Edward H. Koo

Differential expression of amyloid precursor protein mRNAs in cases of Alzheimer's disease and in aged nonhuman primates

Senile plaques are a characteristic feature in brains of individuals with Alzheimers disease (AD) and aged monkeys. The principal component of amyloid in senile plaques is beta/A4, a peptide derived from a larger amyloid precursor protein (APP). To date, several alternatively spliced APP transcripts have been described. The relationship between levels of these APP mRNAs and amyloid deposition is unclear. In this study, we directly measured the relative levels of APP transcripts that lack the protease inhibitor domain (APP-695) and transcripts that encode the inhibitor sequences (APP-751/770). Our results indicate that the expression of APP mRNAs is not selectively altered in AD cortex. Moreover, the differential expression of APP transcripts is not correlated with the deposition of amyloid in cases of AD and aged monkeys. These findings suggest that other factors, not directly related to the relative expression of APP mRNAs, may contribute to amyloidogenesis in the brain.

Aluminum neurotoxicity altered expression of cytoskeletal genes

To better understand perturbations of the neuronal cytoskeleton that occur in several mammalian disorders, we have focused on an animal model in which neurofibrillary pathology follows the administration of aluminum salts. In susceptible species, the injection of aluminum produces accumulations of neurofilaments (NFs) in cell bodies and proximal axons of certain populations of neurons. Mechanisms involved in the production of these abnormalities are unclear; in particular, the role of gene expression in the genesis of this type of neurofibrillary pathology has not been examined. In this study of aluminum-intoxicated rabbits, the expression of genes coding for several cytoskeletal proteins was studied in the spinal cord and dorsal root ganglia (DRG)--tissues with and without neurofibrillary pathology, respectively. In aluminum-treated rabbits, in situ hybridization using a cDNA probe demonstrated the presence of mRNA coding for the 68-kDa NF (NF-L) protein in spinal cord motor neurons with NF accumulations as well in unaffected neurons. On Northern blots, the expression of genes coding for the NF-L protein and tubulin was reduced by approximately 3.5-fold and 3-fold, respectively, in spinal cords of aluminum-intoxicated rabbits as compared to controls. On blots, levels of actin mRNA were not significantly different in spinal cords of aluminum-treated rabbits as compared to controls, but there was a trend for a slight reduction. In DRG of intoxicated animals, the expression of genes coding for these cytoskeletal proteins was not altered.

Alzheimer β/A4-Amyloid Precursor Protein: Evidence for Putative Amyloidogenic Fragment

Abstract: Recombinant baculovirus was used to overexpress human Alzheimer β/A4‐amyloid precursor protein (APP) in Spodoptera frugiperda (Sf9) cells. Lysates of these cells were then analyzed for the presence of carboxyl‐terminal fragments of APP by an immuno‐blotting assay using either an antibody against the APP cytoplasmic domain (rabbit anti‐human 695APP645–694 or an antibody against the amino terminus of β/A4‐amyloid (rabbit anti‐human 695APP586–606). Anti‐human 695APP645–694 identified APP holoprotein, a 25‐kDa species, and a prominent group of carboxyl‐terminal fragments of 17, 16, and 14 kDa, whereas anti‐human 695APP586–606 identified APP holoprotein and a single prominent low‐molecular‐mass protein species comigrating with the 17‐kDa carboxyl‐terminal fragment identified by anti‐human 695APP645–694. No immunoreactive species was detected at these molecular mass positions when either antibody was used for analysis of lysates of either uninfected Sf 9 cells or Sf 9 cells infected with wild‐type Autographa californica baculovirus. For each antibody, specific immunoreactivity was abolished by preabsorption with the corresponding peptide immunogen. The incorporation of a β/A4‐amyloid amino‐terminal epitope into a 17‐kDa fragment of APP suggests that, in the baculoviral overexpression system, the electrophoretic microheterogeneity of APP carboxyl‐terminal fragments is due, at least in part, to alternative proteolysis of APP. If such carboxyl‐terminal fragments of APP containing an intact β/A4‐amyloid domain are produced in human brain, then they may represent intermediates in the conversion of APP to deposited β/A4‐amyloid. The identification of potentially amyloidogenic fragments in recom‐binantly engineered Sf 9 cells may provide a useful experimental system for determination of alternative sites of APP proteolysis and investigation of the processing mechanisms involved.

Developmental expression of α1-antichymotrypsin in brain may be related to astrogliosis

Abstract In the brains of individuals with Alzheimers disease (AD) and aged monkeys, the serine protease inhibitor α 1 -antichymotrypsin (ACT) is selectively associated with deposits of amyloid found in senile plaques and in the walls of blood vessels. The origin of ACT in the brains of these aged subjects is unclear. In this study, ribonucleic acid (RNA) blots of human brains show that ACT messenger RNA (mRNA) increases during development. Levels of mRNA were negligible in fetuses and young adults but were increased slightly in normal aged individuals and highest in individuals with AD. In situ hybridization detected ACT transcripts in astrocytes of the cortex, subependymal region, and superficial white matter. The expression of ACT mRNA was highest in subjects with AD, in an adult with Downs syndrome, in an individual with Picks disease, and in cases of Huntingtons disease. In the brains of adult monkeys, ACT expression was detected primarily in astrocytes of the subependyma and white matter. Thus the presence of ACT appears to be related to the response of astrocytes to the brain abnormalities seen in these conditions.

