P161 PALLIDO‐LUYSO‐RUBRO‐NIGRAL ATROPHY ASSOCIATED WITH FAMILIAL AMYOTROPHIC LATERAL SCLEROSIS: A CLINICAL, GENETIC AND PATHOLOGICAL STUDY
Gamez J1, Corbera‐Bellalta M1, Mila M2, Lopez‐Lisbona R3, Boluda S4, Ferrer I4
1Hospital Vall d'Hebron, UAB, Barcelona, Spain, 2Hospital Clinic, UB, Barcelona, Spain, 3Servei Neumologia, Hospital Universitari de Bellvitge, Hospitalet de Llobregat, Spain, 4Institut Neuropatologia, IDIBELL‐Hospital Universitari de Bellvitge, Hospitalet de Llobregat, Spain
E‐mail address for correspondence: [email protected]
Background: Atypical manifestations in ALS include sensory impairment, dementia, ocular palsy, bladder/bowel dysfunction, decubiti, cerebellar and extrapyramidal signs. These special forms of ALS are defined as ALS Plus. Hyperkinetic movements in ALS are very rare.
Objectives: To report the clinical, genetic, and neuropathological findings in one woman with clinical criteria of familial ALS who, 24 months after onset of motor symptoms, presented hyperkinetic movements consistent with choreoathetosis and ballism.
Methods: A 53‐year‐old woman suffered from progressive weakness and amyotrophy in the lower and then upper extremities, breathing difficulties, neurogenic bladder and speech and swallowing difficulties consistent with ALS. A similar disease had occurred in her father and her older sister. Two years after initial motor symptoms, choreic movements affecting the face, mouth, neck and hands appeared, together with continuous, rotary excursions of the upper limbs at proximal joint level, mimicking ballism. The patient died of respiratory failure 27 months after initial motor deficit. Genetic studies for IT15, DRPLA, and SOD1 gene mutations were performed. Brain and spinal cord were obtained six hours after death and subsequent neuropathological examination was performed.
Results: HD, DRPLA, ALS1, acanthocytosis, Wilson's disease, thyroid and parathyroid disorders were ruled out by appropriate genetic studies, peripheral blood smear examination, ceruloplasmin, TSH and PTH levels.
Neuropathological examination showed neuron loss in the anterior horn of the spinal cord and motor nuclei of the medulla oblongata, and atrophy and myelin pallor of the anterior spinal roots and pyramidal tracts, as well as of the spinocerebellar, rubrospinal and vestibulospinal tracts with preservation of the posterior columns. In addition, neuron loss and gliosis occurred in the internal globus pallidus, subthalamus, substantia nigra pars compacta and red nucleus. Intranuclear inclusions were absent.
Conclusions: The main clinical features in this patient with ALS Plus were the presence of hyperkinetic movements 24 months after the onset of leg weakness. Neuropathological examination showed involvement of the spinocerebellar, rubrospinal, reticulospinal and vestibulospinal tracts, and upper and lower motor degeneration. Most important in the present context was degeneration of the internal globus pallidus, subthalamic nucleus, substantia nigra and red nucleus. This combination together with FALS is unique, although it probably should be considered within the spectrum of ALS Plus with pallido‐luyso‐nigral atrophy. These data support the concept that extrapyramidal symptoms in ALS Plus may be the result of associated pallido‐luyso‐(rubro‐)nigral atrophy. Considering that this association is not unusual in ALS Plus cases, pallido‐luyso‐rubro‐nigral atrophy can be considered as a particular manifestation in certain sporadic and familial ALS cases. The cause of this multisystemic atrophy is not related to HD, DRPLA or SOD1 gene mutations.
Acknowledgements: FIS 05/0776, FIS 06/0502, FIS 05/1570, Fundació La Marató‐TV3 146/2006.
P162 RNA EDITING IN SPORADIC ALS AND OTHER MOTOR NEURON DISEASES
Hideyama T, Nishimoto Y, Ito K, Kakita A, Takahashi H, Tsuji S, Shin K
Department of Neurology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan
E‐mail address for correspondence: [email protected]
Background: Deficient RNA editing of GluR2 mRNA at the Q/R site has been proposed to be the cause of death in spinal motor neurons in sporadic ALS. RNA editing at the GluR2 Q/R site is specifically catalysed by adenosine deaminase acting on RNA type 2 (ADAR2). Therefore, underediting of GluR2 in ALS motor neurons is likely to be due to a decrease of the ADAR2 enzyme activity. The aim of this study is to investigate whether the reduction of RNA editing of the GluR2 Q/R site occurs in motor neuron diseases other than sporadic ALS, and whether the ADAR2 activity is reduced in ALS motor neurons compared with control motor neurons.
Methods: With a laser microdissector, motor neurons were dissected from frozen autopsied spinal cord of spinal and bulbar muscular atrophy (SBMA) patients and symptomatic rats transgenic for mutated human Cu/Zn superoxide dismutase (SOD1) (G93A, H46R). In addition, ventral grey matter tissue was dissected en bloc under a microscope from frozen autopsied human control and ALS spinal cords. Dissected tissues were collected and were subjected to RT‐PCR. The editing efficiency of ADAR2 substrates including the GluR2 Q/R site was determined by the quantitative analysis of the fragments of the PCR products digested by a restriction enzyme, and the expression levels of ADAR2 mRNA and GluR2 mRNA were measured using LightCycler system.
Results: We found that GluR2 mRNA was completely edited in all the motor neurons of SBMA patients and SOD1 transgenic rats examined. The expression level of ADAR2 mRNA was significantly decreased only in the ventral grey matter of the ALS spinal cord. Among the various known ADAR2‐catalysed editing sites we examined, RNA editing was significantly reduced only at the GluR2 Q/R site in ALS ventral grey matter compared with the control ventral grey matter.
Conclusions: Motor neurons in mutated SOD1 transgenic rats and SBMA patients may die via mechanisms other than deficient RNA editing, hence there seem to be multiple death pathways in motor neurons. Furthermore, underediting of the GluR2 Q/R site is the molecular mechanism unique to death of motor neurons in sporadic ALS. A reduction of the ADAR2 activity is likely to be the cause of deficient GluR2 RNA editing. Although the expression level of ADAR2 mRNA is one of the regulatory factors of the ADAR2 activity in vivo, it is likely that there are other factors regulating the ADAR2 activity at the Q/R site of GluR2 mRNA.
P163 ALSIN, A CAUSAL GENE PRODUCT OF AMYOTROPHIC LATERAL SCLEROSIS 2, PHYSICALLY INTERACTS WITH TOLLIP AND REGULATES NF‐κB FUNCTION
Liu T‐L, Kouda T, Nakagawara A
Chiba Cancer Center Research Institute, Chiba, Japan
E‐mail address for correspondence: [email protected]
Background: Amyotrophic lateral sclerosis (ALS) is an adult‐onset fatal neurodegenerative disease. The mutated ALS2 gene has been identified as a cause of the recessive inherited juvenile ALS (ALS2). The ALS2 gene encodes a large protein termed alsin, which contains a number of predicted sequence motifs of cell signalling and protein trafficking. Alsin is expressed in various tissues and cells, including neurons of brain and spinal cord, but the molecular mechanism of the ALS2‐related motor neuronal death remains unclear.
Objectives: To gain insight into the mechanisms of neuronal cell death in ALS2, we searched for the alsin‐interacting protein by using the yeast two‐hybrid system. We hypothesized that the downstream signaling pathway of alsin and its interacting molecules may play a key role in regulating motor neuron death in ALS2.
