Intracellular pathways involved in cell survival are deregulated in mouse and human spinal muscular atrophy motoneurons.

Spinal Muscular Atrophy (SMA) is a severe neuromuscular disorder caused by loss of the Survival Motor Neuron 1 gene (SMN1). Due to this depletion of the survival motor neuron (SMN) protein, the disease is characterized by the degeneration of spinal cord motoneurons (MNs), progressive muscular atrophy, and weakness. Nevertheless, the ultimate cellular and molecular mechanisms leading to cell loss in SMN-reduced MNs are only partially known. We have investigated the activation of apoptotic and neuronal survival pathways in several models of SMA cells. Even though the antiapoptotic proteins FAIM-L and XIAP were increased in SMA MNs, the apoptosis executioner cleaved-caspase-3 was also elevated in these cells, suggesting the activation of the apoptosis process. Analysis of the survival pathway PI3K/Akt showed that Akt phosphorylation was reduced in SMA MNs and pharmacological inhibition of PI3K diminished SMN and Gemin2 at transcriptional level in control MNs. In contrast, ERK phosphorylation was increased in cultured mouse and human SMA MNs. Our observations suggest that apoptosis is activated in SMA MNs and that Akt phosphorylation reduction may control cell degeneration, thereby regulating the transcription of Smn and other genes related to SMN function.

Calpain activation and CaMKIV reduction in spinal cords from hSOD1G93A mouse model.

Amyotrophic Lateral Sclerosis (ALS), a severe neurodegenerative disease, affects the upper and lower motor neurons in the brain and spinal cord. In some studies, ALS disease progression has been associated with an increase in calcium-dependent degeneration processes. Motoneurons are specifically vulnerable to sustained membrane depolarization and excessive elevation of intracellular calcium concentration. The present study analyzed intracellular events in embryonic motoneurons and adult spinal cords of the hSOD1G93A ALS mouse model. We observed activation of calpain, a calcium-dependent cysteine protease that degrades a variety of substrates, and a reduction in calcium-calmodulin dependent protein kinase type IV (CaMKIV) levels in protein extracts from spinal cords obtained at several time-points of hSOD1G93A mice disease progression. However, in cultured embryonic motoneurons these differences between controls and hSOD1G93A mutants are not evident. Our results support the hypothesis that age-dependent changes in calcium homeostasis and resulting events, e.g., calpain activation and CaMKIV processing, are involved in ALS pathogenesis.

SMN deficiency attenuates migration of U87MG astroglioma cells through the activation of RhoA.

Spinal muscular atrophy (SMA) is a neurodegenerative disease that affects alpha motoneurons in the spinal cord caused by homozygous deletion or specific mutations in the survival motoneuron-1 (SMN1) gene. Cell migration is critical at many stages of nervous system development; to investigate the role of SMN in cell migration, U87MG astroglioma cells were transduced with shSMN lentivectors and about 60% reduction in SMN expression was achieved. In a monolayer wound-healing assay, U87MG SMN-depleted cells exhibit reduced cell migration. In these cells, RhoA was activated and phosphorylated levels of myosin regulatory light chain (MLC), a substrate of the Rho kinase (ROCK), were found increased. The decrease in cell motility was related to activation of RhoA/Rho kinase (ROCK) signaling pathway as treatment with the ROCK inhibitor Y-27632 abrogated both the motility defects and MLC phosphorylation in SMN-depleted cells. As cell migration is regulated by continuous remodeling of the actin cytoskeleton, the actin distribution was studied in SMN-depleted cells. A shift from filamentous to monomeric (globular) actin, involving the disappearance of stress fibers, was observed. In addition, profilin I, an actin-sequestering protein showed an increased expression in SMN-depleted cells. SMN is known to physically interact with profilin, reducing its actin-sequestering activity. The present results suggest that in SMN-depleted cells, the increase in profilin I expression and the reduction in SMN inhibitory action on profilin could lead to reduced filamentous actin polymerization, thus decreasing cell motility. We propose that the alterations reported here in migratory activity in SMN-depleted cells, related to abnormal activation of RhoA/ROCK pathway and increased profilin I expression could have a role in developing nervous system by impairing normal neuron and glial cell migration and thus contributing to disease pathogenesis in SMA.

