Neuronal Apoptosis Inhibitory Protein Research Articles

Neuronal apoptosis inhibitory protein is expressed in developing kidney and is regulated by PAX2

During fetal kidney development, the extent of ureteric bud (UB) branching will determine final nephron endowment for life. Nephron number varies widely among normal humans and those who are born at the low end of the nephron number spectrum may be at risk for essential hypertension in adulthood. Little is known about how nephron number is set. However, we previously showed that the transcription factor, Pax2, suppresses apoptosis in UB cells during kidney development and optimizes branching morphogenesis. Here, we report that PAX2 directly binds to a specific recognition motif in the human neuronal apoptosis inhibitory protein (NAIP) gene promoter. NAIP is an endogenous inhibitor of apoptosis, inactivating caspase-3 and caspase-7 in neuronal tissues. PAX2 activates NAIP gene transcription (7-fold) in vitro and NAIP transcript level is increased fourfold in HEK293 cells stably transfected with PAX2. We show that Naip is expressed in embryonic day 15 (E15) fetal kidney tissue (RT-PCR) and NAIP protein is demonstrated by immunohistochemistry in E15 mouse kidney collecting ducts and P1 proximal tubules. Naip mRNA is significantly reduced (50%) in heterozygous Pax2 mutant mice. Finally, we show that an antisense Naip1 cDNA transfected into murine collecting duct cells doubles caspase-3/7 activity induced by Baxalpha. These observations suggest that the powerful effects of PAX2 on renal branching morphogenesis and final nephron number may be mediated by activation of Naip which then suppresses apoptosis in UB cells.

Morphodynamics of ovarian follicles during oogenesis in mice

In the mouse, oogonia enter the prophase of the first meiotic division and differentiate into oocyte while developing in the fetal ovary. Shortly after birth, all oocytes are arrested in the dictyate stage of late prophase in the developing follicles; a small number of follicles reach the ovulatory stage; the rest are lost by apoptosis. The resumption of meiotic division and nuclear progression to metaphase II (oocyte maturation) occur in the ovulatory follicles. In this article we review recent morphological data that have clarified how cytokines and glycosaminoglycans (GAGs) are involved in mouse follicular development, atresia, and maturation during oogenesis, as exogenous/endogenous factors. (1) Microvascular networks and angiogenic factors (epidermal growth factor; GAGs) are deeply involved in selective mouse oocyte growth beyond approximately 20-30 microm in diameter. (2) Gonadotropin-inducible neuronal apoptosis inhibitory protein may indirectly affect oocyte survival as a result of the inhibition of apoptotic granulosa-cell death during folliculogenesis. (3) The pattern of oocyte degeneration depends on follicle and oocyte developmental stages, and follicle stimulating hormone accelerates the process of degeneration of oocytes. (4) The process of degeneration of mouse oocytes/eggs is modulated by tumor necrosis factor-alpha that is accumulated in the expanded cumulus during oocyte maturation. (5) A colloidal iron-positive substance was detected in the intercellular spaces of follicular tissue, especially in the cumulus mass. Cells located where the cumulus mass and granulosa cell layer interwound became enlarged during the resumption of oocyte meiosis. Colloidal iron-positive substances accumulated extensively within the intercellular spaces of the enlarged cells.

Spinal Muscular Atrophies: An Ongoing Diagnostic Dilemma?

Background: Spinal muscular atrophies (SMAs) are a group of autosomal recessive disorders of anterior horn cell degeneration. Three genes-survival motor neuron (SMN), neuronal apoptosis inhibitory protein (NAIP), and, more recently, p44 (subunit of basal transcription factor II)-have been considered as candidate genes. The region spanning these genes has a complex organization, which makes molecular analysis difficult. Methods and Results: Molecular genetic testing of deletions of exons 7 and 8 of the SMN(T) (telomeric copy) gene and exon 5 of the NAIP(T) (telomeric copy) gene was performed in 39 diagnosed SMA patients, 31 cases referred as possible SMA, and 24 cases of prenatal diagnosis of SMA. Linkage analysis using markers flanking the SMA region was also performed. In general, the findings of involvement of SMN and NAIP gene deletions in patients diagnosed with SMA are in agreement with those previously published. One possible SMA case was found to be homozygously deleted only for exon 7 of SMN(T) and one deleted only for exon 5 of the NAIP(T) gene. Conclusions: SMAs exemplify human inherited disorders in which application of a variety of different techniques and a search for mutations in multiple genes are involved. Deletion testing of candidate genes (SMN, NAIP) is a powerful approach in patients affected or suspected of being affected with SMA. It is proposed that the direct SMN gene deletion test can be offered as the only test for prenatal diagnosis of SMA in families in which the clinically affected sibling has also been shown to have the homozygous deletion.

