Neuronal Cell Culture Model Research Articles

Investigation of annexin A5 as a biomarker for Alzheimer's disease using neuronal cell culture and mouse model

Alzheimer's disease (AD) differs from other forms of dementia in its relation to amyloid beta peptide (Abeta). Abeta, a proteolytic product of amyloid precursor proteins (APP), has a toxic effect on neuronal cells, which involves perturbation of their Ca(2+) homeostasis. This effect implies that changes of protein expression in neuronal cells with calcium stress should provide a molecular marker for this disease. In the present study, we used the supernatant from a neuronal cell culture after incubation with or without Abeta and isolated a Ca(2+)-dependent acidic phospholipid binding fraction to perform a proteomic study. Several unique proteins were identified after incubation with Abeta. We focused on annexin A5, among these proteins, because it binds both Ca(2+) and lipids likely to be involved in calcium homeostasis. Tg2576 transgenic mice (AD model) overexpressing mutant human APP showed a significant increase of annexin A5 in the brain cortex but not in other organs, including liver, kidney, lung, and intestine. In human plasma samples, the level of annexin A5 was significantly increased in a proportion of AD patients compared with a control group (P < 0.0001 in the logistic regression analysis). From the receiver operating characteristic (ROC) curve with plasma annexin A5 concentrations, the mean area under the curve (AUC 0.898) suggests that annexin A5 is a favorable marker for AD.

Microtubule Stabilization by Bone Morphogenetic Protein Receptor-Mediated Scaffolding of c-Jun N-Terminal Kinase Promotes Dendrite Formation

Neuronal outgrowth occurs via coordinated remodeling of the cytoskeleton involving both actin and microtubules. Microtubule stabilization drives the extending neurite, yet little is known of the molecular mechanisms whereby extracellular cues regulate microtubule dynamics. Bone morphogenetic proteins (BMPs) play an important role in neuronal differentiation and morphogenesis, and BMP7 in particular induces the formation of dendrites. Here, we show that BMP7 induces stabilization of microtubules in both a MAP2-dependent neuronal cell culture model and in dendrites of primary cortical neurons. BMP7 rapidly activates c-Jun N-terminal kinases (JNKs), known regulators of microtubule dynamics, and we show that JNKs associate with the carboxy terminus of the BMP receptor, BMPRII. Activation and binding of JNKs to BMPRII is required for BMP7-induced microtubule stabilization and for BMP7-mediated dendrite formation in primary cortical neurons. These data indicate that BMPRII acts as a scaffold to localize and coordinate cytoskeletal remodeling and thereby provides an efficient means for extracellular cues, such as BMPs, to control neuronal dendritogenesis.

Inhibition of FK506 Binding Proteins Reduces α-Synuclein Aggregation and Parkinson's Disease-Like Pathology

alpha-Synuclein (alpha-SYN) is a key player in the pathogenesis of Parkinson's disease (PD). In pathological conditions, the protein is present in a fibrillar, aggregated form inside cytoplasmic inclusions called Lewy bodies. Members of the FK506 binding protein (FKBP) family are peptidyl-prolyl isomerases that were shown recently to accelerate the aggregation of alpha-SYN in vitro. We now established a neuronal cell culture model for synucleinopathy based on oxidative stress-induced alpha-SYN aggregation and apoptosis. Using high-content analysis, we examined the role of FKBPs in aggregation and apoptotic cell death. FK506, a specific inhibitor of this family of proteins, inhibited alpha-SYN aggregation and neuronal cell death in this synucleinopathy model dose dependently. Knockdown of FKBP12 or FKBP52 reduced the number of alpha-SYN aggregates and protected against cell death, whereas overexpression of FKBP12 or FKBP52 accelerated both aggregation of alpha-SYN and cell death. Thus, FK506 likely targets FKBP members in the cell culture model. Furthermore, oral administration of FK506 after viral vector-mediated overexpression of alpha-SYN in adult mouse brain significantly reduced alpha-SYN aggregate formation and neuronal cell death. Our data explain previously described neuroregenerative and neuroprotective effects of immunophilin ligands and validate FKBPs as a novel drug target for the causative treatment of PD.

