Katalin Schlett

Glutamate as a Modulator of Embryonic and Adult Neurogenesis

It has been widely accepted that neurogenesis continues throughout life. Neural stem cells can be found in the ventricular zone of the embryonic and in restricted regions of the adult central nervous system, including subventricular and subgranular zones of the hippocampal dentate gyrus. The network of signaling mechanisms determining whether neural stem cells remain in a proliferative state or differentiate is only partly discovered. Recent advances indicate that glutamate (Glu), the predominant excitatory neurotransmitter in mature neurons, can influence immature neural cell proliferation and differentiation, as well. Despite many similarities, Glu actions on neurogenesis in the developing and adult brain show distinct differences and are far from being clear. Due to alterations of Glu transport mechanisms, extracellular Glu level is high in the embryonic CNS. Glu acts non-synaptically on dividing progenitors either by directly activating ionotropic and/or metabotropic Glu receptors or can influence other cells which are located in the vicinity of proliferating cells and produce molecules regulating neural precursor cell proliferation by other mechanisms. Due to the complexity of signaling pathways and to regional differences in neural precursors, Glu can influence proliferation and neuronal commitment as well, and acts as a positive regulator of neurogenesis. Brain injuries like ischemia, epilepsy or stress lead to severe neuronal death and additionally, influence neurogenesis, as well. Glu homeostasis is altered under these pathological circumstances, implying that therapeutic treatments mediating Glu signaling might be useful to increase neuronal replacement after cell loss in the brain.

Retinoic acid induced neural differentiation in a neuroectodermal cell line immortalized by p53 deficiency.

Neuroepithelial cell lines were established from cerebral vesicles of 9‐day‐old mouse embryos lacking functional p53 genes (Livingstone et al: Cell 70:923–935, 1992). All‐trans retinoic acid (RA) induced bulk formation of neurons both in several p53‐deficient neuroepithelial cell lines and in wild‐type neural cells derived from early embryonic (E9–E12) forebrain vesicles. Forty‐eight‐hour treatment with 10−6 MRA was necessary and sufficient to initiate neuron formation by p53‐/‐‐progenitors, but neuronal characteristics appeared with a delay of 3–4 days. The first appearance of cells with astroglial features followed that of neurons with a further delay of 4–5 days. The establishment of neuronal phenotypes involved minimally three rounds of cell cycle. Future neurons were sorted out from substrate‐attached cells and were characterized by a specific rearrangement of nestin‐immunoreactive filaments. The formation of neuronal phenotypes was not synchronized within the RA‐treated cell populations. The data indicate that RA, which promotes the initiation of neural differentiation, cannot function as a direct regulator of cell‐fate decisions made by neural progenitor cells. J. Neurosci. Res. 47:405–415, 1997.

Protein Kinase D Controls the Integrity of Golgi Apparatus and the Maintenance of Dendritic Arborization in Hippocampal Neurons

Protein kinase D (PKD) is known to participate in various cellular functions, including secretory vesicle fission from the Golgi and plasma membrane-directed transport. Here, we report on expression and function of PKD in hippocampal neurons. Expression of an enhanced green fluorescent protein (EGFP)-tagged PKD activity reporter in mouse embryonal hippocampal neurons revealed high endogenous PKD activity at the Golgi complex and in the dendrites, whereas PKD activity was excluded from the axon in parallel with axonal maturation. Expression of fluorescently tagged wild-type PKD1 and constitutively active PKD1(S738/742E) (caPKD1) in neurons revealed that both proteins were slightly enriched at the trans-Golgi network (TGN) and did not interfere with its thread-like morphology. By contrast, expression of dominant-negative kinase inactive PKD1(K612W) (kdPKD1) led to the disruption of the neuronal Golgi complex, with kdPKD1 strongly localized to the TGN fragments. Similar findings were obtained from transgenic mice with inducible, neuron-specific expression of kdPKD1-EGFP. As a prominent consequence of kdPKD1 expression, the dendritic tree of transfected neurons was reduced, whereas caPKD1 increased dendritic arborization. Our results thus provide direct evidence that PKD activity is selectively involved in the maintenance of dendritic arborization and Golgi structure of hippocampal neurons.

