Neuroprotective effect of CuATSM on neurotoxin-induced motor neuron loss in an ALS mouse model.

CuATSM is a PET-imaging agent that has recently received attention for its success in extending the lifespan in animals in several neurodegenerative disease models. In the SOD1G93A model of ALS, CuATSM prolonged mouse longevity far longer than any previously tested therapeutic agents. The mechanism underlying this outcome has not been fully understood, but studies suggest that this copper complex contributes to maintaining copper homeostasis in mitochondria. More specifically for the SOD1 model, the molecule supplies copper back to the SOD1 protein. Additionally, CuATSM demonstrated similar protective effects in various in vivo Parkinson's disease mouse models. In the current pilot study, we utilized a neurodegenerative mouse model of motor neuron degeneration induced by the neurotoxin ß-sitosterol ß-D-glucoside. In this model, slow but distinct and progressive features of sporadic ALS occur. Treatment with CuATSM kept animal behavioural performance on par with the controls and prevented the extensive motor neuron degeneration and microglia activation seen in the untreated animals. These outcomes support a broader neuroprotective role for CuATSM beyond mutant SOD models of ALS.

Aluminum as a CNS and Immune System Toxin Across the Life Span.

In the following, I will consider the impact of aluminum on two major systems, the central nervous system (CNS) and the immune system, across the life span. The article will discuss the presence of aluminum in the biosphere, its history, and the sources of the element. These include food, water cosmetics, some vaccines, and a range of other sources. I will also consider aluminum's unique chemistry. Finally, in humans and animals, I will consider how aluminum may impact the CNS at various levels of organization and how it may be involved in various neurological disease states across the life span. These disorders include those of infancy and childhood, such as autism spectrum disorder (ASD), as well as those in adulthood, such as in Alzheimer's disease. The bidirectional nature of CNS-immune system interactions will be considered and put into the context of neurological disorders that have an autoimmune component. I will argue that the exposure to humans and animals to this element needs to be reduced if we are to diminish some CNS and immune system disorders.

Necroptosis in amyotrophic lateral sclerosis and other neurological disorders.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by the progressive degeneration of upper and lower motor neurons. Cell death in ALS and in general was previously believed to exist as a dichotomy between apoptosis and necrosis. Most research investigating cell death mechanisms in ALS was conducted before the discovery of programmed necrosis thus did not use selective cell death pathway-specific markers. Recently, a new form of programmed cell death, termed "necroptosis", has been characterized and has been recently implicated in ALS as a primary mechanism driving motor neuron cell death in different forms of ALS. The present review is aimed at summarizing cell death pathways that are currently implicated in ALS and highlighting the emerging evidence on necroptosis as a major driver of motor neuron cell death.

Reduction in hSOD1 copy number significantly impacts ALS phenotype presentation in G37R (line 29) mice: implications for the assessment of putative therapeutic agents.

BACKGROUND: In vivo animal models of familial amyotrophic lateral sclerosis (fALS) are widely used to delineate the potential role that genetic mutations play in the neurodegenerative process. While these models are extensively used for establishing the safety and efficacy of putative therapeutics during pre-clinical development, effective clinical translation of pharmacological interventions has been largely unsuccessful. RESULTS: In this report we compare a recent cohort of G37R (line 29) mice generated from mating wild-type females with transgenic males obtained commercially to a previous set of offspring produced with transgenic male breeders from a colony established at a local collaborator's facility. Commercially derived progeny presented with a tightly clustered genomic signature for the mutant human superoxide dismutase1 transgene (hSOD1) locus, and exhibited a greater than two-fold reduction in the number of transgene copies present in the genome compared to offspring derived locally. Decrease in transgene levels corresponded with delayed ALS progression and a significant increase in overall lifespan (146%). CONCLUSIONS: These results highlight some key challenges inherent to the use of G37R (line 29) animals in pre-clinical studies for the development of ALS therapeutics. Without stringent assessment of mutant SOD1 copy number/protein levels, heterogeneity of transgene levels within cohorts may influence the behavioural and pathological presentation of disease and thus calls to question the validity of any detected therapeutic effects. Nuanced changes in mutant SOD1 copy number that currently remain unreported may undermine research endeavours, delay efforts for clinical translation, and compromise the rigor of animal studies by limiting reproducibility amongst research groups.