Effect of peritoneal fluid from endometriosis patients on neuroblastoma cells in culture

Aim. Endometriosis is often associated with lower abdominal pain, dysmenorrhea, dyspareunia, and chronic pelvic pain. There is no correlation between the extent of endometriosis and the intensity of pain. The mechanism of pain in endometriosis is unknown. The aim of our study was to investigate the influence of peritoneal fluid (PF) from endometriosis patients on cultured neural cells that are the morphological basis of nociception, and to determine whether there was a relationship between the rAFS staging and an elevation of TGF-β1 production by these cells.Methods. Different human neuroblastoma cell lines were grown to 3/4 confluence and then cultured in presence of PF pooled according to the presence of no, mild, or severe endometriosis. After 6 and 24 h of incubation, the morphological changes were assessed and the metabolic activity was determined.Results. The different cell lines showed strongly varying proliferation and aggregation patterns. The metabolic activity was also varying between cell lines, but no consistently increased cell turnover in the PF when compared with the control medium nor associated to a particular, endometriosis-derived PF pool could be shown. In this experimental setting, we have observed that the cell proliferation in the presence of PF was inhibited, and not enhanced as it might have been expected. Measurement of TGF-β1 showed higher production rates for this cytokine under exposure to PF than in controls for some but not all tested cell lines, but there was no association with the stage (rAFS) of the disease.Conclusion. The neuronal cell culture model may become a useful tool to investigate the endometriosis-derived pain, but different endpoints and cell lines may have to be introduced.

Reduction in programmed cell death and improvement in functional outcome of transient focal cerebral ischemia after administration of granulocyte-macrophage colony-stimulating factor in rats

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a potent hematopoietic growth factor that both enhances the survival and drives the differentiation and proliferation of myeloid lineage cells. Recent studies have suggested that GM-CSF has a neuroprotective effect against CNS injury. In this paper, the authors investigated the neuroprotective effect of GM-CSF on neuron survival and locomotor behavior in a rat model of focal cerebral ischemic injury. To understand its neuroprotective effect in vitro, GM-CSF was administered to a glutamate-induced excitotoxicity neuronal injury cell culture model that mimics the pathophysiology of focal hypoxic cerebral injury. In the animal study, the authors prepared a rat focal cerebral ischemia model by occluding the unilateral middle cerebral artery. They then examined the effects of GM-CSF administration on changes in infarct volume, apoptosis-related gene expression, and improvement in locomotor behavior. Treatment with GM-CSF significantly increased cell viability in a cell culture model of glutamate-induced neuronal injury. Furthermore, in vivo administration of GM-CSF at 60 microg/kg body weight daily for 5 consecutive days beginning immediately after injury decreased infarction volume, altered the expression of several apoptosis-related genes (Bcl-2, Bax, caspase 3, and p53), and improved locomotor behavior in the focal cerebral ischemia model. The GM-CSF had neuroprotective effects in in vitro and in vivo experiments and resulted in decreased infarction volume and improved locomotor behavior. Although the specific mechanism involved in stroke recovery was not fully elucidated as it was not the primary focus of this study, administration of GM-CSF appeared to decrease the extent of neuronal apoptosis by modulating the expression of several apoptosis-related genes such as Bcl-2, Bax, caspase 3, and p53. Further investigations are necessary to better understand the role of GM-CSF on neural regeneration during the recovery phase of a stroke, as well as the intracellular signal transduction pathways that mediate neuroprotection.

Antioxidant potential of Minocycline in Japanese Encephalitis Virus infection in murine neuroblastoma cells: Correlation with membrane fluidity and cell death

Minocycline is neuroprotective in animal models of a number of acute CNS injuries, neurodegenerative diseases and CNS infection. While anti-inflammatory and anti-apoptotic effects of Minocycline have been characterized, the molecular basis for the neuroprotective effects of Minocycline remains unclear. We report here that Minocycline and two classical antioxidant compounds inhibit the Japanese Encephalitis Virus (JEV)-induced free radical generation in mouse neuroblastoma. In cultures of Neuro2a (N2a) cells infected with JEV for up to 24 h, the number of cells undergoing cell death was also reduced by Minocycline (20 μM). JEV infection resulted in increased oxidative stress, as revealed by an increase in the fluorescence intensity for 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA), a reactive oxygen species (ROS) indicator. Minocycline at 20 μM inhibited this ROS production. Cells were moderately protected from JEV-induced death by diphenyleneiodonium (DPI), an inhibitor of flavon-containing enzyme inhibitor, whereas common antioxidants such as N-acetyl-cysteine (NAC) turned out to be ineffective. Direct antioxidant property of Minocycline and reference antioxidant compounds is evaluated by LDH assay, ROS measurement and mitochondrial membrane potential measurement. Our findings suggest that Minocycline reduces the neuronal damage seen in JEV infection in neuronal cell culture models at least in part through inhibition of oxidative stress.

