Metabolic and Cellular Compartments of Acetyl-CoA in the Healthy and Diseased Brain.

The human brain is characterised by the most diverse morphological, metabolic and functional structure among all body tissues. This is due to the existence of diverse neurons secreting various neurotransmitters and mutually modulating their own activity through thousands of pre- and postsynaptic interconnections in each neuron. Astroglial, microglial and oligodendroglial cells and neurons reciprocally regulate the metabolism of key energy substrates, thereby exerting several neuroprotective, neurotoxic and regulatory effects on neuronal viability and neurotransmitter functions. Maintenance of the pool of mitochondrial acetyl-CoA derived from glycolytic glucose metabolism is a key factor for neuronal survival. Thus, acetyl-CoA is regarded as a direct energy precursor through the TCA cycle and respiratory chain, thereby affecting brain cell viability. It is also used for hundreds of acetylation reactions, including N-acetyl aspartate synthesis in neuronal mitochondria, acetylcholine synthesis in cholinergic neurons, as well as divergent acetylations of several proteins, peptides, histones and low-molecular-weight species in all cellular compartments. Therefore, acetyl-CoA should be considered as the central point of metabolism maintaining equilibrium between anabolic and catabolic pathways in the brain. This review presents data supporting this thesis.

EBI2 is expressed in glial cells in multiple sclerosis lesions, and its knock-out modulates remyelination in the cuprizone model.

EBI2 receptor regulates the immune system, and in multiple, sclerosis is upregulated in the central nervous system infiltrating lymphocytes. In newborn EBI2-deficient mice, myelin development is delayed, and its persistent antagonism inhibits remyelination in chemically demyelinated organotypic cerebellar slices. We used the cuprizone model of multiple sclerosis to elucidate the role of central nervous system-expressed EBI2 in de- and remyelination. The wild-type and EBI2 knock-out mice were fed 0.2% cuprizone in chow for 5 weeks and allowed to recover on a normal diet for 2 weeks. The data showed less efficient recovery of myelin, attenuated oligodendrocyte loss, fewer astrocytes and increased total cholesterol levels in the EBI2 knock-out mice after recovery. Moreover, the wild-type mice upregulated EBI2 expression after recovery confirming the involvement of EBI2 signalling during recovery from demyelination in the cuprizone model. The pro-inflammatory cytokine levels were at comparable levels in the wild-type and EBI2 knock-out mice, with only minor differences in TNFα and IL1ß levels either at peak or during recovery. The neuroinflammatory signalling molecules, Abl1 kinase and NFКB1 (p105/p50) subunit, were significantly downregulated in the EBI2 knock-out mice at peak of disease. Immunohistochemical investigations of EBI2 receptor distribution in the central nervous system (CNS) cells in multiple sclerosis (MS) brain revealed strong expression of EBI2 in astrocytes and microglia inside the plaques implicating glia-expressed EBI2 in multiple sclerosis pathophysiology. Taken together, these findings demonstrate the involvement of EBI2 signalling in the recovery from demyelination rather than in demyelination and as such warrant further research into the role of EBI2 in remyelination.

Early and Late Pathomechanisms in Alzheimer's Disease: From Zinc to Amyloid-ß Neurotoxicity.

There are several systemic and intracerebral pathologic conditions, which limit provision and utilization of energy precursor metabolites in neuronal cells. Energy deficits cause excessive depolarization of neuronal cells triggering glutamate-zinc evoked excitotoxic cascade. The intracellular zinc excess hits several intraneuronal targets yielding collapse of energy balance and impairment functional and structural impairments cholinergic neurons. Disturbances in metabolism of acetyl-CoA, which is a direct precursor for energy, acetylcholine, N-acetyl-L-aspartate and acetylated proteins synthesis, play an important role in these pathomechanisms. Disruption of brain homeostasis activates slow accumulation of amyloid-ß 1-42 , which extra and intracellular oligomeric deposits disrupt diverse transporting and signaling processes in all membrane structures of the cell. Both neurotoxic signals may combine aggravating detrimental effects on neuronal cell. Different neuroglial and neuronal cell types may display differential susceptibility to similar pathogenic insults depending on specific features of their energy and functional parameters. This review, basing on findings gained from cellular and animal models of Alzheimer's disease, discusses putative energy/acetyl-CoA dependent mechanism in early and late stages of neurodegeneration.

