Antisense Knockdown of Glutamate Transporters Alters the Subfield Selectivity of Kainate-Induced Cell Death in Rat Hippocampal Slice Cultures

Organotypic rat hippocampal slice cultures were used to study the role of excitatory amino acid transporters (EAATs) in kainate-induced cell death. Expression of the neuronal (EAAT3) or glial (EAAT2) transporters was inhibited with antisense phosphothioate oligonucleotides, and cytotoxicity was assessed with propidium iodide uptake. In control cultures, a concentration of 10 microM kainate was more cytotoxic in CA3 than in CA1. Treatment for 24 h with EAAT3 antisense oligonucleotide decreased kainate toxicity in CA1 but had an opposite effect in CA3. Neither antisense oligonucleotide to EAAT2 nor mismatch oligonucleotide to EAAT3 decreased kainate toxicity in CA1. Immunoblotting with affinity-purified antibodies showed that EAAT3 antisense oligonucleotide decreased selectively EAAT3 but not EAAT2 protein levels, and vice versa. NMDA was more cytotoxic in CA1 than in CA3, and antisense oligonucleotides to either EAAT3 or EAAT2 did not decrease the NMDA effect in CA1 or CA3. Dihydrokainate and DL-threo-beta-hydroxyaspartic acid were more cytotoxic in CA1 than in CA3, suggesting that the higher vulnerability of CA3 to kainate was not the result of its activity as transporter blocker. We conclude that glutamate transporters differentially regulate excitotoxicity in different hippocampal subfields.     

Inhibition of tau phosphorylating protein kinase cdk5 prevents beta-amyloid-induced neuronal death.

The key target of this study was the tau protein kinase II system (TPK II) involving the catalytic subunit cdk5 and the regulatory component p35. TPK II is one of the tau phosphorylating systems in neuronal cells, thus regulating its functions in the cytoskeletal dynamics and the extension of neuronal processes. This research led to demonstration that the treatment of rat hippocampal cells in culture with fibrillary beta-amyloid (Abeta) results in a significant increase of the cdk5 enzymatic activity. Interestingly, the data also showed that the neurotoxic effect of 1-20 microM Abeta on primary cultures markedly diminished with co-incubation of hippocampal cells with the amyloid fibers plus the cdk5 inhibitor butyrolactone I. This inhibitor protected brain cells against Abeta-induced cell death in a concentration dependent fashion. Moreover, death was also prevented by a cdk5 antisense probe, but not by an oligonucleotide with a random sequence. The cdk5 antisense also reduced neuronal expression of cdk5 compared with the random oligonucleotide. The studies indicate that cdk5 plays a major role in the molecular path leading to the neurodegenerative process triggered by the amyloid fibers in primary cultures of rat hippocampal neurons. These findings are of interest in the context of the pathogenesis of Alzheimer's disease.     

Overexpression of heme oxygenase in neuronal cells, the possible interaction with Tau

Increased expression of heme oxygenase-1 (HO-1) is a common feature in a number of neurodegenerative diseases. Interestingly, the spatial distribution of HO-1 expression in diseased brain is essentially identical to that of pathological expression of tau. In this study, we explored the relationship between HO-1 and tau, using neuroblastoma cells stably transfected with sense and antisense HO-1 constructs as well as with the vector alone. In transfected cells overexpressing HO-1, the activity of heme oxygenase was increased, and conversely, the level of tau protein was dramatically decreased when compared with antisense HO-1 or CEP transfected cells. The suppression of tau protein expression was almost completely reversed by zinc-deuteroporphyrin, a specific inhibitor of heme oxygenase activity. The activated forms of ERKs (extracellular signal-regulated kinases) were also decreased in cells overexpressing HO-1 although no changes in the expression of total ERK-1/2 proteins were observed. These data are in agreement with the finding that the expression of tau is regulated through signal cascades including the ERKs, whose activities are modulated by oxidative stresses. The expression of tau and HO-1 may be regulated by oxidative stresses in a coordinated manner and play a pivotal role in the cytoprotection of neuronal cells.     