Notch signaling pathway is activated in motoneurons of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a neurodegenerative disease produced by low levels of Survival Motor Neuron (SMN) protein that affects alpha motoneurons in the spinal cord. Notch signaling is a cell-cell communication system well known as a master regulator of neural development, but also with important roles in the adult central nervous system. Aberrant Notch function is associated with several developmental neurological disorders; however, the potential implication of the Notch pathway in SMA pathogenesis has not been studied yet. We report here that SMN deficiency, induced in the astroglioma cell line U87MG after lentiviral transduction with a shSMN construct, was associated with an increase in the expression of the main components of Notch signaling pathway, namely its ligands, Jagged1 and Delta1, the Notch receptor and its active intracellular form (NICD). In the SMNΔ7 mouse model of SMA we also found increased astrocyte processes positive for Jagged1 and Delta1 in intimate contact with lumbar spinal cord motoneurons. In these motoneurons an increased Notch signaling was found, as denoted by increased NICD levels and reduced expression of the proneural gene neurogenin 3, whose transcription is negatively regulated by Notch. Together, these findings may be relevant to understand some pathologic attributes of SMA motoneurons.

ERK MAPK signaling pathway inhibition as a potential target to prevent autophagy alterations in Spinal Muscular Atrophy motoneurons.

Spinal Muscular Atrophy (SMA) is a severe genetic neuromuscular disorder that occurs in childhood and is caused by misexpression of the survival motor neuron (SMN) protein. SMN reduction induces spinal cord motoneuron (MN) degeneration, which leads to progressive muscular atrophy and weakness. The link between SMN deficiency and the molecular mechanisms altered in SMA cells remains unclear. Autophagy, deregulation of intracellular survival pathways and ERK hyperphosphorylation may contribute to SMN-reduced MNs collapse, offering a useful strategy to develop new therapies to prevent neurodegeneration in SMA. Using SMA MN in vitro models, the effect of pharmacological inhibition of PI3K/Akt and ERK MAPK pathways on SMN and autophagy markers modulation was studied by western blot analysis and RT-qPCR. Experiments involved primary cultures of mouse SMA spinal cord MNs and differentiated SMA human MNs derived from induced pluripotent stem cells (iPSCs). Inhibition of the PI3K/Akt and the ERK MAPK pathways reduced SMN protein and mRNA levels. Importantly, mTOR phosphorylation, p62, and LC3-II autophagy markers protein level were decreased after ERK MAPK pharmacological inhibition. Furthermore, the intracellular calcium chelator BAPTA prevented ERK hyperphosphorylation in SMA cells. Our results propose a link between intracellular calcium, signaling pathways, and autophagy in SMA MNs, suggesting that ERK hyperphosphorylation may contribute to autophagy deregulation in SMN-reduced MNs.

The canonical nuclear factor-κB pathway regulates cell survival in a developmental model of spinal cord motoneurons.

