THEME 5 HUMAN CELL BIOLOGY & PATHOLOGY

P105 SKELETAL MUSCLE INVOLVEMENT IN SPINAL AND BULBAR MUSCULAR ATROPHY

KATSUNO M¹, BANNO H², SZUZUKI K², TAKEUCHI YU², KAWASHIMA M², SUGA N², ADACHI H², TANAKA F², SOBUE G²

^IInstitute for Advanced Research, Nagoya University, Nagoya, Japan, ²Department of Neurology, Nagoya University Graduate School of Medicine, Nagoya, Japan

E-mail address for correspondence: [email protected]

Keywords: spinal and bulbar muscular atrophy, skeletal muscle, neurotrophic factor

Background: Spinal and bulbar muscular atrophy (SBMA) is a hereditary motor neuron disease caused by an expansion of a trinucleotide CAG repeat encoding the polyglutamine tract in the androgen receptor (AR) gene. Testosterone-dependent nuclear accumulation of the pathogenic AR plays a pivotal role in the pathogenesis of SBMA, resulting in the fact that this disease exclusively occurs in males. The pathogenic AR is expressed not only in the nervous system but in non-neuronal tissues, providing the molecular basis of systemic complications such as liver dysfunction and diabetes. Most patients demonstrate a high serum level of creatinine kinase, implying myopathy in SBMA.

Objectives: The aim of this study is to elucidate the skeletal muscle pathology and its clinical implication in SBMA.

Methods: Histopathological analysis was performed on the autopsy specimens of the skeletal muscle from SBMA patients and that from model mice. The intramuscular levels of trophic factors are measured with immunoblotting and immunohistochemistry. The levels of growth factor receptors in lower motor neurons are analyzed with immunohistochemistry of the spinal cord.

Results: Histopathological analysis demonstrated the mixture of neurogenic and myogenic changes in the skeletal muscle. Anti-polyglutamine immunohistochemistry showed abnormal accumulation of the pathogenic AR containing the expanded polyglutamine tract in the skeletal muscles of the SBMA patients and in those of transgenic mice. Protein levels of neurotrophic factors, such as brain-derived neurotrophic factor (BDNF), are significantly decreased in the skeletal muscles from the SBMA patients, although the mRNA levels of these factors were not reduced. The expression levels of the receptors for neurotrophic factors were not decreased in the motor neurons within the anterior horn.

Discussion and Conclusions: The present study indicates that the accumulation of the pathogenic AR causes the skeletal muscle pathology in SBMA. The muscle involvement leads to a decrease in the protein levels of neurotrophic factors, that might enhance neurodegeneration in this disease. Our observations are likely to be the theoretical basis for trophic factor supplementation in SBMA patients. Therefore, the skeletal muscles appear to be a primary site of pathology as well as an important target of therapy development.

P106 DECREASED SERUM LEVELS OF PROLYL HYDROXYLASE IN AMYOTROPHIC LATERAL SCLEROSIS

ONO S, IRIE T, YAMAZAKI T, WATANABE T, MIKAMI H, NOMURA M

Teikyo University Chiba Medical Center, Ichihara, Chiba, Japan

E-mail address for correspondence: [email protected]

Keywords: prolyl hydroxylase, collagen, skin

Background: Recent studies of skin collagen in amyotrophic lateral sclerosis (ALS) have shown several abnormalities including decreased diameter, increased solubility of collagen, alterations of cross-linking of collagen, and decreased type IV collagen. Prolyl hydroxylase (PH) is an enzyme involved in collagen biosynthesis. It catalyses the hydroxylation of prolyl residues in procollagen which is uniquely rich in hydroxyproline. PH activity in number of experimental and clinical states has indicated that this enzyme activity is usually increased in conditions associated with an enhanced rate of collagen formation. The changes in serum PH levels are thought to reflect changes in this enzyme occurring in tissues. However, there has been no study concerning serum PH in ALS.

Objectives: To study serum levels of PH in ALS.

Methods: For the measurement of collagen contents of skin, two-3mm biopsy specimens of skin overlying the left biceps were obtained from 15 ALS patients and 15 diseased control subjects. Approximately 0.5mg of each sample was hydrolyzed with 6N HCl in vacuo for 24h at 115°C after flushing with N₂. An aliquot of the hydrolysates was analyzed for its hydroxyproline content on a liquid chromatograph configured as an amino acid analyzer, using ninhydrin with color development at 135°C. For the measurement of serum PH levels, blood samples were obtained from 28 ALS patients, 20 diseased control subjects and 21 healthy control subjects. Serum PH levels were measured with a sandwich enzyme immunoassay (EIA) kit that used a mouse monoclonal antibody against beta subunit of human PH.

