α-Methyl-l-tryptophan as a tracer to study brain serotonergic system

Special issue devoted to Dr. Leon S. Wolfe.     

Building a cathedral

This article describes the scientific legacy of Dr. Leon Wolfe, with illustrations from his contributions to insect physiology, glycolipid, and eicosanoid biochemistry as well as to our understanding of neuronal ceroid lipofuscinoses and lysosomal storage diseases. In addition to the written record, Wolfe inspired all who knew him with his boundless imagination and enthusiasm for science and his ability to see the promise and potential of every experiment.     

Batten disease (ceroid-lipofuscinosis): The enigma of subunit c of mitochondrial ATP synthase accumulation

Ceroid-lipofuscinosis is an inherited neurodegenerative disease of human beings and domestic animals characterized by the accumulation in neurons and other cells of a fluorescent lipopigment. In the ovine form of disease, subunit c of mitochondrial ATP synthase is the dominant accumulated metabolite (>50%). It also accumulates significantly in the late infantile and juvenile forms of the human disease and several other animal forms. Evidence is accumulating that the underlying biochemical defect may be associated with mitochondria. The extreme hydrophobicity of subunit c and its propensity to aggregate with lipids into regular multilamellar arrays that cannot be catabolised may reflect an initial defect not necessarily associated with faulty proteolysis. This hypothesis extends an earlier one that subunit c accumulated due to a defect in its catabolic pathway.Special issues dedicated to Dr. Leon S. Wolfe.     

Design and serendipity in the path to prostaglandins

Leon Wolfe was a brilliant scientist who was gifted with curiosity and drive. This personal account touches on salient events in a rewarding collaboration and lifelong friendship, starting from the original identification of prostaglandin as a natural neurohumoral agent and progressing with the unpredictable developments of this research beyond the brain. Design and serendipity are the leitmotifs in an exciting story unfolding over the years.     

Neural membrane phospholipids in alzheimer disease

Phospholipids form the backbone of neural membranes, providing fluidity and permeability. Two plasma membrane fractions, one from synaptosomes (SPM), the other glial and neuronal cell bodies (PM), were prepared from different regions of autopsied Alzheimer disease (AD) brains. Corresponding fractions were prepared from age-matched control brains. All fractions from AD brains showed significantly lower levels of ethanolamine glycerophospholipids and significantly higher levels of serine glycerophospholipids than the control brain. No differences were observed in phosphatidylcholine levels among these membranes. These results suggest that altered phospholipid composition of plasma membranes may be involved in the abnormal signal transduction and neurodegeneration in AD.Special issue dedicated to Dr. Leon S. Wolfe.     

Novel eicosanoid pathways

This article reviews a lecture I was honored to present at the Leon Wolfe Symposium in Montreal on March 25, 2004. The lecture described my research career, which started with my interaction with Wolfe at the Montreal Neurological Institute as a postdoctoral fellow and research associate and was followed by additional research discoveries after I left Montreal for my first academic position at the Research Institute, The Hospital for Sick Children and University of Toronto. The article consists of two parts. The first part involves the discovery (in Wolfe’s laboratory) of a new pathway of arachidonic acid, in which a bicyclic prostanoid structure (later called prostacyclin by John Vane and his group) was described, and its further development in Toronto, which led to the discovery of the conversion of the bicyclic prostanoid into 6-keto prostaglandin F_1α. The second part deals with the hepoxilin pathway, a pathway I discovered during a sabbatical leave in Japan with Professor Shozo Yamamoto, which was followed by a stay of several months in the laboratory of Professor Bengt Samuelsson in Sweden. I deal with the historical aspects of both pathways and end with interesting novel aspects of hepoxilin stable antagonist analogs in the treatment of solid tumors in experimental animals.     

Tay-Sachs disease with atypical chronic course and limited brain storage: Alpha-locus hexosaminidase genetic compound

A 19-year-old Irish-Jewish male had a slow neurologic regression starting at age 4 1/2 years with stuttering. The chronic course resembled that of Spielmeyer-Vogt (juvenile ceroid-lipofuscinosis) disease. The brain was atrophic with neuronal losses and huge compound inclusions in the remaining neurons. Lipid NANA was within normal limits in gray and white matter and G_M2 gangliosides were moderately elevated at 11.5% lipid NANA. Beta-hexosaminidase A activity was lipid composition showed nonspecific abnormalities. Exhaustive tissue extraction ruled out the possibility of tightly bound gangliosides to account for the relatively low G_M2 ganglioside concentration. The extract contained unidentified chromogenic substances interfering with the resorcinol reaction. The similarly affected patient's sister lived to age 26 years and her brain was even more atrophic. No biochemical abnormality to account for progressive neuronal losses and relative lack of G_M2 ganglioside storage was found.Deceased.Special issue dedicated to Dr. Leon S. Wolfe.     

