Contribution of the ICE family to neurodegeneration.

The ICE family of cysteine proteases mediates necrotic or apoptotic events in the nervous system as well as in other tissues. This suggests that inhibitors may be of therapeutic value in acute and, perhaps, chronic neurodegenerative disease. In addition, some members of this family may respond to intercellular signals controlling proliferation or differentiation. This possibility should be kept in mind as therapeutics are pursued.

Reduced ischemic brain injury in interleukin-1 beta converting enzyme-deficient mice.

A variety of recent studies suggest a role for both inflammatory cytokines such as interleukin-1 beta (IL-1 beta), and apoptosis in ischemic brain injury. Because IL-1 beta converting enzyme (ICE) is required for the conversion of proIL-1 beta to its biologically active form, and has homology with proteins that regulate apoptosis in invertebrates, we studied the effect of cerebral ischemia on brain injury in mutant mice deficient in the ICE gene (ICE knockout [KO] mice). Focal cerebral ischemia, produced by occlusion of the middle cerebral artery, resulted in brain edema (increased water and sodium content) at 4 hours and a histologically defined brain lesion at 24 hours. Both of these markers of brain injury were significantly reduced in the ICE KO mice as compared to wild-type C57BL/6 mice. Regional cerebral blood flow, determined using the flow tracer, N-isopropyl [methyl 1,3-(14)C] p-iodoamphetamine (14C-IMP), was similar in the two strains of mice, indicating that the reduced brain injury in the KO mice was not a result of a lesser degree of ischemia. These data show that ICE contributes to the development of ischemic brain damage, and that it plays a role at an early time in the pathologic process. Although the mechanism of this effect is uncertain, our results suggest that pharmacologic inhibition of ICE may be a useful treatment for stroke.

Inhibition of the interleukin-1 beta converting enzyme family rescues neurons from apoptotic death.

The interleukin-1 beta converting enzyme (ICE) family of cysteine proteases has been implicated in apoptosis. This study tested the effects of a novel pan-ICE family inhibitor, bocaspartyl(OMe)-fluoromethylketone (boc-Asp-CH2F), against low potassium-induced apoptosis of cultured rat cerebellar granule neurons (CGN). A single application of this cell-permeant compound (20 microM) inhibited apoptotic cell death up to 48 h. Classical apoptotic changes were monitored by fluorescence microscopy, DNA fragmentation and scanning electron microscopy (SEM). A control peptidic fluoromethylketone (boc-Thr-CH2F), and inhibitors to calpain (Ac-Leu-Leu-norleucinal), cathepsin B (Z-Phe-Ala-CH2F), and CPP32-like proteases (Z-DEVD-CH2F), failed to prevent apoptotic death. An 35S-methionine incorporation assay verified that, unlike cycloheximide, boc-Asp-CH2F did not inhibit protein synthesis, hence excluding this as a rescuing mechanism. Although ICE was not detected by northern blot analysis, both CPP32 and Nedd2 expression were found to increase during apoptosis. Kinetic assays with cell extracts from boc-Asp-CH2F-treated neurons measured reduced rates of cleavage for DEVD-pNA and LEVD-pNA. At present, ICE-like proteases remain viable candidates for mediating neuronal death.

Caspase-3/CPP32-like activity is not sufficient to mediate apoptosis in an IL-2 dependent T cell line.

CTLL cells undergo apoptosis when cultured in the absence of IL-2. The IL-1beta-converting-enzyme (ICE)/ caspase family has been implicated as an integral component of some forms of apoptosis. Numerous members of the caspase family have been identified, and it appears as if caspase-3/CPP32 plays a critical role. Previously we demonstrated that ICE/caspase-1 expression increases in CTLL cells during apoptosis; however, inhibition of ICE activity did not abrogate apoptotic death. The purpose of this report is to determine if other members of the caspase family are involved in T cell apoptosis induced by growth factor starvation. We show that cytosolic CPP32-like activity, as measured by the cleavage of DEVD-pNA and poly(ADP-ribose) polymerase (PARP), increases during apoptosis following growth factor deprivation. Cytosolic CPP32-like activity is inhibited in cells treated with the broad spectrum ICE family inhibitor boc-aspartyl(OMe)-fluoromethylketone (D-FMK) and by VAD-FMK and DEVD-FMK which have greater specificity for CPP32-like ICE homologs; however, only the broad spectrum ICE inhibitor D-FMK inhibited apoptosis. Our results suggest that apoptosis induced by growth factor deprivation involves the caspase family, but increased CPP32-like activity is not sufficient to mediate apoptosis induced by IL-2 starvation.

