Smooth as SILK: Translating Discoveries to New Therapies in Amyotrophic Lateral Sclerosis

No effective therapies exist for the treatment of patients suffering from amyotrophic lateral sclerosis, a devastating neurodegenerative disease characterized by motor neuron loss, paralysis, and death on average 3 Р5 years after diagnosis. Although multiple promising candidates have emerged from preclinical experiments in disease models, failure rates of randomized control trials assessing efficacy in humans are over 95%, demonstrating a need to improve the translation of preclinical findings to human biology. In this dissertation, I present original work investigating amyotrophic lateral sclerosis caused by mutations in SOD1 (ALSSOD1) for the successful translation of SOD1-targeting strategies into meaningful treatment options for patients. Through a retrospective study of ALSSOD1 natural history, my research showed that the most common mutation causing ALSSOD1 in North America, Alanine\u3eValine (A4V), is rapidly progressing, while different mutations show a wide range of variability in clinical outcomes. I then describe initial characterization of SOD1 protein half-life in the cerebrospinal fluid (CSF) of ALSSOD1 patients. Similar to healthy control subjects, I found SOD1 is a long-lived protein in ALS patient CSF, with a half-life on the order of weeks. Moreover, A4V SOD1 mutant protein showed differential behavior compared to wild type SOD1 in CSF in one ALS participant, preliminarily suggesting that CSF SOD1 measures may implicate the pathogenic state of SOD1 protein. Finally, I show that measures of CSF SOD1 protein production serve as a pharmacodynamics biomarker for Antisense Oligonucleotides (ASOs) treatment targeting SOD1 mRNA transcripts in the central nervous system (CNS). Lowering of new SOD1 protein production after ASO treatment preceded lowering of total SOD1 protein concentration, suggesting measures of new protein production are more sensitive for mRNA-targeting therapies and are translatable to human trials to assay drug target engagement. Taken together, these data provide insights that contribute to a further understanding of ALS biology, in addition to the study design and interpretation of randomized control trials in ALS

The SOD1-mediated ALS phenotype shows a decoupling between age of symptom onset and disease duration

Superoxide dismutase (SOD1) gene variants may cause amyotrophic lateral sclerosis, some of which are associated with a distinct phenotype. Most studies assess limited variants or sample sizes. In this international, retrospective observational study, we compare phenotypic and demographic characteristics between people with SOD1-ALS and people with ALS and no recorded SOD1 variant. We investigate which variants are associated with age at symptom onset and time from onset to death or censoring using Cox proportional-hazards regression. The SOD1-ALS dataset reports age of onset for 1122 and disease duration for 883 people; the comparator population includes 10,214 and 9010 people respectively. Eight variants are associated with younger age of onset and distinct survival trajectories; a further eight associated with younger onset only and one with distinct survival only. Here we show that onset and survival are decoupled in SOD1-ALS. Future research should characterise rarer variants and molecular mechanisms causing the observed variability

Protein kinetics of superoxide dismutase-1 in familial and sporadic amyotrophic lateral sclerosis

OBJECTIVE: Accumulation of misfolded superoxide dismutase-1 (SOD1) is a pathological hallmark of SOD1-related amyotrophic lateral sclerosis (ALS) and is observed in sporadic ALS where its role in pathogenesis is controversial. Understanding in vivo protein kinetics may clarify how SOD1 influences neurodegeneration and inform optimal dosing for therapies that lower SOD1 transcripts. METHODS: We employed stable isotope labeling paired with mass spectrometry to evaluate in vivo protein kinetics and concentration of soluble SOD1 in cerebrospinal fluid (CSF) of SOD1 mutation carriers, sporadic ALS participants and controls. A deaminated SOD1 peptide, SDGPVKV, that correlates with protein stability was also measured. RESULTS: In participants with heterozygous SOD1 INTERPRETATION: These results highlight the ability of stable isotope labeling approaches and peptide deamidation to discern the influence of disease mutations on protein kinetics and stability and support implementation of this method to optimize clinical trial design of gene and molecular therapies for neurological disorders. TRIAL REGISTRATION: Clinicaltrials.gov: NCT03449212

In vivo kinetic approach reveals slow SOD1 turnover in the CNS

Therapeutic strategies that target disease-associated transcripts are being developed for a variety of neurodegenerative syndromes. Protein levels change as a function of their half-life, a property that critically influences the timing and application of therapeutics. In addition, both protein kinetics and concentration may play important roles in neurodegeneration; therefore, it is essential to understand in vivo protein kinetics, including half-life. Here, we applied a stable isotope-labeling technique in combination with mass spectrometric detection and determined the in vivo kinetics of superoxide dismutase 1 (SOD1), mutation of which causes amyotrophic lateral sclerosis. Application of this method to human SOD1-expressing rats demonstrated that SOD1 is a long-lived protein, with a similar half-life in both the cerebral spinal fluid (CSF) and the CNS. Additionally, in these animals, the half-life of SOD1 was longest in the CNS when compared with other tissues. Evaluation of this method in human subjects demonstrated successful incorporation of the isotope label in the CSF and confirmed that SOD1 is a long-lived protein in the CSF of healthy individuals. Together, the results of this study provide important insight into SOD1 kinetics and support application of this technique to the design and implementation of clinical trials that target long-lived CNS proteins

The biostratigraphy of the offshore Niger delta during the Late Quaternary: Complexities and progress of dating techniques