Description of Culicoides pseudoheliconiae sp.n. from Peruvian Amazon and revalidation of Culicoides contubernalis Ortiz & Leon (Diptera: Ceratopogonidae)

A new species of the Culicoides hylas species group, Culicoides pseudoheliconiae Felippe-Bauer is described and illustrated based on female specimens from Peruvian Amazon, and Culicoides contubernalis Ortiz & Leon from Ecuador is resurrected and redescribed as a valid species. A systematic key, table with numerical characters of females of species of the Culicoides hylas group are given.     

Myelin abnormalities in mice deficient in galactocerebroside and sulfatide

Myelin sheath formation depends on appropriate axo-glial interactions that are mediated by myelin-specific surface molecules. In this study, we have used quantitative morphological analysis to determine the roles of the prominent myelin lipids galactocerebroside (GalC) and sulfatide in both central and peripheral myelin formation, exploiting mutant mice incapable of synthesizing these lipids. Our results demonstrate a significant increase in uncompacted myelin sheaths, the frequency of multiple cytoplasmic loops, redundant myelin profiles, and Schmidt-Lanterman incisures in the CNS of these mutant mice. In contrast, PNS myelin appeared structurally normal in these animals; however, at post-natal day 10, greater than 10% of the axons withered and pulled away from their myelin sheaths. These results indicate that GalC and sulfatide are critical to the formation of CNS myelin. In contrast, PNS myelin formation is not dependent on these lipids; however, GalC and sulfatide appear to be instrumental in maintaining Schwann cell-axon contact during a specific developmental window.     

Gangliosides inhibit PDGF-induced signal transduction events in U-1242 MG human glioma cells

In this study we investigated the responses of intracellular calcium ([Ca²⁺]_i) and protein kinase C (PKC) to PDGF in U-1242 MG cells. PDGF-BB stimulated [³H]PDBu binding approximately 2–3 fold. This response was inhibited by preincubating the cells with an inhibitor of phospholipase C (PLC), U73122, suggesting that PLC mediates the induction of PKC translocation by PDGF. PDGF also increased the concentration of [Ca²⁺]_i that was attenuated in a calcium-free medium. This indicates that PDGF-induced elevation of [Ca²⁺]_i is mainly due to influx of extracellular calcium. PDGF-stimulated translocation of PKC was inhibited by the intracellular calcium buffer BAPTA/AM. All gangliosides studied except GM3 inhibited these responses with similar efficacy. Collectively, these results indicate that the signal transduction pathway initiated by PDGF leading to PKC translocation in U-1242 MG cells is intact, and this pathway is inhibited by several gangliosides.Special issue dedicated to Dr. Leon S. Wolfe.     

Phospholipase A(2), reactive oxygen species, and lipid peroxidation in CNS pathologies

The importance of lipids in cell signaling and tissue physiology is demonstrated by the many CNS pathologies involving deregulated lipid metabolism. One such critical metabolic event is the activation of phospholipase A(2) (PLA(2)), which results in the hydrolysis of membrane phospholipids and the release of free fatty acids, including arachidonic acid, a precursor for essential cell-signaling eicosanoids. Reactive oxygen species (ROS, a product of arachidonic acid metabolism) react with cellular lipids to generate lipid peroxides, which are degraded to reactive aldehydes (oxidized phospholipid, 4-hydroxynonenal, and acrolein) that bind covalently to proteins, thereby altering their function and inducing cellular damage. Dissecting the contribution of PLA(2) to lipid peroxidation in CNS injury and disorders is a challenging proposition due to the multiple forms of PLA(2), the diverse sources of ROS, and the lack of specific PLA(2) inhibitors. In this review, we summarize the role of PLA(2) in CNS pathologies, including stroke, spinal cord injury, Alzheimer's, Parkinson's, Multiple sclerosis-Experimental autoimmune encephalomyelitis and Wallerian degeneration.     