Methods: pBluescript II SK (+) KIAA1563 contains a part of ALS2. We used human brain cDNA library as a template to create the remainder constructs using amplification by polymerase chain reaction (PCR) to obtain the full length of ALS2 cDNA. We used the yeast two‐hybrid system to screen for proteins interacting with alsin. We constructed the plasmids of each domain of alsin. The physical interaction of alsin with the proteins identified by the yeast two‐hybrid system was confirmed by using immunoprecipitation. The transfection method was used for overexpression of alsin and other proteins in cells. Cell death was measured by Trypan blue dye exclusion assay and by fluorescence activated cell sorter (FACS) analysis. For transcriptional activation assay, we used luciferase reporter assay.
Results: The yeast two‐hybrid system identified Tollip (Toll‐interacting protein) as a binding partner of alsin. By immunoprecipitation assay, Tollip was found to specifically bind to the MORN motifs domain of alsin. Overexpression of Tollip induced cell death in HEK293 and NSC34 cells. Interestingly, the cell death was inhibited after co‐transfection of ALS2 and Tollip in those cells. Furthermore, as a Toll/IL‐1R domain‐containing adaptor protein, Tollip formed a complex with IRAKs (IL‐1RI‐associated protein kinases) family, thereby preventing IRAK‐1‐induced‐NFκB activation. In our studies, Tollip inhibited TNFα‐induced NFκB activation as in IL‐1R/TLR signalling. The decreased NFκB activity was recovered after co‐transfection of ALS2 and Tollip.
Discussion and conclusions: In the present study, we first found that Tollip is a specific binding protein with alsin at its MORN motifs domain. Our present results suggest that alsin interacts with Tollip to inhibit its suppression of NF‐κB activation. The mutated alsin might not be able to inhibit Tollip, thereby neuronal cell death could be induced.
Acknowledgments: The authors are indebted Shigemi Irino, Kou Miyazaki, Chiaki Kato, Toshinori Ozaki (Chiba Cancer Center Research Institute) Masasi Aoki, Yasushi Itoyama (Department of Neurology, Tohoku University School of Medicine) for their participation.
P164 COLLAGEN ABNORMALITIES IN PYRAMIDAL TRACT AND ANTERIOR HORN OF THE SPINAL CORD IN AMYOTROPHIC LATERAL SCLEROSIS
Irie T, Ono S
Department of Neurology, Teikyo University School of Medicine, Chiba, Japan
E‐mail address for correspondence: [email protected]
Background: Studies of skin in patients with amyotrophic lateral sclerosis (ALS) have reported morphological and biochemical abnormalities of collagen. These findings, which were not found in controls, could be one of the characteristics of ALS. However, little is known concerning collagen of the spinal cord in ALS.
Objectives: To clarify collagen alterations in the spinal cord of ALS.
Methods: We measured the amount of collagen and characterized collagen at an electron microscopic level in the posterior funiculus, posterior half of the lateral funiculus and anterior horn of cervical enlargement of the spinal cord obtained from 15 patients with ALS, 15 patients with other neurological diseases (control group A), and 15 patients without neurological disease (control group B).
Results: In the posterior half of the lateral funiculus and anterior horn: 1) ultrastructually, collagen bundles were more fragmented and widely separated, and the fibrils were randomly orientated in the perivascular space of capillaries in ALS patients, which were not observed in any areas of control groups or in the posterior funiculus of ALS patients; and 2) the collagen contents in ALS were significantly lower than those in control groups A and B (p<0.001 and p<0.001, respectively).
Discussion and conclusions: These morphological changes of collagen in the interstitial tissue surrounding capillaries, and markedly decreased amount of collagen in the posterior half of the lateral funiculus and in the anterior horn in ALS, could be related to the degeneration of the upper and lower motor neurons in the spinal cord, i.e. selective neuronal involvement in ALS.
P165 INCREASED EXPRESSION OF NEUROTROPHIC FACTORS IN THE SKIN OF AMYOTROPHIC LATERAL SCLEROSIS PATIENTS
Suzuki M, Ono S
Department of Neurology, Teikyo University School of Medicine, Chiba, Japan
E‐mail address for correspondence: [email protected]
Background: Our previous reports have shown unique morphological and biochemical abnormalities in the skin of patients with amyotrophic lateral sclerosis (ALS). One of the features of patients with ALS is the absence of bedsores. Neurotrophic factors including CNTF, IGF‐I, and LIF have been found to affect the survival and maintenance of a variety of neuronal cell types, including motor neurons. In addition, neurotrophic factors have been implicated in the pathogenesis of various neurodegenerative disorders such as ALS and evaluated as a therapeutic agent for the treatment of ALS. Little is known, however, about whether the expression of neurotrophic factors is altered in skin of ALS patients.
Objectives: To study the expression of ciliary neurotrophic factor (CNTF), insulin‐like growth factor I (IGF‐I), and leukaemia inhibitory factor (LIF) in skin from ALS patients, compared with controls.
Methods: Skin biopsy samples were taken from the left upper arm of 15 patients with ALS (11 men and 4 women, mean age 57.3 years) and from 15 controls with other neurodegenerative diseases matched for sex and age (11 men and 4 women, mean age 60.4 years). Routine formalin‐fixed paraffin‐embedded 6 µm sections were immunostained according to standard techniques. The sections were incubated with anti‐CNTF antibody, anti‐IGF‐I antibody, and anti‐LIF antibody. After washing in phosphate‐buffered saline, biotinylated anti‐IgG was applied. The sections were stained by ABC kit. The immunoreactivity was quantified with an image‐analysis system. Statistical comparisons were made by the two‐tailed Student's t‐ test with p<0.05 as the significance level. Correlation coefficients were calculated by the least‐squares method.
Results: The immunoreactivities of CNTF, IGF‐I, and LIF were strongly positive in the epidermis and in some blood vessels and glands of the reticular dermis in all ALS patients but not in controls. These findings became more conspicuous as ALS progressed. The optical densities of CNTF, IGF‐I, and LIF immunoreactivities in ALS patients were significantly higher than in controls. Furthermore, there was a significant positive relationship between the immunoreactivities of CNTF, IGF‐I, and LIF and duration of illness in ALS patients. There was no relationship between the optical densities of CNTF, IGF‐I, and LIF and the presence of dysphagia, weight loss, muscular atrophy, loss of active movement, and bedridden state in ALS patients or controls.
Discussion and conclusions: CNTF, IGF‐I, and LIF were found to be significantly up‐regulated in skin samples from ALS patients. The results suggest that changes of CNTF, IGF‐I, and LIF in the skin of ALS patients are likely to be related to the disease process. These neurotrophic factors may have a trophic role in the skin of ALS patients and may help to explain why decubitus formation is rare in ALS.
P166 AN INCREASE OF OXIDIZED COENZYME Q‐10 OCCURS IN THE PLASMA OF SPORADIC AMYOTROPHIC LATERAL SCLEROSIS PATIENTS
Sohmiya M1, Shirakura K1, Yamamoto Y2, Okamoto K3
1Gunma University Hospital, Gunma, Japan, 2Tokyo University of Technology, Tokyo, Japan, 3Gunma University Graduate School of Medicine, Gunma, Japan
E‐mail address for correspondence: [email protected]
Background: Oxidative stress has been suggested to underlie pathogenesis of amyotrophic lateral sclerosis (ALS) since missense point mutations occur in the copper/zinc (Cu/Zn) superoxide dismutase (SOD) gene of familial ALS patients. Indeed, elevated levels of thiobarbituric acid reactive substances (TBARS) and 8‐hydroxy‐deoxyguanosine (8‐OHdG) were found in the plasma of sporadic ALS patients compared with controls. Nevertheless, plasma and serum levels of antioxidants such as vitamin E, ascorbic acid, coenzyme Q‐10 and carotenoids were not different between sporadic ALS patients and healthy controls. Conflicting results of antioxidant enzyme activities such as SOD and glutathione peroxidase were also obtained in the plasma or serum of ALS patients.
