Theme 5 Human Cell Biology and Pathology

Background: We recently identified mutations in the CCNF gene as a novel cause of ALS/FTD (1), with the p.Ser621Gly mutation found to segregate across multiple generations in an Australian family. CCNF encodes cyclin F, an E3 ubiquitin ligase that forms a part of a SCF complex that binds to proteins for ubiquitylation and degradation by the UPS. Experimental expression of the CCNF (p.Ser621Gly) mutant protein led to defective protein degradation and features of ALS pathogenesis in vitro (1).We investigated the effect of the CCNF p.Ser621Gly mutation on Lys48-specific ubiquitylation of substrates, and how this mutation alters its E3 ligase activity and stability that contributes to the ubiquitylation of neuronal proteins. We examined the phosphorylation status of cyclin F at Serine621 and how this site regulates the Lys48-specific ubiquitylation activity of the SCF (Cyclin F) complex.

Background: We recently identified mutations in the CCNF gene as a novel cause of ALS/FTD (1), with the p.Ser621Gly mutation found to segregate across multiple generations in an Australian family. CCNF encodes cyclin F, an E3 ubiquitin ligase that forms a part of a SCF complex that binds to proteins for ubiquitylation and degradation by the UPS. Experimental expression of the CCNF (p.Ser621Gly) mutant protein led to defective protein degradation and features of ALS pathogenesis in vitro (1).We investigated the effect of the CCNF p.Ser621Gly mutation on Lys48-specific ubiquitylation of substrates, and how this mutation alters its E3 ligase activity and stability that contributes to the ubiquitylation of neuronal proteins. We examined the phosphorylation status of cyclin F at Serine621 and how this site regulates the Lys48-specific ubiquitylation activity of the SCF (Cyclin F) complex.
Methods: Neuro2A or HEK293 cells were transfected with either mutant cyclin F (p.Ser621Gly) or wild-type cyclin F fused with N-terminal mCherry. Lys48-ubiquitylated proteins were immunoprecipitated from cell lysates (n=5), digested with trypsin and identified by LC-MS/MS with stringent filtering criteria. In a parallel study, phosphorylation sites were identified by immunoprecipitating mCherry-cyclin F, digestion with trypsin or Asp-N, TiO2 enrichment of phosphopeptides, followed by LC-MS/MS. E3 ligase activity of the SCF(Cyclin F) complex was evaluated by an in vitro ubiquitylation ELISA activity assay. Results/Discussion: Mass spectrometric analyses identified seven phosphorylation sites (five unique including Serine621) on cyclin F. Phosphorylation at Serine621 in wild-type cyclin F reduced the Lys48-ubiquitylation activity of SCF(Cyclin F) by $1.35-fold (n=3, p50.01) compared to the cyclin F(p.Ser621Gly) mutant. Due to the differences in E3 ligase activity, RRM2 (a known cyclin F substrate) expression was elevated by $1.3-fold (n=3, p50.05) and ubiquitylated mostly with Lys48ubiquitin in mutant cyclin F(p.Ser621Gly) transfected cells while RRM2 in the wild-type cyclin F control lysates was ubiquitylated with both Lys48-and Lys63-ubiquitin. LC-MS/MS identified 395 and 205 Lys48-immunoprecipitated proteins from wild-type cyclin F and cyclin F(p.Ser621Gly) transfected cells, respectively. Of these, Gene Ontology and IPA analysis identified a higher percentage of Lys48-ubiquitylated proteins in cyclin F(p.Ser621Gly) cells clustered into biological networks that are responsible for cellular survival and maintenance. The Serine621 phosphorylation site is a key regulator of E3 ligase activity, with the glycine mutation preventing phosphorylation and conferring elevated Lys48-ubiquitination activity. We show differences in wild-type cyclin F and cyclin F(p.Ser621Gly) Lys48-ubiquitylated proteins with the mutant form targeting a larger proportion of substrates linked to cell survival. These findings highlight new mechanisms of cyclin F phosphorylation and its convergence to ubiquitin-mediated regulation of substrates responsible for sustaining the cellular milieu.
Background: Amyotrophic lateral sclerosis (ALS) is a devastating progressive neurodegenerative disease characterized by the loss of motor neurons, resulting in muscle weakness and paralysis. The most commonly observed ALS causative genes are SOD1, FUS, TARDBP, and C9ORF72. For ALS studies it is noteworthy that relevant tissues, and particularly live CNS cells from affected individuals, are not readily obtainable. Recent advances in stem cells biology have provided exciting opportunities to develop disease-specific cell types that allow us to understand and explore mechanisms that contribute to pathogenesis of disease. In particular, the ability to reprogram somatic cells into induced pluripotent stem cells (IPSC) provide obvious advantages to generate patient-specific IPSC that carry the exact same genetic makeup, including mutations that may contribute to disease process.
Objectives: The disease relevant cell types will be ALS patients' motor neurons (MN) and astrocytes that are differentiated from human-induced pluripotent stem cells (hiPSC) derived from primary fibroblast cultures established from skin biopsies. Our three ALS related assays will monitor the toxicity of patient derived MN when exposed to: (1) supernatant from astrocyte cultures derived from ALS mouse models; and (2) organophosphates (Ops). Using multiple disease relevant assays and cells from different patients ensures that we will test many of the important therapeutic mechanisms and pathways in ALS.