Opsonization with Antimyelin Antibody Increases the Uptake and Intracellular Metabolism of Myelin in Inflammatory Macrophages

In most demyelinating diseases, macrophages are believed to be active agents of myelin destruction. In experimental encephalomyelitis, these cells appear to strip off and ingest the myelin lamellae, and myelin debris has been observed within the cell body. We show here in vitro conditions in which rat peritoneal macrophages phagocytose and metabolize CNS myelin lipids. Purified rat myelin, prelabeled in vivo with [14C]acetate, was incubated with preimmune serum or rabbit antiserum to rat CNS myelin and added to macrophage monolayers. Myelin opsonized with antimyelin antibodies was more readily phagocytosed and metabolized by cultured macrophages than untreated myelin or that preincubated with preimmune serum. In the presence of macrophages, levels of myelin polar lipids and cholesterol decreased, whereas radioactive cholesterol ester and triglyceride accumulated. Up to five times as much radioactive cholesterol ester and about twice as much triglyceride accumulated in macrophage cultures containing antibody-treated myelin as in cultures fed preimmune serum-treated myelin or in those incubated with untreated myelin. Both the fatty acid and the cholesterol from cholesterol ester contained radioactive label; therefore, both were derived at least partly from the radioactive myelin lipid. Antiserum to myelin purified from peripheral nerve was almost as effective as that to CNS myelin in stimulating cholesterol metabolism, whereas antiserum to galactocerebroside was about 70% as active. Antiserum to basic protein had less effect, whereas antiserum to the myelin-associated glycoprotein and proteolipid protein was inactive. Of the polar lipids, ethanolamine phosphatide was most degraded in both the antiserum- and preimmune serum-treated myelin, with the diacyl form and plasmalogen form degraded about equally. These experiments indicate that myelin-specific antibodies in inflammatory CNS lesions may participate in and stimulate macrophage-mediated demyelination.     

Enzymes of platelet activating factor synthesis in brain

In this review, evidence is summarized for the production of PAF in brain, in response to stimulation associated with pathology. As well, there is a growing literature on the duality of actions of this lipid autocoid upon nervous tissue, indicated by extracellular and intracellular actions and binding sites for PAF in brain. The metabolic routes to PAF can be divided into the de novo and remodelling pathways of synthesis. The de novo route consists of 1-alkyl glycerophosphate acetyltransferase, and the subsequent actions of distinct phosphohydrolase and cholinephosphotransferase activities. This acetyltransferase can be activated by phosphorylation, and inhibited by MgATP and fatty acyl CoA thioesters, inhibitions which have particular relevance to brain ischemia. There is also evidence that the cholinephosphotransferase is controlled by phosphorylation, and regulated by levels of CDP-choline. The remodelling pathway to PAF relies upon the actions of phospholipase A₂ or CoA-independent transacylases to generate the l-alkyl glycerophosphorylcholine, as substrate for a distinct acetyltransferase. Following stimulation, rising intracellular calcium may trigger arachidonate selective cytosolic phospholipase activity which leads to increased PAF synthesis. The l-alkyl glycerophosphocholine acetyltransferase activity is quite small in brain in comparison with the de novo acetyltransferase activity, and is also controlled by phosphorylation. Evidence has been presented for the actions of both pathways in brain, in response to biologically relevant stimulation pertinent to the disease state.Special issue dedicated to Dr. Leon S. Wolfe.     

Region-specific vulnerability to lipid peroxidation and evidence of neuronal mechanisms for polyunsaturated fatty acid biosynthesis in the healthy adult human central nervous system

Lipids played a determinant role in the evolution of the brain. It is postulated that the morphological and functional diversity among neural cells of the human central nervous system (CNS) is projected and achieved through the expression of particular lipid profiles. The present study was designed to evaluate the differential vulnerability to oxidative stress mediated by lipids through a cross-regional comparative approach. To this end, we compared 12 different regions of CNS of healthy adult subjects, and the fatty acid profile and vulnerability to lipid peroxidation, were determined by gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS), respectively. In addition, different components involved in PUFA biosynthesis, as well as adaptive defense mechanisms against lipid peroxidation, were also measured by western blot and immunohistochemistry, respectively. We found that: i) four fatty acids (18.1n-9, 22:6n-3, 20:1n-9, and 18:0) are significant discriminators among CNS regions; ii) these differential fatty acid profiles generate a differential selective neural vulnerability (expressed by the peroxidizability index); iii) the cross-regional differences for the fatty acid profiles follow a caudal-cranial gradient which is directly related to changes in the biosynthesis pathways which can be ascribed to neuronal cells; and iv) the higher the peroxidizability index for a given human brain region, the lower concentration of the protein damage markers, likely supported by the presence of adaptive antioxidant mechanisms. In conclusion, our results suggest that there is a region-specific vulnerability to lipid peroxidation and offer evidence of neuronal mechanisms for polyunsaturated fatty acid biosynthesis in the human central nervous system.     

