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Purpose: This study assessed (1) the experience of the eating disorder voice in people with anorexia nervosa or in remission, and (2) the feasibility of creating and interacting with a computerised representation (i.e., avatar) of this voice. Methods: Twenty-one individuals with anorexia nervosa and 18 individuals who were in remission participated in the study. They reported on the characteristics of their eating disorder voice and created a personalised avatar (a visual and auditory representation of the eating disorder voice), using a computerised software. Participants assessed closeness of match between the voice and the avatar, perceived distress and acceptability of re-exposure to the avatar. Results: Patients felt less powerful than their eating disorder voice and unable to disregard the voice's commands. The experience of the voice was associated with negative, as well as some positive emotions, reflecting the prototypical ambivalence towards the illness. Individuals in remission had an opposite pattern of responses. They attributed only negative emotions to the voice, felt more powerful than the voice, and able to disregard its commands. Overall participants reported that there was a good match between the voice and the sound of the avatar. Patients expressed willingness to repeat exposure to the avatar. Conclusion: Individuals with anorexia can create personalised digital avatars representing the eating disorder voice and are willing to engage therapeutically with the avatar. The next step is to test the feasibility of repeated exposure to the avatar to address the power and distress associated with the eating disorder voice. Level of evidence: Level III.

People with anorexia nervosa often struggle with connecting to their identity beyond the illness. They tend to relate to the eating disorder from a powerless and submissive position. The aim of this study was to test AVATAR therapy for anorexia nervosa. The goal of the intervention was to help patients to assert themselves, their identity, will, power and desires, when confronted with the “voice” of the illness. Twelve patients with anorexia nervosa were recruited and 10 completed one assessment session and five to seven therapy sessions. During the assessment session, patients were guided by the therapist to create a representation of their illness (“the avatar”) by manipulating visual and auditory characteristics through a computer software. During treatment, they were supported by the therapist to assert themselves over the illness, responding to the abusive and critical comments of the eating disorder delivered by “the avatar”. Patients’ qualitative feedback indicated that the therapy was meaningful to them, and that it helped to recognise their own voice as distinct from the illness. The distress perceived in relation to the eating disorder voice decreased over time, and feelings of self-compassion increased at the end of therapy. Findings point towards the acceptability and usefulness of AVATAR therapy for anorexia nervosa. Larger studies are needed expand these results.

ABSTRACTIntroduction: Inappropriate activation of apoptosis may contribute to neurodegeneration, a multifaceted process that results in various chronic disorders, including Alzheimer’s and Parkinson’s diseases. Several in vitroand in vivostudies demonstrated that neuronal apoptosis is a multi-pathway cell-death program that requires RNA synthesis. Thus, transcriptionally activated genes whose products induce cell death can be triggered by different stimuli and antagonized by neurotrophic factors. Systems biology is now unveiling the series of intracellular signaling pathways and key drug targets at the intersection of neuronal apoptosis and survival.Areas covered: This review introduces a genomic approach that can be used to elucidate the systems biology of neuronal apoptosis and survival, and to rationally select drug targets, no longer oriented to emulate the action of growth factors at the membrane receptor level, but rather to modulate their downstream signals.Expert opinion: The advent of genomics is offering an unprecedented opportunity to explore how the delicate balance between apoptosis and survival-inducing signals triggers a transcriptional program. Characterization of this program can be useful to identify potential pharmacological targets for existing drugs. Such knowledge might pave the way towards an innovative pharmacology.

Alzheimer's disease (AD) is an irreversible neurodegenerative disease, clinically characterized by progressive impairments of memory and cognition. The hallmarks of AD are neurofibrillary tangles, mainly constituted by altered phosphorylated and truncated portions of tau protein, and the abnormal extracellular deposition of neurotoxic beta amyloid (Aβ) peptides, derived from the proteolytic processing of amyloid precursor protein (APP). According to the amyloid hypothesis, Aβ is considered to be linked to the selective neurodegeneration seen in AD. Recent evidence points to an increase in voltage-gated potassium (Kv) channel currents in the etiology of Aβ-induced neuronal apoptosis. Substance P (SP) is an 11-aa neuropeptide, member of the tachykinin family, broadly distributed in the Central Nervous System where it acts as a neurotransmitter, neuromodulator, and neurotrophic factor. This peptide may play an important role in neurodegenerative disorders, since reduced levels of SP were found in brain areas and spinal fluid of AD patients. In addition to its neuroprotective properties, it was recently demonstrated that SP is able to stimulate non-amyloidogenic APP processing, thereby reducing the possibility of generation of toxic Aβ peptides in the brain. Recent studies, using in vitro and in vivo models, have also shown that the neuroprotective role of SP against Aβ could be related to its ability of modulate Kv channel currents. In this review, we briefly summarized the current findings on the neurotrophic and neuroprotective effects of SP, providing information about its anti-amyloidogenic and anti-Aβ toxicity role.