Unconventional translation initiation of human trypsinogen 4 at a CUG codon with an N-terminal leucine A possible means to regulate gene expression

Summary Chromosomal rearrangements apparently account for the presence of a primate‐specific gene (protease serine 3) in chromosome 9. This gene encodes, as the result of alternative splicing, both mesotrypsinogen and trypsinogen 4. Whereas mesotrypsinogen is known to be a pancreatic protease, neither the chemical nature nor biological function of trypsinogen 4 has been explored previously. The trypsinogen 4 sequence contains two predicted translation initiation sites: an AUG site that codes for a 72‐residue leader peptide on Isoform A, and a CUG site that codes for a 28‐residue leader peptide on Isoform B. We report studies that provide evidence for the N‐terminal amino acid sequence of trypsinogen 4 and the possible mechanism of expression of this protein in human brain and transiently transfected cells. We raised mAbs against a 28‐amino acid synthetic peptide representing the leader sequence of Isoform B and against recombinant trypsin 4. By using these antibodies, we isolated and chemically identified trypsinogen 4 from extracts of both post mortem human brain and transiently transfected HeLa cells. Our results show that Isoform B, with a leucine N terminus, is the predominant (if not exclusive) form of the enzyme in post mortem human brain, but that both isoforms are expressed in transiently transfected cells. On the basis of our studies on the expression of a series of trypsinogen 4 constructs in two different cell lines, we propose that unconventional translation initiation at a CUG with a leucine, rather than a methionine, N terminus may serve as a means to regulate protein expression.

Schedule of NMDA receptor subunit expression and functional channel formation in the course of in vitro-induced neurogenesis

NE‐7C2 neuroectodermal cells derived from forebrain vesicles of p53‐deficient mouse embryos (E9) produce neurons and astrocytes in vitro if induced by all‐trans retinoic acid. The reproducible morphological stages of neurogenesis were correlated with the expression of various NMDA receptor subunits. RT‐PCR studies revealed that GluRε1 and GluRε4 subunit mRNAs were transcribed by both non‐induced and neuronally differentiated cells. GluRε3 subunit mRNAs were not synthesized by NE‐7C2 cells and increased numbers of messages from the GluRε2 gene were detected only after neural network formation. The presence of the GluRζ1 protein was detected throughout neural induction, whereas retinoic acid‐induced neuron formation elevated the amount of exon 21 (C1)‐ and exon 22 (C2)‐containing GluRζ1 mRNAs and resulted in the appearance of exon 5 (N1)‐containing transcripts. NMDA‐elicited Ca2+‐signals were detected only in cells displaying neuronal morphology, but preceding the appearance of synapsin‐I immunoreactivity. Our findings demonstrated that, in spite of the presence of subunits necessary for channel formation, functional channels were formed by NE‐7C2 cells no sooner than the time of neurite maturation. The data show that the cell line provides a suitable model to analyse the mechanisms involved in NMDA receptor gene expression before the appearance of synaptic communication.

Effects of Vinpocetine on mitochondrial function and neuroprotection in primary cortical neurons

Vinpocetine (ethyl apovincaminate), a synthetic derivative of the Vinca minor alkaloid vincamine, is widely used for the treatment of cerebrovascular-related diseases. One of the proposed mechanisms underlying its action is to protect against the cytotoxic effects of glutamate overexposure. Glutamate excitotoxicity leads to the disregulation of mitochondrial function and neuronal metabolism. As Vinpocetine has a binding affinity to the peripheral-type benzodiazepine receptor (PBR) involved in the mitochondrial transition pore complex, we investigated whether neuroprotection can be at least partially due to Vinpocetines effects on PBRs. Neuroprotective effects of PK11195 and Ro5-4864, two drugs with selective and high affinity to PBR, were compared to Vinpocetine in glutamate excitotoxicity assays on primary cortical neuronal cultures. Vinpocetine exerted a neuroprotective action in a 1-50microM concentration range while PK11195 and Ro5-4864 were only slightly neuroprotective, especially in high (>25microM) concentrations. Combined pretreatment of neuronal cultures with Vinpocetine and PK11195 or Ro5-4864 showed increased neuroprotection in a dose-dependent manner, indicating that the different drugs may have different targets. To test this hypothesis, mitochondrial membrane potential (MMP) of cultured neurons was measured by flow cytometry. 25microM Vinpocetine reduced the decrease of mitochondrial inner membrane potential induced by glutamate exposure, but Ro5-4864 in itself was found to be more potent to block glutamate-evoked changes in MMP. Combination of Ro5-4864 and Vinpocetine treatment was found to be even more effective. In summary, the present results indicate that the neuroprotective action of vinpocetine in culture can not be explained by its effect on neuronal PBRs alone and that additional drug targets are involved.