Levamisole derivatives as immune modulators for the treatment of amyotrophic lateral sclerosis (ALS).

Aim: To discover derivatives of the anthelmintic drug levamisole, which has been reported to possess immune-modulatory properties, as treatments for amyotrophic lateral sclerosis (ALS), which has been suggested to be in part an autoimmune disease. Results: We have synthesized ten analogs of the racemic version of levamisole, tetramisole, as well as eleven analogs on a related system. All of the analogs have been tested for their ability to affect the response of five ALS-relevant cytokines. Conclusion: We have discovered a number of interesting derivatives that have encouraging cytokine response data and good metabolic stability, with the potential to have a positive impact on ALS either as single agents, or in combination.

Aim: To discover derivatives of the antiparasitic worm drug levamisole, which has been reported to be able to modulate the immune response, as treatments for amyotrophic lateral sclerosis (ALS), which has been suggested to be in part an autoimmune disease. Results: We have synthesized ten analogs of a variation of levamisole, tetramisole, as well as 11 analogs on a related system. All of the analogs have been tested for their ability to affect the response of five ALS-relevant immune-modulatory substances (cytokines). Conclusion: We have discovered a number of interesting derivatives that have encouraging cytokine response data and good metabolic stability, with the potential to have a positive impact on ALS either as single agents, or in combination.

Metals and Metalloids Increase in Cycas micronesica Seed Gametophyte Tissue in Shaded Growth Conditions.

Exposure to environmental toxins may be partly responsible for mammal neurodegenerative disorders. Consumption of seeds from Guam's cycad tree has been linked to the disorder known as amyotrophic lateral sclerosis-parkinsonism dementia complex (ALS-PDC). The unambiguous identification of causal agents of ALS-PDC has been elusive. We have examined the levels of eight metals and metalloids in cycad seeds as a function of the ambient shade in which the plants were grown. Of these metals, the data strongly suggest that aluminum (Al) and selenium (Se) are present in washed flour prepared from southern Guam's cycad seed tissues at elevated levels, especially when the trees are grown in shade. Previous authors have speculated that Al and Se are involved in various ALS outcomes, and our results support this interpretation.

Environmental neurotoxin-induced progressive model of parkinsonism in rats.

OBJECTIVE: Exposure to a number of drugs, chemicals, or environmental factors can cause parkinsonism. Epidemiologic evidence supports a causal link between the consumption of flour made from the washed seeds of the plant Cycas micronesica by the Chamorro population of Guam and the development of amyotrophic lateral sclerosis/parkinsonism dementia complex. METHODS: We now report that consumption of washed cycad flour pellets by Sprague-Dawley male rats induces progressive parkinsonism. RESULTS: Cycad-fed rats displayed motor abnormalities after 2 to 3 months of feeding such as spontaneous unilateral rotation, shuffling gait, and stereotypy. Histological and biochemical examination of brains from cycad-fed rats revealed an initial decrease in the levels of dopamine and its metabolites in the striatum (STR), followed by neurodegeneration of dopaminergic (DAergic) cell bodies in the substantia nigra (SN) pars compacta (SNc). alpha-Synuclein (alpha-syn; proteinase K-resistant) and ubiquitin aggregates were found in the DAergic neurons of the SNc and neurites in the STR. In addition, we identified alpha-syn aggregates in neurons of the locus coeruleus and cingulate cortex. No loss of motor neurons in the spinal cord was found after chronic consumption of cycad flour. In an organotypic slice culture of the rat SN and the striatum, an organic extract of cycad causes a selective loss of dopamine neurons and alpha-syn aggregates in the SN. INTERPRETATION: Cycad-fed rats exhibit progressive behavioral, biochemical, and histological hallmarks of parkinsonism, coupled with a lack of fatality.

