Clonal PC12 Research Articles

Short-term incubation of gabapentin or pregabalin does not affect chemically induced injury in neuronal cell models in vitro.

PurposeGabapentinoids are currently the mainstay of pharmacological treatments for patients with neuropathic pain. Little is known about the effects of this therapy on the integrity of neuronal networks, especially in patients with an already-damaged nervous system. Since gabapentinoids can worsen cognitive functions and recent studies have shown alterations in the brains of patients with neuropathic pain, it may be possible that these drugs have neurotoxic effects.MethodsRat clonal PC12 pheochromocytoma (autonomic) and primary sensory dorsal-root ganglion (DRG) neurons from newborn Wistar rats were employed for this study. To mimic neuronal damage, cells were exposed to cytotoxins using either hydrogen peroxide (H2O2) or vincristine.ResultsNo direct cytotoxic effects were observed after incubating PC12 cells for 24 hours with increasing concentrations of gabapentin or pregabalin using MTT cytotoxicity assays. Even a 7-day incubation did not cause cellular damage. Furthermore, in preinjured PC12 and DRG neurons, neither gabapentin nor pregabalin prevented or enhanced the cytotoxic effects of H2O2 or vincristine after incubation for 24 hours and 7 days, respectively. Cell morphology and integrity of the cytoskeleton assessed by employing immunostaining of cytoskeletal proteins (α-tubulin, neurofilament L) remained intact and were not altered by gabapentinoids.ConclusionBased on these results, gabapentinoids are unlikely to be neurotoxic in cultured autonomic (PC12) and sensory DRG cells, even when cells are preinjured. These results are of high clinical relevance, as it seems unlikely that the morphological changes recently observed in the brains of neuropathic pain patients are caused or worsened by gabapentinoids.

REST-Governed Gene Expression Profiling in a Neuronal Cell Model Reveals Novel Direct and Indirect Processes of Repression and Up-Regulation.

The role of REST changes in neurons, including the rapid decrease of its level during differentiation and its fluctuations during many mature functions and diseases, is well established. However, identification of many thousand possible REST-target genes, mostly based on indirect criteria, and demonstration of their operative dependence on the repressor have been established for only a relatively small fraction. In the present study, starting from our recently published work, we have expanded the identification of REST-dependent genes, investigated in two clones of the PC12 line, a recognized neuronal cell model, spontaneously expressing different levels of REST: very low as in neurons and much higher as in most non-neural cells. The molecular, structural and functional differences of the two PC12 clones were shown to depend largely on their different REST level and the ensuing variable expression of some dependent genes. Comprehensive RNA-Seq analyses of the 13,700 genes expressed, validated by parallel RT-PCR and western analyses of mRNAs and encoded proteins, identified in the high-REST clone two groups of almost 900 repressed and up-regulated genes. Repression is often due to direct binding of REST to target genes; up-regulation to indirect mechanism(s) mostly mediated by REST repression of repressive transcription factors. Most, but not all, genes governing neurosecretion, excitability, and receptor channel signaling were repressed in the high REST clone. The genes governing expression of non-channel receptors (G protein-coupled and others), although variably affected, were often up-regulated together with the genes of intracellular kinases, small G proteins, cytoskeleton, cell adhesion, and extracellular matrix proteins. Expression of REST-dependent genes governing functions other than those mentioned so far were also identified. The results obtained by the parallel investigation of the two PC12 clones revealed the complexity of the REST molecular and functional role, deciphering new aspects of its participation in neuronal functions. The new findings could be relevant for further investigation and interpretation of physiological processes typical of neurons. Moreover, they could be employed as tools in the study of neuronal diseases recently shown to depend on REST for their development.

Epigenomics of Neural Cells: REST-Induced Down- and Upregulation of Gene Expression in a Two-Clone PC12 Cell Model.

