Cardiovascular Diseases, Medications, and ALS: A Population-Based Case-Control Study.

INTRODUCTION: We investigated the associations between antecedent all-cause CVD diagnoses, cause-specific CVD diagnosis, and CVD medication prescriptions with the risk of developing amyotrophic lateral sclerosis (ALS). MATERIALS AND METHODS: We conducted a population-based case-control study of U.S. Medicare enrollees from 2006 to 2013. The final sample included 3,714 incident ALS cases and 18,570 controls (matched on age, sex, enrollment length, and county). Information was collected from Medicare Parts A, B, and D administrative claims data on hypertension, ischemic heart disease, heart failure, acute myocardial infarction, atrial fibrillation, prescriptions of angiotensin-converting enzyme inhibitors, angiotensin II receptors blockers, calcium channel blockers, beta blockers, and antiarrhythmics. Associations were evaluated using conditional logistic regression adjusting for age, sex, race/ethnicity, geographical location, alcohol and tobacco use, and socioeconomic status. RESULTS: The odds ratio (OR) for having one or more ICD-9 codes for any cardiovascular disease diagnosis at least 24 months prior to the date of ALS diagnosis was 0.85 (95% confidence interval [CI]: 0.78-0.92). Cardiovascular conditions that were inversely associated with ALS included heart failure (OR = 0.79; 95% CI 0.70-0.89), atrial fibrillation (OR = 0.81; 95% CI 0.77-0.92), and hypertension (OR = 0.91; 95% CI 0.84-0.98). Exposures to several classes of cardiovascular medications were inversely associated with ALS risk even after adjusting for confounding by indication, including ACE inhibitors (OR = 0.84, 95% CI 0.77-0.91), calcium channel blockers (OR = 0.64, 95% CI 0.59-0.70), and beta blockers (OR = 0.76, 95% CI 0.71-0.83). DISCUSSION/CONCLUSION: These findings merit additional research, including animal studies and pilot clinical trials, to further evaluate and evidence the effects of ACEIs, CCBs, and BBs on the risk of developing and clinical expression of ALS.

Lysine acetylation regulates the RNA binding, subcellular localization and inclusion formation of FUS.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the preferential death of motor neurons. Approximately 10% of ALS cases are familial and 90% are sporadic. Fused in sarcoma (FUS) is a ubiquitously expressed RNA-binding protein implicated in familial ALS and frontotemporal dementia (FTD). The physiological function and pathological mechanism of FUS are not well understood, particularly whether post-translational modifications play a role in regulating FUS function. In this study, we discovered that FUS was acetylated at lysine-315/316 (K315/K316) and lysine-510 (K510) residues in two distinct domains. Located in the nuclear localization sequence, K510 acetylation disrupted the interaction between FUS and Transportin-1, resulting in the mislocalization of FUS in the cytoplasm and formation of stress granule-like inclusions. Located in the RNA recognition motif, K315/K316 acetylation reduced RNA binding to FUS and decreased the formation of cytoplasmic inclusions. Treatment with deacetylase inhibitors also significantly reduced the inclusion formation in cells expressing ALS mutation P525L. More interestingly, familial ALS patient fibroblasts showed higher levels of FUS K510 acetylation as compared with healthy controls. Lastly, CREB-binding protein/p300 acetylated FUS, whereas both sirtuins and histone deacetylases families of lysine deacetylases contributed to FUS deacetylation. These findings demonstrate that FUS acetylation regulates the RNA binding, subcellular localization and inclusion formation of FUS, implicating a potential role of acetylation in the pathophysiological process leading to FUS-mediated ALS/FTD.

ALS mutations of FUS suppress protein translation and disrupt the regulation of nonsense-mediated decay.

