Cath.a-differentiated Research Articles

The role of microglial LRRK2 kinase in manganese-induced inflammatory neurotoxicity via NLRP3 inflammasome and RAB10-mediated autophagy dysfunction

Chronic manganese (Mn) exposure can lead to manganism, a neurological disorder sharing common symptoms with Parkinson's disease (PD). Studies have shown that Mn can increase the expression and activity of leucine-rich repeat kinase 2 (LRRK2), leading to inflammation and toxicity in microglia. LRRK2 G2019S mutation also elevates LRRK2 kinase activity. Thus, we tested if Mn-increased microglial LRRK2 kinase is responsible for Mn-induced toxicity, and exacerbated by G2019S mutation, using WT and LRRK2 G2019S knock-in mice and BV2 microglia. Mn (30mg/kg, nostril instillation, daily for 3weeks) caused motor deficits, cognitive impairments, and dopaminergic dysfunction in WT mice, which were exacerbated in G2019S mice. Mn induced proapoptotic Bax, NLRP3 inflammasome, IL-1β, and TNF-α in the striatum and midbrain of WT mice, and these effects were more pronounced in G2019S mice. BV2 microglia were transfected with human LRRK2 WT or G2019S, followed by Mn (250μM) exposure to better characterize its mechanistic action. Mn increased TNF-α, IL-1β, and NLRP3 inflammasome activation in BV2 cells expressing WT LRRK2, which was elevated further in G2019S-expressing cells, while pharmacological inhibition of LRRK2 mitigated these effects in both genotypes. Moreover, the media from Mn-treated G2019S-expressing BV2 microglia caused greater toxicity to the cath.a-differentiated (CAD) neuronal cells compared to media from microglia expressing WT. Mn-LRRK2 activated RAB10 which was exacerbated in G2019S. RAB10 played a critical role in LRRK2-mediated Mn toxicity by dysregulating the autophagy-lysosome pathway and NLRP3 inflammasome in microglia. Our novel findings suggest that microglial LRRK2 via RAB10 plays a critical role in Mn-induced neuroinflammation.

Scutellaria incarnata Vent. root extract and isolated phenylethanoid glycosides are neuroprotective against C2-ceramide toxicity.

Neuronal death is a central process in neurodegenerative diseases and represents a therapeutic challenge for their prevention and treatment. Scutellaria incarnata Vent. roots are used traditionally in Colombia for central nervous system conditions including those affecting cognitive functions, but their chemistry and neuroprotective action remain to be explored to understand the scientific basis for their medicinal uses. In this study, S. incarnata roots are investigated to assess whether they have neuroprotective effects that could provide some explanation for their traditional use in neurodegenerative diseases. To evaluate the neuroprotective effect of S. incarnata roots and its chemical constituents against C2-ceramide-induced cell death in Cath.-a-differentiated (CAD) cells. S. incarnata root ethanol extract was fractionated and compounds were isolated by column chromatography; their structures were elucidated by nuclear magnetic resonance spectroscopy, mass spectrometry and infrared spectroscopy. The cytotoxic and neuroprotective effects against C2-ceramide of S. incarnata root extract, fractions and isolated compounds were assessed in CAD cells. S. incarnata root extract and its n-butanol fraction were not cytotoxic but showed neuroprotective effects against C2-ceramide toxicity in CAD cells. The phenylethanoid glycosides incarnatoside (isolated for the first time) and stachysoside C (12.5, 25 and 50μg/mL) from S. incarnata roots also protected CAD cells against C2-ceramide without inducing cytotoxic effects. The observed neuroprotective effects of S. incarnata root extract and isolated phenylethanoid glycosides in CAD cells provide an ethnopharmacological basis for the traditional use of this species in Colombia for central nervous system disorders.

