Early exposure to environmental toxin contributes to neuronal vulnerability and axonal pathology in a model of familial ALS

Adult onset amyotrophic lateral sclerosis (ALS) arises due to progressive and irreversible functional deficits to the central nervous system, specifically the loss of motor neurons. Sporadic ALS causality is not well understood, but is almost certainly of multifactorial origin involving a combination of genetic and environmental factors. The discovery of endemic ALS in the native Chamorro population of Guam during the 1950s and the co-occurrence of parkinsonism and dementia in some patients led to searches for an environmental toxins that could be responsible. In the present paper, we report that an environmental neurotoxin enhances mutant superoxide dismutase (SOD)-induced spinal motor neuron death and pathology and induces motor axon abnormalities. These results cumulatively confirm earlier findings that exposure to an environmental toxin is sufficient to produce the disease phenotype and indicate a role for gene-environment interaction in some forms of the disease

Cycads, Flying Foxes, and Brain Disease in Humans

Cycads are a group of ancient gymnosperms with a rich fossil history. While they once dominated the world’s forests, their adaptation to warm climates has now restricted them to tropical areas. They are not widely eaten by humans due to their known toxicity, but some indigenous groups of the Pacific Islands, such as the Chamorro people of Guam, have developed methods of washing the seeds for safe consumption. Cycad seeds are a common dietary item of several mammals. One such mammal, the flying fox, is considered a delicacy to several indigenous groups. In the mid-twentieth century, these groups also expressed an extraordinarily high rate of a rare and severe neurodegenerative disease known as the amyotrophic lateral sclerosis-parkinsonism dementia complex, or ALS-PDC. An early hypothesis for this high rate of ALS-PDC was toxicity resulting from the widespread consumption of cycad seeds. This was later rejected due to the finding that the washing techniques removed a sufficient amount of toxins to eliminate the possibility of negative effects. The cycad hypothesis was revived when it was discovered that large quantities of cycad toxins accumulate in the tissues of mammals such as flying foxes. Consuming even a single flying fox would deliver a similar amount of cycad toxins as up to a thousand kilograms of cycad flour. Given the demonstrated neurotoxic effects of several cycad toxins, as well as the correlating decline in rates of ALS-PDC and flying fox consumption, it is likely that an underlying cause of ALS-PDC is consumption of flying fox tissues containing bioaccumulated cycad toxins

In Vivo 3D Digital Atlas Database of the Adult C57BL/6J Mouse Brain by Magnetic Resonance Microscopy

In this study, a 3D digital atlas of the live mouse brain based on magnetic resonance microscopy (MRM) is presented. C57BL/6J adult mouse brains were imaged in vivo on a 9.4 Tesla MR instrument at an isotropic spatial resolution of 100 μm. With sufficient signal-to-noise (SNR) and contrast-to-noise ratio (CNR), 20 brain regions were identified. Several atlases were constructed including 12 individual brain atlases, an average atlas, a probabilistic atlas and average geometrical deformation maps. We also investigated the feasibility of using lower spatial resolution images to improve time efficiency for future morphological phenotyping. All of the new in vivo data were compared to previous published in vitro C57BL/6J mouse brain atlases and the morphological differences were characterized. Our analyses revealed significant volumetric as well as unexpected geometrical differences between the in vivo and in vitro brain groups which in some instances were predictable (e.g. collapsed and smaller ventricles in vitro) but not in other instances. Based on these findings we conclude that although in vitro datasets, compared to in vivo images, offer higher spatial resolutions, superior SNR and CNR, leading to improved image segmentation, in vivo atlases are likely to be an overall better geometric match for in vivo studies, which are necessary for longitudinal examinations of the same animals and for functional brain activation studies. Thus the new in vivo mouse brain atlas dataset presented here is a valuable complement to the current mouse brain atlas collection and will be accessible to the neuroscience community on our public domain mouse brain atlas website

Phosphorylation of tau protein at Thr175 is a toxic event associated with neurodegeneration