Cell epigenomics depends on the marks released by transcription factors operating via the assembly of complexes that induce focal changes of DNA and histone structure. Among these factors is REST, a repressor that, via its strong decrease, governs both neuronal and neural cell differentiation and specificity. REST operation on thousands of possible genes can occur directly or via indirect mechanisms including repression of other factors. In previous studies of gene down- and upregulation, processes had been only partially investigated in neural cells. PC12 are well-known neural cells sharing properties with neurons. In the widely used PC12 populations, low-REST cells coexist with few, spontaneous high-REST PC12 cells. High- and low-REST PC12 clones were employed to investigate the role and the mechanisms of the repressor action. Among 15,500 expressed genes we identified 1,770 target and nontarget, REST-dependent genes. Functionally, these genes were found to operate in many pathways, from synaptic function to extracellular matrix. Mechanistically, downregulated genes were predominantly repressed directly by REST; upregulated genes were mostly governed indirectly. Among other factors, Polycomb complexes cooperated with REST for downregulation, and Smad3 and Myod1 participated in upregulation. In conclusion, we have highlighted that PC12 clones are a useful model to investigate REST, opening opportunities to development of epigenomic investigation.

NGF signaling in PC12 cells: the cooperation of p75NTR with TrkA is needed for the activation of both mTORC2 and the PI3K signalling cascade

SummaryPC12-27, a PC12 clone characterized by high levels of the transcription repressor REST and by very low mTORC2 activity, had been shown to be unresponsive to NGF, possibly because of its lack of the specific TrkA receptor. The neurotrophin receptor repressed by high REST in PC12-27 cells, however, is shown now to be not TrkA, which is normal, but p75NTR, whose expression is inhibited at the transcriptional level. When treated with NGF, the PC12-27 cells lacking p75NTR exhibited a defective TrkA autophosphorylation restricted, however, to the TrkA(Y490) site, and an impairment of the PI3K signaling cascade. This defect was sustained in part by a mTORC1-dependent feed-back inhibition that in wtPC12 cells appeared marginal. Transfection of p75NTR to a level and surface distribution analogous to wtPC12 did not modify various high REST-dependent properties of PC12-27 cells such as high β-catenin, low TSC2 and high proliferation rate. In contrast, the defective PI3K signaling cascade and its associated mTORC2 activity were largely rescued together with the NGF-induced neurite outgrowth response. These changes were not due to p75NTR alone but required its cooperation with TrkA. Our results demonstrate that, in PC12, high REST induces alterations of NGF signaling which, however, are indirect, dependent on the repression of p75NTR; and that the well-known potentiation by p75NTR of the TrkA signaling does not concern all the effects induced by NGF but primarily the PI3K cascade and its associated mTORC2, a complex known to play an important role in neural cell differentiation.

Cellular overexpression of Aquaporins slows down the natural HIF-2α degradation during prolonged hypoxia

Overexpression of cell membrane aquaporins (AQPs) has recently been associated with tumor formation, particularly with angiogenesis, cell migration and proliferation. Additionally, the hypoxia inducible factor (HIF) family has been extensively implicated in tumor growth and recent studies evidence interplay between AQP expression and HIF stability. Therefore, we decided to explore the effect that AQP overexpression has on the long-term stability of HIF-2α in PC12 cells exposed to chronic hypoxia, characteristic of a growing tumor. HIF-2α levels were analyzed in five PC12 clones with stable overexpression of different proteins (AQP1, AQP3, AQP5, G6PD, and GDNF), in PC12 transiently expressing G6PD or Kv4.2, and in wild-type PC12 cells. Overexpression of AQP1, 3 or 5 in PC12 cells (o-AQP-c) prevented the HIF-2α down-expression otherwise observed, after 16h at 1% O2, in wt-PC12 and in PC12 overexpressing non-AQP proteins. Longer HIF-2α stability was also observed in o-AQP-c exposed to cobalt chloride or dimethyloxallyl glycine. Normal proteasome activity was confirmed in all clones analyzed. Levels of HIF target genes (PHD2 and 3, VEGF, and PGK1) were 2–4 fold higher in hypoxic o-AQP-c than in wt-PC12 cells, and morphological changes in colony shape together with higher cell proliferation rates were observed in all o-AQP-c. Interestingly, analysis of PHD levels under normoxia revealed lower (50%) PHD3 expression in o-AQP-c than in controls. Our results indicate that AQP overexpression in PC12 cells prolongs HIF-2α stability during chronic hypoxia, leading to higher level of induction of its target genes and likely conferring to these cells a more tumor-like phenotype.

