C3 MICROGLIA - T CELL - MOTONEURON DIALOGUES INFLUENCE DISEASE PROGRESSION
APPEL SH
Methodist Neurological Institute, Houston, TX, USA
Email address for correspondence: [email protected]
Keywords: neuroinflammation, microglia, T cells
Neuroinflammation is characterized by activated microglia and infiltrating T cells, and is a prominent pathological feature of ALS. Experimental models suggest that activated microglia of the innate immune system and CD4 + T cells of the adaptive immune system contribute significantly to disease progression. In early stages of disease in mSOD1 mice the first response to motor neuron injury appears to be mediated by neuroprotective microglia, while in later stages of disease, this neuroprotective response is transformed into a cytotoxic response. In mSOD1 mice lacking functional CD4+T cells, disease progression is accelerated, suggesting a neuroprotective role for T cells as well as microglia. In mSOD1 mice with functional CD4 + T cells, CD4 + CD25 + FoxP3+ T cells (Tregs) are increased during the early slowly progressing phase of disease; as the rate of disease progression accelerates, these numbers rapidly diminish. During the rapid phase, the microglia transform into a toxic M1 phenotype with increased mRNA levels of NOX2 and IL-1β, marked decrease of Tregs, and increased numbers of infiltrating effector T cells (Teffs). The passive transfer of wild-type CD4 + T lymphocytes into ALS mice lacking functional T lymphocytes lengthened disease duration and prolonged survival (1). The passive transfer of endogenous regulatory T lymphocytes from early disease stage mSOD mice was substantially more immunotherapeutic, sustaining interleukin-4 levels, increasing Ym1 and CX3CR1 neuroprotective microglia, and further lengthening disease duration and prolonging survival. The stable disease phase was extended by 88% using mutant SOD Treg. Thus, in the experimental model the dialogue among microglia, T cells and motor neurons suggests that the immune response is not merely a consequence of injury, but actively influences and significantly contributes to the balance between neuroprotection and neurotoxicity. It is clear that neurodegeneration is non-cell autonomous, with the innate and adaptive immune systems contributing to neuronal viability and disease progression.
When these observations were extended to ALS patients individuals with more rapidly progressing disease had decreased numbers of regulatory T lymphocytes, and the numbers of regulatory T lymphocytes were inversely correlated with disease progression rates. These data suggest that immunotherapeutic interventions must begin early in the pathogenic process since immune dysfunction occurs at later stages. The cumulative mouse and human ALS data suggest that increasing the levels of regulatory T lymphocytes in patients with amyotrophic lateral sclerosis at early stages in the disease process may be of therapeutic value. A greater understanding of what dictates the presence of cytotoxic or neuroprotective immunomodulation and how to limit cytotoxicity and enhance neuroprotection would help identify additional targets for immune-based therapy in ALS.
Reference
- Beers, Henkel, Zhao, et al. Brain, 2011;134(Pt 5):1293–314.
C4 ELIMINATION OF INNATE IMMUNE SYSTEM ADAPTOR TRIF SIGNIFICANTLY ACCELERATES DISEASE PROGRESSION OF ALS MICE
YAMANAKA K1, FUJIMORI TN1, YAMASHITA H1,2
1RIKEN Brain Science Institute, Wako, Saitama, Japan, 2Kyoto University, Kyoto, Japan
Email address for correspondence: [email protected]
Keywords: SOD1, innate immunity, glia
Background: Prior work from us and others indicated that mutant toxicities within microglia and astrocytes accelerated the disease progression of mutant SOD1 mice. Although the recent work demonstrated that acquired immune system is involved in the disease process, the role of innate immune system in motor neuron disease was not fully investigated. Toll-like receptors (TLRs) play a key role in innate immune responses requiring MyD88 (myeloid differentiation factor 88) and TRIF (TIR domain-containing adaptor inducing interferon-β) as essential adaptor proteins for signaling.
