Anne M. Heacock

Inositol Lipids and Signal Transduction in the Nervous System: An Update

Abstract: The role that inositol lipids play in cellular signaling events in eukaryotic cells remains one of the most intensively investigated areas of cell biology. In this respect, phosphoinositide‐mediated signal transduction in the CNS is no exception; major advances have been made since a previous review on this subject (Fisher and Agranoff, 1987). Not only have stimulated phosphoinositide turnover and its physiological sequelae been demonstrated repeatedly in a variety of neural preparations, but, in addition, the detailed molecular mechanisms underlying these events continue to unfold. Here we review the progress that has occurred in selected aspects of this topic since 1987. In the first two sections of this article, emphasis is placed on novel functional roles for the inositol lipids and on recent insights into the molecular characteristics and regulation of three key components of the phosphoinositide signal transduction system, namely, the inositol lipid kinases, phospholipases C (PLCs), and the inositol 1,4,5‐trisphosphate[I(1,4,5)P3] receptor. The metabolic fate of I(1,4,5)P3 in neural tissues, as well as its control, is also detailed. Later we focus on identification of the multiple receptor subtypes that are coupled to inositol lipid turnover and discuss possible strategies for intervention into phosphoinositide‐mediated signal transduction. Due to space limitations, an extensive evaluation of the diacylglycerol/protein kinase C (DAG/PKC) limb of the signal transduction pathway is not included (for reviews, see Nishizuka, 1988; Kanoh et al., 1990).

Protein synthesis and transport in the regenerating goldfish visual system

The nature of the proteins synthesized in the goldfish retina and axonally transported to the tectum during optic nerve regeneration has been examined. Electrophoretic analysis of labeled soluble retinal proteins by fluorography verified our previous observation of a greatly enhanced synthesis of the microtubule subunits. In addition, labeling of a tubulin-like protein in the retinal particulate fraction was also increased during regeneration. Like soluble tubulin, the particulate material had an apparent MW of 53–55K and could be tyrosylated in the presence of cycloheximide and [3H]tyrosine. Comparison of post-crush and normal retinal proteins by two-dimensional gel electrophoresis also revealed a marked enhancement in the labeling of two acidic 68–70K proteins. Analysis of proteins slowly transported to the optic tectum revealed changes following nerve crush similar to those observed in the retina, with enhanced labeling of both soluble and particulate tubulin and of 68–70K polypeptides. The most striking change in the profile of rapidly transported protein was the appearance of a labeled 45K protein which was barely detectable in control fish.

Enhanced Coupling of Neonatal Muscarinic Receptors in Rat Brain to Phosphoinositide Turnover

Abstract: The relationship between the density of the muscarinic receptor in developing rat cerebral cortex and its coupling to phosphoinositide turnover is examined. Tissue slices from rats of various ages were incubated with myo‐[2‐3H]inositol, and the effect of carbamoylcholine on the release of total inositol phosphates was determined. Binding of [3H]quinuclidinyl benzilate was determined in the same tissue. Although muscarinic receptor density in day‐18 embryonic cortex was only 5% of that in the adult, the maximal response of stimulated phosphoinositide turnover to carbamoylcholine (1–10 mM) was at the adult level (i.e., threefold increase). Comparison of the dependence of the turnover on carbamoylcholine concentration revealed that in neonates, the dose‐response curve was shifted to the left, giving a half‐maximal effect at concentrations approximately tenfold lower than that in the adult. In addition, the partial muscarinic agonists oxotremorine‐2 and bethanechol were both more efficacious in young rats than in adults. The differences could not be accounted for either by alterations in agonist affinity for the receptor or by the presence of “spare” muscarinic receptors. These results indicate that muscarinic receptors in fetal and newborn rat cerebral cortex are more efficiently coupled to stimulation of phosphoinositide turnover than in the adult.

