Stefania Pirondi

Thyroid hormone participates in the regulation of neural stem cells and oligodendrocyte precursor cells in the central nervous system of adult rat

Oligodendrocyte development and myelination are under thyroid hormone control. In this study we analysed the effects of chronic manipulation of thyroid status on the expression of a wide spectrum of oligodendrocyte precursor cells (OPCs) markers and myelin basic protein (MBP) in the subventricular zone (SVZ), olfactory bulb and optic nerve, and on neural stem cell (NSC) lineage in adult rats. Hypo‐ and hyperthyroidism were induced in male rats, by propyl‐thio‐uracil (PTU) and l‐thyroxin (T4) treatment, respectively. Hypothyroidism increased and hyperthyroidism downregulated proliferation in the SVZ and olfactory bulb (Ki67 immunohistochemistry and Western blotting, bromodeoxyuridine uptake). Platelet‐derived growth factor receptor alpha (PDGFα‐R) and MBP mRNA levels decreased in the optic nerve of hypothyroid rats; the same also occurred at the level of MBP protein. Hyperthyroidism slightly upregulates selected markers such as NG2 in the olfactory bulb. The lineage of cells derived from primary cultures of NSC prepared from the forebrain of adult hypo‐ and hyperthyroid also differs from those derived from control animals. Although no difference of in vitro proliferation of NSCs was observed in the presence of epidermal growth factor, maturation of oligodendrocytes (defined by process number and length) was enhanced in hyperthyroidism, suggesting a more mature state than in control animals. This difference was even greater when compared with the hypothyroid group, the morphology of which suggested a delay in differentiation. These results indicate that thyroid hormone affects NSC and OPC proliferation and maturation also in adulthood.

The galanin-R2 agonist AR-M1896 reduces glutamate toxicity in primary neural hippocampal cells.

Galanin is a neuropeptide involved in a variety of biological functions, including having a strong anticonvulsant activity. To assess a possible role of galanin in modulation of glutamatergic synapses and excitotoxicity, we studied effects of a galanin receptor 2(3) agonist (AR‐M1896) on several molecular events induced by glutamate administration in primary neural hippocampal cells. Exposure of cells, after 5 days in vitro, to glutamate 0.5 mm for 10 min caused morphological alterations, including disaggregation of β‐tubulin and MAP‐2 cytoskeletal protein assembly, loss of neurites and cell shrinkage. When present in culture medium together with glutamate, 1 and 10 nm of AR‐M1896 reduced these alterations. Moreover, AR‐M1896 counteracted glutamate‐induced c‐fos mRNA and c‐Fos protein up‐regulation after 30–150 min, and 24 h, respectively. Massive nuclear alterations (Hoechst 33258 staining), observed 24 h after glutamate exposure, were also antagonized by AR‐M1896 (0.1–100 nm) in a dose‐dependent manner. These findings indicate that galanin, probably mainly through its type 2 receptor, interferes with events associated with glutamate toxicity.

Prenatal glucocorticoid exposure affects learning and vulnerability of cholinergic neurons.

Prenatal treatment with synthetic glucocorticoids is commonly used as a treatment for women at risk of preterm delivery. However, little is known about the life-long consequences of these treatments on the fetus. In the present study, we evaluated cognitive function as well as susceptibility of cholinergic neurons to (192)IgG-saporin immunolesion in adult rats after prenatal glucocorticoid treatment. Morris water maze results revealed a significant difference in learning and memory function in adult rats that were prenatally exposed to dexamethasone, and further cognitive deficits after (192)IgG-saporin exposure. Choline acetyl transferase activity was decreased in the cortex of dexamethasone-treated rats compared with controls. In addition, rats prenatally exposed to either dexa, or betamethasone revealed a dramatic decrease in choline acetyl transferase activity compared to control rats after (192)IgG-saporin lesion. We report behavioral and biochemical evidence for altered cognitive function and increased susceptibility of cholinergic neurons to (192)IgG-saporin in adult rats after prenatal glucocorticoid treatment. Taken together, these results suggest that prenatal treatment with dexamethasone could affect cognitive functions and render cholinergic neurons more vulnerable to challenges later in life.

Changes in brain cholinergic markers and spatial learning in old galanin-overexpressing mice

The cholinergic forebrain system is involved in learning and memory, and its age-dependent decline correlates with a decrease in cognitive performance. Since the neuropeptide galanin participates in cholinergic neuron regulation, we have studied 19- to 23-month-old male mice overexpressing galanin under the platelet-derived growth factor B promoter (GalOE) and wild-type (WT) littermates by monitoring behavioral, neurochemical and morphological/histochemical parameters. In the Morris water maze test, old transgenic animals showed a significant impairment in escape latency in the hidden platform test compared to age-matched WT animals. The morphological/histochemical studies revealed that cholinergic neurons in the basal forebrain display a slight, age- but not genotype-related, alteration in choline acetyltransferase- (ChAT) immunoreactivity. The neurochemical studies showed an age-related decline in ChAT activity in the cerebral cortex of all mice, whereas in the hippocampal formation this effect was seen in GalOE but not WT animals. Expression of BDNF mRNA in the hippocampal formation, as evaluated by RT-PCR, was reduced in old animals; no age- or genotype-induced variations in NGF mRNA expression were observed. These data suggest that galanin overexpression further accentuates the age-related decline of the cholinergic system activity in male mice, resulting in impairment of water maze performance in old animals.

