James Stoll

Impairment in mitochondrial cytochrome oxidase gene expression in Alzheimer disease

Brains from 5 patients with Alzheimers disease (AD) showed a 50%-65% decrease in mRNA levels of the mitochondrial-encoded cytochrome oxidase (COX, a marker of oxidative metabolism) subunits I and III in the middle temporal association neocortex, but not in the primary motor cortex, as compared to 5 control brains. The amount of mitochondrial-encoded 12S rRNA was not altered, nor was the amount of nuclear-encoded lactate dehydrogenase B mRNA (a marker of glycolytic metabolism). These data suggest that the decrease in COX I and III subunits mRNA in affected brain regions may contribute to reduced brain oxidative metabolism in AD.

Identification of the cationic amino acid transporter (System y+) of the rat blood-brain barrier

Abstract: Cationic amino acids are transported from blood into brain by a saturable carrier at the blood‐brain barrier (BBB). The transport properties of this carrier were examined in the rat using an in situ brain perfusion technique. Influx into brain via this system was found to be sodium independent and followed Michaelis‐Men‐ten kinetics with half‐saturation constants (Km) of 50–100 μM and maximal transport rates of 22–26 nmol/min/g for L‐lysine, L‐arginine, and L‐ornithine. The kinetic properties matched that of System y+, the sodium‐independent cationic amino acid transporter, the cDNA for which has been cloned from the mouse. To determine if the cloned receptor is expressed at the BBB, we assayed RNA from rat cerebral microvessels and choroid plexus for the presence of the cloned transporter mRNA by RNase protection. The mRNA was present in both cerebral microvessels and choroid plexus and was enriched in microvessels 38‐fold as compared with whole brain. The results indicate that System y+ is present at the BBB and that its mRNA is more densely expressed at cerebral microvessels than in whole brain.

On the cause of mental retardation in Down syndrome: extrapolation from full and segmental trisomy 16 mouse models

Down syndrome (DS, trisomy 21, Ts21) is the most common known cause of mental retardation. In vivo structural brain imaging in young DS adults, and post-mortem studies, indicate a normal brain size after correction for height, and the absence of neuropathology. Functional imaging with positron emission tomography (PET) shows normal brain glucose metabolism, but fewer significant correlations between metabolic rates in different brain regions than in controls, suggesting reduced functional connections between brain circuit elements. Cultured neurons from Ts21 fetuses and from fetuses of an animal model for DS, the trisomy 16 (Ts16) mouse, do not differ from controls with regard to passive electrical membrane properties, including resting potential and membrane resistance. On the other hand, the trisomic neurons demonstrate abnormal active electrical and biochemical properties (duration of action potential and its rates of depolarization and repolarization, altered kinetics of active Na(+), Ca(2+) and K(+) currents, altered membrane densities of Na(+) and Ca(2+) channels). Another animal model, the adult segmental trisomy 16 mouse (Ts65Dn), demonstrates reduced long-term potentiation and increased long-term depression (models for learning and memory related to synaptic plasticity) in the CA1 region of the hippocampus. Evidence suggests that the abnormalities in the trisomy mouse models are related to defective signal transduction pathways involving the phosphoinositide cycle, protein kinase A and protein kinase C. The phenotypes of DS and its mouse models do not involve abnormal gene products due to mutations or deletions, but result from altered expression of genes on human chromosome 21 or mouse chromosome 16, respectively. To the extent that the defects in signal transduction and in active electrical properties, including synaptic plasticity, that are found in the Ts16 and Ts65Dn mouse models, are found in the brain of DS subjects, we postulate that mental retardation in DS results from such abnormalities. Changes in timing and synaptic interaction between neurons during development can lead to less than optimal functioning of neural circuitry and signaling then and in later life.

Characterization and chromosomal mapping of a cDNA encoding tryptophan hydroxylase from a mouse mastocytoma cell line.

A cDNA library was constructed from RNA prepared from P815 mouse mastocytoma cells and screened for tryptophan hydroxylase. An essentially full-length clone that recognizes a major mRNA species of 1.9 kb in mastocytoma cell lines and in pineal gland, duodenum, and brainstem of the mouse was obtained. The predicted amino acid sequence of this mouse mastocytoma clone showed 97 and 87% identity, respectively, with tryptophan hydroxylase clones isolated from rat and rabbit pineal glands, but the mouse clone contains an unusual 3-amino-acid duplication near the N-terminus and lacks a phosphorylation site. A fragment of the cDNA produced an enzymatically active protein when expressed in Escherichia coli, thus demonstrating that the catalytic domain is included in the C-terminal 380 amino acids. The mouse tryptophan hydroxylase locus, termed Tph, was mapped by Southern blot analysis of somatic cell hybrids and by an interspecific backcross to a position in the proximal half of chromosome 7. Because TPH has been mapped to human chromosome 11, this assignment further defines regions of homology between these mouse and human chromosomes.

