Yu-Min Kuo

Soluble Amyloid β Peptide Concentration as a Predictor of Synaptic Change in Alzheimer’s Disease

We have characterized amyloid β peptide (Aβ. concentration, Aβ deposition, paired helical filament formation, cerebrovascular amyloid angiopathy, apolipoprotein E (ApoE) allotype, and synaptophysin concentration in entorhinal cortex and superior frontal gyrus of normal elderly control (ND) patients, Alzheimers disease (AD. patients, and high pathology control (HPC) patients who meet pathological criteria for AD but show no synapse loss or overt antemortem symptoms of dementia. The measures of Aβ deposition, Aβ-immunoreactive plaques with and without cores, thioflavin histofluorescent plaques, and concentrations of insoluble Aβ, failed to distinguish HPC from AD patients and were poor correlates of synaptic change. By contrast, concentrations of soluble Aβ clearly distinguished HPC from AD patients and were a strong inverse correlate of synapse loss. Further investigation revealed that Aβ40, whether in soluble or insoluble form, was a particularly useful measure for classifying ND, HPC, and AD patients compared with Aβ42. Aβ40 is known to be elevated in cerebrovascular amyloid deposits, and Aβ40 (but not Aβ42) levels, cerebrovascular amyloid angiopathy, and ApoE4 allele frequency were all highly correlated with each other. Although paired helical filaments in the form of neurofibrillary tangles or a penumbra of neurites surrounding amyloid cores also distinguished HPC from AD patients, they were less robust predictors of synapse change compared with soluble Aβ, particularly soluble Aβ40. Previous experiments attempting to relate Aβ deposition to the neurodegeneration that underlies AD dementia may have failed because they assayed the classical, visible forms of the molecule, insoluble neuropil plaques, rather than the soluble, unseen forms of the molecule.

Morphology and Toxicity of Aβ-(1-42) Dimer Derived from Neuritic and Vascular Amyloid Deposits of Alzheimer's Disease

In the course of analyzing the chemical composition of Alzheimers disease neuritic and vascular amyloid, we have purified stable dimeric and trimeric components of Aβ peptides. These peptides (molecular mass 9.0 and 13.5 kDa) were separated by size exclusion chromatography in the presence of 80% formic acid or 5 M guanidine thiocyanate, pH 7.4. The average ratio of monomers, dimers, and trimers was 55:30:15, respectively. Similar structures were produced over time upon incubation of synthetic Aβ-(1-42) at pH 7.4. The stability of these oligomeric forms was also demonstrated by Western blot and mass spectrometry. Atomic force microscopy and electron microscopy rotary shadowing revealed that the monomers polymerized into 8-10-nm filaments, whereas the dimers generated prolate ellipsoids measuring 3-4 nm in diameter. The pathogenic effects of the dimeric Aβ-(1-40/42) were tested in cultures of rat hippocampal neuron glia cells. Only in the presence of microglia did the dimer elicit neuronal killing. It is possible that these potentially pathogenic Aβ-(1-40/42) dimers and trimers from Alzheimers disease amyloid represent the soluble oligomers of Aβ recently described in Alzheimers disease brains (Kuo, Y.-M., Emmerling, M. R., Vigo-Pelfrey, C., Kasunic, T. C., Kirkpatrick, J. B., Murdoch, G. H., Ball, M. J., and Roher, A. E. (1996) J. Biol. Chem., 271, 4077-4081).

Amyloid beta peptides in human plasma and tissues and their significance for Alzheimer's disease

We evaluated the amounts of amyloid beta (Aβ)) peptides in the central nervous system (CNS) and in reservoirs outside the CNS and their potential impact on Aβ plasma levels and Alzheimers disease (AD) pathology.

Alterations of Alzheimer's disease in the cholesterol-fed rabbit, including vascular inflammation. Preliminary observations.

