Local responses to the Maharashtra gutka and pan masala ban: a report from Mumbai.

BACKGROUND: The Maharashtra government has banned the production, sale, distribution and storage of gutka, and pan masala in the Maharashtra State due to the increasing burden of cancer and reproductive health problems attributable to the use of these products. In view of this, it is important to understand the way producers', sellers' and users' are adapting to the ban. OBJECTIVE: During the two months following the ban (July 19 th through Sept 30, 2012), a research team studying smokeless tobacco use and promotion in a low income community of Mumbai conducted rapid surveillance to assess the impact of the ban in the study community. MATERIALS AND METHODS: Assessment involved documenting new points of sale, informal observations of tobacco use, and interviews with thirteen shop owners and eight gutka users'. Overall changes in accessibility, availability, patterns of use of tobacco products, perception of ban, social norms and surveillance activities were assessed. RESULTS: Tobacco companies were marketing new products that resembled gutka, under similar brand logos. Surveillance, financial and social cost of selling gutka or using it in public have had an immediate effect on reducing local supply, demand and use and increasing stigma associated with its use. There was an increased recognition of ill-effects of gutka on cancer among sellers' but not overall. CONCLUSIONS: To reduce the overall consumption of tobacco in the community, it is critical to include programs that create awareness about effects of smokeless tobacco on health and sustain surveillance levels. This would maintain requirements of the ban, and sustain limits on accessibility, availability and use of these products in the community and other similar communities.

Histone deacetylases: focus on the nervous system.

Neurodegenerative disease strikes millions worldwide and there is mounting evidence suggesting that underlying the onset and progression of these debilitating diseases is inappropriate neuronal apoptosis. Recent reports have implicated a family of proteins known as histone deacetylases (HDACs) in various neuronal processes including the neuronal death program. Initial headway in this field has been made largely through the use of broad-spectrum HDAC inhibitors. In fact, pharmacological inhibition of HDAC activity has been shown to protect neurons in several models of neurodegeneration. The observation that HDAC inhibitors can have opposing effects in different paradigms of neurodegeneration suggests that individual members of the HDAC protein family may play distinct roles that could depend on the specific cell type under study. The purpose of this review is to detail work involving the use of HDAC inhibitors within the context of neurodegeneration and examine the roles of individual HDAC members in the nervous system with specific focus on neuronal cell death.

Inactivation of olfactory sensilla of a single morphological type differentially affects the response of Drosophila to odors.

The olfactory organs on the head of Drosophila, antennae and maxillary palps, contain several hundred olfactory hairs, each with one or more olfactory receptor neurons. Olfactory hairs belong to one of three main morphological types, trichoid, basiconic, and coeloconic sensilla, and show characteristic spatial distribution patterns on the surface of the antenna and maxillary palps. Here we show that targeting expression of the cell-death gene reaper to basiconic sensilla (BS) causes the specific inactivation of most olfactory sensilla of this type with no detectable effect on other types of olfactory sensilla or the structure of the antennal lobe. Our data suggest that BS are required for a normal sensitivity to many odorants with a variety of chemical structures, through a wide range of concentrations. Interestingly, however, in contrast to other odorants tested, the behavioral response of ablated flies to intermediate concentrations of propionic and butyric acids is normal, suggesting the involvement of sensilla unaffected by ectopic reaper expression, probably coeloconic sensilla that respond strongly to these two organic acids. As inactivation of BS causes an underestimation of the concentration of both acids detectable at both the highest and lowest odorants concentrations, our results suggest that concentration coding for these two odorants relies on the integration of signals from different subsets of sensilla, most likely of different morphological types.

Novel genes expressed in subsets of chemosensory sensilla on the front legs of male Drosophila melanogaster.

