THEME 9 HUMAN CELL BIOLOGY AND PATHOLOGY

P159 QUANTIFICATION OF GSK-3ß IN PERIPHERAL BLOOD MONONUCLEAR CELLS IN ALS PATIENTS

GONZALEZ-MUÑOZ M ¹, RODRÍGUEZ-MAHILLO AI ¹, MORÁN Y ¹, GIL AYUSO-GONTÁN C ², MONEO I ¹, MARTÍNEZ A ², MORA JS ¹

¹Hospital Carlos III, Madrid, Spain, ² Instituto de Quimica Medica-CSIC, Madrid, Spain

Email address for correspondence: [email protected]

Keywords: GSK-3β, peripheral blood mononuclear cells, biomarker

Background: The glycogen synthase kinase-3β (GSK-3β) phosphorylates many metabolic, signalling, and structural proteins, thereby regulating the neuronal plasticity, gene expression, and cell survival. GSK-3β is known to be altered in nerve tissue in Alzheimer's and ALS, but little is known about its possible alteration in peripheral blood mononuclear cells (PBMCs) and studies published in Alzheimer's are inconclusive.

Objective: The aim of our work was to quantify the total GSK-3β (tGSK), PSer9-GSK-3β (pGSK) and ratio pGSK/ tGSK in PBMSs from ALS patients.

Methods: Sixteen healthy controls (HC) and 22 ALS patients (PALS) were studied. Among patients, 14 were clinically probable ALS (PpALS) and 8 clinically definite ALS (PdALS). PBMCs were separated from whole blood using BD Vacutainer CPT tubes (Becton Dickinson). Once washed, cells were lysed in 600 μL of RIPA buffer and a cocktail of protease inhibitors. Fifty microliters of the lysate were tested for tGSK and pGSK using commercial tests (Enzo Life Sciences). Total protein was measured using a commercial BCA test (Pierce). Quantitative data are shown as median and interquartile range (IQR), in ug/mg, and compared using the non parametric Mann-Whitney test.

Results: No significant differences were found in tGSK between HC (46.85, IQR = 43.72–50.3) and PALS (44.45, IQR = 39.67–55.72), nor among HC and PpALS (45.6, IQR = 36.7–55.72) and PdALS (44.45, IQR = 40.72–70). We neither found significant differences in pGSK between HC (9.5, IQR = 7.75–12.12) and PALS (10.15, IQR = 8.32–13.8), nor among HC and PpALS (10.15, IQR = 8.62–13.8) and PdALS (10.0, IQR = 5.25–19.47). In pGSK/tGSK we neither found significant differences when comparing HC (0.21, IQR = 0.16–0.26) and PALS (0.24, IQR = 0.19–0.29) or PdALS (0.21, IQR = 0.15–0.26) but, although neither were significant (p = 0.08), we found differences when comparing HC with PpALS (0.25, IQR = 0.19–0.33).

Discussion: We found no significant differences in PBMCs tGSK, pGSK or pGSK/tGSK among healthy controls, PALS, neither in PpALS nor in PdALS, although we found a tendency for increased values of pGSK/tGSK in PpALS. As GSK-3β is inactivated by phosphorylation at Ser9, our results may indicate that the GSK-3β activity is decreased in PpALS, although its total levels are not modified. Our results suggest that PBMC GSK-3β levels do not correlate with the high expression observed in nerve tissue of PALS. To date, no studies on GSK-3β levels in PBMCs of PALS have been reported.

Conclusions: No significant differences in tGSK, pGSK or pGSK/tGSK of PBMCs are found among healthy controls, probable ALS and ALS patients. A non-significant tendency to increased values of pGSK/tGSK is found in early stages of ALS.

P160 GLYCOGEN SYNTHASE KINASE-3ß LOCALIZES TO THE CYTOPLASMIC INCLUSIONS IN THE SPINAL MOTOR NEURONS IN AMYOTROPHIC LATERAL SCLEROSIS

NAGAO M ¹, WATABE K ¹, HAYASHI H ¹, MATSUBARA S ¹

¹Tokyo Metripolitan Neurological Hospital, Fuchu, Japan, ²Tokyo Metropolitan Institute for Neuroscience, Fuchu, Japan

