P80 NEURODEGENERATION-ASSOCIATED TDP-43 INTERACTS WITH FMRP/STAU1 AND REGULATES SIRT1 EXPRESSION IN NEURONAL CELLS
Z Yu1
D Fan1
B Gui1
L Shi2
C Xuan2
L Shan2
Q Wang2
Y Shang2
Y Wang2
aPeking University Third Hospital, Beijing, China
bTianjin Key Laboratory of Medical Epigenetics, Department of Biochemistry and Molecular Biology, Tianjin Medical University, Tianjin, China
Email address for correspondence: [email protected]
Keywords: TDP-43, FMRP, STAU1, SIRT1
Background and objective: Despite the identification of the 43kDa transactive response DNA-binding protein (TDP-43) as a major pathological signatory protein in a wide range of neurodegenerative diseases, the mechanistic role of TDP-43 in neurodegenerative disorders is still poorly understood.
Methods and results: We report that TDP-43 is physically associated with fragile X mental retardation protein (FMRP) and Staufen (STAU1) to form a functional complex. Differential microarray analysis revealed that the expression of a collection of functionally important genes including Sirtuin (SIRT1) is regulated by this complex. RNA-immunoprecipitation (RIP) assays demonstrated that TDP-43/FMRP/STAU1 specifically bind to the 3’UTR of SIRT1 mRNA, leading to the degradation of SIRT1 mRNA. SIRT1 is implicated in double-stranded DNA break repair and is required for cell survival. Indeed, depletion of TDP-43/FMRP/STAU1 sensitizes cells to apoptosis and DNA damages.
Conclusions: Our experiments revealed that TDP-43 is physically associated with FMRP and STAU1 to form a functional coordinated complex to co-regulate the expression of SIRT1. Our results indicate that defects in the TDP-43/FMRP/STUA1 complex sensitize neuronal cells to apoptosis and DNA damages in a SIRT1-dependent manner. These findings may shed new light on the understanding of the biological functions and the mechanistic involvement of TDP-43 in neurodegenerative diseases.
P81 INTRINSIC REGULATION OF FUS SUBCELLULAR LOCALIZATION AND AGGREGATION BY DIFFERENT SEQUENCE FEATURES
J Zhang
L Yang
J Gal
E Kasarskis
H Zhu
University of Kentucky, Lexington, KY, USA
Email address for correspondence: [email protected]
Keywords: FUS, subcellular localization, protein aggregation
Background: Mutations of DNA/RNA binding protein fused in sarcoma/translated in liposarcoma (FUS/TLS) have been recently reported to be responsible for the familial ALS type 6. In neurons, FUS is predominantly a nuclear protein and can shuttle between the cytoplasm and nucleus. Mutated FUS is found to accumulate in the cytoplasm and forms cytoplasmic inclusion in postmortem ALS patient brains. Most of the mutation in FUS is clustered at the C-terminus. The very C-terminus of FUS has been identified to be an effective nuclear localization sequence (PY-NLS), and ALS related C-terminal mutation will compromise the function of nuclear localisation signal (NLS). However, the sequence features determining the subcellular localization of FUS and their implication in the aggregation are not fully understood.
Objectives: To study the sequences involved in the import or export of FUS and their relevance to the aggregation.
Methods: Different mutations or truncations of FUS were made and expressed in neuroblastoma 2A in the presence or absence of export 1 specific inhibitor LMB, and confocal images were took to observe their localization. Mouse primary motor neurons and skin fibroblast cells derived from the familial ALS patients carrying FUS mutations were used in the study to validate the findings from the N2A cells.
Results: We found sequence 301VADYFKQIGI310 is a functional nuclear export signal (NES). In addition to the C-terminal NLS, the RRM domain in the C-terminus may play a functional role of nuclear retention, and N-terminus can also direct the nuclear import of FUS through an unknown mechanism. FUS only forms aggregates in the nucleus, and C-terminus of FUS including RRM domain is critic but not sufficient for the aggregation of FUS.
Discussion and conclusions: The import and export of FUS is regulated by multiple domains. Cytoplasmic localization is critic for the aggregation of FUS. Our data suggest mutated FUS can still shuttle between nucleus and cytoplasm. Further study might identify the underlying molecules driving the shuttling of FUS and develop therapeutic strategy.
P82 ARGININE METHYLATION REGULATES FUS CHROMATIN-ASSOCIATION AND SOLUBILITY
L Yang
J Zhang
J Gal
E Kasarskis
H Zhu
University of Kentucky, Lexington, KY, USA
Email address for correspondence: [email protected]
Keywords: FUS, arginine methylation, protein solubility
Background: Mutations on RNA binding protein FUS account for 4–5% of familial ALS. How FUS mutants cause motor neuron degeneration is unknown. FUS toxicity is closely related to its intracellular localization and extracellular solubility.
Objectives: To understand FUS proteinopathy by studying the regulation of its localization and solubility.
Methods: We transfected HEK 293T cells with plasmid expressing GST-tagged FUS or FUS mutants and did cell fractionation assay to detect the intracellular localization of FUS and FUS mutants. We transfected HEK 293T cells with plasmid expressing GST-tagged FUS and treated the cells with methyltransferase inhibitor to investigate the effect of arginine methylation on FUS localization and solubility. We cotransfected HEK 293T cells with plasmid expressing GST-tagged FUS and plasmid expressing EGFP-tagged TDRD3 (Tudor domain-containing protein 3) to study FUS-TDRD3 interaction and localization.
Results: FUS is predominantly nuclear-located and presents in two fractions: nuclear soluble and chromatin associated. We found ALS-related mutations dissociate FUS from chromatin. FUS with low arginine methylation level tends to accumulate in the chromatin fraction, suggesting arginine methylation facilitates FUS chromatin dissociation. Methylated arginine is a binding substrate of protein tudor domain. In our study, we found FUS interact with TDRD3 and this interaction was weakened by methyltransferase inhibitor treatment. Overexpression of TDRD3 promoted FUS chromatin dissociation and FUS mutants cytoplasmic accumulation. Moreover, we found ALS-related mutations and low arginine methylation level reduces FUS solubility in radioimmunoprecipitation assay (RIPA) buffer.
Discussion and conclusions: Our findings suggest that arginine methylation is an important post-translational modification in the regulation of FUS localization and solubility. Manipulation of FUS arginine methylation level can be applied to reduce FUS toxicity.
P83 HISTONE DEACETYLASE 6 REGULATES FAMILIAL ALS MUTANT SOD1 TURNOVER
J Gal
J Chen
L Yang
R Etherton
W-S Fu
E Brumley
T Frailie
H Zhu
University of Kentucky, Lexington, KY, USA
Email address for correspondence: [email protected]
Keywords: SOD1, HDAC6, autophagy
About 20% of the familial ALS cases are caused by mutations in the Cu/Zn superoxide dismutase (SOD1). Protein inclusions in motor neurons and glial cells are a hallmark of ALS and the ALS mutants of SOD1 are prone to aggregation. HDAC6 is a lysine deacetylase that is almost exclusively localized in the cytoplasm. HDAC6 was implicated in the regulation of protein aggregation and autophagy. The role of HDAC6 in ALS has not been investigated in detail.
We found that the knockdown of HDAC6 resulted in the accumulation of highly aggregated mutant SOD1. HDAC6 selectively interacted with mutant, but not with wild- type SOD1. The ubiquitin-binding Zn-finger and the lysine deacetylase activity of HDAC6 were not necessary for the HDAC6-mutant SOD1 interaction. Deletion of the SE14 repeat domain of HDAC6 impaired the interaction with mutant SOD1. However, the isolated SE14 domain did not interact with mutant SOD1. Instead, we found that a motif very similar to the SOD1 Mutant Interaction Region (SMIR) we described in p62/Sequestosome 1 is present near the SE14 domain in HDAC6 and the SMIR mediates the interaction with mutant SOD1. We also identified a second, less conserved SMIR in the first deacetylase domain of HDAC6. We also found that p62 and HDAC6 interacted with each other and this interaction was at least in part mediated by the SMIR motifs of p62 and HDAC6.
