Santiago Ambrosio

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure flux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular assays, we hope to encourage technical innovation in the field.

The level of purines in the striatum of awake, freely moving rats was studied using microdialysis. The calculated extracellular concentration of adenosine and its metabolites inosine and hypoxanthine was very high immediately after implantation of the dialysis probe but decreased within 24 h to a level which remained stable for two days. Using in vitro calibration to determine the relative recovery of the dialysis probes we estimated resting levels in the striatal extracellular space to be 40, 110 and 580 nM, respectively. Inhibition of adenosine deaminase by deoxycoformycin produced a significant 1.4-fold increase in extracellular adenosine levels and a fall in inosine and hypoxanthine. A combination of three uptake blockers (dipyridamole, lidoflazine and nitrobenzylthioinosine), caused a 4.5-fold increase in extracellular adenosine levels without any change in inosine or hypoxanthine levels. After uptake inhibition deoxycoformycin did not have any significant effect. The present results show that the microdialysis technique can be used to determine levels of purines in the extracellular fluid of defined brain regions in awake animals. The high levels recorded during the first several hours after implantation may be artefactually high and reflect trauma. The results also show that adenosine levels can be altered in vivo by inhibitors of adenosine transport and adenosine deaminase. The present results indicate that the physiological adenosine level in striatal extracellular space is in the range 40-460 nM.

Free cytoplasmic dopamine may be involved in the genesis of neuronal degeneration in Parkinson's disease and other such diseases. We used SH-SY5Y human neuroblastoma cells to study the effect of dopamine on cell death, activation of stress-induced pathways, and expression of alpha-synuclein, the characteristic protein accumulated in Lewy bodies. We show that 100 and 500 microM dopamine causes a 40% and 60% decrease of viability, respectively, and triggers autophagy after 24 hr of exposure, characterized by the presence of numerous cytoplasmic vacuoles with inclusions. Dopamine causes mitochondrial aggregation in adherent cells prior to the loss of functionality. Plasma membrane and nucleus also maintain their integrity. Cell viability is protected by the dopamine transporter blocker nomifensine and the antioxidants N-acetylcysteine and ascorbic acid. Dopamine activates the stress-response kinases, SAPK/JNK and p38, but not ERK/MAPK or MEK, and increases alpha-synuclein expression. Both cell viability and the increase in alpha-synuclein expression are prevented by antioxidants; by the specific inhibitors of p38 and SAPK/JNK, SB203580 and SP600125, respectively; and by the inhibitor of autophagy 3-methyladenine. This indicates that oxidative stress, stress-activated kinases, and factors involved in autophagy up-regulate alpha-synuclein content. The results show that nonapoptotic death pathways are triggered by dopamine, leading to autophagy. These findings should be taken into account in the search for strategies to protect dopaminergic neurons from degeneration.

Alpha-synuclein is a brain presynaptic protein that is linked to familiar early onset Parkinson's disease and it is also a major component of Lewy bodies in sporadic Parkinson's disease and other neurodegenerative disorders. Alpha-synuclein expression increases in substantia nigra of both MPTP-treated rodents and non-human primates, used as animal models of parkinsonism. Here we describe an increase in alpha-synuclein expression in a human neuroblastoma cell line, SH-SY5Y, caused by 5-100 microM MPP+, the active metabolite of MPTP, which induces apoptosis in SH-SY5Y cells after a 4-day treatment. We also analysed the activation of the MAPK family, which is involved in several cellular responses to toxins and stressing conditions. Parallel to the increase in alpha-synuclein expression we observed activation of MEK1,2 and ERK/MAPK but not of SAPK/JNK or p38 kinase. The inhibition of the ERK/MAPK pathway with U0126, however, did not affect the increase in alpha-synuclein. The highest increase in alpha-synuclein (more than threefold) in 4-day cultures was found in adherent cells treated with low concentrations of MPP+ (5 microM). Inhibition of ERK/MAPK reduced the damage caused by MPP+. We suggest that alpha-synuclein increase and ERK/MAPK activation have a prominent role in the cell mechanisms of rescue and damage, respectively, after MPP+ -treatment.

Autophagy is an evolutionarily conserved degradation pathway which primary functions as a cell survival adaptive mechanism during stress conditions. Autophagy is a tumor suppressor process and induction of the autophagic machinery can cause cell demise in apoptosis-resistant cancer. Thus, this metabolic pathway can act either to prevent or to promote carcinogenesis, as well as to modulate the response to anticancer therapies, included drug-induced apoptosis. Conventional therapies exert their cytotoxic activity mainly by inducing apoptosis. Massive activation of the apoptotic program in a tissue can result in cell loss providing a selective advantage for growth to displastic cells and tumor cell subpopulations with high levels of malignancy. This suggests that the activation of autophagy can counteract malignancy. On the contrary, therapeutic intervention-induced apoptosis can eliminate cells with pro-mutational biochemical alterations at risk for initiation, initiated cells and cells of focal and advanced preneoplastic and neoplastic lesions. Thus, pharmacological inhibition of autophagy may enhance apoptosis. Autophagy and apoptosis share common stimuli and signalling pathways, so that the final fate, life or death, depends on the cell response. Recently, accumulating data fuel novel potential therapeutic interventions to modulate autophagy to be beneficial in cancer therapy. This review highlights current knowledges aimed at unraveling the molecular interplay between autophagy and cell death as well as the possible therapeutic exploitation in cancer. Keywords: Autophagy, autophagic cell death, apoptosis, molecular swicth, oncogenesis, cancer therapy