Correlation between deletion patterns of SMN and NAIP genes and the clinical features of spinal muscular atrophy in Indian patients

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder involving degeneration of anterior horn cells of spinal cord resulting in progressive muscle weakness and atrophy. The molecular analysis of two marker genes for spinal muscular atrophy (SMA) i.e, the survival motor neuron gene (SMN) and the neuronal apoptosis inhibitory protein gene (NAIP) was conducted in 39 Indian patients with clinical symptoms of SMA. Out of these, 28 showed homozygous deletions and the phenotypic features of these SMA patients were compared with the corresponding genotypes. A tertiary care teaching Hospital. This is a prospective hospital based study. Polymerase chain reaction (PCR) combined with restriction fragment length polymorphism (RFLP) was used to detect the deletion of exon 7 and exon 8 of SMN1 gene, as well as multiplex PCR for exon 5 and 13 of NAIP gene. Exons 7 and 8 of SMN and NAIP (exon 5) were homozygously deleted in 73% of SMA I and 27% of SMA II patients. SMN exon 7 and 8 deletions without NAIP deletions were seen in 27% of type I SMA and 46% of SMA type II patients. Two patients of type III SMA showed single deletion of SMN exon 7 along with 27% of SMA type II patients. With the advent of molecular biology techniques, SMN gene deletion studies have become the first line of investigation for confirmation of a clinical diagnosis of SMA. The findings of homozygous deletions of exons 7 and/or 8 of SMN1 gene confirms the diagnosis of SMA, even in patients with atypical clinical features. Deletions of NAIP gene were mainly seen in severely affected patients, hence is useful for predicting the prognosis.

Expression of the inhibitors of apoptosis proteins in cisplatin-resistant prostate cancer cells

Acquired multi-drug resistance remains a major obstacle in the management of prostate cancer. The objective of this study was to examine whether chemoresistance could be due in part to the expression of the inhibitors of apoptosis proteins (IAPs). We established cisplatin-resistant LNCaP sublines. We examined the effects of cisplatin on cell growth and apoptosis in LNCaP cells and LNCaP sublines by 2-(4-lodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-1) assay and Hoechst 33258 staining, and analyzed cross-resistance to adriamycin, 5-fluorouracil, taxol, taxotere, and etoposide. In addition, the expression of IAP-1, IAP-2, X-linked IAP (XIAP), neuronal apoptosis inhibitory protein, and survivin was investigated by immunoblot analysis in LNCaP sublines. Although the growth rates were reduced in a dose-dependent manner by cisplatin in LNCaP sublines, the anti-proliferative effects of cisplatin were significantly decreased in LNCaP sublines compared to LNCaP cells. Cisplatin-resistant sublines, LNCaP/C1, LNCaP/ C2, and LNCaP/C3 cells, were 6.3-, 9.1-, and 22.3-fold more resistant to cisplatin than LNCaP cells, respectively, and this resistance was paralleled with reduced induction of apoptosis. LNCaP/C3 cells showed cross-resistance to adriamycin, 5-fluorouracil, and etoposide whereas those cells exhibited no or only weak cross-resistance against taxol and taxotere. With the exception of survivin, all the IAPs were identified in LNCaP cells by immunoblot analysis. Interestingly, the expression of IAP-2, XIAP, and survivin gradually increased with the extent of cisplatin-resistance. Altered expression of IAP-2, XIAP, and survivin was involved in these phenotypes of cisplatin-resistant LNCaP sublines. IAPs may make an important contribution to the resistance to the apoptotic effect of cisplatin in prostate cancer.