Our results suggest that beyond their functional interaction in the regulation of protein aggregation and turnover, HDAC6 and p62 can also participate in common biochemical complexes and share a mechanism to recognize mutant SOD1 independent of their ubiquitin-binding domains. We also investigated the modulation of the tubulin deacetylase activity of HDAC6 by mutant SOD1 and the possible role of tubulin acetylation in mutant SOD1 turnover.
P84 ALTERATED EXPRESSION OF SOD1 AND TDP-43 IN PERIPHERAL BLOOD MONONUCLEAR CELLS FROM SPORADIC ALS PATIENTS
E Leoni1,2
P Orietta1
M Pamela1,2
S Valentina1,2
E Diletta1
G Stella1
C Mauro2,3
C Cristina1
cLaboratory of Experimental Neurobiology, “C. Mondino” National Institute of Neurology Foundation, IRCCS, Pavia, Italy
dDepartment of Public Health, Neuroscience, Experimental and Forensic Medicine, University of Pavia, Pavia, Italy
eDepartment of General Neurology, “C. Mondino” National Institute of Neurology Foundation, IRCCS, Pavia, Italy
Email address for correspondence: [email protected]
Keywords: SOD1, TDP-43, peripheral blood mononuclear cells
Background: Several lines of evidence supported the hypothesis of a toxic role played by wild type (WT-SOD1) in the pathogenesis of SALS (Citation1–2). Nevertheless, TDP-43 has been identified as the major pathological protein due to its mislocalisation in the cytoplasm (Citation3). As recent studies (Citation4,Citation5) support the presence of a relationship between these two proteins here we aim to investigate SOD1 and TDP-43 expression in peripheral blood mononuclear cells (PBMC) from SALS patients.
Objectives: Evaluate SOD1and TDP-43 expression and localization in lymphocytes and monocytes from SALS patients.
Methods: PBMCs were obtained from 20 SALS patients and 20 healthy controls (CTR). Nuclear and cytoplasmic fractions were obtained as previously described by (Citation6). Insoluble pellets were resuspended in lysis buffer as reported by (Citation7). SOD1 and TDP-43 expression were evaluated by western blotting. SOD1 localization was evaluated by immunofluorescence technique followed by confocal microscopy observation.
Results: Western blot analysis showed an overall increased nuclear SOD1 expression in SALS patients. The splitting of patients with higher nuclear SOD1 expression from those with similar to controls’ levels highlighted the differences between the patients’ clustering. No differences were found in the cytosolic compartment. Analysis of insoluble lysates evidenced an increase in SOD1 expression in a group of ALS cases suggesting that a reduction in SOD1 expression in some patients was due to the protein precipitation in the insoluble fraction. A further indirect confirmation of these finding was obtained by immunofluorescence and confocal analysis. The presence of small or large foci of aggregated SOD1 protein was regularly detected in the cytoplasm of patients with nuclear SOD1 levels similar to controls. Patients with high nuclear SOD1 levels showed smaller aggregates in the same compartment. Regarding TDP-43, we observed higher nuclear levels compared to cytoplasm in both SALS patients and control, confirming its distribution predominantly in the nuclear compartment. Interestingly, 6 patients with high levels of nuclear SOD1 showed also high level of cytoplasmic TDP-43.
Conclusions: These preliminary data suggest us the involvement of an altered WT-SOD1 and TDP-43 localization in sporadic cases of ALS highlighting new evidences about pathways which may have implication in the disease pathogenesis.
References
- Bosco DA, Morfini G, Karabacak NM . Nat Neurosci 2010;13:1396–403.
- Ezzi SA, Urushitani M and Julien JP. J Neurochem 2007;102:170–8.
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- Somalinga BR, Day CE, Wei S, . PLoS ONE 2012;7:e35818.
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- Vigilanza P, Aquilano K, Rotilio G, Ciriolo MR. Cell Mol Life Sci 2008;65:991–1004.
P85 GENE EXPRESSION PROFILING OF LYMPHOBLASTOID CELLS FROM AMYOTROPHIC LATERAL SCLEROSIS PATIENTS WITH AND WITHOUT C9ORF72 EXPANSIONS AND CONTROLS
J Cooper-Knock
J Kirby
M Rattray
PR Heath
P Shaw
Sheffield Institute for Translational Neuroscience (SITraN), Sheffield, UK
Email address for correspondence: [email protected]
Keywords: transcriptomics, C9ORF72, RNA processing
Introduction: Intronic hexanucleotide repeat expansions of C9ORF72 are found in approximately 10% of patients with amyotrophic lateral sclerosis (ALS) and represent the most common genetic variant identified to date. The function and dysfunction of C9ORF72 is poorly understood, but it has been proposed that the expansion may mediate pathogenesis via a sequestration of RNA splicing factors.
Metholodology: Gene expression profiling (GEP) was carried out on RNA extracted from lymphoblastoid cells from ALS patients with the C9ORF72 expansion (n = 10), ALS patients without the expansion (n = 16) and neurologically normal controls (n = 10) using Affymetrix HG-U133 Plus 2.0 GeneChips. Data were analysed with the Propagating Uncertainty in Microarray Analysis (PUMA) suite of tools. Aberrantly affected pathways were identified using the Database for Annotation, Visualization and Integrated Discovery (DAVID). Differential expression of certain genes was validated by QRT-PCR. A multiplexed fluorescent bead-based immunoassay of cerebrospinal fluid (CSF) from C9ORF72-ALS (n = 4), non-C9ORF72 ALS (n = 5) and controls (n = 5) was used to confirm changes in gene expression at the protein level.
Results: 319 probe sets were differentially expressed identified between C9ORF72-ALS cases and controls. Significantly enriched pathways included ‘RNA splicing’ and ‘chromatin modification’ (p < 0.01). Consistent with earlier studies, C9ORF72 itself was down-regulated. Successful validation of gene expression changes was performed by QRT-PCR. The top differentially expressed gene, as determined by fold change, between C9ORF72-ALS and controls was down-regulation of a neuroprotective cytokine, which was also the second highest differentially expressed gene between C9ORF72-ALS and non-C9ORF72 ALS. A lower level of this cytokine in C9ORF72-ALS compared to non-C9ORF72 ALS (p < 0.05) and controls was confirmed in CSF.
Discussion: Identified down-regulation of splicing factors is consistent with sequestration which may mediate a broader disruption of RNA splicing. As other splicing associated genes were up-regulated, it is hypothesised that this represents compensation and a potential therapeutic target. A similar up-regulation of these genes has been discovered in frontotemporal lobar degeneration with TDP-43-positive inclusions (FTLD-TDP). Certain results were comparable to gene expression in myotonic dystrophy 1 (DM1), another neuromuscular disease mediated by an intronic expansion. The extent to which disease mechanisms in C9ORF72- ALS and DM1 are similar remains to be determined. The identification of aberrant down-regulation of a neuroprotective cytokine in C9ORF72-ALS compared to non-C9ORF72 ALS and controls suggests that pathways of inflammation are important in the pathogenesis of C9ORF72-ALS. This may represent a novel therapeutic target.
