Joseph A. Bobich

Incubation of nerve endings with a physiological concentration of Aβ1-42 activates CaV2.2(N-Type)-voltage operated calcium channels and acutely increases glutamate and noradrenaline release

We wish to understand the normal function of amyloid-beta peptides (Abeta) and to see if they destabilize neuronal calcium homeostasis [Mattson et al., J. Neurosci. 12 (1992), 376-389]. We observed that a physiological concentration (10 nM) of Abeta1-42 increased both glutamate and noradrenaline exocytosis from rat cortical nerve endings at least in part by activation of N-type Ca2+ channels. Abeta oligomers rather than monomers or fibrils probably are the most active form. Three alternatively-proposed effects of Abeta (reactive oxygen species formation, membrane perforation, and disruption of Ca2+ stores) also were tested by incubating nerve endings with a relatively high (by this studys standards) concentration of Abeta1-42(100 nM). None of the three proposed effects were detected during these incubations. These results support the hypothesis that persistent elevations of Abeta, which normally operates as a modulator of N-type voltage gated calcium channels, could increase internal nerve ending Ca2+ and excitatory neurotransmitter release to produce the early neurotoxic effects that eventually lead to Alzheimers disease.

Neuronal calcium sensor-1 facilitates neuronal exocytosis through phosphatidylinositol 4-kinase

This work tested the theory that neuronal calcium sensor‐1 (NCS‐1) has effects on neurotransmitter release beyond its actions on membrane channels. We used nerve‐ending preparations where membrane channels are bypassed through membrane permeabilization made by mechanical disruption or streptolysin‐O. Nerve ending NCS‐1 and phosphatidylinositol 4‐kinase (PI4K) are largely or entirely particulate, so their concentrations in nerve endings remain constant after breaching the membrane. Exogenous, myristoylated NCS‐1 stimulated nerve ending phosphatidylinositol 4‐phosphate [PI(4)P] synthesis, but non‐myristoylated‐NCS‐1 did not. The N‐terminal peptide of NCS‐1 interfered with PI(4)P synthesis, and with spontaneous and Ca2+‐evoked release of both [3H]‐norepinephrine (NA) and [14C]‐glutamate (glu) in a concentration‐dependent manner. An antibody raised against the N‐terminal of NCS‐1 inhibited perforated nerve ending PI(4)P synthesis, but the C‐terminal antibody had no effects. Antibodies against the N‐ and C‐termini of NCS‐1 caused significant increases in mini/spontaneous/stimulation‐independent release of [3H]‐NA from perforated nerve endings, but had no effect on [14C]‐glu release. These results support the idea that NCS‐1 facilitates nerve ending neurotransmitter release and phosphoinositide production via PI4K and localizes these effects to the N‐terminal of NCS‐1. Combined with previous work on the regulation of channels by NCS‐1, the data are consistent with the hypothesis that a NCS‐1–PI4K (NP, neuropotentiator) complex may serve as an essential linker between lipid and protein metabolism to regulate membrane traffic and co‐ordinate it with ion fluxes and plasticity in the nerve ending.

A sequential view of neurotransmitter release.

Only a few years ago it was thought that a single Ca2+-dependent membrane binding protein might control regulated exocytosis, but it is now clear that the coordinated actions of a large number of proteins and lipids are required for the precise targeting, docking and fusion of vesicles to the plasma membrane. Thinking was focused in 1993 by the SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) hypothesis, which proposed that certain synaptic vesicle membrane proteins combined specifically with particular proteins in the synaptic membrane active zone to form a complex that interacted with synaptoplasmic proteins, ATP and calcium ions to fuse the vesicles with the presynaptic membrane. Much research that has followed has verified the basic predictions of the SNARE hypothesis. However, recent research indicates that SNARE proteins are more widely distributed in secretory systems and that the sequence in which the proteins function may not occur as was originally proposed. That has recently produced a period of deconstruction and reinterpretation of the SNARE hypothesis. Our present state of knowledge is briefly summarized in this review.

ADP-ribosylation factor6 regulates both [3H]-noradrenaline and [14C]-glutamate exocytosis through phosphatidylinositol 4,5-bisphosphate.

GTP phosphohydrolase (cell regulating) (EC 3.6.1.47, ADP-ribosylation factor6, ARF6) has been shown to play an important role in different steps of membrane trafficking. It also regulates chromaffin granule exocytosis through phosphatidylcholine phosphatidohydrolase (EC 3.1.4.14, PLD) activation. In this study, the role of ARF6 in neurotransmitter release from both dense-core granules (DCGs) and synaptic vesicles (SVs) in rat brain cortex nerve endings was investigated. We observed that synaptosomal ARF6 is largely particulate but moves to a less easily pelleted compartment upon nerve ending stimulation. We also found that direct inhibition of ARF6 by a specific antibody or interference with ARF6 downstream effects by a myristoylated N-terminal ARF6 peptide both significantly decreased both [3H]-noradrenaline and [14C]-glutamate exocytosis. Addition of phosphatidic acid (PA) and phosphatidylinositol 4,5-bisphosphate (PIP2) partially or completely restored exocytosis. These findings suggest that ARF6 plays important regulatory roles for both DCG and SV exocytosis by activating PLD and ATP:1-phosphatidyl-1D-myo-inositol 4-phosphate 5-phosphotransferase (EC 2.7.1.68, PI4P-5K) to enhance PIP2 synthesis and nerve ending membrane trafficking.

