Jean-Michel Hermel

CRE and TRE sequences of the rat tyrosine hydroxylase promoter are required for TH basal expression in adult mice but not in the embryo

Tyrosine hydroxylase (TH), the rate‐limiting enzyme in the biosynthesis of catecholamine neurotransmitters, is expressed in a restricted number of areas, and subject to numerous regulations during development and in adulthood. Two transcription factor binding sites present in the proximal region of the TH gene, the TPA‐responsive element (TRE) and the c‐AMP responsive element (CRE), have been shown to play important roles in TH gene regulation in vitro. In order to elucidate in vivo the role of these two sites, we produced transgenic mice bearing a 5.3‐kb fragment from the 5′ flanking sequence of the TH gene with mutations in either the CRE‐ or TRE‐sites. Using the intact 5.3‐kb fragment fused to two different reporter genes (HSV1‐tk and lacZ), we show that this promoter fragment is able to specifically direct expression in catecholaminergic tissues both in adult mice and embryos. Interestingly, the CRE‐ and TRE‐mutated transgenes were not expressed in adult mice, contrary to the situation in embryos where they were specifically expressed in catecholaminergic regions. These results demonstrate that the CRE and TRE play an essential role in basal TH expression in adult tissues in vivo. Moreover, they suggest that distinct transcription factors are involved in TH regulation in developing and adult tissues. In support of this, gel mobility shift experiments revealed a complex present only in embryonic tissues. Taken together, these data highlight the diversity of the mechanisms underlying the establishment and maintenance of the catecholaminergic phenotype.

Transgenic expression of CTLA4-Ig by fetal pig neurons for xenotransplantation.

The transplantation of fetal porcine neurons is a potential therapeutic strategy for the treatment of human neurodegenerative disorders. A major obstacle to xenotransplantation, however, is the immune-mediated rejection that is resistant to conventional immunosuppression. To determine whether genetically modified donor pig neurons could be used to deliver immunosuppressive proteins locally in the brain, transgenic pigs were developed that express the human T cell inhibitory molecule hCTLA4-Ig under the control of the neuron-specific enolase promoter. Expression was found in various areas of the brain of transgenic pigs, including the mesencephalon, hippocampus and cortex. Neurons from 28-day old embryos secreted hCTLA4-Ig in vitro and this resulted in a 50% reduction of the proliferative response of human T lymphocytes in xenogenic proliferation assays. Transgenic embryonic neurons also secreted hCTLA4-Ig and had developed normally in vivo several weeks after transplantation into the striatum of immunosuppressed rats that were used here to study the engraftment in the absence of immunity. In conclusion, these data show that neurons from our transgenic pigs express hCTLA4-Ig in situ and support the use of this material in future pre-clinical trials in neuron xenotransplantation.

Evidence for neural stem cells in the medaka optic tectum proliferation zones

Few adult neural stem cells have been characterized in vertebrates. Although teleosts continually generate new neurons in many regions of the brain after embryogenesis, only two types of neural stem cells (NSCs) have been reported in zebrafish: glial cells in the forebrain resembling mammalian NSCs, and neuroepithelial cells in the cerebellum. Here, following our previous studies on dividing progenitors (Nguyen et al. [ 1999 ]: J Comp Neurol 413:385–404.), we further evidenced NSCs in the optic tectum (OT) of juvenile and adult in the medaka, Oryzias latipes. To detect very slowly cycling progenitors, we did not use the commonly used BrdU/PCNA protocol, in which PCNA may not be present during a transiently quiescent state. Instead, we report the optimizations of several protocols involving long subsequent incubations with two thymidine analogs (IdU and CldU) interspaced with long chase times between incubations. These protocols allowed us to discriminate and localize fast and slow cycling cells in OT of juvenile and adult in the medaka. Furthermore, we showed that adult OT progenitors are not glia, as they express neither brain lipid‐binding protein (BLBP) nor glial fibrillary acidic protein (GFAP). We also showed that expression of pluripotency‐associated markers (Sox2, Musashi1 and Bmi1) colocalized with OT progenitors. Finally, we described the spatio‐temporally ordered population of NSCs and progenitors in the medaka OT. Hence, the medaka appears as an invaluable model for studying neural progenitors that will open the way to further exciting comparative studies of neural stem cells in vertebrates.