Detection of courtship-activating female pheromones by contact chemoreceptors on the front legs of male Drosophila melanogaster is thought to play an important role in triggering courtship behavior. However, the chemosensory organs, cells, and molecules responsible are not known. We have isolated two genes, CheA29a and CheB42a, expressed in nonneuronal auxiliary cells within two nested subsets of chemosensory sensilla on the front legs of sexually mature, adult males. The proteins encoded by the CheA29a and CheB42a genes have no sequence similarity to each other or any other known protein, but they belong to two novel families of proteins encoded by the D. melanogaster genome. Members of the two families are predicted to have a single transmembrane domain at their amino terminus, probably to serve as a signal peptide, suggesting that they are soluble and secreted. Finally, in addition to CheA29a and CheB42a, other genes within each family are expressed preferentially in appendages where chemosensory organs are concentrated, in several cases in a male-specific manner. Our data suggest that CheA29a and CheB42a and other members of these two protein families are involved in male-specific chemical senses, perhaps pheromone response.

Distinct phosphorylation patterns underlie Akt activation by different survival factors in neurons.

The survival of cultured cerebellar granule neurons can be maintained by depolarizing levels of potassium (high K(+), HK), insulin-like growth factor (IGF-1), cyclic AMP or lithium. We examined the possibility that the signaling pathways activated by these different factors converge and that Akt might represent such a point of convergence. Consistent with this possibility, we find that Akt is phosphorylated and activated by all four survival factors. The pattern of Akt phosphorylation induced by the four survival factors, however, shows differences. While IGF-1 induces phosphorylation of Akt at both Ser473 and Thr308, HK and cyclic AMP stimulate phosphorylation at Thr308 only. Lithium increases phosphorylation at Ser473 but not at Thr308. Our results are consistent with the possibility that Akt is a central component of different survival-promoting pathways in granule neurons. The different phosphorylation patterns, however, point to a previously unappreciated complexity in the regulation of Akt activity in neurons. Finally, we provide evidence indicating that SGK, a kinase that is structurally related to Akt, is also activated by the four survival factors.

Aberrant apoptosis in the neurological mutant Flathead is associated with defective cytokinesis of neural progenitor cells.

Flathead is a rat neurological mutant which is phenotypically characterized by a flattened cranium, resting tremor, ataxia, progressive paralysis of the hind limbs, and death at 3-4 weeks after birth. Previous studies showed that rats homozygous for the mutation have a dramatically reduced brain size caused by a burst of apoptosis that begins after embryonic day 16 (E16) and which peaks at about E18. Late-developing structures such as the dentate gyrus, internal granule layer of the cerebellum, and superficial layers of the neocortex are severely depleted of cells. In the present study we have found that neurons and glia are both affected by the mutation. Immunohistochemical analysis with TAG-1, a marker for migratory neurons, revealed reduced staining in Fh neocortex and cerebellum, indicating that the mutation affects neuronal migration or a developmental event prior to it. Analysis of acutely dissociated neocortical cultures showed an accumulation of nestin-positive progenitor cells. Moreover, a substantial proportion of these progenitor cells were multinucleated with the nuclei organized as rosettes. Such multinucleated cells were also found in intact sections of the neocortex and the cerebellum where their presence was restricted to proliferative zones. Within the neocortex, the abundance of multinucleated progenitors is highest at E18 and decreases thereafter, thus correlating with the profile of cell death. This, along with the dramatically higher frequency of apoptosis among multinucleated cells, suggests that the aberrant cell death in Fh is due to defective cytokinesis that occurs in progenitor cells during late stages of brain development.

NF-kappaB is involved in the survival of cerebellar granule neurons: association of IkappaBbeta [correction of Ikappabeta] phosphorylation with cell survival.