In vivo and in vitro motoneuron survival depends on the support of neurotrophic factors. These factors activate signaling pathways related to cell survival or inactivate proteins involved in neuronal death. In the present work, we analyzed the involvement of the nuclear factor-κB (NF-κB) pathway in mediating mouse spinal cord motoneuron survival promoted by neurotrophic factors. This pathway comprises ubiquitously expressed transcription factors that could be activated by two different routes: the canonical pathway, associated with IKKα/IKKß kinase phosphorylation and nuclear translocation RelA (p65)/p50 transcription factors; and the noncanonical pathway, related to IKKα kinase homodimer phosphorylation and RelB/p52 transcription factor activation. In our system, we show that neurotrophic factors treatment induced IKKα and IKKß phosphorylation and RelA nuclear translocation, suggesting NF-κB pathway activation. Protein levels of different members of the canonical or noncanonical pathways were reduced in a primary culture of isolated embryonic motoneurons using an interference RNA approach. Even in the presence of neurotrophic factors, selective reduction of IKKα, IKKß, or RelA proteins induced cell death. In contrast, RelB protein reduction did not have a negative effect on motoneuron survival. Together these results demonstrated that the canonical NF-κB pathway mediates motoneuron survival induced by neurotrophic factors, and the noncanonical pathway is not related to this survival effect. Canonical NF-κB blockade induced an increase of Bim protein level and apoptotic cell death. Bcl-x(L) overexpression or Bax reduction counteracted this apoptotic effect. Finally, RelA knockdown causes changes of CREB and Smn protein levels.

Survival motor neuron protein and neurite degeneration are regulated by Gemin3 in spinal muscular atrophy motoneurons.

Spinal Muscular Atrophy (SMA) is a genetic neuromuscular disorder caused by reduction of the ubiquitously expressed protein Survival Motor Neuron (SMN). Low levels of SMN impact on spinal cord motoneurons (MNs) causing their degeneration and progressive muscle weakness and atrophy. To study the molecular mechanisms leading to cell loss in SMN-reduced MNs, we analyzed the NF-κB intracellular pathway in SMA models. NF-κB pathway activation is required for survival and regulates SMN levels in cultured MNs. Here we describe that NF-κB members, inhibitor of kappa B kinase beta (IKKß), and RelA, were reduced in SMA mouse and human MNs. In addition, we observed that Gemin3 protein level was decreased in SMA MNs, but not in non-neuronal SMA cells. Gemin3 is a core member of the SMN complex responsible for small nuclear ribonucleoprotein biogenesis, and it regulates NF-κB activation through the mitogen-activated protein kinase TAK1. Our experiments showed that Gemin3 knockdown reduced SMN, IKKß, and RelA protein levels, and caused significant neurite degeneration. Overexpression of SMN increased Gemin3 protein in SMA MNs, but did not prevent neurite degeneration in Gemin3 knockdown cells. These data indicated that Gemin3 reduction may contribute to cell degeneration in SMA MNs.

A new model to study spinal muscular atrophy: neurite degeneration and cell death is counteracted by BCL-X(L) Overexpression in motoneurons.

Spinal muscular atrophy (SMA) is a motoneuron disorder characterized by deletions or specific mutations in the Survival Motor Neuron gene (SMN). SMN is ubiquitously expressed and has a general role in the assembly of small nuclear ribonucleoprotein (snRNP) and pre-mRNA splicing requirements. However, in motoneuron axons SMN deficiency results in inappropriate levels of certain transcripts in the distal axon, suggesting that the specific susceptibility of motoneurons to SMN deficiency is related to a specialized function in these cells. Although mouse models of SMA have been generated and are useful for in vivo and in vitro studies, the limited number of isolated MNs that could be obtained from them makes it difficult to perform biochemical, genetic and pharmacological approaches. We describe here an in vitro model of isolated embryonic mouse motoneurons in which the cellular levels of endogenous SMN are reduced. These cells show neurite degeneration and cell death after several days of SMN knockdown. We found that the over-expression of the anti-apoptotic protein Bcl-x(L) into motoneurons rescues these cells from the phenotypic changes observed. This result demonstrates that Bcl-x(L) signaling could be a possible pharmacological target of SMA therapeutics.

Spinal Muscular Atrophy autophagy profile is tissue-dependent: differential regulation between muscle and motoneurons.

