Assessment of feasibility and utility of universal referral to specialty palliative care in a multidisciplinary amyotrophic lateral sclerosis clinic: A cohort study.

INTRODUCTION: Although the value of palliative care integration in multidisciplinary amyotrophic lateral sclerosis (ALS) clinics has been increasingly recognized, limited data are available demonstrating the feasibility and utility of a palliative care specialist embedded in an ALS clinic. The purpose of this study is to describe the feasibility and utility of a universal outpatient referral model for specialty palliative care for patients with amyotrophic lateral sclerosis. METHODS: This is a retrospective cohort study of initial patient visits with a palliative care physician in a multidisciplinary ALS clinic at an academic institution. All patients were referred to an embedded palliative care physician from January to December 2019. RESULTS: Within the study period, 52 of 69 (75%) patients with a confirmed diagnosis of ALS were seen by the palliative care physician. The most common reason patients were not seen by palliative care was a lack of insurance authorization (n = 5). At the visit, 94% of patients discussed at least one advance care planning or goals of care topic. The most common advance care planning topic discussed was code status (40%). The most common goals of care topic discussed was meaning and values (57%). Symptom management was discussed with 40 patients (77%). The most common symptom addressed was pain and/or muscle spasms (33%). DISCUSSION: These data support the feasibility and utility of universal, outpatient palliative care referral for patients with ALS. Further research is necessary to determine the long-term effect on outcomes for this population.

Circuit-Specific Early Impairment of Proprioceptive Sensory Neurons in the SOD1G93A Mouse Model for ALS.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease in which motor neurons degenerate, resulting in muscle atrophy, paralysis, and fatality. Studies using mouse models of ALS indicate a protracted period of disease development with progressive motor neuron pathology, evident as early as embryonic and postnatal stages. Key missing information includes concomitant alterations in the sensorimotor circuit essential for normal development and function of the neuromuscular system. Leveraging unique brainstem circuitry, we show in vitro evidence for reflex circuit-specific postnatal abnormalities in the jaw proprioceptive sensory neurons in the well-studied SOD1G93A mouse. These include impaired and arrhythmic action potential burst discharge associated with a deficit in Nav1.6 Na+ channels. However, the mechanoreceptive and nociceptive trigeminal ganglion neurons and the visual sensory retinal ganglion neurons were resistant to excitability changes in age-matched SOD1G93A mice. Computational modeling of the observed disruption in sensory patterns predicted asynchronous self-sustained motor neuron discharge suggestive of imminent reflexive defects, such as muscle fasciculations in ALS. These results demonstrate a novel reflex circuit-specific proprioceptive sensory abnormality in ALS.SIGNIFICANCE STATEMENT Neurodegenerative diseases have prolonged periods of disease development and progression. Identifying early markers of vulnerability can therefore help devise better diagnostic and treatment strategies. In this study, we examined postnatal abnormalities in the electrical excitability of muscle spindle afferent proprioceptive neurons in the well-studied SOD1G93A mouse model for neurodegenerative motor neuron disease, amyotrophic lateral sclerosis. Our findings suggest that these proprioceptive sensory neurons are exclusively afflicted early in the disease process relative to sensory neurons of other modalities. Moreover, they presented Nav1.6 Na+ channel deficiency, which contributed to arrhythmic burst discharge. Such sensory arrhythmia could initiate reflexive defects, such as muscle fasciculations in amyotrophic lateral sclerosis, as suggested by our computational model.

Long-term edaravone efficacy in amyotrophic lateral sclerosis: Post-hoc analyses of Study 19 (MCI186-19).

