Characterization of dUTPase expression in mouse postnatal development and adult neurogenesis.

The enzyme dUTPase has an essential role in maintaining genomic integrity. In mouse, nuclear and mitochondrial isoforms of the enzyme have been described. Here we present the isoform-specific mRNA expression levels in different murine organs during development using RT-qPCR. In this study, we analyzed organs of 14.5-day embryos and of postnatal 2-, 4-, 10-week- and 13-month-old mice. We demonstrate organ-, sex- and developmental stage-specific differences in the mRNA expression levels of both isoforms. We found high mRNA expression level of the nuclear isoform in the embryo brain, and the expression level remained relatively high in the adult brain as well. This was surprising, since dUTPase is known to play an important role in proliferating cells, and mass production of neural cells is completed by adulthood. Thus, we investigated the pattern of the dUTPase protein expression specifically in the adult brain with immunostaining and found that dUTPase is present in the germinative zones, the subventricular and the subgranular zones, where neurogenesis occurs and in the rostral migratory stream where neuroblasts migrate to the olfactory bulb. These novel findings suggest that dUTPase may have a role in cell differentiation and indicate that accurate dTTP biosynthesis can be vital, especially in neurogenesis.

Region-selective control of the thalamic reticular nucleus via cortical layer 5 pyramidal cells.

Corticothalamic pathways, responsible for the top-down control of the thalamus, have a canonical organization such that every cortical region sends output from both layer 6 (L6) and layer 5 (L5) to the thalamus. Here we demonstrate a qualitative, region-specific difference in the organization of mouse corticothalamic pathways. Specifically, L5 pyramidal cells of the frontal cortex, but not other cortical regions, establish monosynaptic connections with the inhibitory thalamic reticular nucleus (TRN). The frontal L5-TRN pathway parallels the L6-TRN projection but has distinct morphological and physiological features. The exact spike output of the L5-contacted TRN cells correlated with the level of cortical synchrony. Optogenetic perturbation of the L5-TRN connection disrupted the tight link between cortical and TRN activity. L5-driven TRN cells innervated thalamic nuclei involved in the control of frontal cortex activity. Our data show that frontal cortex functions require a highly specialized cortical control over intrathalamic inhibitory processes.

Generation of diverse neuronal subtypes in cloned populations of stem-like cells.

BACKGROUND: The central nervous tissue contains diverse subtypes of neurons with characteristic morphological and physiological features and different neurotransmitter phenotypes. The generation of neurons with defined neurotransmitter phenotypes seems to be governed by factors differently expressed along the anterior-posterior and dorsal-ventral body axes. The mechanisms of the cell-type determination, however, are poorly understood. Selected neuronal phenotypes had been generated from embryonic stem (ES) cells, but similar results were not obtained on more restricted neural stem cells, presumably due to the lack of homogeneous neural stem cell populations as a starting material. RESULTS: In the presented work, the establishment of different neurotransmitter phenotypes was investigated in the course of in vitro induced neural differentiation of a one-cell derived neuroectodermal cell line, in conjunction with the activation of various region-specific genes. For comparison, similar studies were carried out on the R1 embryonic stem (ES) and P19 multipotent embryonic carcinoma (EC) cells. In response to a short treatment with all-trans retinoic acid, all cell lines gave rise to neurons and astrocytes. Non-induced neural stem cells and self-renewing cells persisting in differentiated cultures, expressed "stemness genes" along with early embryonic anterior-dorsal positional genes, but did not express the investigated CNS region-specific genes. In differentiating stem-like cell populations, on the other hand, different region-specific genes, those expressed in non-overlapping regions along the body axes were activated. The potential for diverse regional specifications was induced in parallel with the initiation of neural tissue-type differentiation. In accordance with the wide regional specification potential, neurons with different neurotransmitter phenotypes developed. Mechanisms inherent to one-cell derived neural stem cell populations were sufficient to establish glutamatergic and GABAergic neuronal phenotypes but failed to manifest cathecolaminergic neurons. CONCLUSION: The data indicate that genes involved in positional determination are activated along with pro-neuronal genes in conditions excluding any outside influences. Interactions among progenies of one cell derived neural stem cells are sufficient for the activation of diverse region specific genes and initiate different routes of neuronal specification.

Isolation of radial glia-like neural stem cells from fetal and adult mouse forebrain via selective adhesion to a novel adhesive peptide-conjugate.