Genetic and metabolic status of NGF-deprived sympathetic neurons saved by an inhibitor of ICE family proteases.

Sympathetic neurons undergo programmed cell death (PCD) when deprived of NGF. We used an inhibitor to examine the function of interleukin-1 beta-converting enzyme (ICE) family proteases during sympathetic neuronal death and to assess the metabolic and genetic status of neurons saved by such inhibition. Bocaspartyl(OMe)-fluoromethylketone (BAF), a cell-permeable inhibitor of the ICE family of cysteine proteases, inhibited ICE and CPP32 (IC50 approximately 4 microM) in vitro and blocked Fas-mediated apoptosis in thymocytes (EC50 approximately 10 microM). At similar concentrations, BAF also blocked the NGF deprivation-induced death of rat sympathetic neurons in culture. Compared to NGF-maintained neurons, BAF-saved neurons had markedly smaller somas and maintained only basal levels of protein synthesis; readdition of NGF restored growth and metabolism. Although BAF blocked apoptosis in sympathetic neurons, it did not prevent the fall in protein synthesis or the increase in the expression of c-jun, c-fos, and other mRNAs that occur during neuronal PCD, implying that the ICE-family proteases function downstream of these events during PCD.NGF and BAF rescued sympathetic neurons with an identical time course, suggesting that NGF, in addition to inhibiting metabolic and genetic events associated with neuronal PCD, can act posttranslationally to abort apoptosis at a time point indistinguishable from the activation of cysteine proteases. Both poly-(ADP ribose) polymerase and pro-ICE and Ced-3 homolog-1 (ICH-1) appear to be cleaved in a BAF-inhibitable manner, although the majority of pro-CPP32 appears unchanged, suggesting that ICH-1 is activated during neuronal PCD. Potential implications of these findings for anti-apoptotic therapies are discussed.

IL-1 beta converting enzyme (ICE) is not required for apoptosis induced by lymphokine deprivation in an IL-2-dependent T cell line.

Clonal T cells undergo programmed cell death (PCD) or apoptosis when cultured without the appropriate cytokines. The cysteine protease, IL-1 beta converting enzyme (ICE), is implicated in apoptosis based on its structural similarity to the PCD gene, ced-3, in Caenorhabditis elegans and the induction of PCD in fibroblasts transfected with recombinant ICE. We show that the murine IL-2-dependent CTLL T cell line expresses ICE but not IL-1 beta. Interestingly, ICE mRNA and protein levels increase during apoptosis. Yet inhibition of ICE enzymatic activity (> 90%) with either of two cell-permeable ICE inhibitors does not abrogate or delay apoptosis following IL-2 deprivation, as measured by DNA fragmentation and viability. Our results suggest that ICE is not required for apoptosis in lymphokine-deprived T cells.

Cloning, tissue expression and regulation of rat interleukin 1 beta converting enzyme.

Using oligomer primers based on the cDNA sequence of human interleukin 1 beta converting enzyme (ICE), we have employed the RT-PCR method and rat spleen RNA to clone and sequence rat ICE. We report here that the predicted amino acid sequence of rat ICE proenzyme consists of 402 amino acids (p45) and shares 61% and 90% identity, respectively, with human and mouse ICE amino acid sequences. The active site cysteine (Cys284) and 3 or 3 potential processing sites are conserved suggesting that their the rat ICE heterodimer consists of a p22 (Ser104-Asp296) and a p10 (Gly315-His402) subunit or a cryptic processing site creates a smaller heterodimer. Northern blot analysis has revealed a approximately 2.2 kb and a more abundant approximately 1.45 kb ICE transcript both widely expressed in the rat with the highest expression in spleen and intestine and lowest in brain. IL-1 beta mRNA was similarly distributed. Injection of the immunostimulant, lipopolysaccharide (0.2 mg/kg, i.p.), increased rICE mRNA content between 2- to 3-fold in the rat brain with smaller increases measured in testis and spleen. The structural conservation of this enzyme suggests that rat models of inflammation will be useful for evaluating the therapeutic potential of ICE inhibitors in humans.