BACKGROUND: In a Phase 3 study, amyotrophic lateral sclerosis (ALS) patients experienced significantly less physical functional decline with 24-week edaravone vs placebo, followed by open-label treatment for an additional 24 weeks. METHODS: Outcome (the change in ALS Functional Rating Scale-Revised, ALSFRS-R, from baseline) was projected for placebo patients through 48 weeks and compared with 48-week edaravone or 24-week edaravone after switching from placebo. RESULTS: A total of 123 patients received open-label treatment (65 edaravone-edaravone; 58 placebo-edaravone). The projected ALSFRS-R decline for placebo from baseline through week 48 was greater than for 48-week edaravone (P < .0001). For patients switching from placebo to edaravone, ALSFRS-R slope approached that of continued edaravone for 48 weeks. ALSFRS-R decline did not differ between actual and projected edaravone through week 48. CONCLUSIONS: Compared with placebo, these analyses suggest that edaravone is beneficial in ALS patients even after 6 mo of receiving placebo, and efficacy is maintained for up to 1 year.

Resurgent Na+ Current Offers Noise Modulation in Bursting Neurons.

Neurons utilize bursts of action potentials as an efficient and reliable way to encode information. It is likely that the intrinsic membrane properties of neurons involved in burst generation may also participate in preserving its temporal features. Here we examined the contribution of the persistent and resurgent components of voltage-gated Na+ currents in modulating the burst discharge in sensory neurons. Using mathematical modeling, theory and dynamic-clamp electrophysiology, we show that, distinct from the persistent Na+ component which is important for membrane resonance and burst generation, the resurgent Na+ can help stabilize burst timing features including the duration and intervals. Moreover, such a physiological role for the resurgent Na+ offered noise tolerance and preserved the regularity of burst patterns. Model analysis further predicted a negative feedback loop between the persistent and resurgent gating variables which mediate such gain in burst stability. These results highlight a novel role for the voltage-gated resurgent Na+ component in moderating the entropy of burst-encoded neural information.

Mexiletine for muscle cramps in amyotrophic lateral sclerosis: A randomized, double-blind crossover trial.

INTRODUCTION: More than 90% of amyotrophic lateral sclerosis (ALS) patients have muscle cramps, but evidence-based treatments have not been available. METHODS: A multicenter, double-blind, placebo-controlled crossover trial of mexiletine 150 mg twice daily was conducted in ALS patients requesting treatment of symptomatic muscle cramps. RESULTS: Muscle cramp frequency was reduced in 18 of 20 patients; 13 reductions were attributed to treatment (P < 0.05). The average reduction, based on t tests, was 1.8 cramps per day (a reduction from 5.3 with placebo to 3.5 with mexiletine). The estimated reduction of cramp severity was 15 units on a 100-unit scale (P = 0.01) from a baseline average of 46. No effect on fasciculations was noted. One patient discontinued the study because of dizziness, and another patient discontinued the study to start open-label mexiletine therapy. No serious adverse event occurred. DISCUSSION: Mexiletine is a well tolerated and effective medication for controlling the symptom of muscle cramps in ALS. Muscle Nerve, 2018.

Homeostatic dysregulation in membrane properties of masticatory motoneurons compared with oculomotor neurons in a mouse model for amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative motoneuron disease with presently no cure. Motoneuron (MN) hyperexcitability is commonly observed in ALS and is suggested to be a precursor for excitotoxic cell death. However, it is unknown whether hyperexcitability also occurs in MNs that are resistant to degeneration. Second, it is unclear whether all the MNs within homogeneous motor pools would present similar susceptibility to excitability changes since high-threshold MNs innervating fast fatigable muscle fibers selectively degenerate compared with low-threshold MNs innervating fatigue resistant slow muscle fibers. Therefore, we concurrently examined the excitability of ALS-vulnerable trigeminal motoneurons (TMNs) controlling jaw musculature and ALS-resistant oculomotor neurons (OMNs) controlling eye musculature in a well studied SOD1(G93A) ALS mouse model using in vitro patch-clamp electrophysiology at presymptomatic ages P8-P12. Our results show that hyperexcitability is not a global change among all the MNs, although mutant SOD1 is ubiquitously expressed. Instead, complex changes occur in ALS-vulnerable TMNs based on motor unit type and discharge characteristics. Firing threshold decreases among high-threshold TMNs and increases in a subpopulation of low-threshold TMNs. The latter group was identified based on their linear frequency-current responses to triangular ramp current injections. Such complex changes in MN recruitment were absent in ALS-resistant OMNs. We simulated the observed complex changes in TMN excitability using a computer-based jaw closer motor pool model. Model results suggest that hypoexcitability may indeed represent emerging disease symptomology that causes resistance in muscle force initiation. Identifying the cellular and molecular properties of these hypoexcitable cells may guide effective therapeutic strategies in ALS.