Email address for correspondence: [email protected]. jp

Keywords: GSK3β, TDP-43, inclusion body

We immunohistochemically examined glycogen synthase kinase-3β (GSK-3β) expression in the spinal cord in amyotrophic lateral sclerosis (ALS). Spinal cords were obtained from 13 patients with ALS, and 8 controls without neurological disease. Specimens were fixed in 15% neutral formalin for 2 weeks, and then embedded in paraffin. Axial sections (5-μm) of the lumbar spinal cords were excised for immuno-histochemistry. Immunostaining was done with rabbit polyclonal antibodies against GSK-3β, Ser9-phosphorylated GSK-3β at (pGSK-3β ), TDP-43, Ser409/410-phosphorylated TDP-43 (pTDP43). GSK-3β was localized in the punctate structures in the cytosol of motor neurons in the control spinal cord. GSK-3β-immunoreactive (IR) puncta were reduced in ALS. GSK-3β was present in cytoplasmic inclusions (CIs) such as round inclusions and was less frequent in skein-like inclusions. GSK-3β colocalized with TDP-43 in the CIs. GSK-3β-IR CIs were mainly found in neurons without GSK-3β-IR puncta. Our results suggest that aggregation of GSK-3β-IR CIs correlated with the decrease in GSK-3β-IR puncta. Accumulation of GSK-3β in the CIs may be toxic to ALS motor neurons.

p161 the kynurenine pathway and inflammation in amyotrophic lateral sclerosis

CHEN Y^1,2, STANKOVIC R⁴, CULLEN K⁵, MEININGER V⁶, CHUNG R⁷, GUILLEMIN G¹,²

¹Department of Pharmacology, School of Medical Sciences, University of New South Wales, Sydney, Australia, ²St Vincent's Centre for Applied Medical Research Darlinghurst, Darlinghurst, Australia, ³Department of Neurology, St. Vincent's Hospital, Darlinghurst, Australia, ⁴Departments of Pathology, University of Sydney, Sydney, Australia, ⁵Anatomy and Histology, University of Sydney, Sydney, Australia, ⁶Centre for SLA, Hôpital Pitié-Salpétrière, APHP, Paris, Paris, France, ⁷NeuroRepair Group, Menzies Research Institute University of Tasmania, Hobart, Australia

Email address for correspondence: [email protected]

Keywords: kynurenine pathway, excitotoxicity, neuroinflammation

ALS is a motor neuron degenerative disease and the kynurenine pathway (KP) is emerging as a possible cause. The KP metabolizes tryptophan (TRP) and generates neuroactive compounds, picolinate (PIC) and quinolinate (QUIN). The first enzyme is indoleamine-2,3 dioxygenase (IDO-1). This study aims were to characterize the KP in ALS patients (ex vivo) and NSC34 motor neuron cell line (in vitro).

GC/MS and HPLC were used to analyze CSF and serum for QUIN, PIC, TRP and kynurenine levels of ALS patients (n = 150) and controls (n = 20). Antibodies to HLA-DR, IDO and QUIN were used on paraffin embedded ALS spinal cord and motor cortex. In NSC34 cells, RT-PCR and ICC were used to characterize KP enzymes and catabolites; LDH test assessed the effect of QUIN, with and without inhibitors.

Results showed significant increases in CSF and serum TRP (P < 0.0001), KYN (P < 0.0001) and QUIN (P < 0.05) and decrease serum PIC (P < 0.05) in ALS samples. Significant numbers of activated microglia-expressing HLA-DR (P < 0.0001) and increase in neuronal and microglial expression of IDO and QUIN were detected in ALS motor cortex and spinal cord. NSC34 cells stained positive for KP enzymes and catabolites; RT-PCR showed the presence of most of KP enzymes; and LDH production showed a dose dependant increase with QUIN, partially inhibited by antagonists.

Our results indicate the presence of neuroinflammation in ALS and provide the first strong evidence for the involvement of the KP in the neuropathogenesis of ALS.

P162 ELEVATION OF ALTERNATIVE MACROPHAGE ACTIVATION MARKER PARC/CCL18 IN PLASMA FROM PATIENTS WITH AMYOTROPHIC LATERAL SCLEROSIS

ZHANG R ¹, HONRADA R ¹, MADISON C ², HARRIS W ², KATZ J ², FORSHEW DA ², MILLER RG ², McGRATH MS ¹

¹University of California, San Francisco, San Francisco, CA, USA, ²California Pacific Medical Center, San Francisco, CA, USA

Email address for correspondence: [email protected]