Article19 August 2004free access BMP-2 decreases Mash1 stability by increasing Id1 expression Francesc Viñals Francesc Viñals Departament de Ciències Fisiològiques II, Campus de Bellvitge, Universitat de Barcelona, Feixa Llarga s/n, L'Hospitalet de Llobregat, Spain Search for more papers by this author Julia Reiriz Julia Reiriz Departament d'Infermeria Fonamental i Medicoquirúrgica, Campus de Bellvitge, Universitat de Barcelona, Feixa Llarga s/n, L'Hospitalet de Llobregat, Spain Search for more papers by this author Santiago Ambrosio Santiago Ambrosio Departament de Ciències Fisiològiques II, Campus de Bellvitge, Universitat de Barcelona, Feixa Llarga s/n, L'Hospitalet de Llobregat, Spain Search for more papers by this author Ramon Bartrons Ramon Bartrons Departament de Ciències Fisiològiques II, Campus de Bellvitge, Universitat de Barcelona, Feixa Llarga s/n, L'Hospitalet de Llobregat, Spain Search for more papers by this author Jose Luis Rosa Jose Luis Rosa Departament de Ciències Fisiològiques II, Campus de Bellvitge, Universitat de Barcelona, Feixa Llarga s/n, L'Hospitalet de Llobregat, Spain Search for more papers by this author Francesc Ventura Corresponding Author Francesc Ventura Departament de Ciències Fisiològiques II, Campus de Bellvitge, Universitat de Barcelona, Feixa Llarga s/n, L'Hospitalet de Llobregat, Spain Search for more papers by this author Francesc Viñals Francesc Viñals Departament de Ciències Fisiològiques II, Campus de Bellvitge, Universitat de Barcelona, Feixa Llarga s/n, L'Hospitalet de Llobregat, Spain Search for more papers by this author Julia Reiriz Julia Reiriz Departament d'Infermeria Fonamental i Medicoquirúrgica, Campus de Bellvitge, Universitat de Barcelona, Feixa Llarga s/n, L'Hospitalet de Llobregat, Spain Search for more papers by this author Santiago Ambrosio Santiago Ambrosio Departament de Ciències Fisiològiques II, Campus de Bellvitge, Universitat de Barcelona, Feixa Llarga s/n, L'Hospitalet de Llobregat, Spain Search for more papers by this author Ramon Bartrons Ramon Bartrons Departament de Ciències Fisiològiques II, Campus de Bellvitge, Universitat de Barcelona, Feixa Llarga s/n, L'Hospitalet de Llobregat, Spain Search for more papers by this author Jose Luis Rosa Jose Luis Rosa Departament de Ciències Fisiològiques II, Campus de Bellvitge, Universitat de Barcelona, Feixa Llarga s/n, L'Hospitalet de Llobregat, Spain Search for more papers by this author Francesc Ventura Corresponding Author Francesc Ventura Departament de Ciències Fisiològiques II, Campus de Bellvitge, Universitat de Barcelona, Feixa Llarga s/n, L'Hospitalet de Llobregat, Spain Search for more papers by this author Author Information Francesc Viñals1, Julia Reiriz2, Santiago Ambrosio1, Ramon Bartrons1, Jose Luis Rosa1 and Francesc Ventura 1 1Departament de Ciències Fisiològiques II, Campus de Bellvitge, Universitat de Barcelona, Feixa Llarga s/n, L'Hospitalet de Llobregat, Spain 2Departament d'Infermeria Fonamental i Medicoquirúrgica, Campus de Bellvitge, Universitat de Barcelona, Feixa Llarga s/n, L'Hospitalet de Llobregat, Spain *Corresponding author. Departament de Ciències Fisiològiques II, Unitat de Bioquímica, Campus de Bellvitge, Universitat de Barcelona, Feixa Llarga s/n, 08907 L'Hospitalet de Llobregat, Spain. Tel.: +34 93 402 4281; Fax: +34 93 402 4268; E-mail: [email protected] The EMBO Journal (2004)23:3527-3537https://doi.org/10.1038/sj.emboj.7600360 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info In neural development, bone morphogenetic proteins (BMPs) restrict neuronal differentiation, thereby promoting the maintenance of progenitor cells or even inducing astrocytogenesis. We report that exposure of neuroendocrine lung carcinoma cells to BMP-2 leads to a rapid decline in steady-state levels of Mash1 protein and some neuron-specific markers. BMP-2 induces a post-transcriptional decrease in Mash1 levels through enhanced degradation. We demonstrate that Mash1 protein stability is tightly regulated by the E47/Id1 expression ratio. Transient induction of Id1 by BMP-2 negatively correlates with Mash1 levels. Furthermore, an ectopic increase in Id1 levels is sufficient to induce degradation of either ectopic or endogenous Mash1, whereas expression of Mash1 in Id1-deficient cells or overexpression of E47 makes Mash1 levels refractory to the addition of BMP-2. Furthermore, we show that the E47/Id1 expression ratio also regulates CK2-mediated phosphorylation of Mash1 on Ser152, which increases interaction of Mash1–E47 heterodimers. We propose a novel mechanism in which the balance between Id and E protein levels regulates not only the transcriptional function but also protein stability of the neurogenic bHLH transcription factor Mash1. Introduction During differentiation, progenitor cells develop into more specialized phenotypes by highly specific changes in gene expression. The developmental maturation of precursor cells can be divided into at least two stages: the commitment of undifferentiated cells to a particular lineage, followed by terminal differentiation into a specific phenotype. Generally, the determination of cell fate depends on the activity of transcription factors. One of the best-known regulators of cell differentiation is the family of basic helix–loop–helix (bHLH) transcription factors. Most members of this family play a central role in the development of distinct mammalian cell lineages, including muscle and nerve cells. There is strong evidence that sequential expression of bHLH factors represents regulatory cascades in which early expressed factors modulate the appearance of others that are expressed later. For example, two members of the bHLH family, MyoD and myf-5, execute myogenic lineage determination, while myogenin and MRF4 appear to regulate the differentiation program (Rawls and Olson, 1997). Similarly, the bHLH factors Mash1, Math and neurogenin control neurogenesis in the central (CNS) and peripheral (PNS) nervous systems (Lee, 1997; Cai et al, 2000). Mash1 is expressed in proliferating neural precursors in restricted regions of the CNS and PNS, olfactory epithelium and neuroendocrine cells (Lo et al, 1991; Guillemot et al, 1993; Borges et al, 1997; Casarosa et al, 1999). Mice that are homozygous for targeted disruption of the Mash1 gene show fewer autonomic, enteric and olfactory neurons as well as neuroendocrine cells in the lung (Guillemot et al, 1993; Borges et al, 1997). A variety of neuroendocrine marker genes, such as Phox2a, tyrosine hydroxylase (TH) and dopamine β-hydroxylase (DBH), are directly or indirectly regulated by Mash1 (Lo et al, 1998, 1999). For instance, disrupting Mash1 function by antisense treatment of small cell lung carcinoma (SCLC) cells results in a strong decrease in neuroendocrine markers, whereas overexpression of this gene in transgenic mice induces the appearance of neuroendocrine tumors from pulmonary epithelium (Linnoila et al, 2000). Mash1 and all the members of the bHLH family heterodimerize with ubiquitously expressed bHLH E proteins, such as E2A gene products, E12 and E47, through their HLH domain. Heterodimers bind to DNA through their basic domain and activate the transcription of genes that have a CANNTG sequence (E box) in their promoter region (Lee, 1997). Only one subfamily of HLH factors, known as Id proteins, lacks this basic region. Heterodimerization of Id proteins with bHLH is sufficient to block their DNA binding and function (Norton et al, 1998; Norton, 2000). Thus, Id family members are mainly known as negative regulators of the commitment and/or differentiation that the bHLH factors promote in muscle, lymphoid and neurogenic precursors (Sun, 1994; Spits et al, 2000; Kee et al, 2001). Four mammalian Id proteins, Id1 to Id4, that display partially overlapping expression patterns and certain levels of functional redundancy have been identified (Norton et al, 1998; Norton, 2000). This network of expression and function of transcription factors and their antagonists is ultimately controlled by cell-intrinsic programs and by extracellular signaling factors. Although many studies have focused on positive signals, negative molecular signals are equally important in balancing both the timing of differentiation and cell specification (Ross et al, 2003). Bone morphogenetic proteins (BMPs) are good candidates for molecules that act as negative regulators of neurogenesis (reviewed in Mehler et al, 1997). BMPs belong to the TGF-β superfamily of cytokines (Derynck and Zhang, 2003; Shi and Massagué, 2003). Each ligand of this family exerts its biological function by inducing the formation of heteromeric complexes of specific type I and II serine/threonine kinase receptors, which in turn propagate the signal inside the cell through phosphorylation of the Smad family of transcription factors. Smads1, 5 and 8 are mainly involved in BMP signaling, whereas Smads 2 and 3 function in TGF-β/activin signaling. Following phosphorylation, receptor-specific Smads hetero-oligomerize with Smad4, the only co-Smad isolated from mammals to date, and translocate into the nucleus. The third subfamily includes Smads 6 and 7, which are named I-Smads because of their ability to inhibit receptor-mediated signaling. Upon entry to the nucleus, Smads usually form complexes that contain sequence-specific DNA-binding factors and transcriptional coactivators or corepressors in order to achieve stable binding and transcriptional regulation (ten Dijke et al, 2000; Shi and Massagué, 2003; Derynck and Zhang, 2003). Interestingly, BMP-2 and leukemia inhibitory factor (LIF) synergistically promote neuroepithelial cells to become astrocytes through interaction between Smad1 and STAT3 (Nakashima et al, 1999). During embryonic neural induction, BMPs promote epidermal fate and suppress neural fate in the developing ectoderm (Wilson and Hemmati-Brivanlou, 1995). These cytokines promote glial differentiation in late embryonic or adult CNS precursors and simultaneously inhibit neurogenesis (Gross et al, 1996; Shou et al, 1999; Nakashima et al, 1999, 2001; Lim et al, 2000). However, BMPs also cause apoptosis in CNS precursor cells and induce neurogenesis in neural crest stem cells or cortical progenitors precursors (Shah et al, 1996). The mechanism by which BMPs mediate this wide variety of responses depending on the time of development is unclear. However, it may result from a diversity of signal transduction components that allow distinct cell populations to respond differentially to gradients of BMP concentration. In addition, compared with this relatively strong phenomenological evidence, little is known about the molecular mechanisms that mediate the antineurogenic effects of BMPs. Therefore, we studied the molecular mechanisms involved in the antineurogenic function of BMPs in SCLC cells with a neuroendocrine phenotype. We show that BMP-2 decreases the expression of neuroendocrine markers and causes a post-transcriptional decrease in Mash1 protein levels. Our data indicate that induction of Id proteins by BMP-2 is necessary and sufficient to block stabilization of Mash1 by means of direct competition between Mash1 and Id1 for binding to ubiquitous E proteins. We propose a novel mechanism where the balance between Id and E protein levels regulates not only function but also protein stability of tissue-specific bHLH transcription factors. Results BMP-2 downregulates neuroendocrine markers in small cell lung carcinoma cells To assess the effects of BMP-2 on the maintenance of the neuroendocrine phenotype, we first confirmed relatively high expression levels of several known neural markers in the SCLC cell line DMS53 (Borges et al, 1997). Addition of BMP-2 (5 nM) reduced the expression of TH and synaptophysin and did not affect other neural markers such as β-tubulin III or SNAP-25 (Figure 1A). Dose–response analysis of BMP-2 showed that TH protein levels were strongly decreased by BMP-2 stimulation at 5–10 nM. Similarly, to assess the temporal pattern of this response more accurately, we performed time-course assays indicating that both TH protein and mRNA reached minimal levels after 4–8 h and increased thereafter (Figure 1B). Interestingly, as previously described (Gómez-Santos et al, 2002), the related cytokine TGF-β had the opposite effects, and strongly increased TH expression. To further examine whether the regulation of TH by BMP-2 is mediated at the level of gene transcription, we transfected C17.2 cells with a reporter construct containing 4.3 kb of mouse TH promoter-enhancer region (Suzuki et al, 2002). Reporter activity was significantly reduced (up to 45% decrease with respect to control cells) in BMP-2-treated cells, indicating that the BMP-2 effects on TH protein levels were mainly due to a decreased transcriptional rate (Figure 1C). Figure 1.(A) DMS53 cells were treated with 5 nM BMP-2 for 6 or 12 h. Total extracts were obtained and analyzed by immunoblotting for the neural markers indicated. (B) DMS53 cells were treated for 4 h with the indicated amounts of BMP-2 or TGF-β (upper panel) or with 10 nM BMP-2 for the times indicated (lower panel). TH and actin expressions were analyzed by immunoblotting and/or semiquantitative RT–PCR. (C) C17.2 cells were cotransfected with a TH promoter-driven reporter construct and the combinations of Mash1 and/or E47 expression vectors indicated. Luciferase assay was performed after treatment for 16 h with 5 nM BMP-2 (filled bars) or no addition (empty bars). The results are expressed as mean±s.e.m. of triplicates from four independent transfections. (D) Northern blot analysis was performed with total RNA from DMS53 cells treated for the indicated times with 10 nM BMP-2 as described in Materials and methods. Download figure Download PowerPoint SCLC exhibits a neuroendocrine phenotype in tight association with Mash1 and Phox2 expression (Borges et al, 1997; Lo et al, 1999; Ito et al, 2000; Brosenitsch and Katz, 2002). Thus, we explored the transcriptional regulation of TH transcription by the Mash1/E47 bHLH protein complex and their response to BMP-2 (Figure 1C). As expected, overexpression of Mash1 increased reporter responses, which were further augmented by coexpression of E47. Addition of 5 nM BMP-2 decreased transcriptional responses in both cases, indicating that BMP-2 interferes with the function of bHLH transcriptional complexes. Post-transcriptional regulation of Mash1 expression by BMP-2 We analyzed modulation of endogenous levels of Mash1 mRNAs by BMP-2. We also analyzed the levels of Id1, a negative regulator of neurogenic bHLH transcription factors, which is induced by BMP-2 in several differentiation models (Nakashima et al, 2001; López-Rovira et al, 2002). BMP-2 induced a transient increase in Id1 mRNA levels. However, Mash1 mRNA levels showed no significant differences up to 6 h after BMP-2 addition (Figure 1D). BMP-2 inhibits neurogenesis in olfactory epithelium cultures through enhanced proteolytic degradation of Mash1. These effects are very rapid (2 h) and require new transcription of an early induced gene by BMP (Shou et al, 1999). Immunoblot analysis showed a strong decrease in Mash1 protein levels (up to 60% decrease at 2 h) followed by a recovery after 6 h (Figure 2A). Dose–response assays also indicate that concentrations of BMP-2 higher than 5 nM were required to produce a significant decrease (Figure 2C). These profiles provide a temporal link that indicates that the decrease in Mash1 levels precede those of TH. Interestingly, immunoblot analysis of Id1 protein levels showed a marked negative correlation between Id1 and Mash1 protein levels (Figure 2A and B). To further confirm that the decrease in Mash1 protein levels arose from a post-transcriptional mechanism, we generated DMS53 cells stably overexpressing an epitope-tagged version of Mash1 under a heterologous CMV promoter. Transcription from the CMV promoter is not modulated by BMP-2 up to 6 h (data not shown). BMP-2 effects were analyzed in two independent clones. Similar to endogenous Mash1, ectopic Mash1 expression was downregulated 2 h after BMP-2 addition (Figure 2D). Figure 2.(A) Cell extracts were obtained from DMS53 cells treated with 5 nM BMP-2 for the times indicated and analyzed by immunoblotting. (B) Quantifications of Mash1 (filled circles) and Id1 (filled squares) protein levels from five separate assays were performed using α-tubulin as normalization and were expressed as fold-change over control samples at time zero and expressed as mean±s.e.m. (C) DMS53 cells were treated for 2 h with the indicated amounts of BMP-2 and Mash1 expression was analyzed by immunoblotting. (D) Parental and two independent myc-Mash1 overexpressing clones were treated for 2 h with 5 nM BMP-2 and expressions of ectopic Mash1 (upper panel) or endogenous Mash1 (lower panel) were analyzed by immunoblotting. Download figure Download PowerPoint We then assayed N-Acetyl-Leu-Leu-Norleucinal (LLnL), which inhibits the activity of the 26S proteasome, for its ability to abolish BMP-induced Mash1 degradation. Addition of LLnL blocked the degradation of Mash1 after 2 h incubation with BMP-2 (Figure 3A). In order to confirm the ability of the ubiquitin system to target Mash1 for proteolytic degradation, HEK-293 cells were transfected with distinct combinations of myc-tagged Mash1 and His-tagged ubiquitin. Immunoblotting of Ni2+-NTA-purified complexes from cells cotransfected with these two constructs allowed us to detect higher molecular weight complexes containing ubiquitinated Mash1 (Figure 3B). Figure 3.(A) DMS53 cells were treated with 50 μM LLnL and/or 5 nM BMP-2 for 2 h and expression of Mash1 was analyzed by immunoblotting. (B) Extracts from HEK-293 cells transfected with the indicated expression constructs and treated overnight with 50 μM LLnL were subjected to immunoblotting to detect myc-Mash1 expression before (left panel) or after Ni2+-NTA-agarose purification (right panel). Download figure Download PowerPoint The ratio between Id1 and E47 levels modulates Mash1 protein stability These previous data led us to hypothesize that, in addition to inhibiting Mash1/E47 transcriptional complexes and function, the induction of Id proteins is responsible for the BMP-2-increased degradation of Mash1. To test this, HEK-293 cells were transfected with distinct combinations of myc-tagged Mash1, E47 and Id1 expression vectors. As previously described (Bounpheng et al, 1999; Shou et al, 1999; Sriuranpong et al, 2002), Mash1 and Id1 levels were strongly enhanced by the addition of LLnL, indicating that these proteins are quickly turned over in the cell (Figure 4A). More importantly, coexpression of Mash1 and E47 resulted in a profound stabilization of Mash1 in the absence of the proteasome inhibitor. Furthermore, Mash1 stabilization by E47 was partially lost by coexpression of Id1. We confirmed that E47 heterodimerizes with either Id1 or Mash1, whereas Mash1 and Id1 do not (Norton, 2000; Jögi et al, 2002; data not shown). To analyze the importance of Mash1–E47 heterodimer formation in Mash1 stabilization, we made similar assays using two E47 deletion mutants (E47 HLH, which contains bHLH, and E47 ΔHLH, defective in heterodimer formation; Voronova and Baltimore, 1990). The E47 mutant, which was unable to heterodimerize with Mash1, was also defective in Mash1 stabilization (Figure 4B). Interestingly, expression of a mutant form of Mash1, devoid of the bHLH region, was much less enhanced by the addition of LLnL, suggesting that bHLH might contain determinants for the rapid turnover of Mash1 (Figure 4B). We further confirmed the decreased Mash1 protein turnover in the heterodimeric state by pulse-chase assays in HEK-293 cells. Overexpression of E47 markedly enhanced the half-life of exogenous Mash1, which was about 3 h compared with only 50 min in cells that overexpressed Id1 either in combination with Mash1 alone or with both Mash1 and E47 (Figure 4C). These data indicate that Mash1 protein stability is strongly enhanced when Mash1 forms heterodimers with E proteins, which is competed by the presence of Id1. Figure 4.(A, B) Extracts from HEK-293 cells transfected with the expression constructs indicated, treated or not with 50 μM LLnL for 8 h, were analyzed by immunoblotting. (C) HEK-293 cells, transfected with the constructs indicated, were pulsed with [35S]methionine for 3 h and labeled proteins were chased in unlabeled media for different times. Labeled Mash1 was immunoprecipitated and visualized by SDS–PAGE. Quantification of Mash1 levels from three to four separate assays is expressed as mean±s.e.m. of percentage values of samples at time zero. Download figure Download PowerPoint To modulate Id1 protein levels independently of cytokine addition, we generated DMS53 clones that expressed murine Id1 under a tetracycline-responsive promoter (Tet off) (Chambard and Pognonec, 1998). Several clones resulted positive for tetracycline-regulated expression of exogenous Id1. Exogenous murine Id1 has a slightly higher electrophoretic mobility compared with human Id1, which allows us to discriminate easily between ectopic and endogenous Id1 expression (Figure 5A). We then performed time-course and dose–response analysis of Id1 induction and analyzed endogenous levels of Mash1 expression. When two independent clones were incubated with decreasing amounts of tetracycline, they displayed increasing levels of ectopic Id1 that correlated with decreasing amounts of Mash1 protein (Figure 5B). Similarly, parental and two Id1-overexpressing clones were incubated overnight with 30 ng/ml tetracycline in order to repress ectopic Id1 expression, and then depleted of tetracycline for a range of times. The induction profile of Id1 protein levels correlated with a decrease in Mash1 expression (Figure 5C). Figure 5.(A) Immunoblot analysis of tetracycline (Tet) responsiveness of several stable DMS53-Id1 clones. (B) Id1-overexpressing clones (Id16 and Id17) were incubated in the presence of 30 ng/ml of tetracycline overnight, and then incubated for 8 h in the presence of different concentrations of tetracycline. Expressions of Mash1 and Id1 were analyzed by immunoblotting. (C) Parental and two Id1-overexpressing clones (Id16 and Id17) were incubated overnight with 30 ng/ml of tetracycline in order to block ectopic Id1 expression. Then cells were deprived of tetracycline for the times indicated and expressions of Mash1 and Id1 were analyzed by immunoblotting. Download figure Download PowerPoint DMS53 cell clones, which stably overexpressed E47, were generated and analyzed (Figure 6A). Clones 10 and 17 showed an increase in the steady-state levels of endogenous Mash1 compared with wild-type cells (Figure 6A). More importantly, after addition of BMP-2 for 2 h, overexpressed E47 partially prevented the degradation of Mash1. Figure 6.(A) E47 expression of DMS53 cell clones, stably overexpressing E47, was analyzed by immunoblotting and is shown in the upper panel. Parental and two independent clones (E10 and E17) were treated for 2 or 4 h with 5 nM BMP-2 and expressions of endogenous Mash1 and Id1 were analyzed by immunoblotting (lower panel). (B) Wild-type or Id1−/− MEFs were transfected with the indicated expression constructs. After 24 h, cells were splitted and treated with 10 nM BMP-2 for 3 h. Expressions of Mash1, Id1 and tubulin were analyzed by immunoblotting. Download figure Download PowerPoint To examine the requirement of Id1 for the BMP-induced effects on Mash1 levels, we analyzed BMP-induced Mash1 degradation in Id1−/− mouse embryonic fibroblasts (MEFs). Wild-type as well as Id1−/− MEFs were able to respond to BMP-2. This was observed using a reporter containing the BMP-specific enhancer of the Id1 gene promoter (López-Rovira et al, 2002) (data not shown). When Mash1 was ectopically expressed in wild-type or Id1−/− MEFs, BMP-2-induced degradation of Mash1 was lost in Id1−/− MEFs (Figure 6B). These results indicate that the induction of Id1 is necessary and sufficient to mediate the BMP effects on Mash1 levels. E47-dependent phosphorylation on Ser152 increases Mash1 protein stability SDS–PAGE analysis of cell extracts from HEK-293 cells cotransfected with Mash1 and E47 showed that Mash1 appeared as two major species compared with a single band in extracts from cells transfected with Mash1 alone (Figure 4C). To determine whether the retarded band is due to phosphorylation events, we immunoprecipitated 35S-labeled Mash1 from cells cotransfected with E47 and tested the immunoprecipitates with alkaline phosphatase. Phosphatase treatment converted the upper form into the lower Mash1 species, indicating that the upper band was caused by phosphorylation events (data not shown). We next examined the effects of cotransfection of E47 and Id1 on Mash1 phosphorylation and E47 interaction. Cotransfection of E47 led to increased levels of phosphorylated forms of Mash1 and the appearance of co-immunoprecipitated E47, compared with cells transfected with Mash1 alone (Figure 7A). In contrast, cotransfection of E47 plus Id1 partially reverted the appearance of phosphorylated forms of Mash1 and levels of co-immunoprecipitated E47, which further supports a correlation between the amounts of E47 that can interact with Mash1 and levels of hyperphosphorylated Mash1. Similar to Id1, when a constitutively active form of BMP receptor type I was cotransfected with E47, we also observed a decrease in the hyperphosphorylated forms of Mash1 (Figure 7B). To analyze the requirement of Mash1–E47 heterodimer formation in Mash1 phosphorylation, we made similar assays using two E47 deletion mutants (E47 HLH, which contains bHLH, and E47 ΔHLH, defective in heterodimer formation). The E47 mutant, which was unable to heterodimerize with Mash1, was also unable to induce Mash1 phosphorylation (Figure 7C). Similarly, expression of a mutant form of Mash1, devoid of the bHLH region, was not phosphorylated in the presence of E47. We also analyzed extracts from transfected cells labeled with 32Pi and [35S]methionine and compared the ratios of the major labeled forms of Mash1. Mash1 was present as two main phosphorylated bands and a higher mobility unphosphorylated one. While coexpression of E47 induced the appearance of the higher phosphorylated band, coexpression of Id1 increased the amount of Mash1 in the unphosphorylated form (Figure 7D). Phosphoamino acid analysis revealed that all phosphorylated residues were phosphoserine (data not shown). To determine E47-induced phosphorylation sites on Mash1, tryptic peptides were generated from the two phosphorylated bands, and the resulting peptides were analyzed by Tris–Tricine SDS–PAGE. Comparison of 32P-labeled peptides from these bands indicated that differential phosphorylation was located in a peptide with a molecular mass of about 400–800 Da (data not shown). Sequence analysis revealed that only three distinct peptides of about that mass contain serines conserved in Mash1 orthologs. We generated site-directed mutants corresponding to these sites and confirmed them by sequencing. Analysis of these mutants demonstrated that the mutant in which Ser152 was changed to Ala (S152A) lost E47-dependent phosphorylation (Figure 8A). Examination of the structure of bHLH transcription factors revealed that Ser152 is located in the solvent-exposed loop that joins the two α-helices involved in transcription factor heterodimerization (Chavali et al, 2001). It is noteworthy that Mash1(S152A) co-immunoprecipitated significantly lower levels of E47. To assess the relevance of Ser152 phosphorylation on transcriptional activity, mutants were transfected with the TH reporter construct. The Ser152 mutant showed higher transcriptional activity than wild type or the other mutants when transfected alone, whereas no significant differences were observed when cotransfected with E47 (Figure 8B). On the basis of our previous results, we tested whether Ser152 phosphorylation was involved in the stabilization of Mash1 protein levels by heterodimerization with E47. Pulse-chase assays indicated that, as previously shown, wild-type Mash1 levels were stabilized by coexpression of E47. In contrast, Mash1(S152A) levels were much less stabilized (Figure 8C and D). Figure 7.HEK-293 cells, transfected with the constructs indicated, were labeled with [35S]methionine or 32Pi for 3 h as described in Materials and methods. Labeled Mash1 or E47 was immunoprecipitated and visualized by SDS–PAGE (see text). Download figure Download PowerPoint Figure 8.(A) HEK-293 cells, transfected with the wild-type or mutant Mash1 constructs, with or without E47 expression construct as indicated, were labeled with [35S]methionine and Mash1 was immunoprecipitated as above. (B) C17.2 cells were cotransfected with a TH promoter-driven reporter construct and the indicated combinations of wild-type and mutant Mash1 with (filled bars) or without (empty bars) E47 expression vectors. The results are expressed as mean±s.e.m. of triplicates from five independent transfections. (C, D) Pulse-chase assays and quantification were performed in HEK-293 cells, transfected with the constructs indicated, as described in Figure 4B. Download figure Download PowerPoint Involvement of CK2 in E47-dependent phosphorylation of Mash1 Analysis of the sequence flanking Ser152 of Mash1 from several species revealed consensus sites for PKA (KKx Ser152) and CK2 (Ser152xxE). To assess the involvement of distinct

