Search

Objective:The purpose of this study was to evaluate Canadian amyotrophic lateral sclerosis (ALS) centres with respect to: 1) the prevalence of Non-invasive positive pressure ventilation (NIPPV) and invasive mechanical ventilation via tracheostomy (TV) utilization, 2) the approach to NIPPV use, focusing upon the currently employed initiation criteria and 3) the barriers influencing NIPPV administration.Methods:A descriptive survey research design aimed to obtain quantitative data and open-ended responses from an active physician at each of the 15 multidisciplinary Canadian ALS centres.Results:The principal findings of this study were: 1) NIPPV and TV are used in 18.3% and 1.5% of patients at Canadian ALS centres, respectively, 2) symptoms of respiratory insufficiency, namely orthopnea (clinical significance rated at 9.00/10 ± 1.48), dyspnea (8.27 ± 1.95) and morning headache (7.55 ± 1.21) are the most significant indicators for NIPPV initiation, 3) the primary barriers to NIPPV utilization are patient intolerance (70% of centres) and inaccessibility of respirologists and ventilation technologists (50% of centres).Conclusions:Variability in NIPPV use has an impact upon the management of Canadian ALS patients. The establishment of more definitive NIPPV initiation criteria, emphasizing respiratory symptoms, and the attenuation of barriers to NIPPV use should be targeted so as to ensure optimal care for all ALS patients.

In this review, the role of aberrant RNA metabolism in ALS is examined, including the evidence that a majority of the genetic mutations observed in familial ALS (including mutations in TDP-43, FUS/TLS, SOD1, angiogenin (ANG) and senataxin (SETX)) can impact directly on either gene transcription, pre-mRNA splicing, ribonucleoprotein complex formation, transport, RNA translation or degradation. The evidence that perturbed expression or function of RNA binding proteins is causally related to the selective suppression of the low molecular weight subunit protein (NFL) steady state mRNA levels in degenerating motor neurons in ALS is examined. The discovery that mtSOD1, TDP-43 and 14-3-3 proteins, all of which form cytosolic aggregates in ALS, can each modulate the stability of NFL mRNA, suggests that a fundamental alteration in the interaction of mRNA species with key trans-acting binding factors has occurred in ALS. These observations lead directly to the hypothesis that ALS can be viewed as a disorder of RNA metabolism, thus providing a novel pathway for the development of molecular pharmacotherapies.

Objective: To systematically review evidence bearing on the management of patients with amyotrophic lateral sclerosis (ALS). Methods: The authors analyzed studies from 1998 to 2007 to update the 1999 practice parameter. Topics covered in this section include breaking the news, multidisciplinary clinics, symptom management, cognitive and behavioral impairment, communication, and palliative care for patients with ALS. Results: The authors identified 2 Class I studies, 8 Class II studies, and 30 Class III studies in ALS, but many important areas have been little studied. More high-quality, controlled studies of symptomatic therapies and palliative care are needed to guide management and assess outcomes in patients with ALS. Recommendations: Multidisciplinary clinic referral should be considered for managing patients with ALS to optimize health care delivery and prolong survival (Level B) and may be considered to enhance quality of life (Level C). For the treatment of refractory sialorrhea, botulinum toxin B should be considered (Level B) and low-dose radiation therapy to the salivary glands may be considered (Level C). For treatment of pseudobulbar affect, dextromethorphan and quinidine should be considered if approved by the US Food and Drug Administration (Level B). For patients who develop fatigue while taking riluzole, withholding the drug may be considered (Level C). Because many patients with ALS demonstrate cognitive impairment, which in some cases meets criteria for dementia, screening for cognitive and behavioral impairment should be considered in patients with ALS (Level B). Other management strategies all lack strong evidence.

Objective: In familial amyotrophic lateral sclerosis (fALS) harboring superoxide dismutase (SOD1) mutations (fALS1), SOD1 toxicity has been linked to its propensity to misfold and aggregate. It has recently been proposed that misfolded SOD1 may be causative of all types of ALS, including sporadic cases (sALS). In the present study, we have used a specific antibody to test for the presence of monomer/misfolded SOD1 in sALS. Methods: Sections from lumbar spinal cords of 5 fALS1 cases, 13 sALS cases, and 1 non-SOD1 fALS case were labeled immunocytochemically using SOD1-exposed-dimer-interface (SEDI) antibody, which we have previously validated as being specific for pathological monomer/misfolded forms of SOD1. Results: Monomer/misfolded SOD1 was detected with SEDI antibody in all 5 of the fALS1 cases, localizing predominantly to hyaline conglomerate inclusions, a specific pathological feature of fALS1. In contrast, monomer/misfolded SOD1 was not detected in any of the 13 sALS cases or in the non-SOD1 fALS cases. These results were confirmed by immunoprecipitation. Interpretation: Although SEDI antibody does not necessarily label all misfolded forms of SOD1, these findings show a distinct difference between fALS1 and sALS, and do not support that monomer/misfolded SOD1 is a common disease entity linking all types of ALS. This is important to our understanding of ALS disease pathogenesis and to considerations of the applicability of using therapeutics that target misfolded SOD1 to non-SOD1-related cases.

Background: Primary lateral sclerosis (PLS) is an idiopathic upper motor neuron degenerative disorder. The aim of this study was to compare brain volumes in patients with PLS and controls and determine whether differences were due to loss of gray matter (GM), white matter (WM), or both. Methods: T1-weighted images were acquired in patients with PLS and controls. Freesurfer was used for volumetric segmentation of whole brain, cortical GM, precentral and postcentral cortex, WM, corpus callosum, basal ganglia, thalamus, cerebellum, and CSF. Relationships were sought between disease severity, disease duration, age and brain volumes. Results: Eleven patients with PLS and 10 age-matched healthy controls were included in this study. Compared to control subjects, patients with PLS had significantly smaller whole brain ( p = 0.043), frontal lobe ( p = 0.036), precentral cortex ( p = 0.016), and corpus callosum ( p = 0.036) volumes. There was a trend toward a smaller thalamus ( p = 0.051). Disease severity correlated with ventricular CSF volume (rho = −0.604, p = 0.025) and precentral cortex volume loss (rho = 0.599, p = 0.026). Disease duration tended to correlate with a loss of WM (rho = −0.636, p = 0.063). Conclusions: Our results suggest that there is focal atrophy in patients with primary lateral sclerosis compared with controls especially in the precentral cortex and the corpus callosum, specifically where there is transfer of motor fibers. ALS = amyotrophic lateral sclerosis; ALSFRS-R = ALS Functional Rating Scale; fSPGR = fast spoiled gradient echo sequence; GM = gray matter; PLS = primary lateral sclerosis; TE = echo time; TR = repetition time; WM = white matter.