Spinal muscular atrophy (SMA) is a neuromuscular genetic disease caused by reduced survival motor neuron (SMN) protein. SMN is ubiquitous and deficient levels cause spinal cord motoneurons (MNs) degeneration and muscle atrophy. Nevertheless, the mechanism by which SMN reduction in muscle contributes to SMA disease is not fully understood. Therefore, studies evaluating atrophy mechanisms in SMA muscles will contribute to strengthening current knowledge of the pathology. Here we propose to evaluate autophagy in SMA muscle, a pathway altered in myotube atrophy. We analized autophagy proteins and mTOR in muscle biopsies, fibroblasts, and lymphoblast cell lines from SMA patients and in gastrocnemius muscles from a severe SMA mouse model. Human MNs differentiated from SMA and unaffected control iPSCs were also included in the analysis of the autophagy. Muscle biopsies, fibroblasts, and lymphoblast cell lines from SMA patients showed reduction of the autophagy marker LC3-II. In SMA mouse gastrocnemius, we observed lower levels of LC3-II, Beclin 1, and p62/SQSTM1 proteins at pre-symptomatic stage. mTOR phosphorylation at Ser2448 was decreased in SMA muscle cells. However, in mouse and human cultured SMA MNs mTOR phosphorylation and LC3-II levels were increased. These results suggest a differential regulation in SMA of the autophagy process in muscle cells and MNs. Opposite changes in autophagy proteins and mTOR phosphorylation between muscle cells and neurons were observed. These differences may reflect a specific response to SMN reduction, which could imply diverse tissue-dependent reactions to therapies that should be taken into account when treating SMA patients.

Expression of Candida albicans glutathione transferases is induced inside phagocytes and upon diverse environmental stresses.

Candida albicans has four ORFs for glutathione transferases (GSTs) of the GTT classes, and another one coding for an Omega class member. Under laboratory conditions, only GTT11 (GTT1/2 class) and GTO1 (Omega class) are expressed significantly in exponentially growing cells, particularly when these are subjected to diverse environmental stresses, including oxidative stress. They also become transitorily upregulated at the early stationary phase. Accordingly, the levels of the CaGto1 and CaGtt11 proteins increase after treatment with oxidants and upon osmotic stress, in addition to the early stationary phase. GTT11 and GTO1 transcription shows a complex dependence on the Hog1 and Cap1 factors upon different stresses. Purified CaGtt11 and CaGto1 proteins display enzyme activities similar to the Saccharomyces cerevisiae homologues. Thus, CaGtt11 has activity against standard GST substrates and is also active as peroxidase, while CaGto1 displays thiol oxidoreductase and dehydroascorbate reductase activities. Fluorescence microscopy and subfractionation studies indicate that CaGto1 is cytosolic, while CaGtt11 is associated with a particulate fraction. Under ex vivo conditions, CaGto1 and CaGtt11 become transitorily upregulated inside macrophages and neutrophils. Under these conditions, the promoter of GTT14 (GTT1/2 class) also becomes activated. These observations point to the importance of C. albicans GSTs in the defence against phagocytes.

Calpain system is altered in survival motor neuron-reduced cells from in vitro and in vivo spinal muscular atrophy models.

Spinal muscular atrophy (SMA) is a severe neuromuscular disorder caused by loss of the survival motor neuron 1 (SMN1) gene. SMA is characterized by the degeneration of spinal cord motoneurons (MNs), progressive skeletal muscle atrophy, and weakness. The cellular and molecular mechanisms causing MN loss of function are only partially known. Recent advances in SMA research postulate the role of calpain protease regulating survival motor neuron (SMN) protein and the positive effect on SMA phenotype of treatment with calpain inhibitors. We analyzed the level of calpain pathway members in mice and human cellular SMA models. Results indicate an increase of calpain activity in SMN-reduced MNs. Spinal cord analysis of SMA mice treated with calpeptin, a calpain inhibitor, showed an increase of SMN, calpain, and its endogenous inhibitor calpastatin in MNs. Finally, in vitro calpeptin treatment prevented microtubule-associated protein 1A/1B-light chain 3 (LC3) increase in MNs neurites, indicating that calpain inhibition may reduce autophagosome accumulation in neuron prolongations, but not in soma. Thus, our results show that calpain activity is increased in SMA MNs and its inhibition may have a beneficial effect on SMA phenotype through the increase of SMN in spinal cord MNs.