L1CAM and its cell‐surface mutants: new mechanisms and effects relevant to the physiology and pathology of neural cells

The L1 syndrome, a genetic disease that affects 1/30 000 newborn males, is sustained by numerous missense mutations of L1 cell adhesion molecule (L1CAM), an adhesion surface protein active also in transmembrane signaling, essential for the development and function of neurons. To investigate the cell biology of L1CAM, we employed a high RE1-silencing transcription (factor) clone of the pheochromocytoma PC12 line, defective in L1CAM expression and neurite outgrowth. The clone was transfected with wild-type L1CAM and four missense, disease-inducing point mutants encoding proteins distributed to the cell surface. The mutant-expressing cells, defective in adhesion to extracellular matrix proteins and in migration, exhibited unchanged proliferation. The nerve growth factor (NGF)-induced neurite outgrowth was re-established in defective clone cells transfected with the wild-type and the H210Q and I219T L1CAMs mutants, but not in the others. The stimulated outgrowth was confirmed in a second defective PC12 clone over-expressing the NGF receptor TrkA, treated with NGF and/or a recombinant L1CAM chimera. These results revealed a new function of L1CAM, a positive, robust and dose-dependent modulation of the TrkA receptor activated spontaneously or by NGF. The variable effects observed with the different L1CAM mutants suggest that this function contributes to the marked heterogeneity of symptoms and severity observed in the patients affected by the L1 syndrome.

Outgrowth of neurites is a dual process

In neurons and neurosecretory (nerve) cells, neurite outgrowth requires the enlargement of the plasma membrane sustained by the exocytosis of specific vesicles. The well known, slow canonical form of outgrowth induced in pheochromocytoma PC12 cells by NGF, as well as the outgrowth taking place in neurons, involve vesicles positive for the vSNARE Ti-VAMP. Working in defective PC12 clones expressing high levels of the transcriptional repressor REST, we have identified now a new, rapid form of outgrowth, triggered by activation of a small GTPase, Rac1. This form is sustained by the exocytosis of another type of vesicles, taking place locally at the tip of neurite growth cones, the enlargeosomes (vSNARE: VAMP4). This new form, which is positively controlled by REST, requires the dynamics of microtubules, but not of microfilaments. Its signalling remains undefined because established second messengers, (Ca2+, DAG, cAMP) seem not involved. Using a high REST/enlargeosome-rich PC12 clone transfected with TrkA we have found that the NGF-induced outgrowth is not always slow, but can be fast in cells expressing high levels of the receptor involved, TrkA; that PC12 can express together the two distinct forms of outgrowth, canonical and new, activated independently from each other. Their comparative characterization in terms of changes in the cytoskeleton has now been initiated. The two forms are present also in neurons where the new one seems to predominate in the initial phases of development, the canonical one later on. Our results identify a new aspect of the REST impact in nerve cell specificity/function. The existence of two distinct forms of neurite outgrowth may cope better than a single form with the variable needs of nerve cells in the subsequent stages of their development.

A New Form of Neurite Outgrowth Sustained by the Exocytosis of Enlargeosomes Expressed under the Control of REST

In neurons and neurosecretory (nerve) cells, neurite outgrowth requires surface enlargement sustained by exocytosis of specific but poorly characterized vesicles. A canonical, relatively slow form of outgrowth is known to require the v-SNARE Ti-VAMP. Recently, we have identified a new, rapid form, triggered by activation of Rac1 and sustained by the exocytosis of enlargeosomes (v-SNARE: VAMP4). By parallel study of various pheochromocytoma PC12 cell clones exhibiting either a single or both forms of outgrowth, we show that expression of enlargeosomes, their exocytosis at growth cones and their form of neurite outgrowth are positively governed by the RE-1 silencing transcription factor (REST), a repressor of many nerve cell-specific genes. Using a high REST/enlargeosome-rich PC12 clone transfected with TrkA, we found (i) that nerve growth factor (NGF) can increase the expression of both REST and the enlargeosome maker, Ahnak; and (ii) that outgrowth triggered by NGF, independent from the form triggered by Rac1 and supported mostly by exocytic, Ti-VAMP-positive organelles distinct from enlargeosomes, occurs at slow or fast rates depending on the strength of the TrkA signaling. These results confirm the duality of the outgrowth forms sustained by the two types of exocytic vesicles, reveal their distinct properties and identify new aspects of the REST impact in nerve cell specificity/function.

