Kishore K. Bhakoo

In vitro expression of N-acetyl aspartate by oligodendrocytes : Implications for proton magnetic resonance spectroscopy signal in vivo

Abstract: Magnetic resonance spectroscopy (MRS) provides a noninvasive means of assessing in vivo tissue biochemistry. N‐Acetyl aspartate (NAA) is a major brain metabolite, and its presence is used increasingly in clinical and experimental MRS studies as a putative neuronal marker. A reduction in NAA levels as assessed by in vivo 1H MRS has been suggested to be indicative of neuronal viability. However, temporal observations of brain pathologies such as multiple sclerosis, mitochondrial encephalopathy with lactic acidosis and stroke‐like episodes (MELAS), and hypothyroidism have shown reversibility in NAA levels, possibly reflecting recovery of neuronal function. A knowledge of the cellular localisation of NAA is critical in interpreting these findings. The assumption that NAA is specific to neurones is based on previous immunohistochemical studies on whole brain using NAA‐specific antibodies. The neuronal localisation was further substantiated by cell culture experiments in which its presence in the oligodendrocyte‐type 2 astrocyte progenitors and immature oligodendrocytes, but not in the mature oligodendrocytes, was observed. More recently, studies on oligodendrocyte biology have revealed the requirement for trophic factors to promote the generation, maturation, and survival of oligodendrocytes in vitro. Here, we have used this new information to implement a more pertinent cell cultivation procedure and demonstrate that mature oligodendrocytes can express NAA in vitro. This observation brings into question whether the NAA changes observed in clinical in vivo 1H MRS studies reflect neuronal function alone. The data presented here support the hypothesis that oligodendrocytes may express NAA in vivo and contribute to the NAA signal observed by 1H MRS.

Distribution of mRNAs Encoding the Peroxisome Proliferator-Activated Receptor α, β, and γ and the Retinoid X Receptor α, β, and γ in Rat Central Nervous System

Abstract: We report the isolation, by RT‐PCR, of partial cDNAs encoding the rat peroxisome proliferator‐activated receptor (PPAR) isoforms PPARα, PPARβ, and PPARγ and the rat retinoid X receptor (RXR) isoforms RXRα, RXRβ, and RXRγ. These cDNAs were used to generate antisense RNA probes to permit analysis, by the highly sensitive and discriminatory RNase protection assay, of the corresponding mRNAs in rat brain regions during development. PPARα, PPARβ, RXRα, and RXRβ mRNAs are ubiquitously present in different brain regions during development, PPARγ mRNA is essentially undetectable, and RXRγ mRNA is principally localised to cortex. We demonstrate, for the first time, the presence of PPAR and RXR mRNAs in primary cultures of neonatal meningeal fibroblasts, cerebellar granule neurons (CGNs), and cortical and cerebellar astrocytes and in primary cultures of adult cortical astrocytes. PPARα, PPARβ, RXRα, and RXRβ mRNAs are present in all cell types, albeit that PPARα and RXRα mRNAs are at levels near the limit of detection in CGNs. PPARγ mRNA is expressed at low levels in most cell types but is present at levels similar to those of PPARα mRNA in adult astrocytes. RXRγ mRNA is present either at low levels, or below the level of detection of the assay, for all cell types studied.

Developmental and regional distribution of aspartoacylase in rat brain tissue

The function of N‐acetyl‐aspartate (NAA), a predominant molecule in the brain, has not yet been determined. However, NAA is commonly used as a putative marker of viable neurones. To investigate the possible function of NAA, we determined the anatomical, developmental and cellular distribution of aspartoacylase, which catalyses the hydrolysis of NAA. Levels of aspartoacylase activity were measured during postnatal development in several brain regions. The differential distribution of aspartoacylase activity in purified populations of cells derived from the rat CNS was also investigated. The developmental and anatomical distribution of aspartoacylase correlated with the maturation of white matter tracts in the rat brain. Activity increased markedly after 7 days and coincided with the time course for the onset of myelination in the rat brain. Gray matter showed little activity or developmental trend. There was a 60‐fold excess in optic nerve (a white matter tract) when compared with cortex at 21 days of development. In the adult brain there was a 18‐fold difference in corpus callosum compared with cortex (stripped of corpus callosum). Cellular studies demonstrated that purified cortical neurons and cerebellar granular neurones have no activity. Primary O‐2A progenitor cells had moderate activity, with three‐fold higher activity in immature oligodendrocyte and 13‐fold increase in mature oligodendrocytes (myelinating cells of the CNS). The highest activity was seen in type‐2 astrocytes (20‐fold difference compared with O‐2A progenitors) derived from the same source. Aspartoacylase activity increased with time in freshly isolated astrocytes, with significantly higher activity after 15 days in culture. We conclude that aspartoacylase activity in the developing postnatal brain corresponds with maturation of myelination, and that the cellular distribution is limited to glial cells.