Mass-spectrometry based oxidative lipidomics and lipid imaging: applications in traumatic brain injury

Lipids, particularly phospholipids, are fundamental to CNS tissue architecture and function. Endogenous polyunsaturated fatty acid chains of phospholipids possess cis-double bonds each separated by one methylene group. These phospholipids are very susceptible to free-radical attack and oxidative modifications. A combination of analytical methods including different versions of chromatography and mass spectrometry allows detailed information to be obtained on the content and distribution of lipids and their oxidation products thus constituting the newly emerging field of oxidative lipidomics. It is becoming evident that specific oxidative modifications of lipids are critical to a number of cellular functions, disease states and responses to oxidative stresses. Oxidative lipidomics is beginning to provide new mechanistic insights into traumatic brain injury which may have significant translational potential for development of therapies in acute CNS insults. In particular, selective oxidation of a mitochondria-specific phospholipid, cardiolipin, has been associated with the initiation and progression of apoptosis in injured neurons thus indicating new drug discovery targets. Furthermore, imaging mass-spectrometry represents an exciting new opportunity for correlating maps of lipid profiles and their oxidation products with structure and neuropathology. This review is focused on these most recent advancements in the field of lipidomics and oxidative lipidomics based on the applications of mass spectrometry and imaging mass spectrometry as they relate to studies of phospholipids in traumatic brain injury.     

New insight into lysosomal protein storage disease: Delayed catabolism of ATP synthase subunit c in batten disease

Subunit c is normally present as an inner mitochondrial membrane component of the Fo sector of the ATP synthase complex, but in the late infantile form of neuronal ceroid lipofuscinosis (NCL) it was also found in lysosomes in high concentrations. Mechanism for specific accumulation of subunit c in lysosomes is not known. The rate of degradation of subunit c as measured by pulsechase and immunoprecipitation showed a marked delay of degradation in patients fibroblasts with late infantile form of NCL. There were no significant differences between control cells and cells with disease in the degradation of cytochrome oxidase subunit IV, an inner membrane protein of mitochondria. Measurement of labeled subunit c in mitochondrial and lysosomal fractions showed that the accumulation of labeled subunit c in the mitochondrial fraction can be detected before lysosomal appearance of radioactive subunit c, suggesting that subunit c accumulated as a consequence of abnormal catabolism in the mitochondrion and is transferred to lysosomes, through an autophagic process. There were no large differences of various lysosomal protease activities between control and patient cells. In patient cells sucrose loading caused a marked shift of lysosomal density, but did not a shift of subunit c containing storage body. The biosynthetic rate of subunit c and mRNA levels for P1 and P2 genes that code for it were almost the same in both control and patient cells. These findings suggest that a specific failure in the degradation of subunit c after its normal inclusion in mitochondria and its consequent accumulation in lysosomes.Special issue dedicated to Dr. Leon S. Wolfe.     

Lipids of nervous tissue: composition and metabolism

As indicated in the Introduction, the many significant developments in the recent past in our knowledge of the lipids of the nervous system have been collated in this article. That there is a sustained interest in this field is evident from the rather long bibliography which is itself selective. Obviously, it is not possible to summarize a review in which the chemistry, distribution and metabolism of a great variety of lipids have been discussed. However, from the progress of research, some general conclusions may be drawn. The period of discovery of new lipids in the nervous system appears to be over. All the major lipid components have been discovered and a great deal is now known about their structure and metabolism. Analytical data on the lipid composition of the CNS are available for a number of species and such data on the major areas of the brain are also at hand but information on the various subregions is meagre. Such investigations may yet provide clues to the role of lipids in brain function. Compared to CNS, information on PNS is less adequate. Further research on PNS would be worthwhile as it is amenable for experimental manipulation and complex mechanisms such as myelination can be investigated in this tissue. There are reports correlating lipid constituents with the increased complexity in the organization of the nervous system during evolution. This line of investigation may prove useful. The basic aim of research on the lipids of the nervous tissue is to unravel their functional significance. Most of the hydrophobic moieties of the nervous tissue lipids are comprised of very long chain, highly unsaturated and in some cases hydroxylated residues, and recent studies have shown that each lipid class contains characteristic molecular species. Their contribution to the properties of neural membranes such as excitability remains to be elucidated. Similarly, a large proportion of the phospholipid molecules in the myelin membrane are ethanolamine plasmalogens and their importance in this membrane is not known. It is firmly established that phosphatidylinositol and possibly polyphosphoinositides are involved with events at the synapse during impulse propagation, but their precise role in molecular terms is not clear. Gangliosides, with their structural complexity and amphipathic nature, have been implicated in a number of biological events which include cellular recognition and acting as adjuncts at receptor sites. More recently, growth promoting and neuritogenic functions have been ascribed to gangliosides. These interesting properties of gangliosides wIll undoubtedly attract greater attention in the future.(ABSTRACT TRUNCATED AT 400 WORDS)     