The Use of Epstein–Barr Virus-Based Shuttle Vectors in Rat PC12 Cells

1. The rat pheochromocytoma PC12 cell line has been a commonly used model for studies of neuronal development, function, and death. Thus the abilityto transfect PC12 cells in an efficient manner and to manipulate their gene expression would enhance the usefulness of these cells.2. We demonstrate that EBV-based vectors provide a useful expression system for gene manipulation in rat PC12 cells.3. The EBV-based vectors replicate episomally in PC12 cells for at least 2months, as evidence by their recovery from the transfected cells and by the digestion of the episomal plasmid with the isoschizomer MboI and DpnI restriction enzymes.3. PC12 cells are efficiently transfected by EBV-based vectors both transiently and stably.4. Transfection of PC12 cells with an EBV-based vector containing tau cDNA in the antisense orientation resulted in a decrease in the level of tau protein in the transfected cells.5. The results demonstrate that EBV-based vectors can be a useful expression system for gene manipulation in PC12 cells.     

β-Amyloid-Induced Neurotoxicity of a Hybrid Septal Cell Line Associated with Increased Tau Phosphorylation and Expression of β-Amyloid Precursor Protein

Abstract: Recent evidence suggests that β-amyloid peptide (β-AP) may induce tau protein phosphorylation, resulting in loss of microtubule binding capacity and formation of paired helical filaments. The mechanism by which β-AP increases tau phosphorylation, however, is unclear. Using a hybrid septal cell line, SN56, we demonstrate that aggregated β-AP_1–40 treatment caused cell injury. Accompanying the cell injury, the levels of phosphorylated tau as well as total tau were enhanced as detected immunochemically by AT8, PHF-1, Tau-1, and Tau-5 antibodies. Alkaline phosphatase treatment abolished AT8 and PHF-1 immunoreactivity, confirming that the tau phosphorylation sites were at least at Ser^199/202 and Ser³⁹⁶. In association with the increase in tau phosphorylation, the immunoreactivity of cell-associated and secreted β-amyloid precursor protein (β-APP) was markedly elevated. Application of antisense oligonucleotide to β-APP reduced expression of β-APP and immunoreactivity of phosphorylated tau. Control peptide β-AP_1–28 did not produce significant effects on tau phosphorylation, although it slightly increased cell-associated β-APP. These results suggest that βAP_1–40-induced tau phosphorylation may be associated with increased β-APP expression in degenerated neurons.     

Spinal Neurokinin NK1 Receptor Down-Regulation and Antinociception: Effects of Spinal NK1 Receptor Antisense Oligonucleotides and NK1 Receptor Occupancy

Abstract: To define the effects of antisense oligonucleotides on spinal neurokinin 1 (NK1) receptor function in nociceptive processing, several antisense oligonucleotides directed against the NK1 receptor mRNA were intrathecally injected into rats via an implanted catheter, and their effect on the behavioural response to formalin injected into the paw was assessed. We observed that there was no significant reduction of pain behaviour or immunostaining of spinal NK1 receptors after repeated daily intrathecal treatment with an antisense oligonucleotide. However, spinal application of substance P (SP) in the antisense oligonucleotide-treated animals resulted in a profound and long-lasting reduction in the behavioural response to formalin injection, and a parallel reduction in the NK1 receptor immunoreactivity normally observed in spinal dorsal horn. Intrathecal SP in the control groups, i.e., rats treated with an oligonucleotide containing four mismatched bases, the corresponding sense oligonucleotide, a mixture of the sense and the antisense oligonucleotides, in each case had no effect. The effects of SP were blocked by NK1 receptor antagonists and were not mimicked by NMDA. The mechanism underlying these effects is not clear. It may be due to partial degradation of the internalised receptors, which cannot be replaced by newly synthesised receptors because of the action of the NK1 antisense oligonucleotide.     

Neurons are protected from excitotoxic death by p53 antisense oligonucleotides delivered in anionic liposomes.