A Dopamine D4 Receptor Antagonist Attenuates Ischemia-Induced Neuronal Cell Damage via Upregulation of Neuronal Apoptosis Inhibitory Protein

Neuronal apoptosis inhibitory protein (NAIP/BIRC1), the inhibitor of apoptosis protein (IAP) family member, suppresses neuronal cell death induced by a variety of insults, including cell death from ischemia and stroke. The goal of the present study was to develop an efficient method for identification of compounds with the ability to upregulate endogenous NAIP and to determine the effects on these compounds on the cellular response to ischemia. A novel NAIP-enzyme-linked immunosorbent assay (ELISA)-based in vitro drug-screening system is established. Use of this system identified an antagonist of dopamine D4 receptor, termed L-745,870, with a potent NAIP upregulatory effect. L-745,870-mediated NAIP upregulation in neuronal and nonneuronal cultured cells resulted in decreased vulnerability to oxidative stress-induced apoptosis. Reducing NAIP expression via RNA interference techniques resulted in prevention of L-745,870-mediated protection from oxidative stress. Further, systemic administration of L-745,870 attenuated ischemia-induced damage of the hippocampal CA1 neurons and upregulated NAIP expression in the rescued hippocampal CA1 neurons in a gerbil model. These data suggest that the NAIP upregulating compound, L-745,870, has therapeutic potential in acute ischemic disorders and that our NAIP-ELISA-based drug screening may facilitate the discovery of novel neuroprotective compounds.

Study of Survival of Motor Neuron (SMN) and Neuronal Apoptosis Inhibitory Protein (NAIP) gene deletions in SMA patients

In view of the paucity of deletion studies of survival of motor neuron (SMN) and neuronal apoptosis inhibitor protein (NAIP) genes in Indian SMA patients, this study has been undertaken to determine the status of SMN1, SMN2 and NAIP gene deletions in Indian SMA patients. Clinically and neurophysiologically diagnosed SMA patients were included in the study. A gene deletion study was carried out in 45 proximal SMA patients and 50 controls of the same ethnic group. Both SMN1 and NAIP genes showed homozygous absence in 76% and 31% respectively in proximal SMA patients. It is proposed that the lower deletion frequency of SMN1 gene in Indian patients may be due to mutations present in other genes or population variation, which need further study.

Hippocalcin protects against caspase-12-induced and age-dependent neuronal degeneration

Hippocalcin is a neuronal calcium binding protein, but its physiological function in brain is unknown. We show here that hippocampal neurons from hippocalcin-deficient mice are more vulnerable to degeneration, particularly using thapsigargin, elevating intracellular calcium. Caspase-12 was activated in neurons lacking hippocalcin, while calpain was unchanged. Neuronal viability was accompanied by endoplasmic reticulum (ER) stress and a change in the relative induction of the ER chaperone, BiP/GRP78. Neuronal apoptosis inhibitor protein (NAIP), known to interact with hippocalcin, was not altered, but hippocampal neurons from gene-deleted mice were more sensitive to excitotoxicity caused by kainic acid. In addition, an age-dependent increase in neurodegeneration occurred in the gene-deleted mice, showing that hippocalcin contributes to neuronal viability during aging.

Neuronal Apoptosis-inhibitory Protein Does Not Interact with Smac and Requires ATP to Bind Caspase-9

The neuronal apoptosis-inhibitory protein (NAIP) is the founding member of the mammalian family of inhibitor of apoptosis (IAP) proteins (also known as BIRC proteins) and has been shown to be antiapoptotic both in vivo and in vitro. The 160-kDa NAIP contains three distinct regions: an amino-terminal cluster of three baculoviral inhibitory repeat (BIR) domains, a central nucleotide binding oligomerization domain (NOD), and a carboxyl-terminal leucine-rich repeat (LRR) domain. The presence of the NOD and LRR domains renders NAIP unique among the IAPs and suggests that NAIP activity is regulated in a manner distinct from that of other members of the family. In this report, we examined the interaction of various regions of NAIP with caspase-9 and Smac. Recombinant NAIPs with truncations of the carboxyl-terminal LRR or NOD-LRR regions bound to caspase-9. In contrast, the full-length protein did not, suggesting some form of structural autoregulation. However, the association of the wild type full-length protein with caspase-9 was observed when interaction analysis was performed in the presence of ATP. Furthermore, mutation of the NAIP ATP binding pocket allowed full-length protein to interact with caspase-9. Thus, we conclude that NAIP binds to caspase-9 with a structural requirement for ATP and that in the absence of ATP the LRR domain negatively regulates the caspase-9-inhibiting activity of the BIR domains. Interestingly, and in contrast to the X-chromosome-linked inhibitor of apoptosis protein (XIAP), NAIP-mediated inhibition of caspase-9 was not countered by a peptide containing an amino-terminal IAP binding motif (IBM). Consistent with this observation was the failure of Smac protein to interact with the NAIP BIR domains. These results demonstrate that NAIP is distinct from the other IAPs, both in demonstrating a ligand-dependent caspase-9 interaction and in demonstrating a distinct mechanism of inhibition.