Objectives: The aim of the present study is to examine the levels of several antioxidants and the redox status of coenzyme Q‐10 in the plasma of sporadic ALS patients.
Methods: Plasma samples were collected from 20 sporadic ALS patients and 20 controls. We applied a sensitive and reliable method with high performance liquid chromatography for the simultaneous detection of ubiquinol‐10 (CoQH2‐10, the reduced form of coenzyme Q‐10) and ubiquinone‐10 (CoQ‐10, the oxidized form of coenzyme Q‐10) in plasma samples from sporadic ALS patients and age/sex‐matched controls. Then we calculated the ratio of CoQ‐10 to total coenzyme Q‐10 (%CoQ‐10) as an indicator of oxidative stress.
Results: We found no significant differences in plasma levels of uric acid, ascorbic acid, unconjugated bilirubin, vitamin E, CoQH2‐10, total coenzyme Q‐10 and CoQH2‐10/total cholesterol between sporadic ALS patients and control volunteers. However, plasma levels of CoQ‐10 (p = 0.0002) and CoQ‐10/total cholesterol (p<0.0001) were significantly higher in sporadic ALS patients than those in controls. In addition, %CoQ‐10 values were significantly higher (p<0.0001) in sporadic ALS patients than in controls. A significant correlation was observed between %CoQ‐10 values and the duration of illness (p<0.05)
Conclusion: We demonstrated a significant increase of plasma %CoQ‐10 in sporadic ALS patients, suggesting systemic oxidative stress in the pathogenesis of the disease.
P167 DECREASED LEVELS OF REACTIVE OXYGEN SPECIES, SOD1 EXPRESSION AND ACTIVITY IN PERIPHERAL TISSUES OF SPORADIC AMYOTROPHIC LATERAL SCLEROSIS PATIENTS
Cova E1, Bongioanni P2, Cereda C1, Galli A3, Corato M3, Rossi B2, Ceroni M4
1Neurological Institute IRCCS C. Mondino, Pavia, Italy, 2Unit of Neurorehabilitation, Azienda Ospedaliera Universitaria Pisana, Pisa, Italy, 3Department of Neurology, Policlinico of Monza, Monza, Italy, 4Department of Neurological Sciences, University of Pavia, Pavia, Italy
E‐mail address for correspondence: [email protected]
Background: Pathogenesis of neuronal degeneration in both sporadic and SOD1‐ mutated familial amyotrophic lateral sclerosis (ALS) may involve oxidative stress (1). In a previous paper (2) we demonstrated that lymphocytes from sporadic ALS patients (SALS) are more prone to undergo alteration of cell membrane integrity in both basal condition and following additional oxidative stress. Moreover, SALS lymphocytes showed reduced intracellular SOD1 levels compared to controls. Such results suggest that oxidative stress protection pathways are deregulated in sALS patients.
Objectives: The aim of this work was to assay over time SOD1 levels and activity in SALS lymphocytes and plasma samples. Moreover, plasma oxidative status was evaluated by assaying reactive oxygen species (ROS) levels.
Methods: Intracellular SOD1 expression was evaluated in lymphocytes from 10 SALS patients at the time of diagnosis and after six months. Lymphocytes obtained by Ficoll‐Hystopaque 1077 gradient were processed for protein extraction and SOD1 expression was evaluated by Western blotting. Total blood SOD1 activity and plasma ROS levels were assayed in 79 SALS patients: blood samples from all patients were drawn every two months over six years. SOD1 activity (587 blood samples) was assayed at 30°C by its ability to inhibit superoxide radical‐dependent reactions (3) and expressed as U/g of haemoglobin. ROS levels (468 samples) were evaluated by a colorimetric method (Diacron International, Grosseto, Italy) and expressed as U CARR.
Results: Western blotting experiments showed that intracellular SOD1 expression was significantly (p<0.01, Student's t‐ test) decreased after six months from the first withdrawal. Mean value of SOD1 activity evaluated for each patient was decreased in 71% of cases. Moreover, mean value of plasma ROS levels calculated for each patient was under the normal range in 61% of cases.
Discussion: Our data show that total blood SOD1 activity, reflecting both the activity of extracellular SOD and that of intracellular SOD1 released by damaged cells, is lower compared to normal values. Moreover, SOD1 expression, previously demonstrated to be significantly lower in SALS lymphocytes compared to those from controls, decreases over time in SALS patients. Taken together, these findings reinforce the hypothesis that the wild‐type SOD1 protein may also be implicated in ALS pathogenesis, although the precise mechanisms are still unknown. Surprisingly, in the majority of patients circulating ROS levels were under the normal range, suggesting that ROS increase may be confined to the intracellular compartment.
References
- Robberecht W J. Neurol. 2000;247:I/1.
- Cova E, Cereda C, Galli A, et al Neurosci Lett. 2006;(in press).
- Flohè L, Otting F. Methods Enzymol. 1984;105:93.
P168 A COMMON MOLECULAR SIGNATURE IN SOD1 FOR BOTH SPORADIC AND FAMILIAL AMYOTROPHIC LATERAL SCLEROSIS
Gruzman AL1, Prasad MD1, Wood WL2, Mass J1, Miller RG1, Cleveland DW3, Bowser R4, Otto M5, Spefeld A5, Ludolph AC5, Wood TD2, Lingappa VR1, Liu J1
1California Pacific Medical Center Research Institute, San Francisco, CA, USA, 2University of Buffalo, Buffalo, NY, USA, 3Ludwig Institute for Cancer Research, San Diego, CA, USA, 4University of Pittsburgh School of Medicine, Pittsburgh, PA, USA, 5University of Ulm, Ulm, Germany
E‐mail address for correspondence: [email protected]
Background: Amyotrophic lateral sclerosis (ALS) is a devastating motor neuron degenerative disease whose etiology and pathogenesis remain poorly understood. Most cases of ALS (∼90%) are sporadic (SALS), occurring in the absence of genetic associations. About 20% of familial ALS (FALS) cases are due to mutations in the copper/zinc superoxide dismutase (SOD1) gene. Molecular evidence for a common pathogenesis of SALS and FALS has remained elusive.
Objectives: The objective of this study was to identify a common molecular signature for both SALS and FALS.
Methods: We applied a new approach involving biotinylation of accessible lysine residues in proteins, followed by SDS‐PAGE and Western blot (WB) analysis for SOD1. To verify that the SOD‐immunoreactive (IR) protein species indeed contained the SOD1 molecule, nanospray mass spectrometry technique was used. In addition, column chromatography method was used to determine the source that gave rise to the SOD1‐IR band.
Results: Biotinylation revealed a 32 kDa ALS‐specific protein species immunoreactive to a peptide‐specific rabbit antiserum to human SOD1. This distinctive SOD1‐IR species was observed in both spinal cord and muscle of individuals with ALS, but not present in normal or non‐ALS disease cases. Furthermore, we confirmed that the 32 kDa SOD1‐IR contained the SOD1 molecule and was distinct and independent from normal sized SOD1 during biotinylation. Thus, covalent chemical modification allowed us to identify an attribute of SOD1 common to both SALS and SOD1‐linked FALS.