Anticancer and Neuroprotective Effects of the Triterpene Glycosides From Sea Cucumber Holothuria imitans

Introduction: Sea cucumbers has gained notoriety because possess a wide range of biological and pharmacological activities. In this sense, the aim of this work was to evaluate the anticancer and neuroprotective effects of the triterpene glycosides from sea cucumber Holothuria imitans. Methods: Triterpene glycosides were separated and purified by Reversed-phase high-performance liquid chromatography (RP-HPLC). Their structures were deduced by spectral analysis and chemical evidence. Cytotoxic activity was evaluated using normal African green monkey kidney epithelial cell line (VERO) and three cancer cell lines: cancer gastric (MKN-28), breast adenocarcinoma (MCF-7) and lung carcinoma (A-549). Besides, the neuroprotective effect was studied using the Cath.a-differentiated (CAD) cell line and human glial (Oligodendrocytic) hybrid cell line (MO3.13). Results: Two triterpene glycosides (Fuscocineroside C and Scabraside D) were isolated, which showed low cytotoxic activity against VERO cell line, and high cytotoxic activity against lines MKN-28, MCF-7 and A-549 cells, with IC50 between the ranges of 0.92 μmol/L to 2.61 μmol/L. The isolated triterpene glycosides showed the ability to regain mitochondrial viability in CAD and MO3.13 cells treated with neurotoxin (C2-ceramide) with statistically significant results (p<0.05). Conclusion: The triterpene glycosides Fuscocineroside C and Scabraside D isolated from sea cucumber Holothuria imitans show anticancer and neuroprotective potential and may be considered promising active principles for anticancer and neuroprotective drugs.

A Novel Methodology Using Dexamethasone to Induce Neuronal Differentiation in the CNS-Derived Catecholaminergic CAD Cells.

The Cath.a-differentiated (CAD) cell line is a central nervous system-derived catecholaminergic cell line originating from tyrosine hydroxylase (TH)-producing neurons located around the locus coeruleus area of the mouse brain. CAD cells have been used as an in vitro model for cellular and molecular studies due to their ability to differentiate under serum-free media conditions. However, the lack of serum-derived survival factors, limits the longevity for differentiated CAD cells to be maintained in healthy conditions; thereby, limiting their use in long-term culture studies. Here, we present a novel differentiation method that utilizes dexamethasone (Dex), a synthetic glucocorticoid receptor agonist. Specifically, we discovered that the addition of 100µM of Dex into the 1% fetal bovine serum (FBS)-supplemented media effectively induced neuronal differentiation of CAD cells, as characterized by neurite formation and elongation. Dex-differentiated CAD cells exited the cell cycle, stopped proliferating, extended the neurites, and expressed neuronal markers. These effects were dependent on the glucocorticoid receptors (GR) as they were abolished by GR knockdown. Importantly, Dex-differentiated CAD cells showed longer survival duration than serum-free differentiated CAD cells. In addition, RNA-sequencing and qPCR data demonstrate that several genes involved in proliferation, neuronal differentiation, and survival pathways were differentially expressed in the Dex-differentiated cells. This is the first study to reveal Dex as a novel differentiation methodology used to generate postmitotic neuronal CAD cells, which may be utilized as an in vitro neuronal model for cellular and molecular neurobiology research.

UV-triggered polymerization of polycatecholamines enables the production of organ-on-chips inside a biosafety cabinet

Surface modification of microfluidic chips used for making organ-on-a-chip (OOC) applications is often a time-consuming process, involving chip cleaning, ultraviolet (UV)-exposure, and steam sterilization. This work reports developing a simple, rapid, and cost-effective method that can achieve photo-activated polymerization and patterning of catecholamine materials on microfluidic chips in a single step using the UV light present in a standard biosafety cabinet. Polydimethylsiloxane (PDMS) microfluidic devices were filled with monomers of dopamine and norepinephrine, followed by exposure to UV light triggers polymerization of the material, which creates a highly viable surface for OOC applications. We examined the performance of these UV-triggered surface coatings for creating three different kinds of OOCs, where microfluidic chips were bonded and modified in three different ways: i) conventional oxygen plasma bonded microfluidic chips filled with monomer solutions and then exposed to UV to modify the surface (Plasma bonded, polymer-coated); ii) both the fluidic layer and glass substrate were exposed to UV to coat the functional layer and simultaneously allow adhesive proteins to bind the two pieces together (UV-bonded, polymer-coated); and iii) project the UV light through a mask to create fluid wall microfluidic channels on a polydimethylsiloxane (PDMS) substrate (projection coating). Cath.a.differentiated (CAD) cells seeded on UV-exposed polymer-coated surface in the three techniques showed significantly high cell viability, cell adhesion, proliferation, genetic expression, and they retained the functionality compared to uncoated PDMS. The UV-triggered surface modification technique uses a minimalist approach by using less equipment and existing infrastructure, such as a biosafety cabinet, for creating a functional OOC. This novel, simple, low-cost approach to reproducibly generating an organ-on-a-chip will facilitate the wider adoption of this technique.