Mutation A30P in the alpha-synuclein gene is a cause of familial Parkinson disease. Transgenic mice expressing wild mouse and mutant human A30P alpha-synuclein, Tg5093 mice (Tg), show a progressive motor disorder characterized by tremor, rigidity, and dystonia, accompanied by accumulation of alpha-synuclein in the soma and neurites and by a conspicuous gliosis beginning in the hippocampal formation at the age of 7 to 8 months and spreading throughout the CNS. Impaired short-term changes in synaptic strength have also been documented in hippocampal slices from Tg mice. Alpha-synuclein aggregates of approximately 34 and 70 kDa, in addition to the band of 17 kDa, corresponding to the molecular weight of alpha-synuclein, were recovered in the PBS-soluble fraction of brain homogenates from Tg mice but not from brain samples from age-matched wildtype littermates. MPTP-treated Tg and wildtype mice produced alpha-synuclein aggregates in the PBS-, deoxycholate-, and SDS-soluble fractions. Aggregates of alpha-synuclein, although with different molecular weights, were also observed in rotenone-treated Tg and wildtype mice. Pull-down studies with members of the Rab protein family have shown that alpha-synuclein from Tg mice interacts with Rab3a, Rab5, and Rab8. This binding is not due to the amount of alpha-synuclein (levels of which are higher in Tg mice) and it is not dependent on the amount of Rab protein used in the assay. Rather, alpha-synuclein interactions with Rab proteins are due to mutant alpha-synuclein as demonstrated in Rab pull-down assays with recombinant of wildtype and mutant A30P human alpha-synuclein. Since Rab3a, Rab5, and Rab8 are important proteins involved in synaptic vesicle trafficking and exocytosis at the synapse, vesicle endocytosis, and trans-Golgi transport, respectively, it can be suggested that these functions are impaired in Tg mice. This rationale is consistent with previous data showing that short-term hippocampal synaptic plasticity is altered and that alpha-synuclein accumulates in the cytoplasm of neurons in Tg mice.

The present study examines alpha-synuclein interactions with rab3a and rabphilin by antibody arrays, immunoprecipitation and pull-down methods in the entorhinal cortex of control cases and in diffuse Lewy body disease (LBD) cases. Alpha-synuclein immunoprecipitation revealed alpha-synuclein binding to rabphilin in control but not in LB cases. Immunoprecipitation with rab3a disclosed rab3a binding to rabphilin in control but not in LB cases. Moreover, rab3a interacted with high molecular weight (66 kDa) alpha-synuclein only in LB cases, in agreement with parallel studies using antibody arrays. Results were compared with pull-down assays using His(6)/Flag-tagged rab3, rab5 and rab8, and anti-Flag immunoblotting. Weak bands of 17 kDa, corresponding to alpha-synuclein, were obtained in LB and, less intensely, in control cases. In addition, alpha-synuclein-immunoreactive bands of high molecular weight (36 kDa) were seen only in LB cases after pull-down assays with rab3a, rab5 or rab8. These findings corroborate previous observations showing rab3a-rabphilin interactions in control brains, and add substantial information regarding decreased binding of rab3a to rabphilin and increased binding of rab3a to alpha-synuclein aggregates in LB cases. Since, alpha-synuclein, rab3a and rabphilin participate in the docking and fusion of synaptic vesicles, it can be suggested that exocytosis of neurotransmitters may be impaired in LB diseases.

Prodigiosin is a bacterial red pigment with cytotoxic properties and potential antitumor activity that has been tested against different cancerous cells. In this study we report the effect and mechanisms of action of prodigiosin against different human neuroblastoma cell lines: SH-SY5Y, LAN-1, IMR-32 (N-type) and SK-N-AS (S-type). We compare the anticancerous effect of prodigiosin with that of cisplatin at different concentrations during 24 h of exposure. Prodigiosin is more potent, with IC50 values lower than 1.5 microM in N-type neuroblastoma cells and around 7 microM in the S-type neuroblastoma cell line. We describe prodigiosin as a proton sequestering agent that destroys the intracellular pH gradient, and propose that its main cytotoxic effect could be related to its action on mitochondria, where it exerts an uncoupling effect on the electronic chain transport of protons to mitochondrial ATP synthase. As a result of this action, ATP production is reduced but without decreasing in oxygen consumption. This mechanism of action differs from those induced by conventional chemotherapeutic drugs, suggesting a possible role for prodigiosin to enhance the effect of antitumor agents in the treatment of neuroblastoma.

Neuroblastoma is a malignant embryonal tumor occurring in young children, consisting of undifferentiated neuroectodermal cells derived from the neural crest. Current therapies for high-risk neuroblastoma are insufficient, resulting in high mortality rates and high incidence of relapse. With the intent to find new therapies for neuroblastomas, we investigated the efficacy of low-doses of actinomycin D, which at low concentrations preferentially inhibit RNA polymerase I-dependent rRNA trasncription and therefore, ribosome biogenesis.Neuroblastoma cell lines with different p53 genetic background were employed to determine the response on cell viability and apoptosis of low-dose of actinomycin D. Subcutaneously-implanted SK-N-JD derived neuroblastoma tumors were used to assess the effect of low-doses of actinomycin D on tumor formation.Low-dose actinomycin D treatment causes a reduction of cell viability in neuroblastoma cell lines and that this effect is stronger in cells that are wild-type for p53. MYCN overexpression contributes to enhance this effect, confirming the importance of this oncogene in ribosome biogenesis. In the wild-type SK-N-JD cell line, apoptosis was the major mechanism responsible for the reduction in viability and we demonstrate that treatment with the MDM2 inhibitor Nutlin-3, had a similar effect to that of actinomycin D. Apoptosis was also detected in p53(-/-)deficient LA1-55n cells treated with actinomycin D, however, only a small recovery of cell viability was found when apoptosis was inhibited by a pan-caspase inhibitor, suggesting that the treatment could activate an apoptosis-independent cell death pathway in these cells. We also determined whether actinomycin D could increase the efficacy of the histone deacetylase inhibitor, SAHA, which is in being used in neuroblastoma clinical trials. We show that actinomycin D synergizes with SAHA in neuroblastoma cell lines. Moreover, on subcutaneously-implanted neuroblastoma tumors derived from SK-N-JD cells, actinomycin D led to tumor regression, an effect enhanced in combination with SAHA.The results presented in this work demonstrate that actinomycin D, at low concentrations, inhibits proliferation and induces cell death in vitro, as well as tumor regression in vivo. From this study, we propose that use of ribosome biogenesis inhibitors should be clinically considered as a potential therapy to treat neuroblastomas.

We report the identification of a human 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase gene (PFKFB3) isolated from a human fetal brain cDNA library. The gene was localized to 10p15→p14 by fluorescence in situ hybridization. The entire cDNA (4,322 bp) codes for a polypeptide of 520 amino acid residues (molecular weight, 59.571 kDa). Structural analysis showed the presence of a kinase domain located at the amino terminus and a bisphosphatase domain at the carboxy terminus, characteristic of previously described 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase isozymes. In addition, a phosphorylation site for cAMP-dependent protein kinase was found at the carboxy terminus. Northern blot analysis showed the presence of a unique 4.8-kb mRNA expressed in the different tissues studied. In mammalian COS-1 cells, this cDNA drives the expression of an active isozyme. Taken together, these results identify the presence of a gene coding for a human 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase isozyme which is ubiquitously expressed.

In addition to being a master regulator of cell cycle progression, E2F1 regulates other associated biological processes, including growth and malignancy. Here, we uncover a regulatory network linking E2F1 to lysosomal trafficking and mTORC1 signaling that involves v-ATPase regulation. By immunofluorescence and time-lapse microscopy we found that E2F1 induces the movement of lysosomes to the cell periphery, and that this process is essential for E2F1-induced mTORC1 activation and repression of autophagy. Gain- and loss-of-function experiments reveal that E2F1 regulates v-ATPase activity and inhibition of v-ATPase activity repressed E2F1-induced lysosomal trafficking and mTORC1 activation. Immunoprecipitation experiments demonstrate that E2F1 induces the recruitment of v-ATPase to lysosomal RagB GTPase, suggesting that E2F1 regulates v-ATPase activity by enhancing the association of V0 and V1 v-ATPase complex. Analysis of v-ATPase subunit expression identified B subunit of V0 complex, ATP6V0B, as a transcriptional target of E2F1. Importantly, ATP6V0B ectopic-expression increased v-ATPase and mTORC1 activity, consistent with ATP6V0B being responsible for mediating the effects of E2F1 on both responses. Our findings on lysosomal trafficking, mTORC1 activation and autophagy suppression suggest that pharmacological intervention at the level of v-ATPase may be an efficacious avenue for the treatment of metastatic processes in tumors overexpressing E2F1.