Lack of effects of typical and atypical antipsychotics in DARPP-32 and NCS-1 levels in PC12 cells overexpressing NCS-1

BackgroundSchizophrenia is the major psychiatry disorder, which the exact cause remains unknown. However, it is well known that dopamine-mediated neurotransmission imbalance is associated with this pathology and the main target of antipsychotics is the dopamine receptor D2. Recently, it was described alteration in levels of two dopamine signaling related proteins in schizophrenic prefrontal cortex (PFC): Neuronal Calcium Sensor-1 (NCS-1) and DARPP-32. NCS-1, which is upregulated in PFC of schizophrenics, inhibits D2 internalization. DARPP-32, which is decreased in PFC of schizophrenics, is a key downstream effector in transducing dopamine signaling. We previously demonstrated that antipsychotics do not change levels of both proteins in rat's brain. However, since NCS-1 and DARPP-32 levels are not altered in wild type rats, we treated wild type PC12 cells (PC12 WT) and PC12 cells stably overexpressing NCS-1 (PC12 Clone) with antipsychotics to investigate if NCS-1 upregulation modulates DARPP-32 expression in response to antipsychotics treatment.ResultsWe chronically treated both PC12 WT and PC12 Clone cells with typical (Haloperidol) or atypical (Clozapine and Risperidone) antipsychotics for 14 days. Using western blot technique we observed that there is no change in NCS-1 and DARPP-32 protein levels in both PC12 WT and PC12 Clone cells after typical and atypical antipsychotic treatments.ConclusionsBecause we observed no alteration in NCS-1 and DARPP-32 levels in both PC12 WT and Clone cells treated with typical or atypical antipsychotics, we suggest that the alteration in levels of both proteins in schizophrenic's PFC is related to psychopathology but not with antipsychotic treatment.

Differential repression by the transcription factor REST/NRSF of the various Ca2+ signalling mechanisms in pheochromocytoma PC12 cells

Expression of the nerve cell phenotype is orchestrated by the REST/NRSF transcription repressor, working on hundreds of genes recognized at a specific regulatory binding sequence. Most PC12 clones, the most frequently employed neuronal model, maintain low levels of REST; however a few, defective of neurosecretion, express high levels. To investigate the role of REST in Ca2+ signalling we studied the [Ca2+]i changes in single cells of four clones, two wild-type and two defective, pre-treated for 5 days with NGF. We focused on Ca2+ influxes induced by depolarization and ATP. Only a subpopulation (∼15%) of the defective, high REST cells responded to depolarization (CaV expression ∼10%). The ATP-induced intracellular Ca2+ release was little changed, whereas influx via ionotropic P2X receptors was decreased, in agreement with the decreased expression of P2X2 receptors. The percentage of defective cells expressing store-operated calcium entry (SOCE) following ATP stimulation was also lower. The responses of the defective clones were little affected by their differentiated state. In conclusion, our results revealed important new aspects of REST control of Ca2+ homeostasis, of potential physiological importance. The mechanisms of this control remain to be investigated.

Expression of Dense‐core Vesicles and of Their Exocytosis Are Governed by the Repressive Transcription Factor NRSF/REST

The mechanism by which neurons and neurosecretory cells govern the expression and the exocytic discharge of their clear and dense-core vesicles had remained unclear until recently when studies in the neurosecretory cell model PC12 revealed these processes to be orchestrated by the transcriptional repressor neuron restrictive silencer factor (NRSF)/repressor element-1 silencing transcription factor (REST). In wild-type PC12 fully competent for neurosecretion, NRSF/REST is low. The genes of the proteins involved in neurosecretion [from the secretory to vesicle membrane and plasma membrane proteins, including the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) of exocytosis] were all repressed by increases of NRSF/REST expression to various extents when the increase was only a fewfold but were completely or almost completely repressed when the increase was large, as in spontaneously defective PC12 clones. In the first case the dense-core vesicles were still competent for exocytosis but were smaller and less dense than in wild-type cells; in the second they were no longer visible but did reappear when the repression was attenuated by transfection of a dominant-negative construct of NRSF/REST combined with a secretory chromogranin or strengthened by treatment with a blocker of NRSF/REST-associated enzymes, the histone deacetylases.