In vitro pattern formation during neurogenesis in neuroectodermal progenitor cells immortalized by p53-deficiency

In vitro neural differentiation was induced in a p53‐deficient immortalized neuroectodermal progenitor cell line, NE‐4C, by treatment with retinoic acid [K. Schlett and E. Madarász (1997) J. Neurosci. Res.47, 405–416]. Rearrangement of nestin filaments was an early marker of neuron‐formation. The increase in neurofilament protein content was accompanied by a decrease in the expression of nestin filaments in induced precursors. Cells with astroglial features appeared with a delay of 4–5 days compared to the appearence of neurons.

Dynamics of cell aggregation during in vitro neurogenesis by immortalized neuroectodermal progenitors

Early events of in vitro neuronal development were studied by inducing neuron formation in a neuroectodermal cell line, NE‐4C/A3, derived from the embryonic forebrain vesicles of p53‐deficient mice. Neuronal differentiation was initiated by treating the cells with all‐trans retinoic acid (RA). By the second day of RA treatment compact cell aggregates were formed. The first signs of neuronal cell fate decision were revealed inside the aggregates. To elucidate the process of aggregate formation, the dynamics of cell clustering and the migration of individual cells were investigated by a novel computer‐controlled videomicroscopic system. Besides real‐time observation of cell motility, the system allowed statistical analysis of large sets of data providing quantitative evaluation of cell locomotion during an early, critical phase of RA induced neuron formation. The results showed that chemoattractants did not play a principal role in cell aggregation. Retinoic acid, on the other hand, was found to cause a rapid decrease in the average migratory velocity without changing the randomness of migratory routes. The data indicated that aggregation was facilitated by increased cohesion upon incident collision of randomly encountering cells. The resulting compact cell clusters provided the structural conditions for contact communication apparently needed for the neuronal differentiation of NE‐4C/A3 cells. J. Neurosci. Res. 60:184–194, 2000

Ischemia-induced increase in long-term potentiation is warded off by specific calpain inhibitor PD150606

In the present study, the effect of specific, membrane-permeable calpain inhibitor, PD150606, was analysed on synaptic efficacy in in vitro brain slices experiments after ischemic insult of rats in vivo, and on cell viability in a glutamate excitotoxicity test in mouse cell culture. Bilateral common carotid artery ligation (BCCL) for 24 h markedly increased calpain activity and enhanced LTP induction in rat hippocampus, although the CA1 layer significantly shrank. The enhancement of LTP could be diminished by short-term application of PD150606 (40 microM) into the perfusion solution. Intracerebroventricular administration of PD150606 (100 microM) parallel with ischemic insult prevented LTP and effectively inhibited hippocampal calpain activity. Intracerebroventricularly applied PD150606 inhibited the CA1 layer shrinkage after common carotid ligation. High level of exogenous glutamate caused marked decrease of cell viability in mouse cerebellar granule cell cultures, which could be partly warded off by 20 microM PD150606. Our data witness that calpain action is intricately involved in the regulation of synaptic efficacy.

Changes of KCl sensitivity of proliferating neural progenitors during in vitro neurogenesis

The effects of KCl‐treatment on the survival and proliferation of NE‐4C self‐renewing neural progenitor cells were investigated during early phases of in vitro induced neurogenesis. NE‐4C cells, derived from the anterior brain vesicles of embryonic mouse (E9), divided continuously under non‐inducing conditions, but acquired neuronal features within 6 days, if induced by all‐trans retinoic acid (RA). During the first 2 days of induction, the cells went on proliferating and did not show signs of morphological differentiation. In this stage, the resting membrane potential of RA‐induced cells adopted more negative values in comparison to non‐induced ones. Despite the increased membrane polarity and K+ conductance, addition of 20–50 mM KCl failed to elicit inward Na+ currents and did not induce an increase in the intracellular Ca+ level. Long‐term treatment with 25 mM KCl, on the other hand, resulted in a selective loss of cells committed to neuronal fate by both decreasing the rate of cell proliferation and increasing the rate of cell death. The data indicate that the viability and proliferation of neural progenitors are influenced by extracellular K+‐level in a differentiation stage‐dependent manner.