Proteomic analysis of embryonic stem cell–derived neural cells exposed to the antidepressant paroxetine

Antidepressant drugs can have significant effects on the mood of a patient suffering from major depression or other disorders. The pharmacological actions of these drugs generally affect the uptake or metabolism of the neurotransmitters serotonin, noradrenalin, and, to a lesser extent, dopamine. However, many aspects of antidepressant action are not understood. We conducted a proteomic analysis in a neuronal cell culture model in an attempt to identify molecules important to the operation of pathways functionally relevant to antidepressant action. The model involved generating cultures containing mixed neural and glial cells by controlled differentiation of mouse embryonic stem cells, followed by exposure to 1 microM paroxetine for 14 days. After antidepressant exposure, we observed increased expression or modification of sepiapterin reductase (SPR), heat shock protein 9A, RAS and EF-hand domain containing, and protein disulfide isomerase associated 3 and decreased expression or modification of creatine kinase, actin, prohibitin, a T-cell receptor alpha chain, defensin-related cryptdin 5, and the intermediate filament proteins glial fibrillary acidic protein and vimentin. SPR, the most strongly up-regulated protein observed, controls production of tetrahydrobiopterin, an essential cofactor for the synthesis of many neurotransmitters including serotonin, making it a plausible and intriguing candidate protein for involvement in mood control and antidepressant drug action. SPR and the other proteins identified may represent links to molecular processes of importance to mood dysregulation and control, and their respective genes may be novel candidates for the study of antidepressant pharmacogenetics.

Inhibitors of metabolism rescue cell death in Huntington's disease models

Huntington's disease (HD) is a fatal inherited neurodegenerative disorder. HD is caused by polyglutamine expansions in the huntingtin (htt) protein that result in neuronal loss and contribute to HD pathology. The mechanisms of neuronal loss in HD are elusive, and there is no therapy to alleviate HD. To find small molecules that slow neuronal loss in HD, we screened 1,040 biologically active molecules to identify suppressors of cell death in a neuronal cell culture model of HD. We found that inhibitors of mitochondrial function or glycolysis rescued cell death in this cell culture and in in vivo HD models. These inhibitors prevented cell death by activating prosurvival ERK and AKT signaling but without altering cellular ATP levels. ERK and AKT inhibition through the use of specific chemical inhibitors abrogated the rescue, whereas their activation through the use of growth factors rescued cell death, suggesting that this activation could explain the protective effect of metabolic inhibitors. Both ERK and AKT signaling are disrupted in HD, and activating these pathways is protective in several HD models. Our results reveal a mechanism for activating prosurvival signaling that could be exploited for treating HD and possibly other neurodegenerative disorders.

Protein Kinase Cδ Negatively Regulates Tyrosine Hydroxylase Activity and Dopamine Synthesis by Enhancing Protein Phosphatase-2A Activity in Dopaminergic Neurons

Tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis, can be regulated by phosphorylation at multiple serine residues, including serine-40. In the present study, we report a novel interaction between a key member of the novel PKC family, protein kinase Cdelta (PKCdelta), and TH, in which the kinase modulates dopamine synthesis by negatively regulating TH activity via protein phosphatase 2A (PP2A). We observed that PKCdelta is highly expressed in nigral dopaminergic neurons and colocalizes with TH. Interestingly, suppression of PKCdelta activity with the kinase inhibitor rottlerin, PKCdelta-small interfering RNA, or with PKCdelta dominant-negative mutant effectively increased a number of key biochemical events in the dopamine pathway, including TH-ser40 phosphorylation, TH enzymatic activity, and dopamine synthesis in neuronal cell culture models. Additionally, we found that PKCdelta not only physically associates with the PP2A catalytic subunit (PP2Ac) but also phosphorylates the phosphatase to increase its activity. Notably, inhibition of PKCdelta reduced the dephosphorylation activity of PP2A and thereby increased TH-ser40 phosphorylation, TH activity, and dopamine synthesis. To further validate our findings, we used the PKCdelta knock-out (PKCdelta-/-) mouse model. Consistent with other results, we found greater TH-ser40 phosphorylation and reduced PP2A activity in the substantia nigra of PKCdelta-/- mice than in wild-type mice. Importantly, this was accompanied by an increased dopamine level in the striatum of PKCdelta-/- mice. Collectively, these results suggest that PKCdelta phosphorylates PP2Ac to enhance its activity and thereby reduces TH-ser40 phosphorylation and TH activity and ultimately dopamine synthesis.

Selective inhibitors of death in mutant huntingtin cells

Huntington disease (HD) is an inherited neurodegenerative disorder with unclear pathophysiology. We developed a high-throughput assay in a neuronal cell culture model of HD, screened 43,685 compounds and identified 29 novel selective inhibitors of cell death in mutant huntingtin-expressing cells. Four compounds were active in diverse HD models, which suggests a role for cell death in HD; these compounds are mechanistic probes and potential drug leads for HD.

Neuronal Differentiation Is Accompanied by Increased Levels of SNAP‐25 Protein in Fetal Rat Primary Cortical Neurons

Alzheimer's disease (AD) is accompanied by progressive memory loss and decline in general cognitive abilities. Neuropathological hallmarks include amyloid plaque formation, neurofibrillary tangles, and neuronal and synaptic loss. To improve research models in AD, we have characterized several aspects of the primary rat neuronal cell culture model, which relate directly to the study of neuroexocytosis, neuronal plasticity, and amyloidosis. We have attempted to isolate and assess the neuronal enrichment of a primary cortical cell culture by morphological and molecular techniques, following progress of these cultured cells for up to 18 days in culture to identify the optimum timeframe for the manipulation and data collection in these cells. We observed maximum neuronal differentiation around day 15, which was in decline by day 18, thus, the critical window for manipulations in these cultures seems to be between days 12-15. The substantial increase in neuritic length and density was apparent at very early time points and peaked at day 15. In parallel to morphological assessments, we measured levels of a group of selected neuronally important proteins, including cytoskeletal and synaptic proteins. Change in the levels of cell-associated APP and synaptophysin were not as dramatic as that of SNAP-25 or secreted APP. Notably, this increased differentiation is accompanied by a dramatic increase in levels of SNAP-25 protein, which peaked at day 15 and declined thereafter. These results suggest that this neuronal population contains quantifiable presynaptic terminals. Thus, the establishment and molecular characterization of this neuronal model would have many implications in neuroscience research including synaptic differentiation and neuronal plasticity and for the evaluation of pharmacological interventions in the disease process.

Cellular prion protein modulates the intracellular calcium response to hydrogen peroxide

The physiological function of the cellular prion protein (PrP(C)) is still under intense investigation. It has been suggested that PrP(C) has a protective role in neuronal cells, particularly against environmental stress caused by reactive oxygen species (ROS). Here we analysed the acute effect of a major ROS, hydrogen peroxide (H(2)O(2)), on intracellular calcium homeostasis in cultured cerebellar granule cells and immortalized hippocampal neuronal cells. Both neuronal cell culture models showed that the rise in intracellular calcium following application of H(2)O(2) was strongly dependent on the presence of PrP(C). Moreover, the N-terminal octapeptide repeats of PrP(C) were required for this effect, because neuronal cells expressing a PrP(C) lacking the N-terminus resembled the PrP(C)-deficient phenotype. Neurones deficient of fyn kinase, or pharmacological inhibition of fyn, also abrogated the calcium response to H(2)O(2) treatment, indicating that fyn activation is a critical step within the PrP(C) signalling cascade. Finally, we identified a possible role of this PrP(C) signalling pathway in the neuroprotective response of PrP(C) to oxidative stress. In conclusion, we put forward the hypothesis that PrP(C) functions as a sensor for H(2)O(2), thereby activating a protective signalling cascade involving fyn kinase that leads to calcium release from intracellular stores.