229 Activation of caspases in human t-lineage leukemia cells treated with analogs of leflunomide metabolite

The importance of the ATP binding site of human Neuronal Apoptosis Inhibitory Protein (NAIP) on its ability in prevention of intrinsic apoptotic pathway was investigated. Thus, ATP binding lysine 476 of NAIP, which is located at the Nucleotide Binding Oligomerization Domain (NOD) was mutated to threonine and the effect of this mutation on autoproteolysis of procaspase-9 and the cleavage of procaspase-3 by apoptosome was investigated. Formation of apoptosome was induced by the addition of cytochrome c and dATP to lysates of HeLa cells transfected with pcDNA-NAIP or pcDNA-NAIP (K476T). Full length wild type NAIP prevented the cleavage of both procaspase-9 to caspase-9 and procaspase-3 to caspase-3. However, K476T variant of NAIP did not block autocleavage of procaspase-9 efficiently. Furthermore, cleavage pattern of procaspase-9 was altered in the presence of mutant NAIP. Interestingly no effect on the procaspase-3 cleavage by apoptosome was observed. The presence of NOD domain by itself had no effect on autocleavage of procaspase-9 yet slightly reduced the cleavage of procaspase-3 by apoptosome. Pull down experiment showed direct interaction of the NOD domain of NAIP with the CARD-NOD domain of Apoptotic Protease Activating Factor 1 (APAF-1). The physical association of these domains was confirmed by pull-down assays. These observations taken with previous findings indicate that the integrity of the NOD domain is essential for effective inhibition of procaspase-9 and procaspase-3 cleavage by the NAIP protein.

Adipogenic and Antiapoptotic Protein Levels in Human Adipose Stromal Cells after Weight Loss

Obesity is a major risk factor for type 2 diabetes and cardiovascular disease. However, current strategies to achieve sustained weight loss are often unsuccessful. Fat reaccumulation might be favored by enhanced adipose cell differentiation or survival in the postreduced state. We measured adipogenic and apoptotic protein expression in subcutaneous abdominal adipose stromal-vascular cells from 10 obese patients (7 women and 3 men) that were obtained before and after a 16% weight loss in a medically supervised weight loss program. After weight loss, protein expression was 2.4-fold higher (p < 0.005) for p42 C/CAAT enhancer binding protein alpha, but there was no change for peroxisome proliferator-activated receptor gamma1; both of these are adipogenic regulators. For neuronal apoptosis inhibitory protein, a protein associated with adipose cell apoptotic resistance, there was a rise of 1.7-fold (p < 0.02). Alterations in C/CAAT enhancer binding protein alpha and neuronal apoptosis inhibitory protein expression occurred in human adipose stromal-vascular cells after weight loss in a pilot study of 10 patients. It will be important for future studies to directly examine whether the adipogenic and antiapoptotic capacity of these cells is changed after weight loss.

Heterotypic interactions among NACHT domains: implications for regulation of innate immune responses.