Charcot-Marie-Tooth disease CMT4A: GDAP1 increases cellular glutathione and the mitochondrial membrane potential.

Mutations in GDAP1 lead to recessively or dominantly inherited peripheral neuropathies (Charcot-Marie-Tooth disease, CMT), indicating that GDAP1 is essential for the viability of cells in the peripheral nervous system. GDAP1 contains domains characteristic of glutathione-S-transferases (GSTs), is located in the outer mitochondrial membrane and induces fragmentation of mitochondria. We found GDAP1 upregulated in neuronal HT22 cells selected for resistance against oxidative stress. GDAP1 over-expression protected against oxidative stress caused by depletion of the intracellular antioxidant glutathione (GHS) and against effectors of GHS depletion that affect the mitochondrial membrane integrity like truncated BH3-interacting domain death agonist and 12/15-lipoxygenase. Gdap1 knockdown, in contrast, increased the susceptibility of motor neuron-like NSC34 cells against GHS depletion. Over-expression of wild-type GDAP1, but not of GDAP1 with recessively inherited mutations that cause disease and reduce fission activity, increased the total cellular GHS content and the mitochondrial membrane potential up to a level where it apparently limits mitochondrial respiration, leading to reduced mitochondrial Ca(2+) uptake and superoxide production. Fibroblasts from autosomal-recessive CMT4A patients had reduced GDAP1 levels, reduced GHS concentration and a reduced mitochondrial membrane potential. Thus, our results suggest that the potential GST GDAP1 is implicated in the control of the cellular GHS content and mitochondrial activity, suggesting an involvement of oxidative stress in the pathogenesis of CMT4A.

Puromycin-sensitive aminopeptidase (PSA/NPEPPS) impedes development of neuropathology in hPSA/TAU(P301L) double-transgenic mice.

Accumulation of neurotoxic hyperphosphorylated TAU protein is a major pathological hallmark of Alzheimer disease and other neurodegenerative dementias collectively called tauopathies. Puromycin-sensitive aminopeptidase (PSA/NPEPPS) is a novel modifier of TAU-induced neurodegeneration with neuroprotective effects via direct proteolysis of TAU protein. Here, to examine the effects of PSA/NPEPPS overexpression in vivo in the mammalian system, we generated and crossed BAC-PSA/NPEPPS transgenic mice with the TAU(P301L) mouse model of neurodegeneration. PSA/NPEPPS activity in the brain and peripheral tissues of human PSA/NPEPPS (hPSA) mice was elevated by ∼2-3-fold with no noticeable deleterious physiological effects. Double-transgenic animals for hPSA and TAU(P301L) transgenes demonstrated a distinct trend for delayed paralysis and showed significantly improved motor neuron counts, no gliosis and markedly reduced levels of total and hyperphosphorylated TAU in the spinal cord, brain stem, cortex, hippocampus and cerebellum of adult and aged animals when compared with TAU(P301L) mice. Furthermore, endogenous TAU protein abundance in human neuroblastoma SH-SY5Y cells was significantly reduced or augmented by overexpression or knockdown of PSA/NPEPPS, respectively. This study demonstrated that without showing neurotoxic effects, elevation of PSA/NPEPPS activity in vivo effectively blocks accumulation of soluble hyperphosphorylated TAU protein and slows down the disease progression in the mammalian system. Our data suggest that increasing PSA/NPEPPS activity may be a feasible therapeutic approach to eliminate accumulation of unwanted toxic substrates such as TAU.