Computed tomography reveals hip dysplasia in the extinct Pleistocene saber-tooth cat Smilodon.

Reconstructing the behavior of extinct species is challenging, particularly for those with no living analogues. However, damage preserved as paleopathologies on bone can record how an animal moved in life, potentially reflecting behavioral patterns. Here, we assess hypothesized etiologies of pathology in a pelvis and associated right femur of a Smilodon fatalis saber-toothed cat, one of the best-studied species from the Pleistocene-age Rancho La Brea asphalt seeps, California, USA, using visualization by computed tomography (CT). The pelvis exhibits massive destruction of the right hip socket that was interpreted, for nearly a century, to have developed from trauma and infection. CT imaging reveals instead that the pathological distortions characterize chronic remodeling that began at birth and led to degeneration of the joint over the animal's life. These results suggest that this individual suffered from hip dysplasia, a congenital condition common in domestic dogs and cats. This individual reached adulthood but could not have hunted properly nor defended territory on its own, likely relying on a social group for feeding and protection. While extant social felids are rare, these fossils and others with similar pathologies are consistent with a spectrum of social strategies in Smilodon supported by a predominance of previous studies.

In vitro effects of cholesterol ß-D-glucoside, cholesterol and cycad phytosterol glucosides on respiration and reactive oxygen species generation in brain mitochondria.

The cluster of neurodegenerative disorders in the western Pacific termed amyotrophic lateral sclerosis-parkinsonism dementia complex (ALS-PDC) has been repeatedly linked to the use of seeds of various species of cycad. Identification and chemical synthesis of the most toxic compounds in the washed cycad seeds, a variant phytosteryl glucosides, and even more toxic cholesterol ß-D-glucoside (CG), which is produced by the human parasite Helicobacter pylori, provide a possibility to study in vitro the mechanisms of toxicity of these compounds. We studied in detail the effects of CG on the respiratory activities and generation of reactive oxygen species (ROS) by nonsynaptic brain and heart mitochondria oxidizing various substrates. The stimulatory effects of CG on respiration and ROS generation showed strong substrate dependence, suggesting involvement of succinate dehydrogenase (complex II). Maximal effects on ROS production were observed with 1 µmol CG/1 mg mitochondria. At this concentration the cycad toxins ß-sitosterol-ß-D-glucoside and stigmasterol-ß-D-glucoside had effects on respiration and ROS production similar to CG. However, poor solubility precluded full concentration analysis of these toxins. Cholesterol, stigmasterol and ß-sitosterol had no effect on mitochondrial functions studied at concentrations up to 100 µmol/mg protein. Our results suggest that CG may influence mitochondrial functions through changes in the packing of the bulk membrane lipids, as was shown earlier by Deliconstantinos et al. (Biochem Cell Biol 67:16-24, 1989). The neurotoxic effects of phytosteryl glucosides and CG may be associated with increased oxidative damage of neurons. Unlike heart mitochondria, in activated neurons mitochondria specifically increase ROS production associated with succinate oxidation (Panov et al., J Biol Chem 284:14448-14456, 2009).

Distribution of free and glycosylated sterols within Cycas micronesica plants.