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease characterized by preferential motor neuron death. Approximately 15% of ALS cases are familial, and mutations in the fused in sarcoma (FUS) gene contribute to a subset of familial ALS cases. FUS is a multifunctional protein participating in many RNA metabolism pathways. ALS-linked mutations cause a liquid-liquid phase separation of FUS protein in vitro, inducing the formation of cytoplasmic granules and inclusions. However, it remains elusive what other proteins are sequestered into the inclusions and how such a process leads to neuronal dysfunction and degeneration. In this study, we developed a protocol to isolate the dynamic mutant FUS-positive cytoplasmic granules. Proteomic identification of the protein composition and subsequent pathway analysis led us to hypothesize that mutant FUS can interfere with protein translation. We demonstrated that the ALS mutations in FUS indeed suppressed protein translation in N2a cells expressing mutant FUS and fibroblast cells derived from FUS ALS cases. In addition, the nonsense-mediated decay (NMD) pathway, which is closely related to protein translation, was altered by mutant FUS. Specifically, NMD-promoting factors UPF1 and UPF3b increased, whereas a negative NMD regulator, UPF3a, decreased, leading to the disruption of NMD autoregulation and the hyperactivation of NMD. Alterations in NMD factors and elevated activity were also observed in the fibroblast cells of FUS ALS cases. We conclude that mutant FUS suppresses protein biosynthesis and disrupts NMD regulation, both of which likely contribute to motor neuron death.

Clinical and experimental studies of a novel P525R FUS mutation in amyotrophic lateral sclerosis.

OBJECTIVE: To describe the clinical features of a novel fused in sarcoma (FUS) mutation in a young adult female amyotrophic lateral sclerosis (ALS) patient with rapid progression of weakness and to experimentally validate the consequences of the P525R mutation in cellular neuronal models. METHODS: We conducted sequencing of genomic DNA from the index patient and her family members. Immunocytochemistry was performed in various cellular models to determine whether the newly identified P525R mutant FUS protein accumulated in cytoplasmic inclusions. Clinical features of the index patient were compared with 19 other patients with ALS carrying the P525L mutation in the same amino acid position. RESULTS: A novel mutation c.1574C>G (p.525P>R) in the FUS gene was identified in the index patient. The clinical symptoms are similar to those in familial ALS patients with the P525L mutation at the same position. The P525R mutant FUS protein showed cytoplasmic localization and formed large stress granule-like cytoplasmic inclusions in multiple cellular models. CONCLUSIONS: The clinical features of the patient and the cytoplasmic inclusions of the P525R mutant FUS protein strengthen the notion that mutations at position 525 of the FUS protein result in a coherent phenotype characterized by juvenile or young adult onset, rapid progression, variable positive family history, and female preponderance.

Distinct and shared functions of ALS-associated proteins TDP-43, FUS and TAF15 revealed by multisystem analyses.

The RNA-binding protein (RBP) TAF15 is implicated in amyotrophic lateral sclerosis (ALS). To compare TAF15 function to that of two ALS-associated RBPs, FUS and TDP-43, we integrate CLIP-seq and RNA Bind-N-Seq technologies, and show that TAF15 binds to ∼4,900 RNAs enriched for GGUA motifs in adult mouse brains. TAF15 and FUS exhibit similar binding patterns in introns, are enriched in 3' untranslated regions and alter genes distinct from TDP-43. However, unlike FUS and TDP-43, TAF15 has a minimal role in alternative splicing. In human neural progenitors, TAF15 and FUS affect turnover of their RNA targets. In human stem cell-derived motor neurons, the RNA profile associated with concomitant loss of both TAF15 and FUS resembles that observed in the presence of the ALS-associated mutation FUS R521G, but contrasts with late-stage sporadic ALS patients. Taken together, our findings reveal convergent and divergent roles for FUS, TAF15 and TDP-43 in RNA metabolism.

Cognitive-behavioral screening reveals prevalent impairment in a large multicenter ALS cohort.