Keywords: inflammation, alternative macrophage activation, PARC/CCL18

Background: Inflammation and immune activation are associated with ALS; however, the relevance of these processes to pathogenesis has not been adequately explored. Monocyte/ macrophages (MO) are critical mediators of inflammatory processes, and play a major role in the resolution of inflammation by producing anti-inflammatory cytokines and chem-okines as well as eliminating tissue debris. MO activation comprises two basic patterns: classically activated or type I MO, which are pro-inflammatory effectors, and alternatively activated or type II MO that exhibit anti-inflammatory properties. Involvement of classical MO activation in ALS pathogenesis has been confirmed by various investigations. Our recent studies on gene expression in peripheral blood mono-nuclear cells from patients with ALS showed upregulation of both classic type I interferon-induced genes and type II alternative MO activation genes, suggesting a hybrid activation state that implicates both classical and alternative MO activation in ALS pathogenesis(1). In order to examine whether alternative MO activation observed by gene expression profiling would be confirmed in ALS patients, we evaluated the alternative MO activation marker, PARC/CCL18 in plasma from patients with ALS.

Objectives: 1) To quantify levels of plasma PARC/CCL18 in ALS patients as compared to healthy controls; 2)To determine if plasma levels of PARC/CCL18 correlate with clinical stage of disease in ALS.

Methods: PARC/CCL18 ELISA was performed to quantify plasma levels of PARC/CCL18 in heparinized blood samples from 20 ALS patients (Forbes-Norris MDA/ALS program, CPMC) and 20 healthy controls (HC). Results from this immune study were evaluated in light of the duration of disease or severity of neurological impairment as determined by ALSFRS-R score.

Results: Compared to HC (43.2 ± 26.5ng/ml), significantly elevated levels of plasma PARC/CCL18 were identified in patients with ALS (101.5 ± 83.4ng/ml, p = 0.0069). Plasma PARC/CCL18 levels were independent of ALS disease severity as defined by ALSFRS-R score. However, there was a positive correlation between plasma PARC/CCL18 levels and disease duration in ALS (r = 0.7756, p < 0.0001). Significantly higher levels of plasma PARC/CCL18 were found in ALS patients with the lowest rate of disease progression (DP rate: loss of ALSFRS-R score/month) (DP rate < 1: 119.9 ± 93.4ng/ml, n = 14) as compared to those with the active disease progression (DP rate > 1: 58.5 ± 24.3ng/ml, n = 6) (p = 0.0234).

Conclusions: This study, for the first time, revealed that plasma levels of alternative MO activation marker PARC/ CCL18 were significantly higher in ALS patients, and that increased PARC/CCL18 levels correlated with the duration/ rate of disease progression of ALS. These data suggest that alternative MO activation might be important for survival during ALS disease process by countering pathogenic signaling by classically activated type I MOs. These findings provide new insights into the pathogenesis of chronic MO activation in ALS, and identify a new potential candidate marker for tracking ALS disease progression.

Reference

• Zhang R, Hadlock KG, Do H, et al. J Neuroimmunol 2011;230:114–23.
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P163 MUTANT FUS INDUCES ENDOPLASMIC RETICULUM STRESS IN AMYOTROPHIC LATERAL SCLEROSIS AND INTERACTS WITH PROTEIN DISULPHIDE ISOMERASE

FARG M ¹, SOO K ¹, WALKER A ^1,4, ORIAN J ¹, HORNE M ^3,4, WARRAICH S ^2,5, WILLIAMS K ^2,5, BLAIR I ^2,5, ATKIN J ^1,4

¹Department of Biochemistry, La Trobe University, Victoria, Australia, ²Northcott Neuroscience Laboratory, ANZ AC Research Institute, NSW, Australia, ³Florey Neuroscience Institutes, University of Melbourne, Victoria, Australia, ⁴Centre for Neuro-science, University of Melbourne, Victoria, Australia, ⁵Sydney Medical School, University of Sydney, NSW, Australia

Email address for correspondence: [email protected]

Keywords: FUS fused in sarcoma/ translocated in liposarcoma, endoplasmic reticulum stress, protein disulphide isomerase

Background: Endoplasmic reticulum (ER) stress is increasingly recognized as an important and early pathway to motor neuron death in animal and cellular disease models based on superoxide dismutase 1 (SOD1). ER stress involves upregulation of an important ER chaperone, protein disulphide isomerase (PDI), which is protective against mutant SOD1 aggregation and toxicity.