<h2>Summary</h2> Cancer cells rely on mTORC1 activity to coordinate mitogenic signaling with nutrients availability for growth. Based on the metabolic function of E2F1, we hypothesize that glucose catabolism driven by E2F1 could participate on mTORC1 activation. Here, we demonstrate that glucose potentiates E2F1-induced mTORC1 activation by promoting mTORC1 translocation to lysosomes, a process that occurs independently of AMPK activation. We showed that E2F1 regulates glucose metabolism by increasing aerobic glycolysis and identified the PFKFB3 regulatory enzyme as an E2F1-regulated gene important for mTORC1 activation. Furthermore, PFKFB3 and PFK1 were found associated to lysosomes and we demonstrated that modulation of PFKFB3 activity, either by substrate accessibility or expression, regulates the translocation of mTORC1 to lysosomes by direct interaction with Rag B and subsequent mTORC1 activity. Our results support a model whereby a glycolytic metabolon containing phosphofructokinases transiently interacts with the lysosome acting as a sensor platform for glucose catabolism toward mTORC1 activity.

Lysosomes, as primary degradative organelles, are the endpoint of different converging pathways, including macroautophagy. To date, lysosome degradative function has been mainly studied in interphase cells, while their role during mitosis remains controversial. Mitosis dictates the faithful transmission of genetic material among generations, and perturbations of mitotic division lead to chromosomal instability, a hallmark of cancer. Heretofore, correct mitotic progression relies on the orchestrated degradation of mitotic factors, which was mainly attributed to ubiquitin-triggered proteasome-dependent degradation. Here, we show that mitotic transition also relies on lysosome-dependent degradation, as impairment of lysosomes increases mitotic timing and leads to mitotic errors, thus promoting chromosomal instability. Furthermore, we identified several putative lysosomal targets in mitotic cells. Among them, WAPL, a cohesin regulatory protein, emerged as a novel SQSTM1-interacting protein for targeted lysosomal degradation. Finally, we characterized an atypical nuclear phenotype, the toroidal nucleus, as a novel biomarker for genotoxic screenings. Our results establish lysosome-dependent degradation as an essential event to prevent chromosomal instability.Abbreviations: 3D: three-dimensional; APC/C: anaphase-promoting complex; ARL8B: ADP ribosylation factor like GTPase 8B; ATG: autophagy-related; BORC: BLOC-one-related complex; CDK: cyclin-dependent kinase; CENPE: centromere protein E; CIN: chromosomal instability; ConcA: concanamycin A; CQ: chloroquine; DAPI: 4,6-diamidino-2-penylinole; FTI: farnesyltransferase inhibitors; GFP: green fluorescent protein; H2B: histone 2B; KIF: kinesin family member; LAMP2: lysosomal associated membrane protein 2; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MEF: mouse embryonic fibroblast; MTOR: mechanistic target of rapamycin kinase; PDS5B: PDS5 cohesin associated factor B; SAC: spindle assembly checkpoint; PLEKHM2: pleckstrin homology and RUN domain containing M2; SQSTM1: sequestosome 1; TEM: transmission electron microscopy; ULK1: unc-51 like autophagy activating kinase 1; UPS: ubiquitin-proteasome system; v-ATPase: vacuolar-type H+-translocating ATPase; WAPL: WAPL cohesion release factor.

There is growing evidence that apoptotic mechanisms underlie the neurodegeneration leading to Parkinson's disease. 1-Methyl-4-phenylpyridinium ion (MPP+), the active metabolite of the parkinsonism-inducing drug MPTP, induced apoptosis in cultures of human SH-SY5Y neuroblastoma cells. Nuclear fragmentation, DNA laddering, and a 20% decrease in viability were seen after a 4-day incubation with 5 μM MPP+. Cell viability decreased by 40% at 100 μM MPP+, but the degree of apoptosis was not correlatively increased. The MPP+-induced apoptosis was completely prevented by the broad caspase inhibitor zVAD.fmk but not by the caspase-8 inhibitor IETD.fmk. Furthermore, MPP+ had no effect on the levels of Fas or Fas-L, suggesting lack of activation of the Fas-L/Fas/caspase-8 pathway of apoptosis. There was no evidence of mitochondrial dysfunction at 5 μM MPP+: No differences were seen in transmembrane potential or in cytochrome c release from controls. At 100 μM MPP+, the mitochondrial potential decreased, and cytoplasmic cytochrome c and caspase-9 activation increased slightly. At both low and high concentrations of MPP+, VDVADase and DEVDase activities increased. We conclude that MPP+ can induce caspase-mediated apoptosis, which is prevented by caspase inhibition, at concentrations lower than those needed to trigger mitochondrial dysfunction and closer to those found in the brains of MPTP-treated animals. J. Neurosci. Res. 63:421–428, 2001. © 2001 Wiley-Liss, Inc.

Intraperitoneal administration of galactosamine (400 mg/kg body wt) to rats results in reversible liver cell injury that is related to a dose-dependent depletion of uridine phosphates by formation of UDP-sugar derivatives. This damage was monitored through changes in serum enzymatic activities that increased after the first 6 hr of drug administration. Glycemia and serum albumin remained stable during liver injury, whereas cholesterol and triglycerides decreased. To maintain plasma glucose concentration, the hepatic carbohydrate metabolism was greatly altered. Glycogen dropped during the first hours, remaining low for up to 48 hr. Fructose 2,6-bisphosphate and ATP levels decreased even faster than glycogen, with lactate following a similar diminution and being restored in parallel with both metabolites. The reduction in fructose 2,6-bisphosphate can be explained by changes in the substrates or modulators of the 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase, because neither the cyclic AMP levels nor the activity ratio of the enzyme were modified. Simultaneous administration of galactosamine and fructose 1,6-bisphosphate (2 gm/kg) prevented liver cell death, as monitored by serum enzyme activities. Furthermore, the bisphosphorylated metabolite had protective effects on the changes in liver calcium content and ATP and fructose 2,6-bisphosphate concentrations. In contrast, fructose, fructose-1-phosphate and fructose-6-phosphate had no significant protection. Fructose 1,6-bisphosphate might decrease galactosamine toxicity by increasing fructose 2,6-bisphosphate and ATP levels, the changes in both metabolites probably being related. The significance of these findings with respect to the mechanism of galactosamine-induced liver injury is also discussed. (HEPATOLOGY 1992;15:1147–1153).

Dopamine at 100–500 μM has toxic effects on human SH-SY5Y neuroblastoma cells, manifested as apoptotic cell loss and strong autophagy. The molecular mechanisms and types of dopamine-induced cell death are not yet well known. Their identification is important in the study of neurodegenerative diseases that specifically involve dopaminergic neurons. We looked for changes in expression and content of proteins involved in apoptosis and autophagy after dopamine treatment. All the changes found were prevented by avoiding dopamine oxidation with N-acetylcysteine, indicating a key role for the products of dopamine oxidation in dopamine toxicity. As early as 1–2 h after treatment we found an increase in hypoxia-inducible factor-1α (HIF-1α) and an accumulation of ubiquitinated proteins. Proteins regulated by HIF-1α and involved in apoptosis and/or autophagy, such as p53, Puma and Bnip3, were subsequently increased. However, apoptotic parameters (caspase-3, caspase-7, PARP) were only activated after 12 h of 500 μM dopamine treatment. Autophagy, monitored by the LC3-II increase after LC3-I linkage to autophagic vacuoles, was evident after 6 h of treatment with both 100 and 500 μM dopamine. The mTOR pathway was inhibited by dopamine, probably due to the intracellular redox changes and energy depletion leading to AMPK activation. However, this mechanism is not sufficient to explain the high LC3-II activation caused by dopamine: the LC3-II increase was not reversed by IGF-1, which prevented this effect when caused by the mTOR inhibitor rapamycin. Our results suggest that the aggregation of ubiquitinated non-degraded proteins may be the main cause of LC3-II activation and autophagy. As we have reported previously, cytosolic dopamine may cause damage by autophagy in neuroblastoma cells (and presumably in dopaminergic neurons), which develops to apoptosis and leads to cell degeneration.

Konzo and lathyrism are associated with consumption of cassava and grass pea, respectively. Cassava consumption has also been associated with a third disease, tropical ataxic neuropathy (TAN). This review presents a new unifying hypothesis on the causative agents for these diseases: namely, that they are nitriles, compounds containing cyano groups. The diseases may be caused by different but similar nitriles through direct neurotoxic actions not mediated by systemic cyanide release. Both cassava and Lathyrus contain nitriles, and other unidentified nitriles can be generated during food processing or in the human body. Available data indicate that several small nitriles cause a variety of neurotoxic effects. In experimental animals, 3,3'-iminodipropionitrile (IDPN), allylnitrile and cis-crotononitrile cause sensory toxicity, whereas hexadienenitrile and trans-crotononitrile induce selective neuronal degeneration in discrete brain regions. IDPN also induces a neurofilamentous axonopathy, and dimethylaminopropionitrile is known to cause autonomic (genito-urinary) neurotoxicity in both humans and rodents. Some of these actions depend on metabolic bioactivation of the parental nitriles, and sex- and species-dependent differences in susceptibility have been recorded. Recently, neuronal degeneration has been found in rats exposed to acetone cyanohydrin. Taken together, the neurotoxic properties of nitriles make them excellent candidates as causative agents for konzo, lathyrism and TAN.

Bone morphogenetic proteins (BMPs) are a family of growth differentiation factors which induce bone formation from mesenchymal cells. These proteins are members of the transforming growth factor-β superfamily. The expression of BMPs in the nervous system as well as in other tissues has been reported. In this study, we show that the presence of BMP-2 resulted in a dose-dependent increase in the number of tyrosine hydroxylase-immunoreactive ventral mesencephalic cells after 7 days in serum-free medium cultures. A maximal response was elicited at 10 ng/mL. BMP-2 also increased the number of primary neurites and branch points as well as the length of the longest neurite in a dose-dependent manner, with a maximal effect at 1 ng/mL. In contrast, BMP-2 did not modify the number or the function of GABAergic neurons. On the other hand, we observed stimulation of proliferation and morphological changes in glial cells (astrocytes become more fibrous shaped) in the presence of a high BMP-2 concentration (100 ng/mL), but not with lower doses, suggesting that the neurotrophic effect in dopaminergic neurons is not mediated by astroglial cells. This is consistent with the fact that the BMP-2 effect on dopaminergic neurons was observed even when the cultures were treated with α-aminoadipic acid to exclude the presence of glial cells. In summary, our data indicate that BMP-2 is a potent neurotrophic factor for ventral mesencephalic dopaminergic cells in culture. © 1999 John Wiley & Sons, Inc. J Neurobiol 38: 161–170, 1999

The Fas/Fas-L signalling system plays a role in the control of cell death and the survival of lymphocytes, in the regulation of the immune system, and in the progression of autoimmune diseases. Studies in the nervous system have shown Fas/Fas-L activation in multiple sclerosis and in various paradigms leading to neuronal death. Enhanced Fas and Fas-L expression has also been documented in astrocytomas and glioma cell lines. However, little is known about the possible implication of Fas/Fas-L signals in primary human neurodegenerative diseases. In an attempt to gain understanding of the mechanisms commanding cell death and neurone loss in Huntington's disease (HD) and Parkinson's disease (PD), Fas and Fas-L expression has been examined in the brains of patients with HD and PD with Western blotting and immunohistochemistry. Fas and Fas-L expression levels are reduced in the caudate and putamen, but not in the parietal cortex, in HD, as revealed in Western blots. Moreover, Fas and Fas-L immunoreactivity is reduced in striatal neurones in HD. Fas and Fas-L immunoreactivity is also decreased in neurones of the substantia nigra pars compacta in PD. Reduced Fas and Fas-L expression is observed equally in Lewy body-bearing and non-Lewy body-bearing neurones. Yet increased Fas and Fas-L immunoreactivity occurs in normal astrocytes in control brains and in reactive astrocytes in diseased brains. The meaning of increased Fas and Fas-L expression in astrocytes is still unclear. However, the present results suggest that Fas/Fas-L signals are minimized in sensitive neurones in HD and PD.

The transplantation of fetal mesencephalic cell suspensions into the brain striatal system is an emerging treatment for Parkinson's disease. However, one objection to this procedure is the relatively poor survival of implanted cells. The ability of neurotrophic factors to regulate developmental neuron survival and differentiation suggests they could be used to enhance the success of cerebral grafts. We studied the effects of neurotrophin-3 (NT-3) or glial cell line-derived neurotrophic factor (GDNF) on the survival of dopaminergic neurons from rat fetal ventral mesencephalic cells (FMCs) implanted into the rat striatum. Two conditions were tested: (a) incubation of FMCs in media containing NT-3 and GDNF, prior to grafting, and (b) co-grafting of FMCs with cells engineered to overexpress high levels of NT-3 or GDNF. One week after grafting into the rat striatum, the survival of TH+ neurons was significantly increased by pretreatment of ventral mesencephalic cells with NT-3 or GDNF. Similarly, co-graft of ventral mesencephalic cells with NT-3- or GDNF-overexpressing cells, but not the mock-transfected control cell line, increased the survival of graft-derived dopaminergic neurons. Interestingly, we also found that co-grafting of GDNF-overexpressing cells was less effective than NT-3 at improving the survival of fetal dopaminergic neurons in the grafts, and that only GDNF induced intense TH immunostaining in fibers and nerve endings of the host tissue surrounding the implant. Thus, our results suggest that NT-3, by strongly enhancing survival, and GDNF, by promoting both survival and sprouting, may improve the efficiency of fetal transplants in the treatment of Parkinson's disease.

Expression of CCAAT/enhancer-binding protein β (C/EBPβ) and growth-arrest DNA damage-inducible 153/C/EBPβ homology protein (GADD153/CHOP) increased after incubation of human neuroblastoma SH-SY5Y cells with a range of dopamine concentrations. Dopamine (100 μM) caused an increase in C/EBPβ expression between 2 and 12 h of treatment, with no evident intracellular morphological changes. Dopamine (500 μM) led to the appearance of autophagic-like vacuoles and a marked increase in GADD153/CHOP between 6 and 24 h of treatment. The expression of α-synuclein, the main protein of Lewy bodies in Parkinson's disease and other neurological disorders, increased with a profile similar to C/EBPβ. In addition, overexpression of C/EBPβ caused a concomitant increase in the expression of α-synuclein but not of GADD153. In contrast, the overexpression of GADD153 did not alter the expression of α-synuclein. Inhibition of JNK by SP600125 reduced increases in C/EBPβ and α-synuclein expression, whereas inhibition of both JNK and p38MAPK (with SB203580) blocked the increase in GADD153 expression. We conclude that dopamine, through a mechanism driven by stress-activated MAPKs, triggers C/EBPβ and GADD153 expression in a dose-dependent way. Given that the promoter region of the α-synuclein gene contains distinct zones that are susceptible to regulation by C/EBPβ, this factor could be involved in the increased expression of α-synuclein after dopamine-induced cell stress. GADD153 increase seems to be related with the endoplasmic reticulum stress, autophagy and cell death observed at high dopamine concentrations.