Discussion and conclusions: Our studies used chemical modification as a novel tool for the detection of previously imperceptible differences and/or potential disease‐associated biomarkers in the proteome. Our results suggest a shared molecular event involving a known target gene as a common step in the etiology and pathogenesis between SALS and FALS.
P169 AGGREGATION OF SOD1 IS PRIMARILY NEURONAL, RATHER THAN GLIAL IN ALS PATIENTS AND MOUSE MODELS WITH SOD1 MUTATIONS
Deng H‐X1, Wang L2, Liu Z1, Zhai H1, Fu R1, Hays A3, Grutzendler J1, Roos R2, Siddique T1
1Northwestern University, Chicago, IL, USA, 2University of Chicago, Chicago, IL, USA, 3Columbia University, New York, NY, USA
E‐mail address for correspondence: [email protected]
Background: Mutations in the Cu/Zn superoxide simutase gene (SOD1) are associated with about 20% of familial ALS cases. The pathogenic mechanisms underlying SOD1‐mediated ALS are not well understood. A pathological hallmark of the mutant SOD1‐mediated ALS is SOD1‐immono‐reactive aggregates. These aggregates are found in neurons. In a few cases, SOD1 aggregates were also reported in glial cells. The prevalence and the pathogenic role of the aggregates in glial cells are not known.
Objectives: The objective of this project is to study the cellullar distribution of the SOD1 aggregates in the spinal cords of ALS subjects, including ALS patients with SOD1 mutation and transgenic mice overexpressing ALS‐associated SOD1 mutants.
Methods: The spinal cord sections from ALS patients with SOD1I113T and SOD1G85R mutations, and transgenic mice overexpressing SOD1G93A, SOD1L126Z and a newly developed SOD1G85R mouse line (Lijun & Roos, unpublished) were analysed with immunohistochemistry and confocal microscopy using antibodies against SOD1 and GFAP. We also crossbred the SOD1G93A mice with EGFP (enhanced green fluorescent protein) knock‐in mice at CX3CR1 locus. These mice show strong GFP in microglia.
Results: We observed SOD1‐immunoreactive aggregates in large neuronal bodies and their processes. Although these aggregates were reported primarily in astrocytes in one of the SOD1G85R transgenic mouse lines, we were unable to identify the SOD1 aggregates in the astrocytes in our new SOD1G85R transgenic mice and in an ALS patient with SOD1G85R mutation. We did not find apparent SOD1 aggregates in glial cells in the patients with SOD1I113T either. We did not observe SOD1 aggregates in microglia in SOD1G93A mice. We observed an increased expression of SOD1 in some activated astrocytes in the transgenic mouse samples, but this SOD1 was apparently in a diffused form, not an aggregated form.
Discussion and conclusions: Thus, our data suggest that the SOD1 aggregates in glial cells may not be a common feature of the SOD1‐mediated ALS. Therefore, the aggregates in neurons, rather than glial cells may play a pathogenic role in SOD1‐mediated ALS.
Acknowlegements: These studies were supported by grants from the National Institutes of Health, (NS040308, NS040308, NS050641, NS046535), Les Turner ALS Foundation, Playing to Win 4 Life Foundation, V. E. Schaff ALS Research Fund, H. Post Research Professorship, Wenske Foundation, Falk Medical Research Trust, The Les Turner ALS Foundation/Herbert C. Wenske Foundation Professorship, and The David C. Asselin MD Memorial Fund.
P170 NEDL1, A NOVEL UBIQUITIN‐PROTEIN LIGASE, TARGETS MUTANT SUPEROXIDE DISMUTASE‐1 AND P53 TO INDUCE APOPTOSIS
Li Y, Miyazaki K, Ozaki T, Nakagawara A
Chiba Cancer Center Research Institute, Chiba‐city, Chiba, Japan
E‐mail address for correspondence: [email protected]
Background: Approximately 20% of familial amyotrophic lateral sclerosis (FALS) arises from germ‐line mutations in the Cu/Zn superoxide dismutase (SOD) 1 gene. Recently, ubiquitin‐proteasome system impairment has been implicated in mutant SOD1‐induced toxicity. However, the molecular mechanism underlying the pathogenesis of FALS remains unclear.
We have identified a novel HECT‐type E3 ubiquitin ligase termed as NEDL1 (NEDD4‐like ubiquitin protein ligase 1) from oligo‐capping cDNA libraries generated from anonymous neuroblastoma tissues that were undergoing spontaneous repression. Northern blot analysis revealed that NEDL1 was preferentially expressed in neuronal tissues. Furthermore, NEDL1 was highly expressed in favourable neuroblastomas. These observations indicate that NEDL1 might be involved in neuronal cell death.
Objectives: In this study, we sought to investigate the role of NEDL1 in the pathogenesis of FALS as well as the molecular mechanism of induction of neuronal cell death.
Methods: A yeast two‐hybrid assay was performed to identify the physiological target of NEDL1. Immunoprecipitation and immunoblotting were conducted to detect the interaction of NEDL1 with SOD1s. Immunohistochemical analysis was employed to examine the localization of NEDL1 using spinal cord sections from FALS patients and mutant SOD1 transgenic mice. Ubiquitination assay, colony formation assay, and luciferase reporter assay were performed to unveil the functional role of NEDL1 in the induction of neuronal death.
Results: Dishevelled‐1, a regulatory molecule in the Wnt signaling pathway, was found as the physiological target of NEDL1 for ubiquitination and proteasome‐mediated degradation. NEDL1 bound to and ubiquitinated various mutant (but not wild‐type) SOD1s, in a FALS severity‐dependent manner. Immunohistochemical study showed that NEDL1 aggregated with mutant SOD1s in the Lewy body‐like hyaline inclusions, a typical pathological change in FALS, in the spinal cord anterior motor neurons of both FALS patients and mutant SOD1 transgenic mice. Furthermore, NEDL1 physically bound to p53, enhanced its transcriptional activity and induced apoptosis in a p53‐dependent manner.
Discussion and conclusions: In the present study, we examined the interaction of NEDL1 with mutant SOD1s and characterized the function of NEDL1 in induction of apoptosis. NEDL1 is a quality control E3 ligase that recognizes mutant SOD1 to form a tight complex in motor neurons of FALS patients. Our findings suggest that NEDL1 may play a pivotal role in inducing neuronal cell death through functional interaction with mutant SOD1 and p53.
P171 HIGH INCIDENCE OF UBIQUITINATED INCLUSIONS IN AMYOTROPHIC LATERAL SCLEROSIS FROM AN AGED COHORT
Hiroyuki H1, Saito Y3, Ikemura M1, Sengoku R1, Sakiyama Y1, Sawabe M3, Mori H2, Murayama S1
1Department of Neuropathology, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan, 2Department of Neuroscience, Osaka City University, Osaka, Japan, 3Department of Pathology, Tokyo Metropolitan Geriatric Hospital, Tokyo, Japan
E‐mail address for correspondence: [email protected]
Background: Ubiquitinated neuronal intracytoplasmic inclusion (UI) is interpreted to be a hallmark of frontotemporal dementia with and without amyotrophic lateral sclerosis (ALS).
Objectives: We examined incidence of UI from an aged cohort and its pathological significance.
Materials and methods: We examined 1822 serial autopsy cases (average 80.7 years old, 974 men and 848 women) from a geriatric general hospital since 1995. Dentate gyrus was screened immunohistochemically with anti‐ubiquitin antibody (polyclonal, Sigma‐Aldrich, St. Louis, MO).