Preferential adhesion of neural stem cells to surfaces covered with a novel synthetic adhesive polypeptide (AK-cyclo[RGDfC]) provided a unique, rapid procedure for isolating radial glia-like cells from both fetal and adult rodent brain. Radial glia-like (RGl) neural stem/progenitor cells grew readily on the peptide-covered surfaces under serum-free culture conditions in the presence of EGF as the only growth factor supplement. Proliferating cells derived either from fetal (E 14.5) forebrain or from different regions of the adult brain maintained several radial glia-specific features including nestin, RC2 immunoreactivity and Pax6, Sox2, Blbp, Glast gene expression. Proliferating RGl cells were obtained also from non-neurogenic zones including the parenchyma of the adult cerebral cortex and dorsal midbrain. Continuous proliferation allowed isolating one-cell derived clones of radial glia-like cells. All clones generated neurons, astrocytes and oligodendrocytes under appropriate inducing conditions. Electrophysiological characterization indicated that passive conductance with large delayed rectifying potassium current might be a uniform feature of non-induced radial glia-like cells. Upon induction, all clones gave rise to GABAergic neurons. Significant differences were found, however, among the clones in the generation of glutamatergic and cathecolamine-synthesizing neurons and in the production of oligodendrocytes.

Survival and differentiation of neuroectodermal cells with stem cell properties at different oxygen levels.

Freeze-lesioned regions of the forebrain cortex provide adequate environment for growth of non-differentiated neural progenitors, but do not support their neuron formation. Reduced oxygen supply, among numerous factors, was suspected to impair neuronal cell fate commitment. In the present study, proliferation and differentiation of neural stem/progenitor cells were investigated at different oxygen levels both in vitro and in vivo. Low (1% atmospheric) oxygen supply did not affect the in vitro viability and proliferation of stem cells or the transcription of "stemness" genes but impaired the viability of committed neuronal progenitors and the expression of proneural and neuronal genes. Consequently, the rate of in vitro neuron formation was markedly reduced under hypoxic conditions. In vivo, neural stem/progenitor cells survived and proliferated in freeze-lesioned adult mouse forebrains, but did not develop into neurons. Hypoperfusion-caused hypoxia in lesioned cortices was partially corrected by hyperbaric oxygen treatment (HBOT). HBOT, while reduced the rate of cell proliferation at the lesion site, resulted in sporadic neuron formation from implanted neural stem cells. The data indicate that in hypoxic brain areas, neural stem cells survive and proliferate, but neural tissue-type differentiation can not proceed. Oxygenation renders the damaged brain areas more permissive for tissue-type differentiation and may help the integration of neural stem/progenitor cells.

Astroglia genesis in vitro: distinct effects of retinoic acid in different phases of neural stem cell differentiation.

In the developing CNS, the manifestation of the macro-glial phenotypes is delayed behind the formation of neurons. The "neurons first--glia second" principle seems to be valid for neural tissue differentiation throughout the neuraxis, but the reasons behind are far from clear. In the presented study, the mechanisms of this timing were investigated in vitro, in the course of the neural differentiation of one cell derived NE-4C neuroectodermal stem and P19 embryonic carcinoma cells. The data demonstrated that astrocyte formation coincided in time with the maturation of postmitotic neurons, but the close vicinity of neurons did not initiate astrocyte formation before schedule. All-trans retinoic acid, a well-known inducer of neuronal differentiation, on the other hand, blocked effectively the astroglia production if present in defined stages of the in vitro neuroectodermal cell differentiation. According to the data, retinoic acid plays at least a dual role in astrogliogenesis: while it is needed for committing neural progenitors for a future production of astrocytes, it prevents premature astrogliogenesis by inhibiting the differentiation of primed glial progenitors.

Translocator protein (TSPO 18kDa) is expressed by neural stem and neuronal precursor cells.

Translocator protein 18 kDa, the peripheral benzodiazepine receptor by its earlier name, is a mitochondrial membrane protein associated with the mitochondrial permeability pore. While the function of the protein is not properly understood, it is known to play roles in necrotic and apoptotic processes of the neural tissue. In the healthy adult brain, TSPO expression is restricted to glial cells. In developing or damaged neural regions, however, TSPO appears in differentiating/regenerating neurons. Using immunocytochemical, molecular biological and cell biological techniques, we demonstrate that TSPO mRNA and protein, while missing from mature neurons, are present in neural stem cells and also in postmitotic neuronal precursors. Investigating some distinct stages of in vitro differentiation of NE-4C neural stem cells, TSPO 18 kDa was found to be repressed in a relatively late phase of neuron formation, when mature neuron-specific features appear. This timing indicates that mitochondria in fully developed neurons display specific characteristics and provides an additional marker for characterising neuronal differentiation.