To expand the clinical phenotype of autosomal dominant congenital spinal muscular atrophy with lower extremity predominance (SMA-LED) due to mutations in the dynein, cytoplasmic 1, heavy chain 1 (DYNC1H1) gene.

The development of neuronal apoptosis depends on an intrinsic transcriptional program. By DNA microarray technology, we have previously implicated a number of genes in different paradigms of neuronal apoptosis. In the present study, we investigated the spatiotemporal pattern of expression of two of these genes, gastric inhibitory polypeptide (Gip) and its receptor (Gipr) in the rat central nervous system. The levels of their transcripts were measured with real-time quantitative polymerase chain reaction and in situ-hybridization. Widespread expression of Gip and Gipr was found in adult rat brain, whereas during postnatal cerebellum development, they were highly expressed in the external and internal granule layer, and in Purkinje cells. To investigate the possible biological function of Gip we examined its effects in vitro. Addition of Gip to cultured cerebellar granule neurons reduced the extent of apoptotic death induced by switching the growing medium from 25 to 5 mM K. This neurotrophic effect was mimicked by that of PACAP38 and IGF1. We conclude that Gip acts as an endogenous neurotrophic factor and supports neuronal survival.

Chronic lymphocytic leukemia (CLL) is an incurable adult disease of unknown etiology. Understanding the biology of CLL cells, particularly cell maturation and growth in vivo, has been impeded by lack of a reproducible adoptive transfer model. We report a simple, reproducible system in which primary CLL cells proliferate in nonobese diabetes/severe combined immunodeficiency/γcnullmice under the influence of activated CLL-derived T lymphocytes. By cotransferring autologous T lymphocytes, activated in vivo by alloantigens, the survival and growth of primary CFSE-labeled CLL cells in vivo is achieved and quantified. Using this approach, we have identified key roles for CD4+T cells in CLL expansion, a direct link between CD38 expression by leukemic B cells and their activation, and support for CLL cells preferentially proliferating in secondary lymphoid tissues. The model should simplify analyzing kinetics of CLL cells in vivo, deciphering involvement of nonleukemic elements and nongenetic factors promoting CLL cell growth, identifying and characterizing potential leukemic stem cells, and permitting preclinical studies of novel therapeutics. Because autologous activated T lymphocytes are 2-edged swords, generating unwanted graph-versus-host and possibly autologous antitumor reactions, the model may also facilitate analyses of T-cell populations involved in immune surveillance relevant to hematopoietic transplantation and tumor cytoxicity.

Chronic lymphocytic leukemia (CLL) is an incurable adult disease of unknown etiology. Understanding the biology of CLL cells, particularly cell maturation and growth in vivo, has been impeded by lack of a reproducible adoptive transfer model. We report a simple, reproducible system in which primary CLL cells proliferate in nonobese diabetes/severe combined immunodeficiency/γcnull mice under the influence of activated CLL-derived T lymphocytes. By cotransferring autologous T lymphocytes, activated in vivo by alloantigens, the survival and growth of primary CFSE-labeled CLL cells in vivo is achieved and quantified. Using this approach, we have identified key roles for CD4+ T cells in CLL expansion, a direct link between CD38 expression by leukemic B cells and their activation, and support for CLL cells preferentially proliferating in secondary lymphoid tissues. The model should simplify analyzing kinetics of CLL cells in vivo, deciphering involvement of nonleukemic elements and nongenetic factors promoting CLL cell growth, identifying and characterizing potential leukemic stem cells, and permitting preclinical studies of novel therapeutics. Because autologous activated T lymphocytes are 2-edged swords, generating unwanted graph-versus-host and possibly autologous antitumor reactions, the model may also facilitate analyses of T-cell populations involved in immune surveillance relevant to hematopoietic transplantation and tumor cytoxicity.

During normal nervous system development, physiologically appropriate neuronal apoptosis contributes to a sculpting process that removes approximately one-half of all neurons born during neurogenesis. However, neuronal apoptosis subsequent to this developmental window is physiologically inappropriate for most systems and can contribute to neurodegenerative diseases. Neuronal apoptosis is characterized by specific morphological events and requires the activation of an intrinsic transcriptional program. With the completion of genome sequencing in humans and model organisms, and the advent of DNA microarray technology, the transcriptional cascades and networks regulating neuronal apoptosis are being elucidated providing new potential pharmacological targets. This review will introduce the reader to this genomic approach and illustrate with a few examples a methodological strategy for the rational selection of pharmacological targets and the development of neuroprotective agents.