Levels of neurotransmitter and cytoskeletal protein mRNAs during nerve regeneration in sympathetic ganglia

The present study examines levels of neurotransmitter messenger RNA (mRNA) at various stages after crush of postganglionic nerves in the superior cervical ganglia. Using complementary DNA (cDNA) probes, we demonstrated a reduction in ganglionic mRNA levels for tyrosine hydroxylase (TH) after axotomy. Concomitantly, actin and tubulin mRNA levels in ganglia were increased. Thus, in neurons of sympathetic ganglia, axotomy appears to be associated with a selective reduction in levels of TH mRNA, and, in turn, alters levels of protein and enzyme activities.

β-Amyloid Protein is Higher in Alzheimer's Disease Brains: Description of a Quantitative Biochemical Assay

Deposition of β-amyloid protein (Aβ) in senile plaques and in the walls of cerebral vessels is a pathologic hallmark of Alzheimers disease (AD). The current diagnostic criteria for AD requires the presence of neurofibrillary tangles and a minimum number of senile plaques in cortex. Senile plaques are readily visualized by silver staining or immunocytochemistry using antibodies raised to Aβ. Available histochemical and immunocytochemical methods are sensitive but the results may occasionally be variable and sampling from many brain regions is difficult and impractical. This study describes a simple biochemical method for quantifying the Aβ load in unfixed brain homogenates. The immunoassay recognizes all forms of Aβ deposits (neuritic and diffuse plaques, and cerebrovascular amyloid) and has a sensitivity and specificity comparable to immunocytochemistry. In direct comparisons, results from the dot blot method correspond well with both Western blot analysis of partially purified Aβand plaque counting by immunocytochemistry. In a retrospective series of 39 postmortem AD and control cases, the amount of Aβ in brain by dot blot immunoreactivity effectively separated the two groups. Therefore, this method provides a rapid, sensitive, and accurate quantitation of Aβ in postmortem brain tissue and represents an alternative approach for studying Aβ deposition in aging and AD.

β-Amyloid gene is not present in three copies in autopsy-validated Alzheimer's disease

Recently, it has been suggested that Alzheimers disease is associated with a duplication of the amyloid precursor protein gene localized to chromosome 21q21. In this study, a cloned DNA probe (B2.3), complementary to the sequence coding the beta-amyloid peptide, and DNA polymorphisms adjacent to this sequence were used to determine the number of copies of the beta-amyloid gene in DNA isolated from human blood and brain. Individuals with trisomy 21 (Down syndrome) who were heterozygous for the polymorphisms showed a gene-dosage effect, with one allele exhibiting twice the autoradiographic intensity as the other. Heterozygous individuals with Alzheimers disease and controls showed equal intensities of the two allelic bands, suggesting that there are only two copies of the beta-amyloid gene in these individuals. In individuals with Alzheimers disease and in controls who were homozygous for these polymorphisms, the number of copies of the beta-amyloid gene was determined by comparing the autoradiographic intensity of beta-amyloid alleles to that of DNA fragments detected by a reference probe. No difference was detected between these two groups.

AMYLOID-RELATED PROTEINS AND NERVE GROWTH FACTOR IN ALZHEIMER'S DISEASE AND ANIMAL MODELS

Summary: Alzheimers disease (AD), the most common cause of dementia in adult life, is characterized by the deposition of amyloid in brain parenchyma and the degeneration of specific populations of nerve cells, including cholinergic neurons in the basal forebrain. In this review, we first outline studies of cellular and molecular events that lead to age‐associated deposition of amyloid in the brains of nonhuman primates and then describe investigations of the effect of treatment with nerve growth factor (NGF) on experimentally induced abnormalities in cholinergic neurons of the basal forebrain. These studies of amyloidogenesis and the efficacy of trophic factors on specific groups of experimentally damaged neurons provide information about issues central to understanding the pathogenesis and treatment of human degenerative diseases, including AD.

Neuronal responses to injury and aging: lessons from animal models.

Alzheimers disease (AD), the most common type of adult-onset dementia, is characterized by a variety of brain abnormalities, including degeneration of certain populations of nerve cells, alterations in the neuronal cytoskeleton, and the abnormal deposition of amyloid within brain parenchyma. Pathogenetic processes that lead to these brain abnormalities are difficult to study in humans. Recently, investigators have begun to utilize animal models to examine some of the mechanisms that cause cellular/molecular alterations in transmitter systems, cytoskeletal elements, and APP. These investigations have helped to clarify issues related to the lesions that occur in aged humans and individuals with AD.

Neuronal disorders: Studies of animal models and human diseases

The peripheral nervous system and the central nervous system (CNS) are comprised of assemblies of neurons that communicate via electrical and chemical signals. Different disease processes selectively affect specific populations of neurons and/or specific cell functions (i.e., “selective vulnerability” of neurons is a principal determinant of phenotypes of disease). New cellular and molecular biological approaches have begun to clarify some of the mechanisms of selective cell injury in human diseases and their animal models. Following a brief review of the normal biology of nerve cells, we use illustrations drawn from studies of experimental and human diseases to discuss the mechanisms of structural/chemical abnormalities that occur in a variety of neuronal disorders.