Reduced Geminin levels promote cellular senescence.

Cellular senescence is a permanent out-of-cycle state regulated by molecular circuits acting during the G1 phase of the cell cycle. Cdt1 is a central regulator of DNA replication licensing acting during the G1 phase and it is negatively controlled by Geminin. Here, we characterize the cell cycle expression pattern of Cdt1 and Geminin during successive passages of primary fibroblasts and compare it to tumour-derived cell lines. Cdt1 and Geminin are strictly expressed in distinct subpopulations of young fibroblasts, similarly to cancer cells, with Geminin accumulating shortly after the onset of S phase. Cdt1 and Geminin are down-regulated when primary human and mouse fibroblasts undergo replicative or stress-induced senescence. RNAi-mediated Geminin knock-down in human cells enhances the appearance of phenotypic and molecular features of senescence. Mouse embryonic fibroblasts heterozygous for Geminin exhibit accelerated senescence compared to control fibroblasts. In contrast, ectopic expression of Geminin in mouse embryonic fibroblasts delays the appearance of the senescent phenotype. Taken together, our data suggest that changes in Geminin expression levels affect the establishment of senescence pathways.

Survival motor neuron protein in motor neurons determines synaptic integrity in spinal muscular atrophy.

The inherited motor neuron disease spinal muscular atrophy (SMA) is caused by deficient expression of survival motor neuron (SMN) protein and results in severe muscle weakness. In SMA mice, synaptic dysfunction of both neuromuscular junctions (NMJs) and central sensorimotor synapses precedes motor neuron cell death. To address whether this synaptic dysfunction is due to SMN deficiency in motor neurons, muscle, or both, we generated three lines of conditional SMA mice with tissue-specific increases in SMN expression. All three lines of mice showed increased survival, weights, and improved motor behavior. While increased SMN expression in motor neurons prevented synaptic dysfunction at the NMJ and restored motor neuron somal synapses, increased SMN expression in muscle did not affect synaptic function although it did improve myofiber size. Together these data indicate that both peripheral and central synaptic integrity are dependent on motor neurons in SMA, but SMN may have variable roles in the maintenance of these different synapses. At the NMJ, it functions at the presynaptic terminal in a cell-autonomous fashion, but may be necessary for retrograde trophic signaling to presynaptic inputs onto motor neurons. Importantly, SMN also appears to function in muscle growth and/or maintenance independent of motor neurons. Our data suggest that SMN plays distinct roles in muscle, NMJs, and motor neuron somal synapses and that restored function of SMN at all three sites will be necessary for full recovery of muscle power.

Spinal muscular atrophy: the role of SMN in axonal mRNA regulation.

Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by homozygous mutations or deletions in the survival of motor neuron (SMN1) gene, encoding the ubiquitously expressed SMN protein. SMN associates with different proteins (Gemins 2-8, Unrip) to form a multimeric complex involved in the assembly of small nuclear ribonucleoprotein complexes (snRNPs). Since this activity is essential for the survival of all cell types, it still remains unclear why motor neurons are selectively vulnerable to low levels of SMN protein. Aside from its housekeeping role in the assembly of snRNPs, additional functions of SMN have been proposed. The well-documented localization of SMN in axonal transport granules and its interaction with numerous mRNA-binding proteins not involved in splicing regulation suggest a role in axonal RNA metabolism. This review will focus on the neuropathological and experimental evidence supporting a role for SMN in regulating the assembly, localization, or stability of axonal messenger ribonucleoprotein complexes (mRNPs). Furthermore, how defects in this non-canonical SMN function may contribute to the motor neuron pathology observed in SMA will be discussed. This article is part of a Special Issue entitled RNA-Binding Proteins.

Immunoexpression of gemins 2 and 4 in the rat spinal cord. Is the SMN complex a new target in investigations of sporadic amyotrophic lateral sclerosis pathogenesis?