Clonal evolution and outgrowth of cellular variants with additional chromosomal abnormalities are major causes of disease progression in chronic lymphocytic leukemia (CLL). Because new DNA lesions occur during S phase, proliferating cells are at the core of this problem. In this study, we used in vivo deuterium (2H) labeling of CLL cells to better understand the phenotype of proliferating cells in 13 leukemic clones. In each case, there was heterogeneity in cellular proliferation, with a higher fraction of newly produced CD38+ cells compared with CD38− counterparts. On average, there were 2-fold higher percentages of newly born cells in the CD38+ fraction than in CD38− cells; when analyzed on an individual patient basis, CD38+2H-labeled cells ranged from 6.6% to 73%. Based on distinct kinetic patterns, interclonal heterogeneity was also observed. Specifically, 4 patients exhibited a delayed appearance of newly produced CD38+ cells in the blood, higher leukemic cell CXC chemokine receptor 4 (CXCR4) levels, and increased risk for lymphoid organ infiltration and poor outcome. Our data refine the proliferative compartment in CLL based on CD38 expression and suggest a relationship between in vivo kinetics, expression of a protein involved in CLL cell retention and trafficking to solid tissues, and clinical outcome.

Clonal evolution and outgrowth of cellular variants with additional chromosomal abnormalities are major causes of disease progression in chronic lymphocytic leukemia (CLL). Because new DNA lesions occur during S phase, proliferating cells are at the core of this problem. In this study, we used in vivo deuterium (2H) labeling of CLL cells to better understand the phenotype of proliferating cells in 13 leukemic clones. In each case, there was heterogeneity in cellular proliferation, with a higher fraction of newly produced CD38+cells compared with CD38−counterparts. On average, there were 2-fold higher percentages of newly born cells in the CD38+fraction than in CD38−cells; when analyzed on an individual patient basis, CD38+2H-labeled cells ranged from 6.6% to 73%. Based on distinct kinetic patterns, interclonal heterogeneity was also observed. Specifically, 4 patients exhibited a delayed appearance of newly produced CD38+cells in the blood, higher leukemic cell CXC chemokine receptor 4 (CXCR4) levels, and increased risk for lymphoid organ infiltration and poor outcome. Our data refine the proliferative compartment in CLL based on CD38 expression and suggest a relationship between in vivo kinetics, expression of a protein involved in CLL cell retention and trafficking to solid tissues, and clinical outcome.

We examined copy number changes in the genomes of B cells from 58 patients with chronic lymphocytic leukemia (CLL) by using representational oligonucleotide microarray analysis (ROMA), a form of comparative genomic hybridization (CGH), at a resolution exceeding previously published studies. We observed at least 1 genomic lesion in each CLL sample and considerable variation in the number of abnormalities from case to case. Virtually all abnormalities previously reported also were observed here, most of which were indeed highly recurrent. We observed the boundaries of known events with greater clarity and identified previously undescribed lesions, some of which were recurrent. We profiled the genomes of CLL cells separated by the surface marker CD38 and found evidence of distinct subclones of CLL within the same patient. We discuss the potential applications of high-resolution CGH analysis in a clinical setting.

We examined copy number changes in the genomes of B cells from 58 patients with chronic lymphocytic leukemia (CLL) by using representational oligonucleotide microarray analysis (ROMA), a form of comparative genomic hybridization (CGH), at a resolution exceeding previously published studies. We observed at least 1 genomic lesion in each CLL sample and considerable variation in the number of abnormalities from case to case. Virtually all abnormalities previously reported also were observed here, most of which were indeed highly recurrent. We observed the boundaries of known events with greater clarity and identified previously undescribed lesions, some of which were recurrent. We profiled the genomes of CLL cells separated by the surface marker CD38 and found evidence of distinct subclones of CLL within the same patient. We discuss the potential applications of high-resolution CGH analysis in a clinical setting.

Chronic lymphocytic leukemia (CLL) cells are thought to have diminished cell-cycling capacity, a view challenged by their phenotypic resemblance to activated human B lymphocytes. The present study addresses the cell-cycling status of CLL cells, focusing on those leukemic cells expressing CD38, a molecule involved in signaling and activation that also serves as a prognostic marker in this disease. CD38+ and CD38− members of individual CLL clones were analyzed for coexpression of molecules associated with cellular activation (CD27, CD62L, and CD69), cell-cycle entry (Ki-67), signaling (ZAP-70), and protection from apoptosis (telomerase and Bcl-2). Regardless of the size of the CD38+ fraction within a CLL clone, CD38+ subclones are markedly enriched for expression of Ki-67, ZAP-70, human telomerase reverse transcriptase, and telomerase activity. Although the percentage of cells (approximately 2%) entering the cell cycle as defined by Ki-67 expression is small, the absolute number within a clone can be sizeable and is contained primarily within the CD38+ fraction. Despite these activation/proliferation differences, both CD38+ and CD38− fractions have similar telomere lengths, suggesting that CD38 expression is dynamic and transient. These findings may help explain why high percentages of CD38+ cells within clones are associated with poor clinical outcome.