Objective: Mutations in the gene encoding fused in sarcoma/ tranlocated in liposarcoma (FUS) are linked to amyotrophic lateral sclerosis (ALS), but the mechanisms by which these mutants trigger neurodegeneration remain unknown. FUS is normally located in the nucleus but in sporadic and familial ALS, FUS redistributes to the cytoplasm and forms inclusions, similar to TAR DNA binding protein 43. In this study, we investigated the effect of nuclear and cytoplasmic mutant FUS on ER stress.

Methods: A motor neuron like cell line (NSC-34) was transiently transfected with wildtype and two mutant FUS proteins. Activation of ER stress and ER localisation was assayed by immunocytochemistry and immunoprecipitation. Postmortem spinal cords from two ALS patients and a control patient without any neurological disorders were immunostained with both anti-FUS and anti-PDI antibodies.

Results: We demonstrated that mutant FUS, which mislocalises to the cytoplasm, but not nuclear mutant FUS, specifically triggers ER stress in NSC-34 cells. This was determined by activation of IRE1 and ER stress-specific pro-apoptotic C/ EBP homologous protein (CHOP). Mislocalised mutant FUS co-localised with markers of the ER, indicating that redistribution to the cytoplasm is associated with the ER. Furthermore, mutant FUS also co-localised with PDI in NSC34 cells and PDI was co-localised with FUS inclusions in human ALS lumbar spinal cords, in both sporadic ALS or mutant FUS-linked familial ALS tissues.

Conclusions: These findings implicate ER stress in the pathophysiology of FUS and suggest a possible role for PDI in the misfolding of FUS. This study also provides evidence for common pathogenic pathways in ALS linked to the ER.

P164 AUTOPHAGY-RELATED PROTEINS IMMUNOREACTIVITY IN BASOPHILIC INCLUSION BODY DISEASE

FUJITA K ¹, NISHII M ¹, WATE R ¹, KANEKO S ¹, ITO H ², KUSAKA H ¹

¹ Department of Neurology, Kansai Medical University, Moriguchi, Osaka, Japan, ² Department of Neurology, Kyoto University Graduate School of Medicine, Kyoto, Japan

Email address for correspondence: [email protected]

Keywords: autophagy, basophilic inclusion, stress granules

Background: Basophilic inclusions (BIs) were originally observed in patients with juvenile amyotrophic lateral sclerosis (ALS) and were then found also in adult-onset motor neuron disease with BIs. In a previous immunohistochemical study, we obtained the first evidence for the presence of RNA and certain mRNA-related proteins in BIs, suggesting a link between BIs and stress granules (SGs). However, the exact constituents of BIs and process of formation of these inclusions are yet unknown. Recently, there has been a growing interest in the role of the autophagy-lysosome pathway in neurodegenerative diseases. However, the role of autophagy in BI formation has not yet been studied.

Objective: We studied the involvement of several autophagy-related proteins in BI formation, as well as in several inclusions in other motor neuron disease.

Methods: Applying antibodies specific for autophagy-related proteins, we investigated the neuronal inclusions in the motor cortex and spinal cord in 2 patients with basophilic inclusion body disease (BIBD), in 5 with sporadic amyotrophic lateral sclerosis (SALS), and in 1 with familial ALS with a Cu/Zn superoxide dismutase (SOD1) mutation (FALS) as well as in G93A mutant SOD1 transgenic mice.

Results: In BIBD specimens, BIs showed strong immunoreactivity for microtubule-associated protein 1 light chain 3 (LC3), autophagy-related gene (Atg) proteins, p62, and histone deacetylase 6 (HDAC6). However, BIs were not immunoreactive with antibodies specific for cathepsin D (catD) or lysosomal membrane protein 2 (LAMP2). The other inclusions in other motor neuron diseases, e.g., skein-like inclusions, Bunina bodies, round inclusions, spheroids, conglomerate inclusions or Lewy body-like hyaline inclusions, were not positive for LC3, Atg proteins or HDAC6.

Conclusions and discussion: LC3, p62, Atg, and HDAC6 proteins are effectors essential before the completion of autophagosome formation, whereas catD and LAMP2 are elements that appear after the fusion of autophagosomes with lysosomes. The results of our present study indicate that the autophagy was disrupted before the completion of autophagosome formation in these BIBD patients. Based on the results of our previous and present studies taken together, the following scenario might be plausible: SGs, which aggregate in response to an unidentified stress, are tagged with p62 and initially processed for autophagy. However, defective autophagy accelerates the aggregation of SGs by HDAC6, resulting in the formation of BIs in this disease.