P86 ANALYSIS OF THE C9ORF72 HEXANUCLEOTIDE REPEATS SIZE IN ALS PATIENT DERIVED FIBROBLASTS, IPS CELLS AND POSTMORTEM BRAIN TISSUES
P-W Zhang1
C Donnelly1
R Sattler1,2
U Balasubramanian1
L Ostrow1
J Rothstein1,2
fDepartment of Neurology, Johns Hopkins University School of Medicine, Baltimore, MD, USA
gBrain Science Institute, Baltimore, MD, USA
Email address for correspondence: [email protected]
Keywords: hexanucleotide repeat, C9ORF72, southern blotting
Background: Approximately 10–15% of ALS cases are familiar. Mutations in SOD1 encoding the cooper/zinc superoxide dismutase, TARDBP (TAR DNA-binding protein 43) and FUS (fused in sarcoma) are responsible for 20%–30% of familial ALS disease. Rare mutations in other genes like ANG, VAPB, DAO, OPTN, VCP and UBQLN2 have been reported. Recent studies have identified expanded GGGGCC hexanucleotide repeats in the C9ORF72 gene in ALS, frontotemporal dementia (FTD) and ALS-FTD. This novel mutation is currently the most common genetic cause of familial ALS (23%–47%) as well as sporadic ALS (4%–21%) in the USA, Belgium and Finland. Characterization of the role of repeat length and tissue distribution effects of variation in repeat expansion size in the C9ORF72 gene is suggested to be crucial for the understanding of the mechanisms and pathways of ALS pathology.
Objectives: ALS patient derived fibroblasts(3), iPS cells(3) and postmortem brain tissues(6).
Methods: The size and number of repeats were determined by optimized Southern Blotting approach.
Results: All the expanded repeats are over 3 kb long and one of them was found to contain over 1000 repeats. Interestingly, the size and number of repeats in the iPS cell lines were different from the fibroblasts derived from the same patients. Repeat length in our control group close to the findings of previous reports. We also examined the dynamic changes in the repeat size as cell lines underwent increasing passages. Finally we analyzed repeat length in multiple different brain region from more than six C9ORF72 autopsies.
Discussion and conclusions: Southern Blotting is the only approach to determine GGGGCC hexanucleotide repeats in the C9ORF72 gene once the repeat number is over 100 since it is 100% GC rich region. The size and number of repeats in the iPS cell lines were different from the fibroblasts derived from the same patients based on our result. Here we report data on the repeat size in the gene in different cell types and tissue samples obtained from ALS patients. Using an optimized Southern Blotting approach, the size and number of repeats were determined in fibroblasts and iPS cell lines derived from ALS patients with C9ORF72 repeat mutations.
P87 MICROARRAY ANALYSIS IN SPINAL CORDS OF SPORADIC ALS PATIENTS WITH CELL-TYPE SPECIFIC TRANSCRIPTOME
H Yamashita1,2
N Fujimori2
H Ito1
Y Iguchi3
N Atsuta3
F Tanaka3
G Sobue3
R Takahashi1
K Yamanaka2
hKyoto university, Kyoto, Kyoto, Japan
iRIKEN, Wako, Saitama, Japan
jNagoya university, Nagoya, Aichi, Japan
Email address for correspondence: [email protected]
Keywords: microarray, non-cell autonomous neuronal death, glia
Objective: With DNA microarray, we analyzed the molecular pathomechanism in sporadic ALS spinal cords with a focus on the function of microglia and astrocytes. We analyzed the microarray data in a cell type specific manner to understand the molecular mechanisms within each cell type of ALS spinal cord.
Background: Glial cells including astrocytes and microglia are reported to be actively involved in motor neuron death in ALS, but the precise mechanisms for the ‘non-cell autonomous neuron death’ have not been elucidated.
Design and methods: We profiled using DNA microarray the mRNA expression with cervical spinal cords of 4 sporadic ALS patients and 5 disease-control. To predict the cell type(s) in which each gene was expressed abundantly, we established the cell-type specific transcriptomes using mouse CNS primary culture, then the integrated database was converted to human orthologue. Isolated misregulated genes from microarray were analyzed in terms of glial functions, by using cell-type specific mouse transcriptomes.
Results: We isolated over 200 genes which were significantly changed in the spinal cords of ALS patients. We then classified these genes according to the cells that expressed those genes abundantly, and found nearly half of those genes were expressed abundantly in microglia or astrocytes. Furthermore, many of these genes were also changed in ALS mouse models (SOD1G37R Tg mice, SOD1G85R Tg mice). We confirmed that the predicted gene expression pattern was true by immunohistochemistry for several genes with spinal cords of ALS mouse models. Pathway analysis predicted that innate immunity was one of the significantly altered pathways in glial cells.
Conclusion: We could predict the molecular pathomechamism, especially of glia contributing to the non-cell autonomous motor neuronal death of the ALS in the spinal cord that consists of heterogenous cell types.
P88 REDUCED LEVELS AND ALTERED SUBCELLULAR DISTRIBUTION OF THE INSULINOMA-ASSOCIATED PROTEIN 2 (IA-2) IN ALS AND FTLD
D Riascos
R Bowser
Barrow Neurological Institute, Phoenix, AZ, USA
Email address for correspondence: [email protected]
Keywords: endosomes, membrane trafficking, neuronal vulnerability
Background: Several mutations linked to ALS implicate dysregulated endosomal trafficking as an important pathophysiological mechanism. Changes in the expression of constituents of the regulated secretory pathways in neurons may contribute to selective vulnerability not only in ALS but also in frontotemporal lobar dementia (FTLD). We recently used unbiased proteomics methodologies to identify proteins that exhibit abnormal levels in the CSF of ALS patients. We detected reduced Insulinoma-associated protein 2 (IA-2) in the CSF of ALS patients. IA2 is a transmembrane protein within the protein tyrosine phosphatase superfamily that appears enriched in the secretory vesicles and dense-core granules of various neuroendocrine cell types including central nervous system (CNS) neurons. IA-2 is a major autoantigen in type I diabetes mellitus (T1DM).
Objectives: To characterize the expression and distribution of IA-2 in ALS and FTLD to identify novel mechanisms related to disregulated endosomal trafficking in ALS and FTLD.
Methods: Paraffin tissue and snap frozen tissue from the spinal cord, hippocampus, and frontal cortex of ALS, FTLD, and age-matched control patients were subjected to IA-2 immunohistochemistry (IHC) and immunoblot analysis.
Results: Immunohistochemistry demonstrated decreased IA-2 in spinal cord motor neurons of ALS patients, consistent with our prior proteomic analysis. Furthermore, we observed by immunoblot the loss of the full-length IA-2 isoform in spinal cord tissue from ALS patients compared to controls. In addition, IA-2 was present in distal neuritic processes and neurites in the frontal cortex and hippocampus from FTLD patients.
Conclusions: Our results suggest that reduced levels of IA-2 in ALS (displayed by both IHC and mass spectrometry) and altered subcellular distribution of IA-2 in neurites of FTLD further indicate dysregulation of endosomal trafficking in these neurodegenerative disorders. Moreover, we observe a loss of the full-length IA-2 isoform in ALS. Ongoing studies will clarify whether the loss of full length IA-2 is due to altered mRNA processing or to protein catalysis. Future studies will explore how these changes in IA-2 are related to neuronal loss, and if autoimmune mechanisms resembling T1DM are involved. Motor neurons and cortical motor neurons may be particularly susceptible to this failure because their long axonal processes result in a high demand for turnover of membrane components.
P89 EVALUATION OF TH1/TH2 LYMPHOCYTE BALANCE AND MACROPHAGE SCAVENGER RECEPTOR EXPRESSION IN SPORADIC AMYOTROPHIC LATERAL SCLEROSIS (SALS)
R Zhang1
RO Honrada1
W Harris2
J Katz2
DA Forshew2
RG Miller2
MS Mcgrath1
kUniversity of California, San Francisco, CA, USA
lCalifornia Pacific Medical Center, San Francisco, CA, USA
Email address for correspondence: [email protected]
Keywords: alternative immune activation, CD36 scavenger receptor, Th1/Th2 balance
Background: Involvement of classical monocyte/macrophage (MO) activation and inflammation in ALS pathogenesis has been confirmed by various investigations. Our recent studies on gene expression in peripheral blood mononuclear cells from patients with ALS showed upregulation of both classic type I (M1) interferon-induced genes and type II (M2) alternative MO activation genes, suggesting a hybrid activation state that implicates both classical and alternative MO activation in ALS pathogenesis. Abnormal elevation of alternative MO activation-associated chemokine CCL18 was also observed in ALS plasma. In an attempt to provide further evidence of alternative MO activation in ALS, we evaluated expression of macrophage scavenger receptor CD36, an alternative MO activation marker, and balance of T-helper 1 (Th1) and Th2 subsets of CD4 + lymphocytes in sALS patient blood. Th1 and Th2 cells can be redefined as polarized forms of immune responses; mirroring the Th1/Th2 nomenclature, many refer to polarized macrophages as M1 and M2 cells.