Flour derived from Cycas micronesica seeds was once the dominant source of starch for Guam's residents. Cycad consumption has been linked to high incidence of human neurodegenerative diseases. We determined the distribution of the sterols stigmasterol and ß-sitosterol and their derived glucosides stigmasterol ß-d-glucoside and ß-sitosterol ß-d-glucoside among various plant parts because they have been identified in cycad flour and have been shown to elicit neurodegenerative outcomes. All four compounds were common in seeds, sporophylls, pollen, leaves, stems, and roots. Roots contained the greatest concentration of both free sterols, and photosynthetic leaflet tissue contained the greatest concentration of both steryl glucosides. Concentration within the three stem tissue categories was low compared to other organs. Reproductive sporophyll tissue contained free sterols similar to seeds, but greater concentration of steryl glucosides than seeds. One of the glucosides was absent from pollen. Concentration in young seeds was higher than old seeds as reported earlier, but concentration did not differ among age categories of leaf, sporophyll, or vascular tissue. The profile differences among the various tissues within these organs may help clarify the physiological role of these compounds.

Investigating microglia during motor neuron degeneration using a zebrafish model.

Many different types of pathologies can arise in the central nervous system (CNS), such as neurodegeneration. The incidence of neurodegenerative diseases continues to increase, yet the pathogenesis underlying most neurodegenerative diseases, notably in amyotrophic lateral sclerosis (ALS), remains elusive. Neuronal support cells, or glia, are known to play a crucial role in ALS. Microglia are the resident immune cells of the CNS and also have neurotrophic support functions. These cells have a disease-modifying function in ALS, yet this role is not well understood. A likely reason for this is that the intact CNS is particularly challenging to access for investigation in patients and in most animal models, which has impeded research in this field. The zebrafish is emerging as a robust model system to investigate cells in vivo, and offer distinct advantages over other vertebrate models for investigating neurodegenerative diseases. Live imaging in vivo is a powerful technique to characterize the role of dynamic cells such as microglia during neurodegeneration, and zebrafish provide a convenient means for live imaging. Here, we discuss the zebrafish as a model for live imaging, provide a brief overview of available high resolution imaging platforms that accommodate zebrafish, and describe our own in vivo studies on the role of microglia during motor neuron degeneration. Live in vivo imaging is anticipated to provide invaluable advancements to defining the pathogenesis underlying neurodegenerative diseases, which may in turn allow for more specifically targeted therapeutics.

Early postnatal injections of whole vaccines compared to placebo controls: Differential behavioural outcomes in mice.

The present study was designed to evaluate the possible effects of the paediatric vaccination schedule in the United States on the central nervous system in a murine model. We compared the impact of treatment with the whole vaccines versus true placebo control. Seventy-six pups were divided into three groups: two vaccinated groups and unvaccinated control. The two vaccinated groups were treated between 7 and 21 post-natal days either with one or three times of the vaccine doses per body weight as used in children between newborn and eighteen months of age. The post-vaccination development, neuromotor behaviours and neurobehavioural abnormalities (NBAs) were evaluated in all mouse groups during the 67 post-natal weeks of mouse age. Mouse body weight was affected only in the vaccinated females compared to males and control. Some NBAs such as decreased sociability, increased anxiety-like behaviours, and alteration of visual-spatial learning and memory were observed in vaccinated male and female mice compared to controls. The present study also shows a slower acquisition of some neonatal reflexes in vaccinated female mice compared to vaccinated males and controls. The observed neurodevelopmental alterations did not show a linear relationship with vaccine dose, suggesting that the single dose gave a saturated response. The outcomes seemed to be sex-dependent and transient with age.

Progranulin is expressed within motor neurons and promotes neuronal cell survival.