Results: The collagen content per dry weight of skin in ALS patients (0.77±0.28 nmol/mg) was significantly smaller (p < 0.001) than in diseased control subjects (1.69±0.20 nmol/mg). The collagen content in ALS showed a progressive decrease in relation to duration of illness. This negative correlation was highly significant (r = -0.75, p < 0.01). Serum PH levels were significantly smaller (p < 0.001 and p < 0.001) in ALS patients than in diseased and healthy control subjects. There was a significant negative correlation (r = -0.91, p < 0.001) between serum PH levels and duration of illness in ALS patients. In addition, there was a significant positive correlation (r = 0.61, p < 0.02) between serum PH concentrations and the collagen content of skin in ALS patients.

Discussion and Conclusions: The present study showed that serum PH concentrations were markedly decreased in ALS patients, which became more pronounced in proportion to the duration of illness. These results indicate a shift in the balance of collagen metabolism towards the direction of a low rate of collagen synthesis in ALS patients. These data suggest that decreased serum PH levels reflect decreased collagen content of skin in ALS patients, and that serum PH may be useful in examining the relationship between the formation and the degeneration in skin in ALS.

P107 CIRCULATING ENDOTOXIN AND SYSTEMIC IMMUNE ACTIVATION IN SPORADIC AMYOTROPHIC LATERAL SCLEROSIS (SALS)

ZHANG R¹, MILLER RG², GASCON R¹, CHAMPION S², KATZ J², LANCERO M¹, NARVAEZ A¹, HONRADA R¹, RULVACABA D¹, MCGRATH MS¹

¹University of California, San Francisco, California, United States, ²California Pacific Medical Center, San Francisco, California, United States

E-mail address for correspondence: [email protected]

Keywords: Amyotrophic Lateral Sclerosis (ALS); Monocyte/macrophage activation; Lipopolysaccharide (LPS)

Background: Recent studies on blood specimens from patients with sALS found elevated levels of abnormally activated monocyte/macrophages (MO). Lipopolysaccharide (LPS) is a systemic macrophage activator that when injected into animals leads to acute neuron cell death and chronic neuroinflammation/neurodegeneration. More specific to ALS, the injection of LPS into SOD1G37R ALS mice model caused a dramatic shortening of their lifespan suggesting that LPS mediated macrophage activation may exacerbate the pathogenesis of ALS in vivo. In an attempt to identity why MOs were activated in ALS patient blood, we evaluated levels of circulating endotoxin (LPS) in the plasma of patients with sALS and compared those results with degree of MO activation and stage of ALS disease.

Objectives: 1) To quantify levels of plasma LPS in sALS patients as compared to control groups, 2) To test whether levels of plasma LPS in blood would correlate with MO activation and/or MO IL-10 expression, and 3) To determine if LPS levels in plasma correlated with clinical stage of disease in sALS.

Methods: Flow cytometry and LAL assay were performed to quantify blood monocyte markers and plasma LPS levels in heparinized blood samples from 13 sALS patients, 13 Alzheimer's (AD) patients, and 13 normal controls (NC). Results from immune studies were evaluated in light of the severity of neurological impairment as determined by ALSFRS-R scores.

Results: Compared to normal plasma (21±7 pg/ml), significantly higher levels of plasma LPS were identified in sALS (42±19 pg/ml, p<0.05) and AD (62±41 pg/ml, p< 0.01) patients. Plasma LPS levels between two disease groups of sALS and AD were similar. Higher levels of MO activation were observed in sALS (Mean CD14DR = 738±242, p<0.05) and AD (Mean CD14DR = 1018±445, p<0.001) as compared to NC (Mean CD14DR = 501±113). Analysis of MO activation markers showed that MO HLA-DR expression varied in a direct relationship to plasma LPS levels in disease groups (Pearson r = 0.7093, p<0.0001). There was a significant negative relationship between plasma LPS and levels of MO IL-10 expression in sALS blood (Pearson r= -0.8861, p=0.0001), but not in the AD and NC groups. Levels of plasma LPS were significantly higher in patients with severe impairment (ALSFRS-R score = 25–36, n = 6; LPS = 52±17 pg/ml) compared to NC (p<0.01) and patients with milder impairment (ALSFRS-R score = 37–48, n = 6; LPS = 34±20 pg/ml, p<0.05); but no difference was found between NC and patients with milder impairment.

Conclusions: This study, for the first time, reveals that circulating endotoxin levels in patients with sALS may be associated with ALS disease progression. The coupling of elevated LPS levels with loss of MO IL-10 expression suggests that endogenous anti-inflammatory capabilities are defective in patients with sALS. Systemic LPS levels and LPS activated MOs represent two new co-factors that may play significant roles in the pathogenesis of ALS and as such represent novel targets for therapeutic intervention in patients with ALS.

