Immunoresponsive gene 1 (Irg1) is highly expressed in mammalian macrophages during inflammation, but its biological function has not yet been elucidated. Here, we identify Irg1 as the gene coding for an enzyme producing itaconic acid (also known as methylenesuccinic acid) through the decarboxylation of cis-aconitate, a tricarboxylic acid cycle intermediate. Using a gain-and-loss-of-function approach in both mouse and human immune cells, we found Irg1 expression levels correlating with the amounts of itaconic acid, a metabolite previously proposed to have an antimicrobial effect. We purified IRG1 protein and identified its cis-aconitate decarboxylating activity in an enzymatic assay. Itaconic acid is an organic compound that inhibits isocitrate lyase, the key enzyme of the glyoxylate shunt, a pathway essential for bacterial growth under specific conditions. Here we show that itaconic acid inhibits the growth of bacteria expressing isocitrate lyase, such as Salmonella enterica and Mycobacterium tuberculosis. Furthermore, Irg1 gene silencing in macrophages resulted in significantly decreased intracellular itaconic acid levels as well as significantly reduced antimicrobial activity during bacterial infections. Taken together, our results demonstrate that IRG1 links cellular metabolism with immune defense by catalyzing itaconic acid production.
Parkinsons disease (PD) is a major neurodegenerative chronic disease, most likely caused by a complex interplay of genetic and environmental factors. Information on various aspects of PD pathogenesis is rapidly increasing and needs to be efficiently organized, so that the resulting data is available for exploration and analysis. Here we introduce a computationally tractable, comprehensive molecular interaction map of PD. This map integrates pathways implicated in PD pathogenesis such as synaptic and mitochondrial dysfunction, impaired protein degradation, alpha-synuclein pathobiology and neuroinflammation. We also present bioinformatics tools for the analysis, enrichment and annotation of the map, allowing the research community to open new avenues in PD research. The PD map is accessible at http://minerva.uni.lu/pd_map .
Alpha-synuclein protein is strongly implicated in the pathogenesis Parkinsons disease. Increased expression of ?-synuclein due to genetic multiplication or point mutations leads to early onset disease. While ?-synuclein is known to modulate membrane vesicle dynamics, it is not clear if this activity is involved in the pathogenic process or if measurable physiological effects of ?-synuclein over-expression or mutation exist in vivo. Macrophages and microglia isolated from BAC ?-synuclein transgenic mice, which overexpress ?-synuclein under regulation of its own promoter, express ?-synuclein and exhibit impaired cytokine release and phagocytosis. These processes were affected in vivo as well, both in peritoneal macrophages and microglia in the CNS. Extending these findings to humans, we found similar results with monocytes and fibroblasts isolated from idiopathic or familial Parkinsons disease patients compared to age-matched controls. In summary, this paper provides 1) a new animal model to measure ?-synuclein dysfunction; 2) a cellular system to measure synchronized mobilization of ?-synuclein and its functional interactions; 3) observations regarding a potential role for innate immune cell function in the development and progression of Parkinsons disease and other human synucleinopathies; 4) putative peripheral biomarkers to study and track these processes in human subjects. While altered neuronal function is a primary issue in PD, the widespread consequence of abnormal ?-synuclein expression in other cell types, including immune cells, could play an important role in the neurodegenerative progression of PD and other synucleinopathies. Moreover, increased ?-synuclein and altered phagocytosis may provide a useful biomarker for human PD.
Dementia with Lewy bodies (DLB) and Parkinsons Disease (PD) are common causes of motor and cognitive deficits and are associated with the abnormal accumulation of alpha-synuclein (?-syn). This study investigated whether passive immunization with a novel monoclonal ?-syn antibody (9E4) against the C-terminus (CT) of ?-syn was able to cross into the CNS and ameliorate the deficits associated with ?-syn accumulation. In this study we demonstrate that 9E4 was effective at reducing behavioral deficits in the water maze, moreover, immunization with 9E4 reduced the accumulation of calpain-cleaved ?-syn in axons and synapses and the associated neurodegenerative deficits. In vivo studies demonstrated that 9E4 traffics into the CNS, binds to cells that display ?-syn accumulation and promotes ?-syn clearance via the lysosomal pathway. These results suggest that passive immunization with monoclonal antibodies against the CT of ?-syn may be of therapeutic relevance in patients with PD and DLB.