Integrative gene-tissue microarray-based approach for identification of human disease biomarkers: application to amyotrophic lateral sclerosis.

Advances in genomics and proteomics permit rapid identification of disease-relevant genes and proteins. Challenges include biological differences between animal models and human diseases, high discordance between DNA and protein expression data and a lack of experimental models to study human complex diseases. To overcome some of these limitations, we developed an integrative approach using animal models, postmortem human material and a combination of high-throughput microarray methods to identify novel molecular markers of amyotrophic lateral sclerosis (ALS). We used laser capture microdissection coupled with microarrays to identify early transcriptome changes occurring in spinal cord motor neurons or surrounding glial cells. Two models of familial motor neuron disease, SOD1(G93A) and TAU(P301L), transgenic mice were used at the presymptomatic stage. Identified gene expression changes were predominantly model-specific. However, several genes were regulated in both models. The relevance of identified genes as clinical biomarkers was tested in the peripheral blood transcriptome of presymptomatic SOD1(G93A) animals using custom-designed ALS microarray. To confirm the relevance of identified genes in human sporadic ALS (SALS), selected corresponding protein products were examined by high-throughput immunoassays using tissue microarrays constructed from human postmortem spinal cord tissues. Genes that were identified by these experiments and located within a linkage region associated with familial ALS/frontotemporal dementia were sequenced in several families. This large-scale gene and protein expression study pointing to distinct molecular mechanisms of TAU- and SOD1-induced motor neuron degeneration identified several new SALS-relevant proteins (CNGA3, CRB1, OTUB2, MMP14, SLK, DDX58, RSPO2) and putative blood biomarkers, including Nefh, Prph and Mgll.

Edaravone efficacy in amyotrophic lateral sclerosis with reduced forced vital capacity: Post-hoc analysis of Study 19 (MCI186-19) [clinical trial NCT01492686].

BACKGROUND: Edaravone slowed the rate of functional decline in subjects with amyotrophic lateral sclerosis (ALS) in phase 3 study MCI186-19 (Study 19). One of the Study 19 inclusion criteria was forced vital capacity (FVC) ≥80% of predicted (≥80%p). Therefore, the study provided no information on edaravone efficacy in subjects with FVC <80%p. In Study 19, 24-week, double-blind treatment was followed by open-label treatment where all subjects received edaravone. At 24 weeks, some subjects had FVC <80%p (FVC24 <80%p). This allowed for post-hoc assessment of the effects of edaravone in subgroups of subjects with FVC24 ≥80%p vs <80%p. OBJECTIVE: To address the question of the efficacy of edaravone in ALS patients with FVC <80%p. METHODS: Post-hoc analysis of Study 19 comparing edaravone efficacy at week 48 in subjects with FVC24 ≥80%p vs <80%p. RESULTS: With edaravone treatment, subjects in both the FVC24 ≥80%p and the FVC24 <80%p subgroups experienced a reduction in ALS Functional Rating Scale-Revised (ALSFRS-R) score loss vs placebo subjects through week 48. For the FVC24 ≥80%p subgroup, the changes in ALSFRS-R scores from baseline to week 48 were -7.63 for edaravone-edaravone vs -9.69 for placebo-edaravone, a difference of 2.05 (P = .034; 95% CI: 0.16, 3.94). For the FVC24 <80%p subgroup, the changes in ALSFRS-R scores from baseline to week 48 were -10.26 for edaravone-edaravone vs -15.20 for placebo-edaravone, a difference of 4.94 (P = .0038; 95% CI: 1.64, 8.25). Linear regression analysis indicated that, in the FVC24 <80%p subgroup, there was a notable change in the slope of the ALSFRS-R score-vs-time graph after the start of edaravone treatment. CONCLUSION: ALS subjects in the Study 19 placebo arm had a slowing in disease progression, even when edaravone was added with an FVC of <80%p prior to starting edaravone. A randomized, placebo-controlled study is needed to validate these post-hoc findings.