Methods: Fibroblasts will be prepared from skin biopsies of consenting ALS patients recruited for this study. The fibroblasts will then be used to generate two lines of hiPSCs per patient according to established protocols. A Brain Canada platform makes our hiPSCs, genome-edits them from fibroblasts and assists in the differentiation into different neural cell types. The Laval hub of this platform (directed by Dr Pumyrat) derives and reprograms patients' fibroblasts into hiPSCs and the Montreal hub of the platform (directed by Dr Fon) differentiates them and genome edits them using CRISPR/Cas9. Results: Fibroblasts from 6 C9ORF72, 1 FUS, and 1 SOD1 case and 2 control individuals have already been collected and are ready for the preparation of hiPSC. A three-step strategy is used to induce robust MN differentiation from hiPSCs. The motor neurons' nature was confirmed by testing for characteristic markers (e.g. HB9, ChAT, Tuj1). Astrocytic differentiation is now routinely done by treating hiPSCs with FGF2 and EGF. The astrocytic nature of these hiPSC-derived cells will be confirmed by GFAP immunocytochemistry and a panel of additional markers (BDNF, GDNF).
Discussion and conclusions: hiPSC-derived MN and astrocyte cultures from our deeply phenotyped ALS cases are the ideal model to develop disease relevant assays. These assays can be used to test for molecules that can alter MN cell survival/reduce the presence of pathogenic protein aggregates and/or RNA foci in those MN.  (1). A powerful approach to study the pathomechanism of G4C2 repeats expansion in human MNs is use of human-induced pluripotent stem cells (iPSC) derived MNs from ALS patients carrying C9ORF72 mutation. Previous studies have implicated MN vulnerability to AMPA-receptor (AMPAR)-mediated excitotoxicity (2).
Objective: Gene correction of C9ORF72 mutation in hiPSC and assess if C9orf72 mutation causes MN vulnerability to AMPAR-mediated excitotoxicity.
Methods: MN's were generated from iPSC derived from two healthy donors, two ALS patients harboring C9ORF72 mutation, and one gene corrected iPSC using standard protocol (3). All the lines differentiated into highly enriched spinal neuronal culture that expressed MN marker Isl1/2 (40-60%). MNs demonstrated increased block of AMPAR-mediated currents by NASPM, a selective inhibitor of Ca 2+ AMPAR, and elevated single-channel conductance when compared to control and C9-D MNs.

Results
Discussion and conclusion: Generation of isogenic control iPSC from C9ORF72 mutant line demonstrates the causal link between the repeat expansion mutation and MN vulnerability to AMPAR-mediated excitotoxicity. Our data also shows that excitotoxicity is mediated by elevated expression of Ca 2+ AMPAR in C9ORF72 MNs.
Background: An intronic expansion of a GGGGCC repeat within the C9ORF72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Several downstream mechanisms have been proposed but the exact mechanism underlying the selective motor neuron degeneration is still unknown. Defective axonal transport is an early perturbed event in several neurodegenerative disorders, occurring prior to cell loss.
Objectives: Here, we have used induced pluripotent stem cells (iPSCs) derived from C9ORF72 FTLD/ALS patients and control fibroblasts in order to investigate the consequences of a C9ORF72 expansion in human motor neurons.
Methods: C9ORF72 and control iPSCs have been successfully generated using CytoTune-2 Sendai-virus reprogramming kit and further differentiated into motor neurons using a specific motor neuron differentiation protocol. Immunocytochemistry, gene expression and electrophysiological analysis have been performed to validate that the differentiation protocol yielded mature motor neurons. In order to measure the transport of mitochondria within the axons, MN cultures differentiated from control and C9ORF72 iPSCs were labeled with MitoTracker. Live cell imaging was used to monitor mitochondrial translocation along MN processes and time/distance kymographs were generated to quantify the number of stationary and moving mitochondria. Methods: In different human tissues, in human neuroblastoma SK-N-BE cells treated with retinoic acid and in human iPSCs differentiated into motoneurons, the alternative splicing of TNIKex15 was analyzed by both RT-PCR and WB using a custom antibody recognizing exon 15-encoded region. Regulation of TNIKex15 splicing was also assessed by a minigene splicing assay in HEK cells.

Results
Results: We observed that TNIK alternative splicing is differently regulated in human tissues. TNIKex15 mRNA isoforms were prevalent in all cerebral (frontal cortex, hippocampus, cerebellum) and spinal cord regions analyzed as well as in skeletal muscle, as compared to lung and kidney where these isoforms were absent. When we induced neuronal differentiation in vitro, a significant increase of TNIKex15 isoforms, both at transcript and protein level, was observed in SK-N-BE treated with retinoic acid and in iPSCs differentiated into motoneurons. Immunofluorescence analyses showed a prevalent perinuclear distribution of TNIKex15 protein in neuron/ motoneuron-differentiated cells. Since TDP-43 protein levels remained unchanged during in vitro neuronal differentiation, we focused on the possible involvement of other splicing factors in regulating TNIKex15 splicing. As putative consensus binding sequences for ELAV and NOVA1 proteins were present in intron 15 and as these RNA-binding proteins are specifically expressed during neuronal differentiation, we studied their effect on TNIKex15 splicing using a minigene assay. Upon overexpression of these single-splicing factors and in competition assays with TDP-43, we found that TNIKex15 inclusion is differently regulated by ELAV and NOVA1, suggesting a complex interplay between TDP-43 and these neuronal-specific splicing factors.
