Anne B. Johnson

Mitochondrial Abnormalities in Alzheimer's Disease

Mitochondria from persons with Alzheimers disease (AD) differ from those of age-matched control subjects. Differences in mitochondrial morphology and function are well documented, and are not brain-limited. Some of these differences are present during all stages of AD, and are even seen in individuals who are without AD symptoms and signs but who have an increased risk of developing AD. This chapter considers the status of mitochondria in AD subjects, the potential basis for AD subject mitochondrial perturbations, and the implications of these perturbations. Data from multiple lines of investigation, including epidemiologic, biochemical, molecular, and cytoplasmic hybrid studies, are reviewed. The possibility that mitochondria could potentially constitute a reasonable AD therapeutic target is discussed, as are several potential mitochondrial medicine treatment strategies.

Peroxisomal and mitochondrial defects in the cerebro-hepato-renal syndrome.

The cerebro-hepato-renal syndrome is a rare familial malady with cerebral, renal, and skeletal abnormalities, severe hypotonia, cirrhosis, iron and lipid storage, and death within 6 months. Correlated electron microscopic, histochemical, and biochemical studies demonstrate defects in two oxidative organelles. Peroxisomes cannot be found in hepatocytes and renal proximal tubules. In hepatocytes and cortical astrocytes, mitochondria are distorted in their appearance and glycogen stores are increased. Oxygen consumnption of brain and liver mitochondrial preparations with succinate and with substrates reducing nicotinamide adenine dinucleotide is markedly diminished, but the consumption is normal with ascorbate and tetramethylphenylenediamine, which suggests a defect in electron transport prior to the cytochromes. Histochemical studies of mitochondrial oxidation point to a defect between the succinate dehydrogenase flavoprotein and coenzyme Q, possibly in the region of nonheme iron protein.

Mutations in GFAP, encoding glial fibrillary acidic protein, are associated with Alexander disease.

Alexander disease is a rare disorder of the central nervous system of unknown etiology. Infants with Alexander disease develop a leukoencephalopathy with macrocephaly, seizures and psychomotor retardation, leading to death usually within the first decade; patients with juvenile or adult forms typically experience ataxia, bulbar signs and spasticity, and a more slowly progressive course. The pathological hallmark of all forms of Alexander disease is the presence of Rosenthal fibers, cytoplasmic inclusions in astrocytes that contain the intermediate filament protein GFAP in association with small heat-shock proteins. We previously found that overexpression of human GFAP in astrocytes of transgenic mice is fatal and accompanied by the presence of inclusion bodies indistinguishable from human Rosenthal fibers. These results suggested that a primary alteration in GFAP may be responsible for Alexander disease. Sequence analysis of DNA samples from patients representing different Alexander disease phenotypes revealed that most cases are associated with non-conservative mutations in the coding region of GFAP. Alexander disease therefore represents the first example of a primary genetic disorder of astrocytes, one of the major cell types in the vertebrate CNS.

Microtubule Reduction in Alzheimer's Disease and Aging Is Independent of τ Filament Formation

Biochemical studies show that phosphorylated tau, like that found in paired helical filaments (PHFs), does not promote microtubule assembly leading to the view that PHF formation leads to microtubule deficiency in Alzheimers disease (AD). However, although this issue is one of the most important aspects to further understanding the cell biology of AD, no quantitative examination of microtubule diminution in AD and its relationship with PHFs has been performed. To examine this issue directly, we undertook a morphometric study of brain biopsy specimens from AD and control cases. Ultrastructural analysis of neurons was performed to compare the microtubule assembly state in neurons of diseased and control cases and to examine the effect of PHF accumulation. We found that both number and total length of microtubules were significantly and selectively reduced in pyramidal neurons from AD in comparison to control cases (P = 0.000004) but that this decrement in microtubule density was surprisingly unrelated to PHFs (P = 0.8). Further, we found a significant age-dependent decrease in microtubule density with aging in the control cases (P = 0.016). These findings suggest that reduction in microtubule assembly is not dependent on tau abnormalities of AD and aging.

Glial fibrillary acidic protein mutations in infantile, juvenile, and adult forms of Alexander disease

Alexander disease is a progressive, usually fatal neurological disorder defined by the widespread and abundant presence in astrocytes of protein aggregates called Rosenthal fibers. The disease most often occurs in infants younger than 2 years and has been labeled a leukodystrophy because of an accompanying severe myelin deficit in the frontal lobes. Later onset forms have also been recognized based on the presence of abundant Rosenthal fibers. In these cases, clinical signs and pathology can be quite different from the infantile form, raising the question whether they share the same underlying cause. Recently, we and others have found pathogenic, de novo missense mutations in the glial fibrillary acidic protein gene in most infantile patients examined and in a few later onset patients. To obtain further information about the role of glial fibrillary acidic protein mutations in Alexander disease, we analyzed 41 new patients and another 3 previously described clinically, including 18 later onset patients. Our results show that dominant missense glial fibrillary acidic protein mutations account for nearly all forms of this disorder. They also significantly expand the catalog of responsible mutations, verify the value of magnetic resonance imaging diagnosis, indicate an unexpected male predominance for the juvenile form, and provide insights into phenotype–genotype relations. Ann Neurol 2005;57:310–326

