Autoinhibition of TBCB regulates EB1-mediated microtubule dynamics

Tubulin cofactors (TBCs) participate in the folding, dimerization, and dissociation pathways of the tubulin dimer. Among them, TBCB and TBCE are two CAP-Gly domain-containing proteins that together efficiently interact with and dissociate the tubulin dimer. In the study reported here we showed that TBCB localizes at spindle and midzone microtubules during mitosis. Furthermore, the motif DEI/M-COO^? present in TBCB, which is similar to the EEY/F-COO^? element characteristic of EB proteins, CLIP-170, and α-tubulin, is required for TBCE–TBCB heterodimer formation and thus for tubulin dimer dissociation. This motif is responsible for TBCB autoinhibition, and our analysis suggests that TBCB is a monomer in solution. Mutants of TBCB lacking this motif are derepressed and induce microtubule depolymerization through an interaction with EB1 associated with microtubule tips. TBCB is also able to bind to the chaperonin complex CCT containing α-tubulin, suggesting that it could escort tubulin to facilitate its folding and dimerization, recycling or degradation.     

Cellular therapy to target neuroinflammation in amyotrophic lateral sclerosis

Neurodegenerative disorders are characterized by the selective vulnerability and progressive loss of discrete neuronal populations. Non-neuronal cells appear to significantly contribute to neuronal loss in diseases such as amyotrophic lateral sclerosis (ALS), Parkinson, and Alzheimer’s disease. In ALS, there is deterioration of motor neurons in the cortex, brainstem, and spinal cord, which control voluntary muscle groups. This results in muscle wasting, paralysis, and death. Neuroinflammation, characterized by the appearance of reactive astrocytes and microglia as well as macrophage and T-lymphocyte infiltration, appears to be highly involved in the disease pathogenesis, highlighting the involvement of non-neuronal cells in neurodegeneration. There appears to be cross-talk between motor neurons, astrocytes, and immune cells, including microglia and T-lymphocytes, which are subsequently activated. Currently, effective therapies for ALS are lacking; however, the non-cell autonomous nature of ALS may indicate potential therapeutic targets. Here, we review the mechanisms of action of astrocytes, microglia, and T-lymphocytes in the nervous system in health and during the pathogenesis of ALS. We also evaluate the therapeutic potential of these cellular populations, after transplantation into ALS patients and animal models of the disease, in modulating the environment surrounding motor neurons from pro-inflammatory to neuroprotective. We also thoroughly discuss the recent advances made in the field and caveats that need to be overcome for clinical translation of cell therapies aimed at modulating non-cell autonomous events to preserve remaining motor neurons in patients.     

Pathogenic mutation in the ALS/FTD gene, <Emphasis Type="Italic">CCNF,</Emphasis> causes elevated Lys48-linked ubiquitylation and defective autophagy

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal neurodegenerative disorders that have common molecular and pathogenic characteristics, such as aberrant accumulation and ubiquitylation of TDP-43; however, the mechanisms that drive this process remain poorly understood. We have recently identified CCNF mutations in familial and sporadic ALS and FTD patients. CCNF encodes cyclin F, a component of an E3 ubiquitin–protein ligase (SCF^{cyclin F}) complex that is responsible for ubiquitylating proteins for degradation by the ubiquitin–proteasome system. In this study, we examined the ALS/FTD-causing p.Ser621Gly (p.S621G) mutation in cyclin F and its effect upon downstream Lys48-specific ubiquitylation in transfected Neuro-2A and SH-SY5Y cells. Expression of mutant cyclin F^S621G caused increased Lys48-specific ubiquitylation of proteins in neuronal cells compared to cyclin F^WT. Proteomic analysis of immunoprecipitated Lys48-ubiquitylated proteins from mutant cyclin F^S621G-expressing cells identified proteins that clustered within the autophagy pathway, including sequestosome-1 (p62/SQSTM1), heat shock proteins, and chaperonin complex components. Examination of autophagy markers p62, LC3, and lysosome-associated membrane protein 2 (Lamp2) in cells expressing mutant cyclin F^S621G revealed defects in the autophagy pathway specifically resulting in impairment in autophagosomal–lysosome fusion. This finding highlights a potential mechanism by which cyclin F interacts with p62, the receptor responsible for transporting ubiquitylated substrates for autophagic degradation. These findings demonstrate that ALS/FTD-causing mutant cyclin F^S621G disrupts Lys48-specific ubiquitylation, leading to accumulation of substrates and defects in the autophagic machinery. This study also demonstrates that a single missense mutation in cyclin F causes hyper-ubiquitylation of proteins that can indirectly impair the autophagy degradation pathway, which is implicated in ALS pathogenesis.     