Amyotrophic lateral sclerosis (ALS) is increasingly recognized to be a multisystem disorder which includes both clinical and neuropathological features of a frontotemporal lobar degeneration (FTLD). In order to provide a common framework within which to discuss the characteristics of the cognitive and behavioural syndromes of ALS, and with which to conduct clinical and neuropathological research, an international research workshop on frontotemporal dementia (FTD) and ALS was held in London, Canada in June 2007. The recommendations arising from this research workshop address the requirement for a concise clinical diagnosis of the underlying motor neuron disease (Axis I), defining the cognitive and behavioural dysfunction (Axis II), describing additional non-motor manifestations (Axis III) and identifying the presence of disease modifiers (Axis IV).

In the mouse, p190RhoGEF is a low molecular weight neurofilament (NFL) mRNA stability factor that is involved in NF aggregate formation in neurons. A human homologue of this protein has not been described. Our objective was to identify a human homologue of p190RhoGEF, and to determine its interaction with human NFL mRNA. We used sequence homology searches to predict a human homologue (RGNEF), and RT-PCR to determine the expression of mRNA in ALS and neuropathologically normal control tissues. Gel shift assays determined the interaction of RGNEF with human NFL mRNA in vitro, while IP-RT-PCR and gel shift assays were used to confirm the interaction in tissue lysates. We determined that RGNEF is a human homologue of p190RhoGEF, and that its RNA is expressed in both brain and spinal cord. While RGNEF and NFL mRNA interact directly in vitro, interestingly they only appear to interact in ALS lysates and not in controls. These data add another player to the family of NFL mRNA stability regulators, and raise the intriguing possibility that the mechanism by which p190RhoGEF contributes to murine neuronal NF aggregate formation may be important to human ALS NF aggregate formation.

We have performed sciatic axotomies in adult C57BL/6 mice and observed TDP-43 and progranulin (PGRN) expression patterns over 28 days. TDP-43 expression was markedly upregulated in axotomized motor neurons, with prominent cytosolic immunoreactivity becoming maximal by post-injury day 7 and returning to baseline levels by post-injury day 28. Increased TDP-43 expression was confirmed by western blot. TDP-43 mRNA expression was also increased. This was inversely correlated with neuronal PGRN expression which was clearly reduced by day 7 with a return to baseline by post-injury day 28. In contrast, microglial PGRN expression was dramatically increased, and correlated with the inflammatory response to axotomy. Cytosolic TDP-43 colocalized with Staufen and TIA-1, markers for RNA transport and stress granules respectively. We did not observe colocalization of TDP-43 or PGRN with degradative granules (P-bodies) or activated caspase 3. These results indicate that TDP-43 expression is altered in response to neuronal injury and that normal expression is restored following recovery. These findings suggest that the upregulation of TDP-43 expression with prominent cytosolic localization in motor neurons injured by degenerative processes such as ALS may actually represent an appropriate response to neuronal injury.

The neuronal intermediate filament protein peripherin is a component of ubiquitinated inclusions and of axonal spheroids in amyotrophic lateral sclerosis (ALS). Overexpression of peripherin causes motor neuron degeneration in transgenic mice and variations within the peripherin gene have been identified in ALS cases. We have shown previously the abnormal expression of a neurotoxic peripherin splice variant in transgenic mice expressing mutant superoxide dismutase-1. These findings indicated that abnormalities of peripherin splicing may occur in ALS. In the current study, peripherin splice variants were identified by reverse transcription-PCR of human neuronal RNA and comparisons in expression made between control and ALS spinal cord using Western blot analysis and immunocytochemistry. Using this approach we have identified a novel peripherin transcript retaining introns 3 and 4 that results in a 28 kDa splice isoform, designated Per 28. Using an antibody specific to Per 28, we show that this isoform is expressed at low stoichiometric levels from the peripherin gene, however causes peripherin aggregation when its expression is upregulated. Importantly we show an upregulation of Per 28 expression in ALS compared with controls, at both the mRNA and protein levels, and that Per 28 is associated with disease pathology, specifically round inclusions. These findings are the first to establish that peripherin splicing abnormalities occur in ALS, generating aggregation-prone splice isoforms.

Amyotrophic lateral sclerosis is increasingly recognized to be a complex multisystems disorder both at the level of its pathobiology and in the breadth of non-motor manifestations that can accompany it. Paramount among these are disorders of frontotemporal function which can be associated with syndromes of behavioural, cognitive or executive dysfunction or manifest as a frontotemporal dementia (FTD). While these may occur in isolation and precede the development of motor deficits, more commonly they insidiously onset following the initial neuromuscular dysfunction. The earliest clinical manifestation is a loss of verbal fluency, disproportionate to impairments in oromotor control. There is good correlation between the presence of a syndrome of frontotemporal dysfunction and alterations in brain structure or function as identified with a wide variety of neuroimaging techniques and which reflect a frontotemporal lobar degeneration (FTLD). Although the cause(s) of this process remain to be defined, as with the clinical heterogeneity, there is likely to be significant biochemical heterogeneity. This includes alterations in tau protein metabolism which are present in a proportion of familial and sporadic ALS cases, as well as the western Pacific variant, and recently described alterations in the metabolism of the TAR DNA binding protein 43 (TDP-43).

SummaryIn the study of protein complexes, is there a computational method for inferring which combinations of proteins in an organism are likely to form a crystallizable complex? Here we attempt to answer this question, using the Protein Data Bank (PDB) to assess the usefulness of inferred functional protein linkages from the Prolinks database. We find that of the 242 nonredundant prokaryotic protein complexes shared between the current PDB and Prolinks, 44% (107/242) contain proteins linked at high confidence by one or more methods of computed functional linkages. Similarly, high-confidence linkages detect 47% of known Escherichia coli protein complexes, with 45% accuracy. Together these findings suggest that functional linkages will be useful in defining protein complexes for structural studies, including for structural genomics. We offer a database of inferred linkages corresponding to likely protein complexes for some 629,952 pairs of proteins in 154 prokaryotes and archaea.