Different Outcomes of Unliganded and Liganded Estrogen Receptor-α on Neurite Outgrowth in PC12 Cells

A precise description of the mechanisms by which estrogen receptor-alpha (ERalpha) exerts its influences on cellular growth and differentiation is still pending. Here, we report that the differentiation of PC12 cells is profoundly affected by ERalpha. Importantly, depending upon its binding to 17beta-estradiol (17betaE2), ERalpha is found to exert different effects on pathways involved in nerve growth factor (NGF) signaling. Indeed, upon its stable expression in PC12 cells, unliganded ERalpha is able to partially inhibit the neurite outgrowth induced by NGF. This process involves a repression of MAPK and phosphatidylinositol 3-kinase/Akt signaling pathways, which leads to a negative regulation of markers of neuronal differentiation such as VGF and NFLc. This repressive action of unliganded ERalpha is mediated by its D domain and does not involve its transactivation and DNA-binding domains, thereby suggesting that direct transcriptional activity of ERalpha is not required. In contrast with this repressive action occurring in the absence of 17betaE2, the expression of ERalpha in PC12 cells allows 17betaE2 to potentiate the NGF-induced neurite outgrowth. Importantly, 17betaE2 has no impact on NGF-induced activity of MAPK and Akt signaling pathways. The mechanisms engaged by liganded ERalpha are thus unlikely to rely on an antagonism of the inhibition mediated by the unliganded ERalpha. Furthermore, 17betaE2 enhances NGF-induced response of VGF and NFLc neuronal markers in PC12 clones expressing ERalpha. This stimulatory effect of 17betaE2 requires the transactivation functions of ERalpha and its D domain, suggesting that an estrogen-responsive element-independent transcriptional mechanism is potentially relevant for the neuritogenic properties of 17betaE2 in ERalpha-expressing PC12 cells.

Expression of the neurosecretory process in pc12 cells is governed by rest

The neurosecretory process is acquired during differentiation and can be lost en block by differentiated cells. To investigate the role of REST/NRSF, a transcription repressor, in the maintenance of the process we studied two PC12 clones, one wt and one defective, expressing low and high levels of endogenous RE-1 silencing transcription (factor) (REST), respectively. Stable transfection of constructs demonstrated that REST represses 10 genes coding for proteins of neurosecretory vesicles and their exocytosis, eight including and two lacking the REST-binding sequence, RE-1. Of these genes, those of chromogranins were strongly repressed by fewfold increases of REST, those of VAMP2 and syntaxin1a required much higher levels. Moreover, in wt cells transfected with an active construct the dense-core vesicles, still competent for regulated exocytosis, were much smaller, with lighter cores; in defective cells, the dominant-negative construct induced the rescue of many vesicle/exocytosis genes but not of those of chromogranins. Small dense-core vesicles, exocytized upon stimulation, were rescued when the construct-transfected defective cells were transfected also with chromograninA or treated with trichostatinA, a blocker of histone deacetylases. Our results identify REST, working by direct and indirect mechanisms, as the factor governing the maintenance of the neurosecretory process and the properties of dense-core vesicles in PC12 cells.

Dexamethasone-Induced Down-Regulation of Nerve Growth Factor Receptor p75NTR is Mediated by Glucocorticoid Type II Receptor in PC12 Cell Model

PC12 clones are established neuronal models to investigate mechanisms involved in the cross-talk between the neurotrophins and drugs. Chronic treatment of PC12 cells with the glucocorticoid agonist drug, dexamethasone (Dex), elicited a 50% decrease in the selective binding of 125 I-NGF along with a reduction in the NGF receptor p75 NTR mRNA and protein levels, suggesting a transcriptional mechanism. This down regulation of p75 NTR was antagonized by the glu- cocorticoid type II receptor (GR-2), RU-38486 but not by the minerallocorticoid receptor, RU-28318, antagonists. This process was associated with increased autophosphorylation of the NGF receptor, TrkA. Chronic treatment of PC12 with Dex abolished the NGF-induced proliferation of the cells after 20 hours and inhibited by 45% the neurite elongation after 96 hours. RU-38486 blocked Dex-induced shift of PC12 cells from dopaminergic to noradrenergic phenotype. Dex- induced down regulation of p75 NTR receptor is mediated by GR-2 and is correlated with disruption of NGF induced prolif-

Myotonic dystrophy CTG repeat expansion alters Ca 2+ channel functional expression in PC12 cells

