Phillip E. Kish

Glutamate accumulation into synaptic vesicles.

Publisher Summary This chapter discusses the glutamate accumulation into synaptic vesicles. A critical, but rate-limiting step, in the study of glutamate transport into synaptic vesicles is the preparation of synaptic vesicles that are free of contamination yet retain functional capability. However, this requires rather large quantities of affinity-purified antibodies to a synaptic vesicle protein, synapsin I, and, despite this requirement, the yield of such a vesicle preparation is too low to permit the analysis of vesicular glutamate uptake in small tissues. The chapter describes the assay and properties of the glutamate transport system in the synaptic vesicle in three preparations: (1) bovine brain synaptic vesicles highly purified by the use of antisynapsin I immunoglobulin G (IgG), (2) a bovine brain synaptic vesicle fraction purified by sucrose density gradient centrifugation, and (3) a rat brain crude synaptic vesicle fraction. Beyond the characterization of the properties of glutamate uptake into synaptic vesicles, little research has investigated in vivo changes in vesicular uptake.

Glutamate uptake into synaptic vesicles: competitive inhibition by bromocriptine.

Abstract: The ATP‐dependent uptake of l‐glutamate into synaptic vesicles has been well characterized, implicating a key role for synaptic vesicles in glutamatergic neurotransmission. In the present study, we provide evidence that vesicular glutamate uptake is selectively inhibited by the pep‐tide‐containing halogenated ergot bromocriptine. It is the most potent inhibitor of the agents tested; the IC5o was de‐termined to be 22 μM. The uptake was also inhibited by other ergopeptines such as ergotamine and ergocristine, but with less potency. Ergots devoid of the peptide moiety, however, such as ergonovine, lergotrile, and methysergide, had little or no effect. Although bromocriptine is known to elicit dopaminergic and serotonergic effects, its inhibitory effect on vesicular glutamate uptake was not mimicked by agents known to interact with dopamine and serotonin receptors. Kinetic data suggest that bromocriptine competes with glutamate for the glutamate binding site on the glutamate trans‐locator. It is proposed that this inhibitor could be useful as a prototype probe in identifying and characterizing the vesicular glutamate translocator, as well as in developing a more specific inhibitor of the transport system.

Magnetic resonance imaging of ethyl-nitrosourea-induced rat gliomas: a model for experimental therapeutics of low-grade gliomas.

Human low-grade gliomas represent a population of brain tumors that remain a therapeutic challenge. Preclinical evaluation of agents, to test their preventive or therapeutic efficacy in these tumors, requires the use of animal nobreak models. Spontaneous gliomas develop in models of chemically induced carcinogenesis, such as in the transplacental N-ethyl-N-nitrosourea (ENU) rat model. However, without the ability to detect initial tumor formation, multiplicity or to measure growth rates, it is difficult to test compounds for their interventional or preventional capabilities. In this study Fisher-334 rats, treated transplacentally with ENU, underwent magnetic resonance imaging (MRI) examination in order to evaluate this approach for detection of tumor formation and growth. ENU-induced intracranial cerebral tumors were first observable in T2-weighted images beginning at 4 months of age and grew with a mean doubling time of 0.487 ± 0.112 months. These tumors were found histologically to be predominately mixed gliomas. Two therapeutic interventions were evaluated using MRI, vitamin A (all-trans retinol palmitate, RP), as a chemopreventative agent and the anti-angiogenic drug SU-5416. RP was found to significantly delay the time to first tumor observation by one month (P = 0.05). No differences in rates of tumor formation or growth rates were observed between control and RP-treated groups. MRI studies of rats treated with SU-5416 resulted in reduction in tumor growth rates compared to matched controls. These results show that MRI can be used to provide novel information relating to the therapeutic efficacy of agents against the ENU-induced tumor model.

Ontogeny of glutamate accumulating activity in rat brain synaptic vesicles.

The relationship between the ontogeny of the vesicular glutamate uptake system and synaptogenesis in rats was investigated. For this purpose we have developed a simplified procedure for the preparation of crude synaptic vesicles which are sufficiently pure to demonstrate a highly ATP-dependent glutamate uptake. ATP-dependent glutamate uptake into synaptic vesicles was found to increase dramatically starting on postnatal day 10 and reaching a maximum on day 30 (76 +/- 40 and 657 +/- 40 pmol/mg protein/10 min, respectively), correlating well with the active period of synaptogenesis. Stimulation of uptake by chloride also developed in parallel with the vesicular glutamate uptake. In contrast, combined non-ATP-dependent uptake and non-specific binding remained constant (21 +/- 6 pmol/mg protein/10 min). This development of vesicular glutamate uptake during the period of synaptogenesis supports the notion that synaptic vesicles play an important role in glutamate synaptic transmission.

The Eye as an Organizer of Craniofacial Development

The formation and invagination of the optic stalk coincides with the migration of cranial neural crest (CNC) cells, and a growing body of data reveals that the optic stalk and CNC cells communicate to lay the foundations for periocular and craniofacial development. Following migration, the interaction between the developing eye and surrounding periocular mesenchyme (POM) continues, leading to induction of transcriptional regulatory cascades that regulate craniofacial morphogenesis. Studies in chick, mice, and zebrafish have revealed a remarkable level of genetic and mechanistic conservation, affirming the power of each animal model to shed light on the broader morphogenic process. This review will focus on the role of the developing eye in orchestrating craniofacial morphogenesis, utilizing morphogenic gradients, paracrine signaling, and transcriptional regulatory cascades to establish an evolutionarily‐conserved facial architecture. We propose that in addition to the forebrain, the eye functions during early craniofacial morphogenesis as a key organizer of facial development, independent of its role in vision. genesis 49:222–230, 2011.