Objectives: To assess CD36 expression on circulating MO and Th1/Th2 lymphocyte markers in sALS patients and healthy controls, and determine whether levels of systemic alternative immune activation correlated with degree of classic MO activation, and clinical stage of disease and/or riluzole treatment in ALS.
Methods: Flow cytometry was performed to examine blood MO CD36 expression and Th1/Th2 lymphocyte balance in heparinized blood samples of 40 sALS patients and 36 healthy controls (HC). Results from immune studies were analyzed in relation to riluzole treatment and the severity of neurological impairment as determined by ALSFRS-R score.
Results: Patients with sALS had significantly elevated levels of CD36 expression on blood MO as compared to healthy controls (HC, 33.52 ± 24.15; sALS, 48.85 ± 27.27; p = 0.0083). Analysis of Th1/Th2 lymphocyte markers showed that Th2 marker of CD4 + CCR4 + was significantly higher (HC, 31.01 ± 7.23; sALS, 39.49 ± 9.58; p < 0.0001), and CD4 + CXCR3 + /CD4 + CCR4 + ratio (representing Th1/Th2 balance) was lower (HC, 1.24 ± 0.42; sALS, 1.04 ± 0.29; p = 0.0232) in sALS patients than healthy controls. No relationship was found between MO CD36 expression and classic type I MO activation in ALS. MO CD36 expression and Th1/Th2 imbalance in sALS patients were independent of severity of disease, and did not change with riluzole therapy.
Discussion and conclusions: The current study showed persistent disease-associated increases of MO CD36 expression and imbalance of Th1/Th2 lymphocytes in sALS. Considering that a systemic Th2 shift is part of a necessary central nervous system wound healing reaction after injury, MO alternative activation and Th2-cell anti-inflammatory response in ALS may represent a negative feedback mechanism in modulation of an inflammatory response, limiting the propagation of classical type I MO activation and Th1 inflammatory responses and compensating for the inflammatory compounds in ALS disease process. Further investigation will be needed to characterize the role of alternative MO activation and Th1/Th2 shift in ALS pathogenesis.
P90 NORMAL LEVELS OF GLUCOSE PROTECT FIBROBLASTS FROM PATIENTS WITH ALS BY REDUCING ROS PRODUCTION AND MAINTAINING THE EXPRESSION OF ROS-DETOXIFYING ENZYMES
R Martin1
E Fernández1
J Esteban2
A Guerrero-Sola3
JS Mora4
JL Alvarez1
Y Campos1
mCNM/Instituto de Salud Carlos III, Majadahonda, Madrid, Spain
nHospital 12 de Octubre, Madrid, Spain
oHospital Clínico San Carlos, Madrid, Spain
pHospital Carlos III, Madrid, Spain
Email address for correspondence: [email protected]
Keywords: glucose, hypermetabolism, reactive oxygen species
Background: There are many mechanisms involved in the pathophysiology of amyotrophic lateral sclerosis (ALS): glutamate excitotoxicity, mitochondrial dysfunction, oxidative stress, etc. There are also increasing evidences that the disease has systemic features beyond the nervous system. In fact, abnormalities of carbohydrate metabolism and insulin resistance have been reported in patients with ALS since the 1980s (Citation1). Moreover, a state of hypermetabolism has been documented, probably related with a defective homeostasis of glucose in muscle tissue (Citation2). We have earlier reported that fibroblasts from patients growing in low glucose medium had a significant increase in the activity of mitochondrial respiratory chain complexes, when compared with control cells or with fibroblast from patients growing in high glucose concentration. In low glucose medium, cell viability and duplication time were similar to those of control cells. However, a significant reduction in cell viability was observed when they were grown in a medium with high glucose levels. We proposed that the hyperactivity of the respiratory chain allows to cells maintain their mitochondrial membrane potential and survival (Citation3).
Objectives: Because the increase of radical oxygen species (ROS) production is a hallmark of cellular dysfunction in ALS, our aims are to study how glucose affects ROS generation in cells growing in different concentration of the substrate and determine if there are changes in the expression of genes encoding ROS detoxifying enzymes.
Methods: Skin fibroblast cultures were established from age-matched controls and patients (n = 10 and n = 6, respectively) after their informed consent. ROS production was measured with the fluorescent dye H2DCFDA in cells growing in a medium with low glucose (1 g/L) or high glucose concentration (4.5 g/L) as described (Citation4). Expression of detoxifying enzymes was determined by RT-PCR as reported (Citation5).
Results: Fibroblast from patients had an increased ROS production in low and high glucose medium (1.5-fold, p < 0.01 and 2.2-fold, p < 0.01, respectively) when compared with control cells. Moreover, there was a significant difference between patients’ cells when were grown in low or high glucose concentration media (p < 0.01). There were no differences between expression levels of CuZnSOD, MnSOD, catalase and GPx in control cells when were cultured in both mediums. However, there was a significant reduction in the expression of CuZnSOD and catalase (p < 0.01) in cells from patients when were grown in high glucose concentration.
Conclusion: A low level of glucose reduces ROS production and allows the fibroblasts from patients with ALS to maintain normal expression of ROS-detoxifying enzymes.
References
- Pradat PF . Amyotrop Lat Scler. 2010;11:166–71.
- Dupuis L . PNAS 2004;101:11159–64.
- Campos Y . Amyotrop Lat Scler. 2010;11(Supp. 1):100.
- Chwa M . IOVS 2006;47:1902–10.
- Park SY . J Biol Chem. 2004;279:7512–20.
P91 EFFECTS OF ANTIOXIDANT AGENTS ON MITOCHONDRIAL FUNCTION AND CALCIUM HOMEOSTASIS IN SKIN FIBROBLASTS OF PATIENTS WITH SPORADIC AND FAMILIAL AMYOTROPHIC LATERAL SCLEROSIS
G Debska-Vielhaber1
Z Gizatullina1
I Menin1
A Hermann2
W Zuschratter5
W Kunz3
F Gellerich5
S Vielhaber1,4
qUniversity of Magdeburg, Magdeburg, Germany
rUniversity of Dresden, Dresden, Germany
sUniversity of Bonn, Bonn, Germany
tGerman Center for Neurodegenerative Diseases, Magdeburg, Germany
uLeibniz Institute for Neurobiology, Magdeburg, Germany
Email address for correspondence: [email protected]
Keywords: mitochondria, antioxidants, calcium
Introduction: Amyotrophic lateral sclerosis (ALS) has been commonly regarded as a neurodegenerative disorder primarily involving the pyramidal motor system. There is however some evidence that disease-related degenerative changes also occurs in extraneuronal tissues. Therefore, primary skin fibroblast cultures from ALS with mutations in the SOD1 gene were compared with ALS patients without mutations and healthy controls for mitochondrial anomalies.
Objective: To verify the putative impairment of mitochondrial function in extraneuronal tissue of patients with sporadic or familiar ALS, the oxygen consumption (respiration rate) of fibroblasts was measured using a high resolution oxygraph. Furthermore, putative benefits of antioxidative agents on the mitochondrial function were assessed.
Methods: Flux control analysis was applied for quantification of changed respiratory chain complexes. To check possible metabolic consequences for calcium homeostasis fluorimetric measurement of cytosolic Ca2+concentrations were performed. Furthermore, mitochondrial DNA copy number analysis and deletions screening were performed. Age matched healthy subjects served as controls.