Palliative Care Consults in an Inpatient Setting for Patients With Amyotrophic Lateral Sclerosis.

BACKGROUND: Limited data about the frequency and outcomes of palliative care (PC) specialty consultations for patients with amyotrophic lateral sclerosis (ALS) are available. METHODS: This study was a retrospective chart review. Patients with ALS admitted to 2 academic hospitals from 2013-2018 were included. We compared patients who were seen by an inpatient specialty PC service (PC group) with those who were not (NonPC group). RESULTS: Twenty-four patients met inclusion criteria (9 PC group, 15 NonPC group). Patients in both groups were similar in age and had been diagnosed for a similar amount of time before admission. In the PC group, 6 patients were seen by more than 1 PC multidisciplinary team member (physician, social worker, spiritual care provider, clinical nurse specialist). PC consultations were requested for goals of care (GOC) (n = 7), pain (n = 4), hospice information/referral (n = 2), dyspnea (n = 1), and excessive oral secretions (n = 1). GOC topics addressed for both groups were code status, treatment preferences (tracheostomy placement, percutaneous endoscopic gastrostomy placement, change to comfort care), prognostication, and hospice information/referral. Patients in the PC group were significantly more likely to be discharged with GOC (89%, p = 0.02) and completed advance care planning (ACP) documents (89%, p = 0.04) than patients in the NonPC group (32%; 47%). Despite reason for consultation, at least 1 symptom was addressed for every patient seen by PC specialists. CONCLUSIONS: Inpatient specialty PC consultation for patients with ALS leads to greater documentation of GOC and ACP by discharge. PC consultants participate in symptom management in patients with ALS during hospitalization.

A genomic screen for modifiers of tauopathy identifies puromycin-sensitive aminopeptidase as an inhibitor of tau-induced neurodegeneration.

Neurofibrillary tangles (NFT) containing tau are a hallmark of neurodegenerative diseases, including Alzheimer's disease (AD). NFT burden correlates with cognitive decline and neurodegeneration in AD. However, little is known about mechanisms that protect against tau-induced neurodegeneration. We used a cross species functional genomic approach to analyze gene expression in multiple brain regions in mouse, in parallel with validation in Drosophila, to identify tau modifiers, including the highly conserved protein puromycin-sensitive aminopeptidase (PSA/Npepps). PSA protected against tau-induced neurodegeneration in vivo, whereas PSA loss of function exacerbated neurodegeneration. We further show that human PSA directly proteolyzes tau in vitro. These data highlight the utility of using both evolutionarily distant species for genetic screening and functional assessment to identify modifiers of neurodegeneration. Further investigation is warranted in defining the role of PSA and other genes identified here as potential therapeutic targets in tauopathy.

IL-17A is increased in the serum and in spinal cord CD8 and mast cells of ALS patients.

The contribution of inflammation to neurodegenerative diseases is increasingly recognized, but the role of inflammation in sporadic amyotrophic lateral sclerosis (sALS) is not well understood and no animal model is available. We used enzyme-linked immunosorbent assays (ELISAs) to measure the cytokine interleukin-17A (IL-17A) in the serum of ALS patients (n = 32; 28 sporadic ALS (sALS) and 4 familial ALS (fALS)) and control subjects (n = 14; 10 healthy subjects and 4 with autoimmune disorders). IL-17A serum concentrations were 5767 ± 2700 pg/ml (mean ± SEM) in sALS patients and 937 ± 927 pg/ml in fALS patients in comparison to 7 ± 2 pg/ml in control subjects without autoimmune disorders (p = 0.008 ALS patients vs. control subjects by Mann-Whitney test). Sixty-four percent of patients and no control subjects had IL-17A serum concentrations > 50 pg/ml (p = 0.003 ALS patients vs. healthy subjects by Fisher's exact test). The spinal cords of sALS (n = 8), but not control subjects (n = 4), were infiltrated by interleukin-1ß- (IL-1ß-), and tumor necrosis factor-α-positive macrophages (co-localizing with neurons), IL-17A-positive CD8 cells, and IL-17A-positive mast cells. Mononuclear cells treated with aggregated forms of wild type superoxide dismutase-1 (SOD-1) showed induction of the cytokines IL-1ß, interleukin-6 (IL-6), and interleukin-23 (IL-23) that may be responsible for induction of IL-17A. In a microarray analysis of 28,869 genes, stimulation of peripheral blood mononuclear cells by mutant superoxide dismutase-1 induced four-fold higher transcripts of interleukin-1α (IL-1α), IL-6, CCL20, matrix metallopeptidase 1, and tissue factor pathway inhibitor 2 in mononuclear cells of patients as compared to controls, whereas the anti-inflammatory cytokine interleukin-10 (IL-10) was increased in mononuclear cells of control subjects. Aggregated wild type SOD-1 in sALS neurons could induce in mononuclear cells the cytokines inducing chronic inflammation in sALS spinal cord, in particular IL-6 and IL-17A, damaging neurons. Immune modulation of chronic inflammation may be a new approach to sALS.