OBJECTIVES: To characterize the prevalence of cognitive and behavioral symptoms using a cognitive/behavioral screening battery in a large prospective multicenter study of amyotrophic lateral sclerosis (ALS). METHODS: Two hundred seventy-four patients with ALS completed 2 validated cognitive screening tests and 2 validated behavioral interviews with accompanying caregivers. We examined the associations between cognitive and behavioral performance, demographic and clinical data, and C9orf72 mutation data. RESULTS: Based on the ALS Cognitive Behavioral Screen cognitive score, 6.5% of the sample scored below the cutoff score for frontotemporal lobar dementia, 54.2% scored in a range consistent with ALS with mild cognitive impairment, and 39.2% scored in the normal range. The ALS Cognitive Behavioral Screen behavioral subscale identified 16.5% of the sample scoring below the dementia cutoff score, with an additional 14.1% scoring in the ALS behavioral impairment range, and 69.4% scoring in the normal range. CONCLUSIONS: This investigation revealed high levels of cognitive and behavioral impairment in patients with ALS within 18 months of symptom onset, comparable to prior investigations. This investigation illustrates the successful use and scientific value of adding a cognitive-behavioral screening tool in studies of motor neuron diseases, to provide neurologists with an efficient method to measure these common deficits and to understand how they relate to key clinical variables, when extensive neuropsychological examinations are unavailable. These tools, developed specifically for patients with motor impairment, may be particularly useful in patient populations with multiple sclerosis and Parkinson disease, who are known to have comorbid cognitive decline.

NP001 regulation of macrophage activation markers in ALS: a phase I clinical and biomarker study.

This is a phase I, placebo-controlled, single ascending dose safety and tolerability study of NP001 in patients with ALS. NP001 is a novel regulator of inflammatory macrophages and monocytes. As ALS progression is thought to be related to neuroinflammation, an additional objective of the study was to assess the effects of NP001 administration on monocyte activation markers. Thirty-two ALS patients were enrolled and received either placebo (eight) or one of four (six at each dose) ascending single i.v. doses (0.2, 0.8, 1.6 and 3.2 mg/kg NP001). Patients were monitored for safety, and blood monocyte immune activation markers CD16 and HLA-DR were assessed pre- and 24 h post-dosing. Changes from baseline were calculated. Results showed that NP001 was generally safe and well tolerated. Importantly, a single dose of NP001 caused a dose-dependent reduction in expression of monocyte CD16, a marker of monocyte activation/inflammation. Additionally, monocyte HLA-DR expression was also decreased in those patients with elevated values at baseline. In conclusion, these data indicate that NP001 has an acute effect on inflammatory monocytes in ALS patient blood. The potential for modulation of inflammation in the context of ALS disease progression will require further study with long-term follow-up.

FUsed in sarcoma is a novel regulator of manganese superoxide dismutase gene transcription.

AIMS: FUsed in sarcoma (FUS) is a multifunctional DNA/RNA-binding protein that possesses diverse roles, such as RNA splicing, RNA transport, DNA repair, translation, and transcription. The network of enzymes and processes regulated by FUS is far from being fully described. In this study, we have focused on the mechanisms of FUS-regulated manganese superoxide dismutase (MnSOD) gene transcription. RESULTS: Here we demonstrate that FUS is a component of the transcription complex that regulates the expression of MnSOD. Overexpression of FUS increased MnSOD expression in a dose-dependent manner and knockdown of FUS by siRNA led to the inhibition of MnSOD gene transcription. Reporter analyses, chromatin immunoprecipitation assay, electrophoretic mobility shift assay, affinity chromatography, and surface plasmon resonance analyses revealed the far upstream region of MnSOD promoter as an important target of FUS-mediated MnSOD transcription and confirmed that FUS binds to the MnSOD promoter and interacts with specificity protein 1 (Sp1). Importantly, overexpression of familial amyotropic lateral sclerosis (fALS)-linked R521G mutant FUS resulted in a significantly reduced level of MnSOD expression and activity, which is consistent with the decline in MnSOD activity observed in fibroblasts from fALS patients with the R521G mutation. R521G-mutant FUS abrogates MnSOD promoter-binding activity and interaction with Sp1. INNOVATION AND CONCLUSION: This study identifies FUS as playing a critical role in MnSOD gene transcription and reveals a previously unrecognized relationship between MnSOD and mutant FUS in fALS.