BACKGROUND: Progranulin is a secreted high molecular weight growth factor bearing seven and one half copies of the cysteine-rich granulin-epithelin motif. While inappropriate over-expression of the progranulin gene has been associated with many cancers, haploinsufficiency leads to atrophy of the frontotemporal lobes and development of a form of dementia (frontotemporal lobar degeneration with ubiquitin positive inclusions, FTLD-U) associated with the formation of ubiquitinated inclusions. Recent reports indicate that progranulin has neurotrophic effects, which, if confirmed would make progranulin the only neuroprotective growth factor that has been associated genetically with a neurological disease in humans. Preliminary studies indicated high progranulin gene expression in spinal cord motor neurons. However, it is uncertain what the role of Progranulin is in normal or diseased motor neuron function. We have investigated progranulin gene expression and subcellular localization in cultured mouse embryonic motor neurons and examined the effect of progranulin over-expression and knockdown in the NSC-34 immortalized motor neuron cell line upon proliferation and survival. RESULTS: In situ hybridisation and immunohistochemical techniques revealed that the progranulin gene is highly expressed by motor neurons within the mouse spinal cord and in primary cultures of dissociated mouse embryonic spinal cord-dorsal root ganglia. Confocal microscopy coupled to immunocytochemistry together with the use of a progranulin-green fluorescent protein fusion construct revealed progranulin to be located within compartments of the secretory pathway including the Golgi apparatus. Stable transfection of the human progranulin gene into the NSC-34 motor neuron cell line stimulates the appearance of dendritic structures and provides sufficient trophic stimulus to survive serum deprivation for long periods (up to two months). This is mediated at least in part through an anti-apoptotic mechanism. Control cells, while expressing basal levels of progranulin do not survive in serum free conditions. Knockdown of progranulin expression using shRNA technology further reduced cell survival. CONCLUSION: Neurons are among the most long-lived cells in the body and are subject to low levels of toxic challenges throughout life. We have demonstrated that progranulin is abundantly expressed in motor neurons and is cytoprotective over prolonged periods when over-expressed in a neuronal cell line. This work highlights the importance of progranulin as neuroprotective growth factor and may represent a therapeutic target for neurodegenerative diseases including motor neuron disease.

Parkinson-dementia complex and development of a new stable isotope dilution assay for BMAA detection in tissue.

Beta-methylamino-L-alanine (BMAA) has been proposed as a global contributor to neurodegenerative diseases, including Parkinson-dementia complex (PDC) of Guam and Alzheimer's disease (AD). The literature on the effects of BMAA is conflicting with some but not all in vitro data supporting a neurotoxic action, and experimental animal data failing to replicate the pattern of neurodegeneration of these human diseases, even at very high exposures. Recently, BMAA has been reported in human brain from individuals afflicted with PDC or AD. Some of the BMAA in human tissue reportedly is freely extractable (free) while some is protein-associated and liberated by techniques that hydrolyze the peptide bond. The latter is especially intriguing since BMAA is a non-proteinogenic amino acid that has no known tRNA. We attempted to replicate these findings with techniques similar to those used by others; despite more than adequate sensitivity, we were unable to detect free BMAA. Recently, using a novel stable isotope dilution assay, we again were unable to detect free or protein-associated BMAA in human cerebrum. Here we review the development of our new assay for tissue detection of BMAA and show that we are able to detect free BMAA in liver but not cerebrum, nor do we detect any protein-associated BMAA in mice fed this amino acid. These studies demonstrate the importance of a sensitive and specific assay for tissue BMAA and seriously challenge the proposal that BMAA is accumulating in human brain.

Phenotypic Characteristics as Predictors of Phytosterols in Mature Cycas micronesica Seeds.

The relationship between mature Cycas micronesica K.D. Hill seed sterol concentration and content and plant or seed phenotypic characteristics was established by multiple regression. Combined models were significant for free but not glycosylated sterols. Reduced models revealed leaf number as the only significant predictor. Free and glycosylated sterol concentrations were unaffected throughout the range of several predictors: tree height (1.7 to 5.8 m), seed fresh weight (48 to 120 g), seed load (one to 76 seeds per plant), and estimated tree age (32 to 110 years). The free and glycosylated sterol phenotypes were also not dependent on the presence/absence of developed embryos in mature seeds. The significant response to leaf number was subtle with an increase of 43 leaves associated with a 0.1-mg increase in free sterol per gram seed fresh weight. This is the first report for any cycad that discusses reproductive or physiological traits in the context of allometric relations. Results indicate a highly constrained phenotypic plasticity of Cycas gametophyte sterol and steryl glucoside concentration and seed content in relation to whole plant and organ size variation.

