Screen for mutations affecting development of zebrafish neural crest

The neural crest provides a useful model to learn how cell fate diversification is regulated during vertebrate development. Our approach is to isolate zebrafish mutations in which the development of neural crest derivatives is disrupted, in order to learn about the underlying genetic mechanisms. We describe a screen in which parthenogenetic diploid embryos are examined both for visible phenotypes and for cellular defects in neural crest-derived sensory neurons recognized immunohistochemically. We present preliminary results from this screen and briefly describe a few representative mutations. We also discuss the general utility of our strategy and comment on the future directions of this approach. © 1996 Wiley-Liss, Inc.     

beta-Amyloid-induced neuronal apoptosis requires c-Jun N-terminal kinase activation

beta-Amyloid (A beta) has been strongly implicated in the pathophysiology of Alzheimer's disease (AD), but the means by which the aggregated form of this molecule induces neuronal death have not been fully defined. Here, we examine the role of the c-Jun N-terminal kinases (JNKs) and of their substrate, c-Jun, in the death of cultured neuronal PC12 cells and sympathetic neurons evoked by exposure to aggregated A beta. The activities of JNK family members increased in neuronal PC12 cells within 2 h of A beta treatment and reached 3--4-fold elevation by 6 h. To test the role of these changes in death caused by A beta, we examined the effects of CEP-1347 (KT7515), an indolocarbazole that selectively blocks JNK activation. Inclusion of CEP-1347 (100--300 nM) in the culture medium effectively blocked the increases in cellular JNK activity caused by A beta and, at similar concentrations, protected both PC12 cells and sympathetic neurons from A beta-evoked-death. Effective protection required addition of CEP-1347 within 2 h of A beta treatment, indicating that the JNK pathway acts relatively proximally and as a trigger in the death mechanism. A dominant-negative c-Jun construct also conferred protection from A beta-evoked death, supporting a model in which JNK activation contributes to death via activation of c-Jun. Finally, CEP-1347 blocked A beta-stimulated activation of caspase-2 and -3, placing these downstream of JNK activation. These observations implicate the JNK pathway as a required element in death evoked by A beta and hence identify it as a potential therapeutic target in AD.     

Inhibition of insulin-like growth factor-1 receptor and IRS-2 signaling by ethanol in SH-SY5Y neuroblastoma cells

The effect of ethanol on insulin-like growth factor-1 (IGF-I)-mediated signal transduction and functional activation in neuronal cells was examined. In human SH-SY5Y neuroblastoma cells, ethanol inhibited tyrosine autophosphorylation of the IGF-I receptor. This corresponded to the inhibition of IGF-I-induced phosphorylation of p42/p44 mitogen-activated/extracellular signal-regulated protein kinase (MAPK) by ethanol. Insulin-related substrate-2 (IRS-2) and focal adhesion kinase phosphorylation were reduced in the presence of ethanol, which corresponded to the prevention of lamellipodia formation (30 min). By contrast, ethanol had no effect on Shc phosphorylation when measured up to 1 h, and did not affect the association of Grb-2 with Shc. Neurite formation at 24 h was similarly unaffected by ethanol. The data indicate that the IGF-I receptor is a target for ethanol in SH-SY5Y cells However, there is diversity in the sensitivity of signaling elements within the IGF-I receptor tyrosine kinase signaling cascades to ethanol, which can be related to the inhibition of specific functional events in neuronal activation.     

Analysis of a novel mechanism of neuronal toxicity produced by an apolipoprotein E-derived peptide

The apolipoprotein E (apoE)-derived peptide (141-155)2 has a neurotoxic effect, implying that apoE itself could be a source of toxicity in Alzheimer's disease brain. We characterized the toxicity of this peptide on superior cervical ganglion (SCG) neurons and compared the death with the apoptotic death that occurs after nerve growth factor (NGF) deprivation in these cells. A dose of 10 microM apoE (141-155)2 resulted in the death of approximately 50% of the neurons within 24 h. Nuclear condensation and DNA fragmentation preceded the death. However, most inhibitors of NGF deprivation-induced death, including the caspase inhibitor Boc-aspartyl(O-methyl)fluoromethyl ketone and genetic deletion of bax-/-, had no effect on the toxicity. Inclusion of depolarizing levels of potassium did block the toxicity. Receptor-associated peptide (RAP), an antagonist for apoE receptors, did not protect cells in either SCG or hippocampal cultures. In addition, RAP had no effect on internalization of the apoE peptide. These data support the observation that apoE (141-155)2 is neurotoxic but suggest that the neurotoxicity is distinct from classical apoptosis or necrosis. Furthermore, these results indicate that the toxic effect may occur independently of members of the low-density lipoprotein receptor gene family.     

Increased expression of rat synuclein in the substantia nigra pars compacta identified by mRNA differential display in a model of developmental target injury

Human alpha-synuclein was identified on the basis of proteolytic fragments derived from senile plaques of Alzheimer's disease, and it is the locus of mutations in some familial forms of Parkinson's disease. Its normal function and whether it may play a direct role in neural degeneration remain unknown. To explore cellular responses to neural degeneration in the dopamine neurons of the substantia nigra, we have developed a rodent model of apoptotic death induced by developmental injury to their target, the striatum. We find by mRNA differential display that synuclein is up-regulated in this model, and thus it provides an opportunity to examine directly whether synuclein plays a role in the death of these neurons or, alternatively, in compensatory responses. Up-regulation of mRNA is associated with an increase in the number of neuronal profiles immunostained for synuclein protein. At a cellular level, synuclein is almost exclusively expressed in normal neurons, rather than apoptotic profiles. Synuclein is up-regulated throughout normal postnatal development of substantia nigra neurons, but it is not further up-regulated during periods of natural cell death. We conclude that up-regulation of synuclein in the target injury model is unlikely to mediate apoptotic death and propose that it may be due to a compensatory response in neurons destined to survive.     