P108 EVIDENCE OF ANTIOXIDANT DEFENSE IMPAIRMENT IN LYMPHOCYTES FROM SPORADIC ALS PATIENTS

COVA E¹, CEREDA C¹, BONGIOANNI P², MAZZINI G³, DAVIN A¹, ROSSI B², CERONI M⁴

¹Foundation Neurological Institute “C. Mondino” IRCCS, Pavia, Italy, ²Unit of Neurorehabilitation, Azienda Ospedaliero-Universitaria Pisana, Italy, ³IGM-CNR, Histochemistry and Cytometry, Department of Animal Biology, University of Pavia, Pavia, Italy, ⁴Foundation Neurological Institute “C. Mondino” IRCCS, Department of Neurological Sciences, University of Pavia, Italy

E-mail address for correspondence: [email protected]

Keywords: reactive oxygen species, lymphocytes, Bcl-2/SOD1

Background: Supposed pathogenic mechanisms involved in amyotrophic lateral sclerosis (ALS) have been recently summarized in two main hypotheses. The former proposes that mutated Cu, Zn superoxide dismutase (SOD1) produces increased oxidative stress; the latter suggests that modified SOD1 would undergo aggregation with other proteins [1], such as bcl-2 [2], thus acquiring a toxic activity. Both hypotheses have recently been supported in sporadic cases [3], [4], suggesting a common pathogenic mechanism for both familial and sporadic ALS. In previous work, we provided evidence of a functional impairment of mitochondria, calcium metabolism and SOD1 and bcl-2 expression in lymphocytes from sporadic ALS (SALS) patients, indicating that peripheral tissues may provide information about ALS pathogenesis [5], [6].

Objectives: The aim of this work was to evaluate the amount and the time course of oxidative stress, bcl-2 and SOD1 expression levels in lymphocytes of SALS patients.

Methods: SOD1 and bcl-2 expression, reactive oxygen species (ROS), cell damage and apoptosis were evaluated in lymphocytes isolated by Ficoll gradient from peripheral blood of 15 SALS patients and healthy controls. Western blotting with protein extracts using anti-SOD1 and anti-bcl-2 antibodies was performed. ROS, cell damage and apoptosis were determined by flow cytometry using the fluorescent dyes dihydrorhodamine 123, Hoescht33342/Iodium propide and Annexin V.

Results: At the diagnosis, SOD1 and Bcl-2 expression was significantly lower in patient (SOD1: 1.29±0.12, arbitrary units (A.U.); bcl-2: 1.61±0.29, A.U.) than in control (SOD1: 2.28±0.22, A.U., p < 0.05; bcl-2 3.83±0.53, A.U., p < 0.05) lymphocytes, and decreased during disease progression in a statistically significant way (p < 0.05). Flow cytometry technique demonstrated that SALS lymphocytes had significantly higher ROS level and were more damaged and “apoptotic” compared to control cells (p < 0.05). The same analyses performed after about 12 months from diagnosis showed no significant differences for all three assays.

Discussion: The evidence of a progressively reduced SOD1 and bcl-2 expression suggests that cellular pathways involved in the onset of both familial and sporadic forms may be coincident, as hypothesized by others (3,4). Moreover, a deregulation of the antioxidant pathway and abnormalities typical of ALS are present also in lymphocytes, as already suggested by us (6).

P109 FUNCTIONAL CHARACTERIZATION OF TDP-43 MUTATIONS IN SPORADIC AND FAMILIAL ALS PATIENTS

KABASHI E¹, VALDMANIS PN¹, DION P¹, DAOUD H¹, DURHAM HD², VANDE VELDE C¹, ROULEAU GA¹

¹Center of Excellence in Neuromics, Centre Hospitalier de l'Universite de Montreal, and Department of Medicine, University of Montreal, Montreal, QC, Canada, ²Montreal Neurological Institute, and McGill University, Montreal, QC, Canada

E-mail address for correspondence: [email protected]

Keywords: TDP-43, Aggregation, Animal models

TDP-43 was identified as a key component of ubiquitinated aggregates in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Pathological examinations of autopsy tissue have established TDP-43 labeling in most of the neuronal and glial inclusion bodies observed in these two disorders; and in some other neurological disorders. The TARDPB gene (accession number NM_007375) which encodes TDP-43 has been screened in a cohort of ALS patients originating from either France or Quebec to establish whether genetic variants of this gene existed and to investigate whether these could explain the presence of TDP-43 in pathological aggregates. The coding regions of all five exons and of ∼50 bp of each of the flanking introns of TARDBP were sequenced in each ALS patient. We recently reported eight missense mutations in nine individuals — six cases of sporadic ALS (SALS) and three cases from individuals with a family history of ALS (FALS). Bioinformatics software developed to evaluate the impact of amino acid changes predicts that the mutations are likely to affect post-translational modifications and aggregation propensity of the protein. Also using lymphoblasts cell lines derived from ALS patients with TDP-43 mutations, we observed a significant increase of a smaller TDP-43 product (∼25 kDa) alongside the normal product (43 kDa). We further noted that this smaller TDP-43 product was not only specific to individuals were it was mutated (when compared to wild-type TDP-43), but that it was only observed in the detergent insoluble fraction of protein lysates and not in the soluble fraction. We are now in the process of establishing cellular and in vivo models were mutant TDP-43 will be transiently expressed to i- examine if the mutant form of TDP-43 will lead to the formation of cytoplasmic aggregates which will contain the mutant protein; ii- to test if the role of TDP-43 in splicing is affected by these mutations and iii- investigate if the protein normal interactions are altered. Altogether these experiments will help to establish whether these mutations lead to an abnormal gain function of mutant TDP-43 or whether they lead to a loss of function of the protein.