Under normal conditions, the proline-directed serine/threonine residues of neurofilament tail-domain repeats are exclusively phosphorylated in axons. In pathological conditions such as amyotrophic lateral sclerosis (ALS), motor neurons contain abnormal perikaryal accumulations of phosphorylated neurofilament proteins. The precise mechanisms for this compartment-specific phosphorylation of neurofilaments are not completely understood. Although localization of kinases and phosphatases is certainly implicated, another possibility involves Pin1 modulation of phosphorylation of the proline-directed serine/threonine residues. Pin1, a prolyl isomerase, selectively binds to phosphorylated proline-directed serine/threonine residues in target proteins and isomerizes cis isomers to more stable trans configurations. In this study we show that Pin1 associates with phosphorylated neurofilament-H (p-NF-H) in neurons and is colocalized in ALS-affected spinal cord neuronal inclusions. To mimic the pathology of neurodegeneration, we studied glutamate-stressed neurons that displayed increased p-NF-H in perikaryal accumulations that colocalized with Pin1 and led to cell death. Both effects were reduced upon inhibition of Pin1 activity by the use of an inhibitor juglone and down-regulating Pin1 levels through the use of Pin1 small interfering RNA. Thus, isomerization of lys-ser-pro repeat residues that are abundant in NF-H tail domains by Pin1 can regulate NF-H phosphorylation, which suggests that Pin1 inhibition may be an attractive therapeutic target to reduce pathological accumulations of p-NF-H.

Background: TCH346 exerts antiapoptotic effects by binding to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and blocking the apoptotic pathway in which GAPDH is involved. Apoptosis is considered to be a key pathogenic mechanism in neurodegenerative diseases including ALS. Methods: Patients were randomly assigned in a double-blind fashion to receive either placebo or one of four doses of TCH346 (1.0, 2.5, 7.5, or 15 mg/day) administered orally once daily for at least 24 weeks. The primary outcome measure was the rate of change in the revised ALS functional rating scale (ALSFRS-R). The trial design included a 16-week lead-in phase to determine each patient9s rate of disease progression. The between treatment comparison was adjusted for the individual pretreatment rates of progression. The study was powered to detect a 25% reduction in the rate of decline of the ALSFRS-R as compared with placebo. Secondary outcome measures included survival, pulmonary function, and manual muscle testing (MMT). Results: Five hundred ninety-one patients were enrolled at 42 sites in Europe and North America. There were no differences in baseline variables. There were no significant differences between placebo and active treatment groups in the mean rate of decline of the ALSFRS-R or in the secondary outcome measures (survival, pulmonary function, and MMT). Conclusion: The trial revealed no evidence of a beneficial effect of TCH346 on disease progression in patients with ALS.

Tight coordination of the expression of neurofilament subunits is integral to the normal development and function of the nervous system. Imbalances in their expression are increasingly implicated in the induction of neurodegeneration in which formation of neurofilamentous aggregates is central to the pathology. Neurofilament expression can be controlled not only at the transcriptional level but also through post-transcriptional regulation of mRNA localization, stability, and translational efficiency. The critical role that post-transcriptional mechanisms play in maintaining neurofilament homeostasis is highlighted, for example, by the human disease amyotrophic lateral sclerosis, in which selective destabilization of NF-L mRNA (or failure to stabilize it) is associated with the formation of neurofilamentous aggregates - a hallmark of the disease process. This review discusses the post-transcriptional regulatory mechanisms and associated ribonucleoproteins that have been implicated to date in controlling neurofilament expression during normal development and in disrupting neurofilament homeostasis during neurodegenerative disease.

The human TAR DNA-binding protein (TDP43) colocalizes with ubiquitinated inclusions in motor neurons in amyotrophic lateral sclerosis (ALS). TDP43 is both a DNA-binding protein with a nuclear export sequence that interacts with (TG) n T m elements in DNA and an RNA-binding protein that interacts with (UG) 6–12 motifs in single–stranded RNA. In control motor neurons, TDP43 was almost exclusively nuclear, whereas in ALS spinal motor neurons, TDP43 was predominantly localized to the cytosol and not the nucleus. TDP43 was observed as punctuate immunoreactivity and as dense skeins, with and without ubiquitinization. We observed that TDP43 stabilizes the human low molecular weight ( h NFL) mRNA through a direct interaction with the 3′UTR. TDP43 is a unique h NFL mRNA-binding protein that is altered in its somatotopic localization in ALS spinal motor neurons and potentially contributes to the formation of NF aggregates in ALS through alterations in NF stoichiometry.

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disorder in which excitotoxicity has been implicated as a cause for cell death. To examine neurofilament (NF) aggregate-mediated sensitization of motor neurons to NMDA excitotoxicity, we examined NMDA receptor expression and the impact of NO donors (NOC12 or NOC5) or sodium cyanide (NaCN) on calcium influx and viability in dissociated motor neurons derived from wt and hNFL+/+ (NF aggregate-forming) mice. Alterations in intracellular calcium were assayed using Oregon Green calcium dye and the extent of apoptosis using active caspase-3 immunoreactivity. Although NF aggregate-bearing neurons demonstrated increased intracellular calcium levels and enhanced cell death in response to NMDA receptor activation, this was not associated with increased NMDA receptor expression. The down-regulation of the NMDA receptor using NO donors decreased calcium influx and caspase-3 activation in aggregate-bearing neurons, but had no effect on wt cultures. The converse was observed with NaCN in which intracellular calcium levels increased significantly in wt cultures in association with increased cell death. No effect was observed in aggregate-bearing neurons. These findings suggest that the presence of NF aggregates renders motor neurons more susceptible to NMDA-mediated excitotoxicity, and that this can be reversed by NO.