Therapeutic neuroprotective agents for amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal chronic neurodegenerative disease whose hallmark is proteinaceous, ubiquitinated, cytoplasmic inclusions in motor neurons and surrounding cells. Multiple mechanisms proposed as responsible for ALS pathogenesis include dysfunction of protein degradation, glutamate excitotoxicity, mitochondrial dysfunction, apoptosis, oxidative stress, and inflammation. It is therefore essential to gain a better understanding of the underlying disease etiology and search for neuroprotective agents that might delay disease onset, slow progression, prolong survival, and ultimately reduce the burden of disease. Because riluzole, the only Food and Drug Administration (FDA)-approved treatment, prolongs the ALS patient’s life by only 3 months, new therapeutic agents are urgently needed. In this review, we focus on studies of various small pharmacological compounds targeting the proposed pathogenic mechanisms of ALS and discuss their impact on disease progression.     

Research advances in gene therapy approaches for the treatment of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease of motor neurons that causes progressive muscle weakness, paralysis, and premature death. No effective therapy is available. Research in the motor neuron field continues to grow, and recent breakthroughs have demonstrated the possibility of completely achieving rescue in animal models of spinal muscular atrophy, a genetic motor neuron disease. With adeno-associated virus (AAV) vectors, gene transfer can be achieved with systemic non-invasive injection and minimal toxicity. In the context of this success, we review gene therapy approaches for ALS, considering what has been done and the possible future directions for effective application of the latest generation of vectors for clinical translation. We focus on recent developments in the areas of RNA/antisense-mediated silencing of specific ALS causative genes like superoxide dismutase-1 and other molecular pathogenetic targets, as well as the administration of neuroprotective factors with viral vectors. We argue that gene therapy offers new opportunities to open the path for clinical progress in treating ALS.     

Extracellular wildtype and mutant SOD1 induces ER–Golgi pathology characteristic of amyotrophic lateral sclerosis in neuronal cells

Amyotrophic lateral sclerosis (ALS) is a fatal and rapidly progressing neurodegenerative disorder and the majority of ALS is sporadic, where misfolding and aggregation of Cu/Zn-superoxide dismutase (SOD1) is a feature shared with familial mutant-SOD1 cases. ALS is characterized by progressive neurospatial spread of pathology among motor neurons, and recently the transfer of extracellular, aggregated mutant SOD1 between cells was demonstrated in culture. However, there is currently no evidence that uptake of SOD1 into cells initiates neurodegenerative pathways reminiscent of ALS pathology. Similarly, whilst dysfunction to the ER–Golgi compartments is increasingly implicated in the pathogenesis of both sporadic and familial ALS, it remains unclear whether misfolded, wildtype SOD1 triggers ER–Golgi dysfunction. In this study we show that both extracellular, native wildtype and mutant SOD1 are taken up by macropinocytosis into neuronal cells. Hence uptake does not depend on SOD1 mutation or misfolding. We also demonstrate that purified mutant SOD1 added exogenously to neuronal cells inhibits protein transport between the ER–Golgi apparatus, leading to Golgi fragmentation, induction of ER stress and apoptotic cell death. Furthermore, we show that extracellular, aggregated, wildtype SOD1 also induces ER–Golgi pathology similar to mutant SOD1, leading to apoptotic cell death. Hence extracellular misfolded wildtype or mutant SOD1 induce dysfunction to ER–Golgi compartments characteristic of ALS in neuronal cells, implicating extracellular SOD1 in the spread of pathology among motor neurons in both sporadic and familial ALS.     