Aberrant phosphorylation and pathological deposition of the microtubule associated protein tau (tau protein) is associated with toxicity and cellular death in a number of neurodegenerative diseases (tauopathies). Specific phosphorylation sites are of interest in the processes leading to tau protein toxicity. One site of interest on tau protein is Thr175 (pThr175), which has been identified in diseased brain tissue from individuals with amyotrophic lateral sclerosis with cognitive impairment (ALSci) and Alzheimer’s disease. In vitro, pseudophosphorylation at this residue has been shown to induce the formation of pathological tau fibrils and, apoptotic cell death. In my thesis, I have investigated the mechanism of cellular toxicity following phosphorylation of tau protein at Thr175. After showing that Thr175 pseudophosphorylation alone is insufficient to initiate tau protein fibrillization, I demonstrated that tau phosphorylation at Thr175 directly leads to the induction of kinase glycogen synthase kinase-3β (GSK3β) which in turn phosphorylates tau protein at Thr231. Both of these steps are necessary for the cytotoxicity of pThr175 tau to be manifest. I have shown that the pharmacological inhibition of this process leading to Thr231 phosphorylation prevents both fibril formation and cell death. To determine the extent to which this pathological process of Thr231 phosphorylation was applicable across the tauopathies in general, I characterized the presence of pThr175, activated GSK3b, pThr231tau and oligomeric tau formation across multiple tauopathies. In doing so, I demonstrated that this pathway may play an integral role in the generation of pathological tau deposition beyond that discovered for ALSci. I then characterized pThr175 tau protein pathology in the trauma-associated neurodegenerative disease chronic traumatic encephalopathy (CTE) and CTE with amyotrophic lateral sclerosis (CTE-ALS), demonstrating the presence of pThr175 tau protein in pathology associated with these diseases as well. In order to determine whether the induction of pathological pThr175 tau was a primary event in the induction of this neuropathology, I used a rat model of moderate traumatic brain injury in which I demonstrated that after a single cortical impact, phosphorylation of endogenous tau protein at Thr175 was persistently elevated. pThr175 tau was present in axonal pathology as well as tau protein fibrillar neuronal pathology. In order to definitively prove that pThr175tau was sufficient to induce tau pathology in vivo, I undertook somatic gene transfer of a rAAV9 construct expressing pseudophosphorylated human pThr175 tau (Thr175-Asp tau) in young adult rat hippocampus. I observed that one year following the stereotactic inoculation of this modified viral vector, rats developed tau pathology in construct-expressing hippocampal neurons along with caspase-3 cleavage. While the construct was similarly expressed in control rats, including empty vector and wild-type human tau, none of these latter rats developed pathology. These findings indicate that phosphorylation of human tau at Thr175 triggers the pathological phosphorylation of tau protein at Thr231 through activation of GSK3b, and that this cascade leads to pathological fibril formation in vitro and in vivo. I have further demonstrated that this pathological process may have broader applicability than to the pathogenesis of ALSci, and includes a broad range of tauopathies in addition to CTE and CTE-ALS

Group I metabotropic glutamatergic receptors regulating glutamate release and microglia phenotype in a murine model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by the death of upper and lower motor neurons. Although the aetiology of the disease is still unclear, glutamate (Glu)-mediated excitotoxicity is a major cause. Our previous studies demonstrated that presynaptic Group-I metabotropic Glu receptors (mGluR1 and mGluR5) are over-expressed in spinal cord synaptosomes of 120-day-old SOD1G93A mice, that represent the late stage of the disease, and that their activation by the selective mGluR1/5 agonist (S)-3,5-Dihydroxyphenylglycine (3,5-DHPG) produced abnormal Glu release. The aim of the present study was to investigate whether mGluR1 and mGluR5 also affect Glu release during the pre- and early-symptomatic time-course of the pathlogy (30, 60 and 90 days), in the same animal model. Our results showed that the mGluR1/5 agonist 3,5-DHPG evoked the release of glutamate in a concentration-dependent way and the effects were almost superimposable between 30/60-day-old WT and SOD1G93A mice. At variance, 0.3 \u3bcM 3,5-DHPG significantly increased Glu release (25%, p<0.05) in 90-day-old SOD1G93A mice but not in WT aged controls. The involvement of both metabotropic glutamate receptor subtypes was demonstrated using mGluR1 and mGluR5 selective antagonists/negative allosteric modulators (LY367385, MPEP, respectively). The analysis of the molecular mechanisms underlying the 3,5-DHPG-evoked Glu release revealed that it was of vesicular origin and induced by Ca2+ released from intra terminal stores. Confocal imaging confirmed that both mGluR1 and mGluR5 were co-localized onto glutamatergic nerve terminals and their expression was increased in SOD1G93A mice at the onset of the disease. We have also set up a method to isolate extracellular vesicles enriched in exosomes to investigate whether EVs derived from cultured activated astrocytes, treated with a mGluR5 antagonist, were able to change the the inflammatory pattern of microglia