P165 AN IMMUNOHISTOCHEMICAL STUDY OF UBIQUITIN IN THE SKIN OF SPORADIC AMYOTROPHIC LATERAL SCLEROSIS

WATANABE T, ONO S

Teikyo University Chiba Medical Center, Ichihara, Chiba, Japan

Email address for correspondence: [email protected]

Keywords: immunohistochemical study, ubiquitin, skin

Background: Ubiquitin (UB) is implicated in non-lysosomal degradation of short-lived and abnormal proteins and can be demonstrated as a part of many intraneuronal inclusions such as the Lewy body, the Pick body, the neurofibrillary tangle, and the argyrophilic inclusion in multiple system atrophy. The disease-causing proteins in many chronic degenerative conditions have a propensity to form intracellular aggregates containing the ubiquitinated proteins. UB-immunoreactive filamentous inclusions, previously undetected with routine histological methods, have been found in spinal anterior horn cells of patients with amyotrophic lateral sclerosis (ALS) and it has been suggested that they may be characteristic of these disorders. It is unknown, however, whether UB-positive structures are present in ALS skin.

Objectives: We have carried out an immunohistochemical study of UB in skin from ALS patients.

Methods: Skin biopsy samples were taken from the upper left arm of 19 patients with ALS (mean age 61.5 years) and from 21 controls with other neurodegenerative diseases matched for sex and age (mean age 62.1 years). Routine formalin-fixed paraffin-embedded 6 μm sections were immunostained according to standard techniques. The sections were incubated with anti-UB 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 are expressed as the mean ± SD.

Results: The cytoplasm of UB-positive (UB⁺) cells showed no positive immunoreaction. Numerous UB⁺ cells were observed in the epidermis in ALS patients, which became more marked as ALS progressed, and a small number of cells were seen in controls. UB immunoreactivity of UB⁺ cells was markedly positive in the epidermis and moderately positive in some dermal blood vessels and glands in ALS patients. These findings became more conspicuous as ALS progressed. On the other hand, UB + cells of the epidermis, dermal blood vessels and glands in control subjects showed a weak positive reaction even after repeated antigen-retrieval trials. The proportion of UB + cells in the epidermis in ALS patients (mean ± SD, 80.1 ± 15.1%) was significantly higher (p < 0.001) than in controls (4.4 ± 2.2%). There was a significant positive relationship (r = 0.92, p < 0.001) between the proportion and duration of illness in ALS patients, but there was no such relationship in control subjects.

Conclusions: The conspicuous finding in the skin of ALS was the increased UB immunoreactivity, which was even more significant with a longer duration of illness. These data suggest that changes of UB in ALS skin are related to the disease process and that metabolic alterations of UB may take place in the skin of patients with ALS.

P166 ALTERED TENASCIN-R EXPRESSION AND PERINEURONAL NET MORPHOLOGY IN AMYOTROPHIC LATERAL SCLEROSIS

COLLINS M, BOWSER R

University of Pittsburgh, Pittsburgh, PA, USA

Email address for correspondence: [email protected]

Keywords: tenascin, perineuronal net, extracellular matrix

Background: Changes in the extracellular matrix are implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS), yet the mechanisms leading to these changes are unclear. In the CNS, the extracellular matrix is highly specialized and that which surrounds neurons is referred to as a perineuronal net (PNN). The composition of PNN's is diverse, although previous work has established that the protein Tenascin-R (TNR) is necessary for PNN formation. We identified decreased levels of TNR in ALS patients using an unbiased proteomics approach and thus sought to further characterize levels of the protein in postmortem tissue from ALS patients.

Objectives: This study had two principal goals. The first was to determine TNR levels in post-mortem tissue from ALS patients and normal controls. The second was to examine PNN morphology in control and ALS patients.

Methods: We obtained post-mortem spinal cord tissue (paraffin sections and snap frozen tissue) from the University of Pittsburgh ALS Tissue Bank. Normal controls were age-matched to their ALS counterparts. To determine TNR levels, we used a combination of immunoblot and immunohistochemistry using anti-TNR specific antibodies. PNN morphology was assessed by immunohistochemistry using biotinylated lectin from Wisteria Floribunda, a lectin conjugate commonly used to stain PNN's.