Specific vulnerability of mouse spinal cord motoneurons to membrane depolarization.

Intracellular calcium (Ca(2+)) concentration determines neuronal dependence on neurotrophic factors (NTFs) and susceptibility to cell death. Ca(2+) overload induces neuronal death and the consequences are thought to be a probable cause of motoneuron (MN) degeneration in neurodegenerative diseases. In the present study, we show that membrane depolarization with elevated extracellular potassium (K(+)) was toxic to cultured embryonic mouse spinal cord MNs even in the presence of NTFs. Membrane depolarization induced an intracellular Ca(2+) increase. Depolarization-induced toxicity and increased intracellular Ca(2+) were blocked by treatment with antagonists to some of the voltage-gated Ca(2+) channels (VGCCs), indicating that Ca(2+) influx through these channels contributed to the toxic effect of depolarization. Ca(2+) activates the calpains, cysteine proteases that degrade a variety of substrates, causing cell death. We investigated the functional involvement of calpain using a calpain inhibitor and calpain gene silencing. Pre-treatment of MNs with calpeptin (a cell-permeable calpain inhibitor) rescued MNs survival; calpain RNA interference had the same protective effect, indicating that endogenous calpain contributes to the cell death caused by membrane depolarization. These findings suggest that MNs are especially vulnerable to extracellular K(+) concentration, which induces cell death by causing both intracellular Ca(2+) increase and calpain activation.

Calpain Inhibition Increases SMN Protein in Spinal Cord Motoneurons and Ameliorates the Spinal Muscular Atrophy Phenotype in Mice.

Spinal muscular atrophy (SMA), a leading genetic cause of infant death, is caused by the loss of survival motor neuron 1 (SMN1) gene. SMA is characterized by the degeneration and loss of spinal cord motoneurons (MNs), muscular atrophy, and weakness. SMN2 is the centromeric duplication of the SMN gene, whose numbers of copies determine the intracellular levels of SMN protein and define the disease onset and severity. It has been demonstrated that elevating SMN levels can be an important strategy in treating SMA and can be achieved by several mechanisms, including promotion of protein stability. SMN protein is a direct target of the calcium-dependent protease calpain and induces its proteolytic cleavage in muscle cells. In this study, we examined the involvement of calpain in SMN regulation on MNs. In vitro experiments showed that calpain activation induces SMN cleavage in CD1 and SMA mouse spinal cord MNs. Additionally, calpain 1 knockdown or inhibition increased SMN level and prevent neurite degeneration in these cells. We examined the effects of calpain inhibition on the phenotype of two severe SMA mouse models. Treatment with the calpain inhibitor, calpeptin, significantly improved the lifespan and motor function of these mice. Our observations show that calpain regulates SMN level in MNs and calpeptin administration improves SMA phenotype demonstrating the potential utility of calpain inhibitors in SMA therapy.

Sp1-regulated expression of p11 contributes to motor neuron degeneration by membrane insertion of TASK1.

Disruption in membrane excitability contributes to malfunction and differential vulnerability of specific neuronal subpopulations in a number of neurological diseases. The adaptor protein p11, and background potassium channel TASK1, have overlapping distributions in the CNS. Here, we report that the transcription factor Sp1 controls p11 expression, which impacts on excitability by hampering functional expression of TASK1. In the SOD1-G93A mouse model of ALS, Sp1-p11-TASK1 dysregulation contributes to increased excitability and vulnerability of motor neurons. Interference with either Sp1 or p11 is neuroprotective, delaying neuron loss and prolonging lifespan in this model. Nitrosative stress, a potential factor in human neurodegeneration, stimulated Sp1 expression and human p11 promoter activity, at least in part, through a Sp1-binding site. Disruption of Sp1 or p11 also has neuroprotective effects in a traumatic model of motor neuron degeneration. Together our work suggests the Sp1-p11-TASK1 pathway is a potential target for treatment of degeneration of motor neurons.