Results: Enzymatic and respirometric measurements clearly demonstrate mitochondrial impairments in sALS fibroblast with complex I as the main target. Although we have so far investigated only mitochondria of 5 fALS patients, it seems that the extent of complex I impairment is less than as in sALS mitochondria. In both, sALS and fALS patient groups the Ca2+ release into the cytosol induced by histamine and FCCP was significantly diminished. Furthermore we detected, that decreased as well as elevated stationary basal Ca2+concentrations could occur in ALS fibroblasts indicating differences in the pathophysiological mechanisms of sALS and fALS. Under endogenous conditions the enlarged Ca2+cyt (sALS) could cause an over-energization and the diminished Ca2+cyt at fALS could cause an under-energization of mitochondria with consequences for the requirements of intact cells. Most interestingly, the antioxidants Trolox and CoQ10 ameliorate the functional impairment of mitochondria from both groups of patients, most probably by a direct antioxidative and membrane-stabilizing action on mitochondria. No significant differences in mtDNA copy number or common deletion level between patients and healthy controls were found.
Conclusions: Our results support the viewpoint that mitochondrial impairments and consequently Ca2+-dyshomeostasis are detectable in extraneuronal tissues of patients with ALS. Fibroblasts may serve as a relatively easily accessible read out system to test the anti-oxidative properties of specific substances in ALS.
P92 CELL CYCLE DYSREGULATION IN MOTOR NEURONS OF SPINAL AND BULBAR MUSCULAR ATROPHY (SBMA)
M Katsuno1
H Adachi1
N Kondo1
M Minamiyama1,2
H Doi1
S Matsumoto1
Y Miyazaki1
M Iida1
H Nakatsuji1
F Tanaka1
G Sobue1
vDepartment of Neurology, Nagoya University Graduate School of Medicine, Nagoya, Japan
wNational Center of Geriatrics Gerontology, Obu, Japan
Email address for correspondence: [email protected]
Keywords: androgen receptor, TGF-beta, cell cycle
Background: Our previous study showed that the polyglutamine-expanded AR protein, the causative protein of spinal and bulbar muscular atrophy (SBMA), induces transcriptional dysregulation of type 2 TGF-beta receptor and thereby inhibits TGF-beta signalling (Citation1). Although TGF-beta signaling is known to regulate a diverse range of cellular responses including cell cycle, it is unclear how the disruption of this signaling causes neuronal dysfunction in SBMA.
Objectives: The aim of this study is to elucidate the molecular events downstream to the disruption of TGF-beta signaling, and identify the signal pathway that leads to neuronal cell death in SBMA.
Methods: Expression of cell cycle-specific proteins in motor neurons of a transgenic mouse model of SBMA (AR-97Q) was investigated using immunohistochemistry. The effects of pharmacological inhibition of TGF-beta on cell cycle were analyzed in mouse primary cortical neurons using immunoblot, immunocytochemistry, and cell viability assay. Bromodeoxyuridine (BrdU), an analog of thymidine that can be incorporated into newly synthesized DNA, was intraperitoneally administrated to the AR-97Q mice to assess the levels of DNA synthesis in spinal motor neurons.
Results: Immunohistochemical analysis demonstrated that the spinal motor neurons of AR-97Q mice showed decreased expression levels of TGF-beta-regulated genes, P15 and P21, intracellular accumulation of cell cycle regulators, such as cyclines, E2F1, and PCNA, and hyper-phosphorylation of pRb protein. These phenomena were also observed in autopsy specimens of SBMA patients, but not in non-affected neurons in mice or patients. The uptake of BrdU was increased in spinal motor neurons, but not in Purkinje cells, of AR-97Q mice. TGF-beta inhibitor SD-208 increased the expression levels of cyclines, and intensified the phosphorylation of pRb in primary cortical neurons. These effects are blocked by cycline-dependent kinase inhibitors that suppress cell cycle. Intraventrical administration of SD-208 also increased the expression levels of cycline D1 and induced hyper- phosphorylation of pRb in neurons of the brainstem of AR-97Q mice.
Discussion and conclusion: The present study showed that the inhibition of TGF-beta signaling up-regulates the expression of cell cycle regulators in neurons. The affected motor neurons in mice and patients of SBMA showed increased levels of the proteins that accelerate cell cycle. These findings suggest that cell cycle re-entry is associated with the pathogenesis of neurodegeneration in SBMA.
References
- Katsuno M, Adachi H, Minamiyama M, Waza . J Neurosci. 2010;30:5702–5712.
P93 INVESTIGATING THE ROLE OF CHRONIC AND ADAPTIVE ER STRESS IN SELECTIVE MOTOR NEURON VULNERABILITY USING AN ALS GENETIC RISK FACTOR AS A MODEL SYSTEM
Y Liu1
Z Simmons2
J Connor1
xDepartment of Neurosurgery
yDepartment of Neurology; Penn State University Hershey College of Medicine, Hershey, PA, USA
Email address for correspondence: [email protected]
Keywords: selective neuron vulnerability, endoplasmic reticulum stress, HFE H63D
Background: Accumulating evidence suggests endoplasmic reticulum (ER) stress as an early cause of motor neuron degeneration in ALS. In the presence of chronic ER stress, the cell can adapt and cell survival is favored or the stress may promote apoptosis. In ALS and other neurodegenerative disorders, persistent ER stress can be tolerated for a long time but eventually it will lead to cell death. It is not known how an adaptive response transforms to apoptosis. The HFE gene encodes an iron regulating protein, and the H63D variant of this gene is found in increased frequency in patients with ALS and may increase the risk of ALS four-fold. We have reported that HFE H63D is associated with chronic but non-lethal ER stress.
Objective: To understand how an ALS genetic risk factor HFE H63D increases the susceptibility to the disease through chronic ER stress. We are testing the hypothesis that predisposing chronic ER stress induced by HFE H63D lowers the stress threshold in motor neurons and promotes selective neuronal vulnerability.
Methods: By chronic exposure to low doses of chemical ER stressors, we recapitulated the adaptive ER stress status in a neuronal cell line and in primary neuronal cultures prepared from wild type and HFE H67D (the mouse equivalent of human H63D) knock-in mouse spinal cord. Serum deprivation was applied as an additional insult and the effect on the cells harboring predisposing adaptive ER stress was determined. The measurement included cell death, apoptosis activation and ER stress levels.
Results: We demonstrated that although treatment with mild ER stressor or serum deprivation alone did not reduce cell survival, combining these two stressors resulted in robust caspase activation and massive cell death. Examination of major ER stress markers revealed that an ER-stress-induced apoptosis occurred under this dual-stress insult, at least partially due to an increased level of ER stress. These results indicated that a chronic and adaptive ER stress response can be transformed to apoptosis by non-ER-stress insults from the environment. Primary neuronal cells prepared from HFE H67D knock-in mouse spinal cord were more vulnerable to serum deprivation or additional ER stress than the wild type counterparts.
Discussion and conclusions: When adaptive ER stress is challenged neuronal cells are more vulnerable to non- ER-stress-inducing insults. Transformation of predisposing chronic ER stress lowers the stress threshold in neurons which may explain selective neuronal vulnerability and indentifies the HFE mutation as an agent for lowering the stress threshold that promotes neurodegeneration. This has potential therapeutic implications for patients with ALS who harbor HFE H63D mutations.