蛋白激酶A介导慢性压迫背根节神经元的肾上腺素能兴奋反应

本实验在奶节（ＤＲＧ）慢性压迫模型上,采用离体灌流ＤＲＧ和单纤维记录神经元自发放电的方法研究了初级感觉神经元的交感－感觉耦联作用及其细胞内机制。用外源性支甲肾上腺素（ＮＥ,１０μｍｏｌ／Ｌ）浸浴损伤的ＤＲＧ时,在９５个ＤＲＧ神经元中有８５个自发放电的神经元产生明显反应。其中,４４个呈现单纯兴奋效应,２１个表现先兴奋后抑制效应,６个出现兴奋－抑制交替振荡现象,１４个表现抑制效应。ＮＥ对损伤神经元的兴     

Bone marrow-derived mesenchymal stem cells already express specific neural proteins before any differentiation

Bone marrow mesenchymal stem cells (MSC) are multipotent cells. To explain their plasticity, we postulated that undifferentiated MSC may express proteins from other tissues such as neuronal tissues. MSC are obtained by two different approaches: plastic adhesion or negative depletion (RosetteSep and magnetic beads CD45/glycophorin A). MSC are evaluated through FACS analysis using a panel of antibodies (SH2, SH3, CD14, CD33, CD34, CD45, etc.). To confirm the multipotentiality in vitro, we have differentiated MSC into adipocytes, chondrocytes, osteocytes, and neuronal/glial cells using specific induction media. We have evaluated neuronal and glial proteins (Nestin, Tuj-I, betaIII Tubulin, tyrosine hydroxylase [TH], MAP-2, and GFAP) by using flow cytometry, Western blots, and RT-PCR. We found that MSC constituently express native immature neuronal proteins such as Nestin and Tuj-1. After only five passages, MSC can already express more mature neuronal or glial proteins, such as TH, MAP-2, and GFAP, without any specific induction. We noticed an increase in the expression of more mature neuronal/glial proteins (TH, MAP-2, and GFAP) after exposure to neural induction medium, thus confirming the differentiation of MSC into neurons and astrocytes. The constitutive expression of Nestin or Tuj-1 by MSC suggests that these cells are "multidifferentiated" cells and thus can retain the ability for neuronal differentiation, enhancing their potentiality to be employed in the treatment of neurological diseases.     

Computational, spectroscopic, and resonant mirror biosensor analysis of the interaction of adrenodoxin with native and tryptophan-modified NADPH-adrenodoxin reductase

In steroid hydroxylation system in adrenal cortex mitochondria, NADPH-adrenodoxin reductase (AR) and adrenodoxin (Adx) form a short electron-transport chain that transfers electrons from NADPH to cytochromes P-450 through FAD in AR and [2Fe-2S] cluster in Adx. The formation of [AR/Adx] complex is essential for the electron transfer mechanism in which previous studies suggested that AR tryptophan (Trp) residue(s) might be implicated. In this study, we modified AR Trps by N-bromosuccinimide (NBS) and studied AR binding to Adx by a resonant mirror biosensor. Chemical modification of tryptophans caused inhibition of electron transport. The modified protein (AR*) retained the native secondary structure but showed a lower affinity towards Adx with respect to AR. Activity measurements and fluorescence data indicated that one Trp residue of AR may be involved in the electron transferring activity of the protein. Computational analysis of AR and [AR/Adx] complex structures suggested that Trp193 and Trp420 are the residues with the highest probability to undergo NBS-modification. In particular, the modification of Trp420 hampers the correct reorientation of AR* molecule necessary to form the native [AR/Adx] complex that is catalytically essential for electron transfer from FAD in AR to [2Fe-2S] cluster in Adx. The data support an incorrect assembly of [AR*/Adx] complex as the cause of electron transport inhibition.     

Transgenic expression of Cre recombinase from the tyrosine hydroxylase locus

Catecholaminergic neurons are affected in several neurological and psychiatric diseases. Tyrosine hydroxylase (TH) is the first, rate-limiting enzyme in catecholamine synthesis. We report a knockin mouse expressing Cre-recombinase from the 3'-untranslated region of the endogenous Th gene by means of an internal ribosomal entry sequence (IRES). The resulting Cre expression matches the normal pattern of TH expression, while the pattern and level of TH are not altered in the knockin mouse. Crossings with two different LacZ reporter mice demonstrated Cre-mediated genomic recombination in TH expressing tissues. In addition, LacZ was found in some unexpected cell populations (including oocytes), indicating recombination due to transient developmental TH expression. Our novel knockin mouse can be used for generation of tissue-specific or general knockouts (depending on scheme of crossing) in mice carrying genes flanked by loxP sites. This knockin mouse can also be used for tracing cell lineages expressing TH during development.     

GDNF augments survival and differentiation of TH-positive neurons in neural progenitor cells

GDNF plays an important role in the survival and differentiation of primary dopaminergic neurons, but it requires multiple factors for its entire range of activities. This study investigated the effects of GDNF and its cofactors on the development of bFGF-responsive neural progenitor cells (NPCs), mesencephalic and cortical progenitor cells (MP and CP). Various factors were found to have significant inductive effects on the survival and maintenance of these cells in late developmental stages. MP had greater potential than CP to differentiate into dopaminergic neurons. Treatment of NPCs with GDNF and its cofactors enhanced MAP-2 and TH expression, particularly the latter. These findings suggest that NPCs, particularly MP, could develop into more specific neurons if the appropriate factors were applied during the final cell fate specification. They might thus become beneficial sources of donor cells in the treatment of neurological disorders.