Phosphatidylinositol 4,5-bisphosphate promotes both [3H]-noradrenaline and [14C]-glutamate exocytosis from nerve endings.

Inhibition of phosphatidylinositol 4-phosphate (PI4P) and phosphatidylinositol 4,5-bisphosphate (PI4,5P(2)) synthesis by phenylarsine oxide (PAO) inhibits both [3H]-noradrenaline ([3H]-NA) and [14C]-glutamate ([14C]-glu) exocytosis from streptolysin-O (SLO)-perforated synaptosomes. When PI4,5P(2) is blocked by an antibody or chelated by neomycin, neurotransmitter exocytosis again is inhibited. Also, when phosphoinositide (PI) synthesis is indirectly decreased by shunting phosphatidic acid (PA) synthesis into phosphatidylbutanol production, both [14C]-glutamate and [3H]-noradrenaline exocytosis are inhibited. All of these results indicate that PI4,5P(2) is necessary for exocytosis of both synaptic vesicles (SVs) and dense core vesicles (DCVs).

[3H]-Noradrenaline release from streptolysin-O permeated rat cortical synaptosomes: enhancement of the Ca2+-induced signal

We examined [3H]-noradrenaline ([3H]-NA) release from rat brain cortical synaptosomes permeated with streptolysin-O (SLO) under a variety of conditions. Three temperatures (20 degrees C, 25 degrees C, and 30 degrees C) were tested at different times of permeation. Lowering the incubation temperature to 20 degrees C decreased basal release, but Ca(2+)-induced [3H]-NA release increased slightly. Also, the incubation time to achieve the maximal ratio of Ca(2+)-induced release to basal release shifted to longer times with decreasing incubation temperature. If the synaptosomes were permeated with SLO before release was triggered, similar results were observed. Permeation at 20 degrees C allowed [gamma-32P] ATP and cAMP-dependent protein kinase (PKA) catalytic subunit to rapidly enter the synaptosomes to phosphorylate synapsins. Lactate dehydrogenase (LDH) efflux was time- and SLO-concentration dependent. The fact that 0.1 mM Cd2+ did not inhibit [3H]-NA release from permeabilized synaptosomes indicated that permeabilization by SLO was complete under these conditions. This also suggests that the release machinery involved after Ca2+ entry is not sensitive to Cd2+.

A double-labeled preparation for simultaneous measurement of [3H]-noradrenaline and [14C]-glutamic acid exocytosis from streptolysin-O (SLO)-perforated synaptosomes

We have developed a novel method to examine [3H]-noradrenaline and [14C]-glutamate release from the same sample of streptolysin-O (SLO) perforated rat cortical synaptosomes. Ca2+ -dependent [3H]-noradrenaline and [14C]-glutamate release was examined at different temperatures and was found to be greater at 30 degrees C than at 25 degrees C. Ca2+ -dependent release of [3H]-noradrenaline is more ATP dependent than Ca2+ -dependent release of [14C]-glutamate. No significant reuptake of either neurotransmitter by the perforated synaptosomes was detected, indicating all the synaptosomes were indeed perforated. Incubations with 1 mM ouabain, a specific Na+,K+ -ATPase inhibitor, slightly increased Ca2+ -dependent release of both neurotransmitters. [3H]-noradrenaline is released from large dense-core vesicles and [14C]-glutamate is released from small clear synaptic vesicles, so one can directly compare and contrast neurotransmitter release mechanisms between large dense-core vesicles and small clear synaptic vesicles using this preparation.

[3H]-noradrenaline secretion from rat cortex synaptosomes perforated with Staphylococcus aureus α-toxin

Rat cortex synaptosomes have been successfully perforated with high concentrations (> or = 400 U/ml) of Staphylococcus aureus alpha-toxin. The free Ca2+-concentration dependence of [3H]-noradrenaline release was similar to that observed for PC 12 and chromaffin cells. Release from the alpha-toxin perforated synaptosomes was not significantly inhibited by omega-conotoxin GVIA. Initially, Ca2+-dependent release was independent of MgATP (for 0.5 min), but became increasingly dependent on MgATP with time. Lactate dehydrogenase efflux from alpha-toxin-perforated synaptosomes was not different than efflux from control synaptosomes, and an antibody to N-ethylmaleimide-sensitive fusion protein did not enter the synaptosomes. [3H]-noradrenaline release was temperature and alpha-toxin-concentration dependent. Ca2-dependent release was more resistant to rundown from alpha-toxin- than from streptolysin-O-perforated synaptosomes. This preparation of perforated synaptosomes should be useful for studies of regulated exocytosis from nerve endings.

How the ASBMB recommended curriculum has influenced one sole biochemist.