Intrastriatal sonic hedgehog injection increases Patched transcript levels in the adult rat subventricular zone

The morphogen sonic hedgehog (Shh) is implicated in neural tissue patterning and the growth of brain structures during embryogenesis and postnatal development and is also present in the adult brain. Shh signals through interaction with the tumour suppressor Patched (Ptc). This receptor for Shh is associated with Smoothened (Smo), a protein with high homology to the G‐protein coupled receptors. However, little is known about the transduction mechanisms implicated in Shh signalling in the adult brain. The study described here shows that injection of aminoterminal myristoylated Shh (myrShhN) into the adult rat striatum robustly increases the levels of Ptc transcripts in selective brain areas including the subventricular zone (SVZ). The adult SVZ contains cell progenitors, which can proliferate and differentiate into new neurons and glia. In the myrShhN injected animals, proliferation and differentiation of these SVZ precursor cells were not affected as demonstrated by BrdU incorporation and immunohistochemistry performed with specific antibodies for nestin (uncommitted neural progenitors), PSA‐NCAM (migrating neuroblasts) or GFAP (astrocytes). Together with the presence of Smo expressing cells and amino‐terminal Shh (ShhN) protein in SVZ area of untreated animals, the data presented here supports the hypothesis that the Shh pathway may be activated in the adult brain, and that a niche for Shh signalling exists within the adult SVZ.

Neuroprotective effect of zVAD against the neurotoxin 3-nitropropionic acid involves inhibition of calpain

The contribution of calpains and caspases to cell death has been widely studied using pharmacological inhibitors. Among them, the caspase inhibitor N-benzyloxycarbonyl-valyl-alanyl-aspartyl-fluoromethylketone (zVAD) has been used as a specific caspase inhibitor in nearly 1000 published studies. However, several studies showed that zVAD also behaves as a calpain inhibitor in peripheral cells. The effects of zVAD as a calpain inhibitor have never been assessed in neurodegeneration models. We examined here whether zVAD could reduce neurodegeneration in Huntingtons disease models using the mitochondrial inhibitor 3-nitropropionic acid (3NP). In these models, 3NP toxicity has been shown to require calpain activation. In rats, intra-cerebro-ventricular infusion of zVAD significantly reduced 3NP-induced striatal degeneration, and decreased the 3NP-induced activation of calpain and calpain-dependent cleavage of fodrin. zVAD (100 microM) also blocked 3NP-induced death of cultured striatal neurons. In vitro, zVAD inhibited purified mu-calpain with high affinity (IC50=10 nM). The present data demonstrate that zVAD protects neurons against 3NP through calpain inhibition. This suggests that, in certain models of neuronal death where zVAD showed protective effects, caspases but also calpains may be involved.

Zebrafish midbrain slow-amplifying progenitors exhibit high levels of transcripts for nucleotide and ribosome biogenesis

Investigating neural stem cell (NSC) behaviour in vivo, which is a major area of research, requires NSC models to be developed. We carried out a multilevel characterisation of the zebrafish embryo peripheral midbrain layer (PML) and identified a unique vertebrate progenitor population. Located dorsally in the transparent embryo midbrain, these large slow-amplifying progenitors (SAPs) are accessible for long-term in vivo imaging. They form a neuroepithelial layer adjacent to the optic tectum, which has transitory fast-amplifying progenitors (FAPs) at its margin. The presence of these SAPs and FAPs in separate domains provided the opportunity to data mine the ZFIN expression pattern database for SAP markers, which are co-expressed in the retina. Most of them are involved in nucleotide synthesis, or encode nucleolar and ribosomal proteins. A mutant for the cad gene, which is strongly expressed in the PML, reveals severe midbrain defects with massive apoptosis and sustained proliferation. We discuss how fish midbrain and retina progenitors might derive from ancient sister cell types and have specific features that are not shared with other SAPs.

