Stressed Astrocytes: Insights on the Pathology of Alexander Disease Eileen Guilfoyle Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Graduate School of Arts and Sciences COLUMBIA UNIVERSITY 2013 © 2013 Eileen Guilfoyle All rights reserved ABSTRACT Stressed Astrocytes: Insights on the Pathology of Alexander Disease Eileen Guilfoyle Alexander disease (AxD) is a rare and fatal neurological disorder caused by mutations in the gene that encodes glial fibrillary acidic protein (GFAP), an intermediate filament protein found in astrocytes in the central nervous system. The clinical presentations of AxD are diverse, ranging from onset in infancy to onset in early adulthood, and include seizures, psychomotor retardation, ataxia, and a variety of neurological signs related to abnormal brain stem function. The defining neuropathological hallmark is the presence of cytoplasmic, proteinaceous inclusions called Rosenthal fibers in astrocytes. Although GFAP expression is astrocytic, AxD patients also show de/dysmyelination and variable amounts of neuronal loss, most severely in infantile-onset patients. Astrocytes undergo severe morphological changes, beyond that of typical reactive astrocytes, and develop several forms of cell stress. However, how stressed astrocytes cause the loss of myelin in this disease is unknown. In this work I have conducted a largely immunohistological investigation of AxD patient tissue, model mice, and primary astrocytes cultured from the AxD model mice, focusing on factors that might provide insight into the pathological manifestations of AxD and paying particular attention to those factors which might contribute to de/dysmyelination. To gain insight on the morphological transformation of astrocytes in AxD, I analyzed GFAP in the hippocampus of the most severely affected AxD mouse. Astrocytes in these mice lose their star-like shape, and become hypertrophic and often multinucleated. They accumulate large amounts of GFAP. Subsequent study of primary cultured astrocytes from AxD mice revealed that these cells have perinuclear inclusions of GFAP surrounded by displaced microtubules and displaced Golgi. I next investigated another mechanism of stress that may affect astrocyte function in AxD. Work in our lab and others’ has demonstrated proteasomal inhibition in AxD astrocytes. Because the unfolded protein response in the endoplasmic reticulum (ER) can be enacted by proteasomal inhibition, I examined the immunohistochemical expression of two proteins commonly increased under conditions of ER stress. We found BIP/Grp78, an ER chaperone, increased in AxD patient astrocytes and model mice. Additionally, the CCAAT enhancer binding protein homologous protein (CHOP) was expressed by a small subset of astrocytes in the AxD mouse hippocampus, unveiling ER stress as a potential contributory factor in AxD pathology. Work in other labs has found iron in astrocytes in AxD model mice. To further elucidate mechanisms of cellular stress in AxD, I conducted an immunohistochemical analysis of iron and several regulatory proteins in AxD patients and found, by enhanced Perls’ staining, Fe3+ in Rosenthal fibers and iron and ferritin accumulated in astrocytes. This finding is in marked contrast to what one sees in the normal CNS, with little staining of astrocytes, and easily detectable staining of oligodendrocytes. Finally, I examined the localization of the cell surface glycoprotein CD44, along with several related proteins, including its ligand hyaluronan. I found CD44 protein expression greatly increased in the white matter, cortex and hippocampus of AxD patients and in the hippocampus of AxD mice. Additionally, through use of a biotinylated hyaluronan binding protein, I found abnormally high levels of hyaluronan in the hippocampus of AxD mice in the same areas where increases in CD44 were found. Work elsewhere has found CD44 and hyaluronan in other disorders that affect myelination, and experiments have revealed an inhibitory effect of hyaluronan on oligodendrocyte development and myelination. The studies in this thesis contribute novel stressors to the list of those that impact astrocytes in AxD and, in particular, suggest the accumulation of iron in astrocytes as potentially important to the pathological manifestations of AxD. Additionally, my research has revealed dramatic increases in the expression of CD44 in AxD astrocytes which, in conjunction with widespread increases in hyaluronan, may be critical to understanding the mechanisms underlying the de/dysmyelination that occur in this disease. Table of Contents Chapter 1 Introduction ................................................................................................................................. 1 Alexander Disease: a brief history .................................................................................................. 2 Rosenthal fibers ............................................................................................................................... 3 GFAP protein and GFAP mutation ................................................................................................... 5 AxD and other leukodystrophies ................................................................................................... 12 Age of onset: an important indicator of AxD severity ................................................................... 14 Classifications of AxD – type I and type II ...................................................................................... 15 AxD mouse models ........................................................................................................................ 16 AxD and cellular stress ................................................................................................................... 17 Reactive astrocytes ........................................................................................................................ 21 Thesis overview .............................................................................................................................. 23 Tissue used in these studies .......................................................................................................... 24 Chapter 1: Figures .......................................................................................................................... 26 Chapter 2 Alexander disease astrocytes: morphologically and ER stressed .............................................. 33 Introduction ................................................................................................................................... 34 Results ............................................................................................................................................ 37 GFAP is increased in AxD Tg/KI mice at 2 and 4 weeks .................................................... 37 Severe morphological changes are seen in astrocytes in the str. lac. of 4 week old Tg/KI mice ................................................................................................................................... 38 GFAP and Alpha Tubulin are found together in perinuclear inclusions ............................ 39 Golgi is mislocalized in Tg primary astrocytes with perinuclear inclusions ...................... 39 AxD patients show accumulated BIP expression in white matter astrocytes .................. 40 BIP is significantly elevated at 2 weeks and 4 weeks in AxD Tg/KI mice .......................... 41 Expression of BIP in astrocytes of one year old Tg Mouse ............................................... 41 CHOP is co-expressed with BIP in a subset of astrocytes ................................................. 42 Discussion ...................................................................................................................................... 42 High levels of GFAP expression in AxD mice coincide with morphological transformation of astrocytes ..................................................................................................................... 43 Binucleation of astrocytes ................................................................................................ 44 Perinuclear rings of GFAP and microtubules in primary Tg astrocytes ............................ 45 Aberrant expression of BIP in ER of AxD Patients ............................................................. 46 ER stress is an early and widespread response in Tg/KI animals ...................................... 46 ER stress and KI mouse mortality after Kainic Acid induced stress .................................. 47 BIP in 1 year Tg animal suggests unresolved ER stress ..................................................... 48 i Possible causes of UPR in In AxD 49 BIP loss in patient oligodendrocytes may signal impaired myelination 50 CHOP expression may not be pro-apoptotic .................................................................... 51 Chapter 2: Figures ............................................................................................................ 52 Chapter 3 Characterization of CD44, hyaluronan and Ezrin Radixin Moesin proteins in Alexander disease .................................................................................................................................................................... 72 Introduction 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