A Putative M3 Muscarinic Cholinergic Receptor of High Molecular Weight Couples to Phosphoinositide Hydrolysis in Human SK-N-SH Neuroblastoma Cells

The M1‐selective (high affinity for pirenzepine) muscarinic acetylcholine receptor (mAChR) antagonist pirenzepine displaced both N‐[3H]methylscopolamine ([3H]NMS) and [3H]qui‐nuclidinylbenzilate from intact human SK‐N‐SH neuroblastoma cells with a low affinity (Ki= 869–1,066 nM), a result indicating the predominance of the M2 or M3 (low affinity for pirenzepine) receptor subtype in these cells. Whereas a selective M2 agent, AF‐DX 116 {11–2[[2‐[(diethylamino)methyl]‐1‐piperidinyl]‐acetyl]‐5,11‐dihydro‐6H‐pyrido[2,3‐b][1,4]benzodiazepin‐6‐one} bound to the mAChRs with a very low affinity (Ki= 6.0 μM), 4‐diphenylacetoxy‐N‐methylpiperidine methiodide (4‐DAMP), an agent that binds with high affinity to the M3 subtype, potently inhibited [3H]NMS binding (Ki= 7.2 nM). 4‐DAMP was also 1,000‐fold more effective than AF‐DX 1 16 at blocking stimulated phosphoinositide (PPI) hydrolysis in these cells. Covalent labeling studies (with [3H]propylbenzilylcholine mustard) suggest that the size of the SK‐N‐SH mAChR (Mr= 81.000–98,000) distinguishes it from the predominant mAChR species in rat cerebral cortex (Mr=66,000), an M1‐enriched tissue. These results provide the first demonstration of a neural M3 mAChR subtype that couples to PPI turnover.

Increased tubulin messenger RNA in the goldfish retina during optic nerve regeneration.

Axotomy induces profound morphological changes in the neuronal cell body 2,a,1°. Neurons whose axons lie within the central nervous system of higher vertebrates (intrinsic neurons) show little or no functional regenerative capability 4,8. However, many mammalian neurons whose axons lie outside the CNS, e.g., sensory and motor neuronsS, 15 and some intrinsic CNS neurons of lower vertebrates, such as teleosts and amphibia, can regenerate functional axons and re-establish normal synaptic fieldsT,9,11. This recuperative response of the perikaryon to axotomy provides a model for the study of regeneration as well as synaptogenesis and other events thought to underlie neuronal growth, recognition and plasticity. Regeneration of functional retinotectal fibers following optic nerve crush in goldfish is associated with a number of quantifiable biochemical changes in the retina, including increased nucleoside uptake and phosphorylation 1. There is also an enhanced labeling of cytoplasmic poly(A)-containing RNA 1 and of the microtubule protein tubulin 6. The enhanced radioisotopic labeling of goldfish retinal tubulin following optic nerve crush could be attributed either to its increased synthesis or decreased degradation. Since the former explanation could reflect regulation at the transcriptional or translational level and axotomy had resulted in enhanced labeling of poly(A)containing RNA, we investigated whether retinal poly(A)-RNA obtained following optic nerve crush was enriched in specific messenger RNAs, particularly that for tubulin. Translation of retinal poly(A)-containing RNA in a heterologous, cell-free protein synthesizing system, followed by SDS gel electrophoresis of the products, provided the necessary tools to detect the proposed changes in RNA populations. Cytoplasmic poly(A)-containing RNA preparations were isolated from both normal and from post-crush retinas, then incubated in a wheat germ protein synthesizing system. The protein labeling patterns from 4, 10 and 15 day post-crush incubations and their controls are shown in Fig. 1. A distinct increase in radioautographic density in the tubulin region of the gel was apparent in proteins translated

Reutilization of precursor following axonal transport of [3H]proline-labeled protein

In further studies on axonally transported protein in the goldfish visual system, the turnover of rapidly transported [3H]proline-labeled protein was examined. It was found that: (1) a fraction of the rapidly transported protein has a relatively short half-life; (2) [3H]proline released following proteolysis of transported protein is efficiently reutilized for tectal protein synthesis, as inferred from an increased labeling of nuclear protein in the contralateral tectum (COT) relative to that in the ipsilateral tectum (IOT); (3) a small amount of [3H]proline arrives in the COT by axonal flow of the free amino acid; and (4) [3H]leucine and [3H]asparagine are less efficiently reutilized than [3H]proline. These findings may relate to the phenomenon of transneuronal transfer of radioactivity which has been observed with [3H]proline as precursor. The extensive reutilization of [3H]proline may account for part or all of the labeling at secondary synaptic sites. The results suggest that asparagine may be highly suitable for radioautographic identification of primary neuronal fields.

CDP-DIACYLGLYCEROL SYNTHASE FROM MAMMALIAN TISSUES

CDP-diacylglycerol resides at the branch point of glycerolipid biosynthesis as precursor of both the phosphoinositides and phosphatidylglycerol. The discovery of the phosphoinositide signal transduction pathway and the recognition of its prominent role in intracellular communication has focused new attention on CDP-diacylglycerol synthase. As a rate-limiting step in this pathway, it is a likely target for regulation. Exploration of this possibility will be facilitated by the recent cloning of mammalian CDP-DAG synthase.