Chronic lymphocytic leukemia (CLL) cells are thought to have diminished cell-cycling capacity, a view challenged by their phenotypic resemblance to activated human B lymphocytes. The present study addresses the cell-cycling status of CLL cells, focusing on those leukemic cells expressing CD38, a molecule involved in signaling and activation that also serves as a prognostic marker in this disease. CD38+and CD38−members of individual CLL clones were analyzed for coexpression of molecules associated with cellular activation (CD27, CD62L, and CD69), cell-cycle entry (Ki-67), signaling (ZAP-70), and protection from apoptosis (telomerase and Bcl-2). Regardless of the size of the CD38+fraction within a CLL clone, CD38+subclones are markedly enriched for expression of Ki-67, ZAP-70, human telomerase reverse transcriptase, and telomerase activity. Although the percentage of cells (approximately 2%) entering the cell cycle as defined by Ki-67 expression is small, the absolute number within a clone can be sizeable and is contained primarily within the CD38+fraction. Despite these activation/proliferation differences, both CD38+and CD38−fractions have similar telomere lengths, suggesting that CD38 expression is dynamic and transient. These findings may help explain why high percentages of CD38+cells within clones are associated with poor clinical outcome.

Apoptosis has been postulated to play a possible causal role in the onset of Alzheimer's disease due to shortage of trophic supply, deafferentation and excessive production of free radicals. Many experiments in the past have demonstrated the requirement of de novo gene expression during neuronal apoptosis. In view of the possible involvement of apoptotic processes in Alzheimer's disease and to begin a comprehensive survey of the gene-based molecular mechanisms that underlie these events we have used genome scale screening by DNA microarray technology in cerebellar granule neurons following serum and potassium deprivation. From the 8740 genes interrogated by the microarrays, 423 genes were found regulated both at the transcriptional and post-transcriptional level and segregated into distinct clusters. Functional clustering based on gene ontologies showed coordinated expression of genes with common biological functions and metabolic pathways. Among the genes implicated in apoptotic cerebellar granule neurons, 70 were in common with those differentially expressed in cortical neurons exposed to amyloid andamp;#946;-protein, indicating the existence of common mechanisms responsible of neuronal cell death. This new approach offer a genomic view of the changes that accompany neuronal apoptosis and yield new insights into the molecular basis underlying it.

Doppel and the prion protein (PrP) are two related proteins involved in different aspects of neuronal degeneration. While a structural modification of PrP is necessary and sufficient for its toxic effect, the neurotoxicity of Doppel in the Purkinje cells of the cerebellum relies solely on its overexpression. Understanding the Doppel-related neurotoxicity thus involves the analysis of its developmental and transcriptional regulation. Here we report for the first time that Doppel is expressed in the embryonic neurons of mice dorsal root ganglia and spinal cord and that the closely related Brn-3a and Brn-3b transcription factors are involved in its transcriptional regulation.

In vitrocultured cerebellar granule cells (cgc) undergo apoptotic death when the depolarising concentration of KCl 25 mM is adjusted to 5 mM. We investigated whether the apoptotic message can be transferred from a group of neurons to neighbouring but separated sister cultures by resorting to two different culture systems, one based on the use of concentric dishes, the other employing the transwells. The extent of transferred death was 38.6 in the two dishes system and 31.3 in the transwell system. The transfer of such death-inducing signal(s), accompanied by chromatin condensation, was inhibited by actinomicin D. Preliminary experiments aimed at identifying such apoptosis-inducing signal(s) suggest the involvement of β-amyloid fragment(s).

The excitatory neurotransmitter glutamate plays a major role in determining certain neurological disorders. This situation, referred to as ‘glutamate neurotoxicity’ (GNT), is characterized by an increasing damage of cell components, including mitochondria, leading to cell death. In the death process, reactive oxygen species (ROS) are generated. The present study describes the state of art in the field of GNT with a special emphasis on the oxidative stress and mitochondria. In particular, we report how ROS are generated and how they affect mitochondrial function in GNT. The relationship between ROS generation and cytochrome crelease is described in detail, with the released cytochrome cplaying a role in the cell defense mechanism against neurotoxicity.

The excitatory neurotransmitter glutamate plays a major role in determining certain neurological disorders. This situation, referred to as ‘glutamate neurotoxicity’ (GNT), is characterized by an increasing damage of cell components, including mitochondria, leading to cell death. In the death process, reactive oxygen species (ROS) are generated. The present study describes the state of art in the field of GNT with a special emphasis on the oxidative stress and mitochondria. In particular, we report how ROS are generated and how they affect mitochondrial function in GNT. The relationship between ROS generation and cytochrome crelease is described in detail, with the released cytochrome cplaying a role in the cell defense mechanism against neurotoxicity.