Proteins of the NACHT [NAIP (neuronal apoptosis inhibitory protein), CIITA (MHC class II transcription activator), HET-E (incompatibility locus protein from Podospora anserina) and TP1 (telomerase-associated protein)] family may serve as critical pathogen-sensing and signal-transducing molecules within the innate immune system. In the present paper, we show that CLAN [CARD (caspase-recruitment domain), LRR (leucine-rich repeat) and NACHT domain-containing protein], a NACHT-containing protein originally demonstrated to bind and activate pro-caspase 1, is also capable of influencing the functions of other members of the NACHT family. Through heterotypic NACHT-domain interactions, CLAN was found to associate with Nod1, Nod2 and NAC [nucleotide-binding domain and CARD-containing protein; NALP1 (NACHT, LRR and PYRIN protein 1)] when co-expressed in HEK-293T (human embryonic kidney) cells. NF-kappaB (nuclear factor kappaB) reporter assays demonstrated that co-expression of either full-length CLAN or the NACHT domain of CLAN significantly inhibited NF-kappaB activation induced by Nod1 or Nod2 overexpression. In addition, co-expression of CLAN or the NACHT domain of CLAN with Nod1 or Nod2 inhibited the ability of these proteins to generate active IL-1beta (interleukin 1beta) through their association with pro-caspase 1. The NACHT domain of CLAN was demonstrated by co-immunoprecipitation experiments to bind all NACHT domains that were tested, including the NACHT domains from CLAN itself, Nod1, Nod2, cryopyrin, NAC, PAN2 [PAAD [pyrin, AIM (absent-in-melanoma), ASC (apoptosis-associated speck-like protein containing a CARD) and death-domain-like]- and NACHT-containing protein] and NAIP (neuronal apoptosis inhibitory protein). Finally, monocyte-expressed CLAN was found to associate with Nod2 following exposure to bacterial peptidoglycan, implying a regulatory role for interaction of these NACHT proteins in the innate immune response. These studies suggest that by mediating hetero-oligomerization, NACHT domains provide a means by which various NACHT-containing proteins may interact, creating protein-interaction networks that potentially modulate immune responses to invading pathogens.

Motoneuron resistance to apoptotic cell death in vivo correlates with the ratio between X-linked inhibitor of apoptosis proteins (XIAPs) and its inhibitor, XIAP-associated factor 1.

Apoptotic cell death occurs in motoneurons in the neonate but not in the adult after a lesion of a peripheral nerve. To investigate the molecular basis for this difference, we have analyzed the expression and localization of inhibitors of apoptosis proteins (IAPs) and their inhibitors X-linked IAP (XIAP)-associated factor 1 (XAF1), Smac/DIABLO, and Omi/HtrA2 in motoneurons at both ages. Quantitative immunohistochemical and immunoblotting analysis of these proteins in motoneurons revealed an increase in IAP expression [XIAP, neuronal apoptosis inhibitory protein, human IAP1 (HIAP1), and HIAP2] during postnatal development as opposed to XAF1, which decreased during the same period; there was no significant alteration in either Smac/DIABO or Omi/HtrA2. The regulation of IAPs and XAF1 varied after axotomy of the sciatic nerve; in the neonate, there was a significant loss of IAP in the injured motoneurons as opposed to the adult, in which there was only a moderate decrease. By overexpressing exogenous IAPs in neonatal axotomized motoneurons, it was possible to delay motoneuron cell death (Perrelet et al., 2000, 2002). In opposition, the overexpression of exogenous XAF1 in adult motoneurons totally abrogated the natural resistance of these cells to axotomy. The degradation in the adult, induced by XAF1, could be overcome by simultaneously expressing high levels of exogenous XIAP in adult motoneurons. These experiments suggest that it may be the ratio between XAF1 and XIAP that confers the resistance of adult motoneurons to axotomy. In addition, the regulation in the levels of IAPs and XAF1 may be essential in the cell death mechanism of injured motoneurons.

Kainic acid-induced naip expression in the hippocampus is blocked in mice lacking TNF receptors

Mice that lack tumor necrosis factor-α (TNF) receptors are more susceptible than wild-type animals to brain injury produced by kainic acid or transient focal ischemia suggesting that the rapid production of TNF that occurs after these insults serves a neuroprotective role. The mechanisms by which TNF reduces neuronal loss after brain injury may involve the up-regulation of proteins that maintain calcium homeostasis or reduce free radical generation. We report here that systemic administration of kainic acid rapidly elevates expression of mRNA encoding neuronal apoptosis inhibitor protein (NAIP) in the hippocampus and that this increase does not occur in mice that lack TNF receptors. Given that NAIP overexpression can reduce neuronal injury by blocking apoptosis, our findings suggest that induction of the naip gene may contribute to the neuroprotective properties of TNF.

