Effects of FGF receptor peptide agonists on animal behavior under normal and pathological conditions.

Hexafins are recently identified low-molecular-weight peptide agonists of the fibroblast growth factor receptor (FGFR), derived from the beta6-beta7 loop region of various FGFs. Synthetic hexafin peptides have been shown to bind to and induce tyrosine phosphorylation of FGFR1, stimulate neurite outgrowth, and promote neuronal survival in vitro. Thus, the pronounced biological activities of hexafins in vitro make them attractive compounds for pharmacological studies in vivo. The present study investigated the effects of subcutaneous administration of hexafin1 and hexafin2 (peptides derived from FGF1 and FGF2, respectively) on social memory, exploratory activity, and anxiety-like behavior in adult rats. Treatment with hexafin1 and hexafin2 resulted in prolonged retention of social memory. Furthermore, rats treated with hexafin2 exhibited decreased anxiety-like behavior in the elevated plus maze. Employing an R6/2 mouse model of Huntington's disease (HD), we found that although hexafin2 did not affect the progression of motor symptoms, it alleviated deficits in activity related to social behavior, including sociability and social novelty. Thus, hexafin2 may have therapeutic potential for the treatment of HD.

Detection of early behavioral markers of Huntington's disease in R6/2 mice employing an automated social home cage.

Huntington's disease (HD) is an autosomal-dominant neurodegenerative disorder, for which no known cure or effective treatment exists. To facilitate the search for new potential treatments of HD, an automated system for analyzing the behavior of transgenic HD mice is urgently needed. A recently developed behavior screening system, the IntelliCage, allows automated testing of mouse behavior in the home cage employing individual recognition of animals living in social groups. The present study validates the ability of the IntelliCage system to detect behavioral and cognitive dysfunction in R6/2 mice, an established transgenic model of HD. The results indicate that the IntelliCage is a reliable system for recording exploratory activity, drinking behavior, circadian rhythm, spatial preference, and cognition in mice during prolonged periods of assessment. The system detected early dysfunctional behaviors in R6/2 mice, such as decrease in exploratory activity, sleep disturbances, increased drinking, and repetitive behavior. Additionally, the use of various learning tasks, such as spatial avoidance and spatial patrolling, revealed early cognitive changes in R6/2 mice. The simple learning tasks may be used at both early and late stages of the disease.

An automatic procedure for evaluation of single cell motility.

BACKGROUND: Cell motility is vital in many physiological and pathological processes, such as embryogenesis, inflammation, wound healing, and metastasis. However, the time-consuming step in the evaluation of individual cell motility is the analysis of hundreds of recorded images of cell cultures in general consisting of retrieving images, one at a time, and marking the positions of individual cells by a computer mouse. Therefore, the aim of the present study was to develop a novel automatic procedure for the evaluation of cell motility. MATERIALS AND METHODS: The procedure was tested on fibroblasts and glioma and adenocarcinoma cells engineered to express the green fluorescent protein by either transient transfection or adenovirus transduction, allowing automatic recognition of cell coordinates on retrieved images. RESULTS: The effects of serum growth factors, teratogenic compounds, and overexpression of transcription factors on the motile behavior of cultured cells were determined. Cell motility was estimated by both manual and automatic marking of cell position and subsequently motility parameters were computed. The results obtained by the two procedures were found to correlate significantly. CONCLUSIONS: We developed a procedure allowing automatic video recording of sparsely seeded cells transfected with a plasmid or tranduced with a recombinant virus expressing the enhanced green fluorescent protein (EGFP).

The role of RhoA in the regulation of cell morphology and motility.

Members of the Rho family of small GTPases are key regulators of the actin cytoskeleton, particularly in relation to the cell shape changes and the adhesion dynamic that drive cell migration. Here, we report the effect of activation or inhibition of the function of RhoA on cell motility and morphology. Both in the presence and the absence of serum, expression of constitutively active RhoA dramatically inhibited L929 fibroblasts' cell motility, and induced a rounding of the cells and a decrease in the number of processes per cell. In contrast, expression of a dominant negative mutant of RhoA had no effect on cell motility or morphology in steady-state conditions with or without serum in the medium. Inhibition of p160ROCK, a kinase effector of RhoA, only partially inhibited cell migration. Conversely, when cells were submitted to a period of serum deprivation followed by addition of serum, inhibition of endogenous RhoA by expression of the dominant negative mutant of RhoA impeded cell motility after serum stimulation. Thus, RhoA activity is required for stimulation of cell locomotion by serum factors. It was also observed that the addition of serum factors to quiescent L929 and NR6wtEGFR fibroblasts resulted in a delayed motility response of several hours compared to the immediately induced morphological changes, indicating the absence of a previously assumed direct correlation between changes in cell motility and cell morphology in response to serum addition. The motility response of L929 and NR6wtEGFR fibroblasts to serum stimulation required protein synthesis.