Results: UI was observed exclusively in nine (40.9% of total ALS, average age of 71.9 years, five men and four women) out of 22 ALS cases (average age of 74.3 years, eight men and 14 women) in the series. Seven out of the nine UI‐positive (UI+) cases showed subicular degeneration and presented with clinical dementia rating (CDR) zero (two cases); 0.5 (one case); and equal to or above 1 (four cases), while two UI+ cases without subicular degeneration were categorized to CDR zero (one case) and CDR 1 (one case). In contrast, 13 UI‐negative (UI−) cases were classified into CDR zero (11 cases) and CDR 0.5 (two cases). The total clinical course of UI+ cases was 29.2 months and showed no difference from 30.5 months of UI− cases.
Discussion and conclusion: Our study shows that UI is specifically and frequently present in ALS cases from an aged cohort and significantly contributes to their cognitive decline.
P172 NEURONAL INCLUSIONS IN SPORADIC MOTOR NEURON DISEASE ARE NEGATIVE FOR ALPHA‐SYNUCLEIN
Sasaki S, Iwata M
Tokyo Women's Medical University, Tokyo, Japan
E‐mail address for correspondence: [email protected]
Background: Little is known about the immunoreactivity of α‐synuclein in the inclusions, such as Lewy body‐like inclusions and Bunina bodies, observed in ALS/MND, and in particular, that in skein‐like inclusions and basophilic inclusions is almost untouched.
Objective: To examine whether ALS/MND can be categorized as a α‐synucleinopathy.
Methods: Using anti α‐synuclein antibodies, we studied immunocytochemically those inclusions observed in anterior horn cells of ALS. The spinal cords of 28 autopsied sporadic ALS and one MND (with basophilic inclusions) patients, all of which were obtained within six h after death, were investigated (ages 47–83 years; average 65.0 years). Sixteen age‐matched patients without any neurological disease served as controls (ages 35–81 years; average, 64.2 years). Lumbar spinal cords were immunostained with two antibodies to α‐synuclein (a polyclonal antibody to amino acid residues 121–136: anti‐human α‐synuclein S122, IBL and a polyclonal antibody to amino acid residues 124–134 of human α‐synuclein with phosphoserine 129: anti‐PSer 129, a gift from Dr. T. Iwatsubo) and ubiquitin (polyclonal, rabbit, Dako). Immunoreaction was visualized by the streptavidin‐biotin peroxidase complex method.
Results: Immunostaining with anti‐PSer 129 did not produce background staining or anti‐α‐synuclein‐immunoreactive spheroids, but intensely and specifically labeled Lewy bodies, Lewy neuritis and glial cytoplasmic inclusions. Immunostaining with anti‐S122 produced background staining, but intensely labeled Lewy bodies, Lewy neuritis and glial cytoplasmic inclusions. Lewy body‐like hyaline inclusions, skein‐like inclusions, Bunina bodies, basophilic inclusions, or spheroids were not immunostained for α‐synuclein (PSer 129 or S122), whereas Lewy body‐like hyaline inclusions and skein‐like inclusions were immunostained for ubiquitin. In the controls, these inclusions were not observed in the somata of the anterior horn cells or their neuronal processes.
Conclusion: Our findings do not support the hypothesis that ALS/MND could be classified as one of the diseases grouped as α‐synucleinopathies.
P173 PROTEIN SYNTHESIZING SYSTEM IN THE MOTOR NEURONS IN THE SPINAL CORD IN AMYOTROPHIC LATERAL SCLEROSIS: PURSUING THE BEGINNING OF THE ALTERATIONS
Oyanagi K1, Nagasao J1, Yamazaki M1, Okamoto K2, Aoki M3, Watabe K1, Wada M4, Morita T5, Takahashi H6, Mizutani T7, Hayashi H7
1Tokyo Metropolitan Institute for Neuroscience, Tokyo, Japan, 2Gunma University, Gunma, Japan, 3Tohoku University, Miyagi, Japan, 4Yamagata University, Yamagata, Japan, 5Shin‐rakuen Hospital, Niigata, Japan, 6Niigata University, Niigata, Japan, 7Tokyo Metropolitan Neurological Hospital, Tokyo, Japan
E‐mail address for correspondence: [email protected]
Background: Amyotrophic lateral sclerosis (ALS) is a progressive fatal disease involving the upper and lower motor neurons in human adults. Among the neuropathological findings, central chromatolysis (decrease of cytoplasmic RNA and rough endoplasmic reticulum (rER)), axonal accumulation of phosphorylated neurofilaments and fragmentation of the Golgi apparatus have been reported to be the early changes. However, inter‐relationship or hierarchy of these findings and the other findings reported in the protein synthesizing system have not been elucidated.
Objectives: To pursue the beginning and to clarify the inter‐relationship of the findings, the present authors examined: 1) the transcriptional activity of ribosomal (r)RNA gene, 2) amount of cytoplasmic RNA, 3) ultrastructural alteration of the rER, and 4) inter‐relation of molecular chaperone (KDEL) and fragmentation of Golgi apparatus in the motor neurons in the spinal cord and brainstem.
Methods: 1. Patients with sporadic ALS, and experimentally, rats with the SOD1 gene mutation (H46R) and with facial nerve avulsion were used. 2. Transcription activity of the rRNA gene was measured in the motor neurons in the spinal cord in sporadic ALS, SOD1‐transgenic (Tg) rats and in the motor neurons in the facial nerve avulsed rats by AgNORs (silver staining of nucleolar organizer region‐associated proteins) using formalin‐fixed paraffin‐embedded sections. 3. Ultrastructural findings of the rER of the spinal motor neurons in sporadic ALS patients were examined. 4. Immunohistochemistry for KDEL and TGN 46 was performed.
Results: 1. Transcription activity of the rRNA gene decreased in the motor neurons in the spinal cord in sporadic ALS patients. 2. Transcription activity of the rRNA gene did not decrease in the motor neurons in the spinal cord in SOD1‐Tg rats, although the rats showed marked depletion of the number of motor neurons. 3. Transcription activity of the rRNA gene decreased significantly in the motor neurons of the facial nucleus from 2 h after the avulsion, and the number of the neurons depleted from two weeks after the operation. 4. Ribosomal detachment on the rER was found in the spinal motor neurons in sporadic ALS patients. 5. Abnormal localization of KDEL was found even in the spinal motor neurons showing normal‐looking Golgi apparatus in sporadic ALS patients.
Discussion and conclusions: 1) Decrease of the transcription activity of rER in motor neurons is a sign of the very early stages of the degeneration of the motor neurons, and depletion for a certain period induces loss of the motor neurons. 2) Spinal motor neurons in the SOD1‐Tg rats degenerate in a different way from the sporadic ALS patients. 3) Localization of molecular chaperon KDEL changes earlier than the fragmentation of the Golgi apparatus.
P174 GOLGI APPARATUS OF THE MOTOR NEURONS IN PATIENTS WITH ALS AND OTHER MOTOR NEURON DISORDERS
Fujita Y, Yaguchi M, Okamoto K
Gunma University, Maebashi, Japan
E‐mail address for correspondence: [email protected]
Background: Several studies revealed that the Golgi apparatus (GA) of the anterior horn cells was fragmented in amyotrophic lateral sclerosis (ALS) patients and in asymptomatic transgenic mice expressing the G93A mutation of the gene encoding Cu/Zn SOD1. Therefore, GA is one of the early targets of the pathological processes initiating the neuronal degeneration in ALS.
Objectives: We investigated the GA of motor neurons in patients with sporadic ALS and other motor neuron disorders (MND) including familial ALS, juvenile ALS with basophilic inclusions (BIs) and X‐linked spinal and bulbar muscular atrophy (SBMA). We also examined the relationship of any inclusions in MND and Golgi fragmentation.