Directed differentiation of human-induced pluripotent stem cells generates active motor neurons.

The potential for directed differentiation of human-induced pluripotent stem (iPS) cells to functional postmitotic neuronal phenotypes is unknown. Following methods shown to be effective at generating motor neurons from human embryonic stem cells (hESCs), we found that once specified to a neural lineage, human iPS cells could be differentiated to form motor neurons with a similar efficiency as hESCs. Human iPS-derived cells appeared to follow a normal developmental progression associated with motor neuron formation and possessed prototypical electrophysiological properties. This is the first demonstration that human iPS-derived cells are able to generate electrically active motor neurons. These findings demonstrate the feasibility of using iPS-derived motor neuron progenitors and motor neurons in regenerative medicine applications and in vitro modeling of motor neuron diseases.

Induction of Nrf2 and xCT are involved in the action of the neuroprotective antibiotic ceftriaxone in vitro.

In amyotrophic lateral sclerosis, down-regulation of the astrocyte-specific glutamate excitatory amino acid transporter 2 is hypothesized to increase extracellular glutamate, thereby leading to excitotoxic motor neuron death. The antibiotic ceftriaxone was recently reported to induce excitatory amino acid transporter 2 and to prolong the survival of mutant superoxide dismutase 1 transgenic mice. Here we show that ceftriaxone also protects fibroblasts and the hippocampal cell line HT22, which are not sensitive to excitotoxicity, against oxidative glutamate toxicity, where extracellular glutamate blocks cystine import via the glutamate/cystine-antiporter system x(c)(-). Lack of intracellular cystine leads to glutathione depletion and cell death because of oxidative stress. Ceftriaxone increased system x(c)(-) and glutathione levels independently of its effect on excitatory amino acid transporters by induction of the transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2), a known inducer of system x(c)(-), and the specific x(c)(-) subunit xCT. No significant effect was apparent in fibroblasts deficient in Nrf2 or xCT. Similar ceftriaxone-stimulated changes in Nrf2, system x(c)(-), and glutathione were observed in rat cortical and spinal astrocytes. In addition, ceftriaxone induced xCT mRNA expression in stem cell-derived human motor neurons. We conclude that ceftriaxone-mediated neuroprotection might relate more strongly to activation of the antioxidant defense system including Nrf2 and system x(c)(-) than to excitatory amino acid transporter induction.

Human wild-type tau interacts with wingless pathway components and produces neurofibrillary pathology in Drosophila.

