Identification of inhibitor-of-differentiation 2 (Id2) as a modulator of neuronal apoptosis.

Inhibitor-of-differentiation 2 (Id2) belongs to a family of transcriptional modulators that are characterized by a helix loop helix region but lack the basic amino acid domain. During development, Id2 antagonizes differentiation mediated by the retinoblastoma protein, probably by scavenging downstream E-box basic helix-loop-helix proteins. Here, using differential display RT-PCR, we identify Id2 as an induced gene during serum and potassium deprivation-induced apoptosis of cerebellar granule neurons. Consistent with a biological role for induced Id2 messenger RNA and protein expression in neuronal cell death, expression of Id2 antisense RNA, or targeted deletion of the Id2 gene in neurons from Id2 knock-out mice, protect from apoptosis. Further, gene transfer- mediated overexpression of Id2 induces neuronal cell death both in high potassium and low potassium conditions. Thus, the present study defines a role for Id2 in the modulation of neuronal apoptosis.

Evidence that helix-loop-helix proteins collaborate with retinoblastoma tumor suppressor protein to regulate cortical neurogenesis.

The retinoblastoma tumor suppressor protein (pRb) family is essential for cortical progenitors to exit the cell cycle and survive. In this report, we test the hypothesis that pRb collaborates with basic helix-loop-helix (bHLH) transcription factors to regulate cortical neurogenesis, taking advantage of the naturally occurring dominant-inhibitory HLH protein Id2. Overexpression of Id2 in cortical progenitors completely inhibited the induction of neuron-specific genes and led to apoptosis, presumably as a consequence of conflicting differentiation signals. Both of these phenotypes were rescued by coexpression of a constitutively activated pRb mutant. In contrast, Id2 overexpression in postmitotic cortical neurons affected neither neuronal gene expression nor survival. Thus, pRb collaborates with HLHs to ensure the coordinate induction of terminal mitosis and neuronal gene expression as cortical progenitors become neurons.

A critical temporal requirement for the retinoblastoma protein family during neuronal determination.

In this report, we have examined the requirement for the retinoblastoma (Rb) gene family in neuronal determination with a focus on the developing neocortex. To determine whether pRb is required for neuronal determination in vivo, we crossed the Rb-/- mice with transgenic mice expressing beta-galactosidase from the early, panneuronal Talpha1 alpha-tubulin promoter (Talpha1:nlacZ). In E12.5 Rb-/- embryos, the Talpha1:nlacZ transgene was robustly expressed throughout the developing nervous system. However, by E14. 5, there were perturbations in Talpha1:nlacZ expression throughout the nervous system, including deficits in the forebrain and retina. To more precisely define the temporal requirement for pRb in neuronal determination, we functionally ablated the pRb family in wild-type cortical progenitor cells that undergo the transition to postmitotic neurons in vitro by expression of a mutant adenovirus E1A protein. These studies revealed that induction of Talpha1:nlacZ did not require proteins of the pRb family. However, in their absence, determined, Talpha1:nlacZ-positive cortical neurons underwent apoptosis, presumably as a consequence of "mixed signals" deriving from their inability to undergo terminal mitosis. In contrast, when the pRb family was ablated in postmitotic cortical neurons, there was no effect on neuronal survival, nor did it cause the postmitotic neurons to reenter the cell cycle. Together, these studies define a critical temporal window of requirement for the pRb family; these proteins are not required for induction of neuronal gene expression or for the maintenance of postmitotic neurons, but are essential for determined neurons to exit the cell cycle and survive.

Intraneuronal accumulation and persistence of radiolabel in rat brain following in vivo administration of [3H]-chlorisondamine.