Novel α-Lipoic Acid/3-n-Butylphthalide Conjugate Enhances Protective Effects against Oxidative Stress and 6-OHDA Induced Neuronal Damage.

Neurodegenerative diseases are irreversible conditions that result in progressive degeneration and death of nerve cells. Although the underlying mechanisms may vary, oxidative stress is considered to be one of the major causes of neuronal loss. Importantly, there are still no comprehensive treatments to completely cure these diseases. Therefore, protecting neurons from oxidative damage may be the most effective therapeutic strategy. Here we report a neuroprotective effects of a novel hybrid compound (dlx-23), obtained by conjugating α-lipoic acid (ALA), a natural antioxidant agent, and 3-n-butylphthalide (NBP), a clinical anti-ischemic drug. Dlx-23 protected against neuronal death induced by both H2O2 induced oxidative stress in Cath.-a-differentiated (CAD) cells and 6-OHDA, a toxin model of Parkinson's disease (PD) in SH-SY5Y cells. These activities proved to be more potent than the parent compound (ALA) alone. Dlx-23 scavenged free radicals, increased glutathione levels, and prevented mitochondria damage. In addition, live imaging of primary cortical neurons demonstrated that dlx-23 protected against neuronal growth cone damage induced by H2O2. Taken together these results suggest that dlx-23 has substantial potential to be further developed into a novel neuroprotective agent against oxidative damage and toxin induced neurodegeneration.

Bio-prospecting endemic Mascarene Aloes for potential neuroprotectants.

The Mascarene Aloes are used in the traditional pharmacopoeia against various ailments including cutaneous diseases and as antispasmodics. Scientific evidence to support these claims is non-existent and mainly based on the scientific repute of A.vera. The antioxidant profile of methanolic leaf extracts of A.purpurea Lam., A.tormentorii (Marais) L.E.Newton & G.D.Rowley, A.lomatophylloides Balf.f., A.macra Haw. and A.vera (L.) Burm.f. was studied using the total antioxidant capacity, copper equivalent and superoxide dismutase assays. In vitro cytotoxicity was evaluated on CAD (Cath.-a-differentiated) neuronal cells by the methyl tetrazolium assay, and the neuroprotective profile was assessed using hydrogen peroxide-induced neurotoxicity with the CAD cells. The aloin and vitexin content were determined by high-performance liquid chromatography with diode-array detection. A.purpurea had the highest aloin content (546.6nmol/g), while A.tormentorii had the highest vitexin content (67.3nmol/g). A.macra (concentration <0.1mg/mL) elicited a 10% cytotoxicity effect on CAD cells while other Mascarene Aloes were not cytotoxic. This study validates the antioxidant and neuroprotective potential of Mascarene Aloes focusing on their aloin and vitexin content that are also present in other reputed medicinal Aloes.

Performance assessments of vertically aligned carbon nanotubes multi-electrode arrays using Cath.a-differentiated (CAD) cells

In this work, Cath.a-differentiated (CAD) cells were used in place of primary neuronal cells to assess the performance of vertically aligned carbon nanotubes (VACNTs) multi-electrode arrays (MEA). To fabricate high-performance MEA, VACNTs were directly grown on graphene/Pt electrodes via plasma enhanced chemical deposition technique. Here, graphene served as an intermediate layer lowering contact resistance between VACNTs and Pt electrode. In order to lower the electrode impedance and to enhance the cell adhesion, VACNTs-MEAs were treated with UV–ozone for 20 min. Impedance of VACNTs electrode at 1 kHz frequency exhibits a reasonable value (110 kΩ) for extracellular signal recording, and the signal to noise ratio the is good enough to measure low signal amplitude (15.7). Spontaneous firing events from CAD cells were successfully measured with VACNTs MEAs that were also found to be surprisingly robust toward the biological interactions.

