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Mutations in the human Superoxide dismutase 1 (hSOD1) gene are well-established cause of the motor neuron disease ALS. Patients and transgenic (Tg) ALS model mice carrying mutant variants develop hSOD1 aggregates in the CNS. We have identified two hSOD1 aggregate strains, which both transmit spreading template-directed aggregation and premature fatal paralysis when inoculated into adult transgenic mice. This prion-like spread of aggregation could be a primary disease mechanism in SOD1-induced ALS. Human SOD1 aggregation has been studied extensively both in cultured cells and under various conditions in vitro. To determine how the structure of aggregates formed in these model systems related to disease-associated aggregates in the CNS, we used a binary epitope-mapping assay to examine aggregates of hSOD1 variants G93A, G85R, A4V, D90A, and G127X formed in vitro, in four different cell lines and in the CNS of Tg mice. We found considerable variability between replicate sets of in vitro-generated aggregates. In contrast, there was a high similarity between replicates of a given hSOD1 mutant in a given cell line, but pronounced variations between different hSOD1 mutants and different cell lines in both structures and amounts of aggregates formed. The aggregates formed in vitro or in cultured cells did not replicate the aggregate strains that arise in the CNS. Our findings suggest that the distinct aggregate morphologies in the CNS could result from a micro-environment with stringent quality control combined with second-order selection by spreading ability. Explorations of pathogenesis and development of therapeutics should be conducted in models that replicate aggregate structures forming in the CNS. (Figure presented.)

Mutations in the gene encoding the ubiquitously expressed free radical scavenging enzyme superoxide dismutase-1 (SOD1) are found in 2–6% of amyotrophic lateral sclerosis patients. The most frequent SOD1 mutation worldwide is D90A. Amyotrophic lateral sclerosis caused by this mutation has some unusual features: the heredity is usually recessive, the phenotype is stereotypic with slowly evolving motor symptoms beginning in the legs and may also include sensory, autonomic, and urinary bladder involvement. Furthermore, the mutant protein resembles the wild type, with normal content and enzymatic activity in the central nervous system. Here, we report neuropathological findings in nine patients homozygous for the D90A mutation. All nine had numerous small granular inclusions immunoreactive for misfolded SOD1 in motor neurons and glial nuclei in the spinal cord and brainstem. In addition to degeneration of the corticospinal tracts, all patients had degeneration of the dorsal columns. We also found intense gliosis in circumscribed cortical areas of the frontal and temporal lobes and in the insula. In these areas and in adjacent white matter, there were SOD1 staining neuropil threads. A few SOD1-immunopositive cytoplasmic neuronal inclusions were observed in cortical areas, as were glial nuclear inclusions. As suggested by the symptoms and signs and earlier neurophysiological and imaging investigations, the histopathology in patients homozygous for the D90A SOD1 extends beyond the motor system to include cognitive and sensory cortical areas. However, even in the patients that had a symptomatic disease duration of more than 2 or 3 decades and lived into their 70s or 80s, there were no SOD1-inclusion pathology and no typical dysfunction (apart from the musculature) in non-nervous organs. Thus, only specific parts of the CNS seem to be vulnerable to toxicity provoked by homozygously expressed mutant SOD1.