Results: Our immunohistochemical analysis of TNR in spinal cord tissue revealed a striking loss of normal TNR immunoreactivity around the cell body and processes of motor neurons in ALS patients. In contrast, the control group showed a robust and continuous staining pattern in these areas. This finding was verified by immunoblot analysis, which showed reduced levels of TNR in tissue extracts from ALS patients relative to controls. Our analysis of PNN morphology also revealed aberrant morphology in ALS, reflecting a loss of normal structure.

Discussion: Our immunohistochemistry and immunoblot results validate our CSF based proteomics analysis and demonstrate a distinct loss of TNR protein and immunoreactivity in the spinal cord of ALS patients. This loss of TNR expression is correlated with abnormal PNN morphology. Collectively, these results suggest that alteration to the extracellular matrix surrounding motor neurons is a component of ALS. Future studies will examine the role of TNR for maintenance of PNN's, as previous studies have shown that TNR is necessary for PNN formation. If TNR is essential for PNN maintenance around adult motor neurons, a loss of TNR expression could severely compromise the viability of motor neurons.

Conclusions: TNR protein level is decreased in post-mortem tissue from ALS patients compared to normal control tissues and abnormal PNN morphology is evident surrounding motor neurons. Further investigation is warranted to examine molecular mechanisms that regulate TNR expression and consequences for motor neuron survival.

P167 NEUROPATHOLOGICAL FINDINGS IN A PEDIGREE WITH ALTERNATING FRONTAL LOBE DEMENTIA AND AMYOTROPHIC LATERAL SCLEROSIS

BRÄNNSTRÖM T, BERGH J, FORSBERG K, MARKLUND S, ANDERSEN P

Umeå University, Umeå, Sweden

Email address for correspondence: thomas.brannstrom@medbio. umu.se

Keywords: FALS, FTD, SOD1

Background: Ubiquitinated inclusions are hallmarks of most forms of frontotemporal dementia (FTD) and of amyotrophic lateral sclerosis (ALS). Pedigrees in which these syndromes alternate are also known. Both these facts point towards a shared pathophysiology between these syndromes. However, few autopsy reports are available from such pedigrees. Here we report findings from 6 patients from a pedigree with more than 2000 known members in 14 generations. Of these 39 patients have ALS or ALS-dementia while 12 have FTD.

Objectives: To characterize and systematically investigate clinical and morphological findings in six autopsies of ALS/ FTD patients.

Methods: Brain and spinal cord sections from six autopsies were investigated by immunohistochemistry. Four different anti-peptide antibodies with specificity for misfolded/disordered SOD1were used as well as antibodies directed at GFAP, ubiquitin, TDP-43, NF and cystatin C.

Results: The mean age of symptoms in the ALS/ ALS-dementia cases is 60.9 years and the mean duration of disease is 30 months. ALS patients in this family have the classical Charcot type of the disease. Of the six patients, 5 showed ALS/ALS-dementia and one had a pure FTD. In the ALS cases a loss of motoneurons in the spinal cord and brainstem was seen. In some of remaining motonerons, aggregates immunoreactive of misfolded SOD1 were found in the cytoplasm. Glial cells showed intranuclear reactivity for SOD1 using antibodies directed against misfolded SOD1.

Degeneration of the corticospinal tract and dorsal column was seen, as well as gliosis in the cortical areas of the frontal and temporal lobes and in the insula. Interestingly, these areas showed microvacuolar degeneration of the superficial lamina, especially in layer II and III.

Discussion: Positron emission tomography (PET) studies on D90A patients using [¹¹C]flumazenil binding has revealed changes both in motor areas as well as in non-motor areas such as the left fronto-temporal and anterior cingulated cortices. Our present finding of pathological microvacuolar degeneration in the superficial layers of temporal and frontal cortices supports in the FTD patient support this notion.

Interestingly in this pedigree both TDP-43 and SOD1 inclusions have been found, findings that some authors have regarded as mutually exclusive.

Conclusion: Pathological changes in the ALS patients showed for ALS characteristic changes and the FTD patient show frontotemporal pathology ssociated with FTD.