Macroglossia in amyotrophic lateral sclerosis.

IMPORTANCE: We encountered 2 patients with amyotrophic lateral sclerosis (ALS) with tongue enlargement and protrusion outside the oral cavity (macroglossia). To our knowledge, the relationship between macroglossia and ALS has not been reported in the literature. The objective of this article was to describe the clinical characteristics, imaging, and pathology of macroglossia in ALS and to develop a hypothesis regarding its pathophysiology. OBSERVATIONS: Two patients developed progressive weakness at age 54 and 40 years. Both patients exhibited dysarthria, dysphagia, tongue atrophy, neck extensor weakness, and weakness of jaw closure during a 1-year period. Both required tracheostomy and mechanical ventilation and afterward developed macroglossia. A 3-dimensional-reconstructed sagittal computed tomographic image confirmed tongue protrusion outside the oral cavity with focal compression and showed the transition from the atrophied part of the tongue in the oropharynx to the edematous part outside the mouth. Tongue biopsy demonstrated fatty replacement and fascicles of degenerative muscle. CONCLUSIONS AND RELEVANCE: We are unaware of previous reports of macroglossia in ALS/motor neuron disease. Given the paucity of case material, we speculated that this is an extremely rare complication of ALS. Based on this series, we propose a pathophysiological mechanism by reviewing imaging and tongue biopsy.

Current pathways for epidemiological research in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive neurodegenerative disease. The current status of the epidemiology, challenges to its study, and novel study design options are discussed in this paper. We focus on recent results from large-scale population based prospective studies, case-control studies and population based registries, risk factors, and neuropathologic findings in chronic traumatic encephalomyelopathy. We identify areas of interest for future research, including time-trends in the incidence and prevalence of ALS; the meaning of lifetime risk; the phenotypic description of ALS; the definition of familial versus sporadic ALS, syndromic aspects of ALS; specific risk factors such as military service, life style factors such as smoking, the use of statins, and the presence of ß-N-methylamino-L-alanine (BMAA), an excitotoxic amino acid derivative possibly produced by cyanobacteria found in almost every terrestrial and aquatic habitat; the emergence and disappearance of an endemic ALS in areas of the Pacific; and gene-environment interactions in the etiology of ALS. To move the epidemiology forward, we suggest using well-characterized cohorts of newly diagnosed ALS patients to identify risk and prognostic factors; storing biological material for future studies; building on the National ALS Registry as a resource of future studies; working in multidisciplinary consortia; and addressing the possible early life etiology of ALS.

Unilateral parotid electron beam radiotherapy as palliative treatment for sialorrhea in amyotrophic lateral sclerosis.

When ALS patients experience oropharyngeal weakness, sialorrhea can become a considerable challenge. Drooling has a profound negative impact in patient's quality of life causing embarrassing social situations. Several therapeutic modalities, including anticholinergic drugs, botulinum toxin injection, and radiotherapy have emerged as treatments for drooling in ALS. This retrospective case series study examined the effect of palliative radiotherapy in controlling problematic oral secretions in 10 ALS patients refractory to medical management. External electron beam radiation was targeted to a single parotid gland unilaterally with a total dose of 1500 cGy in 3 fractions at a depth determined by CT scanning. One patient received additional radiotherapy to the contralateral parotid due to persistent secretions. All patients reported improvement with a reduction in the intensity and amount of drooling. In 5 of 10 patients, anticholinergics were discontinued and were reduced in another two. There were no major side effects of treatment. We conclude that unilateral parotid electron radiotherapy provides satisfactory relief from sialorrhea in ALS patients and should be considered as a therapeutic option for patients who are refractory to medical management.