Disruption of calcitonin gene-related peptide signaling accelerates muscle denervation and dampens cytotoxic neuroinflammation in SOD1 mutant mice

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease. Neuronal vacuolization and glial activation are pathologic hallmarks in the superoxide dismutase 1 (SOD1) mouse model of ALS. Previously, we found the neuropeptide calcitonin gene-related peptide (CGRP) associated with vacuolization and astrogliosis in the spinal cord of these mice. We now show that CGRP abundance positively correlated with the severity of astrogliosis, but not vacuolization, in several motor and non-motor areas throughout the brain. SOD1 mice harboring a genetic depletion of the βCGRP isoform showed reduced CGRP immunoreactivity associated with vacuolization, while motor functions, body weight, survival, and astrogliosis were not altered. When CGRP signaling was completely disrupted through genetic depletion of the CGRP receptor component, receptor activity-modifying protein 1 (RAMP1), hind limb muscle denervation, and loss of muscle performance were accelerated, while body weight and survival were not affected. Dampened neuroinflammation, i.e., reduced levels of astrogliosis in the brain stem already in the pre-symptomatic disease stage, and reduced microgliosis and lymphocyte infiltrations during the late disease phase were additional neuropathology features in these mice. On the molecular level, mRNA expression levels of brain-derived neurotrophic factor (BDNF) and those of the anti-inflammatory cytokine interleukin 6 (IL-6) were elevated, while those of several pro-inflammatory cytokines found reduced in the brain stem of RAMP1-deficient SOD1 mice at disease end stage. Our results thus identify an important, possibly dual role of CGRP in ALS pathogenesis.     

Stem cell transplantation for amyotrophic lateral sclerosis: therapeutic potential and perspectives on clinical translation

Amyotrophic lateral sclerosis (ALS) is a fatal neurological disease characterized by degeneration of upper and lower motor neurons. There are currently no clinically impactful treatments for this disorder. Death occurs 3–5 years after diagnosis, usually due to respiratory failure. ALS pathogenesis seems to involve several pathological mechanisms (i.e., oxidative stress, inflammation, and loss of the glial neurotrophic support, glutamate toxicity) with different contributions from environmental and genetic factors. This multifaceted combination highlights the concept that an effective therapeutic approach should counteract simultaneously different aspects: stem cell therapies are able to maintain or rescue motor neuron function and modulate toxicity in the central nervous system (CNS) at the same time, eventually representing the most comprehensive therapeutic approach for ALS. To achieve an effective cell-mediated therapy suitable for clinical applications, several issues must be addressed, including the identification of the most performing cell source, a feasible administration protocol, and the definition of therapeutic mechanisms. The method of cell delivery represents a major issue in developing cell-mediated approaches since the cells, to be effective, need to be spread across the CNS, targeting both lower and upper motor neurons. On the other hand, there is the need to define a strategy that could provide a whole distribution without being too invasive or burdened by side effects. Here, we review the recent advances regarding the therapeutic potential of stem cells for ALS with a focus on the minimally invasive strategies that could facilitate an extensive translation to their clinical application.     

Free amino acids in motor cortex of amyotrophic lateral sclerosis.

Free amino acids were estimated quantitatively in the motor cortex from 3 patients with amyotrophic lateral sclerosis (ALS) and 11 control subjects. Among 7 amino acids which showed statistically significant changes, taurine was the only one which was increased constantly and most markedly in the motor cortex of all the 3 ALS cases. It was suggested that the metabolism of sulfur amino acids might be affected in comparatively early stages of ALS.     

PAP/REG3A favors perineural invasion in pancreatic adenocarcinoma and serves as a prognostic marker

Pancreatic ductal adenocarcinoma (PDA) is a fatal and insidious malignant disease for which clinicians’ tools are restricted by the current limits in knowledge of how tumor and stromal cells act during the disease. Among PDA hallmarks, neural remodeling (NR) and perineural invasion (PNI) drastically influence quality of life and patient survival. Indeed, NR and PNI are associated with neuropathic pain and metastasis, respectively, both of which impact clinicians’ decisions and therapeutic options. The aim of this study was to determine the impact and clinical relevance of the peritumoral microenvironment, through pancreatitis-associated protein (PAP/REG3A) expression, on PNI in pancreatic cancer. First, we demonstrated that, in PDA, PAP/REG3A is produced by inflamed acinar cells from the peritumoral microenvironment and then enhances the migratory and invasive abilities of cancer cells. More specifically, using perineural ex vivo assays we revealed that PAP/REG3A favors PNI through activation of the JAK/STAT signaling pathway in cancer cells. Finally, we analyzed the level of PAP/REG3A in blood from healthy donors or patients with PDA from three independent cohorts. Patients with high levels of PAP/REG3A had overall shorter survival as well as poor surgical outcomes with reduced disease-free survival. Our study provides a rationale for using the PAP/REG3A level as a biomarker to improve pancreatic cancer prognosis. It also suggests that therapeutic targeting of PAP/REG3A activity in PDA could limit tumor cell aggressiveness and PNI.     