Superoxide dismutase-1 is a ubiquitously expressed antioxidant enzyme. Mutations in SOD1 can cause amyotrophic lateral sclerosis, probably via a toxic gain-of-function involving protein aggregation and prion-like mechanisms. Recently, homozygosity for loss-of-function mutations in SOD1 has been reported in patients presenting with infantile-onset motor neuron disease. We explored the bodily effects of superoxide dismutase-1 enzymatic deficiency in eight children homozygous for the p.C112Wfs*11 truncating mutation. In addition to physical and imaging examinations, we collected blood, urine and skin fibroblast samples. We used a comprehensive panel of clinically established analyses to assess organ function and analysed oxidative stress markers, antioxidant compounds, and the characteristics of the mutant Superoxide dismutase-1. From around 8 months of age, all patients exhibited progressive signs of both upper and lower motor neuron dysfunction, cerebellar, brain stem, and frontal lobe atrophy and elevated plasma neurofilament concentration indicating ongoing axonal damage. The disease progression seemed to slow down over the following years. The p.C112Wfs*11 gene product is unstable, rapidly degraded and no aggregates were found in fibroblast. Most laboratory tests indicated normal organ integrity and only a few modest deviations were found. The patients displayed anaemia with shortened survival of erythrocytes containing decreased levels of reduced glutathione. A variety of other antioxidants and oxidant damage markers were within normal range. In conclusion, non-neuronal organs in humans show a remarkable tolerance to absence of Superoxide dismutase-1 enzymatic activity. The study highlights the enigmatic specific vulnerability of the motor system to both gain-of-function mutations in SOD1 and loss of the enzyme as in the here depicted infantile superoxide dismutase-1 deficiency syndrome.

Background The dose–effect of various SOD1 mutations on SOD1 enzymatic activity offers valuable insights into ALS pathogenesis with possible therapeutic implications. Homozygous SOD1 mutations, yet scarce, are of special interest. We report a novel homozygous SOD1 mutation with decreased enzymatic activity and severe early onset ALS phenotype. Methods Whole exome sequencing and targeted screening of commonly implicated genes were conducted. Repeat-primed PCR and fragment length analysis were used for C9orf72. Bi-directional Sanger sequencing was used for SOD1 and other genes. SOD1 activity was measured by direct spectrophotometry. Serum neurofilament light chain level was measured by the ELLA immunoassay system. Results The homozygous patient for a novel SOD1 variant p.Ser69Pro showed poor SOD1 enzymatic activity (16% of controls) and an early onset ALS phenotype predominantly affecting lower motor neurons with rapid involvement of the trunk, upper limbs and bulbar muscles. The asymptomatic heterozygous relatives had at least 68% of normal enzyme activity. Level of serum neurofilament light chain was much higher (148 pg/ml) in the patient than the relatives who had normal levels (6–10 pg/ml). Conclusion This novel mutation adds knowledge to the ALS genotype–phenotype spectrum and supports the strong dose–effect of SOD1 mutations associated with severely decreased enzymatic activity.

ObjectiveA hallmark of amyotrophic lateral sclerosis (ALS) caused by mutations in superoxide dismutase-1 (SOD1) are inclusions containing SOD1 in motor neurons. Here, we searched for SOD1-positive inclusions in 29 patients carrying ALS-linked mutations in six other genes.MethodsA panel of antibodies that specifically recognise misfolded SOD1 species were used for immunohistochemical investigations of autopsy tissue.ResultsThe 18 patients with hexanucleotide-repeat-expansions in C9orf72 had inclusions of misfolded wild type (WT) SOD1WT in spinal motor neurons. Similar inclusions were occasionally observed in medulla oblongata and in the motor cortex and frontal lobe. Patients with mutations in FUS, KIF5A, NEK1, ALSIN or VAPB, carried similar SOD1WT inclusions. Minute amounts of misSOD1WT inclusions were detected in 2 of 20 patients deceased from non-neurological causes and in 4 of 10 patients with other neurodegenerative diseases. Comparison was made with 17 patients with 9 different SOD1 mutations. Morphologically, the inclusions in patients with mutations in C9orf72HRE, FUS, KIF5A, NEK1, VAPB and ALSIN resembled inclusions in patients carrying the wildtype-like SOD1D90A mutation, whereas patients carrying unstable SOD1 mutations (A4V, V5M, D76Y, D83G, D101G, G114A, G127X, L144F) had larger skein-like SOD1-positive inclusions.Conclusions and relevanceAbundant inclusions containing misfolded SOD1WT are found in spinal and cortical motor neurons in patients carrying mutations in six ALS-causing genes other than SOD1. This suggests that misfolding of SOD1WT can be part of a common downstream event that may be pathogenic. The new anti-SOD1 therapeutics in development may have applications for a broader range of patients.