Expression of alpha 1-chimaerin (rac-1 GAP) alters the cytoskeletal and adhesive properties of fibroblasts.

The small GTP-binding protein rac-1, a member of the ras gene superfamily of GTPases, is thought to be a key component of a signal transduction pathway that mediates cell membrane ruffling and actin stress fiber formation induced by growth factors. rac-1 protein is regulated by the interplay of several activities: proteins that enhance GDP dissociation (GDP Dissociation Stimulator, GDS), inhibit nucleotide exchange (GDP Dissociation Inhibitor, GDI), or accelerate GTP hydrolysis (GTPase Activating Protein, GAP). We have assessed the relative contribution of the rac-1/GAP interactions to the overall activity of rac-1 by expressing alpha 1-chimaerin, a rac-1-specific GAP, in fibroblasts. NIH 3T3 cells were transfected with alpha 1-chimaerin cDNA-containing expression vector and stable clones were established. Extracts prepared from alpha 1-chimaerin-expressing cells showed rac-1 GAP activity that was regulated by phosphatidylserine and phorbol ester. The cells expressing alpha 1-chimaerin showed a distinct phenotype. They had altered adhesive properties as measured by their ability to bind to a fibronectin-coated glass surface, suggesting that the expression of a rac-1 GAP alters the assembly of integrin receptors, actin and cytoskeletal proteins such as vinculin and talin. Direct demonstration of this phenomenon was achieved by studying the organization of actin stress fiber and formation of focal adhesions in the alpha 1-chimaerin expressing cells following stimulation by growth factors. Mock transfected cells, upon serum or lysophosphatidic acid stimulation, organize actin as a dense array of parallel fibers running the length of the cell. This process did not take place in the cells expressing rac-1 GAP. Similarly, the formation of focal adhesions as measured by the appearance of vinculin clusters was impaired in the alpha 1-chimaerin expressing cells. These results demonstrate that expression of a GAP for rac-1 in fibroblasts produces profound changes in the cytoskeletal organization and suggest that GAP activity negatively regulates rac-1 function.

Interleukin-1 beta dissociates beta-amyloid precursor protein and beta-amyloid peptide secretion.

A heightened production of interleukin 1 beta (IL-1 beta) has been reported in microglial-associated amyloid deposits in Alzheimer's disease (AD) brains. These plaques are composed predominantly of beta/A4 peptide derived from beta-amyloid precursor protein (beta APP). We demonstrate that short-term (1 h) IL-1 beta-treatment increases beta APPs secretion and concomitantly decreases cell-associated beta APP in human H4 neuroglioma cells. Long-term (5 h) IL-1 beta treatment did not alter secreted or cell-associated beta APP content. In contrast, the secretion of beta/A4-containing epitope was not affected by short-term IL-1 beta stimulation; however, long-term IL-1 beta treatment decreased the amount of beta/A4-containing epitope secreted from the cells. These results show that IL-1 beta modifies the processing and secretion of beta APP to exacerbate perhaps the neuropathology of AD.

Two high affinity binding sites for pituitary adenylate cyclase-activating polypeptide have different tissue distributions.

Two bioactive products of pituitary adenylate cyclase-activating polypeptide (PACAP) prohormone have been isolated from ovine hypothalamus: PACAP38 with 38 residues and PACAP27 corresponding to the N-terminal 27 residues of PACAP38. Immunocytochemical and RIA results support the existence of PACAP in the rat brain, posterior pituitary, and various peripheral tissues. Furthermore, high affinity PACAP-binding sites have been detected in the rat brain, anterior pituitary, and cultured astrocytes which differ from those in lung, liver, and cultured mouse splenocytes. In the present study additional rat tissues were examined to elucidate the location and characteristics of PACAP-binding sites using [125I] PACAP27 with conventional methods of receptor autoradiography and RRA. Binding specificity was established by displacement with unlabeled PACAP27 or a related peptide, vasoactive intestinal polypeptide (VIP). PACAP27-binding sites were localized autoradiographically in the testis, epididymis, adrenal gland, lung, liver, prostate gland, and seminal vesicle; binding sites were not detected in the heart, kidney, or thymus. In the testis and epididymis, a PACAP27-binding site was localized on germinal cells and in the adrenal gland on medullary chromaffin cells. Excess VIP did not displace PACAP27 binding localized in these three tissues. A site with a greater affinity for PACAP27 than for VIP was detected in adrenal gland and epididymis, characteristic of a site recognized previously in hypothalamus, anterior pituitary, and cultured astrocytes. The PACAP-specific site was more abundant in these tissues than a second site to which PACAP27 and VIP bound with similar affinities. Accordingly, the first site has been named type I. In lung, liver, prostate, and seminal vesicle, VIP displaced PACAP27 binding localized autoradiographically. Lung and liver contained an abundant site to which PACAP27 and VIP bound with similar affinities. This binding site, measured previously in lung, liver, and cultured splenocytes, may be shared by PACAP and VIP and has been named type II. Taken together, these data support the existence of two high affinity binding sites for PACAP with different tissue distribution.