Synaptic Vesicular Glutamate Uptake: Modulation by a Synaptosomal Cytosolic Factor

Abstract: We have demonstrated previously that L‐glutamate is taken up into isolated synaptic vesicles in an ATP‐dependent manner, supporting the neurotransmitter role of this acidic amino acid. We now report that a nerve terminal cytosolic factor inhibits the ATP‐dependent vesicular uptake of glutamate in a dose‐dependent manner. This factor appears to be a protein with a molecular weight >100,000, as estimated by size exclusion chromatography, and is precipitated by ammonium sulfate (40% saturation). The inhibitory factor is inactivated by heating to 100°C. Proteolytic digestion of the ammonium sulfate fraction by trypsin or chymotrypsin did not reduce, but rather increased slightly, the inhibition of glutamate uptake. Unlike the native factor, the digest retained inhibitory activity after heating, suggesting that proteolytic digestion may generate active fragments. The inhibition of ATP‐dependent vesicular glutamate uptake is not species‐specific, as the factor obtained from both rat and bovine brains produced an equal degree of inhibition of glutamate uptake into vesicles of each species. These observations raise the possibility that vesicular uptake of glutamate may be regulated by an endogenous factor in vivo.

Calcium-dependent release of accumulated glutamate from synaptic vesicles within permeabilized nerve terminals

We have studied glutamate release from synaptic vesicles in permeabilized synaptosomes, which were preloaded with [3H]glutamate in an ATP-dependent manner. The release was found to be calcium-dependent and to require a heat-labile cytosolic macromolecule factor for maximum activity. Maximal release occurred at 5 microM free Ca2+ and within 5 min. Of the other divalent cations tested, only barium stimulated release of vesicular glutamate. The release was inhibited by N-ethylmaleimide. These results are characteristic of exocytotic release of monoamines and peptides observed in endocrine systems, and constitute direct evidence for the notion that calcium-dependent release of glutamate originates from the vesicular pool.

Natural variability of Kozak sequences correlates with function in a zebrafish model

In eukaryotes, targeting the small ribosomal subunit to the mRNA transcript requires a Kozak sequence at the translation initiation site. Despite the critical importance of the Kozak sequence to regulation of gene expression, there have been no correlation studies between its natural variance and efficiency of translation. Combining bioinformatics analysis with molecular biology techniques, and using zebrafish as a test case, we identify Kozak sequences based on their natural variance and characterize their function in vivo. Our data reveal that while the canonical Kozak sequence is efficient, in zebrafish it is neither the most common nor the most efficient translation initiation sequence. Rather, the most frequent natural variation of the Kozak sequence is almost twice as efficient. We conclude that the canonical Kozak sequence is a poor predictor of translation efficiency in different model organisms. Furthermore, our results provide an experimental approach to testing and optimizing an important tool for molecular biology.

Autophagy regulates cytoplasmic remodeling during cell reprogramming in a zebrafish model of muscle regeneration

ABSTRACT Cell identity involves both selective gene activity and specialization of cytoplasmic architecture and protein machinery. Similarly, reprogramming differentiated cells requires both genetic program alterations and remodeling of the cellular architecture. While changes in genetic and epigenetic programs have been well documented in dedifferentiating cells, the pathways responsible for remodeling the cellular architecture and eliminating specialized protein complexes are not as well understood. Here, we utilize a zebrafish model of adult muscle regeneration to study cytoplasmic remodeling during cell dedifferentiation. We describe activation of autophagy early in the regenerative response to muscle injury, while blocking autophagy using chloroquine or Atg5 and Becn1 knockdown reduced the rate of regeneration with accumulation of sarcomeric and nuclear debris. We further identify Casp3/caspase 3 as a candidate mediator of cellular reprogramming and Fgf signaling as an important activator of autophagy in dedifferentiating myocytes. We conclude that autophagy plays a critical role in cell reprogramming by regulating cytoplasmic remodeling, facilitating the transition to a less differentiated cell identity.

Fgf regulates dedifferentiation during skeletal muscle regeneration in adult zebrafish

Fibroblast growth factors (Fgfs) regulate critical biological processes such as embryonic development, tissue homeostasis, wound healing, and tissue regeneration. In zebrafish, Fgf signaling plays an important role in the regeneration of the spinal cord, liver, heart, fin, and photoreceptors, although its exact mechanism of action is not fully understood. Utilizing an adult zebrafish extraocular muscle (EOM) regeneration model, we demonstrate that blocking Fgf receptor function using either a chemical inhibitor (SU5402) or a dominant-negative transgenic construct (dnFGFR1a:EGFP) impairs muscle regeneration. Adult zebrafish EOMs regenerate through a myocyte dedifferentiation process, which involves a muscle-to-mesenchyme transition and cell cycle reentry by differentiated myocytes. Blocking Fgf signaling reduced cell proliferation and active caspase 3 levels in the regenerating muscle with no detectable levels of apoptosis, supporting the hypothesis that Fgf signaling is involved in the early steps of dedifferentiation. Fgf signaling in regenerating myocytes involves the MAPK/ERK pathway: inhibition of MEK activity with U0126 mimicked the phenotype of the Fgf receptor inhibition on both muscle regeneration and cell proliferation, and activated ERK (p-ERK) was detected in injured muscles by immunofluorescence and western blot. Interestingly, following injury, ERK2 expression is specifically induced and activated by phosphorylation, suggesting a key role in muscle regeneration. We conclude that the critical early steps of myocyte dedifferentiation in EOM regeneration are dependent on Fgf signaling.