Is exposure to aluminium adjuvants associated with social impairments in mice? A pilot study.

BACKGROUND: Our group has shown that significant correlations exist between rates of Autism Spectrum Disorder (ASD) and total aluminum adjuvants given to children through vaccines in several Western countries. These correlations satisfied eight out of nine Hill criteria for causality. Experimental studies have demonstrated a range of behavioural abnormalities in young mice after postnatal exposure to aluminium. To build on our previous work, the current study will investigate the effect of aluminium adjuvants on social behaviour in mice. Anomalies in social interaction are a key characteristic of those with ASD. METHODS: Neonatal CD-1 mice pups were injected with either a total of 550µg of aluminum hydroxide gel (experimental group) or saline (control) spread out during the first two weeks of postnatal life. The mice were then subjected to behavioural tests for social interest and social novelty at postnatal week 8, 17 and 29. p-Values were calculated using the Mann-Whitney and Kruskal Wallis tests. RESULTS: Aluminum injected mice showed diminished social interest compared to controls at week 8 (p=0.016) and 17 (p=0.012). They also demonstrated abnormal social novelty from controls at week 8 (p=0.002) and week 29 (p=0.042). CONCLUSION: This is the first experimental study, to our knowledge, to demonstrate that aluminum adjuvants can impair social behaviour if applied in the early period of postnatal development. The study, however, is insufficient to make any assertive claims about the link between aluminium adjuvants and ASD in humans.

Modeling Environmentally-Induced Motor Neuron Degeneration in Zebrafish.

Zebrafish have been used to investigate motor neuron degeneration, including as a model system to examine the pathogenesis of amyotrophic lateral sclerosis (ALS). The use of zebrafish for this purpose has some advantages over other in vivo model systems. In the current paper, we show that bisphenol A (BPA) exposure in zebrafish embryos results in motor neuron degeneration with affected motor function, reduced motor axon length and branching, reduced neuromuscular junction integrity, motor neuron cell death and the presence of activated microglia. In zebrafish, motor axon length is the conventional method for estimating motor neuron degeneration, yet this measurement has not been confirmed as a valid surrogate marker. We also show that reduced motor axon length as measured from the sagittal plane is correlated with increased motor neuron cell death. Our preliminary timeline studies suggest that axonopathy precedes motor cell death. This outcome may have implications for early phase treatments of motor neuron degeneration.

Animal models of amyotrophic lateral sclerosis: A comparison of model validity.

Animal models are necessary to investigate the pathogenic features underlying motor neuron degeneration and for therapeutic development in amyotrophic lateral sclerosis (ALS). Measures of model validity allow for a critical interpretation of results from each model and caution from over-interpretation of experimental models. Face and construct validity refer to the similarity in phenotype and the proposed causal factor to the human disease, respectively. More recently developed models are restricted by limited phenotype characterization, yet new models hold promise for novel disease insights, thus highlighting their importance. In this article, we evaluate the features of face and construct validity of our new zebrafish model of environmentally-induced motor neuron degeneration and discuss this in the context of current environmental and genetic ALS models, including C9orf72, mutant Cu/Zn superoxide dismutase 1 and TAR DNA-binding protein 43 mouse and zebrafish models. In this mini-review, we discuss the pros and cons to validity criteria in each model. Our zebrafish model of environmentally-induced motor neuron degeneration displays convincing features of face validity with many hallmarks of ALS-like features, and weakness in construct validity. However, the value of this model may lie in its potential to be more representative of the pathogenic features underlying sporadic ALS cases, where environmental factors may be more likely to be involved in disease etiology than single dominant gene mutations. It may be necessary to compare findings between different strains and species modeling specific genes or environmental factors to confirm findings from ALS animal models and tease out arbitrary strain- and overexpression-specific effects.