P110 EXTRANEURONAL TDP-43 PROTEIN AGGREGATES IN INCLUSION BODY MYOSITIS

YAN J, ZAFAR MS, FECTO F, CALIENDO J, SUFIT R, HELLER S, AJROUD-DRISS S, SIDDIQUE T

Northwestern University Feinberg School of Medicine, Chicago, IL, United States

E-mail address for correspondence: [email protected]

Keywords: TDP-43, Inclusion body myositis, autophagy

Background: The pathogenetic role of TDP-43 in neurodegeneration was first suggested from its presence in ubiquitinated inclusions in sporadic and non-SOD1 familial ALS. A genetic aetiology was attributed to ten missense mutations of TDP-43 in ALS. TDP-43 is highly conserved and ubiquitously expressed, but its prevalence in muscle pathology is not known. Inclusion body myositis (IBM) is the most common debilitating muscle disease over the age of 50. Its pathogenesis is unclear; a major issue in IBM pathology is its degenerative nature. The presence of abnormal protein aggregates like beta amyloid and tau protein in IBM strongly supports a degenerative paradigm, but the invasion of cytotoxic T cells and upregulation of MHC I antigen suggest an inflammatory component.

Objectives: To explore the extraneuronal presence of TDP-43 pathology and its possible role in the pathogenesis of IBM

Methods: Muscle biopsy tissues from pathologically confirmed IBM cases were used for immunohistochemistry (IHC), confocal and Western blot analysis. Normal human muscle biopsies were used as controls. Genomic DNA was extracted from muscle samples, amplified with Taq Gold DNA polymerase, then sequenced on the Beckman Coulter CEQ 8000 Series Genetic Analysis System.

Results: Ten IBM cases were studied. TDP-43 aggregates were present in the IBM rimmed vacuoles. IHC of serial muscle sections showed localization of TDP-43 in the same vacuoles with other proteins including beta-amyloid, Tau, valosin-containing protein (VCP), oligo- and multi-ubiquitin, microtubule-associated protein light chain 3 (LC3B) and p62. The colocalization was further validated by confocal microscopic examinations. The sequencing of TDP-43 and western blot analysis are currently underway.

Discussion and Conclusions: This is the first report of extraneuronal presence of TDP-43 pathology and might be of pathological and clinical relevance not only to TDP-43 proteinopathy and also to muscle pathology. The finding of colocalization of TDP-43 with beta-amyloid and tau lends more weight to the degenerative nature of IBM. VCP is causative of hereditary inclusion body myopathy with Paget's disease of the bone and frontotemporal dementia (IBMPFD). The colocalization of TDP-43 and VCP suggests complex pathways in protein regulation in the cell. LC3B, a homologue of yeast Atg8, is an essential component and marker of the autophagy. p62 may link polyubiquitinated protein aggregates to the autophagy machinery. The colocalization of TDP-43 with LC3B and p62 might suggest a role of autophagy in the degradation of TDP-43 and IBM pathogenesis. The undergoing sequencing and Western blot analysis will clarify more on the pathogenic roles of TDP-43 in IBM.

P111 FAMILIAL ALS: CLINICOPATHOLOGICAL CORRELATES

SREEDHARAN J, HORTOBAGYI T, GANESALINGAM J, ROGELJ B, VANCE C, NISHIMURA A, WRIGHT P, AL-CHALABI A, LEIGH PN, SHAW C

¹Institute of Psychiatry, King's College London, London, United Kingdom, ²King's College Hospital, London, United Kingdom

E-mail address for correspondence: [email protected]

Keywords: genetics, pathology, clinical

Background: We have now conducted linkage studies using single nucleotide polymorphism (SNP) DNA microarrays in a number of ALS kindreds and have previously reported our identification of TDP-43 mutations in one family. Since then further groups have identified TDP-43 mutations.

TDP-43 inclusions are seen in both ALS and frontotemporal dementia with ubiquitinated inclusions (FTLD-U) and both conditions have significant clinical overlap as well. It is therefore of note that no mutations have thus far been described in FTLD-U kindreds. Interestingly, none of the published kindreds with TDP-43 mutations have features of dementia. TDP-43 mutations still only account for a small number of fALS cases and, although a key discovery, the continued study of other fALS kindreds is imperative.

Objectives: To correlate clinical and pathological features in fALS kindreds.

Methods: fALS cases were identified and examined by consultant neurologists. Clinical and neurophysiological data were also obtained retrospectively from case notes. Pathological studies were conducted using immunohistochemistry (including TDP-43 and ubiquitin staining) and immunoblotting.

Results: Clinical phenotype varied dramatically within one particular kindred in which typical ALS, PLS and ALS-FTD were all seen, although there was insufficient power to detect linkage and post-mortem tissue was not available. Conversely, in two other kindreds, affected individuals had a strikingly similar clinical phenotype. This is in clear contrast to mutant SOD1 fALS where phenotypic heterogeneity is seen (the D90A mutation being an exception). Pathological studies were successfully conducted on brains and spinal cords from selected cases from these two families.

