Avron D. Spier

Cortistatin : a member of the somatostatin neuropeptide family with distinct physiological functions

Cortistatin is a recently discovered neuropeptide relative of somatostatin named after its predominantly cortical expression and ability to depress cortical activity. Cortistatin-14 shares 11 of the 14 amino acids of somatostatin-14 yet their nucleotide sequences and chromosomal localization clearly indicate they are products of separate genes. Now cloned from human, mouse and rat sources, cortistatin is known to bind all five cloned somatostatin receptors and share many pharmacological and functional properties with somatostatin including the depression of neuronal activity. However, cortistatin also has many properties distinct from somatostatin including induction of slow-wave sleep, apparently by antagonism of the excitatory effects of acetylcholine on the cortex, reduction of locomotor activity, and activation of cation selective currents not responsive to somatostatin. Expression of mRNA encoding cortistatin follows a circadian rhythm and is upregulated on deprivation of sleep, suggesting cortistatin is a sleep modulatory factor. This review summarizes recent advances in our understanding of the neurobiology of cortistatin, examines the similarities and differences between cortistatin and somatostatin, and asks the question: does cortistatin bind to a cortistatin-specific receptor?

From vasoactive intestinal peptide (VIP) through activity-dependent neuroprotective protein (ADNP) to NAP: a view of neuroprotection and cell division.

Accelerated neuronal death brings about cognitive as well as motor and other dysfunctions. A major neuropeptide, vasoactive intestinal peptide (VIP), has been shown to be neuroprotective. However, VIP-based drug design is hampered by the instability of the peptide and its limited bioavailability. Two independent approaches were thus taken to exploit VIP as a lead drug candidate: (1) Potent neuroprotective lipophilic analogs of VIP were synthesized, e.g. [stearyl-norleucine-17] VIP (SNV); and (2) potent neuroprotective peptide derivatives were identified that mimic the activity of VIP-responsive neuroprotective glial proteins. VIP provides neuronal defense by inducing the synthesis and secretion of neuroprotective proteins from astrocytes; activity-dependent neuroprotective protein (ADNP) was discovered as such glial cell mediator of VIP- and SNV-induced neuroprotection. In subsequent studies, an eight-amino-acid peptide, NAP, was identified as the smallest active element of ADNP exhibiting potent neuroprotective activities. This paper summarizes the biological effects of SNV and NAP and further reports advances in NAP studies toward clinical development. An original finding described here shows that NAP, while protecting neurons, demonstrated no apparent effect on cell division in a multiplicity of cell lines, strengthening the notion that NAP is a specific neuroprotective drug candidate.

NAP mechanisms of neuroprotection

An 8-amino-acid peptide, NAPVSIPQ (NAP), was identified as the smallest active element of activity-dependent neuroprotective protein that exhibits potent neuroprotective action. Potential signal transduction pathways include cGMP production and interference with inflammatory mechanisms, tumor necrosis factor-α, and MAC1-related changes. Because of its intrinsic structure, NAP might interact with extracellular proteins and also transverse membranes. NAP-associated protection against oxidative stress, glucose deprivation, and apoptotic mechanisms suggests interference with fundamental processes. This paper identifies p53, a key regulator of cellular apoptosis, as an intracellular target for NAP’s activity.

Calcium changes induced by presynaptic 5-hydroxytryptamine-3 serotonin receptors on isolated terminals from various regions of the rat brain.