P168 OPTINEURIN PATHOLOGY IN ALS, FTLD-TDP, ALZHEIMER'S DISEASE AND HUNTINGTON'S DISEASE

HORTOBÁGYI T, TROAKES C, NISHIMURA AL, KATTUAH W, AL-SARRAJ S, ROGELJ B, SHAW CE

MRC Centre for Neurodegeneration Research, Department of Clinical Neuroscience, Institute of Psychiatry, King's College London, London, UK

Email address for correspondence: [email protected]

Keywords: immunohistochemistry, Optineurin (OPTN), Western blotting

Optineurin (OPTN) is a multifunctional protein involved in vesicular trafficking, signal transduction and gene expression. OPTN mutations were described in eight Japanese patients with familial and sporadic amyotrophic lateral sclerosis (FALS, SALS) (1). OPTN-positive inclusions co-localising with TDP-43 were described in SALS and in FALS with SOD-1 mutations, potentially linking two pathologically distinct pathways of motor neuron degeneration. We have explored the abundance of OPTN inclusions using a range of antibodies in post-mortem tissues from more than 100 cases and controls including sporadic and familial ALS, frontotemporal lobar degeneration (FTLD) and a wide range of neurodegenerative proteinopathies (2). OPTN-positive inclusions were detected in 34% of TDP-43-positive SALS spinal cord and 33% of FTLD-TDP. Western blot of lysates from FTLD-TDP frontal cortex and TDP-43-positive SALS spinal cord revealed decreased levels of OPTN protein compared to controls (p < 0.05), however this correlated with decreased neuronal numbers in the brain. Large OPTN inclusions were not detected in FALS with SOD-1 and FUS-mutation, respectively, or in FTLD-FUS cases. OPTN-positive inclusions were identified in a few Alzheimer's disease (AD) cases. Occasional striatal neurons contained granular OPTN immunopositivity in Huntington's disease (HD) but were absent in spinocerebellar ataxia type 3. No OPTN inclusions were detected in FTLD-tau and alpha-synucleinopathy. We conclude that OPTN inclusions are relatively rare and largely restricted to a minority of TDP-43 positive ALS and FTLD-TDP cases. Our results indicate a role of OPTN in neurodegeneration, however do not support the proposition that OPTN inclusions are crucial in the pathogenesis of ALS, FTLD or other neurodegenerative disorders.

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PI69 CLINICAL FEATURES AND PATHOLOGY OF AUTOSOMAL RECESSIVE AMYOTROPHIC LATERAL SCLEROSIS ASSOCIATED WITH OPTINEURIN MUTATION (P.Q398X)

KAMADA M ¹, IZUMI Y ¹, ITO H ², KAGAWA S ⁴, KUDOU E ⁴, MARUYAMA H ³, NISHIDA Y ⁵, KAWAKAMI H ³, KAJI R ¹

¹ Department of Neurology, Graduate School of Medical Sciences, The University of Tokushima, Tokushima, Japan, ²Department of Neurology, Kyoto University Graduate School of Medicine, Kyoto, Japan, ³Department of Epidemiology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima, Japan, ⁴Department of Human Pathology, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima, Japan, ⁵Department of Neurology, Tokushima, Japan

Email address for correspondence: [email protected]

Keywords: OPTN, autosomal recessive, pathology

Background: OPTN is a causative gene for autosomal dominant, primary open angle glaucoma. We recently found autosomal dominant and recessive forms of optineurin (OPTN ) mutations in patients with ALS.

Objective: We report the clinical features and pathology of ALS patients with the recessive OPTN mutation.

Methods: Clinical data was collected from two patients with the recessive OPTN mutation (p.Q398X). We conducted an autopsy in one patient. Brain and spinal tissue was studied with routine histological examination and immunohistochemical examination.

Result: Case 1: A 54-year-old Japanese woman presented with progressive swallowing difficulty and slurred speech. She was diagnosed bulbar type ALS. She died of respiratory failure at age 61. Duration of symptoms was 7 years. Case 2: A 44-year-old Japanese woman presented with progressive weakness of the right upper limb. She was diagnosed limb type ALS. She died at age 48 of respiratory failure. Duration of symptoms was 4 years. We detected a homozygous Q398X nonsense mutation in the OPTN gene in both patients. Autopsy was performed in case 1. In brain, macroscopic examination showed severe atrophy of bilateral motor cortex. Microscopically, severe loss of Betz cell and pyramidal neurons with gliosis were observed. Neuronal loss in the hypoglossal nucleus and facial motor nucleus was moderate. In spinal cord, macroscopic examination showed bilateral atrophy of the anterior root and the anterior horns of the spinal cord. Microscopically, moderate loss of anterior horn cell and gliosis were observed especially at the level of cervical and thoracic lesions. Bilateral corticospinal tracts were degenerated. Bunina bodies were not found. This case had degeneration in putamen, globus pallidus and substantia nigra. A few ballooned neurons and grain were found in amygdale and ambiens gyrus. OPTN immunohistochemical analysis did not show aggregation of OPTN.