Differential effects of lipopolysaccharide on energy metabolism in murine microglial N9 and cholinergic SN56 neuronal cells.

There are significant differences between acetyl-CoA and ATP levels, enzymes of acetyl-CoA metabolism, and toll-like receptor 4 contents in non-activated microglial N9 and non-differentiated cholinergic SN56 neuroblastoma cells. Exposition of N9 cells to lipopolysaccharide caused concentration-dependent several-fold increases of nitrogen oxide synthesis, accompanied by inhibition of pyruvate dehydrogenase complex, aconitase, and α-ketoglutarate dehydrogenase complex activities, and by nearly proportional depletion of acetyl-CoA, but by relatively smaller losses in ATP content and cell viability (about 5%). On the contrary, SN56 cells appeared to be insensitive to direct exposition to high concentration of lipopolysaccharide. However, exogenous nitric oxide resulted in marked inhibition pyruvate dehydrogenase and aconitase activities, depletion of acetyl-CoA, along with respective loss of SN56 cells viability. These data indicate that these two common neurodegenerative signals may differentially affect energy-acetyl-CoA metabolism in microglial and cholinergic neuronal cell compartments in the brain. Moreover, microglial cells appeared to be more resistant than neuronal cells to acetyl-CoA and ATP depletion evoked by these neurodegenerative conditions. Together, these data indicate that differential susceptibility of microglia and cholinergic neuronal cells to neurotoxic signals may result from differences in densities of toll-like receptors and degree of disequilibrium between acetyl-CoA provision in mitochondria and its utilization for energy production and acetylation reactions in each particular group of cells. There are significant differences between acetyl-CoA and ATP levels and enzymes of acetyl-CoA metabolism in non-activated microglial N9 and non-differentiated cholinergic SN56 neuroblastoma cells. Pathological stimulation of microglial toll-like receptors (TLRs) triggered excessive synthesis of microglia-derived nitric oxide (NO)/NOO radicals that endogenously inhibited pyruvate dehydrogenase complex (PDHC), aconitase, and α-ketoglutarate dehydrogenase complex. However, it caused none or small suppressions of acetyl-CoA and microglial viability, respectively. Microglia-derived NO inhibited same enzymes in cholinergic neuronal cells causing marked viability loss because of acetyl-CoA deficits evoked by its competitive consumption by energy producing and acetylcholine/N-acetyl-l-aspartate (NAA) synthesizing pathways.

Intracellular redistribution of acetyl-CoA, the pivotal point in differential susceptibility of cholinergic neurons and glial cells to neurodegenerative signals.

Intramitochondrial decarboxylation of glucose-derived pyruvate by PDHC (pyruvate dehydrogenase complex) is a principal source of acetyl-CoA, for mitochondrial energy production and cytoplasmic synthetic pathways in all types of brain cells. The inhibition of PDHC, ACO (aconitase) and KDHC (ketoglutarate dehydrogenase complex) activities by neurodegenerative signals such as aluminium, zinc, amyloid ß-peptide, excess nitric oxide (NO) or thiamine pyrophosphate deficits resulted in much deeper losses of viability, acetyl-CoA and ATP in differentiated cholinergic neuronal cells than in non-differentiated cholinergic, and cultured microglial or astroglial cell lines. In addition, in cholinergic cells, such conditions caused inhibition of ACh (acetylcholine) synthesis and its quantal release. Furthermore, cholinergic neuronal cells appeared to be resistant to high concentrations of LPS (lipopolysaccharide). In contrast, in microglial cells, low levels of LPS caused severalfold activation of NO, IL-6 (interleukin 6) and TNFα (tumour necrosis factor α) synthesis/release, accompanied by inhibition of PDHC, KDHC and ACO activities, and suppression of acetyl-CoA, but relatively small losses in their ATP contents and viability parameters. Compounds that protected these enzymes against inhibitory effects of neurotoxins alleviated acetyl-CoA and ATP deficits, thereby maintaining neuronal cell viability. These data indicate that preferential susceptibility of cholinergic neurons to neurodegenerative insults may result from competition for acetyl-CoA between mitochondrial energy-producing and cytoplasmic ACh-synthesizing pathways. Such a hypothesis is supported by the existence of highly significant correlations between mitochondrial/cytoplasmic acetyl-CoA levels and cell viability/transmitter functions respectively.

The Regulatory Effects of Acetyl-CoA Distribution in the Healthy and Diseased Brain.