Objectives: To determine whether the innate immune system is involved in the disease process of amyotrophic lateral sclerosis.
Methods: The gene expression profiles of lumbar spinal cord from symptomatic mutant SOD1 mice were obtained by microarray approach and subsequently analyzed using cell-type specific transcriptome. The mating experiment was carried out by crossing SOD1G93A, MyD88-/-, and TRIF-/- mice. The disease onset, duration, and survival of these cohorts were monitored. The mRNA expression levels of cytokines, chemokines, and neurotrophic factors in the lumbar spinal cords from symptomatic stage were determined by quantitative reverse transcription PCR.
Results: Gene expression profile indicated approximately 70% of upregulated genes were derived from microglia and the pathway analysis indicated the involvement of the innate immune pathway. An accelerated disease progression with shorter survival time was seen only in SOD1G93A/TRIF-/- mice (Mean survival time: SOD1G93A/TRIF-/-: 138 days, SOD1G93A: 162 days, and 50% reduction in disease duration). The duration of disease and survival time of SOD1G93A did not change when MyD88 was eliminated. The expression levels of several chemokines were significantly suppressed in SOD1G93A/TRIF-/- mice as compared with SOD1G93A mice.
Discussion and conclusions: Although MyD88 is responsible for most TLR-signaling except for TLR-3, MyD88 has a marginal effect on disease course in ALS models. To our surprise, the basal activity of innate immune response through TRIF, which resides downstream of TLR-3 and 4, plays an important role to control disease progression with regulation of several chemokines. Further identification of the misregulated molecules to explain the accelerated disease progression in mutant SOD1G93A/TRIF-/- mice is underway.
C5 AGGREGATED WILD TYPE SOD-1 INCREASES SERUM LEVELS OF CYTOKINES IN PATIENTS WITH ALS
WIEDAU-PAZOS M, CHATTOPADHAY M, FIALA M
UCLA, Los Angeles, CA, USA
Email address for correspondence: [email protected]
Keywords: inflammation, SOD-1, disease mechanism
Background: Copper,zinc superoxide dismutase (SOD-1) protein aggregates in neuronal inclusions linked to ALS have toxic properties. They can occur selectively in spinal cord motor neurons in familial and sporadic ALS.
Objectives: We tested the hypothesis that misfolded SOD1 contributes to motor neuron degeneration via inflammatory mechanisms.
Methods: We examined peripheral blood mononuclear cells (PBMC) of patients with sporadic ALS to identify baseline levels of cytokine expression. Next, we studied the cytokine expression in PBMC after they were treated with soluble and aggregated forms of wild type SOD-1. We confirmed the results obtained in PBMC of live patients by studying the infiltration of immune cells in ALS post-mortem spinal cords.
Results: We identified increased levels of several inflammatory cytokines in PBMC of subjects with sporadic ALS, including IL17A, which has previously been linked to Multiple Sclerosis and other immune-modulated disorders, but not ALS. When PBMC were exposed to aggregated forms of wild type SOD-1, the cytokines IL-1β, interleukin-6 (IL-6), and interleukin-23 (IL-23), which induce IL-17A, were elevated. In contrast, the anti-inflammatory cytokine IL-10 was decreased in ALS PBMC. We detected similar results in the anterior horns of ALS subjects, which were infiltrated by macrophages positive for IL-1β and tumor necrosis factor-α macrophages, IL-17A-positive CD8 cells, and IL-17A-positive mast cells. Inflammatory cytokines were induced in ALS macrophages by aggregated SOD-1 through eicosanoid and caspase-1 pathways. In a small subset of patients, the IL-17A levels in PBMC were normalized after treatment with lipid mediators.
Discussion and conclusion: We provide evidence for an inflammatory disease mechanism in ALS. Aggregated wild type SOD-1 protein in ALS neurons could trigger the expression of inflammatory cytokines in invading mononuclear cells, which contribute to the disease progression. Immune-mediating compounds may be of interest in the investigation of novel ALS therapeutics.
