Current Research Interests:
This laboratory is concerned with the biochemistry, function and regulation of synaptic vesicles and protein phosphorylation in the central nervous system.
Glutamate is now recognized as the major excitatory neurotransmitter in the vertebrate central nervous system. As such, proper glutamate synaptic transmission is implicated in learning and memory as well as in basic neuronal communication, and abnormal glutamate transmission is involved in various types of brain diseases, including certain forms of seizures, stroke, Parkinson’s disease, Huntington’s disease, amyloid lateral sclerosis, and potentially schizophrenia. Although glutamate is a common biochemical involved in a number of metabolic pathways in all cells, it enters the neurotransmitter pathway via a specific glutamate transporter in the synaptic vesicle present in the nerve ending. The glutamate molecules thus accumulated in synaptic vesicles are thought to be eventually released into the synaptic cleft, the small gap between the nerve ending and the next neuron, when an electrical impulse propagated along the axon arrives at the nerve terminal. The synaptically released glutamate then excites the postsynaptic neuron through activation of a receptor/channel complex. Thus, the glutamate transporter in the synaptic vesicle membrane plays an important role in initially directing glutamate to the neurotransmitter pathway away from the metabolic pathway.
We discovered that the vesicular glutamate is specific for glutamate among natural amino acids and does not recognize the close analog aspartate. This property is important in selectively allowing glutamate to enter the neurotransmitter pathway. We have studied substrate structural requirements and have shown, using a 3-D structural modeling, why aspartate does not interact with the vesicular glutamate transporter (VGLUT).
This and other laboratories previously provided evidence that glutamate transport into synaptic vesicles is driven by an electrochemical proton gradient generated by a V-type H +-pump ATPase. We obtained evidence that the glutamate transporter and the H +-pump ATPase can be physically separated and, when both are incorporated into liposomes, the ATP-dependent glutamate uptake system is reconstituted. This is the first evidence suggesting that the vesicular glutamate transporter is a physically distinct entity from the V-type H +-pump ATPase. The reconstituted active transport system has revealed characteristics indistinguishable from those observed with the intact synaptic vesicles. This represents a significant step in identification and characterization of the vesicular glutamate transporter.
We have been concerned with the regulation of vesicular glutamate storage. Along this line, we found evidence for the existence of a proteinaceous factor which inhibits glutamate uptake into synaptic vesicles, and have been engaged in the purification of the factor. We have purified to apparent homogeneity a protein (Mr = 138,000) from brain cytosol that inhibits glutamate and γ-aminobutyric acid uptake into synaptic vesicles, and have termed this protein "inhibitory protein factor" (IPF). The hydrodynamic properties of IPF suggest that this is an elongated protein. Partial sequence analysis shows that IPF is derived from α-fodrin, a protein implicated in several diverse cellular activities. IPF a inhibits ATP-dependent glutamate uptake into purified synaptic vesicles with an IC 50 of ~26 nM, while showing no ability to inhibit ATP-independent uptake at concentrations up to 100 nM. Moreover, IPF a inhibited neither norepinephrine uptake into chromaffin vesicles nor Na+-dependent glutamate uptake into synaptosomes. However, IPF a inhibited uptake of γ-aminobutyric acid into synaptic vesicles derived from spinal cord, suggesting that inhibition may not be limited to glutamatergic systems. We propose that IPF could be a novel component of a presynaptic regulatory system. Such a system might modulate neurotransmitter accumulation into synaptic vesicles and thus regulate the overall efficacy of neurotransmission.
Despite the potent action of IPF and its striking sequence homology to α-fodrin, α-fodrin is devoid of the ability to inhibit vesicular glutamate uptake. How IPF is made from α-fodrin is an interesting question. Our sequence data indicate that the N-terminus amino acid of IPF is the 26th amino acid from the α-fodrin N-terminus. It is well known that α-fodrin is cleaved by the calcium-dependent proteolytic enzyme calpain into two fragments, one of which is known as the 155-kDa protein. However, we have observed that the calpain digests of purified α-fodrin exhibit no inhibitory activity, even at high concentrations. This suggests the importance of a distinct, specific proteolytic enzyme which cleaves the bond between the 25th amino acid (Arg) and the 26th amino acid (Tyr), in generating IPF from α-fodrin.
We have obtained evidence that when purified IPF is introduced into isolated nerve endings, a reduction in the amount of exocytotically released glutamate is produced. This suggests that IPF could be operative in the nerve terminal in vivo. We have also been engaged in the search for potent non-proteinaceous compounds which affect vesicular glutamate storage, and have found three classes of remarkably strong inhibitors. We have obtained evidence that a representative compound of one class permeates the plasma membrane, so that this affects vesicular glutamate content within the nerve terminal. Further studies suggested that the amount of exocytotically released neurotransmitter could be affected by alteration of the vesicular transport of neurotransmitters.