PF9601N [N-(2-propynyl) 2-(5-benzyloxyindol) methylamine] is a non-amphetamine type MAO-B inhibitor that has shown neuroprotective properties in vivo using different experimental models of Parkinson's disease. The mechanisms underlying its neuroprotective effects are poorly understood, but appear to be independent of MAO-B inhibition. We have studied its neuroprotective properties using the human SH-SY5Y dopaminergic cell line exposed to 1-methyl-4-phenylpyridinium (MPP(+)), a cellular model of Parkinson's disease. PF9601N pre-treatment significantly reduced MPP(+)-induced cell death and decreased the activation of one of the main executioner caspases, caspase-3. MPP(+) induced stabilization of transcription factor p53, which led to increased levels of this transcription factor, its nuclear translocation and transactivation of p53 response elements. PF9601N prevented this increase, thus reducing its transcriptional activity. Additional results showed that p53 may mediate its pro-apoptotic actions through caspase-2 under our experimental conditions. PUMA-alpha may also contribute to the p53-induced cell death. Since PF9601N significantly reduced MPP(+)-induced caspase-2 activity and PUMA-alpha levels, this reduction may lead to increased cell survival. Thus, PF9601N is a novel molecule with an apparently novel mechanism of action which has a promising potential as a therapeutic agent in the treatment of neurodegenerative diseases.

Neuroblastoma (NB) is the most common solid extracranial tumor in children. Here we showed that trichostatin A, a histone deacetylase inhibitor (HDACi), decreases cell viability in three NB cell lines of different phenotypes. The treatment leads to G2/M-phase arrest, apoptosis and autophagy. Autophagy induction accompanies apoptosis in the most proliferative, N-Myc overexpressing cells. In contrast, autophagy precedes apoptosis and acts as a protective mechanism in the less proliferative, non-N-Myc overexpressing cells. Therefore, the autophagy induction is a relevant event in the NB response to HDACis, and it should be considered in the design of new treatments for this malignancy.

Adenosine has been shown to inhibit dopamine release from striatal slices and synaptosomes. Recently, a direct interaction between the adenosine A2 receptor and dopamine D2 receptor has been provided. Activation of striatal adenosine A1 receptors is known to partially inhibit the release of dopamine (DA), but some aspects of this mechanism remain unclear. We have studied the participation of adenosine A1 receptors in the control of DA release 'in vivo' in awake, freely moving rats using microdialysis. To this end, the effects of 2-chloroadenosine (2-CADO), a non-metabolizable adenosine A1 receptor agonist, were studied on basal and stimulated striatal DA release. Basal levels were found to be slightly decreased by a maximal concentration of 2-CADO without any changes in DA metabolites. Haloperidol stimulated DA release was fully counteracted by 2-CADO. However, high K+ (100 mM) or (+)-amphetamine stimulated DA release was not altered by 2-CADO. Altogether, these data suggest that adenosine acting through A1 receptors possibly localized on striatal dopaminergic nerve terminals can block an induced D2 receptor blockade, but not the releasing effects caused by (+)-amphetamine and high K+ concentration. It is postulated that the increase in DA release by haloperidol is mainly due to an increased firing rate of the DA neurons and that A1 receptor activation can block the DA release observed in response to the action potential activation of DA nerve terminals.

6-Hydroxydopamine (6-OHDA) administered intrastriatally to adult rats in a single injection causes neurodegeneration of the nigrostriatal pathway and loss of > 50% of dopamine neurons in substantia nigra pars compacta 30 days after administration. The death of nigral neurons occurs, at least partially, by a caspase-mediated mechanism. The nigral loss of dopaminergic neurons could be prevented by stereotaxical administration of zVAD.fmk, a caspase inhibitor, into the substantia nigra, indicating that 6-OHDA-induced nigrostriatal degeneration involves caspase activation. These results suggest that caspases are probably involved in neurodegenerative chronic processes such as Parkinson's disease and might be considered as possible targets in the treatment of such neurological disorders.

Parkinson's disease (PD) is one of the most prevalent neurodegenerative disorders, characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta. PD mostly occurs sporadically and its cause remains unknown, nevertheless the discovery of familiar forms of PD, characterized by mutations of genes encoding proteins associated with mitochondria homeostasis, suggests a strong implication of the mitochondrial quality control system in PD. We investigated the effect of dopamine cytosolic accumulation in undifferentiated SH-SY5Y cells, an in vitro model widely used to reproduce impairment of dopamine homeostasis, an early step in PD pathogenesis. A strong depolarization of the mitochondrial membrane was observed after dopamine exposure. Nevertheless, mitochondrial network resulted to assume a peculiar morphology with a distinct pattern of OPA1 and MFN1, key regulators of mitochondrial dynamics. Moreover, selective elimination of dysfunctional mitochondria did not take place, suggesting an impairment of the mitophagic machinery induced by dopamine. Indeed, PINK1 did not accumulate on the outer mitochondrial membrane, nor was parkin recruited to depolarized mitochondria. Altogether, our results indicate that an improper handling of dysfunctional mitochondria may be a leading event in PD pathogenesis. Impaired dopamine (DA) homeostasis and oxidative stress play a key role in the pathogenesis of Parkinson's disease. Free cytosolic dopamine undergoes spontaneous oxidation and generates semiquinonic and quinonic species (DAQ) with the concurrent production of reactive oxygen species (ROS). Dopamine dissipates mitochondrial potential (Δψm ) with a peculiar alteration of the mitochondrial network. However, PINK1-dependent mitophagy is not activated by dopamine toxicity and dysfunctional mitochondria accumulate inside the cell.

MPP(+), the major metabolite of the Parkinsonism-inducing compound MPTP, responsible for the destruction of the nigrostriatal pathway in primates and rodents, has been assayed in isolated rat liver mitochondria in the presence of physiological concentrations of dopamine or analogous concentrations of melanin-dopamine. 5 microM MPP(+) in the presence of 70 microM dopamine or melanin-dopamine, but not alone, decreased the heat production and oxygen consumption of a mitochondrial suspension activated with succinate and ADP. Both dopamine and oxidized dopamine plus MPP(+) also decreased the mitochondrial reductive power measured with MTT. Mitochondrial swelling was observed, associated with an increase in membrane mitochondrial potential, as a synergistic effect between low concentrations of MPP(+) and dopamine. It is suggested that cytosolic dopamine, by itself or via its autooxidation products, may play a relevant role in the mitochondrial toxicity of MPP(+). A failure in the regulation of the storage/release of dopamine could aggravate a mitochondrial damage and trigger the neurodegenerative process underlying MPTP toxicity and Parkinson's disease.

Bovine brain 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase was purified to homogeneity and characterized. This bifunctional enzyme is a homodimer with a subunit molecular weight of 120,000, which is twice that of all other known bifunctional enzyme isozymes. The kinase/bisphosphatase activity ratio was 3.0. The Km values for fructose 6-phosphate and ATP of the 6-phosphofructo-2-kinase were 27 and 55 microM, respectively. The Km for fructose 2,6-bisphosphate and the Ki for fructose 6-phosphate for the bisphosphatase were 70 and 20 microM, respectively. Physiologic concentrations of citrate had reciprocal effects on the enzyme's activities, i.e. inhibiting the kinase (Ki of 35 microM) and activating the bisphosphatase (Ka of 16 microM). Phosphorylation of the brain enzyme was catalyzed by the cyclic AMP-dependent protein kinase with a stoichiometry of 0.9 mol of phosphate/mol of subunit and at a rate similar to that seen with the liver isozyme. In contrast to the liver isozyme, the kinetic properties of the brain enzyme were unaffected by cyclic AMP-dependent protein kinase phosphorylation, and also was not a substrate for protein kinase C. The brain isozyme formed a labeled phosphoenzyme intermediate and cross-reacted with antibodies raised against the liver isozyme. However, the NH2-terminal amino acid sequence of a peptide generated by cyanogen bromide cleavage of the enzyme had no identity with any known bifunctional enzyme sequences. These results indicate that a novel isozyme, which is related to other 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isozymes, is expressed specifically in neural tissues.

Konzo is a neurotoxic motor disease caused by excess consumption of insufficiently processed cassava. Cassava contains the cyanogenic glucoside linamarin, but konzo does not present the known pathological effects of cyanide. We hypothesized that the aglycone of linamarin, acetone cyanohydrin, may be the cause of konzo. This nitrile rapidly decomposes into cyanide and acetone, but the particular exposure and nutrition conditions involved in the emergence of konzo may favor its stabilization and subsequent acute neurotoxicity. A number of preliminary observations were used to design an experiment to test this hypothesis. In the experiment, young female Long-Evans rats were given 10mM acetone cyanohydrin in drinking water for 2 weeks, and then 20mM for 6 weeks. Nutrition deficits associated with konzo were modeled by providing tapioca (cassava starch) as food for the last 3 of these weeks. After this period, rats were fasted for 24h in order to increase endogenous acetone synthesis, and then exposed to 0 (control group) or 50 micromol/kg-h of acetone cyanohydrin for 24h (treated group) through subcutaneous osmotic minipump infusion (n=6/group). Motor activity and gait were evaluated before exposure (pre-test), and 1 and 6 days after exposure. Brains (n=4) were stained for neuronal degeneration by fluoro-jade B. Rats exposed to 50 micromol/kg-h of acetone cyanohydrin showed acute signs of toxicity, but no persistent motor deficits. Two animals showed fluoro-jade staining in discrete thalamic nuclei, including the paraventricular and the ventral reuniens nuclei; one also exhibited labeling of the dorsal endopiriform nucleus. Similar effects were not elicited by equimolar KCN exposure. Therefore, acetone cyanohydrin may cause selective neuronal degeneration in the rat, but the affected areas are not those expected in an animal model of konzo.

3,3'-Iminodipropionitrile (IDPN) exposure causes a neurofilamentous axonopathy and olfactory, audiovestibular and visual toxicity. Many events relevant to these effects and the neurotoxic properties of nitriles as a class remain to be elucidated. We characterized the gliosis associated with the IDPN-induced retinal degeneration in comparison to other effects on the visual and central nervous systems. Gliosis was quantified using an ELISA for the intermediate filament protein, glial fibrillary acidic protein (GFAP). IDPN (0-400 mg kg-1 day-1x3 days, i.p.) caused corneal opacity and dose- and time-dependent increases in retinal GFAP, up to 26-28 fold of control values at 4 weeks post-exposure; a second peak occurred at 16 weeks. In contrast, GFAP peaked at 1 week in olfactory bulbs (OB), cingulate cortex and hippocampus. Cerebellum and striatum showed no gliosis. Retinal dopamine decreased within 2 weeks. Delayed GFAP increases occurred in superior and inferior colliculi. Retina and superior colliculi also showed increased [3H]PK-11195 binding. Histological analysis demonstrated progressive degeneration and gliosis in retina and colliculi. Taken together, the data indicate that primary and secondary degenerative events occur in the retina, and that this retinal degeneration induces GFAP increases in retina and superior colliculus. In addition, GFAP assays demonstrated that the retinal toxicity of IDPN is enhanced by CCl4 hepatotoxicity and blocked by methimazole inhibition of flavin-mono-oxygenases, similarly to its ototoxicity. GFAP assays also indicated that neither vestibulotoxic doses of crotononitrile nor olfatotoxic doses of dichlobenil damage the retina. The data support the use of GFAP assays for assessing the retinal toxicity of IDPN and other nitriles.

In neurons, AMPA receptor (AMPAR) function depends essentially on their constituent components:the ion channel forming subunits and ion channel associated proteins. On the other hand, AMPAR trafficking is tightly regulated by a vast number of intracellular neuronal proteins that bind to AMPAR subunits. It has been recently shown that the interaction between the GluA1 subunit of AMPARs and carnitine palmitoyltransferase 1C (CPT1C), a novel protein partner of AMPARs, is important in modulating surface expression of these ionotropic glutamate receptors. Indeed, synaptic transmission in CPT1C knockout (KO) mice is diminished supporting a positive trafficking role for that protein. However, the molecular mechanisms of such modulation remain unknown although a putative role of CPT1C in depalmitoylating GluA1 has been hypothesized. Here, we explore that possibility and show that CPT1C effect on AMPARs is likely due to changes in the palmitoylation state of GluA1. Based on in silico analysis, Ser 252, His 470 and Asp 474 are predicted to be the catalytic triad responsible for CPT1C palmitoyl thioesterase (PTE) activity. When these residues are mutated or when PTE activity is inhibited, the CPT1C effect on AMPAR trafficking is abolished, validating the CPT1C catalytic triad as being responsible for PTE activity on AMPAR. Moreover, the histidine residue (His 470) of CPT1C is crucial for the increase in GluA1 surface expression in neurons and the H470A mutation impairs the depalmitoylating catalytic activity of CPT1C. Finally, we show that CPT1C effect seems to be specific for this CPT1 isoform and it takes place solely at endoplasmic reticulum (ER). This work adds another facet to the impressive degree of molecular mechanisms regulating AMPAR physiology.

Human neuroblastoma SH-SY5Y cells were used to study the effects of transforming growth factor beta1 (TGF-beta1) and bone morphogenetic protein 2 (BMP-2) on neuronal differentiation and acquisition of a catecholaminergic phenotype. SH-SY5Y cells express the intracellular factors activated through the receptors of the TGFbeta superfamily members, Smad1 and Smad4, as in basal conditions or after differentiation with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or retinoic acid (RA). TGF-beta1 and BMP-2 induce differentiation in SH-SY5Y cells by different pathways: the effect of TGF-beta1 is potentiated by TPA and the effect of BMP-2 is blocked by RA. Cell differentiation due to TGF-beta1 treatment is accompanied by an increase in tyrosine hydroxylase (TH) expression, more pronounced in the presence of TPA or RA and counteracted by BMP-2. BMP-2 and RA both induce noncatecholaminergic cell differentiation, and together they may induce choline acetyltransferase expression in serum-cultured cells. In conclusion, our results suggest that TGF-beta1 and BMP-2 may contribute, in opposite ways, to regulation of the neuronal catecholaminergic phenotype.