Pathologic alterations in the microtubule-associated protein tau have been implicated in a number of neurodegenerative disorders, including Alzheimer's disease (AD), progressive supranuclear palsy (PSP), and frontotemporal dementia (FTD). Here, we show that tau overexpression, in combination with phosphorylation by the Drosophila glycogen synthase kinase-3 (GSK-3) homolog and wingless pathway component (Shaggy), exacerbated neurodegeneration induced by tau overexpression alone, leading to neurofibrillary pathology in the fly. Furthermore, manipulation of other wingless signaling molecules downstream from shaggy demonstrated that components of the Wnt signaling pathway modulate neurodegeneration induced by tau pathology in vivo but suggested that tau phosphorylation by GSK-3beta differs from canonical Wnt effects on beta-catenin stability and TCF activity. The genetic system we have established provides a powerful reagent for identification of novel modifiers of tau-induced neurodegeneration that may serve as future therapeutic targets.

Transcriptome analysis in whole blood reveals increased microbial diversity in schizophrenia.

The role of the human microbiome in health and disease is increasingly appreciated. We studied the composition of microbial communities present in blood across 192 individuals, including healthy controls and patients with three disorders affecting the brain: schizophrenia, amyotrophic lateral sclerosis, and bipolar disorder. By using high-quality unmapped RNA sequencing reads as candidate microbial reads, we performed profiling of microbial transcripts detected in whole blood. We were able to detect a wide range of bacterial and archaeal phyla in blood. Interestingly, we observed an increased microbial diversity in schizophrenia patients compared to the three other groups. We replicated this finding in an independent schizophrenia case-control cohort. This increased diversity is inversely correlated with estimated cell abundance of a subpopulation of CD8+ memory T cells in healthy controls, supporting a link between microbial products found in blood, immunity and schizophrenia.

In vivo peroxidative activity of FALS-mutant human CuZnSODs expressed in yeast.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder leading to loss of motor neurons. We previously characterized the enhanced peroxidative activity of the human familial ALS (FALS) mutants of copper-zinc superoxide dismutase (CuZnSOD) A4V and G93A in vitro. Here, a similar activity is demonstrated for human FALS CuZnSOD mutants in an in vivo model system, the yeast Saccharomyces cerevisiae. Spin trap adducts of alpha-(pyridyl-4-N-oxide)-N-tert-butylnitrone (POBN) have been measured by electron paramagnetic resonance (EPR) in yeast expressing mutant (A4V, L38V, G93A, and G93C) and wild type CuZnSOD upon addition of hydrogen peroxide to the culture. The trapped radical is a hydroxyethyl adduct of POBN, identified by spectral parameters. Mutant CuZnSODs produced greater concentrations of the trapped adduct compared to the wild type enzyme. This observation provides evidence for an oxidative radical mechanism, whereby the mutants of CuZnSOD catalyze the formation of reactive oxygen species that may be related to the development or progression of FALS. This study also presents an in vivo model system to study free radical production in FALS-associated CuZnSOD mutations.

Neuronal phagocytosis by inflammatory macrophages in ALS spinal cord: inhibition of inflammation by resolvin D1.

Although the cause of neuronal degeneration in amyotrophic lateral sclerosis (ALS) remains hypothetical, there is evidence of spinal cord infiltration by macrophages and T cells. In post-mortem ALS spinal cords, 19.8 ± 4.8 % motor neurons, including caspase-negative and caspase-positive neurons, were ingested by IL-6- and TNF-α-positive macrophages. In ALS macrophages, in vitro aggregated superoxide dismutase-1 (SOD-1) stimulated in ALS macrophages expression of inflammatory cytokines, including IL-1ß, IL-6, and TNF-α, through activation of cyclooxy-genase-2 (COX-2) and caspase-1. The lipid mediator resolvin D1 (RvD1) inhibited IL-6 and TNF-α production in ALS macrophages with 1,100 times greater potency than its parent molecule docosahexaenoic acid. ALS peripheral blood mononuclear cells (PBMCs) showed increased transcription of inflammatory cytokines and chemokines at baseline and after stimulation by aggregated wild-type SOD-1, and these cytokines were down regulated by RvD1. Thus the neurons are impacted by macrophages expressing inflammatory cytokines. RvD1 strongly inhibits in ALS macrophages and PBMCs cytokine transcription and production. Resolvins offer a new approach to suppression of inflammatory activation in ALS.