The NF-kappaB transcription factor consists of dimeric complexes belonging to the Rel family, which include p50, p52, p65 (RelA), RelB and c-Rel. NF-kappaB activity is tightly controlled by IkappaB proteins which bind to NF-kappaB preventing its translocation to the nucleus. Activation of NF-kappaB is most often mediated by IkappaB degradation, which permits NF-kappaB to enter the nucleus. We investigated the role of NF-kappaB in the survival of cerebellar granule neurons. We found that survival of these neurons in high potassium medium is blocked by three separate inhibitors of NF-kappaB activity: SN-50, N-tosyl-L-phenylalanine chloromethyl ketone and pyrrolidinedithiocarbamate, indicating that NF-kappaB is required for neuronal survival. Gel-shift assays reveal three complexes that bind to the NF-kappaB binding site in high potassium medium. Switching these cultures to low potassium medium, a stimulus that leads to apoptotic death, causes a reduction in the level of the largest complex, which contains p65. Overexpression of p65 by transfection inhibits low potassium-induced apoptosis, whereas overexpression of IkappaBalpha promotes apoptosis even in high potassium medium. Surprisingly, however, neither the level of endogenous p65 nor that of IkappaBalpha and IkappaBbeta is altered by low potassium treatment. Similarly, no changes are seen in the nuclear or cytoplasmic levels of p50, p52, RelB and c-Rel. Phosphorylation of p65, which can lead to its activation, is unchanged. Phosphorylation of IkappaBbeta is, however, reduced by low potassium treatment. Besides being necessary for high potassium-mediated neuronal survival, NF-kappaB is also involved in the survival-promoting effects of IGF-1 and cAMP as judged by the ability of SN-50 to inhibit the actions of these survival factors and the ability of these factors to inhibit the low potassium-induced alterations in the DNA-binding activity of NF-kappaB. Taken together, our results show that NF-kappaB may represent a point of convergence in the signaling pathways activated by different survival factors and that uncommon mechanisms might be involved in NF-kappaB-mediated survival of cerebellar granule neurons.

The flathead mutation causes CNS-specific developmental abnormalities and apoptosis.

We describe a new mutation, flathead (fh), that arose spontaneously in an inbred colony of Wistar rats. The mutation is autosomal recessive, and the behavioral phenotype of fh/fh rats includes spontaneous seizures, tremor, impaired coordination, and premature death. A striking feature of the fh mutation is a dramatic reduction in brain size (40% of normal at birth). In contrast, no abnormalities are evident in the peripheral nervous system or in other tissues outside of the CNS. Although bromodeoxyuridine incorporation assays indicate that the rate of cell proliferation in the fh/fh cortex is similar to that of unaffected animals, in situ terminal deoxynucleotidyl transferase-mediated dUTP-biotin end-labeling assays reveal a dramatic increase in apoptotic cell death beginning after embryonic day 16 (E16). At E18 there is a 20-fold increase in cell death in the ventricular zone of fh/fh neocortex, and at postnatal day 1 (P1), the number of apoptotic cells is still two times that of normal. However, by P8 the extent of cell death in fh/fh is comparable to that of unaffected littermates, indicating that the reduction in brain growth is caused by abnormally high apoptosis during a discrete developmental period. Late-developing structures such as the cerebellum, neocortex, hippocampus, and retina are most severely affected by the fh mutation. Within these structures, later-generated neuronal populations are selectively depleted. Together, these results suggest that the flathead gene is essential for a developmental event required for the generation and maturation of late-born cell populations in the brain.

Caspase-3 is required for apoptosis-associated DNA fragmentation but not for cell death in neurons deprived of potassium.