A TALEN-Exon Skipping Design for a Bethlem Myopathy Model in Zebrafish

Presently, human collagen VI-related diseases such as Ullrich congenital muscular dystrophy (UCMD) and Bethlem myopathy (BM) remain incurable, emphasizing the need to unravel their etiology and improve their treatments. In UCMD, symptom onset occurs early, and both diseases aggravate with ageing. In zebrafish fry, morpholinos reproduced early UCMD and BM symptoms but did not allow to study the late phenotype. Here, we produced the first zebrafish line with the human mutation frequently found in collagen VI-related disorders such as UCMD and BM. We used a transcription activator-like effector nuclease (TALEN) to design the col6a1ama605003-line with a mutation within an essential splice donor site, in intron 14 of the col6a1 gene, which provoke an in-frame skipping of exon 14 in the processed mRNA. This mutation at a splice donor site is the first example of a template-independent modification of splicing induced in zebrafish using a targetable nuclease. This technique is readily expandable to other organisms and can be instrumental in other disease studies. Histological and ultrastructural analyzes of homozygous and heterozygous mutant fry and 3 months post-fertilization (mpf) fish revealed co-dominantly inherited abnormal myofibers with disorganized myofibrils, enlarged sarcoplasmic reticulum, altered mitochondria and misaligned sarcomeres. Locomotion analyzes showed hypoxia-response behavior in 9 mpf col6a1 mutant unseen in 3 mpf fish. These symptoms worsened with ageing as described in patients with collagen VI deficiency. Thus, the col6a1ama605003-line is the first adult zebrafish model of collagen VI-related diseases; it will be instrumental both for basic research and drug discovery assays focusing on this type of disorders.

17-P005 Molecular characterization of neural stem cells in medaka

While adult neurogenesis in mammals is mainly restricted to two telencephalic zones, in teleost fishes, neurogenesis has been observed in many brain areas. Hence, the medaka (Oryzias latipes) represents an excellent model to study neural stem cells (NSCs). In order to detect NSCs, we have relied on the fact that adult stem cells divide more slowly than quickly amplifying progenitor cells. By using two different thymidine analogs and performing long pulses separated by a long period of time, we have managed to identify cells that are double-labelled in the medaka brain. These slow-proliferating cells are likely to be NSCs. We have looked more precisely at neurogenesis in a cortical structure of the dorsal midbrain, the optic tectum. Cell proliferation occurs at its margin throughout life. With the thymidine analog labeling, we have identified some slow-dividing cells, in the tectum, forming a narrow line at the very periphery of the proliferative zone. This topographic organization allowed us to perform an insitu hybridization screen on medaka embryos and find a dozen of genes whose expression corresponds to this thin line of putative NSCs. By analyzing the functions of these genes, we hope to identify essential genes for adult NSC and embryonic NSC biology. We should be able to bring cues on the conserved features between fish and mammal stem cells and examine whether neurogenesis in adult teleosts is merely the continuation of an embryonic phase or a distinct process involving adult specific mechanisms for stemness maintenance.

Molecular characterization of neural stem cells in medaka fish midbrain

Adult neurogenesis in mammals is restricted to two telencephalic constitutively active zones but appears more widespread in other vertebrates like reptiles, birds, and fish. Without exception, neuronal production in these species occurs throughout life. In teleost, proliferation sites have been found inmany regions of the brain. They are generally restricted to discrete foci, suggesting that niches of stem cells might exist in fish. Adult neural stem cells are characterized by their slow rhythm of cell cycle, correlated with their self-renewal properties. In medaka fish, we have shown that the optic tectum, the dorsal part of the mesencephalon grows throughout life by proliferation of cells at its margin. To provide support to the hypothesis of a stem cell nature of these peripheral cells, we analyzedwhether they express characteristic neural stem cell (NSC) markers. We compared the expression patterns of markers of multipotent neural stem cell genes (Sox2/Musashi/Bmi1) during the development and in adult brain. All thesemarkers are expressed in the brain proliferative zone during the development. In the adult optic tectum these genes are express in the marginal most regions of the proliferativezone. Tocharacterizemitotic activities in this region,we performed analysis basedon the incorporationof nucleoside analogs during cell division in fry and adult fishes. The nucleoside analogs 5iodo-2-deoxyuridine (IdU) and 5-chloro-2-deoxyuridine (CldU), which can be specifically immuno-detected, are used in double labelling experiments. This strategy allows the subset of long-term label retaining cells that are stem cells to be positively identified. We found double labelled cells in themostmarginal zone of the optic tectum. Conclusion: We identified a new population of neural stem cell like progenitors in the medaka adult optic tectum. These cells are slow proliferating, express the neural stem cell markers bmi-1, sox2, musashi 1 and are long lasting progenitors.