Expression of a naturally occurring constitutively active variant of the epidermal growth factor receptor in mouse fibroblasts increases motility.

Tumor cell motility is one of the rate-limiting steps of invasion, which defines progression toward a more malignant phenotype. Elevated expression of epidermal growth factor receptor (EGFR) in many cancers is associated with progression of superficial to invasive forms of the disease. The naturally occuring type III mutant epidermal growth factor receptor (EGFRvIII) is a tumor-specific, ligand-independent, constitutively active variant of the epidermal growth factor receptor. EGFRvIII is expressed frequently by a number of human solid tumours including those of the lung, breast, prostate, brain and ovary. Our study was designed to investigate the effect of EGFRvIII expression on cell motility and compare it to that of ligand-activated EGFR using transfected fibroblasts. We show here using time-lapse video recording that expression of EGFRvIII greatly enhances the motility of fibroblasts independently of ligand stimulation. In addition, expression of EGFRvIII caused a marked increase in the number of cellular protrusions (lamellipodia) and a reduction in the number of stress fibers and focal adhesions. The EGFR tyrosine kinase inhibitor, AG1478, and the MEK inhibitor, U0126, blocked these cellular effects of EGFRvIII. Two cell lines expressing different levels of EGFR were used for comparison. The low-expressing cell line responded to EGF treatment by increasing motility in a manner very similar to the motility induced by EGFRvIII. In contrast, the high-expressing cell line responded to EGF by detachment from the extracellular matrix and decreased motility. Cellular detachment was correlated to a high phosphorylation of PLC-gamma, whereas increased motility was correlated to a high level of ERK phosphorylation. Overall these results indicate that tumor-associated EGFR mutations might be critical for tumor cell motility, invasion and thus progression of disease.

Role of the Fos family members, c-Fos, Fra-1 and Fra-2, in the regulation of cell motility.

The AP-1 transcription factor is composed of members of the Fos, Jun and ATF families, and plays a key role in tumor progression. We investigated whether Fos proteins regulate cell motility, and if so, whether this capacity is related to their transactivation potential. Two cell lines with different expression profiles of AP-1 were employed focusing on the Fos-family members c-Fos, Fra-1 and Fra-2. Transactivation motifs are found in c-Fos, but not in Fra-1 or Fra-2. The adenocarcinoma CSML0 cells display a low motility and do not express Fra-1 or Fra-2, and only very little c-Fos. In contrast, the fibroblastoid L929 cells express both Fra-1 and Fra-2, but no c-Fos, and these cells display a high motility. Transfection with Fra-1 or c-Fos, but not with Fra-2, strongly enhanced the motility of CSML0 cells. The effect of Fra-1 required the presence of the N-terminal domain of this protein. Conversely, transfection with a Fos dominant-negative mutant or with anti-sense fra-1 or fra-2, strongly reduced the motility of L929 cells. Changes in cell motility correlated with the morphological appearance and the degree of contact with the substratum. We conclude that Fos proteins have distinct roles in the regulation of cell motility.

The ability of Fos family members to produce phenotypic changes in epithelioid cells is not directly linked to their transactivation potentials.

Numerous studies have revealed distinct functions of Fos proteins in different mouse tissues and cell lines. Here, we perform a direct comparison of the features of exogenous c-Fos, Fra-1 and Fra-2 proteins expressed in murine tumor cells of epithelial origin, CSML0. Although transactivation potential of c-Fos is much stronger than that of Fra-1 and Fra-2, all three proteins are capable of modulating transcription of target genes. Moreover, there is a certain degree of specificity in the induction of the transcription of AP-1-responsive genes by different Fos proteins. For instance, c-Fos and Fra-1 but not Fra-2 activated genes of the urokinase system. Additionally, not only a strong transcriptional activator c-Fos, but also Fra-1 induced morphological alterations in CSML0 cells. N-terminal domain of Fra-1 was required for this function. On the other hand, Fra-2 failed to change morphology of CSML0 cells. We therefore conclude that c-Fos, Fra-1 and Fra-2 differently activate transcription of target genes and induce morphological changes in epithelioid carcinoma cells in a manner not directly linked to their transactivation potentials.