Methods: Sections from ALS/MND including 16 cases of sporadic ALS, two cases of juvenile ALS with BIs, three cases of familial ALS with SOD1 mutations and five cases of SBMA were immunostained with anti‐MG160, −SOD1 (Cu/Zn) and −1C2 antibodies.
Results: The GA in over 50% of the remaining anterior horn cells and in 13.2% of examined Betz cells was fragmented in the patients with sporadic ALS. In juvenile ALS with BIs, 60% of the preserved large motor neurons showed fragmentation of the GA. Ten of 14 preserved large motor neurons showed fragmentation and reduced numbers of GA. The GA of residual motor neurons in patients with SBMA was normal or reduced in size, and the profiles of small or atrophic GA were different from the fragmented GA. Double immunostaining using anti‐MG160 and ‐1C2 antibodies showed that anterior horn cells bearing an intranuclear inclusion had a normal or atrophic GA. Fragmentation of the GA was not observed in neurons bearing an intranuclear inclusion. On the other hand, the GA of the majority of anterior horn cells containing Bunina bodies, BIs and SOD1‐positive aggregates were fragmented or not strongly stained compared to normal GA.
Discussion and conclusions: We showed abnormal proteinaceous cytoplasmic aggregates may be related to the fragmentation of GA in patients with sporadic ALS, juvenile ALS and familial ALS; however, the fragmented GA was not found in patients with SBMA. The precise molecular mechanisms linking the aggregation of proteins and fragmentation of the GA are not known, and the identification of the molecular composition of the aggregated proteins present in patients with sporadic ALS with a variety of inclusions remains a major challenge.
P175 EVIDENCE FOR SYSTEMIC INFLAMMATION IN PATIENTS WITH ADVANCED AS COMPARED TO MILD AMYOTROPHIC LATERAL SCLEROSIS
Drory VE, Keizman D, Berliner S
Tel‐Aviv Sourasky Medical Centre, Tel‐Aviv, Israel
E‐mail address for correspondence: [email protected]
Background: Inflammatory processes have been described in the brain and spinal cord tissue in ALS. They may be protective to nervous tissues by inducing cell regeneration, but also injurious by causing tissue damage, thereby complicating the assessment of the significance of inflammation for the understanding of the disease, prognosis and treatment options.
Objectives: The aim of the present study was to determine blood levels of parameters of systemic inflammation in patients with amyotrophic lateral sclerosis (ALS) and to correlate findings to clinical variables.
Methods: We determined the intensity of inflammation in the serum of 50 patients with ALS (30 males) without any clinical systemic inflammatory diseases by measuring generally accepted parameters of inflammation (erythrocyte sedimentation rate (ESR), C‐reactive protein (CRP), fibrinogen). Results were compared with the ALS functional rating scale (ALSFRS), which is a sensitive measure of disability.
Results: Patients were 60±14 years old; their ALSFRS was 27±9 (range 7–40). Mean ESR was 14.7±10.7 (range 2–50), CRP was 2.9±3.4 (range 0.04–16.6), fibrinogen was 323±67 (range 184–470). ESR was abnormal in 13 patients; pathologic CRP was found in seven patients. Both ESR and CRP were correlated to ALSFRS (p<0.05), with higher values in more disabled patients. Fibrinogen levels were normal in all patients and were not correlated to ALSFRS.
Conclusions: Although the measured parameters of inflammation were within accepted normal limits in most patients, there was a trend toward higher ESR and CRP in more disabled patients. It is not clear whether this finding is related to a basic inflammatory process in ALS or to disease‐related systemic complications. We intend to follow these patients in order to determine whether the relationship between clinical status and inflammatory measures is maintained over time and whether these simple routine tests might be of prognostic value.
P176 SERUM LEVELS OF SOLUBLE E‐SELECTIN AND L‐SELECTIN IN AMYOTROPHIC LATERAL SCLEROSIS PATIENTS
Bongioanni P1, Metelli MR2, Manzone F2, Fulceri F2, Panicucci E2, Rossi B1, Pietrini P2
1Neurorehabilitation Unit, Department of Neuroscience, Azienda Ospedaliero‐Universitaria Pisana, Pisa, Italy, 2Laboratory of Clinical Biochemistry, Department of Experimental Pathology, Azienda Ospedaliero‐Universitaria Pisana, Pisa, Italy
E‐mail address for correspondence: [email protected]
Background: Immunological abnormalities have been implicated in amyotrophic lateral sclerosis (ALS) pathogenesis. Adhesion molecules are markers of activated endothelial cells up‐regulated by cytokines.
Objectives: The aim of the present study was to investigate whether the vascular cells of ALS patients are activated.
Methods: We assayed repeatedly over a two‐year period soluble E‐selectin (sE‐selectin) and soluble L‐selectin (sL‐selectin) in sera from 69 ALS patients (28 women and 41 men; mean age±SD: 62±10 years). Disease severity was scored by means of the ALS Functional Rating Scale, and patients were subgrouped accordingly into three classes: I (scores between 40 and 31); II (scores from 30 to 11); III (between 10 and 0).
Blood samples were drawn in the morning, and serum was stored immediately at −20°C.
Adhesion molecules were measured by enzyme‐linked immunosorbent assay (ELISA) (R&D systems).
Results: Presented selectin data relate to assays at time of diagnosis (T0) and those at the time of the most recent clinical examination (Tn).
Mean sE‐selectin levels were higher, but not significantly, in class I vs. class II vs. class III patients at To (45.4±31.2, 38.3±20.1, and 23.4±3.1 ng/ml, respectively) and at Tn (34.2±12.7, 30.1±16.6, and 29.6±15.9 ng/ml, respectively).
Mean sL‐selectin levels were mostly in the normal range and did not differ significantly among patients' classes.
Discussion: Our data, although still preliminary, suggest a different role of sE‐selectin and sL‐selectin in ALS: the former might be down‐regulated as disease progresses.
The precise meaning of these findings in terms of vascular cell activation is not clear. Mechanisms involved in such a modulation are currently largely unknown: further studies are much needed to confirm our data and shed light on related molecular events.
P177 THE ACTIVITY OF PROSTAGLANDIN E(2) IN CEREBROSPINAL FLUID FROM AMYOTROPHIC LATERAL SCLEROSIS PATIENTS
Ostrowska M1, Partyka D1, Bielanski W2, Tomik B1, Szczudlik A1
1Department of Neurology, 2Department of Physiology, Jagiellonian University Medical College, Krakow, Poland
E‐mail address for correspondence: [email protected]
Background: Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by selective killing of upper and lower motor neurons. Neuroinflammation is thought to play a role in the pathogenesis of ALS, probably as an accompanying process, contributing to disease progression. Among several inflammatory mediators, prostaglandin E2 (PGE2) has been associated with ALS pathology. PGE2‐induced injury, apart from its pro‐inflammatory and pro‐apoptotic mode of action, occurs through the calcium‐dependent release of glutamate from astrocytes that leads to excitotoxic neuronal cell death. PGE2 has been found to be increased in cerebrospinal fluid (CSF) from ALS patients (1–3).
Objectives: To evaluate the possible involvement of PGE2 in ALS pathomechanism by measurement of the concentration of PGE2 in CSF of Polish ALS patients and comparison with non‐ALS controls.
Methods: Twenty‐one ALS patients (9 females, 11 males, aged 33–73 years, mean 55.19±10.24 years) fulfilling WFN Criteria and 28 diseased, non‐ALS controls (17 females, 11 males, aged 41.92±17.39 years) were enrolled in the study. CSF was obtained from all patients using diagnostic lumbar puncture and stored at −70°C until assayed. The radioimmunoassay (RIA) method was used for PGE2 CSF analysis.