Cloning of CDP-Diacylglycerol Synthase from a Human Neuronal Cell Line

Abstract: A critical step in the supply of substrate for the phosphoinositide signal transduction pathway is the formation of the liponucleotide intermediate, CDP‐diacylglycerol, catalyzed by CDP‐diacylglycerol synthase. Further insight into the regulation of phosphoinositide biosynthesis was sought by cloning of the gene for the vertebrate enzyme. Sequence of the corresponding gene from Drosophila was used to prepare a probe for screening of a human neuronal cell cDNA library. A cDNA was isolated with a predicted open reading frame of 1,332 bases, encoding a protein of 51 kDa. The amino acid sequence showed 50% identity (75% similarity) to that of Drosophila eye CDP‐diacylglycerol synthase and substantial similarity to the Saccharomyces cerevisiae and Escherichia coli homologues. Northern blot analysis, with human cDNA riboprobes, suggested that the corresponding mRNA was expressed in all human tissues examined. Expression of the human cDNA in COS cells resulted in a more than fourfold increase in CDP‐diacylglycerol synthase activity. Knowledge of the sequence of vertebrate CDP‐diacylglycerol synthase should facilitate further investigations into its regulation and the possible existence of distinct isoforms.

Bilateral tectal innervation by regenerating optic nerve fibers in goldfish: a radioautographic, electrophysiological and behavioral study.

Following unilateral enucleation and optic nerve crush in goldfish, the remaining nerve regenerates and innervates both optic tecta. Approximately 5% of the nerve fibers reach the ipsilateral optic tectum (IOT) via the ipsilateral tract at the chiasma. Comparable debris in both tracts was not sufficient to result in an IOT projection since when both nerves were crushed simultaneously the usual pattern was seen, i.e., each nerve innervated a contralateral optic tectum (COT). When the arrival of one nerve at the chiasma was delayed by staggering the nerve crushes, the nerve that first arrived at the chiasma partially innervated the Iot. In most instances the entire IOT was innervated, however, the stratigraphic distribution of fibers in the various tectal lamina was atypical. Electrophysiological analysis indicated that fibers from each area of the retina innervated the IOT visuotopically. The COT was ablated in order to determine whether the IOT projection could mediate behavior. All fish failed to respond to changes in illumination as measured by respiration and failed to swim with or against the stripes in an optomotor drum. Thus, the IOT input, possibly because of its sparseness, could not be shown to be behaviorally functional.

Developmental and Regional Studies of the Metabolism of Inositol 1,4,5-Trisphosphate in Rat Brain

Coupling of CNS receptors to phosphoinositide turnover has previously been found to vary with both age and brain region. To determine whether the metabolism of the second messenger inositol 1,4,5‐trisphosphate also displays such variations, activities of inositol 1,4,5‐trisphosphate 5′‐phosphatase and 3′‐kinase were measured in developing rat cerebral cortex and adult rat brain regions. The 5′‐phosphatase activity was relatively high at birth (∼50% of adult values) and increased to adult levels by 2 weeks postnatal. In contrast, the 3′‐kinase activity was low at birth and reached ∼50% of adult levels by 2 weeks postnatal. In the adult rat, activities of the 3′‐kinase were comparable in the cerebral cortex, hippocampus, and cerebellum, whereas much lower activities were found in hypothalamus and pons/medulla. The 5′‐phosphatase activities were similar in cerebral cortex, hippocampus, hypothalamus, and pons/medulla, whereas 5‐to 10‐fold higher activity was present in the cerebellum. The cerebellum is estimated to contain 50–60% of the total inositol 1,4,5‐trisphosphate 5′‐phosphatase activity present in whole adult rat brain. The localization of the enriched 5′‐phosphatase activity within the cerebellum was examined. Application of a histochemical lead‐trapping technique for phosphatase indicated a concentration of inositol 1,4,5‐trisphosphate 5′‐phosphatase activity in the cerebellar molecular layer. Further support for this conclusion was obtained from studies of Purkinje cell‐deficient mutant mice, in which a marked decrement of cerebellar 5′‐phosphatase was observed. These results suggest that the metabolic fate of inositol 1,4,5‐trisphosphate depends on both brain region and stage of development.