Matthew Schrag, Ph.D. - US grants

SUMMARY Alzheimer?s disease is a burgeoning national epidemic and an effective treatment is urgently needed. It is now widely recognized that vascular disease contributes to Alzheimer?s disease and, in some cases, may be central to the process of neuronal degeneration. Cerebral amyloid angiopathy (CAA) is a vasculopathy produced when ?-amyloid forms a toxic encrustation on cerebral arterioles and capillaries; it independently contributes to cognitive impairment and predisposes elderly patients to intracerebral hemorrhage. CAA has emerged as a critical variable in the search for a treatment for AD, and is particularly important as mechanisms of cerebral ?- amyloid clearance are beginning to be better understood. Never-the-less, the most fundamental mechanisms of how ?-amyloid undermines the structural integrity of vessels remain unknown. We propose to study the microvascular network in AD and CAA by large-scale, three dimensional, microscopic imaging of optically cleared human tissue specimens. We hypothesize that this will show ?-amyloid deposits are closely linked to areas of vascular degeneration. Our main goals are 1) to produce a high-resolution three-dimensional model of the cerebral cortical microvascular network, including capturing changes associated with AD and CAA and 2) to determine whether ?-amyloid is present at sites of microhemorrhage and what morphological features are associated with vascular fragility. This work will answer fundamental questions about the link between vascular integrity and Alzheimer?s disease and provide a foundation for future work to better understand the molecular mechanisms of vascular fragility in CAA.

Alzheimer?s disease (AD) compromises the independence of aging adults and is associated with huge societal burden in healthcare and caregiver costs. An effective treatment is urgently needed to stem the tide of the ongoing and growing epidemic of dementia. Many recent clinical trials have attempted to reduce the level of ?- amyloid in the brain, typically by activating the immune system to promote clearance, yet none of these trials has been successful. We propose to explore this problem in a different way ? we discovered that neuronal axons around deposits of ?-amyloid are swollen and filled with abnormal lysosomes which are deficient in protein-degrading enzymes. Because lysosomes are critical for protein homeostasis, we hypothesize that these abnormal lysosomes contribute to neurodegeneration and if their function could be rescued, it could improve brain function. We will study this hypothesis by focusing on a novel gene, PLD3, which was identified as contributing to AD risk. In our preliminary work, we discovered that PLD3 is robustly enriched on these abnormal lysosomes and brain PLD3 levels correlated inversely with both ?-amyloid burden and the rate of cognitive deterioration in a human cohort. Brain PLD3 levels also correlated inversely with memory performance in a mouse model. We discovered that PLD3 functions as a phospholipase D in acidic environments and is necessary for lysosomal membrane fission. We propose to evaluate to what degree the lysosome dysfunction observed in mice is present in human tissue and to fully evaluate the association of PLD3 with neuronal lysosomal pathology. We will then determine whether the coding variants reported to confer AD-risk impact the function of PLD3 by transfecting plasmids containing PLD3 (which have been mutated to copy these coding variants) and observing the effect on lysosomal membrane fission and on PLD3 enzyme activity. We will determine whether defective neuronal lysosomal fission impacts cognition and AD neuropathology in vivo by crossing the 5xFAD model of genetic AD with a conditional knock-out of Fig4 using a Cre selectively expressed in cerebral neurons. Fig4 is a component of the PIKfyve complex and loss of Fig4 leads to defective lysosomal fission. Importantly, the PIKfyve complex also regulates PLD3 processing and alters PLD3 activity. We will evaluate learning and memory in this mouse and determine whether defective lysosomal fission impacts ?-amyloidosis and dystrophic neurites. These studies will help us understand both how this protein functions in the setting of AD and the role of lysosome membrane fusion in cognitive aging and AD. The parallel training plan will support my training in model animal development and behavioral phenotyping, developing advanced skills in lyosomal neurobiology and refine clinical management of aging related diseases, all of which are critical steps for my career development. Collectively, the outstanding institutional environment, resources, and interdisciplinary mentorship team with broad and complementary areas of expertise will accelerate my acquisition of the necessary expertise to transition to independence.

Abstract Alzheimer?s disease is a burgeoning national epidemic and an effective treatment is urgently needed. It is now widely recognized that vascular disease contributes to Alzheimer?s disease and, in some cases, may be central to the process of neuronal degeneration. Cerebral amyloid angiopathy (CAA) is a vasculopathy produced when ?-amyloid forms a toxic encrustation on cerebral arterioles and capillaries; it independently contributes to cognitive impairment and predisposes elderly patients to intracerebral hemorrhage. CAA has emerged as a critical variable in the search for a treatment for Alzheimer?s disease, and is particularly important as mechanisms of cerebral ?-amyloid clearance are beginning to be better understood. Never-the-less, fundamental mechanisms of whether and how ?-amyloid undermines the structural integrity of vessels remain unknown. We propose to optimize and study a new mouse model of CAA induced by surgically implanting purified ?-amyloid seeds isolated from human cerebral microvessels with CAA into the cerebral ventricles of 5xFAD mice. Previous animal models of CAA are inadequate due to slow development of ?-amyloid pathology and minimal development of microvascular degeneration, whereas the current model develops robust CAA within 3 months of the injection along with other microvascular degeneration features. Our main goals are 1) to optimize this model and 2) use it to definitively assess for a link between vascular ?-amyloid deposition and microvascular degeneration. We will evaluate for microvascular degeneration using advanced microscopy techniques, including 3-dimensional mapping of the microvascular network using CLARITY and assessment of the stiffness of the extracellular matrix in arterioles affected by CAA using atomic force microscopy. We will also assess functional, neurobehavioral outcomes in this new model system. This work will answer fundamental questions about the link between vascular integrity and Alzheimer?s disease and provide a foundation for future work to better understand the molecular mechanisms of vascular fragility in CAA.