Discussion and Conclusions: ALS can present with bulbar or limb, upper or lower motor neuron features, and it is increasingly clear that there is an overlap with FTD. The families we present demonstrate phenotypic variability and stereotypy. Pathological studies are important and our results demonstrate that correlating post-mortem data with clinical phenotype will provide valuable insight into disease pathogenesis.

P112 THE EXPRESSION OF TAU pThr¹⁷⁵ IN ALS AND ALS WITH COGNITIVE IMPAIRMENT (ALSCI)

YANG W, STRONG M

Robarts Research Institute, London, Ontario, Canada

E-mail address for correspondence: [email protected]

Keywords: frontotemporal dementia, tau, phosphorylation, ALSci

Background: We have previously demonstrated the neuropathological hallmark of ALS with cognitive impairment (ALSci) is the presence of a frontotemporal lobar degeneration in association with both intracellular (neuronal and astrocytic) and extracellular tau deposition. We have further identified that tau isolated from ALSci is uniquely phosphorylated at pThr¹⁷⁵ and that tau-aggregate bearing neurons also over-express GSK3β.

Objectives: To determine whether the expression of tau pThr¹⁷⁵ in ALS and ALSci is dependant upon the CNS region of interest, we have developed a rabbit polyclonal antibody to tau pThr¹⁷⁵ and compared its immunoreactivity to either traditional tau antibodies or Gallyas staining.

Method: 6 micrometer paraffin-embedded sections of the superior frontal gyrus, anterior cingulate gyrus, amygdala, hippocampus, entorhinal cortex, basal ganglion and substantia nigra were taken from neuropathologically confirmed ALS [5] and ALSci [5] patients. Sections were immunostained by using rabbit polycolonal tau antibody against pThr¹⁷⁵ without antigen retrieval. Endogenous peroxidase was quenched with 3% hydrogen peroxide (BDH). The primary antibody was incubated at 4^oC overnight. Tissue sections were incubated in 1:200 biotinylated anti-rabbit IgG secondary antibody for 1 hour at room temperature followed by substrate development using the Vectastain ABC kit as per the manufacturers instructions. The extent of pThr¹⁷⁵ immunostaining was described on the basis of both distribution and morphology.

Results: Tau pThr¹⁷⁵ immunoreactive neurons and neurites were observed in the entorhinal, hippocampal, substantia nigra and amygdale of ALS and ALSci. Either punctate, pretangled or tangled neuronal deposition was observed in the entorhinal cortex and the hippocampus, and as a granular deposition in the substantia nigra. In ALci, tau pThr¹⁷⁵ immunoreactive inclusions were mainly distributed in the superficial layers of the entorhinal cortex, in regions CA1 and CA2 of the hippocampus, and throughout the substantia nigra. pThr¹⁷⁵ tau neuronal deposition was occasionally observed in the superior frontal gyrus and the anterior cingulate gyrus of ALSci, but not in ALS. In addition, tau pThr¹⁷⁵ immunoreactive oligodendroglia were occasionally seen in the deeper cortical layers of the superior frontal gyrus of ALSci. No pThr¹⁷⁵ immunoreactive neuritic plaques exist in these given regions. Tau pThr175 immunoreactive pathology was rarely observed in ALS.

Conclusions: Using a polyclonal antibody directed to an ALSci-specific tau phospho-epitope (tau pThr¹⁷⁵), we have observed tau immunoreactive inclusions in a widely distributed pattern in ALS. Of note, only ALSci demonstrated tau pThr175 immunoreactivity in the superior frontal gyrus and anterior cingulate gyrus of ALS.

Research supported by the Scottish Rite Charitable Foundation.

P113 PSEUDOPHOSPHORYLATION OF TAU AT THREONINE 175 (¹⁷⁵THR) IN ALSCI IS ASSOCIATED WITH TAU FIBRIL FORMATION IN VITRO

GOHAR M, YANG W, STRONG W, VOLKENING K, LEYSTRA-LANTZ C, STRONG M

Robarts Research Institute, London, Ontario, Canada

E-mail address for correspondence: [email protected]

Keywords: tau, phosphorylation, frontotemporal dementia

Background: Amyotrophic lateral sclerosis with cognitive impairment (ALSci) is associated with many of the neuropathological features of a frontotemporal lobar degeneration, including tau deposits [1]. We have shown that tau isolated from ALSci patients differs from neurologically normal (control) or Alzheimer's disease (AD) tau in that it is uniquely phosphorylated at ¹⁷⁵Thr (pThr¹⁷⁵ tau)[2]. We have also shown that this is associated with an up-regulation of GSK-3β expression in tau-aggregate containing neurons in ALSci, suggesting that this process may be responsive to pharmacological manipulation (e.g., lithium responsive) [3].