In rat cerebellar granule cells both reactive oxygen species production and release of cytochrome ctake place during glutamate toxicity. This investigation was aimed (i) to ascertain whether and how these two processes are related and (ii) to gain insight into the role played by the released cytochrome cin the onset of neurotoxicity. Cytochrome crelease takes place owing to the generation of reactive oxygen species both in glutamate-treated cerebellar granule cells and in sister control cultures incubated in the presence of the reactive oxygen species-generating system consisting of xanthine plus xanthine oxidase. In the early phase of neurotoxicity (30-min glutamate exposure) about 40% of the maximum (as measured at 3 h of glutamate exposure) cytochrome crelease was found to occur in cerebellar granule cells from mitochondria that were essentially coupled and intact and that had a negligible production of oxygen free radicals. Contrarily, mitochondria from cells treated with glutamate for 3 h were mostly uncoupled and produced reactive oxygen species at a high rate. The cytosolic fraction containing the released cytochrome cwas able to transfer electrons from superoxide anion to molecular oxygen via the respiratory chain and was found to partially prevent glutamate toxicity when added externally to cerebellar neurons undergoing necrosis. In the light of these findings, we propose that in the early phase of neurotoxicity, cytochromecrelease can be part of a cellular and mitochondrial defense mechanism against oxidative stress.

We have recently shown that, in rat cerebellar granule cells, apoptosis triggered by KCl deprivation is associated with an amyloidogenic shift in the processing of amyloid precursor protein (APP) resulting in an increase of amyloid β-protein (Aβ) secretion. To further investigate this issue we studied the relationship between secretion of APP metabolites (Aβ, APPs) and neuronal degeneration. We postulated that the endogenous products of the APP metabolism may modulate neuronal survival by an autocrine loop. Treatment of cerebellar granule cells with various antibodies raised against different epitopes of APPs and Aβ oppositely modulates low potassium apoptotic cell death. Antibodies specific for the N-terminal of Aβ (4G8, 6E10, R3659) increased neuronal survival by 30 over controls. On the contrary, treatment of cultures undergoing apoptosis with the monoclonal antibody 22C11 directed against the APP N-terminus reduced neuronal survival by 53, suggesting that endogenous α-APPs contribute to neuronal survival. Moreover low KCl culture medium, conditioned by cerebellar granule cells, attenuated the apoptotic process. This anti-apoptotic effect was abolished by removal of APPs from the conditioned medium. Western blotting of APPs removed from the conditioned medium confirmed the presence of α-APPs. These data indicate that APP cleavage products oppositely modulate neuronal survival through an autocrine loop and further strengthen an Alzheimer's disease pathogenetic scheme based on altered metabolism of APP.

Cytochrome c(cyt c) release was investigated in cerebellar granule cells used as an in vitro neuronal model of apoptosis. We have found that cyt cis released into the cytoplasm as an intact, functionally active protein, that this event occurs early, in the commitment phase of the apoptotic process, and that after accumulation, this protein is progressively degraded. Degradation, but not release, is fully blocked by benzyloxycarbonyl‐Val‐Ala‐Asp‐fluoromethylchetone (z‐VAD‐fmk). On the basis of previous findings obtained in the same neuronal population undergoing excitotoxic death, it is hypothesized that release of cyt cmay be part of a cellular attempt to maintain production of ATP via cytochrome oxidase, which is reduced by cytosolic NADH in a cytochrome b5‐soluble cyt c‐mediated fashion.

Cellular extracts derived from pheochromocytoma cells (PC12‐) inhibit the assembly of calf brain tubulin, while those derived from nerve growth factor‐differentiated cells (PC12+) do not display this effect. Incubation with RNase abolishes the inhibition by PC12‐ extracts and reveals the presence of an activating effect exerted by PC12+ extracts. Activation of microtubule assembly is enhanced when extracts are prepared from PC12+ cells exposed for 1 day to 1.0 microM taxol and is abolished when PC12+ extracts are: (a) prepared from cells incubated for 1 day with 1 microM colchicine, (b) treated with the non‐ionic detergent Nonidet P‐40 or (c) centrifuged at 100 000 g instead of 80 000 g. 2D gel electrophoresis of the proteins of the 100 000 g pellet responsible for the activating effect (referred to as 100 K g pellet) reveals the presence of 100 K, 88 K and 32 K proteins which are markedly enriched in PC12+ extracts. The 88 K protein is further enriched in taxol‐treated cells and markedly reduced in the same cells incubated with colchicine. A correlation between the differential protein composition of the 100 K g pellets and their effect on microtubule formation is postulated.

