Reversible symptoms and clearance of mutant prion protein in an inducible model of a genetic prion disease in Drosophila melanogaster.

Prion diseases are progressive disorders that affect the central nervous system leading to memory loss, personality changes, ataxia and neurodegeneration. In humans, these disorders include Creutzfeldt-Jakob disease, kuru and Gerstmann-Straüssler-Scheinker (GSS) syndrome, the latter being a dominantly inherited prion disease associated with missense mutations in the gene that codes for the prion protein. The exact mechanism by which mutant prion proteins affect the central nervous system and cause neurological disease is not well understood. We have generated an inducible model of GSS disease in Drosophila melanogaster by temporally expressing a misfolded form of the murine prion protein in cholinergic neurons. Flies accumulating this mutant protein develop motor abnormalities which are associated with electrophysiological defects in cholinergic neurons. We find that, upon blocking the expression of the mutant protein, both behavioral and electrophysiological defects can be reversed. This represents the first case of reversibility reported in a model of genetic prion disease. Additionally, we observe that endogenous mechanisms exist within Drosophila that are capable of clearing the accumulated prion protein.

Adenovirus expressing an NPC1-GFP fusion gene corrects neuronal and nonneuronal defects associated with Niemann pick type C disease.

Niemann Pick type C (NPC) disease is an autosomal recessive disorder characterized by abnormal cholesterol metabolism and accumulation in lysosomal and endosomal compartments. Although peripheral organs are affected, the progressive neurodegeneration in the brain is typically most deleterious, leading to dystonia, ataxia, seizures, and premature death. Although the two genes underlying this disorder in humans and mouse models of the disease have been identified (NPC1 in 95% and NPC2/HE1 in 5% of human cases), their cellular roles have not Been fully defined, and there is currently no effective treatment for this disorder. To help address these issues, we constructed a recombinant adenovirus, Ad(NPC1-GFP), which contains a cDNA encoding a mouse NPC1 protein with a green fluorescent protein (GFP) fused to its C-terminus. Fluorescence microscopy and cholesterol trafficking assays demonstrate that the GFP-tagged NPC1 protein is functional and detectable in cells from different species (hamster, mouse, human) and of different types (ovary-derived cells, fibroblasts, astrocytes, neurons from peripheral and central nervous systems) in vitro. Combined with results from time-lapse microscopy and in vivo brain injections, our findings suggest that this adenovirus offers advantages for expressing NPC1 and analyzing its cellular localization, movement, functional properties, and beneficial effects in vitro and in vivo.

Regional and developmental expression of the Npc1 mRNA in the mouse brain.

Niemann-Pick type C (NP-C) disease is a fatal, autosomal recessive disorder of cholesterol metabolism that results in progressive central nervous system deterioration and premature death. Recently, a gene mutated in NP-C disease (NPC1) was identified in both human patients and in the npc(nih) mouse model. Although the function of the NPC1 gene is at present unknown, determining the pattern of its expression in the brain may facilitate identification of the mechanisms underlying the neuropathology of this disease and in identifying relevant targets for any potential therapeutic intervention. We have used in situ hybridization techniques to characterize the pattern of Npc1 mRNA expression in both the wild-type and the npc(nih) mutant mouse brain. In adult animals of both genotypes, the Npc1 mRNA was detected in the majority of neurons in nearly all regions, but at significantly higher levels in the cerebellum and in specific pontine nuclei. Analysis of Npc1 mRNA levels during development in the wild-type mouse indicated that this transcript was expressed in neurons as early as embryonic day 15 and that a significant region-specific pattern of expression was established by postnatal day 7. Our data suggest that whereas the NPC1 gene is widely expressed in neurons of the brain, the higher levels of expression in the cerebellum and pontine structures established by early postnatal ages may make these regions more susceptible to neuronal dysfunction in NP-C disease.

Embryonic striatal neurons from niemann-pick type C mice exhibit defects in cholesterol metabolism and neurotrophin responsiveness.

Niemann-Pick type C (NP-C) disease is a progressive and fatal neuropathological disorder previously characterized by abnormal cholesterol metabolism in peripheral tissues. Although a defective gene has been identified in both humans and the npc(nih) mouse model of NP-C disease, how this leads to abnormal neuronal function is unclear. Here we show that whereas embryonic striatal neurons from npc(nih) mice can take up low density lipoprotein-derived cholesterol, its subsequent hydrolysis and esterification are significantly reduced. Given the importance of cholesterol to a variety of signal transduction mechanisms, we assessed the effect of this abnormality on the ability of these neurons to respond to brain-derived neurotrophic factor (BDNF). In contrast to its effects on wild type neurons, BDNF failed to induce autophosphorylation of the TrkB receptor and to increase neurite outgrowth in npc(nih) neurons, despite expression of TrkB on the cell surface. The results suggest that abnormal cholesterol metabolism occurs in neurons in the brain during NP-C disease, even at embryonic stages of development prior to the onset of phenotypic symptoms. Moreover, this defect is associated with a lack of TrkB function and BDNF responsiveness, which may contribute to the loss of neuronal function observed in NP-C disease.

