THEME 8 HUMAN CELL BIOLOGY AND PATHOLOGY

P191 THE EFFECT OF HEXANUCLEOTIDE REPEAT EXPANSIONS ON C9ORF72 TRANSCRIPT LEVELS IN PATIENTS WITH AMYOTROPHIC LATERAL SCLEROSIS

De Muynck L^1,2

Diekstra F⁴

Van Den Bosch L^1,2

Van den Berg LH⁴

Medic J⁴

Robberecht W^1,3

Veldink JH⁴

Van Damme P^1,3

^aLaboratory of Neurobiology, Vesalius Research Center, VIB, Leuven, Belgium

^bExperimental Neurology and Leuven Research Institute for Neuroscience & Disease (LIND), University of Leuven, Leuven, Belgium

^cDepartment of Neurology, University Hospital Leuven, Leuven, Belgium

^dDepartment of Neurology, Rudolf Magnus Institute of Neuroscience, University Medical Centre Utrecht, The Netherlands, Belgium

Email address for correspondence: [email protected]

Keywords: C9orf72, quantitative PCR, transcript-specific

Background: A massive expansion of a GGGGCC repeat in the C9ORF72 gene is the most common genetic cause of ALS and causes FTLD and ALS-FTLD as well. C9ORF72 has several transcript isoforms, and the function of the C9ORF72 protein is unknown. Our objective was to study C9ORF72 expression in ALS patients with a C9ORF72 repeat expansion and in controls.

Methods: qPCR specific toC9ORF72 variants 1, 2, and 3 (V1, V2, and V3) and all transcripts of C9ORF72 were performed on a large cohort of blood samples and in fibroblasts of controls and repeat expansion mutation carriers.

Results: In blood and fibroblasts, the V1 transcript was the most abundantly expressed compared to V3, whereas V2 was scarcely expressed. In blood, V1 transcript levels were slightly reduced, but V3 levels were elevated in patients carrying the repeat. There was no difference in the expression of all C9ORF72 transcripts. In fibroblasts from controls and patients from C9ORF72 families, a similar pattern of small reduction in V1 expression and elevation in V3 expression was seen. However, in contrast to blood samples, an increase in all C9ORF72 transcript expression was seen.

Discussion: Our results suggest that expanded GGGGCC repeats in C9ORF72 do no prevent transcription from the affected allele and that haploinsufficiency is not the underlying disease mechanism in patients with C9orf72 repeat expansion mutations.

P192 REDUCTION OF U11/U12 SMALL NUCLEAR RIBONUCLEOPROTEIN IN AMYOTROPHIC LATERAL SCLEROSIS

Ishihara T

Kato T

Shiga A

Ariizumi Y

Koyama A

Yokoseki A

Kakita A

Nishizawa M

Takahashi H

Onodera O

Brain Research Institute, Niigata University, Niigata, Japan

Email address for correspondence: [email protected]

Keywords: SMN, snRNA, splicing

Background: TAR-DNA binding protein 43kDa (TDP-43) has a crucial role in the pathogenesis of ALS. TDP-43 is one of the heterogeneous nuclear ribonucleoproteins and co-localizes with GEM, which is one of the nuclear bodies composed chiefly of survival motor neurons (SMNs) (1). SMN is a causative protein for spinal muscular atrophy, an infantile-onset motor neuron disease. SMN plays an important role in maturation of snRNAs which are components of spliceosome. Spliceosome is the machinery that carries out pre-mRNA splicing, and is classified into two types: the major (U1, U2, U4, U5, and U6) and the minor (U11, U12, U5, U4atac, and U6atac) spliceosomes. Depletion of SMN alters the repertoires of snRNAs by cell-type- and tissue-type-specific manner, particularly reduction of U11/U12 snRNAs in spinal cord (2). We have found that U12 snRNA decreased in affected tissues with ALS (Ishihara, reported in SFN 2012). However, it is unclear whether the reduction of the snRNAs alters the amounts and function of minor spliceosome.

Objective: To investigate whether the repertoires of U11/U12 spliceosome alter in affected neuronal tissues with amyotrophic lateral sclerosis (ALS), whether pre-mRNA splicing associates with U11/U12 spliceosome alters in ALS-affected issues.

Methods: Spliceosome is composed of snRNAs and associated proteins complex, small nuclear ribonucleotide protein (snRNP). Thus, we investigated the amount of U11/U12-type spliceosome in spinal motor neurons with ALS patients and controls (n = 4) using immunofluorescent staining technique with antibody to snRNP of 59 kDa, which is a component of minor spliceosome. Quantitative real-time RT-PCR of snRNAs and splicing efficiency of pre-mRNA were performed with RNA from cultured cells and neuronal tissues (spinal cord, motor cortex, and cerebellum as tissues with or without TDP-43 pathology) from ALS patients (n = 7–10) and control individuals (n = 9–10). Total RNA was extracted using mirVana miRNA isolation kit (Ambion).

Results: The fluorescence intensity of snRNP 59K was decreased to 39% in spinal motor neuron with ALS compared with that in control. The level of mRNA in which minor spliceosome dependent intron was included significantly increased in TDP-43-depleted U-87 MG cells (218%), and the same results were obtained in the motor cortex with ALS but not in spinal cord and cerebellum.

Discussion and conclusion: The decreased amounts of snRNP 59K indicate that the reduction of U12 snRNA reduces the level of minor spliceosome. In addition, some minor spliceosome-dependent introns were retained in affected tissue with ALS. Our results suggest that decreasing the function of minor spliceosome may underline the molecular pathogenesis of ALS.

Acknowledgements:

JSPS KAKENHI Grant Number 23890060.

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P193 RNA PROCESSING FACTORS INTERACT WITH GGGGCC REPEAT EXPANSION RNA IN VITRO AND DISPLAY ALTERED LOCALISATION AND EXPRESSION C9ORF72 ALS CASES

Walsh M¹

Cooper-Knock J¹

Dickman M¹

Higginbottom A¹

Highley JR¹

Rattray M²

Kirby J¹

Hautbergue G¹

Shaw PJ¹

^eUniversity of Sheffield, Sheffield, UK

^fUniversity of Manchester, Manchester, UK

Email address for correspondence: [email protected]

Keywords C9ORF72, sequestration, RNA

Background: Expanded GGGGCC repeats in intron 1 of the C9ORF72 gene represent the most common cause of familial amyotrophic lateral sclerosis (ALS). Other neurodegenerative diseases result from expanded repeat sequences in non-coding regions raising the possibility of a common pathogenic mechanism. Toxic gain-of-function through sequestration of MBNL1 is a major factor underlying myotonic dystrophy types 1 and 2. Identification of protein binders of the GGGGCC repeat expansion therefore represents an important area of study which will help us better understand the pathogenic mechanism of C9ORF72 ALS and potentially open new avenues for drug development in the future.

Objective: To identify GGGGCC RNA binding proteins from human neuronal cell and tissue extracts and investigate their pathological significance at both transcript and protein levels using in vivo models.

Methods: Biotinylated RNA molecules (GGGGCC₅ and AAAAUU₅) were incubated with SHSY-5Y total/nuclear extracts or extracts of human cerebellum. Streptavidin pulldown assays were carried out, and recovered proteins were identified using mass spectrometry (MS).