C6 ABNORMAL AXOGLIAL COMMUNICATION IN THE CORTICOSPINAL TRACTS COULD UNDERLIE UPPER MOTOR NEURON DEGENERATION IN SPORADIC ALS
SONG F1, CHIANG P1, RAVITS J2, LOEB J1
1Hiller ALS Clinic and Research Center, Department of Neurology, Center for Molecular Medicine and Genetics, Wayne State University, Detroit, MI, USA, 2Translational Research Program, Benaroya Research Institute, Seattle, WA, USA
Email address for correspondence: [email protected]
Keywords: corticospinal tract degeneration, demyelination, neuregulin
Background: Sporadic amyotrophic lateral sclerosis (ALS) involves both upper and lower motor systems. Recent observations demonstrate contiguous lower motor neuron disease progression through the spinal column that correlates well with upper motor neuron signs (1). How this progression links the upper with the lower motor system is not known, but could be linked by degeneration within the lateral corticospinal tract (LCST).
Objectives: Here, we have examined the degree of demyelination, axonal loss, microglial activation within the corticospinal tracts of patients with sporadic ALS and linked these to aberrant expression of specific forms of the gliotrophic factor neuregulin1 (NRG1).
Methods: Spinal cords from patients with significant corticospinal tract degeneration and concurrent upper motor neuron signs were compared to patients without significant degeneration as well as controls. The degree of demyelination, axon loss, gliosis and microglial activation were measured. Gene expression (by qPCR) and protein localization of NRG1 isoforms were determined.
Results: Patients who have significant degeneration of the LCST had more pronounced upper motor neuron signs clinically. These patients showed marked demyelination and axonal loss both within the LCST and the anterior corticospinal tract (ACST), raising questions as to whether axonal loss follows an initial demyelinating event. Both corticospinal tracts showed an increase in NRG1 protein, however, with a paradoxical decrease in both type I and III NRG1 mRNA expression. These same regions showed both increased microglial activation as well as sustained NRG1 receptor activation.
Conclusions: Corticospinal tract degeneration in sporadic ALS is associated with aberrant NRG1 expression and signaling, and microglial activation. The observation that NRG1 protein is increased in the absence of increased NRG1 mRNA expression, suggests that enhanced NRG1 signaling comes from descending central motor axons.
Discussion: Activation of microglia by NRG1 from central motor axons could explain the upper motor neuron signs seen in patients with sporadic ALS and may also be a therapeutic target to block disease progression.
Reference
- Ravits J, Laurie P, Fan Y, Moore DH. Neurology 2007; 68:1576–82.
C7 NEUROPATHOLOGICAL FINDINGS OF SIX SCANDINAVIAN PATIENTS HOMOZYGOUS FOR THE D90A SOD1 MUTATION
FORSBERG K, GRAFFMO KS, ANDERSEN PM, MARKLUND SL, BRÄNNSTRÖM T
Umea University, Umea, Sweden
Email address for correspondence: [email protected]
Keywords: SOD1, D90A, frontal lobe dementia
Background: The most common cause of familial amyotrophic lateral sclerosis (ALS) is mutations in superoxide dismutase-1 (SOD1). Currently more than 150 mutations in the SOD1 gene are known and one of the most frequent mutations is the change from aspartate to alanine in position 90 of the gene (D90A). This mutation is unique since it exists in both an autosomal dominant and a recessive form. In the Scandinavian pedigrees, it is inherited in a recessive manner and gives rise to a unique phenotype characterized by a predictive clinical pattern and long disease progression (1). So far, little is known about the neuropathological features of this specific mutation.
Objectives: To characterize and systematically investigate morphological findings in six autopsies of ALS patients homozygous for the D90A mutation in the SOD1 gene.
Methods: Brain and spinal cord sections from six autopsies were investigated by immunohistochemistry. Four different anti-peptide antibodies with specificity for misfolded/disordered SOD1 were used as well as antibodies directed at GFAP, ubiquitin, TDP-43, NF and cystatin C.