P94 UBIQUILIN2-IMMUNOREACTIVE INCLUSIONS REPRESENT A CONVERGENT PATHOLOGY IN A WIDE SPECTRUM OF NEURODEGENERATIVE DISEASES
H-X Deng1
EH Bigio1
G Gorrie1
H Zhai1
J Yan1
F Fecto1
K Ajroud1
MI Mishra1
Q Mao1
N Siddique1
E Mugnaini1
F Tatiana2
R Vassar1
B Ghetti2
T Siddique1
zNorthwestern University, Chicago, IL, USA
aaIndiana University, Indianapolis, IN, USA
Email address for correspondence: [email protected]
Keywords: ubiquilin2, neurodegenerative disease, pathology
Background: Mutations in UBQLN2 have been linked to amyotrophic lateral sclerosis (ALS) and ALS with dementia. The distribution of ubiquilin2 inclusions in ALS and ALS/dementia cases correlates with the clinical symptoms, suggesting a role of ubiquilin2 inclusions in the degenerative process of motor and hippocampal neurons. Mutations in UBQLN2 have been shown to impair the function of ubiquitinated protein degradation, therefore, linking a specific molecular defect in protein degradation to degeneration of motor and hippocampal neurons. The involvement of ubiquilin2 in other neurodegenerative diseases remains to be investigated.
Objectives: This study aims to explore if ubiquilin2 is involved in other neurodegenerative disorders, including Alzheimer's disease, Parkinson's disease, frontotemporal lobar dementia that are the top three prevalent neurodegenerative disorders.
Methods: Immunohistochemistry and confocal microscopy were performed on postmortem brains from 63 patients with clinically distinct types of neurodegenerative disease. Brain sections from the 5XFAD transgenic mouse model of Alzheimer disease were also analyzed. Antibodies included those to ubiquilin2, ubiquitin, p62, FUS, TDP43, tau, Aβ and α-synuclein.
Results: Ubiquilin2-immunoreactive inclusions were observed in the affected regions in all of these types of disease. The ubiquilin2-containing inclusions were also immunoreactive with antibodies to ubiquitin and p62, two other critical proteins involved in protein degradation.
Discussion: Our data demonstrate that ubiquilin2 inclusions are a common pathological hallmark in a wide spectrum of neurodegenerative diseases and provide evidence that defects in ubiquitinated protein clearance underlie the pathogenesis in most of the neurodegenerative diseases, indicating a convergent pathway that may be exploited for therapeutic intervention.
P95 PATHOLOGY ASSOCIATED WITH A NOVEL UBQLN2 MUTATION IN FAMILIAL ALS
S Yang1
S Warraich1,2
K Williams1,2
J Solski1
G Nicholson1,2
I Blair1
abANZAC Research Institute, Sydney, NSW, Australia
acSydney Medical School, University of Sydney, Sydney, NSW, Australia, 3Molecular Medicine Laboratory, Concord Hospital, Sydney, NSW, Australia
Email address for correspondence: [email protected]
Keywords: TDP-43, UBQLN2, ubiquitin-proteasome system
Background: The disease mechanism underlying amyotrophic lateral sclerosis (ALS) is poorly understood. Gene mutations are the only known cause of ALS. Mutations in SOD1, TDP-43 and FUS are present in approximately 25% of familial ALS cases. Expansions of a hexanucleotide repeat in C9ORF72 have been reported in 39.3% of familial and 7% of sporadic ALS cases (Citation1). Recently, mutations in UBQLN2 (encoding ubiquilin-2) have been identified in approximately 2% of familial ALS (Citation2). A pathological hallmark of ALS is ubiquitin-positive inclusions in degenerating motor neurons. The components of these inclusions variably include TDP-43, FUS, p62 and ubiquilin-2, implicating pathogenic roles for these proteins.
Objectives: To identify ubiquilin-2 pathology in post- mortem ALS spinal cord and fibroblasts.
Methods: Immunofluorescent staining in ALS tissues was performed using anti-ubiquilin-2, anti-TDP-43 or anti- ubiquitin antibodies. Control and patient fibroblasts with ubiquilin-2 mutation were treated with 5 μM proteasome inhibitor MG-132 for 24 hr or 0.5 mM oxidative stress inducer sodium arsenite for 3 hr, followed by immunofluorescent staining using anti-ubiquilin-2 and anti-TDP-43 antibodies. Cells were visualised with confocal microscopy. Toxicity was measured by MTT assay.
Results: In a patient with UBQLN2 p.T487I mutation, ubiquilin-2 co-localised with ubiquitin, TDP-43 and FUS in spinal motor neuron inclusions. The spinal motor neurons of a patient with FUS p.R521C mutation showed ubiquilin-2 co-localisation with FUS and ubiquitin. In fibroblast cells, no ALS-like cellular pathology was found under normal conditions. Inhibition of the ubiquitin-proteasome pathway by MG-132 led to cytoplasmic inclusions in both patient and control fibroblasts. Patient fibroblasts with UBQLN2 p.T487I mutation showed significantly more cytoplasmic inclusions that were positive for both ubiquilin-2 and TDP-43. In contrast, oxidative stress (sodium arsenite) induced TDP-43 positive inclusions that were negative for ubiquilin-2. No differences in toxicity were observed between control and patient fibroblasts following either treatment.
Discussion: Ubiquilin-2 positive inclusions have been found in various types of ALS. We found that ubiquilin-2 pathology was also present in a patient with a novel ubiquilin-2 mutation and a patient with a FUS mutation. Ubiquilin-2 is a member of ubiquitin-like protein family. Mutations in UBQLN2 have been shown to disrupt proteasomal degradation (Citation2). We found that treatment with a proteasome inhibitor, but not an oxidative stress inducer, led to recruitment of inclusions that are positive for both ubiquilin-2 and TDP-43. This suggests that ubiquilin-2 pathology is a consequence of ubiquitin-proteasome pathway disruption. Proteasome inhibition did not alter measured toxicity despite inducing the formation of inclusions.
References
- Majounie E, Renton E, Mok K . Lancet Neurol 2012; 11(4): 323–330.
- Deng X, Chen, W, Hong T . Nature 2011;477(7363):211–5.
P96 RBM45 PATHOLOGY IN SPORADIC AND C9ORF72-LINKED AMYOTROPHIC LATERAL SCLEROSIS
M Collins2
R Bowser1,2
adBarrow Neurological Institute, Phoenix, AZ, USA
aeUniversity of Pittsburgh, Pittsburgh, PA, USA
Email address for correspondence: [email protected]
Keywords: RNA binding protein, stress granules, intracellular inclusion
Background: RNA binding protein pathology is a pathological hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Using liquid chromatography tandem mass spectrometry (LC/MS-MS), we identified the RNA binding protein RBM45 as a putative ALS biomarker. Immunohistochemistry revealed the presence of RBM45-positive inclusions, similar in appearance to those containing TDP-43, in ALS patients. These could be detected in ALS cases with the C9ORF72 hexanucleotide repeat expansion. Recent demonstrations of unique patterns of p62 and ubiquilin 2 pathology in C9ORF72-linked ALS cases suggest that this form of the disease has a distinct pathological profile.
Objectives: This study was conducted to further describe the prevalence and characteristics of RBM45 pathology in the central nervous system of sporadic and C9ORF72-linked ALS cases, as well as to assess the normal distribution, expression, and subcellular localization of the protein.
Methods: ALS, C9ORF72-linked ALS, FTLD, and age-matched control patient tissues were obtained from the University of Pittsburgh ALS Tissue Bank. Regions examined included spinal cord, hippocampus, cerebellum, and frontal cortex. RBM45 expression was evaluated by immunohistochemistry and immunoblot. Immunofluorescence was used to assess the colocalization of RBM45 with TDP-43, stress granule markers, ubiquitin, p62, and SC35.
Results: Immunoblot results demonstrated the presence of RBM45 in the CSF of ALS and control patients, with modest increases in ALS cases, consistent with our LC-MS/MS data. Using immunohistochemistry we detected RBM45 inclusions in the spinal cord of both sporadic and C9ORF72-linked ALS cases. The number of surviving motor neurons and percentage of motor neurons harboring RBM45 inclusions was higher in C9ORF72-linked ALS cases than in sporadic ALS cases. Inclusions were also found in glia. Cytoplasmic RBM45 inclusions could be seen in the dentate gyrus of FTLD patients and, more rarely, sporadic ALS cases. No inclusions were detected in control cases. RBM45 inclusions were frequently positive for TDP-43, ubiquitin, and stress granule markers. RBM45 also commonly exhibited a speckled nuclear staining pattern that was negative for the nuclear speckle marker SC35.