Étude et caractérisation du rôle de protéines TDP-43 mutantes dans la pathogénèse de la sclérose latérale amyotrophique (SLA)

Tableau d'honneur de la Faculté des études supérieures et postdoctorales, 2011-2012La sclérose latérale amyotrophique (SLA) est une maladie mortelle caractérisée par une dégénérescence des neurones moteurs supérieurs et inférieurs. La présence d’inclusions ubiquitinylées de la protéine TDP-43 (Transactive response DNA-binding protein 43) est une caractéristique de la SLA. Afin de comprendre le mécanisme pathogène impliquant cette protéine, nous avons généré et étudié des souris transgéniques en utilisant des fragments génomiques codant pour la TDP-43 humain, de type sauvage ou mutant, associés aux cas familiaux de la SLA. Ces souris développent de nombreux changements liés au processus pathologique et biochimique de la SLA chez l’homme : présence d’inclusions de la protéine TDP-43 ubiquitinylées, anomalies au niveau des filaments intermédiaires, axonopathie et neuroinflammation. Pour mieux comprendre le rôle de la protéine TDP-43 dans la régénération des axones, nous avons utilisé des souris pré-symptomatiques et effectué une lésion du nerf sciatique sur celles-ci. Suite à cette intervention, les souris transgéniques ont eu une paralysie marquée du membre lésé, ont démontré une redistribution altéré de TDP-43 et une regénération plus lente des axones distaux par rapport aux souris non transgéniques. De plus, nous avons constaté que la protéine TDP-43 interagit et colocalise avec la sous-unité p65 du facteur nucléaire κΒ (NF-κΒ). Cette interaction se produit dans les cellules gliales et les neurones des souris transgéniques TDP-43 et aussi chez les patients atteints de la SLA. Nous avons démontré que les niveaux d’ARNm des protéines TDP-43 et NF-κΒ p65, sont plus élevés dans la moelle épinière des patients atteints de SLA que chez les individus sains et que la protéine TDP-43 agit comme un coactivateur de p65. Finalement, le traitement des souris transgéniques TDP-43 avec la Withaférine A, un inhibiteur de l’activité NF-κΒ, réduit le niveau de dénervation des jonctions neuromusculaires et des symptômes liés à la SLA. Nous suggérons donc que le dérèglement de la protéine TDP-43 contribue à la pathogenèse de la SLA en partie par l'augmentation de l'activation de NF-κΒ, et que NF-κΒ pourrait constituer une cible thérapeutique pour la maladie.Amyotrophic lateral sclerosis (ALS) is a lethal disease characterized by degeneration of lower and upper motor neurons. Transactive response DNA-binding protein 43 (TDP-43) ubiquitinated inclusions are a hallmark of ALS. In order to understand the pathogenic mechanism caused by TDP-43, we generated transgenic mice with genomic fragments encoding human TDP-43 wild-type or FALS-linked mutants TDP-43G348C and TDP-43A315T. These novel TDP-43 transgenic mice develop many age-related pathological and biochemical changes reminiscent of human ALS including ubiquitinated TDP-43 positive inclusions, intermediate filament abnormalities, axonopathy and neuroinflammation. In order to understand the role of TDP-43 in axon regeneration, we used pre-symptomatic 3-months old mice and performed sciatic nerve crush on them. After axonal crush, TDP-43 transgenic mice were noticeably paralyzed at the injured limb, have altered TDP-43 redistribution and the distal axons regenerated slowly as compared to non-transgenic mice. Moreover, we found that TDP-43 interacts with and colocalizes with p65, a NF-κΒ subunit, in glial and neuronal cells from TDP-43 transgenic mice and also from ALS patients. We report that TDP-43 and NF-κΒ p65 mRNA and protein expression is higher in spinal cords of ALS patients than healthy individuals. TDP-43 acted as a co-activator of p65, and glial cells expressing higher amounts of TDP-43 produced more proinflammatory cytokines and neurotoxic mediators after stimulation with lipopolysaccharide or reactive oxygen species. TDP-43 overexpression in neurons also increased their vulnerability to toxic mediators. Treatment of TDP-43 mice with Withaferin A, an inhibitor of NF-κΒ activity, reduced denervation in the neuromuscular junction and ALS disease symptoms. We propose that TDP-43 deregulation contributes to ALS pathogenesis in part by enhancing NF-κΒ activation, and that NF-κΒ may constitute a therapeutic target for the disease