Our previous studies have shown that the response to the excitotoxic action of glutamate by cultured cerebellar granule cells depends upon the cell density or the volume of medium in which they have been grown: the higher the cell density or the lower the volume, the higher the response to glutamate. We have hypothesized that this variable response is due to the formation in culture of a glutamate‐sensitizing activity GSA more abundantly in conditioned medium derived from high‐density or low‐volume cultures than that present in low‐density or high volume cultures and capable of restoring sensitivity in previously resistant granule cells. In order to elucidate the mechanism of action of glutamate‐sensitizing activity, we measured the extent and function of NMDA receptors in low‐ and high‐volume cultures and assessed the effect of glutamate‐sensitizing activity on the same receptors. We found that under high‐volume conditions the extent of MK‐801 binding, the amount of NMDA receptor type 1, the currents evoked in whole cells after an NMDA pulse and the response of cultured cells to this ligand were markedly reduced compared with low‐volume cultures. Addition of glutamate‐sensitizing activity to high‐volume cultures increased their glutamate sensitivity, the NMDA‐evoked currents, the extent of MK‐801 binding and the amount of NMDA receptor type 1 protein present. The corresponding mRNA transcripts, on the contrary, were unchanged in high‐volume, low‐volume and high‐volume GSA‐treated cultures.

Summary The distribution of nerve growth factor (NGF) in chick embryo sympathetic neurons has been followed by two distinct procedures, that is, indirect immunofluorescence microscopy employing purified NGF antibodies and autoradiography after exposure of cells to [125I]NGF. This study shows that NGF bound to sympathetic neurons is not uniformly distributed but appears in spots over the surface of perikarya and along processes. If the same cells, after incubation with NGF and fixation, are treated with methanol-acetone to allow permeation of immunoglobulins across the plasma membrane, NGF antibody immunoreactive material is also found within the cytoplasm and notably in the paranuclear area only in sympathetic neurons. Analogous findings are obtained when sympathetic neurons are incubated in the presence of [125I]NGF, fixed, sectioned and processed for autoradiography. Also with this technique NGF appears to be localized in the cytoplasmic compartment and is found around the nucleus. These studies are discussed in connection with the results of similar experiments performed on a clonal line of NGF target cells known as PC12.

Cerebellar granule cells grown in the presence of a serum complex differentiate but are resistant to the lethal action of excitatory amino acids. When these cells are grown also in the presence of insulin-like growth factor I (IGF-I) they become fully susceptible to the toxic, lethal action of glutamate. The glutamate-sensitizing action of IGF-I is dependent on concentration (half-maximal effect at 2-4 ng/ml) and time (half-maximal effect at 2-4 days in vitro) and is paralleled by the appearance of functionally active, glutamate-activated, Ca2+ channels and of voltage-gated Na+ and late K+ channels. IGF-I-induced glutamate sensitivity is rapidly reversible (t1/2 = 30-60 min) after removal of this somatomedin. The action of IGF-I is not mimicked by IGF-II, nerve growth factor, basic or acidic fibroblast growth factor, platelet-derived growth factor, or tumor necrosis factor alpha. We postulate that the constitutive phenotype of cerebellar granule cells is glutamate-resistant and becomes responsive to excitatory amino acids under the action of epigenetic cues among which IGF-I may be one of those operative in vivo.

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.

Cellular extracts derived from pheochromocytoma cells (PC12‐) inhibit the assembly of calf brain tubulin, while those derived from nerve growth factor‐differentiated cells (PC12+) do not display this effect. Incubation with RNase abolishes the inhibition by PC12‐ extracts and reveals the presence of an activating effect exerted by PC12+ extracts. Activation of microtubule assembly is enhanced when extracts are prepared from PC12+ cells exposed for 1 day to 1.0 microM taxol and is abolished when PC12+ extracts are: (a) prepared from cells incubated for 1 day with 1 microM colchicine, (b) treated with the non‐ionic detergent Nonidet P‐40 or (c) centrifuged at 100 000 g instead of 80 000 g. 2D gel electrophoresis of the proteins of the 100 000 g pellet responsible for the activating effect (referred to as 100 K g pellet) reveals the presence of 100 K, 88 K and 32 K proteins which are markedly enriched in PC12+ extracts. The 88 K protein is further enriched in taxol‐treated cells and markedly reduced in the same cells incubated with colchicine. A correlation between the differential protein composition of the 100 K g pellets and their effect on microtubule formation is postulated.

Cytochrome c(cyt c) release was investigated in cerebellar granule cells used as an in vitro neuronal model of apoptosis. We have found that cyt cis released into the cytoplasm as an intact, functionally active protein, that this event occurs early, in the commitment phase of the apoptotic process, and that after accumulation, this protein is progressively degraded. Degradation, but not release, is fully blocked by benzyloxycarbonyl-Val-Ala-Asp-fluoromethylchetone (z-VAD-fmk). On the basis of previous findings obtained in the same neuronal population undergoing excitotoxic death, it is hypothesized that release of cyt cmay be part of a cellular attempt to maintain production of ATP via cytochrome oxidase, which is reduced by cytosolic NADH in a cytochrome b5-soluble cyt c-mediated fashion.