Spinal cord sections were prepared, motor neurons were isolated, and RNA was extracted and analysed using Human Exon 1.0ST GeneChip^® microarrays. Data were analysed using the PUMA suite.

Formalin-fixed, paraffin-embedded slides from the cerebellum of C9ORF72 + cases (n = 3), C9ORF72- cases (n = 3) and controls (n = 3) were labelled with antibodies against hnRNPF/H1, SRSF1 and p62 and visualised using fluorescence microscopy.

Results: Using MS from GGGGCC₅ pulldown experiments, 103 unique proteins were identified. Proteins binding to GGGGCC₅ showed significant enrichment of GO classes related to mRNA splicing/transport/stability. Comparison of MS-identified proteins and transcripts upregulated in C9ORF72+ motor neurons revealed enrichment of similar GO classes with significant overlap of several specific factors implicated in ALS (including hnRNPA3/PURA/FUS/TAF15). Immunofluorescence experiments on C9ORF72+ cerebellar sections revealed cytoplasmic co-localisation between P62/SRSF1 and P62/hnRNPF/H1 in granular layer cells. SRSF1 levels were also shown by Western blot to be increased in C9ORF72+ cases relative to control and C9ORF72- cases in extracts from cerebellar granular layer cells.

Discussion and conclusion: The mass spectrometry data obtained are in agreement with previous findings and significantly expands the number of identified GGGGCC binding proteins. We hypothesise that sequestration of these proteins by the expansion may trigger a cellular response in which the transcript level of these proteins or that of proteins with similar functions are upregulated in a compensatory mechanism. Cytoplasmic mislocalisation and co-localisation with P62 are consistent with C9ORF72-specific extra motor pathology. Under normal circumstances, SRSF1 negatively regulates its own protein expression by influencing the splicing and translation of its own transcripts. If SRSF1 function was impaired, by for example a sequestration event, we might expect to see an increase in its protein level. These data support a RNA toxicity using sequestration model of pathogenesis in C9ORF-related ALS.

P194 C9ORF72 HEXANUCLEOTIDE REPEAT EXPANSION PATHOLOGY IN THE OXFORD BRAIN BANK COHORT

Bäumer D¹

East S¹

Hofer M¹

Kent L¹

Turner M¹

Waite A²

Blake D¹

Morris H²

Talbot K¹

Ansorge O¹

^gNuffield Department of Clinical Neurosciences, Oxford University, Oxford, UK

^hMRC Centre for Neuropsychiatric Genetics and Genomics, Cardiff University School of Medicine, Cardiff, UK

Email address for correspondence: [email protected]

Keywords C9orf72, neuropathology, TDP-43

Background: Hexanucleotide repeat expansions in the gene C9orf72 can cause amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD), or both. The underlying neuropathology is a TDP-43 proteinopathy with additional characteristic TDP-43-negative ubiquitinated inclusions which are most pronounced in the cerebellar granule cell layer. Details of regional C9orf72 expression and its correlation with neuropathology and clinical phenotype are still incompletely understood.

Objectives: To characterise the neuropathology and clinical phenotype of C9orf72 repeat expansion positive cases from the Oxford Brain Bank.

Methods: Fifty cases of ALS, FTLD, or both were identified in the Oxford Brain Bank and screened using immunohistochemistry for p62-positive cerebellar granule cell inclusions. Positive cases underwent genetic testing for the C9orf72 hexanucleotide repeat expansion using repeat-primed PCR (1) as well as further detailed immunohistochemistry work-up including semi-quantitative TDP-43 and p62 staining across various brain regions. RNA was extracted from cerebellum, hippocampus, frontal cortex, and spinal cord in cases with available frozen tissue, and C9orf72 isoform-specific semi-quantitative PCR was performed and compared with age-matched controls.

Results: Eleven cases (5 ALS, 3 ALS-FTLD, and 3 FTLD) showed p62-positive and TDP-43-negative cerebellar granule cell inclusions. All tested positive for the hexanucleotide repeat expansion. Mean age of onset was 57 (range, 39–70) years and disease duration of 4.6 (range, 1–10) years. Spinal-onset ALS was more common than bulbar-onset ALS. The cognitive phenotype of dementia cases was behavioural variant (n = 4) and language variant (n = 2) frontotemporal dementia. Semi-quantitative assessment of p62 and TDP-43 pathology revealed that TDP-43 pathology but not p62- positive non-TDP pathology correlated best with the presence of motor neuron disease, while there was no difference in cases with dementia. Semi-quantitative RT-PCR detected all three major C9orf72 transcripts in all examined regions, most abundant in the cerebellum. The C9orf72 expansion did not lead to detectable new splice variants in the 5’ end of the gene, or a significant shift in isoform ratios.

Discussion and conclusion: C9orf72 hexanucleotide expansions are a common cause of FTLD and ALS in the Oxford brain bank; we describe clinicopathologic and genetic features of a new small cohort of FTLD and ALS caused by C9orf72 hexanucleotide expansions. We focused on the anatomical relationship of TDP-43 pathology and non-TDP-43 pathology with clinical phenotype and differential splicing of C9orf72. The severity and distribution of the non-TDP-43 pathology as assessed by p62 staining did not predict the clinical phenotype in this small cohort. While our results argue against a significant splicing change of C9orf72 in expansion carriers, they do not rule out reduced overall C9orf72 transcript or protein levels.

Reference:

• Renton AE, Majounie E, Waite A et al. A hexanucleotide repeat expansion in C9ORF72 is the cause of chromosome 9p21-linked ALS-FTD. Neuron 2011;72(2):257–68.
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P195 P62 PATHOLOGY AND THE FRONTOTEMPORAL SYNDROME OF ALS

Raaphorst J¹

Steentjes K²

Aronica E²

Baas F³

de Visser M¹

de Jong V¹

Troost D²

ⁱDepartment of Neurology

^jDepartment of Neuropathology

^kDepartment of Genome Analysis, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

Email address for correspondence: [email protected]

Keywords P62, frontotemporal syndrome

Background: P62-positive (TDP-43-negative) inclusions have been shown in the hippocampus, cerebellum and frontal cortex in patients with the expansion of the hexanucleotide repeat in C9orf72 and in the prefrontal cortex in a patient with another mutation. The presence of p62 (a ubiquitin-binding protein that facilitates the degradation of polyubiquinated proteins) as compared to TDP-43 inclusions in non-motor regions of sporadic ALS patients, and the association between p62 inclusions and the presence of the frontotemporal syndrome, is currently unknown.

Objective: i) To examine the presence of p62 inclusions as a marker of non-motor neuron degeneration in sporadic ALS as compared to that in TDP-43 and ii) to examine the relation between p62-positive inclusions in non-motor regions and the frontotemporal syndrome, for example, mild cognitive/behavioural changes or frontotemporal dementia (FTD).

Methods: Post-mortem study was carried out with clinicopathological correlations. The frontotemporal syndrome was diagnosed by an experienced clinician based on complaints of cognitive dysfunction, impairment on ‘bedside’ testing or proxy reports of behavioural changes. We used ALS cases from our brain bank with available paraffin blocks of the hippocampus and the spinal cord and sufficient information on cognitive and behavioural disturbances and/or the presence of dementia. Tissue blocks of the motor cortex and prefrontal cortex (PFC, Brodmann area 10), where available, were also investigated. Following tissue preparation, antibody characterisation and immunohistochemistry, a semi-quantitative analysis was performed for p62, ubiquitin, TDP-43 inclusions. The neuropathological diagnosis of frontotemporal lobar degeneration did not depend on p62 inclusions. The presence of inclusions in clinical subgroups was analysed using chi-square or Fisher's exact test. We are currently examining the C9orf72 hexanucleotide repeat expansion in the cases.