Discussion and conclusion: Our data show that alternative splicing of TNIK gene is differently regulated in human tissues and suggest a potential role of TNIK-ex15 isoforms in human brain and during neuronal/ motoneuronal differentiation. Given the different subcellular localization of TNIKex15 protein, the specific function of these isoforms needs to be further investigated also in association to ALS/FTLD diseases. Background: Coiled-coil helix coiled-coil helix domain containing protein 10 (CHCHD10) is a mitochondrial protein encoded in the nucleus. It is enriched at the cristae junctions of mitochondria and possibly plays a role in cristae structure, respiratory chain regulation (1) and mtDNA stability (2). The finding that mutations in CHCHD10 are associated with familial ALS and FTD directly links mitochondrial dysfunction to the pathology of the ALS-FTD continuum. However, the molecular pathogenesis of CHCHD10-related neurodegeneration remained so far enigmatic.
Objectives: In this study, we analyze the impact of three missense variants found in ALS patients (R15L, P34S, and G66V) on the structure and stability of CHCHD10.
Methods: CHCHD10 turnover was analyzed by the inhibition of translation of HEK293 cells overexpressing wild-type or mutant CHCHD10 followed by the determination of CHCHD10 levels by Western blotting. For structural analyses, we purified wild-type and mutant CHCHD10 expressed in E.coli and compared the respective tertiary structures by CD spectroscopy. Additionally, we took advantage of the recombinant CHCHD10 proteins and determined the thermal stability using a Thermofluor assay.
Results: Analysis of CHCHD10 protein turnover and thermal denaturation reveal a decrease in protein stability and an increased degradation of the R15L and G66V, but not the P34S substitution compared to wild-type CHCHD10. These results are in line with CD spectroscopic measurements indicating considerable structural changes of the R15L and G66V mutant proteins. These structural changes are more pronounced under membrane-mimicking conditions.
Discussion and conclusion: These results match the most recent genetic evidence indicating that the R15L and G66V variants of CHCHD10 are pathogenic while P34S is not significantly associated with neurodegenerative diseases (3). We thus hypothesize that mutations of CHCHD10 induce a structural disturbance and loss-offunction of CHCHD10. Consequently, in order to elucidate downstream pathways mediating detrimental effects of CHCHD10 mutations, we performed a mass spectrometry-based screen for differential, disease-relevant binding partners of wild-type or R15L and G66V mutant CHCHD10, respectively. We recently described four mutations in the Matr3 gene encoding the nuclear matrix protein Matrin 3 that are associated with ALS (1). In human spinal cord tissue, Matrin 3 immunostaining is present in both motor neurons and glia, predominantly nuclear, and is stronger in ALS patients than in control cases and strongest in an ALS patient with a mutation in Matrin 3. Matrin 3 does not appear to exhibit gross mislocalization in ALS tissue as is often seen in other proteins involved in ALS; therefore we have chosen to explore its role and protein interactions within the nucleus.

P137 ALS-ASSOCIATED MUTATIONS IN MATRIN 3 ALTER PROTEIN-PROTEIN INTERACTIONS
Objectives: We sought to determine the protein binding partners of Matrin 3 as well as determine which of these interactions are altered by ALS causing mutations.
Methods: Cell lines stably expressing Flag tagged constructs of each of the four mutations and wild-type Matrin 3 were created in NSC-34 motor neuron-like cells. Immunoprecipitation experiments were performed in these cell lines followed by the separation by gel electrophoresis and tryptic digestion of excised bands. Peptides were extracted and analyzed on a Thermo LTQ Orbitrap Velos mass spectrometer. Interactions were confirmed with immunoprecipitation followed by western blot and colocalization analysis. Proteins binding to wildtype Matrin 3 were compared to proteins binding Matrin 3 mutant proteins to yield a list of protein interactions altered by ALS-linked mutations.
Results: In total, we identified 173 proteins meeting our confidence threshold that bound to one or more of the Matrin 3 constructs with hnRNPD having the highest confidence score for all five. After gene ontology analysis the top category found for both wild-type and all four mutations was mRNA metabolic processes. Other top categories included RNA splicing, RNA processing, and mRNA splicing though the order of such categories and the number of proteins found in each differed between wild-type and mutant. Our experiments confirmed Matrin 3 interacting proteins previously published as well as novel interactions with proteins altered in ALS pathogenesis such as FUS, Map1b, and hnRNPA3. which shared some common properties but were distinct from stress granules (1).
Objectives: To assess whether normal cellular levels of cytoplasmically mislocalized FUS are sufficient to promote formation of such granules in cultured human cells.
Methods: CRISPR/Cas9 technology for genome editing was used to produce several single cell derived clones of human neuroblastoma SH-SY5Y cells with deletion of gene sequences causing expression of FUS lacking nuclear localization signal (NLS), or with longer C-terminal truncation, corresponding to a known familial mutation, G466VfsX14. We also obtained a clone with completely abolished FUS production.
Results: In cells expressing FUS with deleted NLS, the protein was redistributed to the cytoplasm where it was readily recruited to stress granules upon stress exposure.