Pathogenic variants in SOD1, encoding superoxide dismutase 1, are responsible for about 20% of all familial amyotrophic lateral sclerosis cases, through a gain-of-function mechanism. Recently, two reports showed that a specific homozygous SOD1 loss-of-function variant is associated with an infantile progressive motor-neurological syndrome. Exome sequencing followed by molecular studies, including cDNA analysis, SOD1 protein levels and enzymatic activity, and plasma neurofilament light chain levels, were undertaken in an infant with severe global developmental delay, axial hypotonia and limb spasticity. We identified a homozygous 3-bp in-frame deletion in SOD1. cDNA analysis predicted the loss of a single valine residue from a tandem pair (p.Val119/Val120) in the wild-type protein, yet expression levels and splicing were preserved. Analysis of SOD1 activity and protein levels in erythrocyte lysates showed essentially no enzymatic activity and undetectable SOD1 protein in the child, whereas the parents had ∼50% protein expression and activity relative to controls. Neurofilament light chain levels in plasma were elevated, implying ongoing axonal injury and neurodegeneration. Thus, we provide confirmatory evidence of a second biallelic variant in an infant with a severe neurological syndrome and suggest that the in-frame deletion causes instability and subsequent degeneration of SOD1. We highlight the importance of the valine residues at positions V119-120, and suggest possible implications for future therapeutics research.

Absence of SOD1 Activity and Motor Neuron Syndrome In a child with a homozygous truncating mutation in SOD1, SOD1 activity in red cells was absent and fibroblasts grew only with oxygen deprivation....

Objective: Cu/Zn superoxide dismutase (SOD1) reduction prolongs survival in SOD1-transgenic animal models. Pyrimethamine produces dose-dependent SOD1 reduction in cell culture systems. A previous p ...

Amyotrophic lateral sclerosis (ALS) is a lethal neurodegenerative disease that is characterized by the loss of motor neurons, which results in progressive muscle atrophy. The pathology spreads from the initial site of onset to contiguous anatomic regions. Mutations in the gene encoding Cu/Zn-superoxide dismutase (SOD1) have been identified in a dominantly inherited form of ALS (ALS-SOD1). A major hallmark of ALS-SOD1 is the abnormal accumulation of conformationally aberrant SOD1 protein (i.e., misfolded SOD1) within motor neurons. Emerging experimental evidence has suggested that misfolded proteins associated with neurodegenerative diseases exhibit prion-like properties, i.e., misfolded proteins act as conformational templates that convert normal proteins into a pathogenic form. Possibly as a result of this prion-like self-propagation property, misfolded forms of pathological proteins are considered to accumulate in the central nervous system and cause neurodegeneration. In this article, we review recent evidence for the role of prion-like mechanisms in ALS-SOD1. In particular, we discuss the propensity of misfolded SOD1 to act as a pathological seed, spread between cells, and propagate neuroanatomically.

Mutations in the superoxide dismutase (SOD1) gene have been linked to amyotrophic lateral sclerosis (ALS). A 50 base pair (bp) deletion of SOD1 has been suggested to reduce transcription and to be associated with later disease onset in ALS. This study was aimed to reveal if the 50 bp deletion influenced SOD1 enzymatic activity, occurrence and phenotype of the disease in a Swedish ALS/control cohort. Blood samples from 512 Swedish ALS patients and 354 Swedish controls without coding SOD1 mutations were analysed for the 50 bp deletion allele. The enzymatic activity of SOD1 in erythrocytes was analysed and genotype-phenotype correlations were assessed. Results demonstrated that the genotype frequencies of the 50 bp deletion were all found to be in Hardy-Weinberg equilibrium. No significant differences were found for age of onset, disease duration or site of onset. SOD1 enzymatic activity showed a statistically significant decreasing trend in the control group, in which the allele was associated with a 5% reduction in SOD1 activity. The results suggest that the 50 bp deletion has a moderate reducing effect on SOD1 synthesis. No modulating effects, however, were found on ALS onset, phenotype and survival in the Swedish population.