The potential of anionic liposomes for oligonucleotide delivery was explored because the requirement for a net-positive charge on transfection-competent cationic liposome-DNA complexes is ambiguous. Liposomes composed of phosphatidylglycerol and phosphatidylcholine were monodisperse and encapsulated oligonucleotides with 40-60% efficiency. Ionic strength, bilayer charge density, and oligonucleotide chemistry influenced encapsulation. To demonstrate the biological efficacy of this vector, antisense oligonucleotides to p53 delivered in anionic liposomes were tested in an in vitro model of excitotoxicity. Exposure of hippocampal neurons to glutamate increased p53 protein expression 4-fold and decreased neuronal survival to approximately 35%. Treatment with 1 microm p53 antisense oligonucleotides in anionic liposomes prevented glutamate-induced up-regulation of p53 and increased neuronal survival to approximately 75%. Encapsulated phosphorothioate p53 antisense oligonucleotides were neuroprotective at 5-10-fold lower concentrations than when unencapsulated. Replacing the anionic lipid with phosphatidylserine significantly decreased neuroprotection. p53 antisense oligonucleotides complexed with cationic liposomes were ineffective. Neuroprotection by p53 antisense oligonucleotides in anionic liposomes was comparable with that by glutamate receptor antagonists and a chemical inhibitor of p53. Anionic liposomes were also capable of delivering plasmids and inducing transgene expression in neurons. Anionic liposome-mediated internalization of Cy3-labeled oligonucleotides by neurons and several other cell lines demonstrated the universal applicability of this vector.     

Physical and functional interaction of NCX1 and EAAC1 transporters leading to glutamate-enhanced ATP production in brain mitochondria

Glutamate is emerging as a major factor stimulating energy production in CNS. Brain mitochondria can utilize this neurotransmitter as respiratory substrate and specific transporters are required to mediate the glutamate entry into the mitochondrial matrix. Glutamate transporters of the Excitatory Amino Acid Transporters (EAATs) family have been previously well characterized on the cell surface of neuronal and glial cells, representing the primary players for glutamate uptake in mammalian brain. Here, by using western blot, confocal microscopy and immunoelectron microscopy, we report for the first time that the Excitatory Amino Acid Carrier 1 (EAAC1), an EAATs member, is expressed in neuronal and glial mitochondria where it participates in glutamate-stimulated ATP production, evaluated by a luciferase-luciferin system. Mitochondrial metabolic response is counteracted when different EAATs pharmacological blockers or selective EAAC1 antisense oligonucleotides were used. Since EAATs are Na(+)-dependent proteins, this raised the possibility that other transporters regulating ion gradients across mitochondrial membrane were required for glutamate response. We describe colocalization, mutual activity dependency, physical interaction between EAAC1 and the sodium/calcium exchanger 1 (NCX1) both in neuronal and glial mitochondria, and that NCX1 is an essential modulator of this glutamate transporter. Only NCX1 activity is crucial for such glutamate-stimulated ATP synthesis, as demonstrated by pharmacological blockade and selective knock-down with antisense oligonucleotides. The EAAC1/NCX1-dependent mitochondrial response to glutamate may be a general and alternative mechanism whereby this neurotransmitter sustains ATP production, since we have documented such metabolic response also in mitochondria isolated from heart. The data reported here disclose a new physiological role for mitochondrial NCX1 as the key player in glutamate-induced energy production.     

Inhibition of endothelin-1 induced myocardial protein synthesis by an antisense oligonucleotide against the early growth response gene-1

We explored the role of the recently discovered "early growth response gene-1 (Egr-1)" in the induction of myocardial protein synthesis by endothelin-1. Endothelin-1 stimulated protein synthesis (i.e. 3H-phenylalanine incorporation) in isolated adult rat cardiomyocytes more than 2-fold. Addition of a 15mer Egr-1 antisense oligodeoxyribonucleotide complementary to the first 5 codons of the Egr-1 mRNA completely blocked endothelin-induced protein synthesis. A single base mismatch in the oligonucleotide sequence abolished the inhibitory effect. T3-induced stimulation of protein synthesis was unaffected by the antisense oligonucleotide. These results indicate that the Egr-1 gene product is involved (putatively as a third messenger) in the signal transduction cascade initiated by endothelin-1 which eventually culminates in the induction of cardiac protein synthesis.     