Discussion and conclusions: The clinical phenotypes of these two patients with recessively inherited OPTN mutations were different in the sites of onset and disease courses. Because both patients have the same mutation and share a haplotype for 0.9Mb on chromosome 10 containing OPTN, these clinical differences may be derived from other factors. OPTN mutations may augment activation of nuclear factor-κB (NF-κB), which regulates genes involved at multiple stages of immune responses. The clinical course and phenotype of OPTN ALS may be influenced by environmental factors, including inflammation or other genetic factors affecting differences in inflammation sensitivity. The clinical features of autosomal recessive ALS patients with OPTN mutation appear to be indistinguishable from those of other sporadic ALS patients. Although the prevalence of pathological OPTN deletion and mutations are low, their phenotype similar to that in sporadic cases suggest that inappropriate activation of NF-κB may have a pivotal role in ALS in general.

P170 CHRONOLOGICAL SHIFT IN NEUROPATHOLOGICAL FINDINGS OF PATIENTS WITH ALS IN WAKAYAMA PREFECTURE ON THE KII PENINSULA

KIHIRA T ¹, HIRONISHI M ², KOBAYASHI K ², YOSHIDA S ¹, KONDO T ², MORI I ², MORIMOTO S ³, MURAYAMA S ³

¹Kansai University of Health Sciences, Osaka, Japan, ²Wakayama Medical University, Wakayama, Japan, ³Tokyo Metropolitan Geriatric Hospital & Institute of Gerontology, Tokyo, Japan

Email address for correspondence: [email protected]

Keywords: Kii-ALS, senile plaque, neurofibrillary tangle

Background: The reported incidence of ALS is quite high in the southern part of Wakayama (W) Prefecture on the Kii Peninsula, Japan, including Koza/Kozagawa/Kushimoto (K) area as well as in Guam. Although the incidence of ALS in these areas has gradually decreased since the 1980s, a relatively high frequency of ALS has persisted in K area in contrast to its disappearance on Guam. In the 1950s and 1960s, the neuropathological hallmark of ALS both on Guam and on the Kii Peninsula was the classical ALS pathology with neurofibrillary tangles (NFT) but without senile plaque (SP). Although the profile of cases on Guam changed to that showing SP in the 1990s, limited information is available regarding the profile of ALS cases on the Kii Peninsula.

Objectives: To clarify whether there have been any clinical and/or pathological changes in the profile of recent ALS cases on the Kii Peninsula.

Methods: In K area, samples from seven ALS patients who died before 1980 (Group I, mean 51.9 years old) and from three patients who died after 2000 (Group II, mean 72.3 years old) were re-examined. Six-μm paraffin sections of cerebral cortices, Ammon's horn, brainstem and spinal cord were stained using anti-PHF, anti-ß-amyloid, anti-human amyloidß, anti-TDP-43 polyclonal and monoclonal antibodies. Immunohistochemical examinations were performed using the ABC system and visualized with 3,3′-diaminoventidine. The appearance and distribution of NFT and SP as well as TDP-43 positivity were compared between Groups I and II.

Results: Clinical findings in these ALS patients showed upper and lower motor neuron signs without either overt dementia or parkinsonism and these findings did not differ between Groups I and II. Moderate amounts of NFTs were found in Ammon's horn and, to a lesser degree, in the cerebral cortex and brainstem in six cases of Group I (Braak stage III/IV). There was no apparent SP in cases of Group I, except for one patient who showed mild diffuse plaques. In Group II, all cases showed SPs (CERAD Criteria B) and NFTs in Ammon's horn and the cerebral cortex (Braak stage III/IV), but the distribution and frequency of NFT were less prominent than those in Group I. In both Groups I and II, some spinal motor neurons showed TDP-43-positive cytoplasmic inclusions.

Discussion and conclusions: The appearance of NFTs in the cerebrum and TDP-43-positive inclusions in the spinal cord were characteristic findings of ALS in K area on the Kii Peninsula both before the 1980s and after 2000. The appearance of SPs was a characteristic of recent ALS cases in K area and might be, in part, related to aging. The unique tau-, TDP-43 and ß-amyloid pathology might be combined in ALS cases in the focus area on the Kii Peninsula.