1. Chlorisondamine (CHL), a bisquaternary amine, produces a remarkably long-lasting blockade of central responses to nicotine. The mechanism underlying this blockade is not known. The main aim of this study was to test for possible accumulation of [3H]-CHL in rat brain during the period of chronic blockade. 2. Rats received CHL, either systemically (10 mg kg-1) or centrally (10 micrograms i.c.v.). Seven days later, striatal synaptosomes prepared from these animals were tested for nicotine-induced [3H]-dopamine release. This experiment showed that i.c.v. administration of CHL was as effective as systemic administration in producing ex vivo blockade of central nicotinic receptors. 3. Rats received bilateral i.c.v. infusions of [3H]-CHL (10 micrograms) and radioactivity was subsequently quantified in dissected cerebral cortex, striatum, hippocampus, midbrain and cerebellum. Radiolabel was detected at all three survival times (1, 7, and 21 days). Regional heterogeneity was apparent at 7 and 21 days survival. Radiolabel was almost exclusively confined to the insoluble subcellular fraction in all areas sampled. 4. The anatomical distribution of radiolabel was also visualized in brain sections. Rats received bilateral i.c.v. infusions of [3H]-CHL (10 micrograms) and were killed at 1, 7, 21 or 84 days. Immediately before they were killed, all rats were tested behaviourally, and central nicotinic blockade was demonstrated at 1, 7 and 21 days; partial recovery was observed at 84 days. Particularly at longer survival times, tritium was found to be heavily concentrated in the substantia nigra pars compacta, ventral tegmental area, dorsal raphé nucleus, and the granular layer of the cerebellum. 5. The possibility of retrograde axonal transport of radiolabel was then examined. Rats received a unilateral intrastriatal infusion of [3H]-CHL (0.34 or 0.034 micrograms) one week before they were killed. Autoradiographic labelling was largely confined to the site of infusion and to the ipsilateral substantia nigra pars compacta and dorsal raphé nucleus. 6. Thus, after i.c.v. administration, CHL (and/or centrally-formed derivatives) is initially widely distributed within the brain and is then selectively retained within a few brain areas. A persistent accumulation occurs within putative dopaminergic and 5-hydroxytryptaminergic neurones, at least partly through uptake by terminals and/or axons followed by retrograde transport. This persistent and anatomically-selective intraneuronal accumulation possibly underlies the long-term central nicotinic blockade associated with chlorisondamine.

Regulation of nicotinic receptors in rat brain following quasi-irreversible nicotinic blockade by chlorisondamine and chronic treatment with nicotine.

1. Chronic administration of nicotinic agonists in vivo increases the density of brain nicotinic binding sites. It has been proposed that this up-regulation results from agonist-induced functional blockade of nicotinic receptors. This hypothesis was tested by examining post mortem [3H]-nicotine and [125I]-alpha-bungarotoxin ([125I]-alpha BTX) binding following treatment in vivo with the quasi-irreversible and insurmountable CNS nicotinic blocker chlorisondamine, given either alone or in combination with chronic nicotine administration. 2. In rats that had not received chlorisondamine pretreatment, chronic nicotine administration (0.6 mg kg-1 s.c., twice daily for 12 days) increased [3H]-nicotine binding density (Bmax) in forebrain tissue sections by 19%, with no change in the apparent dissociation constant (KD). Chlorisondamine (10 mg kg-1, s.c.), given once prior to the chronic treatment phase, neither increased [3H]-nicotine binding by itself, nor altered the extent of nicotine-induced up-regulation. Nevertheless, chlorisondamine pretreatment resulted in a persistent blockade of CNS nicotinic receptors, as demonstrated by complete block of acute locomotor responses to nicotine. 3. In a second experiment, [3H]-nicotine and [125I]-alpha BTX binding was measured in tissue homogenates prepared from several brain regions. In the absence of chlorisondamine pretreatment, chronic nicotine administration (1 mg kg-1 s.c., twice daily for 12 days) increased the Bmax of [3H]-nicotine binding in the cerebral cortex (by 34%), striatum (by 28%), midbrain (by 16%) and hippocampus (by 36%); KD was unchanged. As before, this up-regulation was neither mimicked nor blocked by chlorisondamine pretreatment (10 mg kg-1, s.c., given twice), despite persistent blockade of acute locomotor responses to nicotine. Chronic nicotine treatment also increased the Bmax (but not KD) of [125I]-alpha BTX binding in cerebral cortex (by 35%), hippocampus (by 46%) and midbrain (by 35%). Chlorisondamine altered neither Bmax nor KD when given alone, but significantly attenuated the nicotine-induced up-regulation of toxin binding sites in midbrain, with a similar trend in the other two regions.4. The finding that chronic receptor blockade neither mimicked nor blocked the agonist-induced up-regulation of [3H]-nicotine binding sites suggests that up-regulation of these receptors is not determined by their functional status. In contrast, it appears that chronic nicotine-induced up-regulation of[125I]-alpha BTX binding sites may result from receptor activation.

Blockade of nicotinic responses by physostigmine, tacrine and other cholinesterase inhibitors in rat striatum.