Hexokinases link DJ-1 to the PINK1/parkin pathway

Background

Hyperexcitability of neuronal networks can lead to excessive release of the excitatory neurotransmitter glutamate, which in turn can cause neuronal damage by overactivating NMDA-type glutamate receptors and related signaling pathways. This process (excitotoxicity) has been implicated in the pathogenesis of many neurological conditions, ranging from childhood epilepsies to stroke and neurodegenerative disorders such as Alzheimer’s disease (AD). Reducing neuronal levels of the microtubule-associated protein tau counteracts network hyperexcitability of diverse causes, but whether this strategy can also diminish downstream excitotoxicity is less clear.

Methods

We established a cell-based assay to quantify excitotoxicity in primary cultures of mouse hippocampal neurons and investigated the role of tau in exicitotoxicity by modulating neuronal tau expression through genetic ablation or transduction with lentiviral vectors expressing anti-tau shRNA or constructs encoding wildtype versus mutant mouse tau.

Results

We demonstrate that shRNA-mediated knockdown of tau reduces glutamate-induced, NMDA receptor-dependent Ca²⁺ influx and neurotoxicity in neurons from wildtype mice. Conversely, expression of wildtype mouse tau enhances Ca²⁺ influx and excitotoxicity in tau-deficient (Mapt ^−/−) neurons. Reconstituting tau expression in Mapt ^−/− neurons with mutant forms of tau reveals that the tau-related enhancement of Ca²⁺ influx and excitotoxicity depend on the phosphorylation of tau at tyrosine 18 (pY18), which is mediated by the tyrosine kinase Fyn. These effects are most evident at pathologically elevated concentrations of glutamate, do not involve GluN2B–containing NMDA receptors, and do not require binding of Fyn to tau’s major interacting PxxP motif or of tau to microtubules.

Conclusions

Although tau has been implicated in diverse neurological diseases, its most pathogenic forms remain to be defined. Our study suggests that reducing the formation or level of pY18-tau can counteract excitotoxicity by diminishing NMDA receptor-dependent Ca²⁺ influx.

     

Corticosterone Exacerbates Kainate-Induced Alterations in Hippocampal Tau Immunoreactivity and Spectrin Proteolysis In Vivo

Abstract: Aberrant elevations in intracellular calcium levels, promoted by the excitatory amino acid glutamate, may be a final common mediator of the neuronal damage that occurs in hypoxic-ischemic and seizure disorders. Glutamate and altered neuronal calcium homeostasis have also been proposed to play roles in more chronic neurodegenerative disorders, including Alzheimer's disease. Any extrinsic factors that may augment calcium levels during such disorders may significantly exacerbate the resulting damage. Glucocorticoids (GCs), the adrenal steroid hormones released during stress, may represent one such extrinsic factor. GCs can exacerbate hippocampal damage induced by excitotoxic seizures and hypoxia-ischemia, and we have observed recently that GCs elevate intracellular calcium levels in hippocampal neurons. We now report that the excitotoxin kainic acid (KA) can elicit antigenic changes in the microtubule-associated protein tau similar to those seen in the neurofibrillary tangles of Alzheimer's disease. KA induced a transient increase in the immunoreactivity of hippocampal CA3 neurons towards antibodies that recognize aberrant forms of tau (5E2 and Alz-50). The tau immunoreactivity appeared within 3h of KA injection, preceded extensive neuronal damage, and subsequently disappeared as neurons degenerated. KA also caused spectrin breakdown, indicating the involvement of calcium-dependent proteases. Physiological concentrations of corticosterone (the species-typical GC of rats) enhanced the neuronal damage induced by KA and, critically, enhanced the intensity of tau immunoreactivity and spectrin breakdown. Moreover, the GC enhancement of spectrin proteolysis was prevented by energy supplementation, supporting the hypothesis that GC disruption of calcium homeostasis in the hippocampus is energetic in nature. Taken together, these findings demonstrate that neurofibrillary tangle-like alterations in tau, and spectrin breakdown, can be induced by excitatory amino acids and exacerbated by GCs in vivo.     