Caspases are crucial effectors of the cell death pathway activated by virtually all apoptosis-inducing stimuli within neurons and nonneuronal cells. Among the caspases, caspase-3 (CPP32) appears to play a pivotal role and has been found to be necessary for developmentally regulated cell death in the brain. We have used mice lacking caspase-3 (-/-CPP32) to examine its involvement in cultured cerebellar granule neurons induced to undergo apoptosis by potassium deprivation (K+). We find that, following K+ deprivation, neurons from -/-CPP32 mice die to the same extent as those from normal (+/+) mice. Although a small delay in the induction of cell death is observed in -/-CPP32 neurons, the rate of cell death is generally comparable to that of +/+ cultures. Though not critical for neuronal death, caspase-3 is required for DNA fragmentation and chromatin condensation as judged by the absence of these apoptotic features in -/-CPP32 neurons. Boc.Asp.fmk, a pan caspase inhibitor, partially protects +/+ neurons from low-K+-mediated cell death and does so to the same extent in -/-CPP32 cultures, suggesting the involvement of a caspase other than caspase-3 in cell death. However, the protective effect of boc.Asp.fmk is not seen beyond 24 hr, suggesting that the effect of caspase inhibition is one of delaying rather than preventing apoptosis. The more selective caspase inhibitors DEVD.fmk, IETD.fmk, and VEID.fmk fail to affect cell death, indicating that members inhibited by these agents (such as caspases - 6 ,7, 8, 9 and 10) are also not involved in low-K+-mediated apoptosis.

Characterization of seizures in the flathead rat: a new genetic model of epilepsy in early postnatal development.

PURPOSE: Disorders in normal central nervous system (CNS) development are often associated with epilepsy. This report characterizes seizures in a novel genetic model of developmental epilepsy, the Flathead (FH) rat. METHODS: Animals (n = 76) ages P0-22 were monitored for clinical and electrographic seizure activity. The effects of various AEDs on seizure frequency and duration also were assessed: phenobarbital (PB; 40 mg/kg), valproate (VPA; 400 mg/kg), or ethosuximide (ESM; 600 mg/kg). RESULTS: FHs display episodes of behavior characterized by whole-body tremor, strub tail, alternating forelimb clonus, and complete tonus. EEG recordings from neocortex reveal that FH seizures are bilateral and begin around P7. Seizures occur at a frequency of approximately six per hour from P7 to P18 and the average duration of seizures increases through development. PB, VPA, and ESM failed to prevent seizures; however, PB significantly increased the interval of seizures but had no effects on the duration of seizures, whereas VPA decreased the duration of seizures and not the interval. CONCLUSIONS: Seizures in FH rats occur at a constant and high frequency through a defined period in early postnatal development, and these seizures are not completely blocked by high doses of PB, VPA, or ESM. Because FH is a single-locus mutant displaying a highly regular pattern of seizure activity, it is an ideal model for examining the process of epileptogenesis in the developing brain, evaluating new AED therapies, and determining the identity of a gene essential to the normal development of cortical excitability.

Decreased expression of the metabotropic glutamate receptor-4 gene is associated with neuronal apoptosis.

Cultured cerebellar granule neurons die by apoptosis when switched from medium containing elevated potassium (K+) and serum to serum-free medium containing low K+. Although cell death begins at about 16 hr, commitment to death occurs within 6 hr after the lowering of K+. We have used this paradigm to examine the role of metabotropic glutamate receptor (mGluR) genes in the regulation of neuronal survival. We find that the expression of one of the mGluR genes, the type-4 gene, is associated with increased neuronal survival. Lowering of K+ leads to an 80% decrease in mGluR-4 mRNA expression within 6 hr. Downregulation of mGluR-4 messenger RNA (mRNA) does not occur if low K+-induced death is prevented by treatment with insulin-like growth factor I or adenosine 3',5'-cyclic monophosphate. If transcription is inhibited by actinomycin D, the difference in mGluR4 mRNA expression between cells switched to high-K+ medium and those switched to low-K+ medium is dramatically reduced, suggesting that decreased mGluR-4 gene transcription rather than increased mRNA breakdown is mainly responsible for the apoptosis-associated decrease in mGluR4 levels. Blockade of transcription also reduces mGluR4 mRNA expression in healthy neurons by more than 50% within 4 hr, suggesting that the mGluR4 mRNA has a relatively short half-life. In pharmacological experiments, we observe that the specific group III mGluR agonists such as L-amino-4-phosphobutyric acid and O-phospho-L-serine inhibit low K+-induced apoptosis. On the other hand, a selective mGluR4 antagonist, (RS)-alpha-cyclopropyl-4-phosphono-phenylglycine, induces apoptosis even in the presence of elevated K+. These results indicate that elevated mGluR4 expression or the activation of this receptor promotes survival and that an inhibition of such survival mechanisms contributes to apoptosis.