In addition, we have been investigating whether IPF or vesicular glutamate uptake has (or whether both have) a role in certain types of pathophysiology in the central nervous system. In collaboration with Dr. Thomas Seyfried’s laboratory at Boston College, we have completed our joint work on the relationship between seizure and vesicular glutamate uptake. We have produced statistically significant data indicating that vesicular glutamate uptake is increased, compared to age-matched control non-epileptic mice, in the cerebral cortex of the epileptic EL mouse, a genetic mutant used as a model for human complex partial seizure. Such a change was not observed in other brain regions such as the hippocampus, cerebellum, or brain stem, nor was it detected in young EL mice without seizure history. Thus, such an increase in vesicular glutamate uptake is brain region-specific and dependent on development and/or seizure experience. These observations suggest that enhanced vesicular glutamate uptake may be involved in maintaining seizure activity or could be an effect of seizure. This represents the first evidence suggesting that abnormal vesicular glutamate activity may underlie certain forms of pathophysiology in the central nervous system. Moreover, we have obtained evidence suggesting that certain forms of seizures are associated with reduced IPF content in the nerve ending cytosol in the hippocampus.
In recent years, we have been interested in the role of glucose metabolism in synaptic transmission, particularly aspects of glutamate uptake into and release from synaptic vesicles. My laboratory has obtained evidence that ATP produced by glycolytic enzymes (GAPDH, 3-phosphoglycerate kinase, and pyruvate kinase) on the surface of synaptic vesicles, rather than ATP synthesized in mitochondria, plays a major role in harnessing glutamate transport into synaptic vesicles. The local, glycolytic synthesis of ATP is faster and more efficient than mitochondrial synthesis of ATP in providing energy required for neurotransmitter uptake into synaptic vesicles. Thus, this demonstration would provide fresh insight into the longstanding question of why glucose metabolism is so crucial for synaptic transmission and brain function. This would also account at least in part for the observation that hypoglycemia results in rapid abnormal synaptic transmission without significantly changing average ATP concentration in tissue. We are currently concerned with the nature of association of glycolytic ATP-generating enzymes with synaptic vesicles.
More recently, we have found a new mechanism by which ATP is produced. We plan to investigate the functional role of this mechanism. We are also concerned with clarification of the precursor of the neurotransmitter glutamate.
Synaptic vesicle-bound pyruvate kinase can support vesicular glutamate uptake.
Ishida A, Noda Y, Ueda T.
Neurochem Res. 2009 May;34(5):807-18. Epub 2008 Aug 27.
The glutamate uptake system in presynaptic vesicles: further characterization of structural requirements for inhibitors and substrates.
Winter HC, Ueda T.
Neurochem Res. 2008 Feb;33(2):223-31. Epub 2007 Oct 17.
Inhibition of vesicular glutamate uptake by Rose Bengal-related compounds: structure-activity relationship.
Bole DG, Ueda T.
Neurochem Res. 2005 Mar;30(3):363-9.
Glycolysis and glutamate accumulation into synaptic vesicles. Role of glyceraldehyde phosphate dehydrogenase and 3-phosphoglycerate kinase.
Ikemoto A, Bole DG, Ueda T.
J Biol Chem. 2003 Feb 21;278(8):5929-40. Epub 2002 Dec 17.
Aberrant reduction of an inhibitory protein factor in a rat epileptic model.
Amano T, Matsubayashi H, Ozkan ED, Sasa M, Serikawa T, Ueda T.
Epilepsy Res. 2002 Sep;51(1-2):81-91.
Prolonged depolarization of rat cerebral synaptosomes leads to an increase in vesicular glutamate content.
Bole DG, Hirata K, Ueda T.
Neurosci Lett. 2002 Mar 29;322(1):17-20.
Inhibition of vesicular glutamate storage and exocytotic release by Rose Bengal.
Ogita K, Hirata K, Bole DG, Yoshida S, Tamura Y, Leckenby AM, Ueda T.
J Neurochem. 2001 Apr;77(1):34-42.
IPF, a vesicular uptake inhibitory protein factor, can reduce the Ca(2+)-dependent, evoked release of glutamate, GABA and serotonin.
Tamura Y, Ozkan ED, Bole DG, Ueda T.
J Neurochem. 2001 Feb;76(4):1153-64.
Glutamate transport and storage in synaptic vesicles.
Özkan, E.D., Ueda, T.
Jpn J Pharmacol. 1998;77:1-10.