Background Central to most omic scale experiments is the interpretation and examination of resulting gene lists corresponding to differentially expressed, regulated, or observed gene or protein sets. Complicating interpretation is a lack of functional annotation assigned to a large percentage of many microbial genomes. This is particularly noticeable in mycobacterial genomes, which are significantly divergent from many of the microbial model species used for gene and protein functional characterization, but which are extremely important clinically. Mycobacterial species, ranging from M. tuberculosis to M. abscessus, are responsible for deadly infectious diseases that kill over 1.5 million people each year across the world. A better understanding of the coding capacity of mycobacterial genomes is therefore necessary to shed increasing light on putative mechanisms of virulence, pathogenesis, and functional adaptations. Description Here we describe the improved functional annotation coverage of 11 important mycobacterial genomes, many involved in human diseases including tuberculosis, leprosy, and nontuberculous mycobacterial (NTM) infections. Of the 11 mycobacterial genomes, we provide 9899 new functional annotations, compared to NCBI and TBDB annotations, for genes previously characterized as genes of unknown function, hypothetical, and hypothetical conserved proteins. Functional annotations are available at our newly developed web resource MycoBASE (Mycobacterial Annotation Server) at strong.ucdenver.edu/mycobase. Conclusion Improved annotations allow for better understanding and interpretation of genomic and transcriptomic experiments, including analyzing the functional implications of insertions, deletions, and mutations, inferring the function of understudied genes, and determining functional changes resulting from differential expression studies. MycoBASE provides a valuable resource for mycobacterial researchers, through improved and searchable functional annotations and functional enrichment strategies. MycoBASE will be continually supported and updated to include new genomes, enabling a powerful resource to aid the quest to better understand these important pathogenic and environmental species.

There are a number of publications showing an association between herpes virus infection and idiopathic pulmonary fibrosis (IPF). These reports use immunohistochemistry and/or PCR, which are susceptible to specificity artifacts. Thus, we investigated the possible association between IPF and viral RNA expression using next-generation sequencing, which has the potential to provide both a high degree of sensitivity as well as specificity. We quantified viral RNA expression for 740 viruses in 21 IPF patient lung biopsy samples and 17 age-matched controls. Our RNA-seq results were confirmed using Real-time RT-PCR for select viruses (EBV, herpes Saimiri and HERV-k). HERV-k expression was examined because of reports indicating that it is elevated in other fibrotic diseases, and because conceptually HERV-k could promote fibrogenesis by inducing cellular stress. Moreover, HERV-k expression is reported to be enhanced in response to herpes virus infection. Although we identified sporadic low-level evidence of viral infections in our lung tissue samples, we did not find statistical difference for expression of any virus, including HERV-k, between IPF and control lungs. To our knowledge, this is the first report that employs RNA-seq to assess whether or not viral infections are linked to IPF. Our results do not address the role of viral infection in the so-called acute exacerbation of IPF, however, they clearly do not support a causative connection between herpes virus infection and IPF.

Blood transcriptional signatures are promising for tuberculosis (TB) diagnosis but have not been evaluated among U.S. patients. To be used clinically, transcriptional classifiers need reproducible accuracy in diverse populations that vary in genetic composition, disease spectrum and severity, and comorbidities. In a prospective case-control study, we identified novel transcriptional classifiers for active TB among U.S. patients and systematically compared their accuracy to classifiers from published studies. Blood samples from HIV-uninfected U.S. adults with active TB, pneumonia, or latent TB infection underwent whole-transcriptome microarray. We used support vector machines to classify disease state based on transcriptional patterns. We externally validated our classifiers using data from sub-Saharan African cohorts and evaluated previously published transcriptional classifiers in our population. Our classifier distinguishing active TB from pneumonia had an area under the concentration-time curve (AUC) of 96.5% (95.4% to 97.6%) among U.S. patients, but the AUC was lower (90.6% [89.6% to 91.7%]) in HIV-uninfected Sub-Saharan Africans. Previously published comparable classifiers had AUC values of 90.0% (87.7% to 92.3%) and 82.9% (80.8% to 85.1%) when tested in U.S. patients. Our classifier distinguishing active TB from latent TB had AUC values of 95.9% (95.2% to 96.6%) among U.S. patients and 95.3% (94.7% to 96.0%) among Sub-Saharan Africans. Previously published comparable classifiers had AUC values of 98.0% (97.4% to 98.7%) and 94.8% (92.9% to 96.8%) when tested in U.S. patients. Blood transcriptional classifiers accurately detected active TB among U.S. adults. The accuracy of classifiers for active TB versus that of other diseases decreased when tested in new populations with different disease controls, suggesting additional studies are required to enhance generalizability. Classifiers that distinguish active TB from latent TB are accurate and generalizable across populations and can be explored as screening assays.

Mycobacterium chelonae is a rapidly growing opportunistic nontuberculous mycobacterial (NTM) species that causes infections in humans and other hosts. Here, we report the draft genome sequence of Mycobacterium chelonae type strain ATCC 35752, consisting of 4.89 Mbp, 63.96% G+C content, 4,489 protein-coding genes, 48 tRNAs, and 3 rRNA genes.

The pathogenesis of neurodegenerative diseases prior to the onset of symptoms is generally not clear. The present study has employed a mouse model with a lack of the low-molecular-weight neurofilament subunit (NFL-/-), in which formation of protein aggregates occurs in neurons, to investigate glial cellular reactions in the lumbar cord segments of NFL-/- mice at ages from 1 to 6 months. Age-matched C57BL/6 mice serve as the control. Apparent neurofilament positive aggregates in the cytoplasm of motoneurons have been observed in NFL-/- mice. However, there were no noticeable changes in microglial numbers and GFAP staining of astrocytes. Unexpectedly, a downregulation in expression of complement receptor type 3 alpha subunit (CD11b) was detected in the spinal cord of NFL-/- mice, while there was no obvious difference between NFL-/- and C57BL/6 mice in the CD11b staining intensity of macrophages from livers and spleens. In addition, retardation in morphological transformation from activated to amoeboid microglia in response to sciatic nerve injury, differential expressions of some cytokines in the lumbar cord segments and induction of Iba-1 (ionized calcium-binding adaptor molecule-1) expression in microglia were observed in NFL-/- mice. Our results suggest not only the existence of an inhibitory niche for CD11b expression in microglia in the lumbar cord segments of NFL-/- mice but also differential microglial reactions between earlier and later stages of neuropathogenesis. Although the real cause for such inhibition is still unknown, this effect might play a particular role in the survival of the abnormal protein aggregate-bearing motoneurons in the early development stage of neurodegeneration in the NFL-/- mice.

