocols in the same Tg models, assessing changes in behavioral parameters, biochemical composition of the brain A? peptidome, type and distribution of parenchymal A? lesions and ratio A?4-x to A?40/42. Cognitive changes will be correlated with the preservation/rescue of synaptic markers, electrophysiological parameters, and metabolic / bioenergetic features in isolated synaptosomes, all elements directly linked to synaptic function (Aim 3)

The etiology of Alzheimer's disease (AD) is complex and multifactorial. Multiple risk factors through still not well understood molecular mechanisms are known to influence the individual susceptibility for sporadic AD. One of the often-overlooked contributors to AD pathophysiology is A? accumulation in the cerebrovasculature (CAA), present in >90% of AD cases. CAA imposes restriction in cerebral blood flow resulting in ischemic white matter lesions, microhemorrhages, enhanced neuroinflammation, and synaptic dysfunction. Synaptic damage correlates with loss of cognitive function, is an early event in AD pathogenesis, and worsens with disease progression. Oligomeric A? (oligA?) has emerged as the species capable to selectively disrupt synaptic transmission, triggering cascades of events that primarily affect mitochondrial function, disrupting ATP production, inducing caspase-3 activation, and affecting levels and distribution of synaptic components. Our own preliminary data in APP Tg lines demonstrate profound changes in pre-/post-synaptic markers, low ATP levels and reduced mitochondrial activity in isolated synaptosomes. Highlighting the relevance of interlinked metabolic pathways, we show that hypoxic conditions drastically potentiate the detrimental effects of oligA?, exacerbating ROS production and inducing comparable toxicity by 500-fold lower A? doses than those required under normoxia. The affected mechanisms are in part related to the protective redox sensor Nrf2 ? downregulated in AD ? as small-molecule Nrf2 activators rescue the in vitro phenotype under normoxic conditions. Notably, hypoxia triggers activation of the oxygen sensitive HIF-1? pathway via upregulation of Siah2, a hypoxia-inducible molecule also capable of downregulating Nrf2. We hypothesize that progressive brain hypoperfusion as a result of CAA precipitate A?-induced mitochondrial dysfunction via dysregulation of the Nrf2?HIF-1? oxidative stress/hypoxia protective response resulting in increased synaptic alterations, neuroinflammation, and vascular susceptibility to microhemorrhages, events we postulate are amenable for translational interventions. We propose in vitro studies to identify the protective mechanisms exerted by small molecule Nrf2 activators under conditions mimicking hypoperfusion, assessing changes in global bioenergetics, functional impact in cell-specific biological parameters, and regulatory shifts in Nrf2?HIF-1? paths modulated by the hypoxia-sensor Siah2. Data will be validated in vivo in Tg models with progressive A? CAA using 1HMRS, conventional MRI, behavioral assessments, and LTP measurements, complemented by biochemical dissection of functional components of the mitochondrial machinery and Nrf2?HIF-1 paths, and their impact on synaptic changes and bioenergetics in isolated microvessels and synaptic mitochondria. Induction of hyperhomocysteinemia through a diet that results in cerebrovascular abnormalities, reduced oxygen delivery and cognitive deficits in non-Tg mice while enhancing CAA by relocation of A? deposits in Tg mice, will provide a complementary model to study vascular contribution to synaptic dysfunction independently or synergistically to the presence of CAA.