Results: All six patients showed a profound loss of motoneurons in the spinal cord and brainstem. In the remaining motonerons, numerous aggregates immunoreactive of misfolded SOD1 were found in the cytoplasm and occasionally also in the nucleus. Glial cells showed intranuclear reactivity for SOD1 in all patients but varied in number of glial cells involved.
Degeneration of the corticospinal tract and dorsal column was seen, as well as gliosis in the cortical areas of the frontal and temporal lobes and in the insula. Interestingly, these areas all showed microvacuolar degeneration of the superficial lamina, especially in layer II and III.
Discussion: Positron emission tomography (PET) studies on D90A patients using [11C]flumazenil binding has revealed changes both in motor areas as well as in non-motor areas such as the left fronto-temporal and anterior cingulated cortices (2). Our present finding of pathological microvacuolar degeneration in the superficial layers of temporal and frontal cortices supports this notion.
Biochemcally, there are no significant differences in SOD1 activity between the D90A enzyme and the wild-type SOD1 enzyme (3). The aggregates of misfolded SOD1 that we find in the cytoplasm of motoneurons in D90A patients are morphologically the same type that can be found in the sporadic form of the disease.
Conclusion: Pathological changes in patients with the D90A SOD 1 mutation can be detected not only in motor areas of the central nervous system but also in different non-motor cortical areas. These findings indicate a possible frontotemporal lobar dementia in addition to motor neuron disease in these patients.
References
- Andersen PM, Forsgren L, Binzer M, et al. Brain. 1996 Aug;119 (Pt 4):1153–72.
- Turner MR, Hammers A, Al-Chalabi A, et al. Brain 2005 Jun;128(Pt 6):1323–9.
- Jonsson PA, Graffmo KS, Brännström T, et al. J Neuropathol Exp Neurol 2006 Dec;65(12):1126–36.
C8 PROMINENT RE-DISTRIBUTION OF TDP-43 AND FUS/TLS UNDER CONDITIONS OF CELLULAR INSULT IN PRIMARY NEURONS
KANNINEN K, JAMES J, PARKER S, CROUCH P, WHITE A
University of Melbourne, Department of Pathology, Victoria, Australia
Email address for correspondence: [email protected]
Keywords: TDP-43, FUS, stress granule
Background: TDP-43 and FUS/TLS are nucleic acid binding proteins found to be mutated in sporadic and familial forms of ALS. Both proteins are implicated in multiple aspects of nucleic acid metabolism, including RNA splicing and transcription. In response to cellular stress, TDP-43 and FUS/TLS can relocate from the nucleus to the cytoplasm and accumulate in stress granules (SGs), which are ribonucleoprotein complexes where protein synthesis is temporarily arrested. It is not yet fully clear how TDP-43 and FUS/TLS proteins are redistributed from the nucleus to the cytoplasm under various cellular stresses and what is the significance of altered sub-cellular localization of these proteins in primary neurons.
Objectives: To investigate sub-cellular localization and accumulation of endogenous TDP-43 and FUS/TLS in primary spinal cord and cortical neurons exposed to oxidative and mitochondrial stress.
Methods: Primary neuronal cultures were harvested from mouse spinal cord and cerebral cortex. Neuronal cultures were treated with mitochondrial inhibitor; paraquat and oxidative stress inducer; sodium arsenite. Neuronal viability following treatments was assessed by MTT and LDH release assays. TDP-43, FUS/TLS and the SG marker TIAR were visualized by immunofluorescent staining.
Results: Neuronal viability is compromised in a time and concentration dependent manner in response to oxidative and mitochondrial stress. When treated at sub-lethal doses used to mimic stress conditions in vivo, TDP-43 and FUS/TLS re-distribute from the nucleus to the cytoplasm. In particular, treating spinal cord neurons with sodium arsenite causes changes to the sub-cellular distribution of both TDP-43 and FUS/TLS. In addition to formation of cytosolic aggregates resembling SGs, punctate, condensed staining of TDP-43 also appears in the nucleus of sodium arsenite treated spinal cord neurons.