Objectives: We have determined whether tau phosphorylation at ¹⁷⁵Thr is associated with tau fibril formation ex vivo using tau isolated from ALS, ALSci and Alzheimer's disease (AD) and compared this to tau fibril formation in vitro, and whether this impacts on neuronal survival.

Methods: Both soluble and insoluble tau protein was purified from control, ALS, ALSci and AD by HPLC and fibril formation assayed ex vivo using the thioflavin S fluorescence assay. Using both Neuro2A and HEK293T cells, we expressed full-length EGFP-tagged tau constructs harbouring either a pseudophosphorylation at ¹⁷⁵Thr (Thr¹⁷⁵Asp-tau), loss of the phosphorylation site (Thr¹⁷⁵Ala-tau) or wild-type tau (wt-tau). The extent of tau fibril formation was assayed using confocal microscopy, and correlated with the extent of cell death (assayed using MTT assay and activated caspase 3 immunoreactivity). As well, the association of each with β-tubulin was assayed immunohistochemically using confocal microscopy.

Results: The extent of fibril formation as assessed with the thioflavin S assay was significantly greater in tau derived from ALSci, while tau isolated from ALS showed intermediate fibril formation between that of control and AD-derived tau. We next examined the association between pThr¹⁷⁵ tau (using EGFP-Thr¹⁷⁵Asp/Ala or wt-tau) and the formation of intraneuronal fibrils in vitro. Both fibril formation (as measured by the presence of EGFP-positive fibrils instead of punctate staining by confocal microscopy) and cell death (as measured by MTT and activated caspase 3 immunocytochemistry) were significantly enhanced in the presence of Thr¹⁷⁵Asp-tau, regardless of the tau isoform. We also observed that wt-tau colocalizes with β-tubulin, while Thr¹⁷⁵Asp-tau fails to colocalize.

Conclusions: These data suggest that the unique phosphorylation at ¹⁷⁵Thr in ALSci is associated with increased tau fibril formation, and that phosphorylation at this site can disrupt the normal interaction of tau with β-tubulin.

Research supported by the Scottish Rite Charitable Foundation.

P114 NOVEL ANTIBODIES REVEAL INCLUSIONS CONTAINING MISFOLDED SOD1 IN ALS PATIENTS

NILSSON K, JONSSON A, ANDERSEN PM, GRAFFMO KS, MARKLUND SL, BRANNSTROM T

Umea University, Sweden

E-mail address for correspondence: [email protected]

Keywords: SOD1, sporadic ALS, spinal cord

Background: Mutations in CuZn-superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis (ALS) and are found in 6% of ALS patients. The cause(s) of the disease in the reminder is largely unknown. Misfolded and aggregation-prone forms of mutant SOD1s are thought to trigger the disease. In several other neurodegenerative conditions such as Alzheimer′s, Parkinson′s and Creutzfeldt-Jacob′s diseases, protein that are mutated in some of the familial patients are also involved in the pathogenesis in patients lacking such mutations.

Objective: Could wild-type SOD1, by analogy, be involved in ALS patients lacking SOD1 mutations?

Methods: Two sets of novel antibodies, raised in rabbits and chickens, against peptides spaced along the human SOD1 sequence, were developed and shown to be specific for denatured SOD1. These were used to examine SOD1 in spinal cords of ALS patients lacking mutations in the enzyme.

Results: Small granular SOD1-immunoreactive inclusions were found in spinal motoneurons of all 37 sporadic and familial ALS patients studied, but only sparsely in 3 of 27 neurodegenerative and 2 of 19 non-neurological control patients. The granular inclusions were by confocal microscopy found to partly colocalize with markers for lysosomes but not with inclusions containing TAR DNA binding protein-43, ubiquitin or markers for endoplasmatic reticulum or mitochondria. Granular inclusions were also found in carriers of SOD1 mutations and they were the major type of inclusions detected in ALS patients homozygous for the wild type-like D90A mutation.

Conclusion: The findings suggest that SOD1 may be involved in ALS pathogenesis in patients’ lacking mutations in the enzyme.

P115 ROLE OF THE TRANSCRIPTIONAL CO-ACTIVATOR PGC-1α IN ALS?

SARLETTE A, KRAMPFL K, DENGLER R, PETRI S

Department of Neurology, Hannover Medical School, Hannover, Germany

E-mail address for correspondence: [email protected]

Keywords: PGC-1alpha, oxidative stress, mitochondria

Background: Cell death induced by reactive oxygen and nitrogen species (ROS, RNS) has been shown to be a pathomechanism in both familial and sporadic ALS. Mitochondria are the major source of cellular ROS production, which further increases if mitochondria are damaged ([1], [2]). The transcriptional co-activator peroxisome proliferator-activated receptor-γ co-activator 1α (PGC-1α) regulates mitochondrial metabolism and biogenesis via activation of transcription factors such as nuclear respiratory factor-1 (NRF-1) [3]. It also plays a role in maintaining synaptic function [4]. Alterations in PGC-1α expression and function have previously been described in models of Huntington's and Alzheimer's disease [5], [6].