Amyotrophic Lateral Sclerosis: Molecular Mechanisms to Diagnostics

Amyotrophic lateral sclerosis (ALS) is a relentlessly progressive and fatal motor neuron disease, characterized by loss of motor neurons in the cortex, brainstem and spinal cord. Clinical management is plagued by a lack of biomarkers and effective treatment. In spite of numerous scientific advancements, molecular mechanisms involved in its initiation and progression remain an enigma. At the mechanistic level, ALS is considered multifactorial. Extracellular signals may modulate nuclear events with a possible consequence being the reactivation of cell cycle-related genes and protein alterations in the terminally differentiated motor neurons. In the first specific aim, we hypothesized that re-entry of post-mitotic motor neurons into the cell cycle, concurrent with altered activity or distribution of transcription factors will result in apoptosis of motor neurons during ALS. To address this hypothesis, we utilized archived human autopsy material from the cortical and spinal cord regions of ALS and age-matched control cases. We conclude that surviving ALS motor neurons in these regions exhibited increased levels of G1 to S phase regulators (Cyclin D1, CDK4, hyperphosphorylated -pRb and E2F-1). It also revealed two intriguing results: (i) E2F-1, a transcription factor, was cytoplasmic and (ii) increased nuclear p53 was noted in spinal motor neurons but absent in neurons of the motor cortex. In addition there was increased protein levels of apoptotic death markers (BAX, FAS, Caspases) and DNA fragmentation. Therefore we have identified a potential role for cell cycle proteins in an apoptotic mode of motor neuron death in ALS. In the second specific aim we hypothesized that a mass spectrometry-based proteomics approach will identify diagnostic biomarkers and molecular targets for drug discoveries. We used cerebrospinal fluid (CSF) from ALS and control subjects to identify and validate a biomarker panel specific to ALS. Furthermore, utilizing peptide map fingerprinting and tandem mass-spectrometry, we have identified three of the protein peaks to be a carboxyl-terminal fragment of neurosecretory chaperone protein 7B2 (3.44kDa), Cystatin C (13.3kDa) and monomer of transthyretin (13.78kDa).Taken together, this body of work furthers the understanding of both the mechanisms leading to selective motor neuron loss in ALS and paves the way for diagnostics and therapeutics

Novel Methods for the Prevention of Neurodegeneration around Neural Implants

The recent development of neural prosthetic technology has demonstrated a therapeutic potential for restoring lost sensory or motor functions via a brain-machine interface. Often the devices require direct contact between neural tissue and implanted electrodes to function properly by electrically stimulating or recording neurons on the scale of micro-volts. It is thus critical that the interface between the tissue and the electrode is seamless as well as stable for durations relevant to clinical applications. Unfortunately, the formation of an abiotic/biotic interface is often riddled with host tissue responses that interfere with device function. Neuronal loss due to insertion injury and chronic inflammation and exclusion of recordable cells by an encapsulated glial sheath have all been implicated as potential sources for chronic neural recording failure. Failure to establish a stable interface severely limits the function of implanted neural devices. In this work we rely upon interdisciplinary techniques in chemistry, biology, and physics to develop methods for the formation of a healthy brain-machine interface as well as new tools for the diagnostics of the tissue response. Through the creation of new biomaterials we have improved the neural interface, reduced local neurodegeneration around chronic implants, and demonstrated in vivo improvement of neural recording. In addition, novel conductive and magnetic nanomaterials have been developed for electrochemically detecting reactive oxygen species associated with implant induced tissue damage as well as treatment strategies using on demand drug release. By establishing a method for the formation of healthy and stable interfaces between neural tissue and electrode recording devices, the development of neural prosthetic devices can continue to progress towards clinical acceptance