Inhibition of APP Trafficking by Tau Protein Does Not Increase the Generation of Amyloid‐β Peptides

Amyloid-beta, a peptide derived from the precursor protein APP, accumulates in the brain and contributes to the neuropathology of Alzheimer's disease. Increased generation of amyloid-beta might be caused by axonal transport inhibition, via increased dwell time of APP vesicles and thereby higher probability of APP cleavage by secretase enzymes residing on the same vesicles. We tested this hypothesis using a neuronal cell culture model of inhibited axonal transport and by imaging vesicular transport of fluorescently tagged APP and beta-secretase (BACE1). Microtubule-associated tau protein blocks vesicle traffic by inhibiting the access of motor proteins to the microtubule tracks. In neurons co-transfected with CFP-tau, APP-YFP traffic into distal neurites was strongly reduced. However, this did not increase amyloid-beta levels. In singly transfected axons, APP-YFP was transported in large tubules and vesicles moving very fast (on average 3 microm/s) and with high fluxes in the anterograde direction (on average 8.4 vesicles/min). By contrast, BACE1-CFP movement was in smaller tubules and vesicles that were almost 2x slower (on average 1.6 microm/s) with approximately 18x lower fluxes (on average 0.5 vesicles/min). Two-colour microscopy of co-transfected axons confirmed that the two proteins were sorted into distinct carriers. The results do not support the above hypothesis. Instead, they indicate that APP is transported on vesicles distinct from the secretase components and that amyloid-beta is not generated in transit when transport is blocked by tau.

A pH-dependent increase in neuronal glutamate efflux in vitro: Possible involvement of ASCT1

Efflux of glutamate from intracellular pools during hypoxia–ischemia has been postulated to be mediated by amino acid transporters and can lead to excitotoxicity. In addition, a decrease in pH seen during global hypoxia–ischemia may influence which transporter is responsible for this glutamate efflux. For example, the neutral amino acid transporter ASCT1 is an effective transporter of glutamate at low pH. We have examined the effects of pH, pH and temperature, and hypoxia on glutamate efflux in a rat primary neuronal cell culture model. We observed a marked increase of glutamate efflux as pH was decreased from 7.4 to 5.5. This pH-dependent efflux is likely due to a transporter-mediated process because it was seen in the presence of tetrodotoxin and was blunted by decreasing the temperature to either 35 °C or 33 °C. In addition, no increase in LDH was seen at pH 5.5 suggesting that increased glutamate levels were not due to cellular death. No change in glutamate levels was seen when the oxygen tension of the medium was lowered from 150 mm Hg to either 30 or 15 mm Hg. Given that EAAT transporters are inhibited by low pH, other transporters, such as ASCT1, may be responsible for this pH-dependent efflux of glutamate.

Mutations in the neurofilament light gene linked to Charcot‐Marie‐Tooth disease cause defects in transport

Neurofilament light gene mutations have been linked to a subset of patients with Charcot-Marie-Tooth disease, the most common inherited motor and sensory neuropathy. We have previously shown that Charcot-Marie-Tooth-linked mutant neurofilament light assembles abnormally in non-neuronal cells. In this study, we have characterized the effects of expression of mutant neurofilament light proteins on axonal transport in a neuronal cell culture model. We demonstrated that the Charcot-Marie-Tooth-linked neurofilament light mutations: (i) affect the axonal transport of mutant neurofilaments; (ii) have a dominant-negative effect on the transport of wild-type neurofilaments; (iii) affect the transport of mitochondria and the anterograde axonal transport marker human amyloid precursor protein; (iv) result in alterations of retrograde axonal transport and (v) cause fragmentation of the Golgi apparatus. Increased neuritic degeneration was observed in neuronal cells overexpressing neurofilament light mutants. Our results suggest that these generalized axonal transport defects could be responsible for the neuropathy in Charcot-Marie-Tooth disease.

Ubiquitinated inclusions and neuronal cell death.