Cerebellar granule cells cultured in the presence of a differentiating factor isolated from rabbit serum exhibit, at variance with those cultured in fetal calf serum, an almost complete resistance to excitatory aminoacid (EAA)-induced citotoxicity. We investigated the behaviour of protein kinase C (PKC), strongly implicated in EAA cytotoxicity, in the two types of culture. Phorbol esters, used to monitor the enzyme, enhanced the depolarization-evoked release ofd-[3H]aspartate, but less effectively in factor-conditioned cells. EAAs increased phorbol esters binding in both cultures, but the effect was briefly lasting in factor-conditioned cells. The different behaviour of PKC is postulated to be causally related to different response to EAA of the cultures.

A specific antibody against nerve growth factor (NGF) and indirect immunofluorescence microscopy have been used to follow the in vitro binding of NGF to cells made permeable to large molecules. All cells tested, both target (sensory neurons and PCI2 cells) and nontarget (3T3, BKH 2I, C6 glioma cells), revealed a decoration of cytoskeletal structures which on the basis of their form, reactivity with antibodies, and sensitivity to specific drugs may be identified as microtubules (MTs) and microfilaments (MFs). The decoration of either structure depends on the fixation and permeabilization conditions: MFs, in the form of stress fibers, are stained by NGF when the plasma membrane is permeabilized with methanol/acetone; MTs become intensely stained when the plasma membrane is solubilized with a nonionic detergent in the presence of a MT-stabilizing medium. The two procedures do not affect the staining of these structures with specific antibodies. Binding of 125I-labeled NGF to PCI2 cells was not competitively inhibited by a 100-fold excess of several positively charged proteins but it was markedly decreased in the presence of DNase I. 125I-Labeled NGF interacted with MTs and F-actin (fixed with paraformaldehyde) in a range of concentrations similar to that used for their cellular localization with NGF-anti-NGF. Our studies show that the specificity and affinity of NGF binding to MTs and MFs is in the range of that of antibodies against tubulin and actin. The possible relevance of these findings to the mechanism of action of NGF in target cells is discussed.

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+]oto 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.

We have investigated the effect of the b isoform of S-100 proteins on adenylate cyclase activity of rat skeletal muscle. S-100b inhibits the adenylate cyclase activity in the presence of Mg 2+(5.0–50 mM), while it activates the same enzyme in the presence of Ca 2+(0.1–1.0 mM) dose-dependently in both cases. S-100b counteracts the stimulatory effect of NaF on adenylate cyclase in the presence of Mg 2+and the inhibitory effect of RMI 12330 A in the presence of Ca 2+.

NERVE growth factor and its receptor(s) are present in several parts of the hippocampus (CA1, CA3, dentate gyrus) but nothing is known about their function in this area which plays a fundamental role in learning and memory processes. NGF delivered exogenously to hippocampal slices causes a concentration-dependent, marked reduction in the expression (but not the induction) of long term potentiation (LTP) without altering basal synaptic transmission. The effect is already half maximal at 0.05–0.1 ng ml-1NGF, is reversible after removal of this growth factor, and is also detectable with a modified version of NGF which has lost its neurite outgrowth promoting activity in PC12 cells. These findings point to a role for hippocampal NGF as a possible modulator of learning and memory processes. Such modulation would be mediated by high-affinity receptors functionally distinct from those promoting morphological differentiation of PC12 cells and other NGF target cells.

A 100 μM glutamate pulse administered to rat cerebellar granule cells causes a very rapid and progressive decrease in both cell and mitochondria! oxygen consumption caused by glucose and succinate addition, respectively. The respiratory control ratio, which reflects the ability of mitochondria to produce ATP, is reduced by 50 within the first 30 min after glutamate addition. Subsequent to glutamate exposure, a progressive decrease of respiratory control ratio to almost 1 was found within the following 3–5 h. The addition of extra calcium had no effect per seon oxygen consumption by cell homogenate.

We have investigated the effect of the b isoform of S‐100 proteins on adenylate cyclase activity of rat skeletal muscle. S‐100b inhibits the adenylate cyclase activity in the presence of Mg2+(5.0–50 mM), while it activates the same enzyme in the presence of Ca2+(0.1–1.0 mM) dose‐dependently in both cases. S‐100b counteracts the stimulatory effect of NaF on adenylate cyclase in the presence of Mg2+and the inhibitory effect of RMI 12330 A in the presence of Ca2+.