A gene essential to brain growth and development maps to the distal arm of rat chromosome 12.

A recently discovered, spontaneous, autosomal recessive mutation in rats, flathead (fh), results in greatly reduced brain growth beginning in late fetal development. In this study we have mapped the fh mutation by determining the pattern of segregation of polymorphic microsatellite markers with respect to fh in 51 affected F2 offspring from a single interstrain intercross. Two markers on chromosome 12, D12Rat80 and D12Mgh6, cosegregated with the fh mutation in all 51 affected animals. The distribution of six additional markers in 40 informative meioses further localizes fh approximately 2 cM teleomeric to nos1. There are no known mutations in homologous regions of either mouse or human genomes that result in deficits in late neurodevelopment similar to that observed in fh/fh animals. The unique phenotype of fh/fh animals and the location of fh suggests the presence of a novel gene essential to normal brain development on the distal end of rat chromosome 12.

A DEVD-inhibited caspase other than CPP32 is involved in the commitment of cerebellar granule neurons to apoptosis induced by K+ deprivation.

Cultured cerebellar granule neurons undergo apoptosis when switched from a medium containing depolarizing levels of K+ (25 mM KCl) to medium containing lower levels of K+ (5 mM KCl). We used this paradigm to investigate the role of caspases in the death process. Two broad-spectrum caspase inhibitors, tert-butoxycarbonyl-Asp x (O-methyl) x fluoromethyl ketone and benzyloxycarbonyl-Val-Ala-Asp x fluoromethyl ketone, significantly reduced cell death (90 and 60%, respectively) at relatively low concentrations (10-25 microM), suggesting that caspase activation is involved in the apoptotic process. DNA fragmentation, a hallmark of apoptosis, was also reduced by these caspase inhibitors, suggesting that caspase activation occurred upstream of DNA cleavage in the sequence of events leading to cell death. As a step toward identifying the caspase(s) involved, the effects of N-acetyl Tyr-Val-Ala-Asp x chloromethyl ketone (YVAD x cmk), an interleukin-1beta converting enzyme-preferring inhibitor, and N-acetyl Asp-Glu-Val-Asp x fluoromethyl ketone (DEVD x fmk), a CPP32-preferring inhibitor, were also evaluated. YVAD x cmk provided only modest (<20%) protection and only at the highest concentration (100 microM) tested, suggesting that interleukin-1beta converting enzyme and/or closely related caspases were not involved. In comparison, DEVD x fmk inhibited cell death by up to 50%. Western blot analyses, however, failed to detect an increase in processing/activation of CPP32 or in the proteolysis of a CPP32 substrate, poly(ADP-ribose) polymerase, during the induction of apoptosis in granule neurons. Similarly, the levels of Nedd2, a caspase that is highly expressed in the brain and that is partially inhibited by DEVD x fmk, also remained unaffected in apoptotic neurons undergoing apoptosis. These results suggest that a DEVD-sensitive caspase other than CPP32 or Nedd2 mediates the induction of apoptosis in K+-deprived granule neurons.

Molecular regulation of neuronal apoptosis.

Apoptosis is a fundamental biological process used by all muticellular organisms to eliminate unwanted or superfluous cells, and is a prominent feature of normal neural development. Developmentally occurring neuronal apoptosis serves to match the number of neurons to the requirements of their synaptic targets and to rid the nervous system of inappropriate connections. While it is generally accepted that apoptosis is a "suicide program" inherent in all cells, the molecular basis of this program is just beginning to be unraveled. Evidence from numerous recent studies indicate that a variety of proteins are involved in the transmission of external signals to the cell-death machinery within the cell. This review describes many of the recent findings of the regulatory pathways and genes that have been implicated in the induction or suppression of apoptosis in neurons.