Ubiquitinated inclusions and selective neuronal cell death are considered the pathological hallmarks of Parkinson's disease and other neurodegenerative diseases. Recent genetic, pathological and biochemical evidence suggests that dysfunction of ubiquitin-dependent protein degradation by the proteasome might be a contributing, if not initiating factor in the pathogenesis of these diseases. In neuronal cell culture models inhibition of the proteasome leads to cell death and formation of fibrillar ubiquitin and alpha-synuclein-positive inclusions, thus modeling some aspects of Lewy body diseases. The processes of inclusion formation and neuronal cell death share some common mechanisms, but can also be dissociated at a certain level.

Neuroprotective effects of estrogen and tamoxifen in vitro: a facilitative role for glia?

Selective estrogen receptor modulators (SERMs) are steroidal or nonsteroidal compounds that can exhibit either estrogen-like agonistic effects or estrogen-antagonistic effects depending on the target tissue. While SERM actions in the breast, bone, and uterus have been well characterized, their effects in the brain are considerably less well understood. Previous work by our laboratory has demonstrated a beneficial effect of tamoxifen in the reduction of ischemic stroke damage in ovariectomized female rats. The present study utilized neuronal cell culture models to attempt to understand the mechanisms of tamoxifen-mediated neuroprotection. Neither physiologic doses of 17beta-E2 nor clinically therapeutic doses of tamoxifen directly protected GT1-7 neurons or purified cultures of rat cerebrocortical neurons from several forms of cell death. Reverse transcriptase polymerase chain reaction and Western blot analysis revealed that GT1-7 neurons possessed both estrogen receptor-alpha (ERalpha) and ERbeta mRNA and protein, whereas purified embryonic rat cortical neurons only expressed appreciable levels of ERalpha transcript and protein, with little to no expression of ERbeta. In contrast to the lack of protection in the purified neuronal cultures, both 17beta- E2 and tamoxifen significantly protected mixed glial/ neuronal cortical cultures from cell death, suggesting that glia may facilitate 17beta-E2-and tamoxifen-mediated neuroprotection. Furthermore, astrocyte-conditioned media and exogenous transforming growth factor-beta1, a documented astrocyte-derived cytokine, were shown to rescue purified cortical neurons from cell death. Together, these findings support a role for astrocytes in neuroprotection and raise the intriguing possibility that astrocytes may help mediate the neuroprotective effect of 17beta-E2 and tamoxifen.

Upregulation of preprodynorphin and preproenkephalin mRNA expression by selective activation of group I metabotropic glutamate receptors in characterized primary cultures of rat striatal neurons

Group I metabotropic glutamate receptors (mGluRs) are positively coupled to phosphoinositide hydrolysis, and are expressed in medium spiny neurons of rat striatum in vivo. By modifying intracellular activities, this group of mGluRs is involved in the regulation of gene expression important for neuroplasticity. To characterize the regulatory role of group I receptors in opioid peptide mRNA expression in vitro, primary cultures of striatal cells were prepared from neonatal day-1 rat pups. Cells were cultured in the presence of a mitotic inhibitor, cytosine arabinoside, which generated predominant neuronal cell cultures after 12–14 days in culture as demonstrated by dense immunostaining of more than 90% of cultured cells to a specific marker for neurons (microtubule-associated protein) but not for astroglial cells (glial fibrillary acidic protein). The vast majority of neurons (>90%) were also verified as GABAergic neurons according to their positive immunoreactivity to GABA and glutamic acid decarboxylase-65/67 antibodies. A few large neurons (<5%) showed high levels of choline acetyltransferase immunoreactivity, presumably cholinergic neurons. To confirm group I mGluR expression in cultured neurons, both in situ hybridization and immunocytochemistry were performed, which detected moderate levels of mGluR1 and mGluR5 mRNAs and protein products in most neurons (>70%), respectively. On this culture system, quantitative in situ hybridization was then performed to quantify changes in preprodynorphin (PPD) and preproenkephalin (PPE) mRNA levels in response to mGluR stimulation. Acute incubation of a non-subgroup selective agonist, 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD), increased PPD and PPE mRNA levels in a concentration-dependent manner (176 and 189% over control for PPD and PPE after 100 μM ACPD incubation, respectively). Application of a selective group I agonist, 3,5-dihydroxyphenylglycine (DHPG), produced much greater induction of either mRNA (285 and 289% over control for PPD and PPE after 100 μM DHPG incubation, respectively). Co-incubation of a selective group I antagonist, n-phenyl-7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxamide (PHCCC), blocked both ACPD- and DHPG-induced PPD/PPE expression. These data demonstrate the validity of a neuronal cell culture model for studying the molecular regulation of opioid gene expression in vitro. Selective activation of identified group I mGluRs facilitates constitutive expression of PPD and PPE mRNAs in cultured striatal neurons.