Hypo‐osmotic swelling‐activated release of organic osmolytes in brain slices: implications for brain oedema in vivo

A decrease in the intracellular levels of osmotically active species has invariably been seen after swelling of mammalian brain tissue preparations. The exact identity of the species, and the manner of their decrease, remain to be described. We investigated the swelling‐activated decrease of organic osmolytes in rat cortical brain slices using 1H‐ and 31P‐magnetic resonance spectroscopy. We found that acute hypo‐osmotic shock causes decreases in the levels of a range of intracellular amino acids and amino acid derivatives, N‐acetyl‐aspartate, creatine, GABA, glutamate, hypotaurine, and also in the levels of the methylamines glycerol‐phosphorylcholine, phosphorylcholine and choline. Incubation of cortical slices with the anion channel blockers niflumic acid and tamoxifen caused inhibition of organic osmolyte efflux, suggesting that such osmolyte efflux occurs through anion channels. Intracellular phosphocreatine was also seen to decrease during acute hypo‐osmotic superfusion, although intracellular ATP remained constant. In addition, the acidification of an intracellular compartment was observed during hypo‐osmotic superfusion. Our results suggest a link between brain energy reserve and brain osmoregulation.

Evidence for altered hepatic gluconeogenesis in patients with cirrhosis using in vivo 31-phosphorus magnetic resonance spectroscopy

BACKGROUND AND AIMS Alterations in gluconeogenesis in the diseased liver can be assessed non-invasively using magnetic resonance spectroscopy by measuring changes in phosphomonoester resonance which contains information regarding several metabolites, including the phosphorylated intermediates of the gluconeogenic pathway. METHODS 31P magnetic resonance spectroscopy was used to determine changes in phosphomonoesters following bolus infusions of 2.8 mmol/kgl-alanine in five patients with functionally compensated cirrhosis and in five patients with functionally decompensated cirrhosis. RESULTS Compared with six healthy volunteers, baseline phosphomonoester values were elevated by 35% (p<0.05) in the compensated cirrhosis group and by 57% (p<0.01) in the decompensated cirrhosis group. Following alanine infusion, phosphomonoesters in healthy volunteers increased by 46% from baseline values (p<0.01), in patients with compensated cirrhosis by 27% (p<0.02) but those with decompensated cirrhosis showed no increase from baseline. There was a reduction in the percentage of inorganic phosphate signal in all subjects. CONCLUSIONS By analysing changes in phosphomonoester and inorganic phosphate resonances it is possible to discern clear metabolic differences between healthy volunteers and patients with cirrhosis of varying severity using magnetic resonance spectroscopy. Those patients with functionally decompensated cirrhosis have higher percentage baseline phosphomonoester values but the absence of phosphomonoester elevation following l-alanine infusion suggests that they are unable to mount a significant metabolic response with a progluconeogenic stimulus.

Metabolic changes underlying 31P MR spectral alterations in human hepatic tumours

Magnetic resonance spectroscopy (MRS) remains the technique of choice for observing tumour metabolism non‐invasively. Although initially 31P MR spectroscopy showed much promise as a non‐invasive diagnostic tool, studies of a wide range of hepatic tumours have conclusively shown that this technique cannot be utilized to distinguish between different tumour types. This lack of specificity and sensitivity appears to be a consequence of the fact that hepatic tumours develop with a range of modalities and not as a single abnormal disease process, and also because of the limited availability of MR detectable metabolic markers. This has led, in recent years, to a re‐evaluation of the role of 31P MR spectroscopy, re‐emerging as a non‐invasive tool to follow the efficacy of the treatment regime. Furthermore, since the principal changes observed in tumours by 31P MRS appear to be an elevation in the concentration of phosphorylcholine (PCho) and phosphoethanolamine (PEth), new research using a combination of MRS and tissue culture of cell lines which carry a combination of known inducible oncogenes, are helping to elucidate some of the metabolic pathways that give rise to these metabolic alterations.