Sporadic amyotrophic lateral sclerosis (sALS) is a neurodegenerative disease leading to degeneration and loss of motoneurons in different structures of the nervous system. Although aetiology of the disease is unknown, it is hypothesized that the survival motor neuron (SMN) protein which protects motoneurons in spinal muscular atrophy, may play a similar role in ALS. Relatively little is known about normal expression and functions of the SMN complex compounds, i.e. SMN protein and the related gemins. Therefore, we have decided to examine the physiological expression of SMN and gemins 2 and 4 in spinal cords of healthy Wistar rats at different age using immunofluorescence and immunohistochemical methods. Our study revealed that (1) in rat spinal cord neurons, the immunoexpression of SMN and gemins 2 and 4 is present through the whole animal lifespan although the reactive cells reveal different intensity of the immunolabeling, (2) both SMN and gemin 2, and SMN and gemin 4 are present in the same motoneurons, (3) immunoexpression of gemin 2 and 4 decreases slightly with aging.

Multiple therapeutic effects of valproic acid in spinal muscular atrophy model mice.

Spinal muscular atrophy (SMA) is a progressive disease involving the degeneration of motor neurons with no currently available treatment. While valproic acid (VPA) is a potential treatment for SMA, its therapeutic mechanisms are still controversial. In this study, we investigated the mechanisms of action of VPA in the treatment of type III-like SMA mice. SMA and wild-type mice were treated with VPA from 6 to 12 months and 10 to 12 months of age, respectively. Untreated SMA littermates and age-matched wild-type mice were used for comparison. VPA-treated SMA mice showed better motor function, larger motor-evoked potentials, less degeneration of spinal motor neurons, less muscle atrophy, and better neuromuscular junction innervation than non-treated SMA mice. VPA elevated SMN protein levels in the spinal cord through SMN2 promoter activation and probable restoration of correct splicing of SMN2 pre-messenger RNA. VPA also increased levels of anti-apoptotic factors, Bcl-2 and Bcl-x(L), in spinal neurons. VPA probably induced neurogenesis and promoted astrocyte proliferation in the spinal cord of type III-like SMA mice, which might contribute to therapeutic effects by enhancing neuroprotection. Through these effects of elevation of SMN protein level, anti-apoptosis, and probable neuroprotection, VPA-treated SMA mice had less degeneration of spinal motor neurons and better motor function than untreated type III-like SMA mice.

A preliminary report on spinal muscular atrophy lymphoblastoid cell lines: are they an appropriate tool for drug screening?

INTRODUCTION: Spinal muscular atrophy (SMA) is a neurodegenerative disease of the motor neurons that results in progressive muscle weakness. It is also the leading hereditary cause of infant mortality. Homozygous loss of the survival motor neuron (SMN1) gene causes SMA, and the number of copies of the SMN2 gene modulates the severity of the disease. Increasing the expression of the SMN2 gene by pharmacological agents is one of the therapeutic approaches currently being implemented. METHODS: In this preliminary study, we investigated the effect of phenylbutyrate, a histone deacetylase (HDAC) inhibitor, on SMN2 expression in two SMA type III Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines to understand the suitability of lymphoblastoid cell lines in drug screening. These cell lines are regarded as a good source as they can easily be established from the peripheral leucocytes of patients. Quantitative analysis of SMN2 mRNA was performed on established cell lines treated with various concentrations of phenylbutyrate and for a range of incubation periods using real-time polymerase chain reaction. Western blot analysis was used to determine SMN protein levels. RESULTS: Real-time polymerase chain reaction and Western blot analysis demonstrated that the levels of SMN2 full-length (fl-SMN2) transcripts and protein were not increased in phenylbutyrate-treated cell lines compared to non-treated controls. CONCLUSION: These results suggest that EBV-transformed lymphoblastoid cell lines are not suitable for studying the effect of certain HDAC inhibitors on SMN2 gene expression.