Pharmacological and neuroprotective profile of an essential oil derived from leaves of Aloysia citrodora Palau.

The Jordanian 'Melissa', (Aloysia citrodora) has been poorly studied both pharmacologically and in the clinic. Essential oils (EO) derived from leaves of A. citrodora were obtained by hydrodistillation, analysed by gas chromatography-mass spectrometry (GC-MS) and were investigated for a range of neurobiological and pharmacological properties, as a basis for potential future use in drug discovery. A selection of central nervous system (CNS) receptor-binding profiles was carried out. Antioxidant activity and ferrous iron-chelating assays were adopted, and the neuroprotective properties of A. citrodora EO assessed using hydrogen peroxide-induced and β-amyloid-induced neurotoxicity with the CAD (Cath.-a-differentiated) neuroblastoma cell line. The major chemical components detected in the A. citrodora EOs, derived from dried and fresh leaves, included limonene, geranial, neral, 1, 8-cineole, curcumene, spathulenol and caryophyllene oxide, respectively. A. citrodora leaf EO inhibited [(3) H] nicotine binding to well washed rat forebrain membranes, and increased iron-chelation in vitro. A. citrodora EO displays effective antioxidant, radical-scavenging activities and significant protective properties vs both hydrogen peroxide- and β-amyloid-induced neurotoxicity. A. citrodora EO displays a range of pharmacological properties worthy of further investigation to isolate the compounds responsible for the observed neuroactivities, to further analyse their mode of action and determine their clinical potential in neurodegenerative diseases.

Convergent Replication of Mouse Synthetic Prion Strains

Prion diseases are neurodegenerative disorders characterized by the aberrant folding of endogenous proteins into self-propagating pathogenic conformers. Prion disease can be initiated in animal models by inoculation with amyloid fibrils formed from bacterially derived recombinant prion protein. The synthetic prions that accumulated in infected organisms are structurally distinct from the amyloid preparations used to initiate their formation and change conformationally on repeated passage. To investigate the nature of synthetic prion transformation, we infected mice with a conformationally diverse set of amyloids and serially passaged the resulting prion strains. At each passage, we monitored changes in the biochemical and biological properties of the adapting strain. The physicochemical properties of each synthetic prion strain gradually changed on serial propagation until attaining a common adapted state with shared physicochemical characteristics. These results indicate that synthetic prions can assume multiple intermediate conformations before converging into one conformation optimized for in vivo propagation.

CPAP is required for cilia formation in neuronal cells

SummaryThe primary cilium is a microtubule-based structure protruded from the basal body analogous to the centriole. CPAP (centrosomal P4.1-associated protein) has previously been reported to be a cell cycle-regulated protein that controls centriole length. Mutations in CPAP cause primary microcephaly (MCPH) in humans. Here, using a cell-based system that we established to monitor cilia formation in neuronal CAD (Cath.a-differentiated) cells and hippocampal neurons, we found that CPAP is required for cilia biogenesis. Overexpression of wild-type CPAP promoted cilia formation and induced longer cilia. In contrast, an exogenously expressed CPAP-377EE mutant that lacks tubulin-dimer binding significantly inhibited cilia formation and caused cilia shortening. Furthermore, depletion of CPAP inhibited ciliogenesis and such effect was effectively rescued by expression of wild-type CPAP, but not by the CPAP-377EE mutant. Taken together, our results suggest that CPAP is a positive regulator of ciliogenesis whose intrinsic tubulin-dimer binding activity is required for cilia formation in neuronal cells.