Results: We noticed lower PGE2 levels in CSF obtained from ALS patients than in controls, although the difference was not statistically significant (109.04±132.24 pg/ml and 132.20±114.68 pg/ml, respectively, p>0.05). The mean (±SD) values of PGE2 level in CSF from limb onset and bulbar onset ALS patients were very similar: 107.86±145.40 vs. 112.8±90.16 pg/ml, respectively, p>0.05. The mean (±SD) value of PGE2 level in CSF from ALS patients with predominantly lower motor neuron involvement was higher compared to patients with predominantly upper motor neuron involvement: 140.55±143.33 pg/ml vs. 30.27±44.71 pg/ml, and this difference was statistically significant (p = 0.006).
Discussion and conclusions: Our results do not confirm previous reports of increased levels of PGE2 in CSF from ALS patients. The reason for this might be a different method used for PGE2 measurement. We used radioimmunoassay, whereas former assessments were made by chemiluminescence immunoassay (1) or enzyme‐linked immunoassay (2,3). The latter may give cross‐reactions with particles other than PGE2 and therefore may be less sensitive. Another reason for this discrepancy might be differences in duration of the disease between ALS groups studied: in our study average time of duration of the disease was 27.2 months, while in the previous studies it was 17 months (2) or 11.8 months (1). These results may suggest that PGE2 plays a major role in the earliest phase of ALS, which is in agreement with results of another study (3).
References
- Almer G, Teismann P, Stevic Z, et al. Neurology 2002;58:1277–9.
- Maihofner C, Probst-Cousin S, Bergmann M, et al. Eur J Neurosci. 2003;18:1527–34.
- Ilzecka J. Acta Neurol Scand. 2003;108:125–9.
P178 EVIDENCE FOR ABNORMAL MONOCYTE IMMUNOGLOBULIN RECEPTOR EXPRESSION IN SPORADIC AMYOTROPHIC LATERAL SCLEROSIS (SALS)
Zhang R1, Miller RG2, Gascon R1, Gelinas DF2, Katz J2, Mass J2, Scholtz D2, Lancero M1, Narvaez A1, McGrath MS1
1University of California, San Francisco, San Francisco, CA, USA, 2California Pacific Medical Center, San Francisco, CA, USA
E‐mail address for correspondence: [email protected]
Background: Immunoglobulins (IgG) from ALS patients have been shown to induce microglial activation and motor neuron degeneration in Balb/c mice (1,2) suggesting a pathogenic role for some form of IgG in ALS pathogenesis. In a recent study overall IgG levels were found to decrease with ALS disease progression; however, levels of CD16 (Fcγ receptor III) expression on activated blood monocyte/macrophages (MO) were found to be elevated (3). The systemic modulation of IgG levels and Fc receptors, critical for mediating phagocytosis in macrophages, suggests some critical interplay between macrophages and immune complexes in ALS; no systematic evaluation of Fc receptor expression in ALS has been performed and this was the subject of the current study.
Objectives: 1) To evaluate expression of Fc‐gamma receptors, CD32 (Fcγ receptor II), CD64 (Fcγ receptor I), and CD89 (IgA receptor), on circulating MO in ALS patients and healthy controls. 2) To determine whether expression of immunoglobulin receptors correlated with clinical stage of disease or riluzole treatment in ALS.
Methods: Heparinized blood was obtained from 35 patients with SALS and 30 age‐matched normal controls (N). Flow cytometry was performed to quantitate median fluorescence intensity (MFI) for expression of CD32, CD64, and CD89 on CD14+ MO. Results from immune studies were evaluated in relation to riluzole treatment and severity of neurological impairment as determined by the revised ALS Functional Rating Scale (ALSFRS‐R).
Results: Patients with SALS had significantly elevated levels of CD64 (MFI CD14+CD64+: N, 88.1±26.0; SALS, 121.8±30.8, p<0.0001) and CD89 (MFI CD14+CD89+: N, 113.7±40.5; SALS, 148.4±47.2, p<0.005) expression on blood MO compared to controls. CD32 levels were similar between patients with SALS (MFI CD14+CD32+ = 21.1±21.0) and control subjects (MFI CD14+CD32+ = 1 6.1±16.8). Expression levels of CD64 and CD89 on MO were significantly elevated at all levels of SALS disease severity unrelated to stage of disease. Untreated SALS patients showed significantly higher CD89 MO expression (186.9±44.0, n = 7) compared to patients treated with riluzole (138.0±43.4, n = 23, p<0.05) and normal controls (113.7±40.5, n = 30, p<0.001). Untreated SALS patient MO had lower levels of CD32 than that of riluzole treated, but this difference did not reach statistical significance (untreated = 9.3±13.5, treated = 24.0±22.0, p = 0.107). No difference between riluzole treated and untreated SALS patient MO expression of CD64 was observed, similar to previous findings of unchanged MO CD16 expression.
Conclusions: Patients with SALS showed persistent disease‐associated increases in CD64 and CD89 expression on circulating MO in SALS, which confirms the earlier study of systemic immune alteration in ALS3. Riluzole use was associated with normalization of two MO immune activation parameters (CD89 and CD32), whereas three others (CD16, CD64 and HLA‐DR) remained abnormal. Thus riluzole use, which extends the lifespan of treated patients, may function in part through modulating MO activation suggesting a potential role for MO targeted therapy in ALS.
P179 MONOCLONAL GAMMOPATHY IN PATIENTS WITH (RAPIDLY PROGRESSIVE) MOTOR NEURON DISEASE
Sutedja NA, Notermans NC, Eurelings M, Paf M, Veldink JH, Brugman F, Wokke JHJ, van den Berg LH
University Medical Centre Utrecht, Utrecht, Netherlands
E‐mail address for correspondence: [email protected]
Background: Previous studies have reported higher frequencies of monoclonal gammopathy in patients with motor neuron disease, in particular when clinical symptoms of peripheral motor neuron damage were present. However, these findings should be interpreted with caution due to methodological limitations, such as selection bias and lack of application of a standardized sensitive assay for detection of monoclonal immunoglobulin in serum.
Objective: To establish the frequency of monoclonal immunoglobulin in serum in rapidly progressive motor neuron disease (ALS and PSMA).
Methods: From 1 January 2001 to 1 July 2005, consecutive patients diagnosed with ALS (n = 274) and PSMA (n = 5) were screened for the presence of monoclonal immunoglobulin in serum by agarose electrophoresis and consequent immunofixation of suspected bands.
Results: Clinical characteristics of patients with ALS and PSMA were representative of those reported in previous population‐based studies. Monoclonal immunoglobulin was present in sera of 5.5% of the ALS and 16.9% of the PSMA patients.
Conclusion: The frequency of monoclonal gammopathy in patients with PSMA is higher than in the general population. A recent population‐based study showed that monoclonal gammopathy occurs in 3.2% of persons 50 years of age and older. After adjusting for age and sex, a positive association of monoclonal gammopathy with PSMA remains. (The highest age‐adjusted rate (males age 80 and older) in the general population is 8.3%.) Moreover, monoclonal gammopathy occurs more frequently in PSMA than in ALS patients (16.9% vs. 5.5%).