Binding and fate of the nerve growth factor (NGF) in pheochromocytom a cells (clone PC12) have been measured with the use of iodine-labeled ligand and with 125I-NGF antibodies. With such double approach it is possible to distinguish between surface bound and total NGF bound to PC12 cells. It is found that NGF-receptor complexes undergo down-regulation. This process is noticeable at low but not at high cell densities, and only in PC12 cells never exposed to NGF. Previous incubation with growth factor leads to the disappearance of down-regulation of NGF-receptor complexes. Assuming that this process is an indirect measure of NGF-receptor internalization, it is concluded that it is modulated by the cell density or by previous exposure to the factor. These findings are postulated to be relevant to the mechanism of action of NGF and to its multiple effects on target cells.

Arrest of mitosis and neurite outgrowth induced by nerve growth factor (NGF) in rat pheochromocytoma cells (clone PC12) is accompanied by a progressive inhibition of the synthesis of a protein that binds to single-stranded but not to double-stranded DNA. Time course experiments show that this inhibition is already apparent after a 2-day incubation with NGF and is maximum (85-95%) upon achievement of complete PC12 cell differentiation. Inhibition of the synthesis of this single-stranded DNA binding protein after 48 hr of incubation with NGF is potentiated by concomitant treatment of PC12 cells with antimitotic drugs acting at different levels of DNA replication. Purification on a preparative scale of this protein and analysis of its major physicochemical properties show that: (i) it constitutes 0.5% of total soluble proteins of naive PC12 cells; (ii) its molecular weight measured by NaDodSO4/PAGE is Mr 34,000 (sucrose gradient centrifugation under nondenaturing conditions yields a sedimentation coefficient s20,w of 8.1 S, indicating that the native protein is an oligomer); (iii) amino acid analysis demonstrates a preponderance of acidic over basic residues, while electrofocusing experiments show that it has an isoelectric point around 8.0; (iv) approximately 15% of the protein is phosphorylated in vivo. It is postulated that control of the synthesis of this protein is connected with activation of a differentiative program triggered by NGF in the PC12 neoplastic cell line at some step(s) of DNA activity.

Vinblastine injections in newborn mice produce severe atrophy of sympathetic ganglia; after a 7-day treatment the ganglia are 80% reduced in volume. Histological examinations show that this effect is due to a marked decrease in the neuronal cell population. The most precocious ultra-structural alterations are localized in the nuclear compartment, followed by axonal swelling and microtubule disappearance. Simultaneous injections of nerve growth factor entirely prevent the noxious effects of the vinca alkaloid, and result in partial appearance of the growth effects of nerve growth factor. Such protective action is not due to inhibition of vinblastine uptake which is the same in control mice and in mice pretreated with nerve growth factor. It is suggested that nerve growth factor prevents the vinca alkaloid action by favoring the assembly or organization, or both, of microtubules, which, from in vitro studies, have been proved to be inaccessible to vinblastine.

The nerve growth factor protein (NGF) favors polymerization of brain actin and induces its organization to form paracrystalline structures that activate myosin ATPase (ATP phosphohydrolase, EC 3.6.1.3) to an extent greater than actin alone. Binding studies show that the initial 1:1 stoichiometry of NGF-G-actin complexes decreases to 1:7-10 when polymerization is ended and paracrystalline structures are formed. The ratio becomes even lower when heavy meromyosin is added in the absence of ATP, suggesting that heavy meromyosin displaces NGF bound to actin microfilaments. This conclusion is supported by the finding that when heavy meromyosin is added to NGF-microfilament complexes, under conditions for "decorating" microfilaments, the usual paracrystalline structure of the complexes disappears. The NGF-mediated organization of actin and activation of myosin ATPase is visualized as a self-regulatory and self-propagating mechanism, because progressive displacement of the growth factor induced by heavy meromyosin binding to F actin as ATP consumption proceeds renders an increasingly higher amount of NGF free for new interactions. These findings are discussed in the light of the mechanism of action of NGF in the target cells.

Addition of nerve growth factor to a 105,000 × gsupernatant of mouse brain induces the formation of a precipitate whose main constituent is the microtubule protein(s) (tubulin). The binding of nerve growth factor to purified tubulin is not inhibited by colchicine and does not appear to depend on the presence of GTP or Mg++. GTP, however, and divalent cations, exert a marked effect on the increased turbidity induced by interaction of nerve growth factor with tubulin. These findings are tentatively interpreted with the hypothesis that binding of the factor to tubulin and the induced aggregation is a sequential two-step process; the latter but not the former would be influenced by GTP or divalent cations.

The distribution of nerve growth factor (NGF) in rat pheochromocytoma cells (clone PC12) has been studied with two different techniques: immunofluorescence and autoradiography. It was found that NGF is progressively internalized in the cytoplasmic compartment and eventually accumulates in the form of discrete dots around the nucleus. A fraction of the internalized NGF appears within the nucleoplasm, often contiguous with the nucleolus. It is suggested that cytoplasmic and perinuclear NGF may be be in contact with a pool of tubulin or actin-like proteins in their soluble or organized form and play a key role in the process of arrest of division and neurite growth.