Results: Thirty-two ALS cases (34% bulbar onset; 10 familial, 22 sporadic) were examined of whom seven (22%) had a frontotemporal syndrome (mild in 1 sporadic and 2 familial patients, and consistent with FTD in 3 sporadic and 1 familial patient). The neuropathological diagnosis in all seven patients was ALS in combination with frontotemporal lobar degeneration. Compared to ALS patients without cognitive or behavioural problems, patients with the frontotemporal syndrome more often showed p62 (100 vs. 40%) and TDP-43 (100 vs. 20%) in the hippocampus; and more often p62 (100 vs. 41%), but not TDP-43, in the prefrontal cortex. The latter dissociation between TDP-43 and p62 was seen in both sporadic and familial cases, and was not observed in the spinal cord.

Discussion and conclusion: The more widespread presence of p62 inclusions compared to TDP-43 inclusions in the prefrontal cortex and the relation with the frontotemporal syndrome suggests that non-TDP-43 pathways lead to protein degradation and clinical signs in the prefrontal cortex of familial and sporadic ALS patients.

P196 STRUCTURE, BIOGENESIS, AND CLEARANCE OF ER-DERIVED INCLUSIONS GENERATED BY THE ALS-LINKED MUTANT OF VAPB

Genevini P¹

Papiani G³

Navone F¹

Borgese N^1,2

^lCNR Institute of Neuroscience, Milano, Italy

^mDept. of Health Science Univ. of Catanzaro Magna Graecia, Catanzaro, Italy

ⁿOligomerix Inc., New York, NY, USA

Email address for correspondence: [email protected]

Keywords: endoplasmic reticulum, inclusion bodies, protein degradation

Background: Vesicle-associated membrane protein-associated protein B(VAP-B)is a ubiquitously expressed, ER-resident tail-anchored adaptor protein implicated in inter-organellar lipid exchange, generation of membrane contact sites, and membrane traffic. Its mutant form, P56S-VAPB, has been linked to a dominantly inherited form of amyotrophic lateral sclerosis (ALS8). P56S-VAPB forms intracellular inclusions, whose role in ALS pathogenesis has not yet been elucidated. We recently showed (1) that newly synthesized mutant VAPB is normally targeted to the ER where it rapidly aggregates, generating a restructured ER domain consisting of ribbons of stacked cisternae apparently held together by the mutated cytosolic domain of VAPB. The P56S-VAPB structures are formed also when the mutant protein is expressed at physiological levels, and are continuous with the surrounding normally organized ER, which is, however, devoid of the mutant protein.

Objectives: To investigate the dynamics of inclusion generation and degradation.

Methods: We used stable HeLa-TetOff cell lines inducibly expressing a Myc-tagged version of wild-type VAPB or P56S-VAPB to establish a ‘pulse-chase’ protocol in which cells were first allowed to accumulate the mutant protein in the absence of Doxycyclin; to stop synthesis of VAPB, the antibiotic was reintroduced into the medium and the rate of decay of the two proteins was studied by analysing the levels of Myc-tagged proteins during this chase period.

Results: At variance with most pathological inclusion bodies (IBs), P56S-VAPB inclusions undergo turnover, and the rate of degradation of the aggregated polyubiquitinated mutant is faster than that of the wild-type protein. Degradation of the mutant protein involves extraction of P56S-VAPB from the ER membrane by the p97 ATPase, as suggested by the observation that transfection of a dominant negative p97 stabilizes mutant VAPB. Clearance of the inclusions occurs via the proteasome degradation pathway with no apparent participation of macro-autophagy (2). Involvement of the proteasome in P56S-VAPB elimination does not impair the proteasome's ability to clear a classical ERAD substrate. Autophagocytosis does not appear to be slowed in cells expressing mutant VAPB.

Discussion and conclusion: P56S-VAPB inclusions differ from other IBs, both in the mechanisms of their genesis and in those of their clearance from the cell. Our results reveal surprisingly efficient extraction from the ER and proteasomal degradation of this severely aggregated mutant protein. Furthermore, they suggest that the slow onset of P56S-linked familial ALS is not a consequence of the progressive accumulation of the mutant protein over time, and that the dominant inheritance of the mutant allele may rather be related to haploinsufficiency.

Acknowledgements:

This work was supported by Cariplo Foundation, Regione Lombardia (TERDISMENTAL) and PNR-CNR Aging Program.

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P197 OPTINEURIN AND MYOSIN VI-ASSOCIATED CELLULAR TRAFFICKING DEFECTS IN ALS

Sundaramoorthy V¹

Walker A^2,1

Farg M¹

Soo KY¹

Atkin J^1,3

^oLatrobe university, Melbourne, Australia

^pUniversity of Pennsylvania, Philadelphia, USA

^qFlorey Neuroscience Institute, Melbourne, Australia

Email address for correspondence: [email protected]

Keywords: optineurin, myosin VI, cellular trafficking

Background: Optineurin is a multifunctional protein that plays an important role in cellular trafficking events in conjunction with molecular motors such as Hungtinton, Rab8 and myosin VI. Optineurin in complex with myosin VI also plays a role in the trafficking of autophagosomes. Recently mutations in optineurin were identified to be a cause of familial amyotrophic lateral sclerosis (ALS), and optineurin-positive inclusions were described in sporadic ALS patient's motor neurons.

Objective: In this study, we aimed to define the role of optineurin mutations in the pathogenesis of ALS.

Methods: We analysed secretory protein trafficking in NSC-34 neuronal cell lines expressing optineurin, by using vesicular viral stomatitis protein (VSVG). Autophagy was examined using immunoblotting and immunocytochemistry for LC3. The formation of the optineurin–myosin VI motor complex was examined in sporadic ALS patient spinal cord lysates using immunoprecipitation.

Results: Immunocytochemical studies demonstrated that the ALS mutations triggered morphological changes to the expression of optineurin in neuronal cells. Wild-type (WT) optineurin and a glaucoma-causing mutant (E5OK) formed vesicular structures (defined as < 3 μm²) in NSC-34 cells. In contrast, the ALS optineurin mutants (E478G, Q398X) did not form these structures, but instead resulted in diffused cytoplasmic accumulation and increased optineurin-positive inclusions (defined as > 3 μm²). Furthermore, immunoprecipitation studies revealed that the ALS optineurin mutants did not bind to myosin VI, unlike WT and the Glaucoma mutant optineurin. Consistent with this observation, in neuronal cells expressing ALS mutant optineurin, protein trafficking from the Golgi to the plasma membrane was significantly inhibited in comparison with control cells expressing the WT optineurin or Glaucoma mutant. We further observed by immunoprecipitation that ALS optineurin mutations disrupt the complex formation of myosin VI with secretory proteins (VSVG). These results indicate that optineurin acts as a linker, connecting myosin VI with secretory vesicles during protein trafficking from the Golgi apparatus to plasma membrane. We also observed that ALS optineurin mutants induced ER stress and Golgi fragmentation in NSC-34 cells, consistent with the inhibition of protein trafficking via the classical secretory pathway. Furthermore, in cells expressing ALS optineurin mutants, there was an accumulation of autophagosomes, causing defective autophagy, suggesting that disruption of the optineurin-myosin VI complex impairs autophagy. Moreover, immunoprecipitation studies revealed that myosin VI did not co-precipitate with optineurin in sporadic ALS patient tissues in contrast to control patients, revealing that the optineurin-myosin VI complex is also disrupted in sporadic ALS.

