Effects of Forskolin on Trefoil factor 1 expression in cultured ventral mesencephalic dopaminergic neurons.

Trefoil factor 1 (TFF1) belongs to a family of secreted peptides that are mainly expressed in the gastrointestinal tract. Notably, TFF1 has been suggested to operate as a neuropeptide, however, its specific cellular expression, regulation and function remain largely unknown. We have previously shown that TFF1 is expressed in developing and adult rat ventral mesencephalic tyrosine hydroxylase-immunoreactive (TH-ir) dopaminergic neurons. Here, we investigated the expression of TFF1 in rat ventral mesencephalic dopaminergic neurons (embryonic day 14) grown in culture for 5, 7 or 10 days in the absence (controls) or presence of either glial cell line-derived neurotrophic factor (GDNF), Forskolin or the combination. No TFF1-ir cells were identified at day 5 and only a few at day 7, whereas TH was markedly expressed at both time points. At day 10, several TFF1-ir cells were detected, and their numbers were significantly increased after the addition of GDNF (2.2-fold) or Forskolin (4.1-fold) compared to controls. Furthermore, the combination of GDNF and Forskolin had an additive effect and increased the number of TFF1-ir cells by 5.6-fold compared to controls. TFF1 expression was restricted to neuronal cells, and the percentage of TH/TFF1 co-expressing cells was increased to the same extent in GDNF and Forskolin-treated cultures (4-fold) as compared to controls. Interestingly, the combination of GDNF and Forskolin resulted in a significantly increased co-expression (8-fold) of TH/TFF1, which could indicate that GDNF and Forskolin targeted different subpopulations of TH/TFF1 neurons. Short-term treatment with Forskolin resulted in an increased number of TFF1-ir cells, and this effect was significantly reduced by the MEK1 inhibitor PD98059 or the protein kinase A (PKA) inhibitor H89, suggesting that Forskolin induced TFF1 expression through diverse signaling pathways. In conclusion, distinct populations of cultured dopaminergic neurons express TFF1, and their numbers can be increased by factors known to influence survival and differentiation of dopaminergic cells.

Accumulation of Ym1/2 protein in the mouse olfactory epithelium during regeneration and aging.

A unique feature of the olfactory system is its efficiency to produce new neurons in the adult. Thus, destruction of the olfactory receptor neurons (ORNs) using chemical (intranasal perfusion with ZnSO4) or surgical (axotomy or bulbectomy) methods, leads to an enhanced rate of proliferation of their progenitors and to complete ORNs regeneration. The aim of our study was to identify new factors implied in this regenerative process. Using an electrophoretic method, we observed the accumulation of a 42 kDa protein after axotomy in the olfactory mucosa, but not in the olfactory bulb. Its expression started after a few days following injury and increased massively during the phase of ORN regeneration. The purification and the sequence characterization revealed that this protein was Ym1/2, recently identified in activated macrophages present in various tissues during inflammation. Western blotting analysis of Ym1/2 confirmed the accumulation of this protein in the regenerating olfactory mucosa consecutively to olfactory axotomy or bulbectomy but also after ZnSO4 irrigation of the nasal cavity. In the olfactory mucosa of control mice, Ym1/2 was hardly detectable in young animals and became more and more abundant with increasing age. In injured and aged mice, Ym1/2 mainly accumulates in the cytoplasm of supporting cells as well as in other cells located throughout the olfactory epithelium. Our results suggest that Ym1/2 is involved in olfactory epithelium remodeling following several kinds of lesions of the adult olfactory mucosa and support the view of a critical role of inflammatory cues in neurodegeneration and aging.

Cell cycle regulation during mouse olfactory neurogenesis.

The development of the nervous system requires a strict control of cell cycle entry and withdrawal. The olfactory epithelium (OE) is noticeable by its ability to yield new neurons not only during development but also continuously during adulthood. The aim of our study was to investigate, by biochemical and immunohistochemical methods, which cell cycle regulators are involved in the control of neuron production during OE development and maturity. At birth, olfactory neural progenitors, the basal cells, exhibited a high mitogenic and neurogenic activity, decreasing in the following weeks together with the drop in expression of several cell cycle regulators. p27Kip1 and p18Ink4c, at birth, were expressed in the whole basal cell layer, whereas p16Ink4a, p19Ink4d, and p21Cip1 were rather located in differentiating or mature neurons. CDK inhibitors may thus act sequentially during this developmental neurogenic process. By comparison, in the adult OE, in which most neural precursors were quiescent, these cells still exhibited p18Ink4c expression but only occasionally p27Kip1 expression. It suggests that p18Ink4c may contribute to maintain basal cells in a quiescent state, whereas p27Kip1 expression in these cells may be rather linked to their neurogenic activity, which declines with age. In keeping with this hypothesis, transgenic mice that lacked p27Kip1 expression displayed a higher rate of cell proliferation versus differentiation in their OE. In these mice, a down-regulation of positive cell cycle regulators was observed that may contribute to compensate for the absence of p27Kip1. Taken together, the present data suggest distinct functions for CDK inhibitors, either in the control of cell cycle exit and differentiation during neurogenesis (respectively, p27Kip1 and p19Ink4d) or in the maintenance of a quiescent state in neural progenitors (p18Ink4c) or neurons (p21Cip1) in adults.

Region-specific expression of cell cycle inhibitors in the adult brain.

In the adult brain, neural proliferation is almost absent and neurons are generally not renewed. By contrast, in the olfactory organ, olfactory neurons are produced continuously throughout life. To investigate whether specific cell cycle inhibitors are involved in the control of neural quiescence in adulthood, we compared their expression either in different regions of the adult brain weakly or non neurogenic or, for comparison, in the olfactory mucosa. We show that numerous cell cycle inhibitors are expressed in the adult brain either in an ubiquitous fashion (as p19Ink4d) or in specific brain regions (p15Ink4b in the forebrain, p27Kip1 and p21Cip1 in the cerebellum). By contrast p18Ink4c was expressed detectably only in the highly neurogenic olfactory epithelium. The present data suggest that various CDK inhibitors may be involved in a region-specific fashion in the maintenance of nerve cell quiescence in adults.

Cytotoxic effects of long-term circulating ultrafiltrable platinum species and limited efficacy of haemodialysis in clearing them.

We applied haemodialysis to clear platinum (Pt) circulating species following renal insufficiency due to an accidental cisplatin overdosage (205 mg/m2 instead of 100 mg/m2). Serum samples were repeatedly obtained during this clinical episode from day 5 up to day 30 after cisplatin dosing. A serum aliquot taken at day 22 after cisplatin administration was tested to assess the possible cytotoxicity exhibited by the circulating Pt species on a head and neck tumour cell line. The profile of ultrafiltrable (UF) Pt during successive haemodialysis cycles was striking. After each haemodialysis cycle, a marked decrease in UF Pt, occurred but was followed by more or less pronounced rebounds. Cisplatin concentration-cytotoxic effect curves obtained in vitro from patient serum before cisplatin administration and healthy control serum exhibited very similar concentration effect profiles. In contrast, the patient serum taken at day 22 after cisplatin administration resulted in marked cytotoxic effects, which were much greater than those which could have been anticipated considering the Pt concentration of this serum sample. The present report underlines the limited usefulness of haemodialysis for rescuing cisplatin treated patients, exhibiting unanticipated postinfusion renal failure with overexposure to the drug. The in vitro investigations suggest that pharmacological effects of Pt derivatives may not only be attributable to short-term effects of the drug diffusion into tissues, but also to more delayed effects from Pt circulating species.