Mutations in superoxide dismutase 1 (SOD1) cause amyotrophic lateral sclerosis (ALS). Disease pathogenesis is linked to destabilization, disorder and aggregation of the SOD1 protein. However, the non-genetic factors that promote disorder and the subsequent aggregation of SOD1 have not been studied. Mainly located to the reducing cytosol, mature SOD1 contains an oxidized disulfide bond that is important for its stability. Since O2 is required for formation of the bond, we reasoned that low O2 tension might be a risk factor for the pathological changes associated with ALS development. By combining biochemical approaches in an extensive range of genetically distinct patient-derived cell lines, we show that the disulfide bond is an Achilles heel of the SOD1 protein. Culture of patient-derived fibroblasts, astrocytes, and induced pluripotent stem cell-derived mixed motor neuron and astrocyte cultures (MNACs) under low O2 tensions caused reductive bond cleavage and increases in disordered SOD1. The effects were greatest in cells derived from patients carrying ALS-linked mutations in SOD1. However, significant increases also occurred in wild-type SOD1 in cultures derived from non-disease controls, and patients carrying mutations in other common ALS-linked genes. Compared to fibroblasts, MNACs showed far greater increases in SOD1 disorder and even aggregation of mutant SOD1s, in line with the vulnerability of the motor system to SOD1-mediated neurotoxicity. Our results show for the first time that O2 tension is a principal determinant of SOD1 stability in human patient-derived cells. Furthermore, we provide a mechanism by which non-genetic risk factors for ALS, such as aging and other conditions causing reduced vascular perfusion, could promote disease initiation and progression. Electronic supplementary material The online version of this article (10.1007/s00401-019-01986-1) contains supplementary material, which is available to authorized users.

Phosphatidylethanol (PEth) is a recently introduced biomarker with high specificity, high sensitivity, and response correlating with alcohol consumption. It has the potential to be a valuable biomarker in population studies on the health effects of alcohol, however its stability in long-term stored blood is not known. We used LC-MS/MS to assess the stability of PEth-16:0/18:1 in blood samples (packed erythrocytes) that were stored between 1 and 19 years at −80 °C in a biobank from a large population survey. The participants answered a life-style questionnaire that included questions on alcohol consumption. For analysis, we selected blood samples from seven homogenous ethanol consumption cohorts collected at intervals from 1997 to 2015. Despite the narrow stated alcohol consumption range, 10–15 g/day, there were large differences in PEth values between individuals in the cohorts, from below the limit of detection of 0.005 µmol/L to 1.40 µmol/L. The median was 0.08 µmol/L. Neither generalized linear modeling, nor principal component analysis revealed a statistically significant association between time of storage and PEth levels. The PEth results indicate that the participants had, on average, under-reported their alcohol consumption several-fold. The findings suggest that PEth in blood has a sufficient long-term stability for use as an alcohol biomarker in prospective case-control studies. Analysis of blood stored in biobanks could significantly improve the validity of assessments exploring the health effects of alcohol.

Amyotrophic lateral sclerosis (ALS) and Parkinson's disease (PD) are protein-aggregation diseases that lack clear molecular etiologies. Biomarkers could aid in diagnosis, prognosis, planning of care, drug target identification and stratification of patients into clinical trials. We sought to characterize shared and unique metabolite perturbations between ALS and PD and matched controls selected from patients with other diagnoses, including differential diagnoses to ALS or PD that visited our clinic for a lumbar puncture. Cerebrospinal fluid (CSF) and plasma from rigorously age-, sex- and sampling-date matched patients were analyzed on multiple platforms using gas chromatography (GC) and liquid chromatography (LC)-mass spectrometry (MS). We applied constrained randomization of run orders and orthogonal partial least squares projection to latent structure-effect projections (OPLS-EP) to capitalize upon the study design. The combined platforms identified 144 CSF and 196 plasma metabolites with diverse molecular properties. Creatine was found to be increased and creatinine decreased in CSF of ALS patients compared to matched controls. Glucose was increased in CSF of ALS patients and α-hydroxybutyrate was increased in CSF and plasma of ALS patients compared to matched controls. Leucine, isoleucine and ketoleucine were increased in CSF of both ALS and PD. Together, these studies, in conjunction with earlier studies, suggest alterations in energy utilization pathways and have identified and further validated perturbed metabolites to be used in panels of biomarkers for the diagnosis of ALS and PD.