Conclusion: This study demonstrates that optineurin is essential for Myosin VI motor function. ALS-causing mutations in optineurin disrupt the normal function of optineurin-myosin VI complex resulting in cellular pathogenic events. Interestingly, myosin VI motor complex defects were also present in sporadic ALS patient tissues, implicating optineurin-myosin VI trafficking defects in the pathogenesis of sporadic ALS.

P198 SOD1, TARDBP, AND FUS: A LINK BETWEEN GENE EXPRESSION LEVELS AND PROTEIN AGGREGATION IN NON-MUTATED AND MUTATED ALS PATIENTS

Gagliardi S¹

Milani P¹

Pansarasa O¹

Diamanti L^2,3

Polveraccio F²

La Salvia S²

Drufuca L²

Ceroni M^2,3

Cereda C¹

^r“C. Mondino” National Institute of Neurology Foundation, IRCCS, Laboratory of Experimental Neurobiology, Pavia, Italy

^sUniversity of Pavia, Department of Brain and Behavioral Sciences, Pavia, Italy

^t“C. Mondino” National Institute of Neurology Foundation, IRCCS, Division of General Neurology, Pavia, Italy

Email address for correspondence: [email protected]

Keywords: protein aggregation, mRNA, ALS patients

Background: Alterations in RNA metabolism have been shown in ALS opening new research perspectives on its pathogenesis (1). In this context, we have already demonstrated the increase in SOD1 mRNA level in SALS patients tissues compared to controls (2).

Objectives: Here in, we described SOD1, TARDBP and FUS mRNA levels and protein aggregation in peripheral blood mononuclear cells (PBMCs) of mutated, non-mutated sporadic ALS patients (SALS) and in sex- and age-matched healthy subjects.

Methods: We analyzed 70 sporadic ALS (SALS) and 70 controls for SOD1, TARDBP, FUS, using real-time PCR. We also included SALS patients mutated in SOD1 (L106F), TARDBP (A382T), and FUS (R521C).

We also performed immunofluorescence experiments to morphologically evaluate the subcellular distribution and appearance of the three proteins in lymphoblasts of mutated, non-mutated patients, and matched controls.

Results: We confirmed that SOD1 mRNA level was up- regulated in ALS patients compared to controls and, interestingly, SOD1 expression in mutated patients was higher than in non-mutated patients. We demonstrated that the higher level is determined by the mutated allele.

Immunofluorescence of lymphoblasts showed the presence of SOD1 cytoplasmatic inclusions in SOD1-mutated patient and sporadic cases. Small aggregates were also observed in patients mutated in FUS. The levels of TARDBP mRNA expression were similar between SALS and controls, but we evidenced that the TARDBP expression in the mutated ALS patient was higher than that in controls and non-mutated SALS cases. As SOD1, the higher mRNA level was regulated by the mutated allele. Large round-shaped cytoplasmatic speckles were evident in the patient with mutation in TDP-43, while smaller inclusions were present in sporadic cases.

Finally, we measured FUS mRNA level in SALS patients, in mutated cases and controls, and no difference was detectable among these groups. Immunofluorescence experiments showed a diffuse distribution of FUS protein in the nuclear compartment of lymphoblasts from all ALS cases as well as from control subjects.

Discussion and conclusion: We demonstrated that genetic mutations can impact both gene expression and protein aggregation. In fact, mutations in SOD1 and TARDBP gene directly correlate with mRNA level and distribution of the protein aggregation.

We hypothesize that FUS involvement in ALS is not related to gene expression and protein aggregation, but, as shown by the mutated patients, a distinct clinical phenotype seems to characterize cases carrying the R521C mutation (3). Only samples with different FUS mutations may clarify these data. We will amplify our study by recruiting SALS patients with different mutations in SOD1, TARDBP, and FUS genes to confirm our data.

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P199 FUNCTIONAL ANALYSES OF MOTOR NEURONS DIFFERENTIATED FROM ALS PATIENT-DERIVED-INDUCED PLURIPOTENT STEM CELLS (IPSC) WITH FUS AND SOD1 MUTATIONS

Naujock M¹

Stanslowsky N¹

Hermann A²

Wegner F¹

Kim KS³

Petri S¹

^uHannover Medical School, Department of Neurology, Hannover, Germany

^vDresden University of Technology, Department of Neurology, Dresden, Germany

^wMcLean Hospital/Harvard Medical School, Molecular Neurobiology Laboratory, Belmont, USA

Email address for correspondence: [email protected]

Keywords: iPSC, pathophysiology, motor neurons

Background: For the study of functional deficiencies in neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) patient-derived-induced pluripotent stem cell (iPSC) could become valuable in vitro disease models. As proof of principle, it has been demonstrated that motor neurons (MNs) differentiated from such ALS-iPSC indeed reproduce certain disease-specific abnormalities (1).

Objectives: The aim of the present study is to unravel pathophysiological mechanisms underlying ALS in MNs differentiated from iPSC lines carrying mutations in the SOD1 and FUS gene in comparison with each other and with iPSC derived from healthy controls.

Methods: Differentiation was induced based either on a novel approach starting from an expandable population of neural precursor cells generated from iPSC (2) or on a rather classical approach starting from iPSC (3). By this, MNs were generated after 18 or 42 days of differentiation, respectively. Markers of differentiation were studied using qPCR and immunocytochemistry. To demonstrate that ALS-iPSC can be differentiated into functional MNs, cells were characterized by calcium-imaging and patch-clamp analysis. Calcium-imaging detected the expression of functional voltage-dependent calcium and ligand-gated channels such as glutamate-receptors. Whole-cell patch-clamp recording was used to confirm functional neuronal properties like sodium-inward currents and action potentials.

Results: We successfully differentiated ALS-iPSC and healthy control iPSC into Tuj1/SMI32/Islet1-positive MNs. These cells show basic neural properties such as steady membrane potential, Na+ K+ currents, spontaneous activity, and action potentials when being target of patch clamp analysis and calcium imaging. Calcium imaging further revealed responses to the application of neurotransmitters such as acetylcholine, glutamate, and GABA.

Discussion: Differentiation of patient- and control-derived iPSC into functional MNs paves the way to detailed comparison of potential mutation-specific functional properties regarding voltage and ligand-gated ion channels. These studies could shed more light on pathophysiological processes involved in disease onset and disease progression.

Conclusions: Having established a reliable strategy to differentiate functional MNs from ALS-iPSC and healthy controls, we now focus on detailed molecular physiological analysis of disease mechanisms in different ALS-related mutations.