Glycoprotein IIb/IIIa antagonists - from bench to practice

The central role played by the α_IIbβ₃ receptor in platelet aggregation, and hence in platelet thrombosis, has led to the development of a number of parenteral and oral glycoprotein (GP) IIb/IIIa inhibitors for use in cardiovascular disease states, such as acute coronary syndromes and stroke. The predominant effect of these agents is to inhibit platelet aggregation, although studies of α_IIbβ₃ receptor function and various GP IIb/IIIa inhibitors have demonstrated the potential for these agents to produce effects on other aspects of platelet function, in addition to non-platelet effects. Overall, clinical studies have demonstrated an impressive beneficial effect for parenteral agents in reducing ischemic complications following percutaneous intervention, and a more modest beneficial effect in the treatment of patients with acute coronary syndromes. Trials with oral GP IIb/IIIa inhibitors in similar patient populations have demonstrated toxicity, manifested by an increased mortality in treated patients. Increased understanding of molecular aspects of both α_IIbβ₃ receptor function and the effects of GP IIb/IIIa inhibition may help explain some of the inconsistency in recently reported clinical studies with parenteral agents, and the frank toxicity of oral agents. Such studies may also hold the key to the development of newer agents with enhanced therapeutic benefit. Received 10 September 2001; received after revision 22 October 2001; accepted 2 November 2001     

Free amino acids in motor cortex of amyotrophic lateral sclerosis

Summary Free amino acids were estimated quantitatively in the motor cortex from 3 patients with amyotrophic lateral sclerosis (ALS) and 11 control subjects. Among 7 amino acids which showed statistically significant changes, taurine was the only one which was increased constantly and most markedly in the motor cortex of all the 3 ALS cases. It was suggested that the metabolism of sulfur amino acids might be affected in comparatively early stages of ALS.Acknowledgments. The authors are grateful to Dr M. Uono, Department of Neurology, Tokyo Metropolitan Hospital of Fuchu, and Dr K. Hirayama, Department of Neurology, Brain Research Institute, School of Medicine, Chiba University, for their generous cooperation.     

The multifaceted role of glial cells in amyotrophic lateral sclerosis

Despite indisputable progress in the molecular and genetic aspects of amyotrophic lateral sclerosis (ALS), a mechanistic comprehension of the neurodegenerative processes typical of this disorder is still missing and no effective cures to halt the progression of this pathology have yet been developed. Therefore, it seems that a substantial improvement of the outcome of ALS treatments may depend on a better understanding of the molecular mechanisms underlying neuronal pathology and survival as well as on the establishment of novel etiological therapeutic strategies. Noteworthy, a convergence of recent data from multiple studies suggests that, in cellular and animal models of ALS, a complex pathological interplay subsists between motor neurons and their non-neuronal neighbours, particularly glial cells. These observations not only have drawn attention to the physiopathological changes glial cells undergo during ALS progression, but they have moved the focus of the investigations from intrinsic defects and weakening of motor neurons to glia–neuron interactions. In this review, we summarize the growing body of evidence supporting the concept that different glial populations are critically involved in the dreadful chain of events leading to motor neuron sufferance and death in various forms of ALS. The outlined observations strongly suggest that glial cells can be the targets for novel therapeutic interventions in ALS.     

Effects of antithymic reticuloepithelial cells serum on the levels of circulating thymic factor and on the sensitivity to azathioprine of spleen spontaneous rosette-forming cells

Summary Swiss mice treated with an antithymic reticuloepithelial cells serum (ATRES) showed a drastic and prolonged depression of the serum thymic factor. A similar but less pronounced effect was also observed following the administration of the antithymocyte (ATS) and the antilymphocyte (ALS) sera. Conversely, the azathioprine sensitivity of spleen spontaneous rosette-forming cells was highly modified by the ATRES but not by the ATS or the ALS. The probable mechanisms of such effects are discussed.     