Activation of TRPC6 channels promotes endocannabinoid biosynthesis in neuronal CAD cells

Calcium influx activates biosynthesis of the endogenous cannabinoids 2-arachidonyl glycerol (2-AG) and anandamide (AEA). The calcium channel involved with endocannabinoid synthesis and release in neurons is still unknown. The canonical TRP (TRPC) channels are calcium-permeable channels that are a homology-based subdivision of the broader class of TRP channels. TRPC3, 6, and 7 are G-protein-gated non-selective cation channels that have been localized to lipid rafts and shown to colocalize with caveolin 1. Because endocannabinoid synthesis has been found to occur “on demand” in a calcium-dependent manner and has been linked to lipid rafts, we explored the potential role of transient receptor potential (TRP) channels in this process. Previously, we observed that after metabolism AEA and arachidonic acid (ArA) can be recycled into new endocannabinoid molecules. Consistent with these previous findings, we found that Cath.a differentiated (CAD) cells pretreated with radiolabeled ArA exhibited a robust increase in 2-AG release in response to TRPC stimulation with the diacylglycerol (DAG) analogue, 1-oleoyl-2-acetyl- sn-glycerol (OAG). Furthermore, cells pretreated with [ 3H]AEA produced a significant amount of AEA and 2-AG upon stimulation of TRPC channels. This process was not mediated through protein kinase C activation. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed that only TRPC6 was present in the CAD cells. siRNA-induced knockdown of TRPC6 in the CAD cells abolished OAG-stimulated production of the endocannabionids. This evidence suggests that TRPC6 may be capable of promoting endocannabinoid synthesis in neuronal cells.

Lack of stabilized microtubules as a result of the absence of major maps in CAD cells does not preclude neurite formation

In many laboratories, the requirement of microtubule-associated proteins (MAPs) and the stabilization of microtubules for the elongation of neurites has been intensively investigated, with controversial results being obtained. We have observed that the neurite microtubules of Cath.a-differentiated (CAD) cells, a mouse brain derived cell, are highly dynamic structures, and so we analyzed several aspects of the cytoskeleton to investigate the molecular causes of this phenomenon. Microtubules and microfilaments were present in proportions similar to those found in brain tissue and were distributed similarly to those in normal neurons in culture. Neurofilaments were also present. Analysis of tubulin isospecies originating from post-translational modifications revealed an increased amount of tyrosinated tubulin, a diminished amount of the detyrosinated form and a lack of the Delta2 form. This tyrosination pattern is in agreement with highly dynamic microtubules. Using western blot analyses with specific antibodies, we found that CAD cells do not express several MAPs such as MAP1b, MAP2, Tau, doublecortin, and stable-tubule-only-peptide. The presence of the genes corresponding to these MAPs was verified. The absence of the corresponding mRNAs confirmed the lack of expression of these proteins. The exception was Tau, whose mRNA was present. Among the several MAPs investigated, LIS1 was the only one to be expressed in CAD cells. In addition, we determined that neurites of CAD cells form and elongate at the same rate as processes in a primary culture of hippocampal neurons. Treatment with nocodazol precluded the formation of neurites, and induced the retraction of previously formed neurites. We conclude that the formation and elongation of neurites, at least in CAD cells, are dependent on microtubule integrity but not on their stabilization or the presence of MAPs.

RE1 silencing transcription factor is involved in regulating neuron‐specific expression of α‐internexin and neurofilament genes

Alpha-internexin and the neurofilament triplet proteins (NF-L, NF-M, and NF-H) co-assemble into intermediate filament networks in neurons. We have found that the RE1 silencing transcription factor (REST) plays a contributory role in the neuron-specific expression of the alpha-internexin, NF-H and NF-M genes. Chromatin immunoprecipitation and transient transfection experiments performed with catecholaminergic neuronal Cath a.-differentiated (CAD) cells and non-neuronal NIH3T3 cells demonstrated that REST repressed transcription of these genes in NIH3T3 cells by binding and recruiting mSin3A, CoREST, histone deacetylase (HDAC) 1 and MeCP2 to the RE1 sites in the intron-1 of alpha-internexin and the 5' flanking regions of NF-H and NF-M. No repression effect of the RE1 sites was observed in CAD cells, which express these neuronal genes but not REST. Treatment of NIH3T3 cells with trichostatin A, a HDAC inhibitor, relieved the REST-mediated repression and induced ectopic activation of alpha-internexin, NF-H and NF-M. The trichostatin A treatment did not affect the levels of REST occupancy but caused coordinated changes in acetylation and methylation of histones around the RE1 sites of these genes in NIH3T3 cells consistent with a transition from transcriptional repression to transcriptional activation. Thus, REST regulates expression of these neuronal genes, partly by a HDAC-dependent epigenetic mechanism.