Monoamine oxidase B (MAO-B) inhibitors are potentially useful in the therapeutic treatment of Parkinson's disease. L-Deprenyl has been shown to slow nigrostriatal tract degeneration in human idiopathic Parkinsonism and to be an effective neuroprotector in experimental 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toxicity models. However, L-amphetamine and (-)methamphetamine, the metabolites generated by L-deprenyl, can have adverse and severe side-effects. Therefore, the search for new MAO-B inhibitors without potential amphetamine-like properties is a matter of great therapeutic interest. The present report is the first to describe the neuroprotective effect--following chronic intraperitoneal (i.p.) treatment--of a novel and non-amphetaminic MAO-B inhibitor, [N-(2-propynyl)-2-(5-benzyloxy-indolyl) methylamine] (PF 9601N), on the neurodegeneration of nigral dopaminergic neurons caused by administration of intrastriatal 6-hydroxydopamine (6-OHDA). Two groups of six animals were unilaterally injected with 6-OHDA in the right striatum. One group was treated daily with 60 mg/kg PF 9601N i.p., starting before stereotaxic lesion and continuing for 18 days thereafter. The other group was treated with vehicle solution. Coronal slabs including the substantia nigra pars compacta (SNpc) were processed for tyrosine hydroxylase immunohistochemistry (TH). The number of TH positive (TH+) neurons in the SNpc was 60% lower in 6-OHDA lesioned rats. However, the loss of TH+ neurons in the SNpc was only 30% in PF 9601N i.p.-treated animals. Therefore, treatment with the specific MAO-B inhibitor significantly reduced the 6-OHDA-induced degeneration to about 50%.

The transplantation of foetal mesencephalic cells (FMC) into the brain striatal system is an emerging treatment for Parkinson's disease, despite of the relatively poor survival of implanted cells. The ability of neurotrophic factors to regulate neurone survival and differentiation suggests they could be used to enhance the success of cerebral grafts. We analyzed the effect of pre-treatment of FMC suspensions with bone morphogenetic protein-2 (BMP-2) (50 ng/ml) prior to grafting into the striatum of 6-hydroxydopamine lesioned rats. The viability of a FMC suspension was enhanced in vitro by BMP-2. Four weeks after transplantation, the number of dopaminergic neurones was higher and their morphology more developed in grafts pre-treated with BMP-2, compared with non-pre-treated grafts and rats showed a significant reduction in the turning behaviour test. Thus, the pre-treatment of FMCs with BMP-2 should be considered, together with other neurotrophic factors, as a procedure for transplantational treatment of Parkinson's disease.

Extracellular dopamine (DA) and its main cerebral metabolites, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), were measured by bilateral striatal microdialysis in rats at different times (2, 7, 15 and 60 days) after unilateral administration into the right striatum of 1-methyl-4-phenylpyridinium ion (MPP+) or 6-hydroxydopamine (6-OHDA). In both cases the decrease in extracellular dopamine did not exceed 40% of control values. The response of DOPAC and HVA depended on the treatment: MPP+ caused a marked acute decrease in the dopamine metabolites but allowed a progressive recovery that was very evident after 60 days; 6-OHDA caused a progressive decrease in the dopamine metabolites throughout the two months of the study. Tyrosine hydroxylase immunostaining revealed severe neuronal loss in substantia nigra two months after striatal administration of 6-OHDA, whereas no significant neuronal loss was found at the same time after MPP+ administration. A bilateral challenge infusion of MPP+ through the microdialysis probe was used to assess the dopaminergic capacity of both striata: at all the times studied there was a sharp depletion of DA on the non-lesioned side; both MPP+- and 6-OHDA-treated striata were unresponsive after a short time (2 days); after 2 months the response in MPP+-lesioned rats was similar on both sides, whereas 6-OHDA-lesioned striata were still unresponsive to MPP+. In rats, then, the effects of MPP+ could be partly reversed whereas the effects of 6-OHDA were not. These results suggest that neurotoxins causing striatal dopamine loss may act through different mechanisms, which could be significant for the etiopathogenic development of Parkinson's disease.

A cDNA encoding the catalytic core of a novel brain 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isoenzyme was isolated from a λgt10 bovine brain library. This brain cDNA begins and ends in an open reading frame encoding a peptide of 476 amino acids. This peptide contains both the catalytic kinase and bisphosphatase domains and has an overall 65% and 67% identity with the bovine heart and liver isozymes, respectively, whereas the NH2 and COOH-termini are divergent. An active catalytic core brain bifunctional enzyme was expressed in E. coli using a T7 RNA polymerase-based expression system. These results support the presence of a distinct gene coding for the protein in bovine brain.

We characterized dopamine toxicity in human neuroblastoma SH-SY5Y cells as a direct effect of dopamine on cell reductive power, measured as NADH and NADPH cell content. In cell incubations with 100 or 500 μM dopamine, the accumulation of dopamine inside the cell reached a maximum after 6 h. The decrease in cell viability was 40% and 75%, respectively, after 24 h, and was not altered by MAO inhibition with tranylcypromine. Dopamine was metabolized to DOPAC by mitochondrial MAO and, at 500 μM concentration, significantly reduced mitochondrial potential and oxygen consumption. This DA concentration caused only a slight increase in cell peroxidation in the absence of Fe(III), but a dramatic decrease in NADH and NADPH cell content and a concomitant decrease in total cell NAD(P)H/NAD(P)+ and GSH/GSSG and in mitochondrial NADH/NAD+ ratios. Dopaminechrome, a product of dopamine oxidation, was found to be a MAO-A inhibitor and a strong oxidizer of NADH and NADPH in a cell-free system. We conclude that dopamine may affect NADH and NADPH oxidation directly. When the intracellular concentrations of NAD(P)H and oxidized dopamine are similar, NAD(P)H triggers a redox cycle with dopamine that leads to its own consumption. The time-course of NADH and NADPH oxidation by dopamine was assessed in cell-free assays: NAD(P)H concentration decreased at the same time as dopamine oxidation advanced. The break in cell redox equilibrium, not excluding the involvement of free oxygen radicals, could be sufficient to explain the toxicity of dopamine in dopaminergic neurons.

In this study we demonstrate that accumulation of reactive oxygen species (ROS) is essential for E2F1 mediated apoptosis in ER-E2F1 PC12 pheochromocytoma, and SH-SY5Y and SK-N-JD neuroblastoma stable cell lines. In these cells, the ER-E2F1 fusion protein is expressed in the cytosol; the addition of 4-hydroxytamoxifen (OHT) induces its translocation to the nucleus and activation of E2F1target genes. Previously we demonstrated that, in ER-E2F1 PC12 cells, OHT treatment induced apoptosis through activation of caspase-3. Here we show that caspase-8 activity did not change upon treatment with OHT. Moreover, over-expression of Bcl-xL arrested OHT-induced apoptosis; by contrast, over-expression of c-FLIP, did not have any effect on OHT-induced apoptosis. OHT addition induces BimL expression, its translocation to mitochondria and activation of Bax, which is paralleled by diminished mitochondrial enrichment of Bcl-xL. Treatment with a Bax-inhibitory peptide reduced OHT-induced apoptosis. These results point out the essential role of mitochondria on the apoptotic process driven by E2F1. ROS accumulation followed E2F1 induction and treatment with the antioxidant N-acetylcysteine, inhibited E2F1-induced Bax translocation to mitochondria and subsequent apoptosis. The role of ROS in mediating OHT-induced apoptosis was also studied in two neuroblastoma cell lines, SH-SY5Y and SK-N-JD. In SH-SY5Y cells, activation of E2F1 by the addition of OHT induced ROS production and apoptosis, whereas over-expression of E2F1 in SK-N-JD cells failed to induce either response. Transcriptional profiling revealed that many of the genes responsible for scavenging ROS were down-regulated following E2F1-induction in SH-SY5Y, but not in SK-N-JD cells. Finally, inhibition of GSK3β blocked ROS production, Bax activation and the down regulation of ROS scavenging genes. These findings provide an explanation for the apparent contradictory role of E2F1 as an apoptotic agent versus a cell cycle activator.

Effects of the parkinsonism inducing neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on striatal dopamine metabolism and the influence of sex on the recovery were investigated in adult (2-month-old) male and female C57/BL mice. We present here evidence that MPTP treatment (2 doses of 30 mg/kg i.p., each at 24 h interval) produced a similar reduction (-65% to -70%) of striatal dopamine in both sexes 24 h after the last injection of MPTP, and a greater loss of the metabolites in the female group. In contrast to the partial recovery observed in the male group, an increased dopamine loss occurred in the female group within 10 days following the last injection of MPTP. This impairment in recovery appears to be different to the one already observed in aged (24-month-old) male mice treated in similar conditions. As the neurotoxic effects of MPTP depend on its conversion to the 1-methyl-4-phenylpyridinium ion (MPP+) by monoamine oxidase B (MAO B), the presence of a different peripheral or central MAO B type in female mice could be in part responsible for these sex related effects. To investigate this possibility, MAO A and B activities were characterized in liver and brain of adult female control mice during the different steps of the oestrous cycle and compared to those of adult control male mice. Significant differences in MAO A and MAO B activities could be detected during the oestrous cycle and between the adult male and female groups. It is concluded that MAO B may be involved in the sex related effects of MPTP.

A significant loss of dopamine was found in rat striatal slices incubated with 1–methyl–4–phenylpyridinium ion (MPP+) at a concentration of 2 mM or higher. The addition of 7–nitroindazole, a specific inhibitor of neuronal nitric oxide synthase (nNOS), prevented this effect on dopamine when the concentration of MPP+ was between 2–5 M, but not at higher concentrations. This protection was reproduced with other less specific NOS–inhibitors, such as nitro–arginine and nitro–arginine methylester. 7–nitroindazole did not protect against the dopamine depletion caused by the non–specific mitochondrial chain blocker rotenone. Neither MPP+ nor rotenone significantly increased the nitrite concentration in striatal slices, measured as an index of nitric oxide production. The basal production of nitric oxide may be enough to trigger the dopamine depletion at very low concentrations of MPP+ probably acting synergistically with cytosolic calcium increase. Higher concentrations of MPP+ are toxic by themselves without the mediation of nitric oxide. The inhibition of nNOS may protect against dopamine loss at early stages of a neurodegenerative process, and it could then be considered in the treatment or prevention of neurodegenerative human processes such as Parkinson’s disease. © 1998 Elsevier Science Ltd. All rights reserved.

The concentration of fructose 2,6-bisphosphate in the brain remained stable during starvation and early stages of ischaemia, but decreased in diabetes or after lengthened ischaemia. 6-Phosphofructo-1-kinase activity was also decreased in diabetic and ischaemic animals, whereas 6-phosphofructo-2-kinase was not modified. The concentration of the bisphosphorylated metabolite seems to be remarkably constant under a wide variety of experimental conditions, suggesting that it plays an essential role in the basal activation of 6-phosphofructo-1-kinase. Purified 6-phosphofructo-2-kinase also showed fructose-2,6-bisphosphatase activity with an activity ratio similar to that of the purified heart isoenzyme. The brain enzyme also has a net charge similar to that of the heart isoenzyme. Its activity is not modified by sn-glycerol 3-phosphate, and it is more sensitive to citrate than the liver or muscle isoenzyme. Moreover, the enzyme from brain, similarly to that from heart and muscle, is not modified by the cyclic AMP-dependent protein kinase or protein kinase C. A near-full-length cDNA probe from liver hybridized with RNA from brain and heart. In both cases, a major band of 6.8 kb of RNA and a minor one of 4 kb of RNA were detected. All these properties support the hypothesis that brain contains a different isoenzymic form from that of liver and muscle, and it is probably related to the heart isoform.

Citicoline, or CDP-choline, is an essential endogenous intermediate in the biosynthesis of phosphatidylcholine that may act as a neuroprotector in several models of neurodegeneration. The present study analyses the effects of citicoline in the paradigm of staurosporine-induced cell death in human SH-SY5Y neuroblastoma cells. Citicoline reduces apoptosis induced by 100 nM staurosporine for 12 h in SH-SY5Y cells. This effect is higher with pre-treatment of 60 mM citicoline for 24 h after staurosporine challenge. Moreover, citicoline treatment restores glutathione redox ratio diminished after staurosporine challenge. Finally, citicoline also reduces the expression levels of active caspase-3 and specific PARP-cleaved products of 89 kDa resulting from staurosporine exposure when citicoline is added to the culture medium 24 h before staurosporine. These findings demonstrate that citicoline affects the staurosporine-induced apoptosis cell-signalling pathway by interacting with the glutathione system and by inhibiting caspase-3 in SH-SY5Y human neuroblastoma cells.

Cytotoxic concentrations of dopamine (100–500 μM DA) induce expression of tumour necrosis factor receptor-1 (TNF-R1) and tumour necrosis factor-α (TNFα) in SH-SY5Y human neuroblastoma cells. TNFα expression is dose-dependent and can also be detected after 6-hydroxydopamine (6-OHDA) or 1-methyl-4-phenylpyridinium iodide (MPP+) treatment. The expression of TNF-R1 is also dose-dependent, but was not observed in 6-OHDA or MPP+-treatment. Cells not expressing TNF-R1 were insensitive to TNFα, whereas those treated with DA showed a further decrease in viability when subsequently treated with TNFα. Thus, DA treatment confers sensitivity to TNFα. The decrease of cell viability caused by DA was in part prevented by neutralizing TNFα with anti-TNFα. As TNF-R1 is increased in substantia nigra of Parkinsonian brains, we suggest that nonvesiculated DA might also play a role in inducing TNF-R1 expression and predispose the neuron to the action of cytokines released in a microglia-mediated inflammatory response.

The effect of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its main metabolite 1-methyl-4-phenyl-pyridinium ion (MPP+) on catecholamine concentrations was tested in some peripheral organs of the rat. Acute injection of either compound caused a marked depletion of heart noradrenaline (NA). MPP+ had more effect than the parent compound. MPTP had no effect on adrenal catecholamines while its metabolite was effective, but only after several hours. Repeated MPTP and MPP+ injections (20 mg/kg X 5) resulted in marked depletion of heart NA and adrenal gland adrenaline. Clear recovery was observed in both organs 10 days after treatment.