Effects of storage and processing on the nutritive value of certain Nigerian foods

The effects of storage and processing on the nutritive value of two Nigerian foods--raw cowpea (Vigna unguiculata) flour and palm oil, have been investigated. Increased retention of both thiamine and riboflavin as a result of increased water activity (Aw) were recorded for cowpea flour stored for 6 months. A storage temperature of 5 degrees C led to minimal losses of both vitamins in the stored flour. Differences in physico-chemical characteristics were observed between palm oil produced by the traditional method and that produced by a modern commercial method. Increasing Aw led to decreasing loss of unsaturation in the traditionally-produced palm oil during 4-week storage at ambient temperature (25 degrees C).     

Joining S100 proteins and migration: for better or for worse,in sickness and in health

The vast diversity of S100 proteins has demonstrated a multitude of biological correlations with cell growth, cell differentiation and cell survival in numerous physiological and pathological conditions in all cells of the body. This review summarises some of the reported regulatory functions of S100 proteins (namely S100A1, S100A2, S100A4, S100A6, S100A7, S100A8/S100A9, S100A10, S100A11, S100A12, S100B and S100P) on cellular migration and invasion, established in both culture and animal model systems and the possible mechanisms that have been proposed to be responsible. These mechanisms involve intracellular events and components of the cytoskeletal organisation (actin/myosin filaments, intermediate filaments and microtubules) as well as extracellular signalling at different cell surface receptors (RAGE and integrins). Finally, we shall attempt to demonstrate how aberrant expression of the S100 proteins may lead to pathological events and human disorders and furthermore provide a rationale to possibly explain why the expression of some of the S100 proteins (mainly S100A4 and S100P) has led to conflicting results on motility, depending on the cells used.     

Cathepsin A/protective protein: an unusual lysosomal multifunctional protein

Cathepsin A/protective protein [3.4.16.5], carboxypeptidase A, is a lysosomal serine protease with structural homology to yeast (Saccharomyces cerevisiae) carboxypeptidase Y. Cathepsin A is a member of the alpha/beta hydrolase fold family and has been suggested to share a common ancestral relationship with other alpha/beta hydrolase fold enzymes, such as cholinesterases. Several lines of evidence indicate that cathepsin A is a multicatalytic enzyme with deamidase and esterase in addition to carboxypeptidase activities. Cathepsin A was recently identified in human platelets as deamidase. In vitro, it hydrolyzes a variety of bioactive peptide hormones including tachykinins, suggesting that extralysosomal cathepsin A plays a role in regulation of bioactive peptide functions. Recent reports emphasize the lysosomal protective function of cathepsin A rather than its protease function. The protective function of cathepsin A is distinct from its catalytic function. Human lysosomal beta-galactosidase and neuraminidase exist as a high molecular weight enzyme complex, in which there is a 54-kDa glycoprotein termed 'lysosomal protective protein'. Based on cell culture studies, protective protein was found to protect both beta-galactosidase and neuraminidase from intralysosomal proteolysis by forming a multienzyme complex and was shown to be deficient in patients with galactosialidosis, a combined deficiency of beta-galactosidase and neuraminidase. Molecular cloning and gene expression studies have disclosed that protective protein is cathepsin A. The cathepsin A precursor has the potential to restore both beta-galactosidase and neuraminidase activities in fibroblasts from patients with galactosialidosis. Cathepsin A knockout mice showed a phenotype similar to human galactosialidosis and the deficient phenotype found in the mutant mice was corrected by transplanting erythroid precursor cells overexpressing cathepsin A. Collectively, these findings demonstrate the significance of cathepsin A as a key molecule in the onset of galactosialidosis and also highlight the therapeutic potential of the cathepsin A precursor for patients with galactosialidosis.     

Structural Time-series Modelling of Monetary Aggregates: A Case Study for Eleven European Countries

Monetary aggregates for eleven European countries are analysed using the structural time-series methodology, paying special attention to unit root issues. Estimation of the parameters of the models is carried out by applying the asymptotic least squares (ALS) procedure. A comparison with the maximum likelihood estimates obtained via the Kalman filter shows that ALS is an alternative to Kalman filter estimation. The empirical results show that for only a small number of series the four variance parameters of the basic structural model are strictly positive. For the majority of the series the variance of the irregular component is equal to 0.©1997 John Wiley & Sons, Ltd.