NGF mediates the neuroprotective effect of the beta2-adrenoceptor agonist clenbuterol in vitro and in vivo: evidence from an NGF-antisense study.

Previous studies in our laboratory suggested that neuroprotective effects of the beta2-adrenoceptor agonist clenbuterol in vitro and in vivo occurred due to enhanced synthesis of nerve growth factor. The aim of the present study was to evaluate the effects of a phosphothioated NGF oligodeoxynucleotide on neuroprotection by clenbuterol in vitro and in vivo. After clenbuterol treatment (1-100 microM) an increase in nerve growth factor mRNA and protein levels (200-300% of control) was observed in primary cultures of rat cortical astrocytes. Nerve growth factor antisense oligonucleotide (0.3-1 microM for 3 days) reduced the content of nerve growth factor protein in the medium of the astrocytes concentration-dependently to 20% of control level. Nerve growth factor content in the medium of mixed hippocampal cells was reduced to 55% of sister cultures receiving the vehicle or a random control oligonucleotide. In mixed hippocampal cultures pretreated with random oligonucleotide (1 microM, 30 h), clenbuterol (10 microM) reduced the percentage of damaged neurons after glutamate exposure (0.5 mM, 1 h) to 17%. Pretreatment with nerve growth factor antisense oligonucleotide (1 microM) for 30 h before glutamate incubation blocked the protective effect of clenbuterol. In vivo, clenbuterol (0.01-0.1 mg/kg) reduced the infarct volume in a rat model of permanent focal cerebral ischemia dose-dependently. Nerve growth factor antisense oligonucleotides injected into the cortical tissue before ischemia abolished the cerebroprotective effect of clenbuterol. Our results indicate that the nerve growth factor antisense oligonucleotide presented in this study is a useful tool to investigate the effects of nerve growth factor knock down. By using the nerve growth factor antisense oligonucleotide we could demonstrate that nerve growth factor mediated the neuroprotective effects of the beta2-adrenoceptor agonist clenbuterol in vitro and in vivo.     

Phosphorylation inhibits turnover of the tau protein by the proteasome: influence of RCAN1 and oxidative stress

Hyperphosphorylated tau proteins accumulate in the paired helical filaments of neurofibrillary tangles seen in such tauopathies as Alzheimer's disease. In the present paper we show that tau turnover is dependent on degradation by the proteasome (inhibited by MG132) in HT22 neuronal cells. Recombinant human tau was rapidly degraded by the 20 S proteasome in vitro, but tau phosphorylation by GSK3beta (glycogen synthase kinase 3beta) significantly inhibited proteolysis. Tau phosphorylation was increased in HT22 cells by OA [okadaic acid; which inhibits PP (protein phosphatase) 1 and PP2A] or CsA [cyclosporin A; which inhibits PP2B (calcineurin)], and in PC12 cells by induction of a tet-off dependent RCAN1 transgene (which also inhibits PP2B). Inhibition of PP1/PP2A by OA was the most effective of these treatments, and tau hyperphosphorylation induced by OA almost completely blocked tau degradation in HT22 cells (and in cell lysates to which purified proteasome was added) even though proteasome activity actually increased. Many tauopathies involve both tau hyperphosphorylation and the oxidative stress of chronic inflammation. We tested the effects of both cellular oxidative stress, and direct tau oxidative modification in vitro, on tau proteolysis. In HT22 cells, oxidative stress alone caused no increase in tau phosphorylation, but did subtly change the pattern of tau phosphorylation. Tau was actually less susceptible to direct oxidative modification than most cell proteins, and oxidized tau was degraded no better than untreated tau. The combination of oxidative stress plus OA treatment caused extensive tau phosphorylation and significant inhibition of tau degradation. HT22 cells transfected with tau-CFP (cyan fluorescent protein)/tau-GFP (green fluorescent protein) constructs exhibited significant toxicity following tau hyperphosphorylation and oxidative stress, with loss of fibrillar tau structure throughout the cytoplasm. We suggest that the combination of tau phosphorylation and tau oxidation, which also occurs in tauopathies, may be directly responsible for the accumulation of tau aggregates.     