Swelling-activated Taurine and Creatine Effluxes from Rat Cortical Astrocytes are Pharmacologically Distinct

Primary cultures of rat cortical astrocytes undergo a swelling-activated loss of taurine and creatine. In this study, the pharmacological characteristics of the taurine and creatine efflux pathways were compared, and significant differences were shown to exist between the two. Both taurine and creatine effluxes were rapidly activated upon exposure of astrocytes to hypo-osmotic media, and rapidly inactivated upon their return to iso-osmotic media. The relative rates of taurine and creatine efflux depended upon the magnitude of the hypo-osmotic shock. Anion-transport inhibitors strongly inhibited taurine efflux, with the order of potency being NPPB > DIDS > niflumic acid. DIDS and NPPB had less of an inhibitory effect on creatine efflux, whereas tamoxifen and niflumic acid actually stimulated creatine efflux. These data are consistent with separate pathways for taurine and creatine loss during astrocyte swelling.

Hormonal Regulation of the mRNA Encoding the Ketogenic Enzyme Mitochondrial 3‐Hydroxy‐3‐Methylglutaryl‐CoA Synthase in Neonatal Primary Cultures of Cortical Astrocytes and Meningeal Fibroblasts

Abstract: We have previously identified cerebellum to contain significantly higher levels, compared with other brain regions, of the mRNA encoding the key ketogenic enzyme mitochondrial 3‐hydroxy‐3‐methylglutaryl‐CoA synthase (mHS). In this report, we extend these observations, using primary cultures of cerebellar astrocytes and cerebellar granule neurons, and show that mHS mRNA was not readily detected in these cell types, suggesting that other cerebellar cell types account for mHS mRNA abundances observed in cerebellum. In contrast, we report, for the first time, the ready detection of mHS mRNA together with the mRNAs encoding the remaining enzymes of the 3‐hydroxy‐3‐methylglutaryl‐CoA cycle, namely, mitochondrial acetoacetyl‐CoA thiolase and 3‐hydroxy‐3‐methylglutaryl‐CoA lyase, in primary cultures of neonatal meningeal fibroblasts. Based on observations of the effects of fetal calf serum in the culture medium and the documented effects of various hormones on mHS mRNA levels in liver, we show that the glucocorticoid hydrocortisone effects a selective fourfold increase in mHS mRNA abundances in both neonatal meningeal fibroblasts and neonatal cortical astrocytes cultured in a serum‐free/hormone‐free medium.

Differential Expression of Carnosine, Homocarnosine and N-Acetyl-L-Histidine Hydrolytic Activities in Cultured Rat Macroglial Cells

N-acetyl-L-histidine (NAH) and N-acetyl-L-aspartate (NAA) are representatives of two series of substances that are synthesized by neurons and other cells in the vertebrate central nervous system (CNS). Histidine containing homologs of NAH are β-alanyl-L-histidine or carnosine (Carn) and γ-aminobutyrl-L-histidine or homocarnosine (Hcarn). A homolog of NAA is N-acetylaspartylglutamate (NAAG). These substances belong to a unique group of osmolytes in that they are synthesized in cells that may not to be able to hydrolyze them, and are released in a regulated fashion to a second compartment where they can be rapidly hydrolyzed. In this investigation, the catabolic activities for NAH, Carn, and Hcarn in cultured macroglial cells and neurons have been measured, and the second compartment for NAH and Hcarn has been identified only with astrocytes. In addition, oligodendrocytes can only hydrolyze Carn, although Carn can also be hydrolyzed by astrocytes. Thus, astrocytes express hydrolytic activity against all three substrates, but oligodendrocytes can only act on Carn. The cellular separation of these hydrolytic enzyme activities, and the possible nature of the enzymes involved are discussed.

N‐acetyl aspartate estimation: a potential method for determining neuronal loss in the transmissible spongiform encephalopathies

Neurodegenerative pathology is typical of the transmissible spongiform encephalopathies (TSEs), and is thought to underlie clinical disease. Some morphometric studies have shown early focal neurone loss, but the full extent of TSE induced neuronal loss in the central nervous system is not known, and can only be accurately estimated using intensive morphometric techniques. We have used a murine scrapie model in which we determined the levels of N‐acetyl aspartate (NAA), a putative neuronal marker, by both high‐performance liquid chromatography and high resolution, proton magnetic resonance spectroscopy in samples taken sequentially from the hippocampus. This scrapie model develops severe neuronal loss in the hippocampus, and the NAA levels showed a significant positive correlation with our previous morphometric estimates of neurone number. NAA measurement may therefore provide a practical alternative to intensive morphometric techniques in the investigation of neurodegeneration in the TSEs.