A consensus about the functions of human wild-type or mutated ?-synuclein (?SYN) is lacking. Both forms of ?SYN are implicated in Parkinsons disease, whereas the wild-type form is implicated in substance abuse. Interactions with other cellular proteins and organelles may meditate its functions. We developed a series of congenic mouse lines containing various allele doses or combinations of the human wild-type ?SYN (hw?SYN) or a doubly mutated (A30P*A53T) ?SYN (hm(2) ?SYN) in a C57Bl/6J line spontaneously deleted in mouse ?SYN (C57BL/6JOla). Both transgenes had a functional role in the nigrostriatal system, demonstrated by significant elevations in striatal catecholamines, metabolites and the enzyme tyrosine hydroxylase compared with null-mice without a transgene. Consequences occurred when the transgenes were expressed at a fraction of the endogenous level. Hemizygous congenic mice did not exhibit any change in the number or size of dopaminergic neurons in the ventral midbrain at 9 months of age. Human ?SYN was predominantly located in neuronal cell bodies, neurites, synapses, and in intraneuronal/intraneuritic aggregates. The hm(2) ?SYN transgene resulted in more aggregates and dystrophic neurites than did the hw5 transgene. The hw?SYN transgene resulted in higher expression of two striatal proteins, synaptogamin 7 and UCHL1, compared with the levels of the hm(2) ?SYN transgene. These observations suggest that mutations in ?SYN may impair specific functional domains, leaving others intact. These lines may be useful for exploring interactions between h?SYN and environmental or genetic risk factors in dopamine-related disorders using a mouse model.
The lipid transport protein apolipoprotein E (apoE) is abundantly expressed in the brain. Its main isoforms in humans are apoE2, apoE3, and apoE4. ApoE4 is the major known genetic risk factor for Alzheimers disease and also contributes to the pathogenesis of various other neurological conditions. In the central nervous system, apoE is synthesized by glial cells and neurons, but it is unclear whether the cellular source affects its biological activities. To address this issue, we induced excitotoxic injury by systemic kainic acid injection in transgenic Apoe knockout mice expressing human apoE isoforms in astrocytes or neurons. Regardless of its cellular source, apoE3 expression protected neuronal synapses and dendrites against the excitotoxicity seen in apoE-deficient mice. Astrocyte-derived apoE4, which has previously been shown to have detrimental effects in vitro, was as excitoprotective as apoE3 in vivo. In contrast, neuronal expression of apoE4 was not protective and resulted in loss of cortical neurons after excitotoxic challenge, indicating that neuronal apoE4 promotes excitotoxic cell death. Thus, an imbalance between astrocytic (excitoprotective) and neuronal (neurotoxic) apoE4 expression may increase susceptibility to diverse neurological diseases involving excitotoxic mechanisms.
Several anti-amyloid ? (A?) antibodies are under evaluation for the treatment of Alzheimers disease (AD). Clinical studies using the N-terminal-directed anti-A? antibody bapineuzumab have demonstrated reduced brain PET-Pittsburg-B signals, suggesting the reduction of A? plaques, and reduced levels of total and phosphorylated tau protein in the CSF of treated AD patients. Preclinical studies using 3D6 (the murine form of bapineuzumab) have demonstrated resolution of A? plaque and vascular burdens, neuritic dystrophy, and preservation of synaptic density in the transgenic APP mouse models. In contrast, few studies have evaluated the direct interaction of this antibody with synaptotoxic soluble A? species. In the current report, we demonstrated that 3D6 binds to soluble, synaptotoxic assemblies of A?(1-42) and prevents multiple downstream functional consequences in rat hippocampal neurons including changes in glutamate AMPA receptor trafficking, AD-type tau phosphorylation, and loss of dendritic spines. In vivo, we further demonstrated that 3D6 prevents synaptic loss and acutely reverses the behavioral deficit in the contextual fear conditioning task in transgenic mouse models of AD, two endpoints thought to be linked to synaptotoxic soluble A? moieties. Importantly C-terminal anti-A? antibodies were ineffective on these endpoints. These results, taken with prior studies, suggest that N-terminal anti-A? antibodies effectively interact with both soluble and insoluble forms of A? and therefore appear particularly well suited for testing the A? hypothesis of AD.
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