Apoptosis of human abdominal preadipocytes before and after differentiation into adipocytes in culture

Differentiation of murine 3T3-L1 preadipocytes into adipocytes is associated with the acquisition of apoptotic resistance accompanied by the upregulation of cell survival genes. We have now examined the effect of adipogenesis on apoptotic susceptibility of human abdominal preadipocytes in primary culture. To induce apoptosis, human preadipocytes, or their differentiated counterparts, were serum-deprived for 24 or 48 hours. When indicated, ceramide was also used as an apoptotic trigger. Cell death was assessed by enumeration of adherent viable cells, and its apoptotic nature was verified by Hoechst staining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). After 48 hours of serum withdrawal, cell death was 26% ± 4% in preadipocytes and was increased to 41% ± 4% in differentiated adipocytes (mean ± SE; n = 7 patients; P < .002). Under serum-free conditions for 24 hours, ceramide-induced cell death was 40% ± 6% in preadipocytes and increased to 68% ± 8% in adipocytes (mean ± SE; P < .01; n = 8 patients). Neuronal apoptosis inhibitor protein (NAIP), an antiapoptotic protein cell survival that increases upon 3T3-L1 adipogenesis, was reduced in human preadipocytes undergoing differentiation (n = 6 patients). Preadipocytes derived from omental versus subcutaneous abdominal fat were more susceptible to apoptosis induced by serum deprivation, 16% ± 4% versus 31% ± 3% cell death, respectively (mean ± SE; P < .02; n = 7 patients). Although the murine 3T3-L1 preadipocyte cell line is a useful model that approximates primary preadipocyte cell biology, our data derived from human preadipocyte studies suggest important differences with respect to the regulation of apoptosis.

NODs: intracellular proteins involved in inflammation and apoptosis.

NOD (nucleotide-binding oligomerization domain) proteins are members of a family that includes the apoptosis regulator APAF1 (apoptotic protease activating factor 1), mammalian NOD-LRR (leucine-rich repeat) proteins and plant disease-resistance gene products. Several NOD proteins have been implicated in the induction of nuclear factor-kappaB (NF-kappaB) activity and in the activation of caspases. Two members of the NOD family, NOD1 and NOD2, mediate the recognition of specific bacterial components. Notably, genetic variation in the genes encoding the NOD proteins NOD2, cryopyrin and CIITA (MHC class II transactivator) in humans and Naip5 (neuronal apoptosis inhibitory protein 5) in mice is associated with inflammatory disease or increased susceptibility to bacterial infections. Mammalian NOD proteins seem to function as cytosolic sensors for the induction of apoptosis, as well as for innate recognition of microorganisms and regulation of inflammatory responses.

Endogenous expression of inhibitor of apoptosis proteins in facial motoneurons of neonatal and adult rats following axotomy

The inhibitor of apoptosis protein family members inhibit cell death resulting from a variety of apoptotic stimuli. However, the endogenous expression of neuronal inhibitor of apoptosis proteins following axonal injury has not been thoroughly examined. Neonatal facial motoneurons are highly susceptible to axotomy-induced apoptosis, whereas adult facial motoneurons survive axotomy. We hypothesized that the endogenous expression of inhibitor of apoptosis proteins may be involved in the differential susceptibility of adult and neonatal facial motoneurons to axonal injury. In this study, we examined the expression of two endogenous inhibitor of apoptosis proteins, neuronal apoptosis inhibitory protein and x-linked inhibitory apoptosis protein, in adult and neonatal rat facial motoneurons following axotomy. Analyses using reverse-transcription polymerase chain reaction and in situ hybridization indicated that neuronal apoptosis inhibitory protein mRNA was increased in neonatal facial nuclei 24 h post axotomy. In the adult, neuronal apoptosis inhibitory protein mRNA expression increased at 1, 3, 7 and 14 days post axotomy, while little change in the expression of X-linked inhibitory apoptosis protein mRNA was detected at any age or time point time point analyzed. Interestingly, immunohistochemistry using antibodies for neuronal apoptosis inhibitory protein and X-linked inhibitory apoptosis protein, revealed the level of these proteins was higher in the neonatal motoneurons when compared with the adult. Furthermore, immunohistochemistry and western blot for neuronal apoptosis inhibitory protein revealed, in contrast to the observed increase in neuronal apoptosis inhibitory protein mRNA, a decline in the expression of neuronal apoptosis inhibitory protein following axotomy in the adult, whereas no change in neuronal apoptosis inhibitory protein was detected in neonatal facial motoneurons. X-linked inhibitory apoptosis protein, as analyzed by immunohistochemistry and western blot, remained unchanged by axotomy in neonatal motoneurons and adult motoneurons. These results indicate differential expression and/or turnover of inhibitor of apoptosis proteins in neonatal versus adult facial motoneurons, and suggest the level of inhibitor of apoptosis protein expression alone is not an indicator of cell fate following axotomy.