References:

• Bilican B, Serio A, Barmada SJ et al. Mutant induced pluripotent stem cell lines recapitulate aspects of TDP-43 proteinopathies and reveal cell-specific vulnerability. PNAS 2012;109(15):5803–5808.
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P200 INDUCED PLURIPOTENT STEM CELLS FROM ALS PATIENTS

Deng M¹

Liu X¹

Zhang S³

^xMedical Research Center, Peking University Third Hospital, Beijing, 100191, China

^yDepartment of Neurology, Peking University Third Hospital, Beijing, 100191, China

^zNational Institute of Biological Sciences, NIBS, Beijing, 102206, China

Email address for correspondence: [email protected]

Keywords: induced pluripotent stem, gene mutation, familial ALS

Aim: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease which lacks effective treatment. Induced pluripotent stem (iPS) cells are the perfect source for cell replacement therapy. We establish ALS-patient-specific iPS cell lines to study the mechanism and lay the foundation for future treatment.

Methods: We introduce four transcription factors including Oct4, Sox2, Klf4, c-Myc into dermal fibroblasts derived from three familial ALS patients carrying FUS P525L, SOD1 C11Y and SOD1 V14M mutations through retrovirus.

Results: These ALS-iPS cells display the morphology of tightly packed and flat colonies. Immunocytochemistry showed that these iPS cells express human ES cell surface antigens such as OCT4, SOX2, SSEA-4, TRA-1-81, and Nanog. Real-time PCR analysis suggested that ALS-iPS cells express human ES cell marker genes including OCT3/4; SOX2; NANOG; GDF3; FGF4; REX1; ESG1; DPPA2; DPPA4; and hTERT. Bisulfite genomic sequencing analyses showed that the CpG in the promoter regions of OCT4, NANOG, and REX1 were highly unmethylated. They have normal karyotypes of 46. Furthermore, they can form EBs by floating cultivation and differentiated into kinds of cell types. And they have the ability to form teratomas when injected into immunodeficient mice and to differentiate into three germ layers in vivo.

Conclusions: These ALS-iPS cells we generated have pluripotency which is similar to human ES cells. Therefore, we successfully established three familial ALS patient's specific iPS cell lines and are the first team to generate ALS-iPS cell lines of FUS gene mutation. These results lay the foundation of establishing an ALS-patient-specific iPS cell bank including all ALS patients and clinical applications.

Acknowledgements:

We thank all the individuals with ALS and healthy control subjects whose contributions made this work possible. This study was funded by National Natural Science Foundation of China (81072374, 31171048, 30973043, 30700906), Beijing Municipal Natural Science Foundation (7102159, 7112146), Beijing Nova Program (2009A04 and 2007A008).

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P201 MITOCHONDRIAL RESPIRATORY CHAIN ENZYMATIC ACTIVITIES AND UCP3 EXPRESSION IN MUSCLES OF PATIENTS WITH HEREDITARY AND SPORADIC AMYOTROPHIC LATERAL SCLEROSIS

Spinazzi M¹

Casarin A²

Tasca E³

Salviati L²

Cima V¹

Gavassini B¹

Pegoraro E¹

Soraù G¹

Angelini C³

^aaDepartment of Neurosciences, Padova, Italy

^abHuman Genetics, Padova, Italy

^acIRCSS San Camillo, Venice, Italy

Email address for correspondence: [email protected]

Keywords: mitochondrial dysfucntion, UCP3, muscle

Objective: We wished to verify whether mitochondrial respiratory chain dysfunction, decreased SOD1, and elevated UCP3 expression occur in the muscles of patients with sporadic and hereditary ALS.

Background: Amyotrophic lateral sclerosis is a complex neuromuscular disease characterized by progressive neurodegeneration of motor neurons with an unclear pathogenesis. A primary dysfunction of the mitochondrial respiratory chain (RC), more prominent at the level of CIV, has been previously reported in the muscles of ALS patients (1), but the results are controversial (2). Moreover, a dramatic increase in the mitochondrial uncoupling protein 3 (UCP3) has been reported in the muscles of ALS patients by a single study (3).

Methods: Activities of the mitochondrial RC enzymes (complex I, II, III, IV, I+ III, and II+ III) as well as of the matrix enzyme citrate synthase, were measured using the optimized spectrophotometric assays recently described by our group (4)in muscles from controls (n = 15), and different types of sporadic (n = 11, of which five with a predominant upper motor neuron phenotype, six with significant denervation) and hereditary ALS patients (two with a SOD1 gene mutation and three with a C9ORF72 mutation). Enzymatic activities of superoxide dismutase activity were also measured according to the previous protocols. Muscle sections of patients and controls were processed with a combined SDH/COX staining technique for the quantification of COX-negative fibers and ragged-blue fibers. The examiner evaluated over 350 fibers for each individual, blinded to the diagnosis. Protein expression of UCP3, the subunit 2 of Complex IV, and SOD1 was measured by western blotting.

Results: The activities of the mitochondrial RC enzymes did not differ between ALS patients and controls except in the coupled assay for complex II+ III (p < 0.05).The frequency of COX-negative and ragged red fibers was also not different between patients and controls. Superoxide dismutase activities were specifically decreased only in patients carrying a heterozygous mutation in the SOD1 gene, but not in other ALS groups.

Expression of the muscle mitochondrial uncoupling protein 3 was variably expressed in both patients and controls, without any significant difference.

Conclusions: Our data argue against a significant mitochondrial dysfunction as a unifying pathogenetic mechanism in the muscles of sporadic and hereditary ALS patients. UCP3 is not a useful biomarker for ALS. We speculate that the isolated mild defect of the coupled assay for complex II+ III could arise from a secondary reduction in coenzyme Q pools in the muscles of ALS patients, but this would require appropriate testing.

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P202 DJ-1 AND PINK1 IN SPORADIC ALS AND IN THE SOD1G93A ALS MOUSE MODEL: ROLE IN MITOCHONDRIAL DYSFUNCTION IN SKELETAL MUSCLES?

Knippenberg S

Sipos J

Thau-Habermann N

Körner S

Rath K J

Dengler R

Petri S

Hannover Medical School, Hannover, Germany

Email address for correspondence: [email protected]

Keywords: DJ-1, PINK1, muscle, mitochondrial dysfunction

Background: Mitochondrial dysfunction is an important mechanism in the pathogenesis of neurodegenerative diseases such as Parkinson’s disease and ALS. DJ-1 and PTEN- induced putative kinase 1 (PINK1) are important proteins for the maintenance of mitochondrial function and the protection against cell death. Mutations in the genes coding for these proteins cause familial forms of PD. Recent studies have described changes in the expression of both proteins in ALS mouse models.

Objectives: While experiments in ALS mice reveal involvement of DJ-1 and PINK1 in the pathogenesis of SOD1^G93A-related ALS, tissue of human patients with sporadic ALS has not yet been analyzed for DJ-1 and PINK1 expression before. We therefore performed a comparative analysis in human postmortem tissue and in SOD1^G93A mice.

Methods: mRNA and protein expression of PINK1 and DJ-1 were studied using quantitative real-time PCR and immunohistochemistry in postmortem brain and spinal cord tissue, as well as in muscle biopsy samples of ALS patients, and in brain, spinal cord and gastrocnemius muscle of SOD1^G93A ALS mice at different disease stages.