D1 and D2 Dopamine Receptor Expression Is Regulated by Direct Interaction with the Chaperone Protein Calnexin

As for all proteins, G protein-coupled receptors (GPCRs) undergo synthesis and maturation within the endoplasmic reticulum (ER). The mechanisms involved in the biogenesis and trafficking of GPCRs from the ER to the cell surface are poorly understood, but they may involve interactions with other proteins. We have now identified the ER chaperone protein calnexin as an interacting protein for both D(1) and D(2) dopamine receptors. These protein-protein interactions were confirmed using Western blot analysis and co-immunoprecipitation experiments. To determine the influence of calnexin on receptor expression, we conducted assays in HEK293T cells using a variety of calnexin-modifying conditions. Inhibition of glycosylation either through receptor mutations or treatments with glycosylation inhibitors partially blocks the interactions with calnexin with a resulting decrease in cell surface receptor expression. Confocal fluorescence microscopy reveals the accumulation of D(1)-green fluorescent protein and D(2)-yellow fluorescent protein receptors within internal stores following treatment with calnexin inhibitors. Overexpression of calnexin also results in a marked decrease in both D(1) and D(2) receptor expression. This is likely because of an increase in ER retention because confocal microscopy revealed intracellular clustering of dopamine receptors that were co-localized with an ER marker protein. Additionally, we show that calnexin interacts with the receptors via two distinct mechanisms, glycan-dependent and glycan-independent, which may underlie the multiple effects (ER retention and surface trafficking) of calnexin on receptor expression. Our data suggest that optimal receptor-calnexin interactions critically regulate D(1) and D(2) receptor trafficking and expression at the cell surface, a mechanism likely to be of importance for many GPCRs.

Electrohydrodynamic jetting of mouse neuronal cells

CAD (Cath.a-differentiated) cells, a mouse neuronal cell line, were subjected to electrohydrodynamic jetting at a field strength of 0.47-0.67 kV/mm, corresponding to an applied voltage of 7-10 kV. After jetting, the cells appeared normal and continued to divide at rates similar to those shown by control samples. Jetted cells, when placed in serum-free medium, underwent differentiation that was sustained for at least 1 month. Some of the droplets produced by jetting contained only a few cells. These results indicate that the process of jetting does not significantly perturb neuronal cells and that this novel approach might in the future be a useful way to deposit small numbers of living nerve cells on to surfaces.

Metabolic Activity: A Novel Indicator of Neuronal Survival in the Murine Dopaminergic Cell Line CAD

Apoptosis is implicated in many neurodegenerative diseases, including Parkinson's disease (PD). Neuroprotective strategies targeting apoptosis need to preserve functional integrity of the saved cells to be effective. The aim of the present study was to evaluate a novel approach for analyzing neuronal function that monitors cellular metabolic responses to receptor activation using the microphysiometer. N-Acetyl-sphingosine (C2-ceramide) induced cell death of the neuronal cell line, Cath.a-differentiated (CAD) cells, which resemble catecholaminergic cells of the CNS, and provide a useful in vitro model for the cells affected in PD. C2-ceramide also suppressed the metabolic response of CAD cells to muscarinic receptor activation. Pretreatment with the caspase inhibitor Boc-Asp-(OMe)-fluoromethylketone (BAF) plus neurotrophin-3 (NT-3) reduced C2- ceramide-induced CAD cell death, delaying cell death more effectively than either agent alone; and, most significantly, BAF and NT-3 enabled the cells remaining 24 h after toxin treatment to generate a normal metabolic response to the muscarinic agonist carbachol. On the basis of these results, we suggest that measuring metabolic responses to receptor activation is a useful method for following neuronal viability after toxin treatment and that the combination of caspase inhibitors and neurotrophic factors might be a plausible strategy for improving neuronal survival, with critical preservation of metabolic function.

Differentiation-induced alterations in cyclic AMP signaling in the Cath.a differentiated (CAD) neuronal cell line.