Texas Christian University (TCU) is affiliated with the Disciples of Christ Church to the extent that it has a divinity school and, until 5 years ago, the Chancellor always had been a Minister of the Disciples. It has, since its opening in 1873, gradually increased its enrollment to the current 7500 undergraduates and approximately 1000 graduate students. It is classified as a “Doctoral/Research University-Intensive” by the Carnegie Classification of Institutes of Higher Education. The TCU School of Science and Engineering offers Ph.D. degrees in chemistry, physics and astronomy, and psychology, and M.S. degrees in biology, chemistry, geology, mathematics, and psychology. The Department of Chemistry also offers a B.A., an American Chemical Society (ACS)-certified B.S. degree in Chemistry and a B.S. in biochemistry. Roughly half of our undergraduates get ACS-certified degrees. Unfortunately, the department does not yet collect data about what all of our undergraduates do after graduation. There are 10 faculty members in the Department of Chemistry plus the (currently vacant) Robert A. Welch Chair, along with 17 graduate students. Chemistry majors in the last 3 years have numbered 24, 29, and 25, respectively, but biochemistry majors have grown from five to eight to 11, respectively, perhaps because a 4-year B.S. degree plan in Biochemistry is sent to all entering freshmen. The fact that the numbers of chemistry majors has not decreased, although the number of biochemistry majors has increased, suggests that adding the B.S. in biochemistry may help someday to increase the total number of departmental majors. As seems typical, these numbers are dwarfed by the 327–342 biology majors each year in the same time period (2000–2002). The TCU biochemistry program has had a tortuous history. Thirty years ago the Chemistry and Biology Departments independently each hired a biochemist, bringing the university’s complement of biochemists to three, and a B.S. degree in biochemistry was offered in the Chemistry Department. Gradually, the number of biochemists in the Biology Department was reduced to one and then zero, and it was felt that one biochemist was not enough to offer a viable B.S. degree, so the degree was ended. The undergraduate curriculum endorsed by the American Society of Biochemistry and Molecular Biology (ASBMB) and published in Biochemistry and Molecular Biology Education journal in 1992, was a revelation. By examining the combined offerings of the Chemistry and Biology Departments, TCU could prepare a series of courses that fulfilled the recommended criteria for a viable and valid degree. Shortly thereafter, the B.S. in biochemistry was reinstituted at TCU. The required science and mathematics courses are listed in Table I (note, however, that the five-digit undergraduate catalog numbers have been dropped). Obviously, the degree program closely mirrors the suggestions of the American Chemical Society because it had to be approved by the Chemistry Department, but it was not examined by the Biology Department, hence the requirement for inorganic chemistry, for example. Biochemistry majors are supposed to take the Honors sections of Introductory Biology, but this requirement can be, and frequently is, waived for transfer students. The first 2 years of biology and chemistry are fairly typical, as far as I can observe. The Honors chemistry sequence includes one 3-h laboratory every week. Organic chemistry is a course used to suggest to lazy or otherwise undesirable students that they should consider other educational opportunities. The physics sequence is calculus-based, and the genetics course and laboratory consist of 3 and 6 contact h, respectively, per week. Introductory physical chemistry course content is more biochemistry-friendly than the alternative first-semester physical chemistry course. The research requirement can be performed with any chemistry faculty member. Students in our Honors program must deliver an oral presentation of their research in the Spring during Honors Week. Undergraduates not in the Honors program have no such requirement for a formal presentation of their work. It seems to me that the newly revised ASBMB-recommended curriculum [1] will be more beneficial to solefaculty biochemists and molecular biologists than any other group of biochemists and molecular biologists. A * Professor of Chemistry. To whom correspondence should be addressed. E-mail: j.bobich@tcu.edu. 1 The abbreviations used are: TCU, Texas Christian University; ASBMB, American Society of Biochemistry and Molecular Biology.

Cation effects on the conformations of muscle and non-muscle α-actinins

We examined the effects of changing KCl concentration on the secondary structures of α-actinins using circular dichroism (CD), 1,1′-bis(4-anilino) naphthalene-5,5′-disulfonic acid (bisANS) fluorescence and proteolysis experiments. Under near-physiological conditions, divalent cations also were added and changes in conformation were investigated. In 25 mm KH2PO4, pH 7.5, increasing KCl from 0 to 120 mm led to decreases in α-helix conformation for brain, platelet and heart α-actinins (40.5-30.2%, 65.5-37.8% and 37.5-27.8%, respectively). In buffered 120 mm KCl, 0.65 mm calcium produced small changes in the CD spectra of both brain and platelet α-actinin, but had no effect on heart α-actinin. bisANS fluorescence of all three α-actinins also showed significant changes in conformation with increasing KCl. However, in buffered 120 mm KCl increasing concentrations of Ca2+ or Mg2+ did not have significant effects on the bisANS fluorescence of any α-actinin. Digestion of brain, platelet and heart α-actinins with α-chymotrypsin showed an increase of proteolytic susceptibility in 120 mm KCl. These experiments also showed that increasing the concentration of Ca2+ or Mg2+ led to greater changes in digestion fragment patterns in the absence of KCl than in the presence of 120 mm KCl. The results suggest that α-actinins exist in different conformations depending on the ionic strength of the medium, which could explain the differences in calcium and F-actin binding results obtained from different α-actinins.