Studies on Brain Biopsies of Patients with Huntington's Chorea

Frontal cortex brain biopsies of four patients with Huntingtons chorea were studied by histological, histochemical, and electron microscopic methods. The changes observed were evaluated in regard to the age of the patients and stage of the disease process. One of the most outstanding features was the large and generalized accumulation of lipofuscin in neurons and glial cells. This increase in lipofusein was detectable in paraffin sections, but was most striking in frozen section examined for fluorescence and in acid-phosphatase preparations. Preliminary extractions of lipofusein demonstrated a higher content of the pigment in Huntingtons chorea than in age-matched control brains. The glial lipofusein was associated with very high acid-phosphatase activity, which was found to be localized in the dense component of the granule. At the fine structural level, neurons evidenced several abnormalities including proliferation of Golgi membranes and Golgi-associated vesicles, disorganization of the rough endoplasmic reticulum with shedding of membrane-bound ribosomes; abnormally dense mitochondria with sparse cristae, occasionally containing crystalline fibrillar arrays within the matrix; abnormal nuclei with chromatin condensed in numerous dense granules and with increased porosity of the nuclear membrane. Increased numbers of microglial cells in the neuropil and ear vessels, often containing remnants of neuronal cytoplasm with lipofuscin, suggest that active removal of irreversibly damaged neurons by phagocytosis is taking place. Astrocytes were proliferated and hypertrophic with a fair increase in glial filaments. They contained large quantities of lipopigment granules in their cell bodies and their processes. Neuronal death and axonal and synaptic degeneration account for the atrophy of the frontal cortex. The abnormalities found were present in all cases, and were of increasing severity with more prolonged disease.

Demyelination in vitro

Myelinated cultures of mouse spinal cord have been exposed to sera raised in rabbits against whole white matter (anti-WM), myelin basic protein (anti-MBP) and galactocerebroside (anti-GC), the major glycolipid of CNS myelin, to determine which factor in central nervous system (CNS) tissue in vitro is the target of serum demyelinating and myelin swelling antibodies. The sera were tested by radioimmunoassay for activity against MBP and against GC and were also specifically absorbed with MBP, GC and control antigens. Studies were also performed with and without active complement. The findings show that demyelination and myelin swelling in vitro are caused by antibodies against GC and not against MBP. Ultrastructurally, the effects of anti-WM and anti-GC sera with and without complement were indistinguishable. This study demonstrates that GC is a major target in antibody-mediated demyelination.

Unusual variants of Alexander's disease.

The purpose of this study was to describe unusual variants of Alexanders disease. We studied 10 patients who did not meet previously established magnetic resonance imaging (MRI) criteria for Alexanders disease, but for whom this diagnosis was considered because of Rosenthal fibers at histological examination or presence of some MRI features suggestive of Alexanders disease. Sequence analysis of the GFAP gene was performed. In eight patients, MRI results showed predominantly posterior fossa lesions, especially multiple tumor‐like brainstem lesions. One patient had asymmetrical frontal white matter abnormalities and basal ganglia abnormalities. One patient (Patient 10) developed degeneration of the frontal white matter. In nine patients, a mutation was found that was concluded to be pathogenic, because the mutation was de novo (five patients), a known mutation was found (two patients), or assembly of the glial fibrillary acidic protein was abnormal in cultured cells (two patients). In Patient 10, a DNA variation was found that was also present in the patients clinically unaffected father and was concluded to be a polymorphism. In conclusion, DNA diagnostics is warranted in patients who display MRI features suggestive of Alexanders disease, even if they do not meet the full set of previously established MRI criteria. Ann Neurol 2005;57:327–338

Purification, Characterization, and Localization of Aspartoacylase from Bovine Brain

Abstract: Canavan disease, an autosomal recessive disorder, is characterized biochemically by N‐acetylaspartic aciduria and aspartoacylase (N‐acyl‐L‐aspartate amidohydrolase; EC 3.5.1.15) deficiency. However, the role of aspartoacylase and N‐acetylaspartic acid in brain metabolism is unknown. Aspartoacylase has been purified to apparent homogeneity with a specific activity of ∼ 19,000–20,000 nmol of aspartate released/mg of protein. The native enzyme is a 58‐kDa monomer. The purified aspartoacylase activity is enhanced by divalent cations, nonionic detergents, and dithiothreitol. Low levels of dithiothreitol or β‐mercaptoethanol are required for enzyme stability. Aspartoacylase has a Km of 8.5 × 10−4M and a Vmax of 43,000 nmol/min/mg of protein. Inhibition of aspartoacylase by glycyl‐L‐aspartate and amino derivatives of D‐aspartic acid suggests that the carbon backbone of the substrate is primarily involved in its interaction with the active site and that a blocked amino group is essential for the catalytic activity of aspartoacylase. Biochemical and immunocytochemical studies revealed that aspartoacylase is localized to white matter, whereas the N‐acetylaspartic acid concentration is threefold higher in gray matter than in white matter. Our studies so far indicate that aspartoacylase is conserved across species during evolution and suggest a significant role for aspartoacylase and N‐acetylaspartic acid in normal brain biology.

Vesicular Disruption of Myelin in Autoimmune Demyelination

A pattern of autoimmune demyelination in EAE and EAN has been described which was encountered consistently and was sometimes more common than the better known phenomenon of active stripping of myelin by macrophages. This pattern involved the rapid dissolution of myelin into a vesicular network which was later degraded by macrophages. It occurred early in the disease, was not accentuated perivascularly, and was usually associated with the presence of macrophases. The underlying mechanisms are not known but several alternatives have been discussed, viz., activity of locally released antibody, cytotoxic factors, or hydrolytic enzymes.