A novel synthetic oleanane triterpenoid, 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid, with potent differentiating, antiproliferative, and anti-inflammatory activity.

The new synthetic oleanane triterpenoid 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO) is a potent, multifunctional molecule. It induces monocytic differentiation of human myeloid leukemia cells and adipogenic differentiation of mouse 3T3-L1 fibroblasts and enhances the neuronal differentiation of rat PC12 pheochromocytoma cells caused by nerve growth factor. CDDO inhibits proliferation of many human tumor cell lines, including those derived from estrogen receptor-positive and -negative breast carcinomas, myeloid leukemias, and several carcinomas bearing a Smad4 mutation. Furthermore, it suppresses the abilities of various inflammatory cytokines, such as IFN-gamma, interleukin-1, and tumor necrosis factor-alpha, to induce de novo formation of the enzymes inducible nitric oxide synthase (iNos) and inducible cyclooxygenase (COX-2) in mouse peritoneal macrophages, rat brain microglia, and human colon fibroblasts. CDDO will also protect rat brain hippocampal neurons from cell death induced by beta-amyloid. The above activities have been found at concentrations ranging from 10(-6) to 10(-9) M in cell culture, and these results suggest that CDDO needs further study in vivo, for either chemoprevention or chemotherapy of malignancy as well as for neuroprotection.

Selective regulation of agrin mRNA induction and alternative splicing in PC12 cells by Ras-dependent actions of nerve growth factor.

The extracellular matrix protein agrin plays an important role in the formation and maintenance of the neuromuscular junction. However, regulation of agrin gene expression and pre-mRNA splicing, important in determining the biological actions of agrin, is not well understood. To begin to identify mechanisms controlling agrin expression, quantitative polymerase chain reaction techniques were used to analyze the effect of growth factors on the expression of agrin mRNA isoforms in rat pheochromocytoma (PC12) cells. Agrin transcripts in untreated cells lacked inserts in the Y and Z sites (agriny0z0), encoding agrin isoforms with low acetylcholine receptor aggregating activity and a primarily non-neuronal tissue distribution. Transcripts encoding isoforms with high aggregating activity and neuronal tissue distribution (agriny4z8, agriny4z11, and agriny4z19) were not detected. Treatment of PC12 cells with nerve growth factor (NGF) caused a significant increase in total agrin mRNA. In contrast, exposure to epidermal growth factor had no effect. Analysis of alternative splicing of agrin mRNA revealed that NGF elicited a specific increase in agriny4 and agrinz8 mRNAs that did not occur in the presence of epidermal growth factor, insulin, dexamethasone, or retinoic acid. Analysis of PC12 sublines stably overexpressing a dominant inhibitory form of p21 Ras indicated that NGF induced changes in levels of agrin mRNA and alternative splicing required Ras activity. The results show that NGF can influence important aspects of neuronal differentiation by regulating alternative splicing. Furthermore, these data provide insight into the mechanisms governing agrin gene expression and suggest that neurotrophic factors may play a role in regulating agrin expression in vivo.

Analysis of mutant platelet-derived growth factor receptors expressed in PC12 cells identifies signals governing sodium channel induction during neuronal differentiation.

The mechanisms governing neuronal differentiation, including the signals underlying the induction of voltage-dependent sodium (Na+) channel expression by neurotrophic factors, which occurs independent of Ras activity, are not well understood. Therefore, Na+ channel induction was analyzed in sublines of PC12 cells stably expressing platelet-derived growth factor (PDGF) beta receptors with mutations that eliminate activation of specific signalling molecules. Mutations eliminating activation of phosphatidylinositol 3-kinase (PI3K), phospholipase C gamma (PLC gamma), the GTPase-activating protein (GAP), and Syp phosphatase failed to diminish the induction of type II Na+ channel alpha-subunit mRNA and functional Na+ channel expression by PDGF, as determined by RNase protection assays and whole-cell patch clamp recording. However, mutation of juxtamembrane tyrosines that bind members of the Src family of kinases upon receptor activation inhibited the induction of functional Na+ channels while leaving the induction of type II alpha-subunit mRNA intact. Mutation of juxtamembrane tyrosines in combination with mutations eliminating activation of PI3K, PLC gamma, GAP, and Syp abolished the induction of type II alpha-subunit mRNA, suggesting that at least partially redundant signaling mechanisms mediate this induction. The differential effects of the receptor mutations on Na+ channel expression did not reflect global changes in receptor signaling capabilities, as in all of the mutant receptors analyzed, the induction of c-fos and transin mRNAs still occurred. The results reveal an important role for the Src family in the induction of Na+ channel expression and highlight the multiplicity and combinatorial nature of the signaling mechanisms governing neuronal differentiation.