Results: We found significant decreases in PINK1 and DJ-1 mRNA levels in muscle tissue of mice. PINK1 mRNA levels in human muscle tissue were also significantly decreased, and DJ-1 mRNA was reduced without reaching statistical significance. Immunohistochemistry of brain and spinal cord sections revealed neuronal up-regulation of both proteins in ALS.

Discussion and conclusion: Our results demonstrate a pathophysiological role of both proteins in mutant SOD1^G93A transgenic mice and in human sporadic ALS. They provide evidence for involvement of PINK1 and DJ-1 in mitochondrial dysfunction and muscle degeneration in both ALS patients and the SOD1^G93A mouse model. These results warrant for further development of therapeutic approaches aiming to increase PINK1 and DJ-1 expression.

P203 SOD1 MUTATION LEADS TO ALTERED METABOLIC PATHWAYS FOR ENERGY GENERATION IN ALS PATIENT FIBROBLASTS

Allen S

Rajan S

Duffy L

Mortiboys H

Higginbottom A

Grierson A

Shaw PJ

University of Sheffield, Sheffield, UK

Email address for correspondence: [email protected]

Keywords: SOD1, mitochondria, metabolism

Background: Metabolic dysfunction plays a key role in amyotrophic lateral sclerosis (ALS) disease progression and has been observed in several cellular and animal models of the disease (1,2). Peripheral tissues such as fibroblasts have been observed to recapitulate pathophysiological abnormalities observed in the CNS (3). The ease of obtaining and culturing of fibroblasts makes them a valuable model system to study pathophysiological processes relevant to neurodegeneration.

Objectives: Our objectives were to ascertain whether fibroblasts isolated from patients with SOD1-related familial ALS show the defects in energy metabolism reported in the CNS, and to identify the specific biochemical pathways underlying these changes.

Methods: Using a Seahorse XF24 bioanalyser, which simultaneously measures the two major energy-producing pathways of the cell, mitochondrial respiration, and glycolysis in real-time, we assessed the effect of the I113T SOD1 mutation on fibroblast mitochondrial respiration, glycolytic flux, and ATP levels. The effect of SOD1 mutation on fibroblast mitochondrial function was assessed when switching from glucose to a more oxidative state under galactose. Using galactose as the sole sugar source forces the cell to rely on oxidative phosphorylation, enhancing any mitochondrial dysfunction observed when using glucose. The contribution of long-chain fatty acid oxidation (FAO) to mitochondrial respiration was also measured to ascertain whether this major energy pathway was dysfunctional in SOD1^I113T fibroblasts.

Results: SOD1 mutation caused a reduction in mitochondrial-coupled respiration and spare respiratory capacity in fibroblasts (19% and 32% respectively, p ≤ 0.05). This caused a 35% reduction in ATP levels (p ≤ 0.05) produced via oxidative phosphorylation. Concomitantly, mutant SOD1 fibroblasts upregulated glycolytic flux by 73% (p ≤ 0.01) restoring cellular ATP levels. Forcing the fibroblasts to rely on oxidative phosphorylation using galactose as an energy source increased uncoupling in mutant SOD1^I113T fibroblasts indicating damaged mitochondria. Assessment of the contribution of FAO to total respiration suggested that FAO was reduced in SOD1 patient fibroblasts, which could be mimicked by starving the control cells of glucose.

Discussion: As in neurones, SOD1 mutation in fibroblasts leads to mitochondrial dysfunction either directly or as a product of increased glycolytic flux, the mutant SOD1 fibroblasts cannot rely on FAO to meet the required energy demands as SOD1 mutation leads to FAO metabolic dysregulation in fibroblasts.

References:

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P204 METABOLIC SIGNATURES OF AMYOTROPHIC LATERAL SCLEROSIS: INSIGHTS INTO DISEASE PATHOGENESIS

Dodge J¹

Treleaven C¹

Fidler J¹

Tamsett T¹

Bao C¹

Searles M¹

Taksir T¹

Sidman R²

Cheng S¹

Shihabuddin L¹

^adGenzyme, a Sanofi Company, Framingham, MA, USA

^aeHarvard Medical School, Boston, MA, USA

Email address for correspondence: [email protected]

Keywords: acidosis, metabolism, glycogen

Background: Acidosis has recently been reported to be an important modulator of disease course in amyotrophic lateral sclerosis (ALS). Metabolic acid–base disorders are classically identified by measuring the concentrations of ions that affect H⁺ concentration. Strong base and acid ion concentrations are used to determine the strong ion difference (SID) and the strong ion gap (SIG). The SIG represents the contribution of unidentified ions and is a strong predictor of mortality in individuals exposed to pathological acidosis. Pathological acidosis can be averted through several compensatory mechanisms, such as lowering acid production (e.g., inhibiting lactate synthesis from glycogen), promoting acid elimination, and re-synthesizing of acids back into substrate stores.

Objectives: The present study investigated whether SOD1^G93A mice display disease-related changes in pH within the CNS. In addition, we also determined whether the disease course affects the SID and the SIG, and whether ALS mice display compensatory mechanisms (ie increased glycogen storage and modifications in lysosomal alpha-glucosidase activity) to avert acidosis. Human ALS cervical spinal cord samples were also evaluated for changes in glycogen content and alpha-glucosidase activity to determine the relevance of our preclinical observations in humans.

Methods: In vivo pH measurements were made using the pH Optica system. A VetACE^® clinical chemistry system was used to determine the concentration of ions for calculating SID and SIG. Glycogen levels were measured using an amplex red oxidase assay kit. Tissue alpha-glucosidase activities were determined using 4-MU-α-D-glucopyranoside as the artificial substrate. Human cervical spinal cord segments were obtained from nine ALS patients and seven age-matched normal individuals.

Results: ALS mice displayed progressive acidosis within the CNS. Furthermore, significant differences in SID and SIG values were also noted. Aberrant accumulation of glycogen in spinal cord and peripheral (muscle, liver, and kidney) tissues was observed in ALS mice. CNS glycogen levels significantly correlated (r² = 0.7714) with changes in pH. In addition, significant alternations in alpha-glucosidase activities were also found. Similarly, glycogen levels were significantly elevated in both grey and ventral white matters of human ALS tissue homogenates. Alternations in alpha-glucosidase activity were also found in human ALS spinal cord homogenates.

Discussion and conclusion: SOD1^G93A mice and ALS patients develop several metabolic abnormalities indicative of pathological acidosis. SID and SIG values observed in ALS mice strongly suggest that the development of acidosis was due in part to the presence of an unidentified anion. SIG values detected in ALS mice were highly predictive of imminent mortality. Furthermore, SOD1^G93A mice displayed metabolic changes indicative of a compensatory response to avert pathological acidosis. Increased glycogen and fluctuations in alpha-glucosidase activity within the spinal cord are also robust features of human ALS. Collectively, our work provides novel insight into the pathogenesis of ALS as well as potential biomarkers and targets for drug development.