Regulation of intracellular cyclic AMP is critical to the modulation of many cellular activities, including cellular differentiation. Moreover, morphological differentiation has been linked to subsequent alterations in the cAMP signaling pathway in various cellular models. The current study was designed to explore the mechanism for the previously reported enhancement of adenylate cyclase activity in Cath.a differentiated cells following differentiation. Differentiation of Cath.a differentiated cells stably expressing the D2L dopamine receptor markedly potentiated both forskolin- and A2-adenosine receptor-stimulated cAMP accumulation. This enhancement was accompanied by a twofold increase in adenylate cyclase 6 (AC6) expression and a dramatic loss in the expression of AC9. The ability of Ca2+ to inhibit drug-stimulated cAMP accumulation was enhanced following differentiation, as was D2L dopamine receptor-mediated inhibition of Galphas-stimulated cAMP accumulation. Differentiation altered basal and drug-stimulated phosphorylation of the cAMP-response element-binding protein, which was independent of changes in protein kinase A expression. The current data suggest that differentiation of the neuronal cell model, Cath.a differentiated cells induces significant alterations in the expression and function of both the proximal and distal portions of the cAMP signaling pathway and may impact cellular operations dependent upon this pathway.

Heterologous sensitization of adenylate cyclase is protein kinase A-dependent in Cath.a differentiated (CAD)-D2L cells.

Persistent activation of Galphai/o-coupled receptors results in a paradoxical enhancement of subsequent drug-stimulated adenylate cyclase activity. The exact mechanism of this up-regulation in the cyclic AMP signaling pathway, known as heterologous sensitization, remains undefined. The present study was designed to investigate the involvement of cyclic AMP-dependent protein kinase in D2L receptor-mediated sensitization in a neuronal cellular environment. The current studies were conducted in the Cath.a differentiated (CAD) cell line transfected stably with the D2L dopamine receptor (CAD-D2L). Long-term 18 h treatment with the D2 receptor agonist, quinpirole, resulted in a two-fold enhancement of forskolin-stimulated cyclic AMP accumulation. Similarly, long-term treatment with the PKA inhibitors, H89 or Rp-8Br-cAMP, also enhanced adenylate cyclase activity. In contrast, long-term activation of protein kinase A (PKA) by forskolin, isobutylmethylxanthine (IBMX), or dibutyryl cyclic AMP caused a significant reduction in subsequent forskolin-stimulated cyclic AMP accumulation and reduced both quinpirole- and H89-induced heterologous sensitization. The effects of PKA inhibitors and activators did not involve changes in PKA subunit expression. RT-PCR analysis of adenylate cyclase isoform expression patterns revealed the expression of mRNA for ACVI and ACIX in CAD-D2L cells. The ability of ACVI to be negatively regulated by PKA is consistent with the observation that inhibition of PKA results in heterologous sensitization of adenylate cyclase activity in CAD-D2L cells.

Characterization of a CNS Cell Line, CAD, in which Morphological Differentiation Is Initiated by Serum Deprivation

A CNS catecholaminergic cell line, Cath.a, was established by targeted oncogenesis in transgenic mice. Cath.a cells express neuronal properties but lack neuronal morphology. Here, we describe a variant of Cath.a, called CAD (Cath.a-differentiated), in which reversible morphological differentiation can be initiated by removal of serum or exogenously added protein from the medium. In serum- or protein-free media, CAD cells stop proliferating and extend long processes. Differentiated CAD cells can be maintained without serum or protein for at least 6 weeks. CAD cells are distinct from Cath.a cells; most significant, the original immortalizing oncogene, SV40 T antigen, was spontaneously lost. By immunostaining or immunoblotting, we show that CAD cells express neuron-specific proteins, such as class III beta-tubulin, GAP-43, SNAP-25, and synaptotagmin, but not GFAP. Ultrastructurally, processes from differentiated CAD cells have abundant parallel microtubules and intermediate filaments, and bear varicosities that contain both large dense-core vesicles/granules (120-160 nm) and smaller clear vesicles (60-80 nm). Additionally, CAD cells express enzymatically active tyrosine hydroxylase and accumulate L-DOPA. CAD cells exhibit biochemical and morphological characteristics of primary neurons and provide an unique tool for studying neuronal differentiation.