GTPase-deficient G alpha 16 and G alpha q induce PC12 cell differentiation and persistent activation of cJun NH2-terminal kinases.

Persistent stimulation of specific protein kinase pathways has been proposed as a key feature of receptor tyrosine kinases and intracellular oncoproteins that signal neuronal differentiation of rat pheochromocytoma (PC12) cells. Among the protein serine/threonine kinases identified to date, the p42/44 mitogen-activated protein (MAP) kinases have been highlighted for their potential role in signalling PC12 cell differentiation. We report here that retrovirus-mediated expression of GTPase-deficient, constitutively active forms of the heterotrimeric Gq family members, G alpha qQ209L and G alpha 16Q212L, in PC12 cells induces neuronal differentiation as indicated by neurite outgrowth and the increased expression of voltage-dependent sodium channels. Differentiation was not observed after cellular expression of GTPase-deficient forms of alpha i2 or alpha 0, indicating selectivity for the Gq family of G proteins. As predicted, overexpression of alpha qQ209L and alpha 16Q212L constitutively elevated basal phospholipase C activity approximately 10-fold in PC12 cells. Significantly, little or no p42/44 MAP kinase activity was detected in PC12 cells differentiated with alpha 16Q212L or alpha qQ209L, although these proteins were strongly activated following expression of constitutively active cRaf-1. Rather, a persistent threefold activation of the cJun NH2-terminal kinases (JNKs) was observed in PC12 cells expressing alpha qQ209L and alpha 16Q212L. This level of JNK activation was similar to that achieved with nerve growth factor, a strong inducer of PC12 cell differentiation. Supportive of a role for JNK activation in PC12 cell differentiation, retrovirus-mediated overexpression of cJun, a JNK target, in PC12 cells induced neurite outgrowth. The results define a p42/44 MAP kinase-independent mechanism for differentiation of PC12 cells and suggest that persistent activation of the JNK members of the proline-directed protein kinase family by GTPase-deficient G alpha q and G alpha 16 subunits is sufficient to induce differentiation of PC12 cells.

Differential expression of sodium channels and nicotinic acetylcholine receptor channels in nnr variants of the PC12 pheochromocytoma cell line.

An important component of neuronal differentiation is the tightly controlled expression of a spectrum of ion channel proteins. Ion channels play a critical role in the generation and propagation of action potentials as well as in the cellular response to neurotransmitters, and thus are central in the transfer and integration of information in the nervous system. A model system amenable to analysis of ion channel expression and neuronal differentiation is the rat pheochromocytoma (PC12) cell line. Here, we have used electrophysiological and molecular biological approaches to analyze the expression of voltage-dependent sodium (Na) channels and nicotinic acetylcholine receptors (nAChR) in mutagenized variants (nnr cells) of the PC12 cell line. Our data reveal striking differences in the expression of these channels when compared to wild-type PC12 cells. Even in the absence of nerve growth factor (NGF), nnr cells express functional Na channels and Na channel mRNA at levels exceeding those in wild-type PC12 cells differentiated with NGF. In contrast, acetylcholine-induced currents were evident in only a small proportion of cells, presumably due to the altered pattern of expression of mRNAs encoding individual nAChR subunits. The altered ion channel expression in these variants provides an avenue for analyzing Na channel and nAChR channel function, as well as for identifying mechanisms governing their expression.

Differential regulation of neuronal sodium channel expression by endogenous and exogenous tyrosine kinase receptors expressed in rat pheochromocytoma cells.