In one of the lines, FUS-positive cytoplasmic granules were formed which possessed characteristics similar to granules previously observed in cells overexpressing ALSlinked FUS variants. Cytoplasmic accumulation and formation of FUS granules by FUS mutants could be promoted by proteasomal inhibition. Moreover, FUS granules could cluster together in response to stress and such higher-order assemblies persisted longer than normal stress granules after removal of stress.

Discussion and conclusions:
These data indicate that the deficiency in FUS nuclear import alone, without an increase in the cellular protein levels, is sufficient to trigger and maintain spontaneous RNA-dependent aggregation of FUS protein in the cytoplasm.

P139 SERUM MICRORNA-PROFILES IMPLICATE NEW RNA-BINDING PROTEINS IN ALS
A Freischmidt 1 , JMB Simbuerger 2 , J Reinders 2 , AC Ludolph 1 , JH Weishaupt 1 disease (1). Furthermore, similar miRNA-profiles were found in a majority ($60%) of sporadic ALS patients (2). Most strikingly, a common 5-nucleotide-sequence motif (GDCGG; D = G, A or U) was highly significantly enriched in the downregulated miRNAs. We therefore assume that the ALS-related downregulation of miRNAs is due to the deregulation/malfunction of one or several specific RNA-binding protein(s).
Objectives: To identify RNA-binding proteins associated with the GDCGG-motif of ALS-related miRNAs and to determine their role in ALS-pathogenesis.
Methods: We used miRNA-pulldown experiments in lysates of HEK293 cells followed by mass spectrometric identification and quantification of binding proteins. MiRNAs were designed to allow discrimination between unspecific RNA-binding proteins and proteins specifically binding the GDCGG-motif. Candidate proteins were purified from E. coli and HEK293 cells and analyzed for their RNA-binding properties using different biochemical assays. MiRNA-pulldown experiments in human serum were used to confirm the results in a physiological context.
Results: Mass spectrometry revealed 37 proteins closely associated with the GDCGG-motif. Biochemical analyses of the top three candidate proteins show direct binding of two of them to miRNAs containing the GDCGGsequence. Physiological validation in human serum confirms binding of these two proteins to the ALS-related miRNAs in vivo.

Discussion and conclusion:
We could identify two RNA-binding proteins interacting directly and specifically with the ALS-related miRNAs containing the GDCGGmotif. Downregulation of these miRNAs in ALS might indicate a deregulation/malfunction of these two proteins in ALS. As the miRNA-profiles were already evident in pre-clinical ALS mutation carriers, these findings might be related to a very early event in the pathogenic cascade. Further studies are needed to determine the roles of these RNA-binding proteins in ALS pathogenesis. Background: Several diverse proteins are linked genetically or pathologically to neurodegeneration in amyotrophic lateral sclerosis (ALS) including superoxide dismutase (SOD1), fused in sarcoma (FUS), and Tar-DNA binding protein-43 (TDP-43). Mutant forms of these proteins inhibit protein transport between the endoplasmic reticulum (ER) and Golgi apparatus in neuronal cells (1), and cells expressing ALS-associated mutant SOD1, TDP, and FUS display autophagy defects. Rab1 has a pivotal role in mediating intracellular membrane trafficking events, including ER-Golgi trafficking (2,3) and autophagosome formation (4). However, the function of Rab1 in ALS remains unclear.
Objectives: To investigate the effect of Rab1 overexpression on (i) ER-Golgi trafficking and (ii) autophagy, in neuronal cells expressing ALS-associated mutant proteins, (iii) the distribution of Rab1 in motor neurons of human spinal cord tissues from patients with sporadic ALS, (iv) and to identify novel Rab1-mimetic compound that could have therapeutic benefit in ALS.
Results: Rab1 overexpression rescues inhibition of ER-Golgi transport and ER stress triggered by mSOD1, mTDP-43 and mFUS, and apoptosis and inclusion formation triggered by mSOD1. However, the inactive mutant Rab1S25N did not rescue ER stress, and the constitutively active Rab1Q70L was more protective relative to WTRab1. Rab1 also restored the inhibitory effects of mFUS on autophagosome and autolysosome formation. Rab1 formed inclusions in motor neurons of spinal cords from sporadic ALS patients and approximately 40% of the Rab1 inclusion-positive motor neurons colocalized with TDP-43. We are currently investigating whether novel compounds that mimic Rab1 activity are protective in neuronal cells expressing mutant SOD1, TDP43, and FUS. Background: Proteostasis inhibition, inducing endoplasmic reticulum (ER) stress, protein misfolding, and proteasomal dysfunction, are all typical cellular pathologies associated with ALS. They are implicated in the formation of misfolded protein inclusions, which in almost all ALS patients contain misfolded TDP-43. These pathologies can be imitated in neuronal cultures by the overexpression of mutant TDP-43 or SOD1, thus creating a model for the study of proteostasis disruption in cells.
Using this model, it has been established that an ER chaperone, protein disulfide isomerase (PDI), is protective against ER stress, protein misfolding, and apoptosis.
Objectives: A series of other ER chaperones, also from the PDI family, were examined for protection against misfolded proteins linked to ALS. These included ERp57, ERp72, and PDIA2.