Objectives: In the present study, we investigated the mRNA and protein expression of PGC-1α and NRF-1 in human post mortem tissue of ALS patients as well as in the G93A-ALS mouse model in presymptomatic, early –and late symptomatic stages.

Methods: We performed in situ hybridization histochemistry, quantitative real time PCR, immunohistochemistry, and Western blot experiments on human post mortem tissue of ALS patients and age matched controls and on spinal cord tissue from the G93A mouse model. We also studied PGC-1α expression and subcellular localization in primary motor neuron cultures derived from transgenic ALS mice and non-transgenic littermates.

Results: Both in post mortem ALS tissue and in G93A mice, we observed a reduction of PGC-1α and NRF-1 at the mRNA and protein level, in the animal model already detectable before symptom onset.

Discussion and Conclusion: We therefore conclude that a decrease in PGC-1α could contribute to reduced antioxidant defense mechanisms in familial and sporadic ALS and that it may represent an interesting therapeutic target.

P116 IMMUNOHISTOCHEMICAL EXPRESSION OF IGF-I AND GSK IN THE SPINAL CORD OF KII AND GUAM ALS PATIENTS

KIHIRA T², SUZUKI AI¹, KONDO T¹, WAKAYAMA I², YOSHIDA S², HASEGAWA K³, GARRUTO R⁴

¹Kansai University of Health Science, Kumatori-Cho, Japan, ²Wakayama Medical University, Wakayama-City, Japan, ³Sagamihara National Hospital, Sagamihara-City, Japan, ⁴The State University of New York, Binghamton, Japan

E-mail address for correspondence: [email protected]

Keywords: IGF, GSK-3α/β; focus ALS

Background: Insulin-like growth factor-I (IGF-I) is a potent survival factor for motor neurons in animals and glycogen synthase kinase-3β (GSK-3β) ??is suspected to have a role in inflammation, apoptosis and tau phosphorylation. However, little information is available on changes in IGF and GSK signalling pathways in ALS patients.

Objectives: We examined the immunological expression for IGF-I, GSK-3β and phospho (p)-GSK-3α/β in the spinal cord and the hippocampus of Kii and Guam ALS patients.

Methods: Sixteen ALS patients (10 sporadic Japanese ALS patients, 3 Kii ALS patients, 3 Guam ALS patients) and 12 neurological controls (8 Japanese patients, 4 Guam patients) were examined. Paraffin embedded sections 8-µ m thick were stained with the immunohistochemical technique using the ABC system (Vector Laboratories, Inc., Burlingame, CA, USA). The immunoreactivity for each antibody was scored as negative (0), slightly positive (1), mildly positive (2) and marked positive (3). Antibodies used in the present study were mouse anti-IGF-I monoclonal antibody (CEHMICON INTERNATIONAL, USA), anti-GSK-3β ?monoclonal and rabbit anti-GSK-3α/β polyclonal antibodies (CELL SIGNALLING TECHNOLOGY, USA).

Results: Motor neurons in the spinal cords from the Japanese neurological controls were mildly positive with anti-IGF-I and anti-GSK-3β antibodies and were slightly positive for anti-p-GSK-3α/β antibody. Those from Guam controls also showed mildly positive for anti-IGF-I and anti-p-GSK-3α/β antibodies. Spinal motor neurons from the sporadic Japanese ALS patients were almost negative for anti-IGF-I antibody, but mildly positive for anti-GSK-3β and anti-p-GSK-3α/β antibodies; however, those from Kii ALS patients with a long clinical duration showed marked positive for anti-IGF-I and p-GSK-3α/β antibodies. Spinal motor neurons from Guam ALS patients with short clinical durations were almost negative for anti-IGF-I and anti-GSK-3β, but marked positive for anti-p-GSK-3α/β antibodies. Ratios for immunoreactivity scores of GSK-3β to p-GSK-3α/β in the spinal motor neurons were different between Kii and Guam ALS patients and Japanese sporadic ALS patients (p < 0.01). Immunological co-localization of IGF-I and p-GSK-3α/β was seen in spinal motor neurons from Kii ALS patients using confocal laser scanning. Neurofibrillary tangles in the hippocampus from Kii and Guam ALS patients were positive for anti-tau and anti-p-GSK-3α/β? antibodies, while negative for anti-GSK-3β?antibody.

Discussion and Conclusion: Positive immunoreactivity for IGF-I might be related to the clinical duration of ALS. Kii and Guam ALS patients showed a similar immuno-staining pattern for GSK-3β and p-GSK-3α/β? which was different from the Japanese sporadic ALS patients. The predominant expression of p-GSK-3α/β compared to GSK-3β in the spinal motor neurons and NFT-laden neurons in the hippocampus was a characteristic finding in Kii and Guam ALS patients. The role of IGF signal pathway and GSK-3β?phosphorylation? in the focus ALS patients should be pursued.