The Late Quaternary marine sediments from the Niger Delta lacks an age model using conventional radiocarbon dating due to the rarity of calcareous macrofossils. The proprietary nature of material drilled by companies prospecting for hydrocarbons in the Niger Delta basin, and in the rare cases when samples are available for study as well as freshwater dilution from continental runoff have contributed to this dearth of knowledge. The availability of three shallow marine (∼3 m) gravity cores obtained from the eastern, central, and western parts of the Niger Delta provides the opportunity for biostratigraphy utilising well-preserved marker species of planktonic foraminifera and calcareous nannofossils in the sediments. The last occurrence (LO) of planktonic foraminiferal species Globorotalia truncatulinoides (late Pleistocene) (MIS 2) and the first occurrence (FO) of Globorotalia tumida (Holocene) (MIS 1) are used to identify two interval zones in the gravity cores. The presence of the calcareous nannofossil Gephyrocapsa oceanica (all <3 μm in size) supports a late Pleistocene age (NN19 Zone) for the lower interval. In addition, an increase in the abundance of Emiliania huxleyi up-section is an indication of early Holocene age (NN20-NN21) for the upper interval

Alzheimers Dement

Disturbances in the brain's capacity to meet its energy demand increase the risk of synaptic loss, neurodegeneration, and cognitive decline. Nutritional and metabolic interventions that target metabolic pathways combined with diagnostics to identify deficits in cerebral bioenergetics may therefore offer novel therapeutic potential for Alzheimer's disease (AD) prevention and management. Many diet-derived natural bioactive components can govern cellular energy metabolism but their effects on brain aging are not clear. This review examines how nutritional metabolism can regulate brain bioenergetics and mitigate AD risk. We focus on leading mechanisms of cerebral bioenergetic breakdown in the aging brain at the cellular level, as well as the putative causes and consequences of disturbed bioenergetics, particularly at the blood-brain barrier with implications for nutrient brain delivery and nutritional interventions. Novel therapeutic nutrition approaches including diet patterns are provided, integrating studies of the gut microbiome, neuroimaging, and other biomarkers to guide future personalized nutritional interventions

The SOD1-mediated ALS phenotype shows a decoupling between age of symptom onset and disease duration

Superoxide dismutase (SOD1) gene variants may cause amyotrophic lateral sclerosis, some of which are associated with a distinct phenotype. Most studies assess limited variants or sample sizes. In this international, retrospective observational study, we compare phenotypic and demographic characteristics between people with SOD1-ALS and people with ALS and no recorded SOD1 variant. We investigate which variants are associated with age at symptom onset and time from onset to death or censoring using Cox proportional-hazards regression. The SOD1-ALS dataset reports age of onset for 1122 and disease duration for 883 people; the comparator population includes 10,214 and 9010 people respectively. Eight variants are associated with younger age of onset and distinct survival trajectories; a further eight associated with younger onset only and one with distinct survival only. Here we show that onset and survival are decoupled in SOD1-ALS. Future research should characterise rarer variants and molecular mechanisms causing the observed variability

Effects of fluoxetine on functional outcomes after acute stroke (FOCUS): a pragmatic, double-blind, randomised, controlled trial

Background Results of small trials indicate that fluoxetine might improve functional outcomes after stroke. The FOCUS trial aimed to provide a precise estimate of these effects. Methods FOCUS was a pragmatic, multicentre, parallel group, double-blind, randomised, placebo-controlled trial done at 103 hospitals in the UK. Patients were eligible if they were aged 18 years or older, had a clinical stroke diagnosis, were enrolled and randomly assigned between 2 days and 15 days after onset, and had focal neurological deficits. Patients were randomly allocated fluoxetine 20 mg or matching placebo orally once daily for 6 months via a web-based system by use of a minimisation algorithm. The primary outcome was functional status, measured with the modified Rankin Scale (mRS), at 6 months. Patients, carers, health-care staff, and the trial team were masked to treatment allocation. Functional status was assessed at 6 months and 12 months after randomisation. Patients were analysed according to their treatment allocation. This trial is registered with the ISRCTN registry, number ISRCTN83290762. Findings Between Sept 10, 2012, and March 31, 2017, 3127 patients were recruited. 1564 patients were allocated fluoxetine and 1563 allocated placebo. mRS data at 6 months were available for 1553 (99·3%) patients in each treatment group. The distribution across mRS categories at 6 months was similar in the fluoxetine and placebo groups (common odds ratio adjusted for minimisation variables 0·951 [95% CI 0·839–1·079]; p=0·439). Patients allocated fluoxetine were less likely than those allocated placebo to develop new depression by 6 months (210 [13·43%] patients vs 269 [17·21%]; difference 3·78% [95% CI 1·26–6·30]; p=0·0033), but they had more bone fractures (45 [2·88%] vs 23 [1·47%]; difference 1·41% [95% CI 0·38–2·43]; p=0·0070). There were no significant differences in any other event at 6 or 12 months. Interpretation Fluoxetine 20 mg given daily for 6 months after acute stroke does not seem to improve functional outcomes. Although the treatment reduced the occurrence of depression, it increased the frequency of bone fractures. These results do not support the routine use of fluoxetine either for the prevention of post-stroke depression or to promote recovery of function. Funding UK Stroke Association and NIHR Health Technology Assessment Programme