Methods: Recombinant, tagged SOD1, TDP-43, and PDI family members were overexpressed in mouse motor neuron-like NSC-34 cells. Markers of cellular pathologies including cell death, inclusion formation, and ER stress were assessed and measured using fluorescence microscopy.
Results: Results suggested that clear specificities existed in the protective activity. ERp57 was protective against cell death, ER stress, inclusion formation and ubiquitin proteasome system dysfunction in mutant SOD1 overexpressed cells. ERp72 and PDIA2 were protective against cell death but not against ER stress and inclusion formation. Similar protection against cellular pathologies induced by mutant TDP-43 was seen with ERp57, while in lumbar spinal cords from sporadic ALS patients, possible ERp57 colocalization with TDP-43-positive inclusions warrants further investigation.
Discussion and conclusion: These results suggest that, like PDI, ERp57 may have a protective role against ALS-like pathological events induced by both mutant SOD1 and TDP-43. This suggests that the PDI family has a broader therapeutic role in ALS than previously considered, and specific protein activities are associated with individual PDI members. The design of novel therapeutics based on the structure of PDI therefore may benefit from considering characteristics of the wider PDI family.
Background: Amyotrophic lateral sclerosis (ALS) is the most common motor neuron disease and Frontotemporal dementia (FTD) is the second most common form of early-onset dementia. Interestingly, both of these devastating neurodegenerative diseases share a common genetic mutation in chromosome 9 open reading frame 72 (C9orf72). An expanded hexanucleotide repeat (GGGGCC) in intron 1 of the C9orf72 gene is the most common genetic cause of familial and sporadic ALS and FTD, along with Huntington's disease phenocopies. Until recently, very little was known about the underlying mechanisms by which this expanded repeat causes neurodegeneration until five independent labs published three papers including one from our own group simultaneously showing that dysfunction in Nucleocytoplasmic Transport (NCT) may be a fundamental pathway for C9orf72 ALS-FTD pathogenesis. NCT, the trafficking of protein and RNA between the nucleus and cytoplasm, is critical for signal transduction and is especially arduous for neurons due to their highly polarized biology. Efficient regulation of this process is mediated by the Nuclear Pore Complex (NPC), an extraordinary molecular machine that serves as the main gateway to the nucleus. In order for any cell to function properly, it is imperative that RNA and protein be efficiently and selectively exchanged between the nucleus and the cytoplasm. This critical task is achieved by the $2000 NPCs that span the entire nuclear envelope. Each NPC consists of multiple copies of 30 different proteins called Nucleoporins (NUPs) that differ in anatomical location, function, domain, post-translational modification and residence time. Mutations in various NUPs result in tissue-specific diseases. Additionally, some of the longest-lived proteins in the mammalian brain are specific NUPs and may represent the ''weakest link'' in the aging proteome.
Results: We now present data using human brain and iPS neurons that the NPC may also be compromised in sporadic ALS (sALS). We have surveyed the majority of NUPs in transgenic and BAC C9orf72 mice, iPS neurons/ astrocytes, HEK293 cells and human post mortem brain tissue using IF, IHC, super resolution imaging, western blot, FRAP, shRNA, overexpression constructs, and proteomic analysis. We have identified a unique set of NUPs with critical and disease relevant functions that are consistently affected across not only models of C9orf72 but also sALS indicating that NPC dysfunction may be a common insult and pathogenic mechanism in the majority of ALS.
Discussion: This suggests that NPC dysfunction may be a critical global mechanism of neurodegeneration and what distinguishes one disease from another is the unique set of NUPs that are differentially affected. Background: Growing evidence suggests a link between changes in sphingolipid metabolism and ALS. We have recently reported drastic deregulation of glycosphingolipid species in the muscle and spinal cord of SOD1 mice, an animal model of ALS. In addition, we have found that UGCG, the gene coding for the enzyme responsible for the synthesis of glucosylceramide, was strongly upregulated in muscle biopsies and forms aggregates in neurogenic muscle fibers of ALS patients.
Objectives: The present work aimed to study the regulation of glycosphingolipids in the central nervous system and at the neuromuscular junctions in ALS. Results: We found that the central nervous system showed an abnormal distribution of gangliosides at disease onset in ALS patients. The cerebrospinal fluid of ALS patients was characterized by an increased content in glucosylceramide (+16%) and gangliosides from the aseries. Loss of glycosphingolipids was detected at disease onset at the motor axons and also in up to 80% of the neuromuscular junctions in the SOD1 animal model. Background: Autophagy, an essential homeostatic mechanism, eliminates damaging entities from the cytoplasm. Interestingly, autophagy does not randomly degrade cytoplasmic content; autophagy receptors such as p62, NDP52, and NBR1 link specific cargoes to the nascent autophagosome membrane for degradation. Evidence suggests that mutations in selective autophagy pathways specialized in elimination of specific substrates cause distinct neurodegenerative diseases including Amyotrophic Lateral Sclerosis (ALS). Pathology is presumably mediated by accumulation of toxic autophagic substrates, but which autophagy substrates lead to ALS pathology remains uncertain.

Methods
Objectives: Historically, the field of autophagy has focused on proteins and organelles as the substrates for degradation, despite evidence that a major fraction of RNA is degraded by autophagy, particularly in stress. Mutated versions of several RNA-binding proteins linked to ALS (e.g. FUS, TDP-43) cause formation of RNA-rich granules called stress granules (SGs). We hypothesized that SGs and another type of RNA granule -processing bodies (PBs) are selectively targeted for the degradation by autophagy receptors.