Abstract: We determined the levels of endothelial inflammation using MECA‐32 antibody and a4 nicotinic receptor subunit densities employing [3H]epibatidine binding in the brains of Alzheimers disease (AD) patients, cholesterol‐fed rabbits, and appropriate controls. We also assessed rabbit brain for β‐amyloid levels and immunohistochemical localization, and for evidence of blood‐brain barrier breach using normally‐excluded Evans Blue dye. Dietary cholesterol induced a twofold increase in β‐amyloid concentration in rabbit hippocampal cortex, which may be related to the appearance of β‐amyloid immunoreactivity in the neuropil. Epibatidine binding was significantly decreased in AD superior frontal cortex, but unchanged in the superior frontal cortex of cholesterol‐fed rabbits. Increased vascular MECA‐32 immunoreactivity occurred in AD and cholesterol‐fed rabbit brain. Evans Blue dye could be found in the parenchyma of cholesterol‐fed rabbits only, and appeared as pockets of dye surrounding small blood vessels. The data suggest that vascular inflammation can lead to breach of the blood‐brain barrier, which may produce biochemical derangements in surrounding brain tissue that are conducive to production of β‐amyloid.

APP Transgenic Mice Tg2576 Accumulate Aβ Peptides That Are Distinct from the Chemically Modified and Insoluble Peptides Deposited in Alzheimer's Disease Senile Plaques†

The amyloid (Abeta) peptides generated in Hsiaos APP Tg2576 transgenic (Tg) mice are physically and chemically distinct from those characteristic of Alzheimers disease (AD). Transgenic mouse Abeta peptides were purified using sequential size-exclusion and reverse-phase chromatographic systems and subjected to amino acid sequencing and mass spectrometry analyses. The mouse Abeta peptides lacked the extensive N-terminal degradations, posttranslational modifications, and cross-linkages abundant in the stable Abeta peptide deposits observed in AD. Truncated Abeta molecules appear to be generated in vivo by hydrolysis at multiple sites rather than by post-mortem C-terminal degradation. In contrast to AD amyloid cores, the Tg mice peptides were soluble in Tris-SDS-EDTA solutions, revealing both monomeric and SDS-stable oligomeric species of Abeta. In contrast to our report on Novartis Pharma APP23 Tg mice [Kuo et al. (2001) J. Biol. Chem. 276, 12991], which maintain high levels of soluble Abeta early on with later development of extensive vascular amyloid, Tg2576 mice exhibited an age-related elevation of soluble Abeta with relatively limited vascular amyloid deposition. The transgenic mouse levels of carboxy-terminal (CT) APP fragments were nearly 10-fold greater than those of human brains, and this condition may contribute to the unique pathology observed in these animals. Immunization of transgenic mice may act to prevent the pathological effects of betaAPP overproduction by binding CT molecules or halting their processing to toxic forms, in addition to having any effects on Abeta itself. Thus, differences in disease evolution and biochemistry must be considered when using transgenic animals to evaluate drugs or therapeutic interventions intended to reduce the Abeta burden in Alzheimers disease.

Exercise enhances the proliferation of neural stem cells and neurite growth and survival of neuronal progenitor cells in dentate gyrus of middle-aged mice

Aging is an important determinant of adult hippocampal neurogenesis as the proliferation of neural stem/precursor cells (NSCs) declines dramatically before middle age. Contrary to this, physical exercise is known to promote adult hippocampal neurogenesis. The objective of this study is to investigate the effects of mandatory treadmill running (TR) on neurogenesis, including 1) NSCs proliferation, 2) neurite outgrowth of neuronal progenitor cells, and 3) the survival of newborn neurons in dentate area of middle-aged animals. Compared with 3-mo-old mice, numbers of mitotic cells and neuronal progenitor cells decreased dramatically by middle age and remained at low levels after middle age. Five weeks of TR not only increased NSC proliferation and the number of immature neurons but also promoted the maturation and survival of immature neurons in middle-aged mice. The neurogenic and neurotrophic effects of TR were not due to the reduction of the age-related elevation of serum corticosterone. Significantly, 5 wk of TR restored the age-dependent decline of brain-derived neurotrophic factor and its receptor, TrkB, which are known to promote neuronal differentiation and survival. Taken together, mandatory running exercise alters the brain chemistries of middle-aged animals toward an environment that is favorable to NSC proliferation, survival, and maturation.