Behavioral and neurochemical studies on brain aging in galanin overexpressing mice

To study possible involvement of galanin in brain aging quality, we have investigated behavioral, neurochemical and morphological parameters in aged mice overexpressing galanin under the platelet-derived growth factor B promoter (GalOE mice) compared to wild-type littermates (WT mice). The behavioral analysis in the forced swim test showed that old GalOE animals spent more time in immobility compared to WT. In the activity cage test, galanin overexpression counteracted the age-induced decrease in exploratory behavior. The neurochemical analysis showed a 30% decrease in noradrenaline overflow in the cerebral cortex of WT old mice that was not present in age-matched GalOE mice. Our results indicate that overexpression of galanin can influence several behavioral and neurochemical parameters in old mice.

The galanin receptor 2/3 agonist Gal2-11 protects the SN56 cells against β-amyloid25–35 toxicity

The neuropeptide galanin is a modulator of cholinergic function and may play a role in Aβ peptide‐induced degeneration of cholinergic forebrain neurons. We have studied the effect of galanin and its galanin receptor subtype 2/3 agonist Gal2‐11on toxicity induced by freshly‐prepared β‐amyloid25–35 in the cholinergic cell line SN56. Both nuclear fragmentation and caspase‐3 expression were analysed. β‐amyloid25–35‐exposure induced a significant increase in caspase‐3 mRNA expression after 30, 60, 90 or 150 min of β‐amyloid25–35 exposure. These effects were abolished in the presence of Gal2‐11 (10 nM). Similarly, β‐amyloid25–35‐induced nuclear fragmentation was prevented by the galanin agonist at all time points studied. These findings indicate that the galanin 2/3 agonist Gal2‐11 protects SN56 cholinergic cells from β‐amyloid25–35‐induced cell death and that this action is mediated by an early reduction of caspase‐3 expression.

Role of c-Fos protein on glutamate toxicity in primary neural hippocampal cells

The hippocampus is extremely sensitive to microenvironmental signals and toxic events, including massive glutamate release. Despite the extensive literature related to the cascade of molecular events triggered in postsynaptic neurons, the distinction between proapoptotic and survival pathways is still being discussed. In this study, we have investigated the role of c‐Fos in glutamate‐induced toxicity in primary cultures of hippocampal neurons by using antisense oligonucleotide (ASO) technology. Exposure of cells (5 days in vitro; DIV) to glutamate 0.5 mM for 24 hr caused massive nuclear alteration. An increase in the number of caspase‐3‐positive cells was also observed 24 hr after glutamate treatment. The expression of c‐fos and c‐jun immediate‐early genes was increased 30 min after glutamate exposure. The study of c‐Fos and c‐Jun protein expression revealed an increase in the number of cells positive for both antibodies. To investigate whether the expression of c‐Fos protein after glutamate treatment was related to cell death activation or cell survival pathways, cells were exposed to 5 μM of c‐fos ASO at 4 DIV, 24 hr before glutamate treatment. The presence of the ASO in the medium significantly decreased the number of altered nuclei, and this was associated with a significant reduction in the number of c‐Fos‐positive cells after glutamate treatment. Exposure of cells to the c‐fos ASO under the conditions described above decreased caspase‐3 immunostaining induced by glutamate. These results suggest that the synthesis of c‐Fos protein after glutamate exposure favors cell death pathway activation in which caspase‐3 is also involved.

Stem cells and nervous tissue repair: from in vitro to in vivo

Recent development in stem cell biology has indicated a new possible approach for the treatment of neurological diseases. However, in spite of tremendous hope generated, we are still on the way to understand if the use of stem cells to repair mature brain and spinal cord is a reliable possibility. In particular, we know very little on the in situ regulation of adult neural stem, and this also negatively impact on cell transplant possibilities. In this chapter we will discuss issues concerning the role and function of stem cells in neurological diseases, with regard to the impact of features of degenerating neurons and glial cells on in situ stem cells. Stem cell location and biology in the adult brain, brain host reaction to transplantation, neural stem cell reaction to experimental injuries and possibilities for exogenous regulation are the main topics discussed.

Isolation of rat embryonic stem-like cells: A tool for stem cell research and drug discovery

The establishment of rat embryonic stem cells constitutes a precious tool since rat has been extensively used in biomedical research, in particular for the generation of human neurodisease animal models. Up to now only a few studies have described the isolation of rat embryonic stem‐like cells. One out of 9 isolated rat embryonic stem‐like cell lines (B1‐RESC) obtained from a 4.5‐day post‐coitum blastocyst were extensively characterized and kept in culture for up to 80 passages on feeders with LIF. The stable growth of these cells and the expression of pluripotent markers were confirmed up to a high number of passages in culture, also in the absence of feeders and LIF. B1‐RESC expresses the three germ layers markers both in vitro, within differentiating embryoid bodies, and in vivo through teratoma formation. Collectively, the B1‐RESC line with a stable near‐diploid karyotype can be used as a highly sensitive tool for testing anti‐proliferative molecules. Developmental Dynamics 240:2482–2494, 2011.

P3-299: Galanin and its agonist AR-M1896 protects the SN56 cholinergic cell line against beta amyloid 25-35 toxicity

E4/4 mice and found that the APOE2/4 mice displayed an intermediate phenotype compared to APOE3/3 and E4/4 mice. Conclusions: This work shows that APOE4 carriers are not born with reduced synaptic integrity, and that the APOE alleles function in a co-dominant fashion.For the first time we have an animal model which can be used to test the effects of the APOE2 allele on cognition and AD. This work is being confirmed using immunocytochemical and morphological analysis. ApoE (-/-) and murine apoE controls are included. We hope to determine if apoE plays an important role in the assembly and maintenance of synaptic connections and how this influences disease onset.