The biological activity of growth factors that act through receptor tyrosine kinases (RTKs) can differ dramatically, depending on both the properties of the RTKs and the cellular environment in which the RTKs are expressed. To determine the ability of different RTKs to elicit ras-independent responses central to neuronal differentiation, we analyzed voltage-dependent sodium (Na) channel expression in rat pheochromocytoma (PC12) cells after activation of a variety of endogenously and exogenously expressed RTKs. In PC12 cells expressing trkB (Ip et al., 1993), the increase in Na current density caused by brain-derived neurotrophic factor (BDNF) was similar to that observed upon activation of endogenous trkA by NGF. BDNF also increased type II Na channel mRNA expression, as did neurotrophin-3 in PC12 cells expressing trkC (Tsoulfas et al., 1993). In contrast, insulin did not increase type II Na channel mRNA expression or Na current density in PC12 cells, while epidermal growth factor (EGF) elicited small, yet reproducible increases in type II Na channel mRNA and Na current density when compared to NGF, even upon coexpression of an EGF receptor/p75 receptor chimera (Yan et al., 1991). Finally, in PC12 cells expressing beta-platelet-derived growth factor (PDGF) receptors (Heasley and Johnson, 1992), PDGF increased type II Na channel mRNA and Na current density to the same extent as NGF. The results show the capabilities of these RTKs in eliciting Na channel expression and the specificity arising due to differences in their intrinsic properties.

Nerve growth factor increases nicotinic ACh receptor gene expression and current density in wild-type and protein kinase A-deficient PC12 cells.

Although neuronal nicotinic ACh receptors (nAChR) play a key role in synaptic transmission and information transfer in the nervous system, little is known about the molecular mechanisms that govern the expression of the multiple subunits that form the receptors and determine their functional properties. Using electrophysiological and molecular biological approaches, we have investigated the NGF-mediated regulation of nAChR expression in rat pheochromocytoma (PC12) cells and protein kinase A (PKA)-deficient PC12 cells. We report that NGF treatment increases steady state levels of mRNA encoding the alpha 3, alpha 5, alpha 7, beta 2, and beta 4 subunits, increases the occurrence of ACh-induced single-channel activity in excised patches, and increases ACh-induced macroscopic current density, all by mechanisms independent of PKA activity.

Suppression of sodium channel function in differentiating C2 muscle cells stably overexpressing rat androgen receptors.

Differentiation of skeletal muscle and the formation of the neuromuscular junction are regulated by steroid hormones. The effects of androgens on ion channel proteins central to neuromuscular signalling have been investigated in differentiating mouse muscle C2 cells and in C2 cells that stably overexpress the rat androgen receptor (AR) cDNA. Neither the expression nor function of ACh receptors was regulated by androgenic actions in these cells. However, voltage-dependent sodium (Na) current density was decreased by androgen treatment of C2 cells and was abolished, even in the absence of androgens, in C2 cells that overexpress the AR. The decrease in functional Na current was not accompanied by concomitant decreases in Na channel mRNA, suggesting that AR influence posttranscriptional processing of Na channels in differentiating C2 cells.

ras-independent induction of rat brain type II sodium channel expression in nerve growth factor-treated PC12 cells.

Nerve growth factor (NGF) plays an important role in the development of the nervous system, and there is considerable interest in understanding the molecular mechanisms underlying its effects on neuronal differentiation. To determine if the activity of proteins of the ras gene family is necessary for the NGF-mediated induction of sodium channel expression in pheochromocytoma (PC12) cells, sodium channel expression was analyzed in PC12 sublines stably overexpressing the dominant inhibitory mutant c-Ha-ras(Asn-17). Northern blot analysis, RNase protection assays, and whole-cell patch clamp recordings indicate that the NGF-mediated increase in type II sodium channel mRNA and sodium current density can occur independent of ras activity and by doing so provide strong evidence for the importance of ras-independent mechanisms in NGF-mediated neuronal differentiation.

The activity of cAMP-dependent protein kinase is required at a posttranslational level for induction of voltage-dependent sodium channels by peptide growth factors in PC12 cells.

The synthesis and expression of voltage-dependent sodium (Na) channels is a crucial aspect of neuronal differentiation because of the central role these ion channels play in the generation of action potentials and the transfer of information in the nervous system. We have used rat pheochromocytoma (PC12) cell lines deficient in cAMP-dependent protein kinase (PKA) activity to examine the role of PKA in the induction of Na channel expression by nerve growth factor (NGF) and basic FGF (bFGF). In the parental PC12 cell line both NGF and bFGF elicit an increase in the density of functional Na channels, as determined from whole-cell patch clamp recordings. This increase does not occur in two PC12 cell lines deficient in both isozymes of PKA (PKAI and PKAII), and is strongly reduced in a third line deficient in PKAII, but not PKAI. Despite the inability of the neurotrophic factors to induce functional Na channel expression in the PKA-deficient cells, Northern blot hybridization studies and saxitoxin binding assays of intact cells indicate that NGF and bFGF are still capable of eliciting increases in both Na channel mRNA and Na channel protein in the membrane. Thus, PKA activity appears to be necessary at a posttranslational step in the synthesis and expression of functional Na channels, and thereby plays an important role in determining neuronal excitability.