Biotechnology offers extensive possibilities to incorporate new traits into organisms. Genetically modified (GM) traits relevant for agro-ecosystems include traits such as pest resistance and herbicide tolerance in crop plants, increased growth rate in fish and livestock, and enhanced nitrogen-fixation capabilities of soil microbes. In this review, we evaluated the direct and indirect trait-specific effects of GM plants, microbes, and animals on ecosystem processes and found that most of the effects of genetically modified organisms (GMOs) on ecosystem processes are indirect and are the result of associated changes in management strategy rather than a direct effect of the GMOs. Conflicting results on the performance and effects of GMOs are frequently reported, especially regarding crop yield and impacts on soil organisms. This is partly because methods with different levels of resolution have been used in different ecological contexts. Overall, there is little evidence that the effects of GM traits on ecosystem processes act with different mechanisms from those of traits modified using conventional methods. However, little is known about trait-specific effects of GMOs on ecosystem processes even though GMOs have been used for more than three decades. In particular, studies linking genetically modified traits to ecosystem processes at longer time scales are rare, but needed for evaluating trait effects, especially in an evolutionary context. In addition, biotechnology may provide a unique tool for gaining insights into the links between traits and ecosystem processes when integrated into basic ecological research.

Despite considerable progress in uncovering the molecular details of protein aggregation in vitro, the cause and mechanism of protein-aggregation disease remain poorly understood. One reason is that the amount of pathological aggregates in neural tissue is exceedingly low, precluding examination by conventional approaches. We present here a method for determination of the structure and quantity of aggregates in small tissue samples, circumventing the above problem. The method is based on binary epitope mapping using anti-peptide antibodies. We assessed the usefulness and versatility of the method in mice modeling the neurodegenerative disease amyotrophic lateral sclerosis, which accumulate intracellular aggregates of superoxide dismutase-1. Two strains of aggregates were identified with different structural architectures, molecular properties, and growth kinetics. Both were different from superoxide dismutase-1 aggregates generated in vitro under a variety of conditions. The strains, which seem kinetically under fragmentation control, are associated with different disease progressions, complying with and adding detail to the growing evidence that seeding, infectivity, and strain dependence are unifying principles of neurodegenerative disease.

Aggregation of mutant superoxide dismutase 1 (SOD1) is a pathological hallmark of a subset of familial ALS patients. However, the possible role of misfolded wild type SOD1 in human ALS is highly debated. To ascertain whether or not misfolded SOD1 is a common pathological feature in non-SOD1 ALS, we performed a blinded histological and biochemical analysis of post mortem brain and spinal cord tissues from 19 sporadic ALS, compared with a SOD1 A4V patient as well as Alzheimer’s disease (AD) and non-neurological controls. Multiple conformation- or misfolded-specific antibodies for human SOD1 were compared. These were generated independently by different research groups and were compared using standardized conditions. Five different misSOD1 staining patterns were found consistently in tissue sections from SALS cases and the SOD1 A4V patient, but were essentially absent in AD and non-neurological controls. We have established clear experimental protocols and provide specific guidelines for working, with conformational/misfolded SOD1-specific antibodies. Adherence to these guidelines will aid in the comparison of the results of future studies and better interpretation of staining patterns. This blinded, standardized and unbiased approach provides further support for a possible pathological role of misSOD1 in SALS.