1. The acetylcholinesterase inhibitors physostigmine, neostigmine, tetrahydroaminoacridine (tacrine; THA) and diisopropylfluorophosphate (DFP) were tested for possible direct nicotinic actions in rat striatal synaptosomes preloaded with [3H]-dopamine. In this preparation, nicotinic cholinoceptor activation evoked [3H]-dopamine release. 2. Antagonist activity was examined by giving a brief nicotine (1 microM) challenge after 30 min superfusion with an acetylcholinesterase (AChE) inhibitor (0.3-300 microM). Physostigmine, neostigmine and tacrine produced a concentration-dependent blockade. Physostigmine and tacrine were particularly potent (IC50S approx. 10 microM and 1 microM, respectively). DFP reduced nicotinic responses only at the highest concentration tested (300 microM). 3. Nicotinic blockade produced by superfusion with physostigmine (30 microM) was insurmountable when tested against nicotine (0.1-100 microM). 4. Physostigmine (30 microM) also reduced responses to the nicotinic agonists 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP) and cytisine, but did not alter responses to high K+ or (+)-amphetamine. A higher concentration of physostigmine (300 microM) completely blocked responses to nicotine, somewhat reduced responses to amphetamine, and did not alter responses to high K+. Tacrine (3 microM) reduced responses to nicotine and to high K+ but did not affect responses to amphetamine. 5. Physostigmine (0.3-300 microM), given as a brief pulse, did not produce a nicotinic agonist-like effect. 6. Physostigmine, neostigmine, tacrine and DFP (all at 30 microM) each produced near-total (> 96%) inhibition of AChE activity. However, DFP at a concentration (60 microM) that produced a degree of AChE inhibition equal to that of physostigmine 30 microM, did not significantly reduce nicotine-induced dopamine release. 7. It thus appears that physostigmine blocks CNS nicotinic receptors in an insurmountable and pharmacologically selective manner, independent of its ability to inhibit acetylcholinesterase. Tacrine reduced nicotinic responses, quite possibly by an indirect mechanism. The possibility of direct or indirect blockade of nicotinic receptor-mediated actions may complicate the interpretation of preclinical studies that have employed physostigmine and tacrine.

Blockade of nicotinic receptor-mediated release of dopamine from striatal synaptosomes by chlorisondamine administered in vivo.

1. The chronic nicotinic blockade produced following in vivo administration of chlorisondamine was investigated in vitro. Nicotine-induced [3H]-dopamine release from striatal synaptosomes was used as a measure of central nicotinic receptor function. 2. In synaptosomal preparations from rats pretreated with a single administration of chlorisondamine (10 mg kg-1, s.c.), 1, 7, 21, 42, 63 or 84 days before they were killed, responses to (-)-nicotine (10(-6) M) were blocked. 3. In vivo administration of chlorisondamine (10 mg kg-1, s.c.), 7 days before rats were killed, produced a nicotinic blockade in vitro that was insurmountable even with a high concentration of (-)-nicotine (10(-4) M). 4. Both in vitro and in vivo administration of chlorisondamine blocked nicotinic responses to acetylcholine (10(-4) M). In contrast, neither in vitro nor in vivo administration of chlorisondamine reduced [3H]-dopamine release induced by high K+ (20 x 10(-3) M) or (+)-amphetamine (10(-6) M). 5. Nicotinic blockade resulting from in vitro administration of chlorisondamine (10(-5) M) recovered partially after 60 min wash-out, and completely by 90 min. In contrast, no recovery was seen in synaptosomes prepared from rats pretreated with chlorisondamine (10 mg kg-1, s.c.) in vivo. 6. Thus, in vivo treatment with chlorisondamine results in a quasi-irreversible, insurmountable block of CNS nicotinic receptors. The persistence of this block ex vivo indicates that physical trapping by the blood brain barrier is not solely responsible for the persistent blockade seen in vivo. The resistance of this blockade to prolonged in vitro wash-out suggests that the underlying mechanism differs from that associated with in vitro administration.

Blockade of nicotinic receptor-mediated release of dopamine from striatal synaptosomes by chlorisondamine and other nicotinic antagonists administered in vitro.

1. Central nicotinic receptor function examined in vitro, by measuring nicotine-induced [3H]-dopamine release from rat striatal synaptosomes. 2. The agonists (-)-nicotine, acetylcholine, 1,1-dimethyl-4-phenylpiperazinium (DMPP) and cytisine (10(-7)-10(-4) M) all increased [3H]-dopamine release in a concentration-dependent manner. Cytisine did not produce a full agonist response, compared to the other agonists. 3. The actions of nicotine, acetylcholine and cytisine were largely dependent on external Ca2+. In contrast, DMPP (10(-5) and 10(-4) M) evoked a marked release of [3H]-dopamine even in the absence of Ca2+. Nevertheless, in the presence of external Ca2+, responses to DMPP were completely blocked by the nicotinic antagonists chlorisondamine and mecamylamine (5 x 10(-5) M); in the absence of external Ca2+, blockade was only partial. 4. Chlorisondamine, mecamylamine and dihydro-beta-erythroidine (10(-8)-10(-4) M) produced a concentration-dependent block of responses to nicotine (10(-6) M). Approximate IC50 values were 1.6, 0.3 and 0.2 x 10(-6), respectively. Chlorisondamine and mecamylamine blocked responses to nicotine (10(-7)-10(-4) M) insurmountably, whereas dihydro-beta-erythroidine behaved in a surmountable fashion. 5. The occurrence of use-dependent block was tested by briefly pre-exposing the synaptosomes to nicotine during superfusion with antagonist, and determining the response to a subsequent nicotine application. Consistent with a possible channel blocking action, brief pre-exposure to agonist increased the antagonist potency of chlorisondamine (approximately 25 fold). No significant use-dependent block was detected with dihydro-beta-erythroidine.