Neuron-specific expression of the rat brain type II sodium channel gene is directed by upstream regulatory elements.

Genetic elements involved in cell-specific expression of the type II sodium channel gene were revealed using transient expression assays. A chimeric reporter gene containing 1051 bp of the sodium channel 5' flanking region was active in neuroblastoma and PC12 cells, but inactive in nonneuronal cell types. Deletion of upstream sequences resulted in an 80-fold increase in reporter gene activity in skeletal muscle cells, suggesting the presence of negative elements. Although no homologies were found between sequences in the type II 5' flanking region and other negative elements or "silencers," a small region common to the type II gene and other genes expressed in the nervous system was identified and may be involved in transcriptional regulation of neuronal genes.

Fertilization events induced by neurotransmitters after injection of mRNA in Xenopus eggs.

Fertilization initiates in the egg a dramatic increase in intracellular calcium that opens ion channels and causes exocytosis. To explore the possibility that these events might involve a receptor-mediated pathway, receptors for serotonin or acetylcholine (M1 muscarinic) were expressed in the Xenopus egg; serotonin or acetylcholine then could initiate a series of responses similar to those normally initiated by sperm. Thus, there may be an endogenous receptor in the egg membrane that is activated by sperm, and the serotonin or M1 muscarinic receptor may replace the sperm receptor in this pathway.

Selective induction of brain type II Na+ channels by nerve growth factor.

Cells derived from a rat pheochromocytoma (PC12 cells) can generate an action potential only upon treatment with nerve growth factor. Using electrophysiological methods, we found that the appearance of action potentials in nerve growth factor-treated PC12 cells can be explained by an increase in the density of Na+ channels. The functional properties of Na+ channels in PC12 cells are similar to those described for peripheral nerves but appear to be different from Na+ channels synthesized in Xenopus oocytes injected with brain type II Na+ -channel mRNA. To determine if PC12 cells express the brain type II Na+ -channel gene, we performed RNase-protection analyses using probes that can distinguish between the brain type I and type II Na+ -channel mRNAs. The results from these studies indicate that undifferentiated PC12 cells express the type II but not the type I Na+ -channel gene. Treatment with nerve growth factor increases expression of the type II Na+ -channel gene but has no effect on type I gene expression. Our findings suggest that Na+ -channel excitability in PC12 cells is due to the specific induction of the brain type II gene by nerve growth factor.

Preparation of isolated mouse olfactory receptor neurons.

A method is described for producing large numbers of isolated olfactory receptor neurons from adult mouse nasal epithelium. The dissociated neurons and other cell types isolated from nasal epithelium retain their morphology and can be identified visually. The neurons were judged to be intact and viable by trypan blue dye exclusion, the presence of olfactory marker protein (OMP), and a variety of electrophysiological measurements indicating the presence of substantial membrane potentials, low levels of intracellular Ca2+, and the ability to fire action potentials. The receptor neurons and other cell types produced by this method are amenable to the patch-clamp technique and to immunohistochemical studies.

Patch-clamp studies of isolated mouse olfactory receptor neurons.

Olfactory receptor neurons isolated from embryonic, neonatal, and adult mice were studied using the patch-clamp technique. Several distinct types of ion channels were characterized in patches of membrane from the neuronal soma and the dendritic knob of receptor neurons, including a 130-pS Ca++-activated K+ channel with voltage-dependent kinetics, an 80-pS Ca++-activated K+ channel with voltage-insensitive kinetics, a 25-pS K+ channel with properties similar to inward rectifiers, and a 40-pS K+ channel that was activated and then inactivated by rapid depolarization. Evidence of large-conductance (greater than 200 pS) Cl- channels, which were Ca++ insensitive and increasingly active at depolarizing membrane potentials, and voltage-activated Ca++ channels (16 pS) was also obtained. From K+ channel activity recorded from cell-attached patches, the intracellular [Ca++] was inferred to be below 0.1 microM, and the membrane potential was inferred to be approximately -50 mV. The receptor neurons had high input resistances, and action potentials could be elicited by picoampere amounts of depolarizing current. The receptor neurons responded to applied odorant molecules and to forskolin with increases in membrane conductance. These results provide a description of the membrane properties of olfactory receptor neurons and a basis for understanding their electrical activity and response to odorants.