Myasthenia gravis associated with etanercept therapy.

Etanercept is an antagonist of tumor necrosis factor alpha that was developed to treat rheumatoid arthritis. In this report we present a patient who developed myasthenia gravis while taking etanercept and had resolution of symptoms after stopping it. This is the first report of this potential side effect and is of additional interest, because etanercept has been proposed as a treatment for myasthenia gravis.

Mutations in the SOD2 promoter reveal a molecular basis for an activating protein 2-dependent dysregulation of manganese superoxide dismutase expression in cancer cells.

A primary antioxidant enzyme in mitochondria, manganese superoxide dismutase (MnSOD), plays a critical role in the survival of aerobic life. It is well documented that, compared with normal cell counterparts, MnSOD level is decreased in neoplastic transformed cells but is increased in aggressive cancers. However, the underlying mechanism for the observed dysregulation of MnSOD in cancer is unknown. We have identified previously a unique set of mutations located in the promoter region of the SOD2 gene in several types of cancer cells. We found that a C-to-T transition at -102 and an insertion of A at -93 down-regulate MnSOD transcription by interrupting the formation of a single-stranded loop that is essential for a high level of promoter activity. Here, we show that the additional downstream mutation, C-to-G transversion at -38, creates a binding site for the transcription factors specificity protein 1 (Sp1) and activating protein 2 (AP-2). The promoter function is regulated by the relative levels of Sp1 and AP-2. In cytokine-induced expression of the SOD2 gene, Sp1 cooperates with a transcriptional complex containing nuclear factor-kappaB and nucleophosmin. The presence of AP-2 attenuates this induction. Our results suggest that the high level of MnSOD observed in aggressive cancer cells may be due, in part, to the absence of AP-2 transcriptional repression.

Retrograde axonal transport and motor neuron disease.

Transport of material between extensive neuronal processes and the cell body is crucial for neuronal function and survival. Growing evidence shows that deficits in axonal transport contribute to the pathogenesis of multiple neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Here we review recent data indicating that defects in dynein-mediated retrograde axonal transport are involved in ALS etiology. We discuss how mutant copper-zinc superoxide dismutase (SOD1) and an aberrant interaction between mutant SOD1 and dynein could perturb retrograde transport of neurotrophic factors and mitochondria. A possible contribution of axonal transport to the aggregation and degradation processes of mutant SOD1 is also reviewed. We further consider how the interference with axonal transport and protein turnover by mutant SOD1 could influence the function and viability of motor neurons in ALS.

Tamoxifen enhancement of TNF-alpha induced MnSOD expression: modulation of NF-kappaB dimerization.

Manganese superoxide dismutase (MnSOD) has been shown to suppress the development of cancer. Tamoxifen (TAM), a nonsteroidal anti-estrogen that is widely used in chemotherapy, is known to be a modulator of antioxidant status. However, the mechanism by which TAM mediates antioxidant enzyme induction remains unclear. In this study we investigated TAM enhancement of MnSOD induction by TNF-alpha. The results show that co-treatment with TAM and TNF-alpha increases the MnSOD promoter/enhancer driven luciferase activity, MnSOD mRNA and protein levels. Interestingly, co-treatment with TAM and TNF-alpha drastically decreases the binding activity of the p50/p50 homodimer and increases that of the p50/p65 heterodimer compared to TNF-alpha alone. This change in DNA binding could not be attributed to a decrease in the level of p50, its precursor, p105, or its inhibitors. Furthermore, TAM did not enhance degradation of IkappaB-alpha. These results suggest that p50/p50 homodimer may act as an inhibitory complex of MnSOD expression. Modulation of the DNA binding activity in favor of the p50/p65 complex may enhance NF-kappaB mediated induction of MnSOD by TAM. These findings reveal a potential novel mechanism for the induction of the human MnSOD gene.