Objective: To characterise stabilities in erythrocytes of mutant SOD1 proteins, compare SOD1 enzymatic activities between patients with different genetic causes of ALS and search for underlying causes of deviant SOD1 activities in individuals lacking SOD1 mutations. Methods: Blood samples from 4072 individuals, ALS patients with or without a SOD1 mutation, family members and controls were studied. Erythrocyte SOD1 enzymatic activities normalised to haemoglobin content were determined, and effects of haemoglobin disorders on dismutation assessed. Coding SOD1 sequences were analysed by Sanger sequencing, exon copy number variations by fragment length analysis and by TaqMan Assay. Results: Of the 44 SOD1 mutations found, 75% caused severe destabilisation of the mutant protein but in 25% it was physically stable. Mutations producing structural changes caused halved erythrocyte SOD1 activities. There were no differences in SOD1 activities between patients without a SOD1 mutation and control individuals or carriers of TBK1 mutations and C9orf72HRE. In the low and high SOD1 activity groups no deviations were found in exon copy numbers and intron gross structures. Thalassemias and iron deficiency were associated with increased SOD1 activity/haemoglobin ratios. Conclusion: Adjunct erythrocyte SOD1 activity analysis reliably signals destabilising SOD1 mutations including intronic mutations that are missed by exon sequencing.

Motor neurons containing aggregates of superoxide dismutase 1 (SOD1) are hallmarks of amyotrophic lateral sclerosis (ALS) caused by mutations in the gene encoding SOD1. We have previously reported that two strains of mutant human (h) SOD1 aggregates (denoted A and B) can arise in hSOD1-transgenic models for ALS and that inoculation of such aggregates into the lumbar spinal cord of mice results in rostrally spreading, templated hSOD1 aggregation and premature fatal ALS-like disease. Here, we explored whether mutant hSOD1 aggregates with prion-like properties also exist in human ALS. Aggregate seeds were prepared from spinal cords from an ALS patient carrying the hSOD1G127Gfs*7 truncation mutation and from mice transgenic for the same mutation. To separate from mono-, di- or any oligomeric hSOD1 species, the seed preparation protocol included ultracentrifugation through a density cushion. The core structure of hSOD1G127Gfs*7 aggregates present in mice was strain A-like. Inoculation of the patient- or mouse-derived seeds into lumbar spinal cord of adult hSOD1-expressing mice induced strain A aggregation propagating along the neuraxis and premature fatal ALS-like disease (p

We report an apparently sporadic amyotrophic lateral sclerosis patient carrying a heterozygous novel frameshift SOD1 mutation (p.Ser108LeufsTer15), predicted to cause a premature protein truncation. RT-PCR analysis of SOD1 mRNA and SDS-PAGE/Western blot analysis of PBMC demonstrated that mRNA from the mutant allele is expressed at levels similar to those of the wild-type allele, but the truncated protein is undetectable also in the insoluble fraction and after proteasome inhibition. Accordingly, the dismutation activity in erythrocytes is halved. Thus, the pathogenic mechanism associated with this mutation might be based on an insufficient activity of SOD1 that would make motor neurons more vulnerable to oxidative injury. However, it cannot be excluded that p.Ser108LeufsTer15 SOD1 is present in the nervous tissue and, being less charged and hence having less repulsive forces than the wild-type protein, may trigger toxic mechanisms as a consequence of its propensity to aggregate.