Discussion: In this study we have further characterized RBM45 pathology in ALS. Moreover, we have added to existing evidence suggesting that sporadic ALS and C9ORF72-linked ALS differ pathologically. Nevertheless, the colocalization of RBM45 inclusions with TDP-43 and stress granule markers suggests common mechanisms of inclusion formation. The speckled nuclear staining pattern observed for RBM45 distinguishes it from TDP-43. This pattern was also separate from SC35-positive nuclear speckles, implying that RBM45 is a component of a distinct subnuclear structure.
Conclusions: We have characterized the expression, distribution, and subcellular localization of RBM45 and RBM45 inclusions in ALS and control subjects. Ongoing studies will determine RBM45's role in RNA metabolism, stress response, and how these processes might contribute to neurodegeneration.
P97 NUCLEAR RNA FOCI IN HEXANUCLEOTIDE EXPANDED C9FTD/ALS
M Baughn1
S Sun2,3
C Lagier-Tourenne2,3
D Pizzo4
M Simpkinson
R Baloh6
S Vandenberg4,5
D Cleveland2,3
J Ravits1
afNeuroscience Department, University of California San Diego, La Jolla, CA, USA
agLudwig Institute for Cancer Research, San Diego, CA, USA
ahDepartment of Cellular and Molecular Medicine
aiDepartment of Pathology
ajDepartment of Neuropathology; University of California San Diego, La Jolla, CA, USA
akDepartment of Neurology, Cedars Sinai Medical Center, Los Angeles, CA, USA
Email address for correspondence: [email protected]
Keywords: RNA, foci, hexanucleotide
Background: Nuclear RNA foci of the expanded hexanucleotide repeat were reported as a feature of c9FTD/ALS in the landmark DeJesus-Hernandez et al. paper of 2011. This finding is fundamental in addressing whether disease biology is mechanistically mediated through haploinsufficiency or RNA toxicity. As yet, this critical finding has not been reproduced.
Objectives: To seek and characterize nuclear RNA foci in c9FTD/ALS.
Methods: Custom fluorescent locked nucleic acid (LNATM) probes (Exiqon) were designed against the GGGGCC expansion and a control sequence. Probes were hybridized using a customized protocol, and examined at 100X magnification on a fluorescent confocal microscope. Specificity of probe binding was confirmed by competition with non-fluorescent probe and by RNase-treated controls. FFPE tissue sections from confirmed C9ORF72 repeat expansion nervous systems (n = 3) were tested at multiple CNS levels, sALS (n = 5), and non-neurologic controls (n = 5) were tested in parallel. In addition, we studied multiple primary fibroblast and immortalized lymphoblast cell lines.
Results: We found nuclear RNA foci exclusively in the c9FTD/ALS tissues. The foci are dumbell shaped and uniform in size, with fluorescent signals measuring approximately 0.5 um across and 0.2 um wide. The foci were seen in about 20–50% of the motor neuron nuclei contained in a histological cross-section (6um) of lumbar spinal cord. The usual count was 1 focus per nucleus, but occasionally 2 or more foci were observed (up to a maximum of 12). Foci were found in the all three spinal levels, the motor cortex and cerebellum. They were observed in both neurons and glial cells. In addition, they were seen in up to ˜30% of cultured primary fibroblasts and immortalized lymphoblast cells, where many foci (> 10) were commonly noted within a single nucleus.
Discussion and conclusions: We confirm that nuclear foci of hexanucleotide RNA repeats are a hallmark feature of c9FTD/ALS. This supports the hypothesis that the expanded repeats exert their toxicity at the RNA level. Additionally, we validated two non-neuronal cell types which can be easily manipulated in a laboratory setting as containing this RNA feature. Since the RNA foci appear in multiple cell lines, including non-neural cells, their presence alone does not appear to explain select neuronal vulnerability.
P98 GAMMA-SYNUCLEIN PATHOLOGY IN ALS
O Peters1
T Hortobágyi2
C Troakes2
N Ninkina1
V Buchman1
alSchool of Biosciences, Cardiff University, Cardiff, UK
amMRC London Neurodegenerative Diseases Brain Bank, Institute of Psychiatry, King's College London, London, UK
Email address for correspondence: [email protected]
Keywords: axonopathy, protein aggregation, phagocyting glial cells
Background: γ-synuclein, a small, cytosolic, aggregation prone protein expressed by select populations of neurons is not currently considered a common contributor to neurological disease. We have previously demonstrated that mice expressing high pan-neuronal levels of γ-synuclein progressively accumulate the protein throughout the CNS, accompanied by the selective degeneration of motor neurons and their axons, in a pathomorphological pattern resembling that typical of ALS mouse models.
Objectives: Due to the auspicious combination of γ-synucleins normal presence in motor neurons, high aggregation propensity and the ALS-like phenotype in mice overexpressing the protein, we aimed to explore the possibility that it might play a role in human motor neuron disease pathogenesis.
Methods: Human tissue samples and detailed pathology reports were obtained from the MRC London Neurodegenerative Diseases Brain Bank: sALS (n = 16), f SOD1-fALS (n = 3), FUS-fALS (n = 3), Alzheimer's disease (n = 6), Lewy body dementia (n = 1), Parkinson's disease case (n = 1) and control cases (n = 8). Spinal cord sections were immunostained with two separate antibodies specific to human γ-synuclein. A set of antibodies against markers of specific cell types were used for co-localisation studies. Sequential protein extraction was also carried out upon several samples.
Results: Immunohistochemical screening revealed a subset of sporadic (8/16) and familial (2/6) ALS cases with a novel pathology characterized by the accumulation of γ-synuclein within the dorsolateral corticospinal tracts, associated with all stages of upper motor neuron atrophy. A portion of these structures was found in association with phagocytic glial cells positive for HLA-DRα and Mac-2/Galectin-3. Sequential fractionation of proteins from spinal cord tissues revealed detergent-insoluble γ-synuclein species specifically in the dorsolateral corticospinal tracts of ALS patients with γ-synuclein-positive profiles in this region.
Discussion: We show a novel type of pathological profile in the descending motor columns of approximately half of ALS patients included in this study, revealed by immunostaining with antibodies specific to γ-synuclein, but not with antibodies to other proteins typically associated with ALS pathology. The specific presence of detergent insoluble γ- synuclein species in the extracts from the profile-positive dorsolateral column but not from the profile-negative anterior horn suggests these structures contain aggregated γ-synuclein. Interestingly, a portion of these structures was associated with Mac-2/galectin-3 positive phagocytic glia, a novel class of which were recently associated with γ-synucleins dependent neurodegeneration of the optic nerve in a mouse model of glaucoma (Nguyen et al., 2011), suggesting the a role for the protein in a common mechanism of axonal degeneration.
Conclusion: Our observations suggest that in some cases the pathological aggregation of γ-synuclein might contribute to the pathogenesis of ALS.
P99 H63D HFE MUTATION MAY CONTRIBUTE TO ALS PATHOGENESIS VIA MITOCHONDRIAL PATHWAYS
X Su
S Lee
R Mitchell
HE Stephens
E Neely
W Nandar
Z Simmons
J Connor
Penn State University, Hershey, PA, USA
Email address for correspondence: [email protected]
Keywords: mitochondria, H63D HFE, SOD1
Background: The H63D polymorphism in the HFE gene, which regulates iron metabolism, is present in about 30% of sporadic ALS cases. In vitro research suggests H63D HFE causes mitochondrial dysfunction, a process separately implicated in ALS pathogenesis. Investigating the effects of H63D polymorphism in ALS patient muscle biopsies, as well as relevant cell culture and mouse models, in the context of mitochondrial processes may provide insights into ALS pathophysiology.