Protective effect of l-Deprenyl against apoptosis induced by okadaic acid in cultured neuronal cells

l-Deprenyl, an irreversible MAO-B (monoamine oxidase B, EC 1.4.3.4) inhibitor, is used for the treatment of Parkinson’s disease and to delay the progression of Alzheimer’s disease. l-Deprenyl also exhibits protective effects against neuronal apoptosis which are independent of its ability to inhibit MAO-B. The purpose of this study was to compare the antiapoptotic efficacy of l-deprenyl against different types of apoptotic inducers in three neuronal cell culture models. The level of apoptosis was quantified by measuring the activation of caspase-3 enzyme, which is the main apoptotic executioner in neuronal cells. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and LDH (lactate dehydrogenase, EC 1.1.1.27) assays were used to demonstrate the cytotoxic response of apoptotic treatments. Our results showed that okadaic acid, an inhibitor of protein phosphatase 1 and 2A, induced a prominent increase in caspase-3 activity both in cultured hippocampal and cerebellar granule neurons as well as in Neuro-2a neuroblastoma cells. Interestingly, l-deprenyl offered a significant protection against the apoptotic response induced by okadaic acid in all three neuronal models. The best protection appeared at the concentration level of 10 −9 M. l-Deprenyl also provided a protection against apoptosis after AraC (cytosine β- d-arabinoside) treatment in hippocampal neurons and Neuro-2a cells and after etoposide treatment in Neuro-2a cells. However, l-deprenyl did not offer any protection against apoptosis caused by serum withdrawal or potassium deprivation. Okadaic acid treatment in vivo is known to induce an Alzheimer’s type of hyperphosphorylation of tau protein, formation of β-amyloid plaques, and a severe memory impairment. Our results show that the okadaic acid model provides a promising tool to study the molecular basis of Alzheimer’s disease and to screen the neuroprotective capacity of l-deprenyl derivatives.

Developmental Expression of Wild-Type and Mutant Presenilin-1 in Hippocampal Neurons from Transgenic Mice: Evidence for Novel Species-Specific Properties of Human Presenilin-1

Presenilins 1 (PS1) and 2 (PS2) are multispanning transmembrane proteins associated with familial Alzheimer disease (FAD). They are developmentally regulated, being expressed at highest levels during neuronal differentiation and are sustained at a lower level throughout life. We investigated the distribution and metabolism of endogenous murine PS1 as well as human wild-type (wtPS1) and the familial AD Met146Leu (M146L) mutant presenilins in dissociated cultures of hippocampal neurons derived from control and transgenic mice. We found that the PS1 endoproteolytic fragments and, to a lesser extent, the full-length protein, were expressed as early as day 3 post-plating. Both species increased until the cells were fully differentiated at day 12. Confocal microscopy revealed that presenilin is present in the Golgi and endoplasmic reticulum and, as in punctate, vesicle-like structures within developing neurites and growth cones. Using a human-specific PS1 antibody, we were able to independently examine the distribution of the transgenic protein which, although similar to the endogenous, showed some unique qualities. These included (i) some heterogeneity in the proteolytic fragments of human PS1; (ii) significantly reduced levels of full-length human PS1, possibly as a result of preferential processing; and (iii) a more discrete intracellular distribution of human PS1. Colocalization with organelle-specific proteins revealed that PS1 was located in a diffuse staining pattern in the MAP2-positive dendrites and in a punctate manner in GAP43-positive axons. PS1 showed considerable overlap with GAP43, particularly at the growth cones. Similar patterns of PS1 distribution were detected in cultures derived from transgenic animals expressing human wild-type or mutant presenilins. The studies demonstrate that mutant presenilins are not grossly different in their processing or distribution within cultured neurons, which may represent more physiological models as compared to transfection systems. Our data also suggest that the molecular pathology associated with PS1 mutations results from subtle alterations in presenilin function, which can be further investigated using these transgenic neuronal cell culture models.