Methods and results: We found that p62 and NDP52 are preferentially recruited to SGs and PBs, respectively, as detected by expressing mCherry -TIA-1 and GFP-Dcp1a. Further NDP52 preferentially colocalized with endogenous markers of PBs rather than SGs, while p62 colocalized with endogenous SGs induced by oxidative stress. A third autophagy receptor -NBR1, colocalized with a subset of these SGs and PBs. Proteomic analyses of p62 interactors during oxidative stress revealed an extensive network of RNA-binding proteins localized to SGs. Association of p62 with RNA-binding proteins localized to SGs was confirmed by immunoprecipitation in ALS patients as well. Further, differential biochemical profiling of ALS cerebellum tissues enriched for SG proteins along with p62 in the insoluble urea fraction. PBs and SGs frequently colocalized with autophagosomes and depletion of NDP52 and p62 increased the number of PBs and SGs per cell, respectively. Overall, this strongly suggests that individual autophagy receptors preferentially recognize distinct RNA granules (PBs or SGs) for selective degradation by autophagy (1).
Discussion: Autophagy may be an effective mechanism to regulate bulk turnover of RNA regrouped in granules. Intriguingly, emerging evidence suggests that pathology in ALS involves inefficient autophagic clearance of mutant proteins in RNA-rich SGs. Our identification of p62 as a selective autophagy receptor for degradation of SGs suggests a mechanism for the genetic implication of p62 in ALS and underscores the physiological relevance of RNA degradation by autophagy. Results: We were able to demonstrate enhanced mRNA as well as protein levels of TGF-ß2 exclusively in postmortem SC tissue of ALS patients compared to healthy controls. Within the MC and OL tissue, there was no alteration in the expression levels if TGF-ß1, TGF-ß2, and the TGF-ßRII were detectable.
Discussion and conclusion: Taken together, the results of the current study might shed some light on possible pathways mediating disease progression and provide possible purchases for the treatment options in ALS. Background: Neurodegeneration in amyotrophic lateral sclerosis (ALS) is accompanied by a well characterized neuroinflammatory reaction within the central nervous system (CNS) and importantly, also cells of the peripheral immune system (1). Particularly, circulating monocytes have been implicated in ALS pathogenesis (2). Exosomes are membrane-enclosed vesicles secreted by various cell types with a diameter of 50-150 nm (3).
Objectives: We hypothesize that circulating blood exosomes are putative mediators of the monocytic deregulation observed in ALS patients. The aims of this study are to shed light on the interplay of peripheral blood monocytes and exosomal vesicles in the context of ALS.
Methods: We characterized the immunogenic content of serum exosomes isolated from healthy donors as well as ALS patients. Further, we applied exosomes isolated from human serum and TDP-43 containing exosomes produced in cell culture to cultures of primary human monocytes isolated from the blood of ALS patients and healthy age-matched controls. Subsequently we analyzed the monocytic uptake of the applied exosomes as well as their consecutive immunologic activation.
Results: Here, we found the uptake of serum exosomes by CD14++ monocytes to be independent of the exosome donor. Further, we show that pro-inflammatory cytokine secretion by ALS monocytes upon exosomal stimulation is impaired compared to control monocytes. Moreover, we show that both the exosome uptake and the consecutive monocytic cytokine secretion are dependent on the exosomal TDP-43 cargo.

Discussion and conclusions:
We show that ALS monocytes exhibit an impaired pro-inflammatory reaction upon contact to serum exosomes at similar exosomal uptake efficiencies implicating a distinct functional impairment of ALS monocytes (1,2). Further, we provide evidence that the exosomal content of TDP-43 significantly affects the consecutive monocytic activation. Thus, this study underlines the distinct functional deregulation of peripheral monocytes in ALS (2) and the impact of circulating blood exosomes on monocyte activation. Background: The blood-CSF (BCSF) barrier is structurally comprised of an endothelial cell layer and a polarized epithelial cell layer named the choroid plexus (CP). The BCSF barrier lines the inside of the brain ventricles, separating the blood in the vascular system from the cerebrospinal fluid (CSF) that is in direct contact with neurons. The CP mainly functions in CSF production and turnover as well as selective transport of nutrients from the systemic compartment, removal of metabolic products out of the brain, regulation of cell trafficking into the CSF, and the prevention of diffusion of harmful blood solutes into the brain. Under inflammatory conditions, the CP epithelium expresses many factors involved in mediating and facilitating an immune response thus serving as a primary point of entry of immune cells into the nervous system. Aging and neurodegeneration, specifically Alzheimer's disease, have been reported to greatly affect CP morphology and function, decreasing CSF production and turnover by as much as 50%, altering levels of proteins involved in energy production and free radical scavenging, and increasing protein leakage from blood to the CSF. ALS is similarly associated with increased oxidative stress markers, neuronal loss, and metabolic disturbances that may also impede CP function. Our group and others have reported increased levels of many proteins in CSF from ALS patients, including inflammatory targets, cytoskeletal and extracellular matrix proteins, as well as aggregated proteins. Having these proteins and factors abnormally localized to the CSF compartment suggests impaired BCSF barrier permeability, increased protein leakage from blood to the CSF and decreased clearance of metabolic products. These in turn point to a dysfunctional BCSF barrier. To date, there have been few studies investigating BCSF barrier or CP alterations in ALS patients. We hereby have investigated BCSF integrity in ALS.
