To examine the diversity of astrocytes in the human brain, we immunostained surgical specimens of temporal cortex and hippocampus and autopsy brains for CD44, a plasma membrane protein and ...extracellular matrix receptor. CD44 antibodies outline the details of astrocyte morphology to a degree not possible with glial fibrillary acidic protein (GFAP) antibodies. CD44+ astrocytes could be subdivided into two groups. First, CD44+ astrocytes with long processes were consistently found in the subpial area ("interlaminar" astrocytes), the deep isocortical layers, and the hippocampus. Many of these processes ended on blood vessels. Some were also found adjacent to large blood vessels, from which they extended long processes. We observed these CD44+, long-process astrocytes in every brain we examined, from fetal to adult. These astrocytes generally displayed high immunostaining for GFAP, S100β, and CD44, but low immunostaining for glutamine synthetase, excitatory amino-acid transporter 1 (EAAT1), and EAAT2. Aquaporin 4 (AQP4) appeared distributed all over the cell bodies and processes of the CD44+ astrocytes, while, in contrast, AQP4 localized to perivascular end feet in the CD44- protoplasmic astrocytes. Second, there were CD44+ astrocytes without long processes in the cortex. These were not present during gestation or at birth, and in adult brains varied substantially in number, shape, and immunohistochemical phenotype. Many of these displayed a "mixed" morphological and immunocytochemical phenotype between protoplasmic and fibrous astrocytes. We conclude that the diversity of astrocyte populations in the isocortex and archicortex in the human brain reflects both intrinsic and acquired phenotypes, the latter perhaps representing a shift from CD44- "protoplasmic" to CD44+ "fibrous"-like astrocytes.
Alexander Disease (AxD) is a primary disorder of astrocytes, caused by heterozygous mutations in GFAP, which encodes the major astrocyte intermediate filament protein, glial fibrillary acidic protein ...(GFAP). Astrocytes in AxD display hypertrophy, massive increases in GFAP, and the accumulation of Rosenthal fibers, cytoplasmic protein inclusions containing GFAP, and small heat shock proteins. To study the effects of GFAP mutations on astrocyte morphology and physiology, we have examined hippocampal astrocytes in three mouse models of AxD, a transgenic line (GFAP(Tg)) in which the normal human GFAP is expressed in several copies, a knock-in line (Gfap(+/R236H)) in which one of the Gfap genes bears an R236H mutation, and a mouse derived from the mating of these two lines (GFAP(Tg); Gfap(+/R236H)). We report changes in astrocyte phenotype in all lines, with the most severe in the GFAP(Tg);Gfap(+/R236H), resulting in the conversion of protoplasmic astrocytes to cells that have lost their bushy-like morphology because of a reduction of distal fine processes, and become multinucleated and hypertrophic. Astrocytes activate the mTOR cascade, acquire CD44, and lose GLT-1. The altered astrocytes display a microheterogeneity in phenotypes, even neighboring cells. Astrocytes also show diminished glutamate transporter current, are significantly depolarized, and not coupled to adjacent astrocytes. Thus, the accumulation of GFAP in the AxD mouse astrocytes initiates a conversion of normal, protoplasmic astrocytes to astrocytes that display severely "reactive" characteristics, many of which may be detrimental to neighboring neurons and oligodendrocytes.
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. 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. (Abstract shortened by UMI.)
CAMTA, the Canadian Association of Medical Teams Abroad, is a group of medical and lay individuals dedicated to helping underprivileged people suffering from orthopedic problems in Ecuador. The ...informal group conducted two missions prior to officially incorporating in 2001 and has, since its official founding, conducted 3 more missions in Ecuador. During their surgical missions the CAMTA team also aims to share information and provide teaching to local nurses and physicians.
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.
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