We have previously reported that supernatant derived from LPS-activated BV-2 cells, an immortalized microglial cell line, induces death of NSC-34 cells (a motor neuron hybridoma) through a TNFα and nitric oxide synthase (NOS) dependant mechanism. In this study, we have observed that LPS-activated BV-2 supernatant induces NSC-34 cell death in association with an upregulation of the TNF receptor 1 (TNFR1) expression on NSC-34 cells, both at the transcription level and at the cell surface protein level. The upregulation of TNFR1 receptor was independent of TNFα, and could be partly inhibited by the inhibition of iNOS activation in the BV-2 cells. The TNFR2 receptor was not involved. These observations have important implications in understanding the mechanism by which microglial activation contributes to the motor neuron degeneration.

Although tuberculosis remains a substantial global threat, the mechanisms that enable mycobacterial persistence and replication within the human host are ill defined. This study represents the first genome-wide expression analysis of Mycobacterium tuberculosis from clinical lung samples, which has enabled the identification of M. tuberculosis genes actively expressed during pulmonary tuberculosis. To obtain optimal information from our DNA array analyses, we analyzed the differentially expressed genes within the context of computationally inferred protein networks. Protein networks were constructed using functional linkages established by the Rosetta stone, phylogenetic profile, conserved gene neighbor, and operon computational methods. This combined approach revealed that during pulmonary tuberculosis, M. tuberculosis actively transcribes a number of genes involved in active fortification and evasion from host defense systems. These genes may provide targets for novel intervention strategies.

Among the motor neuron diseases, three share the clinical features of prominent upper motor neuron signs--amyotrophic lateral sclerosis (ALS), primary lateral sclerosis (PLS) and the hereditary spastic paraplegias (HSP). While genetic testing can assist in the identification of several variants of the latter, in the remaining cases, including those in which spasticity may be associated with amyotrophy, clinical differentiation of the three disorders may prove difficult. In this paper we review the evidence that these are distinct disorders and conclude that, for ALS and PLS particularly, there may be justification in considering them as single points along a continuum of multisystem disorders with conspicuous motor neuron involvement. Only through the development and application of exacting clinical diagnostic criteria to epidemiological studies, along with greater numbers of post-mortem examinations, however, will these questions be answered fully.

The mechanism by which mutated copper-zinc superoxide dismutase (SOD1) causes familial amyotrophic lateral sclerosis is believed to involve an adverse gain of function, independent of the physiological antioxidant enzymatic properties of SOD1. In this study, we have observed that mutant SOD1 (G41S, G85A, and G93A) but not the wild type significantly reduced the stability of the low molecular weight neurofilament mRNA in a dosage-dependent manner. We have also demonstrated that mutant SOD1 but not the wild type bound directly to the neurofilament mRNA 3'-untranslated region and that the binding was necessary to induce mRNA destabilization. These observations provide an explanation for a novel gain of function in which mutant SOD1 expression in motor neurons alters an intermediate filament protein expression.

Neurofilament (NF) aggregate formation within motor neurons is a pathological hallmark of both the sporadic and familial forms of amyotrophic lateral sclerosis (ALS). The relationship between aggregate formation and both microglial and astrocytic proliferation, as well as additional neuropathological features of ALS, is unknown. To examine this, we have used transgenic mice that develop NF aggregates, through either a lack of the low-molecular-weight NF subunit [NFL (-/-)] or the overexpression of human NFL [hNFL (+/+)]. Transgenic and wild-type C57bl/6 mice were examined from 1 month to 18 months of age, and the temporal pattern of motor neuron degeneration, microglial and astrocytic proliferation, and heat shock protein-70 (HSP-70) expression characterized. We observed three overlapping phases in both transgenic mice, including transient aggregate formation, reactive microgliosis, and progressive motor neuron loss. However, only NFL (-/-) mice demonstrated significant astrogliosis and HSP-70 upregulation in both motor neurons and astrocytes. These in vivo models suggest that the development of NF aggregates in motor neurons leads to motor neuron death, but that the interaction between the degenerating motor neurons and the adjacent non-neuronal cells may differ significantly depending on the etiology of the NF aggregate itself.

Among the neurodegenerative diseases associated with ageing, amyotrophic lateral sclerosis (ALS) remains the most devastating. The disease inexorably progresses, the vast majority of pharmacotherapies have failed to modify the disease course, death ensues on average within 5 years of symptom onset and increasing numbers of individuals are afflicted with the disease. However, significant advances in our understanding of the natural history of ALS and of the fundamental nature of the biological defect underlying motor neuron degeneration have been gained, providing hope for the development of novel pharmacotherapies for ALS. Among these is the recognition that ALS is a biologically heterogeneous disorder in which genetics, environment and ageing all interrelate. The observation of clinical heterogeneity, with initial clinical manifestations serving as predictors of survivorship, is of considerable importance in designing therapeutic trials. The presence of frontotemporal dysfunction in a subset of patients has led to increased interest in the relationship between ALS and the degenerative tauopathies. Ultimately, the degenerating motor neurons do not die alone. The contribution of both microglia and astrocytes to the degenerative process are increasingly recognised. Understanding how these processes interrelate has become critical to understanding the pharmacotherapy of ALS and in the design of clinical trials. This review will highlight recent epidemiological and neurochemical advances in our understanding of ALS, and place them into the context of understanding the development of novel treatment avenues for this devastating disease.

In the spring of 2015, investigators in Switzerland reported a cluster of six patients with invasive infection with Mycobacterium chimaera, a species of nontuberculous mycobacterium ubiquitous in soil and water. The infected patients had undergone open-heart surgery that used contaminated heater-cooler devices during extracorporeal circulation (1). In July 2015, a Pennsylvania hospital also identified a cluster of invasive nontuberculous mycobacterial infections among open-heart surgery patients. Similar to the Swiss report, a field investigation by the Pennsylvania Department of Health, with assistance from CDC, used both epidemiologic and laboratory evidence to identify an association between invasive Mycobacterium avium complex, including M. chimaera, infections and exposure to contaminated Stöckert 3T heater-cooler devices, all manufactured by LivaNova PLC (formerly Sorin Group Deutschland GmbH) (2). M. chimaera was described as a distinct species of M. avium complex in 2004 (3). The results of the field investigation prompted notification of approximately 1,300 potentially exposed patients.* Although heater-cooler devices are used to regulate patients' blood temperature during cardiopulmonary bypass through water circuits that are closed, these reports suggest that aerosolized M. chimaera from the devices resulted in the invasive infections (1,2). The Food and Drug Administration (FDA) and CDC have issued alerts regarding the need to follow updated manufacturer's instructions for use of the devices, evaluate the devices for contamination, remain vigilant for new infections, and continue to monitor reports from the United States and overseas (2).