Results: We hypothesized that this physical barrier is disrupted in ALS possibly via excess metalloproteinase (MMP) activation, thus allowing an influx of immune cells into the CSF and conversely into the nervous system. We have investigated the morphology and distribution of various cell junction and cell adhesion proteins such as cadherins, occludins, and claudins in ALS CP compared to control CP by immunohistochemistry. We show that levels and distribution of many of these markers is altered in ALS. Results were confirmed by real-time PCR analysis. In addition, we have examined levels of various matrix metalloproteinases (MMP) in ALS CP compared to controls, as a potential cause for BCSF barrier breakdown. Background: Approximately 50% of amyotrophic lateral sclerosis (ALS) patients exhibit cognitive and behavioral deficits. Most common, are deficits in verbal fluency and executive function, which are mediated by the frontal lobe. These deficits are similar to those observed in a seemingly disparate disorder, frontotemporal dementia (FTD), a disease caused by breakdown of the frontal and temporal lobes. Genetic studies suggest that ALS and FTD may lie on a disease spectrum. Repeat expansions in the C9orf72 gene result in either ALS or FTD and TDP-43 + ve protein aggregates are found in both diseases. Furthermore, significant overlap in cognitive dysfunction is observed in both patient cohorts and some FTD patients exhibit motor deficits, reinforcing the hypothesis of a disease spectrum.
Objective: Synapse loss is a common pathogenic feature of many neurodegenerative disorders, including FTD and we aimed to discover if this was also true for ALS and whether synapse loss in the frontal cortex associates with cognitive decline.

Methods:
We used the high-resolution imaging technique array tomography, to analyze approximately half a million synapses in the frontal cortex, from 11 control cases and 17 ALS cases. Of the 17 ALS cases analyzed, 11 were cognitively tested, 7 were unimpaired, and 4 cognitively impaired. Cognitive testing was performed using the Edinburgh Cognitive and Behavioral ALS Screen (ECAS), to give us an accurate representation of ALSspecific cognitive changes.
Results: There was a significant decrease (p50.05) in mean synapse density in ALS frontal cortices (n=17) compared to control (n=11). When the ALS group was split into impaired (n=4) and unimpaired (n=7) groups, the impaired group had a lower synapse density in the frontal cortex than control nondemented brains (p50.05).
No change in cortical thickness was noted between groups (p=0.4). In the motor cortex, no change in synapse density was observed between groups, however, the ALS motor cortices were significantly thinner (p=0.004). This suggests cortical atrophy of the motor cortex occurs in ALS cases and the tighter packing of synapses in the smaller cortex likely leads to no change in synapse density when compared to control. In a separate group of control (n=6) and ALS cases (n=20, not cognitively defined) electron microscopy revealed a decrease in synapses from the frontal cortex of ALS brains compared to controls (p=0.003). Furthermore, an increase in degenerating (electron dense) synapses was discovered in the ALS brains (p=0.025).
Discussion: Taken together, our data suggests that synapse loss in the frontal cortex may play an important role in the pathogenesis of cognitive decline in ALS patients.
Background: Senescent cells-associated secretory phenotype (SASP) may contribute to neurodegeneration. Recent findings in vivo suggest neurons undergo a senescent-like state associated with persistent DNA damage (1). Moreover, increased expression of a neuronal DNA damage response (DDR) associates with cognitive decline in an aging cohort (2), suggesting the neuronal SASP in response to persistent DNA damage may contribute to neurodegeneration.
Objectives: To determine whether neuronal senescence is a feature of motor neuron disease (MND) and to establish an in vitro neuronal model of persistent DNA damage.
Methods: Using immunohistochemistry, markers of DNA damage (H2AX) and senescence (p16, p21, and SA-b-gal) were investigated in frontal cortex, motor cortex, and spinal cord sections of 10 human autopsy MND and 10 control cases. A model of acute and chronic DNA damage was developed in vitro by stressing postmitotic LUHMES cells with hydrogen peroxide. Changes in the expression of DNA damage and senescence-related genes were investigated by qRT-PCR. GFP-LUHMES were cocultured with stressed LUHMES or incubated with their conditioned media (SCM) for 24 h; neurite outgrowth impairment of GFP-LUHMES was investigated.
Results: SA-b-gal activity was present in glia and neurons of both MND and control cases. Expression of p16 was detected in glia, whereas p21 was detected in glia and neurons. Glial p16 expression and neuronal p21 expression were significantly elevated in MND versus control cases (p=0.017 and p=0.029) in the frontal cortex. In the in vitro model, the detection of H2AX foci in stressed neurons confirmed oxidative damage in the form of DNA double-strand breaks (DSBs). Damage was repaired 24h post-stress in the acute model, whereas in the persistent model, H2AX foci were detectable 96h post-stress. Changes in the relative expression of senescence related genes were detected in both models, including upregulation of CDKN1A (p21). A reduction in the neurite length of GFP-LUHMES was detected when incubated with SCM (p 0.05); however, a more intense effect in neurite outgrowth impairment was seen under coculture conditions (p 0.0001).