P205 INCREASED EXPRESSION OF VALOCIN-CONTAINING PROTEIN IN THE SKIN OF PATIENTS WITH AMYOTROPHIC LATERAL SCLEROSIS: AN IMMUNOHISTOCHEMICAL STUDY

Mikami H

Watanabe T

Oketa Y

Suzuki M

Ono S

Teikyo University Chiba Medical Center, Ichihara, Chiba, Japan

Email address for correspondence: [email protected]

Keywords: valocin-containing protein, skin, immunohistochemistry

Background: So far studies of the skin of amyotrophic lateral sclerosis (ALS) have shown unique pathological and biochemical abnormalities in collagen, elastic fibers, and the ground substance. The lack of bedsore formation even in the terminal stages in ALS patients is considered characteristic. The valosin-containing protein (VCP) is one of the most evolutionarily conserved proteins that is ubiquitous and abundant in cells accounting for more than 1% of total cellular proteins. VCP was implicated in the pathogenesis of neurodegenerative diseases. Specifically VCP was found in the pathologic lesions in Alzheimer’s disease, Parkinson’s disease, ALS, and polyglutamine repeat diseases. It is unknown, however, whether VCP-positive (VCP+) structures are present in ALS patients’ skin.

Objectives: We have carried out immunohistochemical study of VCP in the skin of ALS patients.

Methods: Skin biopsy specimens were taken from the left biceps from 20 sporadic ALS patients (61.0 ± 9.4 years) and 20 control subjects with other neurologic disorders (62.3 ± 9.8 years). Routine formalin-fixed paraffin-embedded 6-μm sections were immunostained according to standard techniques. A densitometric analysis was performed using an image analysis system.

Results: Numerous VCP+ 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. VCP immunoreactivity of VCP+ 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, VCP+ 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 VCP+ cells in the epidermis in ALS patients (64.0 ± 8.8%) was significantly higher (p < 0.001) than in controls (16.6 ± 16.0%). There was a significant positive relationship (r = 0.59, p < 0.01) between the proportion and duration of illness in ALS patients. The optical density of VCP+ cells in the epidermis in ALS patients (17.7 ± 4.0) is markedly stronger (p < 0.001) than in controls (12.4 ± 4.2). A significant positive relation (r = 0.61, p < 0.01) was observed between the immunoreactivity and duration of illness in ALS patients.

Discussion and conclusion: This study provides the first demonstration of an increased expression of VCP in the skin of patients with ALS. These findings suggest that there may be a common process that leads to the accumulation of VCP in the motor neuron and the skin in patients with ALS. The data suggest that changes in VCP in ALS skin are likely to be related to the disease process and that metabolic alterations of VCP may take place in the skin of patients with ALS.

P206 CATALASE ACTIVITY IN DISTINCT PARTS OF BLOOD TISSUE OF PATIENTS WITH SPORADIC ALS

Golenia A¹

Leskiewicz M²

Regulska M²

Budziszewska B²

Szczesny E²

Jagiella J¹

Wnuk M¹

Ostrowska M¹

Lason W²

Basta – Kaim A²

Slowik A¹

^afJagiellonian University Medical College, Department of Neurology, Krakow, Poland

^agDepartment of Experimental Neuroendocrinology, Institute of Pharmacology, Polish Academy of Sciences, Krakow, Poland

Email address for correspondence: [email protected]

Keywords: oxidative stress, catalase activity

Background: Oxidative stress may be a key element of sporadic amyotrophic lateral sclerosis (SALS) pathogenesis (1). Several data proved that markers of protein, lipids or nucleic acids peroxidation are increased in postmortem tissue of SALS individuals (1). Meanwhile, much less is known about enzymatic antioxidant defense mechanism in SALS patients (2–6).

Objectives: The aim of the study was to assess the activity of catalase (CAT), which is implicated in the defense against oxidative stress, in erythrocytes, plasma, or serum of SALS patients and healthy controls.

Methods: Altogether 46 SALS patients and 54 controls were enrolled in the study. CAT activity was estimated using the commercially available assay kit (Oxford Biomedical Research).

Results: CAT activity in erythrocytes of SALS patients was significantly decreased compared to that in neurologically intact controls (p = 0.04). CAT activity in plasma and serum was similar in both studied groups.

Discussion and conclusion: Erythrocyte CAT activity, in contrast to other parts of blood tissue, is reduced in SALS cases as compared to that in controls, which may indicate that antioxidant defense system plays a role in pathogenesis of SALS. However, it is difficult to conclude which parts of blood tissue are optimal for antioxidative enzyme activity assessments. At present, erythrocytes are suspected to be more sensitive to assess antioxidative enzyme activity because they are highly vulnerable to oxidative stress for the sake of their specialized function and structure (7).

Acknowledgements:

This work received the financial support from the project Interdisciplinary PhD Studies “Molecular sciences for medicine” (co-financed by the European Social Fund within the Human Capital Operational Programme). Additionally, it was supported by Statutory Funds of the Institute of Pharmacology PAS.

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P207 HUMAN MESENCHYMAL STROMAL CELL INCREASES REGULATORY T LYMPHOCYTES/CD4 LYMPHOCYTES RATIO IN PBMC OF PATIENTS WITH ALS

Kwon M²

Noh MY¹

Cho KA¹

Oh K¹

Kang B¹

An J¹

Kim S¹

^ahHanyang University, Seoul, Republic of Korea

^aiCHA university, Seongnam,Gyeonggi-do, Republic of Korea

Email address for correspondence: [email protected]

Keywords: mesenchymal stromal cell, regulatory T lymphocyte, Immunomodulation

Background: Although there have been several attempts to develop treatment for the amyotrophic lateral sclerosis (ALS), the progress has been unsuccessful and ALS has remained as an untreatable disease until now. However, several clinical trials are progressing to investigate the effect of human mesenchymal stromal cells (hMSCs) on ALS patients based on the reports that hMSCs can be a candidate for the treatment of intractable neurological disorder. In addition, recent studies have demonstrated that the positive effect of hMSCs on ALS may be due to their immunomodulatory properties.

Objectives: Based on this positive effect of the bone marrow-derived hMSCs (BM-hMSCs) on ALS patients, we investigated the immunomodulatory effect of BM-hMSCs on peripheral blood mononuclear cells (PBMCs) of ALS patients, focusing on the previous reports of alteration of regulatory T lymphocytes (CD4⁺ CD25^hiFoxP3⁺, T_regs) slowing ALS progression.

Methods: Peripheral blood samples were donated from 20 healthy volunteers and 20 ALS patients diagnosed with definite or probable sporadic ALS. The PBMCs were separated using histoqpaue density-gradient method from whole blood samples. After separation, the PBMCs were cultured immediately with or without BM-hMSCs (5X10⁴/well) in 24-well culture plates for 24 h or 72h. The ratio of BM-hMSC to PBMC was 1:10 in one well. BM-hMSCs was donated from healthy volunteer. After 24 h or 72 h of culturing, the PBMCs were isolated and the FACS analysis was performed in the isolated PBMCs.

Results: In the FACS analysis, we found that BM-hMSCs increased T_regs/total T lymphocytes ratio significantly in both healthy volunteers (HC) and ALS patients regardless of culture duration. The ratio of T_regs to total T lymphocytes was calculated to be 0.093 ± 0.014 (mean± SEM) in PBMCs (HC) only; 0.569 ± 0.07 in PBMCs+ BM-hMSCs; 0.112 ± 0.014 in PBMCs (ALS); and 0.599 ± 0.081 in PBMCs (ALS)+ BM-hMSC in 24-h culture system. The ratio of T_regs to total T lymphocytes was calculated to be 0.340 ± 0.068 in PBMCs (HC) only; 1.155 ± 0.114 in PBMCs+ BM-hMSCs; 0.291 ± 0.0040 in PBMCs (ALS); and 1.360 ± 0.156 in PBMCs (ALS)+ BM-hMSC in 72-h culture system.