Insulin-like growth factor and potassium depolarization maintain neuronal survival by distinct pathways: possible involvement of PI 3-kinase in IGF-1 signaling.

Cultured cerebellar granule neurons die by apoptosis when switched from a medium containing an elevated level of potassium (K+) to one with lower K+ (5 mM). Death resulting from the lowering of K+ can be prevented by insulin-like growth factor (IGF-1). To understand how IGF-1 inhibits apoptosis and maintains neuronal survival, we examined the role of phosphoinositide 3-kinase (PI 3-kinase). Activation of PI 3-kinase has been shown previously to be required for NGF-mediated survival in the PC12 pheochromocytoma cell line. We find that in primary neurons, IGF-1 treatment leads to a robust activation of PI 3-kinase, as judged by lipid kinase assays and Western blot analysis. Activation of PI 3-kinase is likely to occur via tyrosine phosphorylation of the insulin receptor substrate protein. Treatment with two chemically distinct inhibitors of PI 3-kinase, wortmannin and LY294002, reduces PI 3-kinase activation by IGF-1 and inhibits its survival-promoting activity, suggesting that PI 3-kinase is necessary for IGF-1-mediated survival. Death resulting from PI 3-kinase blockade is accompanied by DNA fragmentation, a hallmark of apoptosis. Furthermore, neurons subjected to PI 3-kinase blockade can be rescued by transcriptional and translation inhibitors, suggesting that IGF-1-mediated activation of PI 3-kinase leads to a suppression of "killer gene" expression. In sharp contrast to IGF-1, elevated K+ does not activate PI 3-kinase and can maintain neuronal survival in the presence of PI 3-kinase inhibitors. Therefore, survival of granule neurons can be maintained by PI 3-kinase dependent (IGF-1-activated) and independent (elevated K+-activated) pathways.

Left ventricular morphology in chronic renal failure by echocardiography.

M-mode, two-dimensional, and Doppler echocardiography were performed in 38 chronic renal failure (CRD) patients on conservative management, 35 patients on hemodialysis, and 36 matched controls. The controls were matched for age, sex, and comorbidities. The incidence of hypertension, left ventricular (LV) end diastolic volume, LV end systolic volume, and LV mass index were significantly higher in patients on hemodialysis compared to the controls. The LV parameters in the predialysis patients were not significantly different from the controls, except the LV end systolic internal dimensions were significantly higher in the CRF patients. Multiple regression analysis underscored the strong association between increase in LV mass index (LVMI) and hypertension. The diabetic patients with renal failure had large LV internal diameter and end diastolic volume compared to non-diabetics. Systolic function was well preserved even in hypertensive and diabetic patients with uremia. The incidence of diastolic dysfunction and asymmetrical septal hypertrophy were not significantly different in the three groups of patients.

Opposing effects of thapsigargin on the survival of developing cerebellar granule neurons in culture.