Recent advances in adenovirus-mediated gene therapy for cerebral ischemia.

Cerebral ischemia induces many degenerative cellular reactions, including the release of excitatory amino acids, the formation of oxygen free radicals, Ca2+ overload, the activation of several cellular enzyme systems such as Ca2+ dependent proteases, and the initiation or genomic responses that can affect the tissue outside the area of reduced blood flow. Furthermore, increasing evidence indicates that apoptosis contributes to the death of brain cells following cerebral ischemia. Several studies have shown that cerebral ischemia alters the expression of genes, some of which may play protective or harmful roles. Although many genes have the potential to treat cerebral ischemia, target genes or their translated products are often difficult to express, if at all, in brain cells. However, adenovirus-mediated gene transfer can overcome this disadvantage. To date, many treatment strategies have been developed for cerebral ischemia using target genes such as neuronal apoptosis inhibitory protein (NAIP), glial cell line-derived neurotrophic factor (GDNF), sensitive to apoptosis gene (SAG), 150-kDa oxygen-regulated protein (ORP150), etc. Moreover, new vectors and gene delivery systems are constantly being invented although there is no perfect vector to date. Gene therapy could constitute a powerful strategy to treat cerebral ischemia in the near future.

Cloning and characterization of rat neuronal apoptosis inhibitory protein cDNA

The human neuronal apoptosis inhibitory protein (NAIP) gene was originally discovered because of its deletion in infantile spinal muscular atrophy (SMA), a childhood genetic disorder characterized by motor neuron loss and progressive paralysis with muscular atrophy. Although SMA is now known to be caused by deletions of survival motor neuron (SMN), the fact that NAIP is an anti-apoptotic protein is consistent with the NAIP gene modifying SMA severity. Here we report the cloning of a 1.5 kb rat NAIP cDNA fragment which contains BIR-3 (third baculovirus inhibitory repeat) domain. This fragment shows 78% homology to the human NAIP and 86% homology to the murine counterpart. We have investigated the distribution of NAIP mRNA expressing neurons by in situ RT-PCR technique in the rat central nervous system (CNS). Although all of the neurons appeared to express NAIP mRNA ubiquitously, pronounced elevation of NAIP mRNA expression was observed in the areas innervated by glutamatergic neurons after kainic acid (KA) injection. We have raised an anti-rat NAIP antiserum in rabbits using NAIP cDNA and recombinant rat NAIP, and carried out an immunohistological investigation. We observed highly immunoreactive neuronal subpopulations in the retinal ganglion, cerebral cortex, hippocampus, basal forebrain, thalamus, areas of midbrain, Purkinje cells of the cerebellum, and motor neurons in the spinal cord. Increased immunoreactivity of glutamatergic neurons was also observed broadly in the CNS after KA treatment. This study provides additional evidence that expression of mRNA and gene products of NAIP seem to be regulated in response to excessive stimuli or injuries in rat CNS, and these results are compatible with an anti-apoptotic role of NAIP in acute SMA as well as in brain injuries.

Genetic diagnosis of Werdnig-Hoffmann disease: a problem for application to prenatal diagnosis.

We report a floppy infant with Werdnig-Hoffmann disease (spinal muscular atrophy: SMA type 1) and Klinefelter syndrome. After genetic counseling with parents, a genetic diagnosis using DNA from the infant's peripheral blood mononuclear cells was performed. The parents' deletion of exons 7 and 8 of the survival motor neuron (smn) gene and exons 4 and 5 of the neuronal apoptosis inhibitory protein (naip) gene were noted in the infant, so he was confirmed to have SMA type 1. The parents wanted to receive a prenatal diagnosis on the next pregnancy. However this genetic test is achieved by confirming that a specific band can not be detected by PCR. Therefore, this method should be applied with great care to prenatal diagnosis using chorionic villi, which may be contaminated with maternal tissue.