Cerebral Amyloid Angiopathy: Accumulation of Aβ in Interstitial Fluid Drainage Pathways in Alzheimer's Disease

Abstract: Cerebral amyloid angiopathy (CAA) is characterized by the accumulation of β‐amyloid (Aβ) peptides in the walls of arteries both in the cortex and meninges. Here, we test the hypothesis that CAA results from the progressive accumulation of Aβ in the perivascular interstitial fluid drainage pathways of the brain. Experimental studies have shown that interstitial fluid (ISF) from the rat brain flows along periarterial spaces to join the cerebrospinal fluid (CSF) to drain to cervical lymph nodes. Such lymphatic drainage plays a key role in B‐cell and T‐cell mediated immunity of the brain. Anatomical studies have defined periarterial ISF drainage pathways in the human brain that are homologous with the lymphatic pathways in the rat brain but are largely separate from the CSF. Periarterial channels in the brain in man are in continuity with those of leptomeningeal arteries and can be traced from the brain to the extracranial portions of the internal carotid arteries related to deep cervical lymph nodes. The pattern of deposition of Aβ in senile plaques and in CAA suggests that Aβ accumulates in pericapillary and periarterial ISF drainage pathways. Aβ could accumulate in CAA due to either (i) increased production of Aβ, (ii) reduced solubility of Aβ peptides, or (iii) impedance of drainage of Aβ along periarterial ISF drainage pathways within the brain and leptomeninges due to aging factors in cerebral arteries. Elucidation of factors that reduce elimination of Aβ via perivascular drainage pathways may lead to their rectification and to new strategies for treatment of Alzheimers disease.

Differential effects of treadmill running and wheel running on spatial or aversive learning and memory: roles of amygdalar brain-derived neurotrophic factor and synaptotagmin I

Chronic exercise has been reported to improve cognitive function. However, whether and how different types of exercise affect various learning and memory tasks remain uncertain. To address this issue, male BALB/c mice were trained for 4 weeks under two different exercise protocols: moderate treadmill running or voluntary wheel running. After exercise training, their spatial memory and aversive memory were evaluated by a Morris water maze and by one‐trial passive avoidance (PA), respectively. Levels of neural plasticity‐related proteins, i.e. brain‐derived neurotrophic factor (BDNF), tropomyosin‐related kinase B (TrkB) and synaptotagmin I (Syt I), in hippocampus and amygdala were determined by ELISA or immunoblotting. Finally, the functional roles of these proteins in the basolateral amygdala were verified by locally blocking them with K252a (a TrkB kinase inhibitor), or lentivirus expressing Syt I shRNA. We found that (1) although both moderate treadmill running and wheel running improved the Morris water maze performance, only the former improved PA performance; (2) likewise, both exercise protocols upregulated the BDNF–TrkB pathway and Syt I in the hippocampus, whereas only treadmill exercise upregulated their expression levels in the amygdala; (3) local injection of K252a abolished the treadmill exercise‐facilitated PA performance and upregulation of amygdalar TrkB and Syt I; and (4) local administration of Syt I shRNA abolished the treadmill exercise‐facilitated PA performance and upregulation of amygdalar Syt I. Therefore, our results support the notion that different forms of exercise induce neuroplasticity changes in different brain regions, and thus exert diverse effects on various forms of learning and memory.

Compulsive exercise acutely upregulates rat hippocampal brain-derived neurotrophic factor

Summary.This study was to examine the effects of treadmill exercise on the expression of brain-derived neurotrophic factor (BDNF) in rat hippocampus. After 1-wk treadmill familiarization, animals in exercise groups received a 4-wk exercise training or an acute exercise. They were sacrificed 2 h or 2 d after exercise and their hippocampal BDNF mRNA and protein levels were determined. We demonstrated that 1) hippocampal BDNF mRNA and protein levels were both elevated in response to exercise training at 2 h after the last run but not after 2 d; 2) an acute moderate exercise (1 or 3 d) increased BDNF protein levels; 3) acute severe exercise increased BDNF protein and mRNA levels in animals under a familiarization regimen, while suppressed the BDNF mRNA level in rats without treadmill familiarization, paralleling the stress effect of immobilization/water exposure. We conclude that compulsive treadmill exercise with pre-familiarization acutely upregulates rat hippocampal BDNF gene expression.

Complement activation by neurofibrillary tangles in Alzheimer's disease

Brain inflammation is widely documented to occur in Alzheimers disease (AD), but its sources are still incompletely understood. Here, we present in vitro and in situ evidence that, like amyloid beta peptide (Abeta), tau, the major protein constituent of the neurofibrillary tangle, is a potent, antibody-independent activator of the classical complement pathway. Complement activation, in turn, is known to drive numerous inflammatory responses, including scavenger cell activation and cytokine production. Because Abeta deposits and extracellular tangles are present from early preclinical to terminal stages of AD, their ability to activate complement provides a ready mechanism for initiating and sustaining chronic, low-level inflammatory responses that may cumulate over the disease course.