Background: The authors compared tau protein deposition in the frontal cortex of patients with cognitive impairment of amyotrophic lateral sclerosis (ALSci) (n = 6), cognitively intact patients with ALS (n = 6), and age-matched controls (n = 6) in order to determine the pathologic substrate of ALSci. Methods: Archival paraffin-embedded tissue was examined using Gallyas staining and immunostaining for tau-1 (phosphorylation-dependent tau epitope), tau-2 (phosphorylation independent), Alzheimer-specific tau phosphoepitopes (AT 8; ser396 phosphorylation), β-amyloid, glial fibrillary acid protein, SMI 31 (recognizing phosphorylated NFH), α-synuclein, or ubiquitin. Results: Tau immunoreactive astrocytic and dense neuronal inclusions were found in both ALS and ALSci, although to a greater extent in ALSci. Superficial linear spongiosis and Gallyas-positive intraneuronal aggregates, immunoreactive with tau-1 and AT 8 but rarely to ser396 tau, were unique to ALSci. Dense extracellular aggregates were observed by both Gallyas staining and tau-1 immunostaining. Tufted degenerating astrocytes containing tau-1 and AT 8 immunoreactive aggregates and, rarely, dense Gallyas positive neuritic plaques immunoreactive with tau-1 and AT 8, but not with ser396 tau or β-amyloid, were observed in ALSci. Tau positive glial coiled bodies were observed in the deep cortical layers and adjacent subcortical white matter in ALSci. Although 3R and 4R tau mRNA isoforms were expressed to similar levels in the frontal cortex of all cases, the total amount of tau mRNA was increased in both ALS and ALSci. Both gray and white matter soluble tau protein expression was similar among control, ALS, and ALSci cases. Conclusions: Cognitive dysfunction in ALS may reflect abnormal tau protein metabolism.

Genome-wide functional linkages among proteins in cellular complexes and metabolic pathways can be inferred from high throughput experimentation, such as DNA microarrays, or from bioinformatic analyses. Here we describe a method for the visualization and interpretation of genome-wide functional linkages inferred by the Rosetta Stone, Phylogenetic Profile, Operon and Conserved Gene Neighbor computational methods. This method involves the construction of a genome-wide functional linkage map, where each significant functional linkage between a pair of proteins is displayed on a two-dimensional scatter-plot, organized according to the order of genes along the chromosome. Subsequent hierarchical clustering of the map reveals clusters of genes with similar functional linkage profiles and facilitates the inference of protein function and the discovery of functionally linked gene clusters throughout the genome. We illustrate this method by applying it to the genome of the pathogenic bacterium Mycobacterium tuberculosis, assigning cellular functions to previously uncharacterized proteins involved in cell wall biosynthesis, signal transduction, chaperone activity, energy metabolism and polysaccharide biosynthesis.

Neurofilament (NF) aggregates in motor neurons are a key neuropathological feature of amyotrophic lateral sclerosis (ALS). We have previously observed an alteration in the stoichiometry of NF subunit steady state mRNA levels in ALS spinal motor neurons using in situ hybridization and proposed that this led to aggregate formation. We have now examined the levels of NF mRNA in whole tissue homogenates of spinal cord using the RNase protection assay and real time reverse transcriptase-PCR and observed significant elevations of NF mRNA level in ALS. Compared with age-matched control, we observed a greater stability of heterogeneously expressed NFL mRNA in the presence of ALS spinal cord homogenates. Heat denaturing or protease K digestion of the control homogenates increased the stability of the NFL mRNA to levels observed in ALS homogenate. Increased NFL mRNA stability was also induced by increasing the percentage of ALS homogenate in an admixture of control and ALS homogenates. These observations suggest the presence of trans-acting NFL mRNA-destabilizing elements in control but not in ALS spinal cord homogenates. This was confirmed in gel retardation assays. We also observed that the destabilizing elements interact with the 3'-untranslated region of NFL mRNA. These findings suggest that the trans-acting NFL-destabilizing elements are selectively suppressed in ALS homogenates, resulting in an increased stability and level of NFL mRNA.

Glutamatergic-mediated nitric oxide (NO) production occurs via the N-methyl-D-aspartic acid (NMDA) postsynaptic density protein 95 (PSD95)-neuronal nitric oxide synthase (NOS1) ternary complex. To determine whether NOS1 is targeted to the membrane subsequent to NMDA receptor activation, we examined the effect of NMDA on NOS1 subcellular localization in nerve growth factor (NGF) differentiated PC12 cells. No effect on cell viability was observed using a range of NMDA concentrations from 500 to 1000 microM. Within 3 min of stimulation with 750 microM NMDA, increased cytoplasmic NOS1 immunostaining was observed with rapid membrane staining thereafter. This was inhibited by NMDAR inhibition with MK801. This observation was confirmed using subcellular fractionation and immunoblotting. Using 4, 5-diaminofluorescein diacetate (DAF2-DA) staining and a diazotization assay, concurrent NO production was observed. When PC 12 cells were co-treated with either NMDA and N(6)-nitro-L-arginine methyl ester hydrochloride (L-NAME) or (5R, 10S)-(+)-5-methyl-10, 11-dihydro-5H-dibenzo [a, d] cyclohepten-5, 10-imine hydrogen maleate (MK-801), nitric oxide (NO) generation was inhibited. Stimulation in a calcium-free medium did not increase NO levels. Although no evidence of cytotoxicity was observed utilizing either the MTT assay or measures of apoptosis within the maximal interval of NOS1 translocation, cell viability was reduced following 10 h of continuous NMDA exposure. While it has been shown that NMDA triggers NOS1 activation, these results indicate that NMDAR activation also mediates NOS1 targeting to the membrane. Our data validate that NGF-differentiated PC12 cells may be employed as a useful in vitro model to further study the regulation of NOS1 subsequent to NMDAR activation.