Regulation of Survival Motor Neuron Protein by the Nuclear Factor-Kappa B Pathway in Mouse Spinal Cord Motoneurons.

Survival motor neuron (SMN) protein deficiency causes the genetic neuromuscular disorder spinal muscular atrophy (SMA), characterized by spinal cord motoneuron degeneration. Since SMN protein level is critical to disease onset and severity, analysis of the mechanisms involved in SMN stability is one of the central goals of SMA research. Here, we describe the role of several members of the NF-κB pathway in regulating SMN in motoneurons. NF-κB is one of the main regulators of motoneuron survival and pharmacological inhibition of NF-κB pathway activity also induces mouse survival motor neuron (Smn) protein decrease. Using a lentiviral-based shRNA approach to reduce the expression of several members of NF-κB pathway, we observed that IKK and RelA knockdown caused Smn reduction in mouse-cultured motoneurons whereas IKK or RelB knockdown did not. Moreover, isolated motoneurons obtained from the severe SMA mouse model showed reduced protein levels of several NF-κB members and RelA phosphorylation. We describe the alteration of NF-κB pathway in SMA cells. In the context of recent studies suggesting regulation of altered intracellular pathways as a future pharmacological treatment of SMA, we propose the NF-κB pathway as a candidate in this new therapeutic approach.

Saccharomyces cerevisiae cells have three Omega class glutathione S-transferases acting as 1-Cys thiol transferases.

The Saccharomyces cerevisiae genome encodes three proteins that display similarities with human GSTOs (Omega class glutathione S-transferases) hGSTO1-1 and hGSTO2-2. The three yeast proteins have been named Gto1, Gto2 and Gto3, and their purified recombinant forms are active as thiol transferases (glutaredoxins) against HED (beta-hydroxyethyl disulphide), as dehydroascorbate reductases and as dimethylarsinic acid reductases, while they are not active against the standard GST substrate CDNB (1-chloro-2,4-dinitrobenzene). Their glutaredoxin activity is also detectable in yeast cell extracts. The enzyme activity characteristics of the Gto proteins contrast with those of another yeast GST, Gtt1. The latter is active against CDNB and also displays glutathione peroxidase activity against organic hydroperoxides such as cumene hydroperoxide, but is not active as a thiol transferase. Analysis of point mutants derived from wild-type Gto2 indicates that, among the three cysteine residues of the molecule, only the residue at position 46 is required for the glutaredoxin activity. This indicates that the thiol transferase acts through a monothiol mechanism. Replacing the active site of the yeast monothiol glutaredoxin Grx5 with the proposed Gto2 active site containing Cys46 allows Grx5 to retain some activity against HED. Therefore the residues adjacent to the respective active cysteine residues in Gto2 and Grx5 are important determinants for the thiol transferase activity against small disulphide-containing molecules.

Smn-Deficiency Increases the Intrinsic Excitability of Motoneurons.

During development, motoneurons experience significant changes in their size and in the number and strength of connections that they receive, which requires adaptive changes in their passive and active electrical properties. Even after reaching maturity, motoneurons continue to adjust their intrinsic excitability and synaptic activity for proper functioning of the sensorimotor circuit in accordance with physiological demands. Likewise, if some elements of the circuit become dysfunctional, the system tries to compensate for the alterations to maintain appropriate function. In Spinal Muscular Atrophy (SMA), a severe motor disease, spinal motoneurons receive less excitation from glutamatergic sensory fibers and interneurons and are electrically hyperexcitable. Currently, the origin and relationship among these alterations are not completely established. In this study, we investigated whether Survival of Motor Neuron (SMN), the ubiquitous protein defective in SMA, regulates the excitability of motoneurons before and after the establishment of the synaptic contacts. To this end, we performed patch-clamp recordings in embryonic spinal motoneurons forming complex synaptic networks in primary cultures, and in differentiated NSC-34 motoneuron-like cells in the absence of synaptic contacts. Our results show that in both conditions, Smn-deficient cells displayed lower action potential threshold, greater action potential amplitudes, and larger density of voltage-dependent sodium currents than cells with normal Smn-levels. These results indicate that Smn participates in the regulation of the cell-autonomous excitability of motoneurons at an early stage of development. This finding may contribute to a better understanding of motoneuron excitability in SMA during the development of the disease.