Brain neurons, to support their neurotransmitter functions, require a several times higher supply of glucose than non-excitable cells. Pyruvate, the end product of glycolysis, through pyruvate dehydrogenase complex reaction, is a principal source of acetyl-CoA, which is a direct energy substrate in all brain cells. Several neurodegenerative conditions result in the inhibition of pyruvate dehydrogenase and decrease of acetyl-CoA synthesis in mitochondria. This attenuates metabolic flux through TCA in the mitochondria, yielding energy deficits and inhibition of diverse synthetic acetylation reactions in all neuronal sub-compartments. The acetyl-CoA concentrations in neuronal mitochondrial and cytoplasmic compartments are in the range of 10 and 7 µmol/L, respectively. They appear to be from 2 to 20 times lower than acetyl-CoA Km values for carnitine acetyltransferase, acetyl-CoA carboxylase, aspartate acetyltransferase, choline acetyltransferase, sphingosine kinase 1 acetyltransferase, acetyl-CoA hydrolase, and acetyl-CoA acetyltransferase, respectively. Therefore, alterations in acetyl-CoA levels alone may significantly change the rates of metabolic fluxes through multiple acetylation reactions in brain cells in different physiologic and pathologic conditions. Such substrate-dependent alterations in cytoplasmic, endoplasmic reticulum or nuclear acetylations may directly affect ACh synthesis, protein acetylations, and gene expression. Thereby, acetyl-CoA may regulate the functional and adaptative properties of neuronal and non-neuronal brain cells. The excitotoxicity-evoked intracellular zinc excess hits several intracellular targets, yielding the collapse of energy balance and impairment of the functional and structural integrity of postsynaptic cholinergic neurons. Acute disruption of brain energy homeostasis activates slow accumulation of amyloid-ß1-42 (Aß). Extra and intracellular oligomeric deposits of Aß affect diverse transporting and signaling pathways in neuronal cells. It may combine with multiple neurotoxic signals, aggravating their detrimental effects on neuronal cells. This review presents evidences that changes of intraneuronal levels and compartmentation of acetyl-CoA may contribute significantly to neurotoxic pathomechanisms of different neurodegenerative brain disorders.

Short-term effects of zinc on acetylcholine metabolism and viability of SN56 cholinergic neuroblastoma cells.

Excessive accumulation of zinc in the brain is one of putative factors involved in pathomechanism of cholinergic encephalopathies. The aim of this work was to investigate whether short-term increase of zinc concentration in the extracellular space may affect energy and acetylcholine metabolism in SN56 cholinergic cells of septal origin. Short 30 min exposition of SN56 cells to increasing zinc levels caused greater loss of viability of differentiated (DC, [EC(0.4)] 0.09 mM) than nondifferentiated cells (NC, [EC(0.4)] 0.14 mM). Concentration-dependent accumulation of zinc displayed exponential non-saturable kinetics. Zinc accumulation caused the decrease of calcium accumulation in mitochondria and its increase in cytoplasmic compartment of SN56 cells. Significant inverse and direct correlations were found between zinc accumulation and calcium levels in mitochondrial (r=-0.96, p=0.028) and cytoplasmic (r=0.97, p=0.028) compartments of DC, respectively. Zinc exerted similar inhibition of pyruvate dehydrogenase, aconitase and isocitrate dehydrogenase both in NC and DC homogenates, at Ki values equal to about 0.07, 0.08 and 0.005 mM, respectively. On the other hand, ketoglutarate dehydrogenase activity in DC was inhibited by zinc (Ki 0.0005 mM) 8 times stronger that in NC (Ki 0.004 mM). Also zinc-evoked decreases in acetylcholine content and its release were significantly greater in DC than in NC. Same conditions caused suppression of cytoplasmic and mitochondrial content of acetyl-CoA, that positively correlated with inhibition of transmitter functions (r=0.995, p=005) and loss of cell viability (r=0.990, p=0.0006), respectively. Significant correlations were also found in zinc-challenged cells between pyruvate dehydrogenase activity and both mitochondrial acetyl-CoA content and cell viability. These data indicate that pyruvate dehydrogenase-dependent acetyl-CoA synthesis in neuronal mitochondria may be a primary target for short-term neurotoxic effects of zinc. In consequence, shortages of acetyl-CoA in the mitochondrial compartment would cause fast loss of functional and structural integrity of cholinergic neurons.