P180 INVOLVEMENT OF TUMOUR NECROSIS FACTOR SYSTEM IN SPORADIC AMYOTROPHIC LATERAL SCLEROSIS PATIENTS
Cereda C1, Ceroni M2, Cova E1, Corato M3, Rossi B4, Bongioanni P4
1Neurological Institute IRCCS C. Mondino, Pavia, Italy, 2Department of Neurological Sciences, University of Pavia, Pavia, Italy, 3Department of Neurology, Policlinico of Monza, Monza, Italy, 4Unit of Neurorehabilitation, Azienda Ospedaliera Universitaria Pisana, Pisa, Italy
E‐mail address for correspondence: [email protected]
Background: Many hypotheses have been formulated on the pathogenesis of amyotrophic lateral sclerosis (ALS): modifications of dismutase activity of SOD1 protein, aggregates of proteins causing proteasome impairment, excitotoxicity, and autoimmunity. The implication of the immune system in ALS is supported by data on activated T‐cells in spinal cord and brain of sporadic (SALS) patients (1). Our previous findings demonstrated that SALS lymphocytes stimulated with a reactive oxygen species donor show traits similar to those of ALS motor neurons (2). Abnormal levels of interleukin‐6 and tumour necrosis factor (TNFα) have been found in cerebrospinal fluid and sera from ALS patients (3). TNFα and its soluble receptor (sTNF‐Rs) have been found at significantly higher levels in ALS patients' sera (4).
Objectives: The aim of our study was to assay TNFα system expression and describe its time course during disease progression.
Methods: Eighty‐eight ALS patients diagnosed according to the El Escorial criteria were involved in this study. Blood samples from all patients were drawn every two months over six years: on 540 samples 533 TNFalpha, 501 sTNF‐RI and 518 sTNF‐RII assays were carried out by ELISA method using a commercial kit (Bender MedSystems, Vienna, Austria).
Results: TNFα, sTNF‐RI and sTNF‐RII levels were determined in each patient by calculating the mean values of all their assays over time. Such mean values were compared with the range of normal plasma values (TNFα: <14 pg/ml; sTNF‐RI: 0.3–2.9 ng/ml; sTNF‐RII: 1.9–8.5 ng/ml). TNFα levels were higher in 86.5% of SALS patients, whereas only 13.4% and 9.7% of SALS patients showed higher sTNF‐RI and sTNF‐RII levels, respectively. Interestingly, the time course of TNFα, sTNF‐RI and sTNF‐RII expression defined two patients' subgroups: the former had a well‐defined typical time curve for both TNFα and its soluble receptors; the latter displayed a scattered value distribution over time. No correlation was found between the two groups and with any marker of the disease severity.
Discussion: Circulating levels of antigenic TNFα are significantly increased in plasma from ALS patients according to the published data (4). On the other hand, we did not find enhanced sTNF‐Rs levels in our ALS population. A possible explanation could be that we considered a higher number of assays and included not only probable/definite ALS, but also possible ALS patients according to the El Escorial criteria. Our findings suggest that there might not be TNF system modulation in ALS and that increased amounts of TNFα might be a non‐specific inflammatory response.
References
- Smith RG, et al. Neurology 1996;47:S40.
- Cova E, et al. Neurosci Lett. 2006;399:186–90
- Moreau C, et al. Neurology 2005;65:1958.
- Poloni, et al. Neurosci Lett. 2000;287:211.
P181 CHANGES IN GRANULOCYTE COLONY STIMULATING FACTOR AND ITS RECEPTOR IN THE CNS OF AMYOTROPHIC LATERAL SCLEROSIS
Tanaka M, Kikuchi H, Tateishi T, Motomura K, Kira J
Department of Neurology, Neurological Institute, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
E‐mail address for correspondence: [email protected]
Background: Neuro‐glial inflammation is being paid keen interest in the study of ALS pathogenesis. In particular, cytokines/chemokines may play an important role in motor neuron cell death through immune‐mediated neuro‐glial inflammation.
Objectives: The aim of this study was to characterize multiple cytokine profiles in ALS cerebrospinal fluid (CSF) using multiplexed fluorescent bead‐based immunoassay and to clarify the biological significance of relevant cytokines through immunohistochemical analyses of autopsied ALS spinal cords and cell culture assays.
Methods: We simultaneously measured 16 cytokines/chemokines (interleukin (IL)–1β, IL–2, IL–4, IL–5, IL–6, IL–7, IL–8, IL–10, IL–12 (p70), IL–13, IL–17, interferon–γ, tumor necrosis factor‐α, granulocyte colony stimulating factor (G‐CSF), macrophage chemoattractant protein‐1 (MCP–1) and macrophage inflammatory protein‐1β) in CSF and sera from 37 sporadic ALS patients and 33 controls using a multiplexed fluorescent bead‐based immunoassay, a recently developed powerful technology. We also conducted immunohistochemical analyses of the spinal cords from eight autopsied ALS cases and six non‐neurological disease controls as well as cell culture analyses of relevant cytokines and their receptors.
Results: We found that concentrations of G‐CSF and MCP‐1 were significantly increased in ALS CSF compared with controls, but not sera, compared with controls. In the autopsied spinal cords, G‐CSF was expressed in reactive astrocytes in ALS cases but not controls, whereas G‐CSF receptor expression was significantly decreased in large motor neurons of spinal cords from ALS cases as assessed by densitometry. Biologically, G‐CSF had a protective effect on the motor neuron cell line NSC34 under conditions of both oxidative and nutritional stress.
Conclusion: We consider that G‐CSF has a potentially neuroprotective effect on motor neurons in ALS and that down‐regulation of its receptor might contribute to ALS pathogenesis.
P182 INCREASED IL‐13 BUT NOT IL‐4 PRODUCTION BY CD4‐POSITIVE T‐ CELLS AND CD8‐POSITIVE T‐CELLS IN AMYOTROPHIC LATERAL SCLEROSIS
Shi N, Tateishi T, Kikuchi H, Kira J
Department of Neurology, Kyushu University Graduate School of Medicine, Fukuoka, Japan
E‐mail address for correspondence: [email protected]
Background: Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that selectively destroys both upper and lower motor neurons. Although the etiology of the disease is still not clearly understood, recent reports suggest that neuroinflammation may be involved in motor neuron degeneration.
Objective: The aim of this study was to investigate the involvement of immune mechanisms in the pathogenesis of ALS by measuring the intracellular cytokine production profile of peripheral blood CD4+ and CD8+ T‐cells.
Methods: Twenty‐one ALS patients and 16 healthy controls were enrolled in this study. Peripheral blood mononuclear cells were treated for 4 h with phorbol 12‐myristate 13‐acetate and ionomycin in the presence of brefeldin A. After staining the surface antigens, cells were permeabilized and stained for the intracellular cytokines. Finally, the cells were analysed by three‐color flow cytometry using Epics XL System II. In this study, intracellular production of Th1 cytokines (IFN‐γ, TNF‐α and IL‐2), and Th2 cytokines (IL‐4, and IL‐13) in peripheral blood CD4+ and CD8+ T‐cells was measured. We also evaluated the severity of the disease using the ALS Functioning Rating Scale (ALS FRS).
Results: The ALS patients showed significantly higher CD4+IL‐13+ and CD8+IL‐13+‐cell percentages than controls (p<0.05). However, the same group showed no significant change in levels of either Th1 cytokines, or the number of IL‐4‐producing cells. In addition, there was a negative correlation between CD4+IL‐13+ cell percentages and ALS FRS.
Discussion and conclusion: We found here a selective up‐regulation of IL‐13 producing cells in ALS patients. As IL‐13 upregulates MCP‐1 expression in monocytes and microglias, increased IL‐13 producing cells may contribute to the disease process of ALS, as up‐regulation of MCP‐1 has been reported in CSF.