Inhibition of autophagy delays motoneuron degeneration and extends lifespan in a mouse model of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a recessive autosomal neuromuscular disease, due to homozygous mutations or deletions in the telomeric survival motoneuron gene 1 (SMN1). SMA is characterized by motor impairment, muscle atrophy, and premature death following motor neuron (MN) degeneration. Emerging evidence suggests that dysregulation of autophagy contributes to MN degeneration. We here investigated the role of autophagy in the SMNdelta7 mouse model of SMA II (intermediate form of the disease) which leads to motor impairment by postnatal day 5 (P5) and to death by P13. We first showed by immunoblots that Beclin 1 and LC3-II expression levels increased in the lumbar spinal cord of the SMA pups. Electron microscopy and immunofluorescence studies confirmed that autophagic markers were enhanced in the ventral horn of SMA pups. To clarify the role of autophagy, we administered intracerebroventricularly (at P3) either an autophagy inhibitor (3-methyladenine, 3-MA), or an autophagy inducer (rapamycin) in SMA pups. Motor behavior was assessed daily with different tests: tail suspension, righting reflex, and hindlimb suspension tests. 3-MA significantly improved motor performance, extended the lifespan, and delayed MN death in lumbar spinal cord (10372.36 ± 2716 MNs) compared to control-group (5148.38 ± 94 MNs). Inhibition of autophagy by 3-MA suppressed autophagosome formation, reduced the apoptotic activation (cleaved caspase-3 and Bcl2) and the appearance of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive neurons, underlining that apoptosis and autophagy pathways are intricately intertwined. Therefore, autophagy is likely involved in MN death in SMA II, suggesting that it might represent a promising target for delaying the progression of SMA in humans as well.

Anchorage of Candida albicans Ssr1 to the cell wall, and transcript profiling of the null mutant.

Incorporation into the wall of Candida albicans Ssr1, a GPI-dependent protein, was investigated by construction of different truncated genes for which the three potential omega sites (S199, S215 and G216) and the corresponding omega+1 and omega+2 were eliminated or modified. Cells of the C. albicans ssr1Delta mutant were transformed with pADH-pl harboring the truncated versions of CaSSR1, pADH-DeltaCaSSR1t(217-234) (lacking a C-terminal hydrophobic stretch of 18 aa including the putative omega+2 and omega+1, omega+2 of S215 and G216) or pADH-DeltaCaSSR1t(199-201) (lacking three serine residues), and their walls were analyzed for the protein. Results suggested that the three serine residues are essential for incorporation of CaSsr1 into the wall beta-glucan. This interpretation was confirmed when the truncated protein CaSsr1pt(199-201) was found in the spent medium. The transcription profile of the 6039 genes in C. albicans ssr1Delta showed that seven genes are upregulated (1.4-fold), including SRP54 (a signal recognition particle subunit), IPF29 (a zinc finger protein) and PTR3 (a transcriptional regulator), whereas 27 genes are downregulated (0.7-fold), including IPF6318 (a beta-glucosidase) and SOU1 (a sorbitol utilization protein). Additional genes showed a reduced increase, or decreased expression, suggesting that some current orphan genes may have unknown cell wall functions. In addition, a compensatory mechanism would appear to occur, as a substantial increase in the amount of beta-1,3-glucan (2.34-fold) was detected in the cell wall of the mutant cells.