A GGGGCC-repeat expansion in C9orf72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) among Caucasians. However, little is known about the variability of the GGGGCC expansion in different tissues and whether this correlates with the observed phenotype. Here, we used Southern blotting to estimate the size of hexanucleotide expansions in C9orf72 in neural and non-neural tissues from 18 autopsied ALS and FTD patients with repeat expansion in blood. Digitalization of the Southern blot images allowed comparison of repeat number, smear distribution and expansion band intensity between tissues and between patients. We found marked intra-individual variation of repeat number between tissues, whereas there was less variation within each tissue group. In two patients, the size variation between tissues was extreme, with repeat numbers below 100 in all studied non-neural tissues, whereas expansions in neural tissues were 20-40 times greater and in the same size range observed in neural tissues of the other 16 patients. The expansion pattern in different tissues could not distinguish between diagnostic groups and no correlation was found between expansion size in frontal lobe and occurrence of cognitive impairment. In ALS patients, a less number of repeats in the cerebellum and parietal lobe correlated with earlier age of onset and a larger number of repeats in the parietal lobe correlated with a more rapid progression. In 43 other individuals without repeat expansion in blood, we find that repeat sizes up to 15 are stable, as no size variation between blood, brain and spinal cord was found.

Objective Cu/Zn superoxide dismutase (SOD1) reduction prolongs survival in SOD1‐transgenic animal models. Pyrimethamine produces dose‐dependent SOD1 reduction in cell culture systems. A previous phase 1 trial showed pyrimethamine lowers SOD1 levels in leukocytes in patients with SOD1 mutations. This study investigated whether pyrimethamine lowered SOD1 levels in the cerebrospinal fluid (CSF) in patients carrying SOD1 mutations linked to familial amyotrophic lateral sclerosis (fALS/SOD1). Methods A multicenter (5 sites), open‐label, 9‐month‐duration, dose‐ranging study was undertaken to determine the safety and efficacy of pyrimethamine to lower SOD1 levels in the CSF in fALS/SOD1. All participants underwent 3 lumbar punctures, blood draw, clinical assessment of strength, motor function, quality of life, and adverse effect assessments. SOD1 levels were measured in erythrocytes and CSF. Pyrimethamine was measured in plasma and CSF. Appel ALS score, ALS Functional Rating Scale–Revised, and McGill Quality of Life Single‐Item Scale were measured at screening, visit 6, and visit 9. Results We enrolled 32 patients; 24 completed 6 visits (18 weeks), and 21 completed all study visits. A linear mixed effects model showed a significant reduction in CSF SOD1 at visit 6 (p

Amyotrophic lateral sclerosis (ALS) is an adult-onset degeneration of motor neurons that is commonly caused by mutations in the gene encoding superoxide dismutase 1 (SOD1). Both patients and Tg mice expressing mutant human SOD1 (hSOD1) develop aggregates of unknown importance. In Tg mice, 2 different strains of hSOD1 aggregates (denoted A and B) can arise; however, the role of these aggregates in disease pathogenesis has not been fully characterized. Here, minute amounts of strain A and B hSOD1 aggregate seeds that were prepared by centrifugation through a density cushion were inoculated into lumbar spinal cords of 100-day-old mice carrying a human SOD1 Tg. Mice seeded with A or B aggregates developed premature signs of ALS and became terminally ill after approximately 100 days, which is 200 days earlier than for mice that had not been inoculated or were given a control preparation. Concomitantly, exponentially growing strain A and B hSOD1 aggregations propagated rostrally throughout the spinal cord and brainstem. The phenotypes provoked by the A and B strains differed regarding progression rates, distribution, end-stage aggregate levels, and histopathology. Together, our data indicate that the aggregate strains are prions that transmit a templated, spreading aggregation of hSOD1, resulting in a fatal ALS-like disease.

Introduction: Amyotrophic lateral sclerosis (ALS) and Parkinson’s disease (PD) are two severe neurodegenerative disorders for which the disease mechanisms are poorly understood and reliable biomark ...