Subunit selectivity and epitope characterization of mAbs directed against the GABAA/benzodiazepine receptor.

mAbs bd 17, bd 24, and bd 28 raised against bovine cerebral gamma-aminobutyric acid (GABAA)/benzodiazepine receptors were analyzed for their ability to detect each of 12 GABAA receptor subunits expressed in cultured mammalian cells. Results showed that mAb bd 17 recognizes epitopes on both beta 2 and beta 3 subunits while mAb bd 24 is selective for the alpha 1 subunit of human and bovine, but not of rat origin. The latter antibody reacts with the rat alpha 1 subunit carrying an engineered Leu at position four, documenting the first epitope mapping of a GABAA receptor subunit-specific mAb. In contrast to mAbs bd 17 and bd 24, mAb bd 28 reacts with all GABAA receptor subunits tested but not with a glycine receptor subunit, suggesting the presence of shared epitopes on subunits of GABA-gated chloride channels.

Ultrastructural characterization of prolactin-like immunoreactivity in rat medial basal hypothalamus.

Prolactin-like immunoreactivity has been reported in the medial basal hypothalamus at the light microscopic level, in hypophysectomized rats. Here, with preembedding immunocytochemistry at the electron microscopic level, we have observed prolactin-immunoreactive neurons and synapses in the hypothalamus. Reaction product was discovered in medial basal hypothalamic neurons, which had typical large nucleoli and received axosomatic synapses. In the cytoplasm, reaction product was distinctly granular. Immunoreactive neurons were usually surrounded by nonreactive cells. Reaction product was also seen in dendrites, some of which had spines. Some axons in the hypothalamus contained reaction product, usually surrounded by nonreactive axons, and immunopositive synapses were detected both in the hypothalamus and in the midbrain. In a small number of cases immunoreactive axons could be seen synapsing on immunoreactive dendrites.

Chemical characterization of neuroendocrine targets for progesterone in the female rat brain and pituitary.

The secretory products of some of the cell types which respond directly to actions of progesterone in the female rat brain and pituitary were determined by combining immunocytochemistry with autoradiography following systemic administration of the synthetic progestin ligand [3H]-R5020. Four major findings are reported: (1) Approximately 90% of the tyrosine hydroxylase (TH)-immunoreactive neurons in the hypothalamic arcuate nucleus have progesterone receptors, while TH-immunoreactive neurons in other portions of the hypothalamus (e.g. the periventricular region and the zona incerta) do not. (2) Approximately 30% of the beta-endorphin neurons in the hypothalamus have progesterone receptors. (3) None of the luteinizing hormone-releasing hormone neurons examined have progesterone receptors. (4) Approximately 98% of the cells in the anterior pituitary that have progesterone receptors contain luteinizing hormone. Lactotrophs do not contain progesterone receptors. Many progestin targets in the brain remain to be characterized chemically. The implications for progesterone-inducible genes and neuroendocrine control systems are discussed.

The Adhesion Molecule on Glia (AMOG) Is Widely Expressed by Astrocytes in Developing and Adult Mouse Brain.