The serotonin 5-hydroxytryptamine-3 receptor is a ligand-gated ion channel that is distributed widely in the nervous system. Within the CNS, a significant portion of the 5-hydroxytryptamine-3 receptors appears to be present on presynaptic nerve terminals and, using an imaging approach, it was shown previously that presynaptic 5-hydroxytryptamine-3 receptors on individual isolated nerve terminals (synaptosomes) from rat corpus striatum display a distinctive set of properties-slow onset, little desensitization and high apparent permeability for Ca2+-when compared to those observed for 5-hydroxytryptamine-3 receptors localized at postsynaptic sites on neuronal cell bodies. To consider whether their characteristic nature is a common feature of presynaptic 5-hydroxytryptamine-3 receptors across the brain, we used confocal microscopy to measure changes in intracellular Ca2+ concentration resulting from 5-hydroxytryptamine-3 agonist-induced responses in synaptosomes from representative rat brain regions, ranging in expression of overall levels of 5-hydroxytryptamine-3 receptors from relatively low (cerebellum) to intermediate (corpus striatum and hippocampus) to high (amygdala). Application of 100 nM m-chlorophenyl biguanide, a specific 5-hydroxytryptamine-3 receptor agonist, induced changes in relative intracellular Ca2+ concentration in subsets of synaptosomes from the corpus striatum (approximately 6% of total), hippocampus (approximately 3% of total), amygdala (approximately 30% of total) and cerebellum (approximately 32% of total). In order to assure the viability of the synaptosomes that did not respond to 5-hydroxytryptamine-3 agonist stimulation, KCl (45 mM) was subsequently added to depolarize the same population of synaptosomes, and increases in intracellular Ca2+ concentration were then seen in 80-90% of the synaptosomes from all four regions. The kinetics of the intra synaptosomal Ca2+ changes produced by K+-evoked depolarization were similar in all regions, showing a rapid rise to a peak followed by an apparent plateau phase. In contrast, the changes in intracellular Ca2+ concentration evoked by m-chlorophenyl biguanide displayed substantially slower kinetics, similar to previous findings, but which varied among responding synaptosomes from one region to another. In particular, m-chlorophenyl biguanide-induced changes were notably slower in synaptosomes from the amygdala (rise time constant, tau = 25 s), when compared to responses in synaptosomes from other regions (striatum, tau = 12 s; hippocampus, tau= 9.6 s; cerebellum, tau = 7 s). To independently demonstrate the presence of 5-hydroxytryptamine-3 receptors on nerve terminals in the various regions using a molecular approach, we double-immunostained the synaptosomes for the 5-hydroxytryptamine-3 receptor and the synaptic vesicle protein synaptophysin, using, respectively, a polyclonal antibody raised against an N-terminal peptide of the 5-hydroxytryptamine-3 receptor and a monoclonal anti-synaptophysin antibody, and observed 5-hydroxytryptamine-3 receptors in varying subsets of the synaptosomes from each region, providing direct support for the results obtained in our functional experiments. These results suggest that the distinctive properties of presynaptic 5-hydroxytryptamine-3 receptors are found throughout the brain, with evident differences in the kinetics of the responses to agonist stimulation observed across the brain regions studied. As expected, the proportion of the synaptosomal population that responded on application of 5-hydroxytryptamine-3 agonist varied in preparations from one region to another; however, the presence of a relatively high proportion of presynaptic 5-hydroxytryptamine-3 receptors in the cerebellum contrasts with previous binding studies demonstrating a relatively low overall density of 5-hydroxytryptamine-3 receptors in this region. We hypothesize that presynaptic 5-hydroxytryptamine-3 receptors present on nerve terminals regulate the

Antibodies against the extracellular domain of the 5-HT3 receptor label both native and recombinant receptors.

We have developed polyclonal antibodies (pAb120) against a peptide corresponding to a region within the extracellular domain of the 5-hydroxytryptamine3 (5-HT3) receptor subunit, thus permitting, for the first time, localization of 5-HT3 receptors at the cell surface in intact (non-permeabilized) systems. The antibodies are both specific and sensitive: pAb120 recognized as little as 63 ng of protein from HEK293 cells expressing recombinant 5-HT3 receptors, whilst Western blots of recombinant 5-HT3 receptors purified from Sf9 cells revealed two bands at 48 and 54 kDa, and native 5-HT3 receptors from N1E-115 cell membranes produced a broad band at 50-54 kDa with a smaller band at 35 kDa. These bands were also labelled by antibodies against the intracellular loop of the 5-HT3 receptor. Immunofluorescent labelling revealed a ring of intense fluorescence in the plasma membrane of non-permeabilized HEK293 cells expressing recombinant 5-HT3 receptors. Studies on native 5-HT3 receptors revealed that pAb120 could recognize 5-HT3 receptors on presynaptic terminals isolated from rat striatum, and immunohistochemical studies in rat brain sections revealed labelling of cell bodies, dendrites and varicose axons in hippocampus, cortex and lateral hypothalamus; all of these areas have been reported to express 5-HT3 receptors. We conclude that pAb120 is a highly specific and sensitive antiserum that will assist in clarifying fundamental questions about 5-HT3 receptor neurobiology.