Introduction The motor system is selectively vulnerable to mutations in the ubiquitously expressed aggregation-prone enzyme superoxide dismutase-1 (SOD1). Results Autophagy clears aggregates, and factors involved in the process were analyzed in multiple areas of the CNS from human control subjects (n = 10) and amyotrophic lateral sclerosis (ALS) patients (n = 18) with or without SOD1 mutations. In control subjects, the key regulatory protein Beclin 1 and downstream factors were remarkably scarce in spinal motor areas. In ALS patients, there was evidence of moderate autophagy activation and also dysregulation. These changes were largest in SOD1 mutation carriers. To explore consequences of low autophagy capacity, effects of a heterozygous deletion of Beclin 1 were examined in ALS mouse models expressing mutant SOD1s. This caused earlier SOD1 aggregation, onset of symptoms, motor neuron loss, and a markedly shortened survival. In contrast, the levels of soluble misfolded SOD1 species were reduced. Conclusions The findings suggest that an inherent low autophagy capacity might cause the vulnerability of the motor system, and that SOD1 aggregation plays a crucial role in the pathogenesis. Electronic supplementary material The online version of this article (doi:10.1186/s40478-016-0274-y) contains supplementary material, which is available to authorized users.

Mutations in superoxide dismutase-1 (SOD1) are a common known cause of amyotrophic lateral sclerosis (ALS). The neurotoxicity of mutant SOD1s is most likely caused by misfolded molecular species, but disease pathogenesis is still not understood. Proposed mechanisms include impaired mitochondrial function, induction of endoplasmic reticulum stress, reduction in the activities of the proteasome and autophagy, and the formation of neurotoxic aggregates. Here we examined whether perturbations in these cellular pathways in turn influence levels of misfolded SOD1 species, potentially amplifying neurotoxicity. For the study we used fibroblasts, which express SOD1 at physiological levels under regulation of the native promoter. The cells were derived from ALS patients expressing 9 different SOD1 mutants of widely variable molecular characteristics, as well as from patients carrying the GGGGCC-repeat-expansion in C9orf72 and from non-disease controls. A specific ELISA was used to quantify soluble, misfolded SOD1, and aggregated SOD1 was analysed by western blotting. Misfolded SOD1 was detected in all lines. Levels were found to be much lower in non-disease control and the non-SOD1 C9orf72 ALS lines. This enabled us to validate patient fibroblasts for use in subsequent perturbation studies. Mitochondrial inhibition, endoplasmic reticulum stress or autophagy inhibition did not affect soluble misfolded SOD1 and in most cases, detergent-resistant SOD1 aggregates were not detected. However, proteasome inhibition led to uniformly large increases in misfolded SOD1 levels in all cell lines and an increase in SOD1 aggregation in some. Thus the ubiquitin-proteasome pathway is a principal determinant of misfolded SOD1 levels in cells derived both from patients and controls and a decline in activity with aging could be one of the factors behind the mid-to late-life onset of inherited ALS.

Deregulation of microRNAs (miRNAs) contributes to the development and progression of many cancer types; however, their functions in the pathogenesis of testicular germ cell tumor (TGCT) remain unclear. Here, we determined miRNA expression profiles of TGCTs and normal testes using small RNA sequencing, and identified several deregulated miRNAs in TGCTs, including the miR-506~514 cluster. In functional studies in vitro we demonstrated that miR-514a-3p induced apoptosis through direct regulation of the paternally expressed gene 3 (PEG3), and ectopically expressed PEG3 could rescue the apoptotic effect of miR-514a-3p overexpression. Silencing of PEG3 or miR-514a-3p overexpression reduced nuclear accumulation of p50 and NF-κB reporter activity. Furthermore, PEG3 was co-immunoprecipitated with tumor necrosis factor receptor-associated factor 2 (TRAF2) in TGCT cell lysates. We propose a model of PEG3-mediated activation of NF-κB in TGCT. Loss of miR-514a-3p expression in TGCT increases PEG3 expression that recruits TRAF2 and activates the NF-kappa B pathway, which protects germ cells from apoptosis. Importantly, we observed strong expression of PEG3 and nuclear p50 in the majority of TGCTs (83% and 78%, respectively). In conclusion, our study describes a novel function for miR-514a-3p in TGCT and highlights an unrecognized mechanism of PEG3 regulation and NF-κB activation in TGCT.