Adhesion molecule on glia (AMOG) is a 45 - 50 kD cell surface glycoprotein structurally similar to the Na, K-ATPase beta-subunit and associated with the catalytic subunit of this enzyme. Previous immunofluorescence results had suggested that AMOG is transiently expressed on Bergmann glia during mouse cerebellar development, and antibody-inhibition results have implicated it in the migration of granule neurons. We report that, while AMOG mRNA is detected in Bergmann glia during the migratory period, this astrocyte derivative continues to express AMOG mRNA at similar levels in adult mice suggesting a functional role for AMOG in adulthood. Evidence from RNA and protein blot analyses that AMOG is present before birth, increasing about ten fold in adult mouse brain and cerebellum is also provided. RNA blot analysis of astrocyte-enriched cell populations and in situ hybridization results show that astrocytes synthesize AMOG mRNA in all regions of the developing and adult brain. In the adult, AMOG mRNA is more abundant in grey than white matter and, among grey matter regions, highest in cerebellar cortex. These results indicate a relationship between density of neuronal elements and AMOG expression. It is further speculated that AMOG is part of a Na,K-ATPase complex expressed preferentially by astrocytes in mouse brain.

The role of receptor subtype diversity in the CNS.

The molecular characterization of neuroreceptors and voltage-gated ion channels has revealed that receptor subtype heterogeneity is a common feature of chemical and electrical signal reception. The use of distinct genes encoding receptor subtypes is a favoured mechanism for generation of this diversity. We propose that the significance of the multiplicity and diversity of signal reception proteins is to increase the information-handling capacity of neurons. This may contribute to neural plasticity.

The dopamine D2 receptor: two molecular forms generated by alternative splicing.

Cloned human dopamine D2 receptor cDNA was isolated from a pituitary cDNA library and found to encode an additional 29 amino acid residues in the predicted intracellular domain between transmembrane regions 5 and 6 relative to a previously described rat brain D2 receptor. Results from polymerase chain reactions as well as in situ hybridization revealed that mRNA encoding both receptor forms is present in pituitary and brain of both rat and man. The larger form was predominant in these tissues and, as shown in the rat, expressed by dopaminergic and dopaminoceptive neurons. Analysis of the human gene showed that the additional peptide sequence is encoded by a separate exon. Hence, the two receptor forms are generated by differential splicing possibly to permit coupling to different G proteins. Both receptors expressed in cultured mammalian cells bind [3H]spiperone with high affinity and inhibit adenylyl cyclase, as expected of the D2 receptor subtype.

Two novel GABAA receptor subunits exist in distinct neuronal subpopulations.

Two cDNAs encoding novel GABAA receptor subunits were isolated from a rat brain library. These subunits, gamma 2 and delta, share approximately 35% sequence identity with alpha and beta subunits and form functional GABA-gated chloride channels when expressed alone in vitro. The gamma 2 subunit is the rat homolog of the human gamma 2 subunit recently shown to be important for benzodiazepine pharmacology. Cellular localization of the mRNAs encoding the gamma 2 and delta subunits in rat brain revealed that largely distinct neuronal subpopulations express the two subunits. The delta subunit distribution resembles that of the high affinity GABAA receptor labeled with [3H]muscimol; the gamma 2 subunit distribution resembles that of GABAA/benzodiazepine receptors labeled with [3H]flunitrazepam. These findings have implications for the composition of two different GABAA receptor subtypes and for information processing in networks using GABA for signaling.

Importance of a novel GABAA receptor subunit for benzodiazepine pharmacology.

Neurotransmission effected by GABA (gamma-aminobutyric acid) is predominantly mediated by a gated chloride channel intrinsic to the GABAA receptor. This heterooligomeric receptor exists in most inhibitory synapses in the vertebrate central nervous system (CNS) and can be regulated by clinically important compounds such as benzodiazepines and barbiturates. The primary structures of GABAA receptor alpha- and beta-subunits have been deduced from cloned complementary DNAs. Co-expression of these subunits in heterologous systems generates receptors which display much of the pharmacology of their neural counterparts, including potentiation by barbiturates. Conspicuously, however, they lack binding sites for, and consistent electrophysiological responses to, benzodiazepines. We now report the isolation of a cloned cDNA encoding a new GABAA receptor subunit, termed gamma 2, which shares approximately 40% sequence identity with alpha- and beta-subunits and whose messenger RNA is prominently localized in neuronal subpopulations throughout the CNS. Importantly, coexpression of the gamma 2 subunit with alpha 1 and beta 1 subunits produces GABAA receptors displaying high-affinity binding for central benzodiazepine receptor ligands.