Establishment of mouse-immortalized hybrid clones expressing characteristics of differentiated neurons derived from the cerebellar and brain stem regions.

Two clonal immortalized neurons designated CL8c4.7 and CL8a5.2 were established by somatic cell fusion between a hypoxanthine phosphoribosyltransferase-(HPRT-) deficient neuroblastoma N18TG2 and newborn mouse cerebellar/brain stem neurons. In the serum-containing medium without extra differentiating agents, both clones exhibited a morphology of differentiated neurons. They contained high levels of glutamate but no gamma-aminobutyric acid (GABA). The CL8a5.2 clone synthesized choline acetyltransferase and serotonin. In immunocytochemical studies, both clones expressed 200 kD neurofilament protein, neuron-specific enolase, microtubule-associated protein 2 (MAP2), tau protein, neuronal cell adhesion molecule (N-CAM), HNK-1, Thy-1.2, saxitoxin-binding sodium channel protein, and glutamate. Synaptophysin immunoreactivity was identified in the neuritic terminals of CL8c4.7 cells. Most of these antigens were barely detectable on N18TG2 cells. Electrophysiologically, both clones generated action potentials in response to electrical stimuli. The hybrid clones that express characteristics of differentiated neurons derived from the cerebellar and brain stem regions might be invaluable for the study of the molecular basis of neuronal differentiation and degeneration in these regions.     

Differential Changes in Phosphorylation of Tau at PHF-1 and 12E8 Epitopes During Brain Ischemia and Reperfusion in Gerbils

Cortical neurons are vulnerable to ischemic insult, which may cause cytoskeletal changes and neurodegeneration. Tau is a microtubule-associated protein expressed in neuronal and glial cells. We examined the phosphorylation status of tau protein in the gerbil brain cortex during 5 min ischemia induced by bilateral common carotid artery occlusion followed by reperfusion for 20 min to 7 days. Control brain homogenates contained 63, 65 and 68 kD polypeptides of tau immunoreactive with Alz 50, Tau 14 and Tau 46 antibodies raised against non-phosphorylated tau epitopes. Gerbil tau was also immunoreactive with some (PHF-1 and 12E8) but not all (AT8, AT100, AT180 and AT270) antibodies raised against phosphorylated tau epitopes. PHF-1 recognized a single 68 kD polypeptide and 12E8 bound the 63 kD polypeptide. During 5 min ischemia, PHF-1 immunoreactivity declined to 6%, then recovered to control levels after 20 min of blood recirculation and subsequently increased above control values 3 and 7 days later. In contrast, 12E8 immunoreactivity remained stable during ischemia and reperfusion. Our results suggest that the two phosphorylated epitopes of tau are regulated by different mechanisms and may play different roles in microtubule dynamics. They may also define various pools of neuronal/glial cells vulnerable to ischemia. Special issue dedicated to John P. Blass.     