We previously reported that expression of myotonic dystrophy (DM1) expanded CUG repeats impedes NGF-induced differentiation in a PC12 clone (CTG90 cells). Here, we present evidence for changes in the fractional contribution of distinct voltage-gated Ca 2+ channels, key elements in neurotrophin-promoted differentiation, to the total Ca 2+ current in the CTG90 cells. Patch-clamp recordings showed that the relative proportion of pharmacologically isolated Ca 2+ channel types differed between control and CTG90 cells. Particularly, the functional expression of N-type channels was significantly reduced. Though quantitative real-time RT-PCR revealed that transcripts for the pore-forming subunit encoding the N-type channels remained unchanged, the protein level analyzed by semi-quantitative Western blotting was down-regulated in the CTG90 cells. These data suggest modifications in the processing of N-type Ca 2+ channels in PC12 cells expressing the DM1 mutation.

Enlargeosome Traffic: Exocytosis Triggered by Various Signals Is Followed by Endocytosis, Membrane Shedding or Both

Enlargeosomes are cytoplasmic organelles discharged by regulated exocytosis, identified by immunofluorescence of their membrane marker, desmoyokin/Ahnak, but never revealed at the ultrastructural level. Among the numerous enlargeosome-positive cells, the richest and most extensively characterized are those of a PC12 clone, PC12-27, defective of classical neurosecretion. By using ultrastructural immunoperoxidase labeling of formaldehyde-fixed, Triton-X-100-permeabilized PC12-27 cells, we have now identified the enlargeosomes as small vesicles scattered in the proximity of, but never docked to, the plasma membrane. Upon stimulation, these vesicles undergo exocytosis [rapid after the Ca(2+) ionophore, ionomycin, much slower after either the phorbol ester, phorbol myristate acetate (PMA), or ATP, working through a P2Y receptor], with appearance in the plasma membrane of typical desmoyokin/Ahnak (d/A)-positive, Omega-shaped and open profiles evolving into flat patches. Postexocytic removal of the exocytized d/A-positive membrane occurs by two processes: generation of endocytic vesicles, predominant after ionomycin and ATP 100-500 microM; and shedding of membrane-bound cytoplasmic bodies, predominant after PMA and 1 mM ATP, containing little or no trace of endoplasmic reticulum, Golgi, endo/lysosomes and also of a plasma membrane marker. Depending on the stimulation, therefore, the cell-surface expansion by enlargeosome exocytosis is not always recycled but can induce release of specific membranes, possibly important in the pericellular environment.

Bex1, a novel interactor of the p75 neurotrophin receptor, links neurotrophin signaling to the cell cycle

A screening for intracellular interactors of the p75 neurotrophin receptor (p75NTR) identified brain-expressed X-linked 1 (Bex1), a small adaptor-like protein of unknown function. Bex1 levels oscillated during the cell cycle, and preventing the normal cycling and downregulation of Bex1 in PC12 cells sustained cell proliferation under conditions of growth arrest, and inhibited neuronal differentiation in response to nerve growth factor (NGF). Neuronal differentiation of precursors isolated from the brain subventricular zone was also reduced by ectopic Bex1. In PC12 cells, Bex1 overexpression inhibited the induction of NF-kappaB activity by NGF without affecting activation of Erk1/2 and AKT, while Bex1 knockdown accelerated neuronal differentiation and potentiated NF-kappaB activity in response to NGF. Bex1 competed with RIP2 for binding to the p75NTR intracellular domain, and elevating RIP2 levels restored the ability of cells overexpressing Bex1 to differentiate in response to NGF. Together, these data establish Bex1 as a novel link between neurotrophin signaling, the cell cycle, and neuronal differentiation, and suggest that Bex1 may function by coordinating internal cellular states with the ability of cells to respond to external signals.