Objectives: To determine if H63D polymorphism contributes to ALS etiology by measuring: 1) levels of soluble superoxide dismutase (SOD1) protein in muscle biopsies from ALS patients with H63D HFE; 2) levels of mitochondrial electron transport chain (ETC) proteins in human neuroblastoma cells stably transfected with H63D HFE; and 3) ETC protein levels in transgenic mice with H67D HFE (analogous to human H63D HFE).
Methods: Muscle biopsy samples which had previously been obtained from ALS patients with definite, probable, probable laboratory-supported, or possible ALS were separated into two groups: wild type (WT) HFE or H63D HFE (heterozygous or homozygous). Immunoassay was used to determine levels of soluble SOD1 protein. SH-SY5Y human neuroblastoma cell lines stably transfected with empty vector, WT HFE or H63D HFE were collected, and mitochondria were isolated using differential centrifugation. Mitochondrial fractions were analyzed for protein levels of complex IV using Western blot. Quadriceps muscle tissue from 6- month-old WT or transgenic mice hetero- or homozygous for H67D mutation was collected, mitochondria were isolated using differential centrifugation, and mitochondrial complex IV levels were analyzed using Western blot. The study of all human samples was approved by our IRB.
Results: Muscle biopsy results demonstrated that H63D HFE ALS patients (n = 16) had 40.7% lower expression of soluble SOD1 protein versus ALS patients with WT HFE (n = 22, p < 0.005). In vitro results demonstrated that neuroblastoma cells stably transfected with H63D HFE had 61.7% lower expression of mitochondrial complex IV protein versus cells transfected with WT HFE (p < 0.05). Mice either hetero- (n = 3) or homozygous (n = 6) for H67D HFE had approximately 20% lower expression of mitochondrial complex IV protein versus WT mice (n = 6) (p < 0.01 for both comparisons).
Discussion and conclusions: Because previous research suggests expression of soluble SOD1 is lowered in lymphocytes and affected CNS regions in sporadic ALS, the finding that H63D HFE polymorphism appears to decrease levels of soluble SOD1 in muscle is clinically relevant. H63D HFE also appears to disrupt mitochondrial function, as reflected by lowered expression of complex IV, a terminal ETC complex. One possible mechanism for the observed mitochondrial changes may be accumulation of insoluble SOD1 aggregates targeting mitochondrial processes. Given the role of altered non-mutant SOD1 levels as well as mitochondrial dysfunction in ALS, our study further implicates the H63D polymorphism in ALS pathogenesis.
P100 MRNA EXPRESSION PATTERN OF AXON GUIDANCE PROTEINS IN HUMAN POST MORTEM MOTOR CORTEX IN AMYOTROPHIC LATERAL SCLEROSIS (ALS)
S Körner
K Wilzek
N Thau
S Knippenberg
R Dengler
S Petri
Medical School Hannover, Hannover, Germany
Email address for correspondence: [email protected]
Keywords: axon guidance proteins, semaphorins, plexins, neuropilins
Background: Several findings support the hypothesis of early subclinical involvement of neuromuscular junctions and terminal axons followed by subsequent disease progression towards motor neuron cell bodies (‘dying back’-hypothesis) in ALS. Axon guidance proteins have first been described as steering molecules for developing axons but are also important for maintenance of neuronal structure, axonal transport and synaptic function in the adult nervous system. Aberrant function or expression of axon guidance proteins may therefore contribute to the pathological changes in motor neuron connectivity in ALS.
Objectives: In the present study we assess the distribution and expression levels of the different axon guidance proteins (semaphorin 3A, 3B, 3C, 3D, 3E, 3F) and their receptors (plexin PlxA1, PLxA2, PlxA3, PlxA4 und neuropilin Np1, Np2) in human post mortem motor cortex of ALS patients and age matched controls.
Methods: Quantitative real time PCR (qRT-PCR) and in situ hybridization histochemistry (ISH) were used for analysis of the mRNA expression patterns of different axon guidance proteins and their receptors in human post mortem motor cortex of ALS patients (n = 5) and age matched controls (n = 5). In ISH experiments, mRNA expression was quantified macroscopically by densitometric analysis of digitized film autoradiograms, the expression at the cellular level was studied by liquid emulsion autoradiography. Corresponding rotein expression patterns were studied by immunohistochemistry.
Results: mRNA expression of the semaphorin receptors plexin A3 and neuropilin 1 was significantly lower in ALS motor cortex as measured by qRT-PCR. These results could be confirmed by ISH and immunohistochemistry. Immunohistochemistry in addition revealed higher expression of the axon guidance protein semaphorin 3A, which is known as an axon growth inhibitor.
Discussion and conclusion: These results suggest increased expression of the axon growth inhibitor semaphorin 3A with possibly reactive reduced expression of the related receptors in ALS tissue. It has already been shown in a rat model of spinal cord injury and an in vitro axotomy model that semaphorin inhibitors can promote axonal regeneration. According to our results, they should be further evaluated as novel therapeutic option in ALS.
P101 CSF TRACE-ELEMENTAL ANALYSIS OF AMYOTROPHIC LATERAL SCLEROSIS PATIENTS BY X-RAY MICROFLUORESCENCE WITH SYNCHROTRON RADIATION
D Mendonça1
R Serpa2
E De Jesus3
R Higashi4
L Chimelli5
A Martinez6
anDepartamento de Biociências, Universidade Federal de Sergipe, Sergipe, Brazil
aoInstituto de Pesquisas Energéticas e Nucleares, IPEN-CNEN/SP, São Paulo, Brazil
apDepartamento de Engenharia Nuclear, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
aqInstituto de Neurologia Deolindo Couto, Rio de Janeiro, Brazil
arDepartamento de Patologia, Hospital Universitário Clementino Fraga Filho, Rio de Janeiro, Brazil
asDepartamento de Histologia e Embriologia, ICB, CCS, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
Email address for correspondence: [email protected]
Keywords: cerebrospinal fluid, trace elements, protein aggregates
Background and objectives: Biochemical analysis of the Cerebrospinal Fluid (CSF) has immense potential for diagnosis and prognosis in many neurological diseases (Citation1). CSF can provide a repository of ALS pathological information. Histopathologically, in ALS, there is marked neuronal loss and inclusion bodies containing protein aggregates (Citation2). Abnormal protein-protein interactions remain uncertain and a role for trace elements has been suggested. The aim of this study was to investigate trace element levels of ALS CSF samples.
Methods: ALS (10) and control (6) CSF samples were treated for trace elemental analysis by X-ray microfluorescence with synchrotron radiation. Measurements were carried out at the XRF beam line at the Synchrotron Light National Laboratory (Campinas, Brazil). The intensity of X-Ray specter for each element was obtained with AXIL software (distributed by International Agency of Atomic Energy). These obtained data were analyzed statistically by General Linear Models, in a multivariate based method.
Results: In this study, the following elements were analyzed: aluminium, bromine, calcium, chlorine, copper, chromium, iron, potassium, phosphorus, nickel, rubidium, silicon, sulfur and zinc. In ALS CSF samples we observed significant increased concentration of calcium, chlorine and potassium when compared to control samples.
Discussion: Due to its close proximity to the central nervous system parenchyma, CSF is likely to be used for detecting dynamic information about pathological processes of the brain. Excitotoxicity is one of many factors implicated in ALS pathogenic process. The intracellular calcium influx seems to contribute to neurodegeneration in multiple pathways, conducing cell to death (Citation3). Activation of potassium and chlorine channels by increased calcium level was already described and prolonged efflux of both could be involved in apoptosis (Citation4). The elevated levels of these three elements in CSF can reflect an abnormal activation of potassium and chlorine channels, indicating a possible molecular pathway involved in ALS pathogenic process.
Conclusion: Analysis of biological samples provides a powerful strategy for investigating pathological processes. Moreover, extensive studies indicated an important role of trace elements in ALS disease and our data are in agreement with these. The role of trace elements in ALS pathogenesis must be more investigated since the identification of altered elements can be a significant step in ALS research and future therapy.
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