Introduction to the Blood–Brain Barrier: Blood–brain barrier amino acid transport

Introduction Amino acids serve critical roles in brain and are required for normal brain development and function. However, marked differences exist among amino acids, in the roles they serve and metabolic handling within the nervous system. For example, glutamate, aspartate, glycine, and GABA operate as neurotransmitters in brain and may, in fact, be the predominant neurotransmitters at over 90% of central nervous system synapses (Smith and Cooper, 1992). As a result, they are synthesized locally in neurons and their release and re-uptake are regulated closely to maintain synaptic efficiency. In contrast, most large neutral and basic amino acids, including arginine, lysine, tryptophan, leucine, isoleucine, valine, methionine and phenylalanine, cannot be synthesized in brain, yet are required for brain protein synthesis and as precursors for serotonin (tryptophan), nitric oxide (arginine) and the catecholamines (tyrosine). These ‘essential’ amino acids must be delivered to the brain from the circulation to ensure normal cerebral growth and metabolism. The handling of amino acids at the bloodbrain barrier reflects this dichotomy. Most dietary ‘nonessential’ small neutral and anionic (acidic) amino acids have very slow rates of uptake into brain, and, in fact, may be shuttled out of brain by active transport (Oldendorf, 1971; Pardridge, 1983; Al-Sarraf et al ., 1995).

Downregulation of oxidative phosphorylation in Alzheimer disease: loss of cytochrome oxidase subunit mRNA in the hippocampus and entorhinal cortex

Messenger RNA (mRNA) for cytochrome oxidase subunit II (COX II) was localized by in situ hybridization in the entorhinal cortex and hippocampal formation of postmortem brain tissue from normal human subjects and from patients with Alzheimer disease (AD). In the control entorhinal cortex, COX II mRNA was detected mainly in neuronal cell bodies of layers II and IV. In control hippocampal formation, highest levels were localized in neuronal cell bodies of the dentate gyrus and the CA3 and CA1 regions, neurons that are involved in the major input and output pathways of the hippocampal formation. In AD brain, COX II mRNA was markedly reduced in the entorhinal cortex and the hippocampal formation compared with control brain. In the AD hippocampal formation, reductions were in regions severely affected by AD pathology as well as in regions that were relatively spared. These results are consistent with the hypothesis that reduced mitochondrial energy metabolism reflects loss of neuronal connections in AD.

Localization of cytochrome oxidase cox activity and cox mrna in the hippocampus and entorhinal cortex of the monkey brain correlation with specific neuronal pathways

Cytochrome oxidase (COX) activity and COX II mRNA expression were localized in the hippocampal formation and entorhinal cortex of the rhesus monkey brain by means of enzyme histochemistry and in situ hybridization, respectively. Within the hippocampal formation, the terminal field of the perforant pathway showed the highest levels of COX activity, whereas COX II mRNA was localized mainly in neuronal cell bodies. In the entorhinal cortex. COX II mRNA was detected in neuronal cell bodies of layers II and IV. These results indicate that the pattern of localization of COX and its mRNA in entorhinal cortex correlates with the input and output pathways of the hippocampus.

Long-term transplants of mouse trisomy 16 hippocampal neurons, a model for down's syndrome, do not develop Alzheimer's disease neuropathology

Hippocampal tissue from embryonic day 15-17 fetal mice, euploid or trisomic for chromosome 16, was transplanted into the striatum or the lateral ventricle of 6-8 week old female C57B1/6 mice. After 6-14 months of survival, host brains were sectioned and the grafts were examined by histochemical techniques and by immunocytochemistry for antigens present in pathological brain structures of Alzheimers disease (AD) patients. Nissl-stained grafts contained aggregations of neurons similar to the pyramidal or the granule cell layers of the normal adult mouse hippocampus. No obvious morphological difference was detected between trisomic and control transplants. The monoclonal antibody Alz-50, which recognizes the paired helical filaments characteristic of AD, or an antibody raised to beta-amyloid peptide, did not reveal neurodegeneration in these grafts. Antibodies against ubiquitin, 200 kDa subunit of neurofilament, alpha 1-antichymotrypsin and tau also did not demonstrate AD-type immunoreactivity in the trisomic or control grafts. Thioflavin S- or silver stained-sections were also negative. We conclude that transplanted hippocampal tissue from the trisomy 16 mouse does not represent an animal model for AD-type neurodegeneration. These results differ from those of Richards et al., EMBO J. (10) (1991) 297-303, who reported AD-type degeneration in trisomy 16 hippocampal transplants.

Localization of cytochrome oxidase (COX) activity and COX mRNA in the perirhinal and superior temporal sulci of the monkey brain

Cytochrome oxidase (COX) activity and COX II mRNA expression were localized in the perirhinal and superior temporal sulci of the rhesus monkey brain. In both regions, a laminar distribution of COX activity and COX II mRNA was observed. COX activity was intense in layers I and IV and were localized to the neuropil. In contrast, COX II mRNA was localized to neuronal cell bodies. In the prorhinal region, highest levels of COX II mRNA was detected in cell bodies of layers II and IV, and in the perirhinal region, in cell bodies of layers III and V-VI. In the superior temporal sulcus, COX II mRNA was detected in cell bodies of layers III and V-VI. Thus, COX II mRNA and COX activity are uniquely localized in the cortical layers and to those neurons that support cortico-cortical connections.

Reduced expression of voltage-gated sodium channels in neurons cultured from trisomy 16 mouse hippocampus.

Voltage‐gated sodium channels are responsible for the initial depolarizing phase of the action potential. In hippocampal neurons cultured from trisomy 16 (Ts16) mice (a model for Downs syndrome), the maximum inward conductance mediated by these channels was reduced 47% relative to control diploid neurons. This reduced conductance was reflected in a 35% decrease in binding of radiolabeled saxitoxin, a sodium channel‐specific ligand, indicating expression of fewer channels in these neurons. The mRNAs encoding the α and β1 subunits were, however, present at the same levels in Ts16 neurons and control diploid neurons. Thus, the altered regulation of voltage‐gated sodium channels in Ts16 neurons is apparently a post‐transcriptional event and possible mechanisms are discussed.