Oligodendrocyte-derived transcellular signaling regulates axonal energy metabolism

The unique morphology and functionality of central nervous system (CNS) neurons necessitate specialized mechanisms to maintain energy metabolism throughout long axons and extensive terminals. Oligodendrocytes (OLs) enwrap CNS axons with myelin sheaths in a multilamellar fashion. Apart from their well-established function in action potential propagation, OLs also provide intercellular metabolic support to axons by transferring energy metabolites and delivering exosomes consisting of proteins, lipids, and RNAs. OL-derived metabolic support is crucial for the maintenance of axonal integrity; its dysfunction has emerged as an important player in neurological disorders that are associated with axonal energy deficits and degeneration. In this review, we discuss recent advances in how these transcellular signaling pathways maintain axonal energy metabolism in health and neurological disorders.     

Alterations in brain lipid composition of mice made physically dependent to ethanol

The ability of chronic ethanol treatment to alter CNS membrane lipids was tested. Adult male C57/BL6 mice were given a liquid diet containing ethanol for eight days. This regimen produced strong physical dependence as judged by withdrawal seizures, tremors and concomitant hypothermia. Analyses were performed on cholesterol, total phospholipid content and total phospholipid acyl composition of myelin, crude (P2), light and heavy synaptosomes as well as synaptosomal plasma membranes. Chronic ethanol treatment had no effect on total phospholipid levels nor phospholipid acyl composition in any of the above subcellular fractions. In ethanol dependent mice, significant increases in cholesterol content and cholesterol/ phospholipid ratios were observed only in synaptosomal plasma membranes.     

GM2 ganglioside and pyramidal neuron dendritogenesis

GM2 ganglioside, although scarce in normal adult brain, is the predominant ganglioside accumulating in several types of lysosomal disorders, most notably Tay-Sachs disease. Pyramidal neurons of cerebral cortex in Tay-Sachs, as well as many other types of neuronal storage disorders, are known to exhibit a phenomenon believed unique to storage disorders: growth of ectopic dendrites. Recent studies have shown that a common metabolic abnormality shared by storage diseases with ectopic dendrite growth is the abnormal accumulation of GM2 ganglioside. The correlation between increased levels of GM2 and the presence of ectopic dendrites has been found in both ganglioside and nonganglioside storage disorders, the latter including sphingomyelin-cholesterol lipidosis, mucopolysaccharidosis, and -mannosidosis. Quantitative HPTLC analysis has shown that increases in GM2 occur in proportion to the incidence of ectopic dendrite growth, whereas, other gangliosides, including GM1, lack similar increases. Immunocytochemical studies of all nonganglioside storage diseases which exhibit ectopic dendritogenesis have revealed heightened GM2 ganglioside-immunoreactivity in the cortical pyramidal cell population, whereas neurons in normal adult brain exhibit little or no staining for this ganglioside. Further, studies examining disease development have consistently shown that accumulation of GM2 gangliosideprecedes growth of ectopic dendrites, indicating that it is not simply occurring secondary to new membrane production. These findings have prompted an examination for a similar relationship between GM2 ganglioside and dendritogenesis in cortical neurons of normal developing brain. Results show that GM2 ganglioside-immunoreactivity is consistently elevated in immature neurons during the period when they are undergoing active dendritic initiation, but this staining diminishes dramatically as the dendritic tress of these cells mature. Collectively, these studies on diseased and normal brain offer compelling evidence that GM2 ganglioside plays a pivotal role in the regulation of dendritogenesis in cortical pyramidal neurons.Special issue dedicated to Dr. Leon S. Wolfe.