Evaluation of putative CSF biomarkers in paediatric spinal muscular atrophy (SMA) patients before and during treatment with nusinersen

Spinal muscular atrophy (SMA) is a genetic neurodegenerative disorder leading to immobilization and premature death. Currently, three alternative therapeutic options are available. Therefore, biomarkers that might reflect or predict the clinical course of the individual patient with treatment are of great potential use. Currently, the antisense oligonucleotide nusinersen is the prevalent and longest validated therapy for SMA. We analysed CSF candidate biomarkers for degenerative CNS processes (namely phosphorylated heavy chain (pNf-H), light-chain neurofilaments (NfL), total tau protein (T-Tau), neurogranin, β-secretase BACE-1 and alpha-synuclein) in 193 CSF samples of 44 paediatric SMA types 1, 2 and 3 patients before and under nusinersen treatment and related them to standardized clinical outcome scores in a single-centre pilot study. pNf-H and NfL correlated with disease severity and activity, emphasizing their relevance as marker of neuronal loss and clinical outcome. T-Tau was significantly correlated with motor function scores in SMA type 1 making it an interesting marker for treatment response. Additionally, baseline T-Tau levels were elevated in most SMA patients possibly reflecting the extension of neuronal degeneration in paediatric-onset SMA. Further investigations of these CSF proteins might be beneficial for paediatric SMA subtypes and treatment modalities as an indicator for clinical outcome and should be analysed in larger cohorts.     

Suppression of MAP2 in cultured cerebellar macroneurons inhibits minor neurite formation.

We show here that antisense MAP2 oligonucleotides inhibit neurite outgrowth in cultured cerebellar macroneurons. Unlike control neurons, which first extend a lamellipodial veil followed by a consolidation phase during which the cells extend minor neurites, MAP2-suppressed cells persist with lamellipodia and later become rounded. The induction of microtubules containing tyrosinated tubulin, which parallels neurite outgrowth in control neurons, was blocked under antisense conditions. The small but significant increase in acetylated microtubules was not affected. In contrast, the suppression of tau, which selectively blocks axonal elongation, completely prevented the increase of acetylated microtubules, but did not modify the induction of labile microtubules. These results suggest that MAP2 and tau have different functions: the initial establishment of neurites depends upon MAP2, whereas further neurite elongation depends upon tau and microtubule stabilization.     

Neurite Differentiation Is Modulated in Neuroblastoma Cells Engineered for Altered Acetylcholinesterase Expression

Abstract: Previous observations from several groups suggest that acetylcholinesterase (AChE) may have a role in neural morphogenesis, but not solely by virtue of its ability to hydrolyze acetylcholine. We tested the possibility that AChE influences neurite outgrowth in nonenzymatic ways. With this aim, antisense oligonucleotides were used to decrease AChE levels transiently, and N1E.115 cell lines were engineered for permanently altered AChE protein expression. Cells stably transfected with a sense AChE cDNA construct increased their AChE expression 2.5-fold over the wild type and displayed significantly increased neurite outgrowth. Levels of the differentiation marker, tau, also rose. In contrast, AChE expression in cell lines containing an antisense construct was half of that observed in the wild type. Significant reductions in neurite outgrowth and tau protein accompanied this effect. Overall, these measures correlated statistically with the AChE level ( p < 0.01). Furthermore, treatment of AChE-overexpressing cells with a polyclonal antibody against AChE decreased neurite outgrowth by 43%. We conclude that AChE may have a novel, noncholinergic role in neuronal differentiation.     

Development of the glutamate system in rabbit retina

We have investigated two characteristics of the glutamate system in the developing rabbit retina. 1) Glutamate immunoreactivity was observed at birth within developing processes of four cell types; two of which, photoreceptors and ganglion cells, are known to be glutamatergic in the adult. Two other cell types, type A horizontal cells and amacrine cells, are immunoreactive to both glutamate and GABA at birth, suggesting that endogenous pools of glutamate in GABAergic neurons serve as precursor for GABA synthesis. Thus it appears that endogenous glutamate pools are present within neurons prior to synaptogenesis as part of the early expression of either the glutamate or GABA transmitter phenotype. 2) Analysis of³H-glutamate metabolism during retinal development showed that rapid conversion of glutamate to glutamine does not occur until the second postnatal week, coincident with the expression of Muller (glial) cell activity. In the absence of glial metabolism in the neonate, extracellular concentrations of glutamate remain relatively high and are likely to have major effects on neuronal maturation.Special issue dedicated to Dr. Frederick E. Samson