The short-term (24 h) effects of 10 mg/kg of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on cat catecholamine levels of heart, adrenal gland, retina, and caudate nucleus were studied. Significant differences in catecholamines, including adrenomedullar adrenaline, heart noradrenaline, and retinal dopamine, were observed. No differences were found in caudate nucleus dopamine. In this organ, the levels of dopamine metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were significantly lower after MPTP treatment. Biochemical changes caused by MPTP with respect to its effects on the peripheral catecholaminergic organs are discussed.

Although generally considered a prototypical movement disorder, Parkinson's disease is commonly associated with a broad-spectrum of non-motor symptoms, including autonomic dysfunctions caused by significant alterations in catecholaminergic neurons of the peripheral sympathetic nervous system. Here we present evidence that α-synuclein is highly expressed by sympathetic ganglion neurons throughout embryonic and postnatal life and that it is found in tyrosine hydroxylase-positive sympathetic fibers innervating the heart of adult mice. However, mice deficient in α-synuclein do not exhibit any apparent alterations in sympathetic development. Sympathetic neurons isolated from mouse embryos and early postnatal mice are sensitive to the parkinsonian drug MPTP/MPP+ and intoxication requires entry of the neurotoxin through the noradrenaline transporter. Furthermore, recovery of noradrenaline from cardiac sympathetic fibers is reduced in adult mice treated with MPTP systemically. However, MPP+-induced sympathetic neuron loss in vitro or MPTP-induced cardiac noradrenaline depletion in vivo is not modified in mice lacking α-synuclein. This is in clear contrast with the observation that dopaminergic neurons of the central nervous system are significantly less vulnerable to MPTP/MPP+ in the absence of α-synuclein, suggesting different actions of this molecule in central and peripheral catecholaminergic neurons.

Dopamine (DA) and its main cerebral metabolites, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were measured in striatum and cerebrospinal fluid (CSF) from cisterna magna in rats bilaterally lesioned by intrastriatal administration of 6-hydroxydopamine (6-OHDA) or 1-methyl-4-phenylpyridinium ion (MPP+). 6-OHDA caused a progressive lesion in striatum that is only moderately reflected in the decrease in dopamine metabolite concentration in CSF. MPP+ caused an acute but less selective lesion in the dopamine striatal system, as indicated by a significant reduction in striatal GABA content, followed by a slow recovery in dopamine striatal metabolism and content. The locomotor activity was dramatically reduced in both groups 48 hours after the treatment but remained significantly decreased after two months only in 6-OHDA lesioned animals. A positive correlation was found between HVA CSF concentration and striatal DA content in MPP+ lesioned rats, but not in 6-OHDA lesioned rats. It is concluded that the concentration of dopamine metabolites in CSF can be altered only after a severe striatal lesion: reduction of striatal dopamine content below 50% of normal values and involvement of neuronal or non-neuronal elements other than the dopaminergic system, similarly to the lesions caused by MPP+. These results may partly explain why CSF dopamine metabolites concentrations were significantly decreased both in advanced stages of parkinsonism and in other neurodegenerative disorders.

In the present work we report the generation of a new line of alpha-synuclein (alpha-SYN) transgenic mice in which the human wild-type alpha-SYN cDNA is expressed under the control of a tyrosine hydroxylase (TH) promoter. We provide evidence that the ectopic protein is found in TH expressing neurons of both central and peripheral nervous systems. The transgene is expressed very early in development coinciding with the activity of the TH promoter and in the adult brain the human protein distributes normally to the nerve endings and cell bodies of dopaminergic nigral neurons without any evidence of abnormal aggregation. Our results indicate that expression of human wild-type alpha-SYN does not affect normal development or maintenance of TH immunoreactive nigral neurons, striatal dopamine content, or locomotor activity. Systemic administration of the parkinsonian neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induces a loss of TH immunoreactive nigral neurons and terminals and of dopamine levels to the same degree in both transgenic and non-transgenic adult mice. Intoxication also results in a similar loss of cardiac noradrenaline in both genotypes. Surprisingly, cultured transgenic ventral mesencephalic fetal dopaminergic neurons exhibit complete resistance to cell death induced by 1-methyl-4-phenylpyridinium ion (MPP(+)) intoxication, without changes in dopamine transporter (DAT) surface levels. Interestingly, this protection is not observed in other populations of catecholaminergic neurons such as peripheral sympathetic neurons, despite their high sensitivity to MPP(+)in vitro.

Abstract: Fructose 2,6‐bisphosphate has been studied during hypoglycemia induced by insulin administration (40 IU/kg). No changes in content of cerebral fructose 2,6‐bisphosphate were found in mild hypoglycemia, but the level of this compound was markedly decreased in hypoglycemic coma and recovered after 30 min of glucose administration. To correlate a possible modification of the concentration of the metabolite with selective regional damage occurring during hypoglycemic coma, we have analyzed four cerebral areas (cortex, striatum, cerebellum, and hippocampus). Fructose 2,6‐bisphosphate concentrations were similar in the four areas analyzed; severe hypoglycemia decreased levels of the metabolite to the same extent in all the brain areas studied. The decrease in content of fructose 2,6‐bisphosphate was not always accompanied by a parallel decrease in ATP levels, a result suggesting that the low levels of the bisphosphorylated metabolite during hypoglycemic coma could be due to the decreased 6‐phosphofructo‐2‐kinase activity, mainly as a consequence of the fall in concentration of its substrate (fructose 6‐phosphate). These results suggest that fructose 2,6‐bisphosphate could play a permissive role in cerebral tissue, maintaining activation of 6‐phosphofructo‐l‐kinase and glycolysis.

This study reports the expression of the ubiquitous 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene (PFKFB3) (PFK-2/FBPase-2) in different stages of rat brain development. Northern blot and RT-PCR analysis demonstrated that ubiquitous PFK-2/FBPase-2 is expressed in rat brain from embryonic to adult life and shows a transient increase 1 day before birth, coincident with the maximum concentration of Fru-2,6-P(2) and PFK-2 activity. The levels of brain PFK-2/FBPase-2 gene expression as well as the enzymatic activity and the concentration of Fru-2,6-P(2) appear to be remarkably constant during adult life, without significant differences in the brain hippocampus, cortex, cerebellum or striatum areas.

In order to obtain further insight into the interactions between the purinergic and dopaminergic pathways in the striatum, we studied both metabolisms simultaneously, using a microdialysis technique in 1-methyl-1,4-phenylpyridinium ion (MPP+) unilaterally-denervated conscious rats. In these rats the contralateral side was used as control. The perfusates were collected every 20-25 min using 4 mm dialysis probes, implanted in each striatum, and assayed for dopamine and purine metabolites. After MPP+ administration, all adenosine metabolites - with the exception of uric acid - and dopamine levels were significantly increased in the extracellular medium. However, the time-course change in dopamine level did not correlate with the adenosine and inosine time-courses, suggesting a different mechanism of liberation in response to MPP+ administration.

The influence of MPTP (30 mg/kg × 2) on dopamine content and tyrosine hydroxylase activity in striatal, limbic and cortical areas of C-57/B16 mice was studied. General dopamine depletion was observed 24 h after the treatment, but 30 days after administration recovery was complete in all but the striatum. At 24 h, tyrosine hydroxylase activity was decreased only in striatum. It is proposed that MPTP acts differently in striatum and in other dopaminergic areas.

Abstract: The Stereotaxic administration of 1‐methyl‐4‐phenylpyridinium ion (MPP + ) into the neostriatum of male rats caused a lesion that resulted in a large dose‐dependent loss of striatal fructose 2,6‐bisphosphate; initial values were restored 5 days after the treatment. This effect was not protected by systemic administration of MK‐801 or by nitroarginine. The content of hexose 6‐phosphates and ATP was also reduced by MPP + treatment, whereas lactate was increased. Biochemical and histological results suggested that MPP + caused a nonselective cell death, followed by a pronounced astroglial response, parallel to fructose 2, 6‐bisphosphate recovery. The Stereotaxic administration of rotenone showed a different time effect on fructose 2,6‐bisphosphate cerebral content, with a significantly faster recovery. These results indicate that cerebral fructose 2,6‐bisphosphate may be a sensitive metabolite related to brain damage caused by potent neurotoxins such as MPP + . On the other hand, they show that MPP + acts in the brain through a quick, strong cytotoxic mechanism, which probably involves mechanisms other than mitochondria! chain blockage

El modelo social de la discapacidad, centrado en los derechos humanos, está impregnando la normativa y las recomendaciones en el sector turístico, fortaleciendo la necesidad de promocionar el llamado “turismo accesible”. Esta promoción se justifica no sólo por motivos sociales, sino también por un aumento muy signifi cativo del número de personas que en algún momento de su vida presentan necesidades de accesibilidad. En el ámbito del turismo cultural, la accesibilidad se ha eludido frecuentementeescudándose en la necesidad de preservar el patrimonio. No obstante, los establecimientos culturales pueden y deben ser accesibles,ya que existen fórmulas para que esta accesibilidad no vaya en detrimento de la conservación. En el caso de los museos -componentes esenciales del turismo cultural- son ya varias las herramientas de planifi cación que se han articulado para aumentar el compromiso delos gestores y lograr una red museística más accesible. El Museo Reina Sofía se toma como estudio de caso, desgranando las accionesque se han emprendido en los últimos diez años, tanto en materia de accesibilidad espacial y arquitectónica como de accesibilidad a los contenidos, elemento clave para atraer al público no visitante. El establecimiento de redes de colaboración con organizaciones del sector de la discapacidad es fundamental para desarrollar la responsabilidad social del Museo, fortalecer su imagen y consolidarlo como referencia en el mercado del turismo cultural accesible.

Fructose 2,6-bisphosphate (Fru-2,6-P2) levels and 6-phosphofructo-1-kinase and 6-phosphofructo-2-kinase activities have been studied in rat brain during development from embryonal to adult state. Fru-2,6-P2 increases slightly from day 16 of gestation, reaching a maximum 24 h after birth, remaining quite constant during postnatal development. In contrast with 6-phosphofructo-1-kinase, which increases progressively after the first week of age, 6-phosphofructo-2-kinase remains unaltered throughout the period studied. The role of Fru-2,6-P2 in controlling cerebral glycolysis is discussed.

Abstract: The effect of the neurotoxin 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) on D 1 and D 2 dopamine receptors was assayed using in vitro quantitative autoradiography. D 1 receptor subtype was labeled using 1 nM of 3 H‐SCH 23390 and D 2 receptor subtype was labeled using 0.4 nM of 3 H‐spiroperidol. The results are compared to the effect of MPTP on the striatal levels of dopamine and its metabolites, in BL C57 mice. While 2 and 5 doses of MPTP 30 mg/kg/day intraperitoneally reduced the content of striatal dopamine and its metabolites, no modifications were detected in D 2 receptor subtype in any cerebral area studied. However, D 1 receptors were reduced in the substantia nigra 24 hr after the last of 2 doses, but not later. We suggest a compensatory mechanism of the surviving dopaminergic neurones as well as the participation of spare receptors. This would explain the lack of receptor modification after the lesion obtained as seen by the striatal reduction of dopamine and metabolites content, after MPTP administration.

Fructose-1,6-bisphosphate has been shown to reduce ischaemic-induced brain damage in rabbits and gerbils. In view of these findings, we investigated the effects of fructose-1,6-bisphosphate on delayed neuronal death, following bilateral forebrain ischaemia, in the gerbil hippocampus at the fourth day of reperfusion. We subjected gerbils to bilateral forebrain ischaemia for 20 min. Fructose-1,6-bisphosphate was administered: intraperitoneally at a dose of 1 g/kg in saline in hr before the occlusion or at a dose of 1 g/kg 1 hr before the occlusion and every 24 hr for 3 days; or intraventricularly at a dose of 0.1 g/kg just after the carotid occlusion. No significant differences in the number of dying cells in the CA1 area were found between each group of treated animals when compared with controls. This study suggests that fructose-1,6-bisphosphate, administered according to these three different schedules, fails to ameliorate delayed neuronal death after 20 min of bilateral forebrain ischaemia in the CA1 area of the gerbil hippocampus.

Thirty young-adult mice were treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) 30 mg/kg/day for 2 days and sacrificed 24 hours later in order to determine striatal catecholamines and to study morphological changes in the nigrostriatal pathway. Immunohistological techniques were also used with polyclonal antibodies for glial fibrillary acidic protein (GFAP) and tyrosine hydroxylase (TH), and monoclonal antibodies for two subunits of neurofilaments. Silver impregnation demonstrated conspicuous neuronal changes affecting cellular processes from substantia nigra in all treated mice. Terminal and axonal degeneration were also observed in striata. These changes were associated with a moderate to marked gliosis. The TH immunoreactivity was normal in cell bodies of substantia nigra but was decreased in striata from MPTP-treated mice. These data indicate that in mice the deterioration of dendritic and axonal neuropil may constitute a significant causal factor of MPTP neurotoxicity.

The effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its main metabolite 1-methyl-4-phenylpyridinium ion (MPP+) on the peripheral catecholaminergic system of the rat were investigated. MPTP and MPP+ injections (20 mg/kg i.p.) caused a marked acute depletion of heart noradrenaline, up to 75% twelve hours after the administration, and a decrease of adrenal gland adrenaline. The time-course of the effect of MPTP and MPP+ is reported, together with a decrease in the tyrosine hydroxylase activity after MPTP treatment, more evident in the adrenal glands. Pargyline (50 mg/kg i.p.) is not able to prevent such a neurotoxic peripheral effect.