Discussion and conclusions: Oxidative and mitochondrial stress affect viability and induce changes to TDP-43 and TLS/FUS sub-cellular localization in primary neurons. The cytosolic accumulation of TDP-43 and FUS/TLS induced by cellular stress demonstrates that oxidative or mitochondrial stress in vivo may induce aggregation of these proteins as a precursor to neuronal degeneration. We are now investigating the processes involved in cytosolic accumulation of the proteins. To our knowledge this study is the first to have reported aggregation of endogenous TDP-43 in the nucleus of spinal cord neurons exposed to an oxidative stress-inducing agent, sodium arsenite. Further studies will be required to delineate the nature of the nuclear aggregates of TDP-43 and assess their significance in the pathology of ALS.
C9 DEVELOPING A TRANSGENIC ZEBRAFISH MODEL FOR TARDBP MUTATIONS IN ALS
LISSOUBA A, KABASHI E, CHAMPAGNE N, MAIOS C, BRUSTEIN E, DRAPEAU P
Universite de Montreal, Montreal, Quebec, Canada
Email address for correspondence: alexandra.lissouba@umon treal.ca
Keywords: zebrafish, tardbp, development
Background: Mutations of the gene TARDBP, coding for the RNA binding protein TDP-43, have been identified in ALS patients, but the molecular mechanisms that cause the disease still remain uncovered. To assess the biopathology and develop therapeutic approaches of ALS, we use the zebrafish as a genetic model. We have previously functionally characterized a number of ALS-related TARDBP mutations by transient transgenesis in zebrafish for motor deficits (1).
Objectives: To develop a stable transgenic line overexpressing either mutant or WT TARDBP gene in zebrafish embryos and assess how this could cause or contribute to motoneuron degeneration linked to ALS. These lines will be useful for both understanding molecular mechanisms of disease and identification of modifiers of TARDBP functions as well as to screen small molecules that could eventually be used for the development of new ALS therapeutics.
Methods: We have generated two stable, inducible transgenic zebrafish lines expressing either the wild-type human TARDBP gene or the ALS-related mutation G348C. Our genes of interest were myc-tagged and placed under the control of the heat-shock inducible Hsp70 promoter to control the time of the expression and its level.
Results: In the absence of heat-shock, no myc-tagged protein expression was detectable. Heat-shocking embryos at 19 hours post-fertilization at 38.5°C for 30 min was sufficient to induce ubiquitous expression of TDP-43 in both lines, as assayed by RT-PCR, anti-myc immunoblotting and immunolabelling. Western blot analysis revealed an appropriate 50kD band for the myc-tagged protein in both lines and in addition revealed lower molecular weight bands only in the mutant line, presumably due to cleavage or degradation. As shown previously by immunolabelling in embryos transiently expressing TDP-43(1), stably induced mutant embryos showed significantly shorter and hyperbranched motor neuron axons compared to the control embryos. Moreover, the mutant embryos responded to touch stimuli but unlike WT embryos, the majority was unable to swim away, displaying weak and uncoordinated muscular contractions. Other neural cell types have been examined, and molecular partners of TDP-43 have been determined. In a preliminary screen of compounds used to treat neurological disorders, we have tested these for their effectiveness in treating the motor behavior and have identified several compounds which partially rescue the mutant phenotype.
Discussion and conclusions: These results indicate that zebrafish expressing a human TARDBP mutation related to ALS is a promising model for understanding ALS biopathology and advancing therapeutic drug discovery. The perturbation of motor neuron outgrowth may be an early indicator of mutant TDP-induced morphological anomalies causing functional disruption. These transgenic lines will be used for an in depth in vivo proteomic and genomic analysis and for a larger screen of small compounds.
Reference
- E. Kabashi, et al. Hum Mol Genet 2010;19:671.