Conclusion: Our results suggest that BM-hMSCs may slow a disease progression of ALS patients via T_regs/total lymphocytes ratio elevation.

Acknowledgement:

This study was supported by grants from the Korea Healthcare Technology R&D Project, Ministry for Health & Welfare Affairs, Republic of Korea (A101712).

P208 IMMUNE ACTIVATION MARKER PRODUCTION BY CULTURED SPORADIC ALS (SALS) PATIENT BLOOD-DERIVED MACROPHAGES IMPLICATES PRO- AND ANTI-INFLAMMATORY MACROPHAGE ACTIVATION IN ALS PATHOGENESIS

Zhang R¹

Miller RG²

Katz J²

Forshew DA²

Harris W²

McGrath MS¹

^ajUniversity of California, San Francisco, San Francisco, CA, USA

^akCalifornia Pacific Medical Center, San Francisco, CA, USA

Email address for correspondence: [email protected]

Keywords: immune activation, proinflammatory macrophage (M1), anti-inflammatory macrophage (M2)

Background: Macrophages (MOs) dominate sites of CNS injury promoting both injury and repair. These divergent effects may be caused by distinct MO subsets, that is, classically activated proinflammatory (M1) or alternatively activated anti-inflammatory (M2) MOs. Involvement of M1 activation and inflammation in ALS pathogenesis has been confirmed by various investigations. Our recent studies of SALS patient peripheral blood mononuclear cell gene expression showed upregulation of both M1 interferon-induced genes and M2 alternative activation genes (1). Abnormal elevation of M2 activation-associated cellular and secreted factors, MO scavenger receptor CD36 and chemokine CCL18 were also observed in the studies of ALS patient blood (2,3). These data suggest a hybrid activation state that implicates both M1 and M2 MO activation in ALS pathogenesis. The current study was performed to extend the earlier observations and evaluate production of both M1 and M2 MO activation markers in SALS patient blood.

Objectives: To assess C-reactive protein (CRP), matrix metallopeptidase-9 (MMP-9), and cytokine/chemokine secretions of cell culture from blood-derived MOs to define expression pattern of M1 and M2 activation markers in ALS.

Methods: Ten SALS patients and 10 healthy controls were recruited. Presence and levels of MO activation markers produced by blood-derived MOs were measured using ELISA after overnight culture. Results represent mean ± SED (pg/ml).

Results: After overnight culture, production of MO inflammatory markers CRP and MMP-9 were significantly elevated in sALS as compared to healthy control MO cultures (CRP: 3000 ± 611 vs. 1215 ± 363, p = 0.0259; MMP-9: 24822 ± 7242 vs. 4765 ± 1171, p = 0.0231). Significantly higher levels of M1 activation-related cytokine/chemokines, interferon-γ-inducible protein (CXCL11), IL-6, IL-8, MCP-1, and TNF-α were observed in ALS MO culture supernatants (CXCL11: 92.7 ± 41.7 vs. 6.9 ± 4.0, p = 0.0125; IL-6: 473 ± 182 vs. 29 ± 12, p < 0.0001; IL-8: 7312 ± 1193 vs. 2412 ± 695, p = 0.0023; MCP-1: 4933 ± 2627 vs. 757 ± 312, p = 0.0147; TNF-α: 62.5 ± 28.1 vs. 6.0 ± 0.8, p = 0.0025). Moreover, ALS MO also showed increased secretions of IL-1ra, a M2 activation marker (IL-1ra: 1280 ± 326 vs. 181 ± 61, p = 0.0090).

Discussion and conclusion: The current study showed a disease-associated secretion of both M1 and M2 activation markers in cell culture by blood-derived MOs from patients with sALS. These results were consistent with those of our previous studies implicating M1 and M2 MO activation in ALS pathogenesis. With M1 MO-associated inflammation potentially playing a significant role in ALS pathogenesis, it is likely that M2 MO alternative activation/anti-inflammatory responses seen in ALS may represent a negative feedback mechanism modulating inflammation-associated damage. Further investigation will be needed to characterize the role of both M1 and M2 MO activation in ALS pathogenesis.

References:

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P209 ANDROGEN-DEPENDENT IMPAIRMENT OF MYOGENESIS IN SPINAL AND BULBAR MUSCULAR ATROPHY

Soraù G¹

Malena A¹

Pennuto M²

Silani V³

Cenacchi G⁴

Romito S⁵

Morandi L⁶

Russel A⁷

Pegoraro E¹

Vergani L¹

^alDepartment of Neuroscience SNPSRR, University of Padova, Padova, Italy

^amDepartment of Neuroscience and Brain Technologies, Istituto Italiano di Tecnologia, 16163 Genoa, Genova, Italy

^anIRCCS Istituto Auxologico Italiano, 20100 Milan, Milano, Italy

^aoDepartment of Radiological and Histocytopathological Sciences, University of Bologna, Bologna, Italy

^apNeurology Unit, Verona Hospital, Verona, Italy

^aqNeurological Institute C. Besta, Milano, Italy

^arResearch, School of Exercise and Nutrition Sciences, Deakin University, Burwood, Australia

Email address for correspondence: [email protected]

Keywords: SBMA, androgen, muscle

Background: Spinal and bulbar muscular atrophy (SBMA) is an inherited neuromuscular disease caused by expansion of a polyglutamine (polyQ) tract in the androgen receptor (AR). SBMA is triggered by the interaction between polyQAR and its natural ligands, testosterone and dihydrotestosterone (DHT). SBMA is characterized by the loss of lower motor neurons and skeletal muscle fasciculations, weakness, and atrophy.

Aim and methods: To test the hypothesis that the interaction between polyQ-AR and androgens exerts cell-autonomous toxicity in skeletal muscle, we characterized the process of myogenesis and polyQ-AR expression in DHT-treated satellite cells obtained from SBMA patients and age-matched healthy control subjects.

Results: Treatment with androgens increased the size and number of myonuclei in myotubes from control subjects, but not from SBMA patients. Myotubes from SBMA patients had a reduced number of nuclei, suggesting impaired myotube fusion and altered contractile structures. The lack of anabolic effects of androgens on myotubes from SBMA patients was not due to the defects in myoblast proliferation, differentiation, or apoptosis. DHT treatment of myotubes from SBMA patients increased nuclear accumulation of polyQ-AR and decreased the expression of interleukin-4 (IL-4) when compared to that of myotubes from control subjects. Following DHT treatment, exposure of myotubes from SBMA patients with IL-4 treatment rescued myonuclear number and size to control levels. This supports the hypothesis that androgens alter the fusion process in SBMA myogenesis.

Conclusions: These results provide evidence of an androgen-dependent impairment of myogenesis in SBMA that could contribute to disease pathogenesis.

Acknowledgments:

Work supported by Association Française contre les Myopathies (14073 and 14927 to GS, 14631 to LV), Telethon-Italy (GGP10145 to LV; GGP10037 to MP), Progetto d’Ateneo-Università di Padova (to GS). AM was supported by University of Padova, Italy.