Induction of cellular prion protein gene expression by copper in neurons

Prion diseases are caused by the conformational transition of the native alpha-helical cellular prion protein (PrPC) into a beta-sheet pathogenic isoform. However, the normal physiological function of PrPC remains elusive. We report herein that copper induces PrPC expression in primary hippocampal and cortical neurons. PrPC induced by copper has a normal glycosylation pattern, is proteinase K-sensitive and reaches the cell surface attached by a glycosyl phosphatidylinositol anchor. Immunofluorescence analysis revealed that copper induces PrPC levels in the cell surface and in an intracellular compartment that we identified as the Golgi complex. In addition, copper induced the activity of a reporter vector driven by the rat PrPC gene (Prnp) promoter stably transfected into PC12 cells, whereas no effect was observed in glial C6 clones. Also cadmium, but not zinc or manganese, upregulated Prnp promoter activity in PC12 clones. Progressive deletions of the promoter revealed that the region essential for copper modulation contains a putative metal responsive element. Although electrophoretic mobility shift assay demonstrated nuclear protein binding to this element, supershift analysis showed that this is not a binding site for the metal responsive transcription factor-1 (MTF-1). The MTF-1-independent transcriptional activation of Prnp is supported by the lack of Prnp promoter activation by zinc. These findings demonstrate that Prnp expression is upregulated by copper in neuronal cells by an MTF-1-independent mechanism, and suggest a metal-specific modulation of Prnp in neurons.

Serial induction of mutations by ethylnitrosourea in PC12 cells: a new model for a phenotypical characterization of the neurotoxic response to 6-hydroxydopamine

Here we show that the serial generation of allelic mutations by treatment with the mutagen ethylnitrosourea (ENU) in PC12 cells provides a new model for the phenotypical dissection of biological properties. We tested this approach in the neurotoxic 6-OHDA model of Parkinson’s disease in PC12 cells which has been widely used as an in vitro model for the investigation of the molecular pathogenesis of neuronal cell death and for novel treatment approaches. ENU treatment at doses of 0.2 and 0.3 mg/ml for 1 h resulted in 35 and 25% surviving PC12 cells, respectively, which showed mutation frequencies of approximately 10 mutations per genome. Clones derived from single ENU treated PC12 cells showed marked differences in their resistance against 6-OHDA. The phenotypical analysis of resistant and sensitive clones showed a differential transcriptional regulation of multiple genes. The applicability of this approach could be demonstrated by the identification of the rat TM9SF1 gene coding for a transmembrane protein of the nonaspanin superfamily as a regulated gene in PC12 clones resistant against 6-OHDA. Our data demonstrate the suitability of this model for the investigation of the molecular pathogenesis of neurodegeneration and for high-throughput analysis, e.g. for drug discovery.

Dense-core granules: a specific hallmark of the neuronal/neurosecretory cell phenotype.

Expression of dense-core granules, a typical exocytic organelle, is widely believed to be controlled by coordinate gene expression mechanisms specific to neurones and neurosecretory cells. Recent studies in PC12 cells, however, have suggested the number of granules/cells depends on the levels of only one of their cargo proteins, chromogranin A, regulating the metabolism of the other proteins, and thus the composition of the organelles, by an on/off switch mechanism. In addition, transfection of chromogranin A was reported to induce appearance of dense-core granules in the non-neurosecretory fibroblasts of the CV-1 line. Here the role of chromogranin A has been reinvestigated using not the heterogeneous PC12 line but several clones isolated therefrom. In these clones, investigated as such or after transfection with chromogranin A antisense sequences, the ratio between chromogranin A and its secretory protein mate, chromogranin B, was not constant but highly and apparently randomly variable. Variability of the chromogranin A/chromogranin B ratio was seen by confocal immunofluorescence also among the cells of single clones and subclones and among the granules of single cells. Moreover, stable and transient transfections of chromogranin A in a PC12 clone characterised by a low number of dense-core granules (one fifth of the reference clone) failed to modify significantly the number of the organelles, despite the several-fold increase of the granin. Finally, in three types of non-neurosecretory cells (CV-1, adenocarcinoma TS/A and a clone of PC12 incompetent for secretion) the transfected chromogranin A accumulated mostly in the Golgi/transGolgi area and was released rapidly from resting cells (constitutive secretion) as revealed by both immunofluorescence during cycloheximide treatment and pulse-chase experiments. Only a minor fraction was sorted to discrete organelles that were not dense-core granules, but primarily lysosomes because they contained no chromogranin B, and were largely positive for the late endosomal-lysosomal markers, lamp1 and lamp3. Dense-core granules are therefore true hallmarks of neurones and neurosecretory cells. Their number/cell appears independent of chromogranin A and their composition does not appear to be constant; in particular, they exhibit considerable, and so far unexplained variability in the chromogranin A/chromogranin B ratio.