Abstract Differences in purine metabolism produced by three preservation solutions were studied by determining the adenine nucleotide (ATP, ADP, AMP, and IMP) and nucleoside (adenosine, inosine, and hy-poxanthine) levels in human kidney cortical biopsies. Forty kidney allo-grafts were studied using University of Wisconsin (UW) solution (n=20), Euro-Collins (EC) solution (n=12), and modified EC solution with mannitol (M;n=8). No significant differences were found between the three solutions studied with regard to ATP, ADP, or AMP changes. The mean ATP level (nmol/mg prot ± SEM) at the end of preservation in the UW group was 2.7 ± 0.3 nmol/mg, in the EC group 3.8 ± 0.7 nmol/mg, and in the M group 2.3 ± 0.4 nmol/mg. ATP 30 min after reperfusion in the UW, EC, and M groups was 5.7 ± 0.8 nmol/mg, 6.4 ± 1.0 nmol/mg, and 4.6 ± 0.5 nmol/mg, respectively. However, an important difference appeared in the catabolic products determined. Kidneys perfused with UW solution had a significantly higher level of adenosine (2.6 ± 0.6 nmol/mg), inosine (11.8 ± 2.2 nmol/mg), and hypoxan-thine (18.1 ± 2.1 nmol/mg) at the end of cold storage than those perfused with EC (0.4 ± 0.1 nmol/mg, 2.0 ± 0.8 nmol/mg, and 7.1 f 1.4 nmol/mg) and M solutions (0.2 ± 0.05 nmol/mg, 0.5 5 0.1 nmol/mg, and 5.2 ± 0.6 nmol/mg; P < 0.05). These levels returned to initial values 30 min postreperfusion and there were no differences with the EC or M solution groups at that time. Thus, the adenosine present in UW solution does not appear to be useful in recovering the adenine nucleotide pool at reperfusion. Moreover, it produces a marked increase in degradation products. Our findings do not support the beneficial metabolic effect of UW solution in terms of adenine nucleotide metabolism in comparison with simpler and less expensive preservation solutions like EC.

The aim of this work is to demonstrate that the value of the mean transit time (MTT) obtained from the 99mTc-MAG3 renogram deconvolution is related to the levels of adenine nucleotides determined in cortical biopsies from transplanted kidneys.The functional state was estimated by means of the MTT and the initial height (H0) of the renal retention function obtained from the 99mTc-MAG3 renogram deconvolution and by the measure of adenine nucleotides obtained from biopsies. We studied 30 kidney graft recipients, 25 normal functioning grafts (NFG) and 5 with acute tubular necrosis (ATN).The MTT is significantly longer for ATN (p < 0.001). The initial uptake values (H0) are significantly lower for ATN (p < 0.001). The sum of adenine nucleotides (SAN) is significantly greater for NFG than for ATN (p < 0.001). The values of the MTT seem to reflect the energy state of the cells in transplanted kidney.The analysis of MTT may be indicative of the functional metabolic recovery and thus it may be predictive of the renal graft function at least in the same extent than the biochemical analysis of a cortical renal biopsy immediately after blood reperfusion of the tissue.

Introduction Bipolar disorder (BD) is associated with high morbidity and mortality. Patients are symptomatic almost half of their lives and experience significant disability. One subtype of BD is associated with a more chronic course, refractoriness to treatment and poor outcome. Diabetes mellitus type 2 (T2D) and insulin resistance (IR) have been identified as risk factors for this more severe form of BD. Objectives and aims We investigated the rates of IR and T2D in patients with BD and whether this comorbidity is associated with specific clinical features of BD such as rapid cycling or treatment resistance. Methods IR and T2D were screened in patients with BD types I or II, who were on stable treatment with mood stabilizers. The response to treatment was assessed by means of the Alda scale. Results In a preliminary sample, we made a new diagnosis of IR in 40% of patients. The 1% of this sample had a diagnosis of T2D. The treatment response was worse in BD patients with comorbid IR or T2D as compared to those without metabolic abnormalities. Conclusions These findings show that IR and T2D have high prevalence in BD patients and have negative impact on treatment response. Disclosure of interest The authors have not supplied their declaration of competing interest.

Time course of H2A.X phosphorylation after SAHA and Actinomycin D treatment. LA1-55n cells were treated (+) or not (-) with 0.1 nM of actinomycin D (ActD) in the presence (+) or in the absence (-) of 1 μ M SAHA. Indicated protein expression was determined by Western blot analysis at the indicated times after the treatment. (DOCX 710 kb)

Mesencephalic cell suspensions are used, experimentally but also clinically, to compensate for neurological deficiencies, by implantation into the striatum. Here, we have studied the metabolism of mesencephalic cell suspensions obtained from rat embryos by measuring heat dissipation, oxygen consumption, ATP and lactate production. The effect of basic fibroblast growth factor (bFGF) at a 50 ng/ml concentration on these parameters was studied in order to assess the effect of in vitro exposure of cell suspensions to this trophic factor. Heat production and oxygen consumption were low, as could be expected from an immature nervous tissue, and they further decreased after addition of bFGF. This trophic factor decreased the total ATP concentration and increased the lactate production. The viability of the cell suspensions was reduced by nearly a half, 2 h after the addition of bFGF, and numerous fragmented nuclei were observed. It seems that, in contrast to the neuroprotective effect of bFGF on mesencephalic cultures and nigrostriatal neurons, this factor could have an initial sorting effect in the development of mesencephalic structures.

Galactosamine is quickly metabolized to galactosamine 1-phosphate in rats treated with this compound. An HPLC method to quantify hexosamine phosphates in biological samples is described, modified from the o-phthaldialdehyde amino acid analysis procedure. o-Phthaldialdehyde derivatives of hexosamines and hexosamine-phosphates can be eluted from a reverse-phase column at different retention times, with a total analysis time of 30 min and without overlapping with free amino acids at physiological concentrations. The standard curves are linear between 1 and 40 nmol. This simple method is more selective and sensitive than previous enzymatic analyses of hexosamine phosphorylation.

The density and distribution of dopamine D1 and D2 receptors visualized by in vitro autoradiography were investigated in adult and senescent BL C57 mice. A significant decrease was observed in regions of the basal ganglia of senescent animals, which was more pronounced for the D1 subtype. Chronic treatment with cinnarizine, an organic Ca2+ channel antagonist, alters both D1 and D2 receptor densities, with a higher sensitivity of the Dl subtype. These results could indicate that the interactions between dopamine receptor subtypes may be necessary for the full expression of behavioral events mediated by the D2 receptors.

Se presenta el programa educativo �Explora Guernica�, del Museo Nacional Centro de Arte Reina Sofia, de Madrid, una iniciativa innovadora en nuestro pais para favorecer el acceso de las personas con discapacidad visual a los museos mediante la combinacion de informaciones verbales, la exploracion de diagramas tactiles y la realizacion de talleres de creacion. En el diseno de la actividad han primado una concepcion plurisensorial que amplia y potencia el acercamiento a las obras de arte y el convencimiento de que estas deben ser accesibles a todos, en tanto que sus valores sobrepasan con mucho la mera percepcion visual. Ademas, �Explora Guernica� permite la participacion conjunta de personas con y sin discapacidad visual, favoreciendo la integracion y la visibilidad. En el articulo se exponen los retos que se afrontaron durante la programacion de la actividad, el desarrollo de las sesiones educativas, y los recursos didacticos empleados durante las mismas. Para concluir, se incluye una primera valoracion de la experiencia y de la participacion

Bone morphogenetic proteins (BMPs) are a family of growth differentiation factors which induce bone formation from mesenchymal cells. These proteins are members of the transforming growth factor-β superfamily. The expression of BMPs in the nervous system as well as in other tissues has been reported. In this study, we show that the presence of BMP-2 resulted in a dose-dependent increase in the number of tyrosine hydroxylase-immunoreactive ventral mesencephalic cells after 7 days in serum-free medium cultures. A maximal response was elicited at 10 ng/mL. BMP-2 also increased the number of primary neurites and branch points as well as the length of the longest neurite in a dose-dependent manner, with a maximal effect at 1 ng/mL. In contrast, BMP-2 did not modify the number or the function of GABAergic neurons. On the other hand, we observed stimulation of proliferation and morphological changes in glial cells (astrocytes become more fibrous shaped) in the presence of a high BMP-2 concentration (100 ng/mL), but not with lower doses, suggesting that the neurotrophic effect in dopaminergic neurons is not mediated by astroglial cells. This is consistent with the fact that the BMP-2 effect on dopaminergic neurons was observed even when the cultures were treated with α-aminoadipic acid to exclude the presence of glial cells. In summary, our data indicate that BMP-2 is a potent neurotrophic factor for ventral mesencephalic dopaminergic cells in culture. © 1999 John Wiley & Sons, Inc. J Neurobiol 38: 161–170, 1999

ABSTRACT Lysosomes, as primary degradative organelles, are the end-point of different converging pathways including macroautophagy. To date, lysosome function has mainly focused on interphase cells, while their role during mitosis remains controversial. Mitosis dictates the faithful transmission of genetic material among generations, and perturbations of mitotic division lead to chromosomal instability, a hallmark of cancer. Heretofore, correct mitotic progression relies on the orchestrated degradation of mitotic factors, which was mainly attributed to ubiquitin-triggered proteasome-dependent degradation. Here, we show that mitotic transition does not only rely on proteasome-dependent degradation, as impairment of lysosomes increases mitotic timing and leads to mitotic errors, thus promoting chromosomal instability. Furthermore, we identified several putative lysosomal targets in mitotic cells. Among them, WAPL, a cohesin regulatory protein, emerged as a novel p62-interacting protein for targeted lysosomal degradation. Finally, we characterized an atypical nuclear phenotype, the toroidal nucleus, as a novel biomarker for genotoxic screenings. Our results establish lysosome-dependent degradation as an essential event to prevent genomic instability.

Cancer cells rely on mTORC1 activity to coordinate mitogenic signaling with nutrients availability for growth. Based on the metabolic function of E2F1, we hypothesize that glucose catabolism driven by E2F1 could participate on mTORC1 activation. Here, we demonstrate that glucose potentiates E2F1-induced mTORC1 activation by promoting mTORC1 translocation to lysosomes, a process that occurs independently of AMPK activation. E2F1 regulates glucose metabolism by increasing anaerobic glycolysis and identified the key regulatory enzyme, PFKFB3, as E2F1 target gene responsible for mTORC1 activation. PFKFB3 and PFK1 were found associated to lysosomes and we demonstrated that modulation of PFKFB3 activity, either by subtract accessibility or gene expression, regulates the translocation of mTORC1 to lysosomes by direct interaction with Rag B and further mTORC1 activity. These results support a model where a glycolytic metabolon containing phosphofructokinases is present in the lysosome and acts as a sensor platform for glucose catabolism towards mTORC1 activity.

Lysosomes, as primary degradative organelles, are the endpoint of different converging pathways, including macroautophagy. To date, lysosome degradative function has been mainly studied in interphase cells, while their role during mitosis remains controversial. Mitosis dictates the faithful transmission of genetic material among generations, and perturbations of mitotic division lead to chromosomal instability, a hallmark of cancer. Heretofore, correct mitotic progression relies on the orchestrated degradation of mitotic factors, which was mainly attributed to ubiquitin-triggered proteasome-dependent degradation. Here, we show that mitotic transition also relies on lysosome-dependent degradation, as impairment of lysosomes increases mitotic timing and leads to mitotic errors, thus promoting chromosomal instability. Furthermore, we identified several putative lysosomal targets in mitotic cells. Among them, WAPL, a cohesin regulatory protein, emerged as a novel SQSTM1-interacting protein for targeted lysosomal degradation. Finally, we characterized an atypical nuclear phenotype, the toroidal nucleus, as a novel biomarker for genotoxic screenings. Our results establish lysosome-dependent degradation as an essential event to prevent chromosomal instability. <b>Abbreviations:</b> 3D: three-dimensional; APC/C: anaphase-promoting complex; ARL8B: ADP ribosylation factor like GTPase 8B; ATG: autophagy-related; BORC: BLOC-one-related complex; CDK: cyclin-dependent kinase; CENPE: centromere protein E; CIN: chromosomal instability; ConcA: concanamycin A; CQ: chloroquine; DAPI: 4,6-diamidino-2-penylinole; FTI: farnesyltransferase inhibitors; GFP: green fluorescent protein; H2B: histone 2B; KIF: kinesin family member; LAMP2: lysosomal associated membrane protein 2; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MEF: mouse embryonic fibroblast; MTOR: mechanistic target of rapamycin kinase; PDS5B: PDS5 cohesin associated factor B; SAC: spindle assembly checkpoint; PLEKHM2: pleckstrin homology and RUN domain containing M2; SQSTM1: sequestosome 1; TEM: transmission electron microscopy; ULK1: unc-51 like autophagy activating kinase 1; UPS: ubiquitin-proteasome system; v-ATPase: vacuolar-type H⁺-translocating ATPase; WAPL: WAPL cohesion release factor.

Differences in purine metabolism produced by three preservation solutions were studied by determining the adenine nucleotide (ATP, ADP, AMP, and IMP) and nucleoside (adenosine, inosine, and hypoxanthine) levels in human kidney cortical biopsies. Forty kidney allografts were studied using University of Wisconsin (UW) solution (n = 20), Euro-Collins (EC) solution (n = 12), and modified EC solution with mannitol (M; n = 8). No significant differences were found between the three solutions studied with regard to ATP, ADP, or AMP changes. The mean ATP level (nmol/mg prot +/- SEM) at the end of preservation in the UW group was 2.7 +/- 0.3 nmol/mg, in the EC group 3.8 +/- 0.7 nmol/mg, and in the M group 2.3 +/- 0.4 nmol/mg. ATP 30 min after reperfusion in the UW, EC, and M groups was 5.7 +/- 0.8 nmol/mg, 6.4 +/- 1.0 nmol/mg, and 4.6 +/- 0.5 nmol/mg, respectively. However, an important difference appeared in the catabolic products determined. Kidneys perfused with UW solution had a significantly higher level of adenosine (2.6 +/- 0.6 nmol/mg), inosine (11.8 +/- 2.2 nmol/mg), and hypoxanthine (18.1 +/- 2.1 nmol/mg) at the end of cold storage than those perfused with EC (0.4 +/- 0.1 nmol/mg, 2.0 +/- 0.8 nmol/mg, and 7.1 +/- 1.4 nmol/mg) and M solutions (0.2 +/- 0.05 nmol/mg, 0.5 +/- 0.1 nmol/mg, and 5.2 +/- 0.6 nmol/mg; P < 0.05). These levels returned to initial values 30 min postreperfusion and there were no differences with the EC or M solution groups at that time.(ABSTRACT TRUNCATED AT 250 WORDS)