P117 IN SITU DETECTION OF DPP6 EXPRESSION IN THE CENTRAL NERVOUS SYSTEM WITH A NOVEL EFFICIENT IN SITU HYBRIDISATION TECHNIQUE USING 2OME AND LNA MODIFIED DETECTION PROBES.

FLUITER K, TEN ASBROEK-ANNELOOR L, M, A, STA M, TROOST D, ARONICA E, BAAS F

Academic Medical Center, Amsterdam, Netherlands

E-mail address for correspondence: [email protected]

Keywords: gene expression, in situ hybridization, DPP6

Background: LNA (Locked Nucleic Acid) modified DNA oligonucleotides are used to detect miRNA in situ hybridisations. Short oligonucleotides which are fully modified with alternating LNA and 2’-O-Me-RNA (2OME) RNA moieties have very high affinity for matching RNA strands much stronger than the corresponding LNA-modified DNA oligos. We hypothesized that the strong and specific RNA binding of these 2OME/LNA modified oligos would make them ideal for microRNA (miRNA) and mRNA in situ detection. We have used these oligos to develop a “one day” in situ hybridisation protocol allowing detection of both miRNA and mRNA expression in paraffin embedded archival material.

Objectives: To setup a simple hybridisation protocol which can be performed in one day to detect either miRNA or mRNA in archival paraffin embedded samples using the new LNA 2OME modified short oligonucleotides as probes.

Methods: The probes are constructed as 19mer LNA-modified 2OME-RNA oligos with every third base as LNA and the remaining nucleotides as 2OME-RNA. The combination of LNA with 2OME RNA raises the Tm of the oligo with nearly 1.5°C per LNA modification as compared with LNA-DNA oligonucleotides. The oligo sequences were designed to be specific for the target sequences. The LNA/2OME oligo's are 5’ labelled with FAM. The FAM label allows detection and amplification of the signal using a secondary antibody against FAM. For this study we designed LNA/2OME RNA oligo's against a miRNA (mir-134) and a mRNA (DPP6). We tested the specificity of these LNA/2OME RNA oligo's in archival paraffin embedded brain and spinal cord samples. The high affinity of the LNA/2OME probes allows for one hour hybridisations, meaning that the whole protocol can be easily done within one day.

Results: Both the mir-134 probe and the DPP6 probe showed strong and specific staining patterns in brain and spinal cord tissue using the short hybridisation protocols. Both paraffin and frozen sections can be used for this protocol. The signals could be competed for by unlabeled competitor probe indicating specificity. The Mir-134 expression could be detected in pyramidal neurons in the cortex and we could detect high DPP6 levels in neurons in spinal cord and hippocampus.

Discussion and Conclusions: We have set up a convenient and fast in situ hybridisation protocol using new LNA/2OME modified RNA oligo nucleotides. This protocol can be used to detect miRNA and mRNA expression in archival paraffin material.

P118 MICROARRAY ANALYSIS IDENTIFIES THE GENE SIGNATURE OF SPARED VERSUS VULNERABLE MOTOR NEURONE GROUPS UNDERLYING SELECTIVE VULNERABILITY IN MOTOR NEURONE DISEASE (MND)

HEATH P, WOOD E, HOLDEN H, SHAW P

University of Sheffield, United Kingdom

E-mail address for correspondence: [email protected]

Keywords: Selective vulnerability, gene expression, Laser capture

Background: Amyotrophic lateral sclerosis is characterised by the progressive degeneration of upper and lower motor neurones in the motor cortex, brain stem and spinal cord. It leads to skeletal muscle atrophy and weakness, before eventually causing death. The motor neurones affected are primarily alpha motor neurones in the spinal cord and brainstem innervating limb and bulbar muscles, and neurones forming descending pathways of the corticospinal tract. However, other motor neurone groups such as those in the oculomotor nucleus, which innervate the extraocular muscles and Onuf's nucleus in the sacral spinal cord, are spared in the disease [1].

Objectives: This study aims to investigate the differential gene expression profile of spared and vulnerable motor neurone groups, to give insight into the molecular basis of selective vulnerability of motor neurones in the disease process associated with MND.

Methodology: Using human post-mortem tissue from neurologically normal control cases, motor neurones from the lumbar and cervical spinal cord and from the oculomotor nucleus in the brainstem were extracted using laser capture micro-dissection. Total RNA was isolated and amplified using the two step amplification method. Biotin labelled antisense RNA was applied to Affymetrix U133 plus 2 GeneChips and hybridisation, washing and staining were carried out as described previously [2].

Results: The gene expression profiles generated have been compared using the PUMA software programme and differentially expressed genes identified [3]. Thus some 102 genes were upregulated and 185 downregulated in the oculomotor nucleus motor neurones compared to spinal motor neurones. These have been further characterised according to gene ontology terms. Hence genes involved in calcium binding, neurotransmitter receptors, transcription factors and cytoskeletal proteins are differentially regulated in the oculomotor nucleus.

Conclusions: The results indicate that the oculomotor motor neurones are indeed different to spinal cord motor neurones and that these differences may account for their reduced vulnerability to death during ALS.