We utilized fetal spinal motor neurons isolated from either NFL (-/-) or hNFL (+/+) transgenic mice to determine whether the loss of the low molecular weight neurofilament protein (NFL) places spinal motor neurons at a greater risk for cell death triggered by reactive nitrating species (RNS). After 21 days in serum-free, antibiotic-free medium, both the NFL (-/-) and hNFL (+/+) motor neurons developed neurofilamentous aggregates. Cultures were then exposed to nitric oxide(100 microM NOC 5, 100 microM NOC 12, or 2 mM sodium nitroprusside) or to peroxynitrite (250 mM SIN-1) forvarying intervals. NFL (-/-) cultures demonstrated extensive numbers of apoptotic neurons within six hours and complete cell loss by 24 hours in response to NOC 5 and NOC 12. In contrast, apoptosis was only observed in the motor neurons derived from control (C57bl/6) or hNLF (+/+) mice at 24 hours. In response to 2 mM sodium nitroprusside, necrosis was induced in all cells within 60 minutes. In response to 250 mM SIN-1, both C57bl/6 and hNFL (+/+) cells survived to six hours with only minimal evidence of degeneration while NFL (-/-) motor neurons were necrotic by 60 minutes. These observations suggest that NFL deficient motor neurons are at an enhanced risk of cell death mediated by RNS.

Peripherin, a neuronal intermediate filament (nIF) protein found associated with pathological aggregates in motor neurons of patients with amyotrophic lateral sclerosis (ALS) and of transgenic mice overexpressing mutant superoxide dismutase-1 (SOD1G37R), induces the selective degeneration of motor neurons when overexpressed in transgenic mice. Mouse peripherin is unique compared with other nIF proteins in that three peripherin isoforms are generated by alternative splicing. Here, the properties of the peripherin splice variants Per 58, Per 56, and Per 61 have been investigated in transfected cell lines, in primary motor neurons, and in transgenic mice overexpressing peripherin or overexpressing SOD1G37R. Of the three isoforms, Per 61 proved to be distinctly neurotoxic, being assembly incompetent and inducing degeneration of motor neurons in culture. Using isoform-specific antibodies, Per 61 expression was detected in motor neurons of SOD1G37R transgenic mice but not of control or peripherin transgenic mice. The Per 61 antibody also selectively labeled motor neurons and axonal spheroids in two cases of familial ALS and immunoprecipitated a higher molecular mass peripherin species from disease tissue. This evidence suggests that expression of neurotoxic splice variants of peripherin may contribute to the neurodegenerative mechanism in ALS.

We have studied the interactions between activated microglia and injured motor neurons using an immortalized murine microglial cell line (BV-2) stimulated with either lipopolysaccharide (LPS) (Escherichia coli) or supernatant from serum-deprived motor neurons (NSC-34 cell line). Both stimuli induced BV-2 activation. Although both BV-2 supernatants induced a subsequent increase in NO generation in otherwise healthy NSC-34 cells, only LPS-activated microglial supernatant induced NSC-34 cell death through a TNF-alpha-dependent pathway. However, we observed a 20-fold increase in the amount of TNF-alpha required to kill NSC-34 cells in the absence of LPS-activated BV-2 cell supernatant, indicating that microglia secrete factor(s) that facilitate TNF-alpha-mediated motor neuron death in vitro.

We have previously observed in vivo that chronic AlCl3 exposure in young adult New Zealand white rabbits induces a reversible motor neuron degeneration. In addition to the development of neurofilamentous aggregates within degenerating neurons, we have also observed a reduction of microglial number and activation. To determine if aluminum could directly effect microglial function in vivo, we used an immortalized murine microglial cell line (BV-2) to study the effect of organic (aluminum lactate) and inorganic (AlCl3) compounds on microglial function. We have observed a dose-dependent inhibition of phagocytosis, proliferation, migration, and release of TNF-α and nitric oxide in the absence of inducing microglial death. Both organic and inorganic aluminum decreased the extent of LPS-activated microglia-mediated death of a motor neuron hybridoma cell line (NSC 34). These findings demonstrate that aluminum compounds can directly affect microglial in vitro. J. Trace Elem. Exp. Med. 15:141–152, 2002. © 2002 Wiley-Liss, Inc.

The high level of accuracy and sensitivity of next generation sequencing for quantifying genetic material across organismal boundaries gives it tremendous potential for pathogen discovery and diagnosis in human disease. Despite this promise, substantial bacterial contamination is routinely found in existing human-derived RNA-seq datasets that likely arises from environmental sources. This raises the need for stringent sequencing and analysis protocols for studies investigating sequence-based microbial signatures in clinical samples.

Repeat expansions in chromosome 9 open reading frame 72 (C9ORF72) are an important cause of both motor neuron disease (MND) and frontotemporal dementia (FTD). Currently, little is known about factors that could account for the phenotypic heterogeneity detected in C9ORF72 expansion carriers. In this study, we investigated 4 genes that could represent genetic modifiers: ataxin-2 (ATXN2), non-imprinted in Prader-Willi/Angelman syndrome 1 (NIPA1), survival motor neuron 1 (SMN1), and survival motor neuron 2 (SMN2). Assessment of these genes, in a unique cohort of 331 C9ORF72 expansion carriers and 376 control subjects, revealed that intermediate repeat lengths in ATXN2 possibly act as disease modifier in C9ORF72 expansion carriers; no evidence was provided for a potential role of NIPA1, SMN1, or SMN2. The effects of intermediate ATXN2 repeats were most profound in probands with MND or FTD/MND (2.1% vs. 0% in control subjects, p = 0.013), whereas the frequency in probands with FTD was identical to control subjects. Though intermediate ATXN2 repeats were already known to be associated with MND risk, previous reports did not focus on individuals with clear pathogenic mutations, such as repeat expansions in C9ORF72. Based on our present findings, we postulate that intermediate ATXN2 repeat lengths may render C9ORF72 expansion carriers more susceptible to the development of MND; further studies are needed, however, to validate our findings. (C) 2014 Elsevier Inc. All rights reserved.