Nicotinic receptors co-localize with 5-HT3 serotonin receptors on striatal nerve terminals

Nicotinic acetylcholine receptors and 5-HT(3) serotonin receptors are present on presynaptic nerve terminals in the striatum, where they have been shown to be involved in the regulation of dopamine release. Here, we explored the possibility that both receptor systems function on the same individual nerve terminals in the striatum, as assessed by confocal imaging of synaptosomes. On performing sequential stimulation, nicotine (500 nM) induced changes in [Ca(2+)](i) in most of the synaptosomes ( approximately 80%) that had previously responded to stimulation with the 5-HT(3) receptor agonist m-chlorophenylbiguanide (mCPBG; 100 nM), whereas mCPBG induced [Ca(2+)](i) responses in approximately half of the synaptosomes that showed responses on nicotinic stimulation. The 5-HT(3) receptor-specific antagonist tropisetron blocked only the mCPBG-induced responses, but not the nicotinic responses on the same synaptosomes. Immunocytochemical staining revealed extensive co-localization of the 5-HT(3) receptor with the alpha4 nicotinic receptor subunit on the same synaptosomes, but not with the alpha3 and/or alpha5 subunits. Immunoprecipitation studies indicate that the 5-HT(3) receptor and the alpha4 nicotinic receptor subunit do not interact on the nerve terminals. The presence of nicotinic and 5-HT(3) receptors on the same presynaptic striatal nerve terminal indicates a convergence of cholinergic and serotonergic systems in the striatum.

5-HT3A receptor subunits in the rat medial nucleus of the solitary tract: subcellular distribution and relation to the serotonin transporter

The 5-hydroxytryptamine 3 (5HT3) receptor is a serotonin-gated ion channel implicated in reflex regulation of autonomic functions within the nucleus of the solitary tract (NTS). To determine the relevant sites for 5-HT3 receptor mediated transmission in this region, we used electron microscopic immunocytochemistry to examine the subcellular distribution of the 5HT3 receptor subunit A (5HT3A) in relation to the serotonin transporter (SERT) in the intermediate medial NTS (mNTS) of rat brain. The 5HT3A immunolabeling was detected in many axonal as well as somatodendritic and glial profiles. The axonal profiles included small axons and axon terminals in which the 5HT3A immunoreactivity was localized to membranes of synaptic vesicles and extrasynaptic plasma membranes. In dendrites and glia, the 5HT3A immunoreactivity was located on the plasma membranes or in association with membranous cytoplasmic organelles. The dendritic plasmalemmal 5HT3A labeling was prominent within and near excitatory-type synapses from terminals including those that resemble vagal afferents. The 5HT3A-labeled glial processes apposed 5HT3A-immunoreactive axonal and dendritic profiles, some of which also contained SERT. Terminals containing 5-HT3A and/or SERT were among those providing synaptic input to 5HT3A-labeled dendrites. Thus, 5HT3A has a subcellular distribution consistent with the involvement of 5-HT3 receptors in modulation of both presynaptic release and postsynaptic responses of mNTS neurons, some of which are serotonergic. The results further suggest that the neuronal as well as glial 5HT3 receptors can be activated by release of serotonin from presynaptic terminals or by diffusion facilitated by SERT distribution at a distant from the synapse.

Cortistatin and somatostatin mRNAs are differentially regulated in response to kainate

Cortistatin (CST) is a presumptive neuropeptide that shares 11 of its 14 amino acids with somatostatin (SST). CST and SST are expressed in partially overlapping but distinct populations of cortical interneurons. In the hippocampal formation, most CST-positive cells are also positive for SST. In contrast to SST, administration of CST into the rat brain ventricles reduces locomotor activity and specifically enhances slow wave sleep. Intracerebroventricular injection of CST or SST has been shown to protect against the neurotoxic effects of kainic acid. Here, we show that CST and SST mRNAs respond differently to kainate-induced seizures. Furthermore, comparison of the upstream sequences from the CST and SST precursor genes reveal that they contain binding motifs for different transcriptional regulatory factors. Our data demonstrate that CST and SST, which are often co-expressed in the same neurons, are regulated by different stimuli.

Neuropeptides: brain messengers of many faces

Neuropeptides 2001, 2nd Joint Meeting of the European Neuropeptide Club and the American Summer Neuropeptide Conference (11th Annual Meeting). 6-11 May 2001 with Satellite Symposium, Israeli-French Symposium, Israel Ministry of Science, Culture and Sport, 6 May 2001, held at Maale Hachmicha and Tel Aviv University, Israel.

Overexpression of the human β-amyloid precursor protein downregulates cortistatin mRNA in PDAPP mice

We measured preprocortistatin mRNA expression in young and aged transgenic (Tg) mice overexpressing the human beta-amyloid precursor protein (hbetaAPP) under the platelet-derived growth factor-beta promoter. Our findings suggest that the significant increase in hippocampal cortistatin mRNA expression during normal aging is significantly attenuated in Tg mice at an age known to exhibit beta-amyloid protein (Abeta) deposition. These deficits in cortistatin expression may play a role in the deficits in hippocampal-dependent spatial learning and sleep/wake states previously demonstrated in aged Tg mice.