A Study on Molecular Mechanism of Novel Mitophagy Regulators

학위논문 (박사)-- 서울대학교 대학원 : 생명과학부, 2015. 2. 정용근.CHDH (choline dehydrogenase) is an enzyme catalyzing the dehydrogenation of choline to betaine aldehyde in mitochondria. Apart from this well-known activity, I report here a pivotal role of CHDH in mitophagy. Knockdown of CHDH expression impairs CCCP-induced mitophagy and PARK2/parkin-mediated clearance of mitochondria in mammalian cells, including HeLa cells and SN4741 dopaminergic neuronal cells. Conversely, overexpression of CHDH accelerates PARK2-mediated mitophagy. CHDH is found on both the outer and inner membranes of mitochondria in resting cells. Interestingly, upon induction of mitophagy, CHDH accumulates on the outer membrane in a mitochondrial potential-dependent manner. I found that CHDH is not a substrate of PARK2 but interacts with SQSTM1 independently of PARK2 to recruit SQSTM1 into depolarized mitochondria. The FB1 domain of CHDH is exposed to the cytosol and is required for the interaction with SQSTM1, and overexpression of the FB1 domain only in cytosol reduces CCCP-induced mitochondrial degradation via competitive interaction with SQSTM1. In addition, CHDH, but not the CHDH FB1 deletion mutant, forms a ternary protein complex with SQSTM1 and MAP1LC3 (LC3), leading to loading of LC3 onto the damaged mitochondria via SQSTM1. Further, CHDH is crucial to the mitophagy induced by MPP+ in SN4741 cells. Overall, my results suggest that CHDH is required for PARK2-mediated mitophagy for the recruitment of SQSTM1 and LC3 onto the mitochondria for cargo recognition.ABSTRACT TABLE OF CONTENTS LIST OF FIGURES 1. INTRODUCTION 2. MATERIALS AND METHODS 2.1. Cell culture and transfection 2.2. Plasmids and siRNA 2.3. Measurement of Mito-GFP intensities 2.4. Measurement of enzyme activity and LC-MS 2.5. Immunoprecipitation, western blot and antibodies 2.6. Immunofluorescence and colocalization coefficient 2.7. Flow cytometry 2.8. Mitochondrial DNA quantification 2.9. Mitochondria fractionation 2.10. Proteinase K degradation assay 3. RESULTS 3.1. CHDH is required for PARK2-mediated mitophagy 3.2. Mitophagic activity of CHDH is independent of enzyme activity 3.3. CHDH accumulates on the outer membrane following mitochondrial damage 3.4. CHDH interacts with SQSTM1 independently of PARK2 during mitophagy 3.5. The interaction of CHDH with SQSTM1 brings LC3 to damaged mitochondria for cargo recognition during mitophagy 3.6. CHDH is implicated in MPP+-induced mitophagy in SN4741 dopaminergic cells 4. DISCUSSION 5. REFFERENCES 국문 초록Docto

Study of Cyclophilin A Function in Models of Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease targeting preferentially motor neurons. Cyclophilin A (CypA) was identified as a hallmark of disease in mutant SOD1 (mSOD1) animal models of familial ALS (fALS) at a presymptomatic stage, and in sporadic (sALS) patients (Massignan 2007; Nardo 2011). Moreover, CypA was enriched in the spinal cord aggregates of mSOD1 mice and sALS patients (Basso 2009). CypA is an ubiquitous protein with multiple functions relevant to the CNS, where it is abundantly expressed. Insights into CypA function in ALS were provided via a proteomic analysis of its interacting proteins, that functionally associated CypA with different proteins networks. In particular, it extensively binds proteins regulating RNA metabolism, including several hnRNPs and TDP-43, a major disease protein in ALS. TDP-43 and CypA interact in the nucleus, in an RNA-dependent way. CypA has a key role in the stabilization of TDP-43/hnRNP A2/B1 interaction, and TDP-43-mediated HDAC6 expression regulation, properties impaired in TDP-43 ALS-mutants, possibly because of a loss-of-interaction with CypA. CypA interacts also with mSOD1, suggesting a gain-of-interaction specifically linked to fALS. Mice expressing mSOD1 and lacking CypA show increased levels of insoluble mSOD1 and hyperphosphorylated TDP-43 in the spinal cord at the onset. This thesis work shows that CypA has a protective role in ALS: as a chaperone (for mSOD1) and in maintenance of multi-protein (TDP-43/hnRNPs) complex stability. Regardless the cause of the disease, mSOD1 or alterations in TDP-43, the interaction with CypA is impaired and it is cosequestered in proteinaceous aggregates, altering its protective activities. The net effect is the formation of pathological inclusions that may lead to a compromised RNA metabolism. CypA being a key interacting partner of both mSOD1 and TDP-43 can represent the "missing link" of these two patho-mechanisms in ALS and an interesting target for therapeutic interventions