Small GTPases participate in a broad range of cellular processes such as proliferation, differentiation, and migration. The exchange of GDP for GTP resulting in the activation of these GTPases is catalyzed by a group of enzymes called guanine nucleotide exchange factors (GEFs), of which two classes: Dbl-related exchange factors and the more recently described dedicator of cytokinesis proteins family exchange factors. Increasingly, deregulation of normal GEF activity or function has been associated with a broad range of disease states, including neurodegeneration and neurodevelopmental disorders. In this review, we examine this evidence with special emphasis on the novel role of Rho guanine nucleotide exchange factor (RGNEF/p190RhoGEF) in the pathogenesis of amyotrophic lateral sclerosis. RGNEF is the first neurodegeneration-linked GEF that regulates not only RhoA GTPase activation but also functions as an RNA binding protein that directly acts with low molecular weight neurofilament mRNA 3′ untranslated region to regulate its stability. This dual role for RGNEF, coupled with the increasing understanding of the key role for GEFs in modulating the GTPase function in cell survival suggests a prominent role for GEFs in mediating a critical balance between cytotoxicity and neuroprotection which, when disturbed, contributes to neuronal loss.

Nontuberculous mycobacterial infections caused by Mycobacterium abscessus are responsible for a range of disease manifestations from pulmonary to skin infections and are notoriously difficult to treat, due to innate resistance to many antibiotics. Previous population studies of clinical M. abscessus isolates utilized multilocus sequence typing or pulsed-field gel electrophoresis, but high-resolution examinations of genetic diversity at the whole-genome level have not been well characterized, particularly among clinical isolates derived in the United States. We performed whole-genome sequencing of 11 clinical M. abscessus isolates derived from eight U.S. patients with pulmonary nontuberculous mycobacterial infections, compared them to 30 globally diverse clinical isolates, and investigated intrapatient genomic diversity and evolution. Phylogenomic analyses revealed a cluster of closely related U.S. and Western European M. abscessus subsp. abscessus isolates that are genetically distinct from other European isolates and all Asian isolates. Large-scale variation analyses suggested genome content differences of 0.3 to 8.3%, relative to the reference strain ATCC 19977 T . Longitudinally sampled isolates showed very few single-nucleotide polymorphisms and correlated genomic deletion patterns, suggesting homogeneous infection populations. Our study explores the genomic diversity of clinical M. abscessus strains from multiple continents and provides insight into the genome plasticity of an opportunistic pathogen.

In an attempt to explore infectious agents associated with nasopharyngeal carcinomas (NPCs), we employed our high-throughput RNA sequencing (RNA-seq) analysis pipeline, RNA CoMPASS, to investigate the presence of ectopic organisms within a number of NPC cell lines commonly used by NPC and Epstein-Barr virus (EBV) researchers. Sequencing data sets from both CNE1 and HONE1 were found to contain reads for human papillomavirus 18 (HPV-18). Subsequent real-time reverse transcription-PCR (RT-PCR) analysis on a panel of NPC cell lines identified HPV-18 in CNE1 and HONE1 as well as three additional NPC cell lines (CNE2, AdAH, and NPC-KT). Further analysis of the chromosomal integration arrangement of HPV-18 in NPCs revealed patterns identical to those observed in HeLa cells. Clustering based on human single nucleotide variation (SNV) analysis of two separate HeLa cell lines and several NPC cell lines demonstrated two distinct clusters with CNE1, as well as HONE1 clustering with the two HeLa cell lines. In addition, duplex-PCR-based genotyping showed that CNE1, CNE2, and HONE1 do not have a HeLa cell-specific L1 retrotransposon insertion, suggesting that these three HPV-18 + NPC lines are likely products of a somatic hybridization with HeLa cells, which is also consistent with our RNA-seq-based gene level SNV analysis. Taking all of these findings together, we conclude that a widespread HeLa contamination may exist in many NPC cell lines, and authentication of these cell lines is recommended. Finally, we provide a proof of concept for the utility of an RNA-seq-based approach for cell authentication. IMPORTANCE Nasopharyngeal carcinoma (NPC) cell lines are important model systems for analyzing the complex life cycle and pathogenesis of Epstein-Barr virus (EBV). Using an RNA-seq-based approach, we found HeLa cell contamination in several NPC cell lines that are commonly used in the EBV and related fields. Our data support the notion that contamination resulted from somatic hybridization with HeLa cells, likely occurring at the point of cell line establishment. Given the rarity of NPCs, the long history of NPC cell lines, and the lack of rigorous cell line authentication, it is likely that the actual prevalence and impact of HeLa cell contamination on the EBV field might be greater. We therefore recommend cell line authentication prior to performing experiments using NPC cell lines to avoid inaccurate conclusions. The novel RNA-seq-based cell authentication approach reported here can serve as a comprehensive method for validating cell lines.

The pathophysiology of tethered cord syndrome (TCS) is uncertain; however, it has been suggested that fibrous and fatty elements within the filum terminale (FT) play a role. The objective of this study was to describe the radiological and histological features of the FT in TCS and determine if there are associations between those features and clinical outcomes, complications, and urodynamics.In this retrospective study, histological, MRI, and clinical data obtained in 293 patients with TCS who underwent FT transection were reviewed and analyzed in a multivariate analysis.The median patient age was 4.9 years (range 0.3-64.3 years). On MRI, a fatty filum was present in 65% of patients and a thickened filum (2 mm) was seen in 45%. Histologically, the FT contained prominent fibrous tissue in 95%, nerve twigs in 79%, adipose tissue in 59%, and vascular tissue in 36%. Histological features associated with a thickened filum on MR images were adipose tissue (OR 3.5, p0.001), nerve twigs (OR 2.2, p = 0.028), and vascular tissue (OR 0.5, p = 0.025). Adipose tissue was associated with a conus level below the L2-3 disc space (OR 2.3, p = 0.031) and with a fatty filum on imaging (OR 9.8, p0.001). Nerve twigs were associated with abnormal urodynamics (OR 10.9, p = 0.049). The only variable predictive of clinical improvement was conus level; patients with conus levels caudal to L-2 were less likely to improve postoperatively (OR 0.3, p = 0.042).Fibrous tissue was ubiquitous and may be important in the pathophysiology of TCS. Nerve twigs and adipose tissue were associated with abnormal urodynamics and low-lying coni, respectively. Although the majority of patients clinically improved, patients with normal conus levels had significantly better outcomes.