The pharmacology of the nicotinic antagonist, chlorisondamine, investigated in rat brain and autonomic ganglion.

1. A single administration of the ganglion blocker, chlorisondamine (10 mg kg-1, s.c.) is known to produce a quasi-irreversible blockade of the central actions of nicotine in the rat. The mechanism of this persistent action is not known. It is also unclear whether chlorisondamine can block neuronal responses to excitatory amino acids and whether chronic blockade of nicotinic responses also occurs in the periphery. 2. Acute administration of chlorisondamine (10 mg kg-1, s.c.) to rats resulted in a blockade of central nicotinic effects (ataxia and prostration) when tested 1 to 14 days later, but caused no detectable cell death in tissue sections sampled throughout the rostrocaudal extent of the brain which were stained in order to reveal neuronal degeneration. 3. Long-term blockade of central nicotinic effects by chlorisondamine was not associated with significant alterations in the density (Bmax) of high-affinity [3H]-nicotine binding to forebrain cryostat-cut sections. 4. In cultured dissociated mesencephalic cells of the foetal rat, chlorisondamine and mecamylamine inhibited [3H]-dopamine release evoked by N-methyl-D-aspartate (NMDA, 10(-4) M), but only at high concentrations (IC50 approx. 600 and 70 microM, respectively). A high concentration of chlorisondamine (10(-3) M) had no effect on responses to quisqualate (10(-5) M) and only slightly reduced responses to kainate (10(-4) M). Mecamylamine (10(-3) M) was ineffective against both agonists. 5. In adult rat hippocampal slices, chlorisondamine depressed NMDA receptor-mediated synaptically-evoked field potentials, but again only at high concentrations (10(-4)-10(-3) M). Synaptic responses that were mediated by non-NMDA excitatory amino acid receptors were less affected. 6. In rat isolated superior cervical ganglion, electrically-evoked synaptic transmission was reduced 1 h after acute in vivo administration of chlorisondamine (0.1 mg kg-1, s.c.). However, in vivo administration of a higher dose (10 mg kg-1, s.c.) did not significantly affect ganglionic transmission when tested two weeks later, despite the continued presence of central nicotinic blockade.7. These results indicate that the persistent CNS nicotinic blockade by chlorisondamine is not accompanied by changes in nicotinic [3H]-nicotine binding site density or by neuronal degeneration in the brain; that at doses sufficient to produce nicotinic receptor blockade, chlorisondamine acts in a pharmacologically selective manner; and that chronic central blockade is not accompanied by long-term peripheral ganglionic blockade.

Thymopoietin, a potent antagonist at nicotinic receptors in C2 muscle cell cultures.

Recent work has shown that thymopoietin, a polypeptide with actions in the immune and nervous systems, potently binds to the alpha-bungarotoxin (alpha-BGT) receptor. The present study was done to characterize the interaction of thymopoietin at the nicotinic alpha-BGT binding site in cultured muscle cells and to correlate these findings with the effects of the polypeptide on nicotinic receptor-mediated function. Inhibition studies showed that thymopoietin potently inhibited 125I-alpha-BGT binding in C2 muscle cells in culture, with an IC50 of 1.1 nM, a value similar to that for alpha-BGT. Thymopoietin bound to the alpha-BGT receptor in the cells in culture relatively slowly; at 10(-8) M thymopoietin, maximal inhibition occurred after 45 to 75 min of exposure to the polypeptide. Dissociation of thymopoietin from the receptor exhibited a much longer time course; recovery of alpha-BGT binding to control values after exposure to 10(-8) M thymopoietin occurred approximately 16 hr after removal of the polypeptide. The effects of thymopoietin on 125I-alpha-BGT binding correlated well with those on nicotinic function. Thymopoietin potently inhibited nicotinic receptor-mediated 22Na uptake in muscle cells in culture, with an IC50 of 2 nM. This effect was dependent on the length of the preincubation period with thymopoietin, with maximal inhibition occurring after 60 min of exposure to the polypeptide. Recovery of the functional response after thymopoietin (10(-8) M) exposure required about 16 hr. The mode of inhibition of receptor-mediated ion flux by thymopoietin was similar to that observed with alpha-BGT but distinct from that obtained with d-tubocurarine and gallamine. To conclude, thymopoietin, a thymic polypeptide associated with the immune system, potently inhibited both 125I-alpha-BGT binding and nicotinic receptor-mediated function in C2 muscle cells. These findings may have implications for myasthenia gravis and/or other neuromuscular disorders.