Elevated levels of potassium (K+) promote maturation and survival of cerebellar granule neurons in culture. When switched from a culture medium containing high K+ (25 mM) to one with low K+ (5 mM) mature granule neurons undergo death by apoptosis. The mechanism by which high K+ promotes neuronal survival (and conversely inhibits apoptosis) is unclear. Several pieces of evidence indicate that an increase in intracellular calcium (Ca2+) resulting from depolarization mediated-influx of extracellular Ca2+ is necessary. We examined the effect of thapsigargin on granule neuron cultures. Thapsigargin is an inhibitor of the endoplasmic reticular Ca2+ ATPase causing a depletion of Ca2+ from internal stores. This treatment would therefore be expected to raise intracellular cytosolic Ca2+ without membrane depolarization. We find that treatment of mature neurons with thapsigargin at doses > or = 5 nM inhibits death resulting from the lowering of extracellular K+. The survival effect of thapsigargin was not affected by inhibitors of extracellular Ca2+ influx including nifedipine, verapamil, methoxyverapamil, Mg2+, and Ni2+, nor was it inhibited by the NMDA receptor antagonist, MK801. We have further examined whether thapsigargin could substitute for elevated K+ during the maturation of granule cells. Unexpectedly, treatment of younger (immature) neuronal cultures with the same dose of thapsigargin (5 nM) induced cell death. DNA fragmentation analysis suggested that death was due to apoptosis and not toxicity. As observed with the survival effect on mature neurons, the lethal effect of thapsigargin on immature granule cells was not prevented by inhibitors of Ca2+ influx.

Lithium induces apoptosis in immature cerebellar granule cells but promotes survival of mature neurons.

Lithium (Li+) has been used in the treatment of manic-depressive disorders for several decades. More recently, Li+ has been shown to affect the signaling pathway of various neurotransmitters and growth/neurotrophic factors. We examined the effect of Li+ on the survival of cerebellar granule neurons in culture. Treatment of immature granule cells with Li+ resulted in programmed cell death (apoptosis). The death process is accompanied by DNA fragmentation, a hallmark of apoptosis. Following maturation in vitro, granule neurons are dependent on elevated concentrations of extracellular potassium ([K+]o) for survival. Lowering of [K+]o to physiological levels induces apoptosis. Surprisingly, Li+ prevents death of mature neurons caused by low [K+]o. Moreover, the concentration range at which Li+ exerts its protective effect is the same as that at which it induces apoptosis in immature neurons. Thus, a single agent under similar extracellular conditions has opposing effects on survival, depending on the developmental status of the neuron.

Induction of apoptosis in cerebellar granule neurons by low potassium: inhibition of death by insulin-like growth factor I and cAMP.

High levels of extracellular K+ ensure proper development and prolong survival of cerebellar granule neurons in culture. We find that when switched from a culture medium containing high K+ (25 mM) to one containing a low but more physiological K+ concentration (5 mM), differentiated granule neurons degenerate and die. Death induced by low K+ is due to apoptosis (programmed cell death), a form of cell death observed extensively in the developing nervous system and believed to be necessary for proper neurogenesis. The death process is accompanied by cleavage of genomic DNA into internucleosome-sized fragments, a hallmark of apoptosis. Inhibitors of transcription and translation suppress apoptosis induced by low K+, suggesting the necessity for newly synthesized gene products for activation of the process. Death can be prevented by insulin-like growth factor I but not by several other growth/neurotrophic factors. cAMP but not the protein kinase C activator phorbol 12-myristate 13-acetate can also support survival in low K+. In view of the large numbers of granule neurons that can be homogeneously cultured, our results offer the prospect of an excellent model system to study the mechanisms underlying apoptosis in the central nervous system and the suppression of this process by survival factors such as insulin-like growth factor I.

SGP2, ubiquitin, 14K lectin and RP8 mRNAs are not induced in neuronal apoptosis.

Cultured embryonic sympathetic neurones and differentiated PC12 cells undergo apoptosis when deprived of nerve growth factor (NGF). Apoptosis caused by NGF deprivation can be prevented by inhibitors of RNA and protein synthesis suggesting the involvement of newly synthesized gene products in the death process. We have examined in these neurones, the expression of four genes known to be stimulated in non-neuronal cells, specifically during apoptosis. The levels of SGP-2, ubiquitin, and RP-8 mRNAs are not altered during neuronal death. Expression of 14-K lectin mRNA is down-regulated 3-4 fold. These results show that the four genes examined do not serve as 'death genes' in the induction of apoptosis in neuronal cells and raise the possibility that other genes and mechanisms are involved.