Conclusions: A persistent DNA damage response is a feature of neurons and glia in MND. The differential expression of senescence-associated markers suggests that pathways differ between cell types. Acute and persistent DNA damage can be induced in postmitotic neurons in culture. Oxidative DNA damage induces senescencerelated genes and these changes depend on the chronicity of the damage. Persistent DNA damage may impact cell functionality, including the release of damaging factors.
Background: Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are progressive neurodegenerative diseases with substantial clinical, pathological, and genetic overlap. A hexanucleotide repeat expansion in the C9orf72 gene is the most frequent reported genetic cause of ALS and FTD. The objective of this study was to identify pathways involved in disease pathogenesis that are unique to each clinical phenotype and those that are similar across this spectrum.

Methods:
We performed an unbiased, quantitative proteomic screen using post-mortem brain tissue from patients clinically diagnosed with ALS (n=19), FTD (n=12), and patients who had both ALS and FTD (n=10), compared to normal (without neurological disease) controls (n=10). Patient tissue included those with and without the C9orf72 expansion mutation.
Results: Our data identified several pathways that differentiated these four clinically defined groups using weighted correlation network analysis. These included RNA-binding proteins, astrocytic markers, proteins involved in nucleocytoplasmic transport, and synaptic proteins among other pathways. Using principal component analysis, we found that the proteomic signatures segregated out by clinical diagnosis. The presence of a C9orf72 expansion mutation was not identified as an independent variable associated with proteomic differences. Future detailed validation of these observed differences will clue us into the molecular underpinnings of these overlapping diseases.
Discussion: Based on our findings, we conclude that there are overlapping cellular pathways implicated in the diseases along this spectrum but also specific differences unique to clinical phenotypes separating ALS, FTD, and ALS-FTD. Remarkably, there is no direct correlation between phenotype, genotype, and pathology.
Objectives: We use a novel method to examine the proteomic composition of intraneuronal inclusions in human FTLD cases to identify unifying mechanism of FTLD. Determining FTLD proteomes may also provide insight into the mechanisms of ALS/MND, where diseased tissues contain inclusions composed of proteins also found in FTLD.
Methods: Currently, there are no comprehensive proteomic studies of FTLD inclusions. Methods that isolate inclusions by centrifugation tend to exclude soluble proteins, while laser capture microdissection is prone to sample contamination by the surrounding tissue due to the small size of the inclusions. We have developed an unbiased approach to elucidate FTLD inclusion proteomics in formalin-fixed brains called spatially targeted optical micro-proteomics (STOMP). In STOMP, the tissue section is saturated with a ''photo-tag'' compound. The tissue is stained with an antibody (anti-tau or antiubiquitin) to reveal the inclusions and imaged by standard confocal microscopy. The resulting image is a 3D coordinate file indicating the location of inclusions in the tissue. That image file in combination with two-photon excitation is used to deliver UV light specifically to the protein inclusions, which results in the exclusive attachment of the ''photo-tag'' to all proteins present in the inclusion bodies. The tissue section is then solubilized and the photo-tagged proteins are affinity-purified and identified by mass spectrometry. STOMP provides high resolution microproteomics for protein inclusions in FTLD at a resolution that far exceeds current methods.

Results:
We have acquired 12 FTLD-ubiquitin cases, one case of FTD-tau, and 12 control cases from the Maritime Brain Tissue bank. As a preliminary result, we have performed STOMP on the FTLD-tau case and generated a list of proteins that associate with tau-positive inclusions. The list of proteins is pending statistical analysis and validation by immunohistochemistry, but the preliminary data show that STOMP is an effective method to interrogate FTLD inclusion proteomics.
Conclusions: Understanding the composition of protein inclusions in FTLD will increase our understanding of this heterogeneous group of disorders and our STOMP technique is likely the only existing technique that is capable of achieving this goal. Recently, we and others identified a significant downregulation of miR-1234-3p and miR-1825 in the serum of sporadic ALS (sALS) patients (1,2). Additionally, downregulation of miR-1825 was evident in the serum of familial ALS (fALS) cases (3). However, miR-1825 and miR-1234-3p are not well characterized and their mRNA targets have not been investigated so far.
Objectives: We aimed to examine if the extracellular downregulation of miR-1825 and miR-1234-3p in ALS patients is the consequence of reduced intracellular expression levels and to determine the possible role of miR-1825 in the pathogenesis of ALS.
Methods: Using RT-qPCR we measured the relative levels of miR-1825 and miR-1234-3p in various postmortem tissue (brainstem, spinal cord, liver, lymph node, skeletal muscle) and ALS patient-derived keratinocytes. Additionally, we identified the mRNA targets of miR-1825 by combining both proteomic (mass spectrometry) and transcriptomic profiling (microarrays) in HEK293 cells transfected with a miR-1825-mimic. Targets of miR-1825 relevant for ALS were validated using a luciferaseapproach.
Results: A significant downregulation of miR-1825 was detected in postmortem tissue of ALS patients while miR-1234-3p showed less prominent intracellular alterations. Several of the targets of miR-1825 were related to ALS and could be confirmed in vitro.

Discussion and conclusion:
Our results confirm previous studies showing a systemic downregulation of miR-1825 in ALS (1,3). Knowledge of the targets of miR-1825 will help to clarify its role in ALS pathogenesis.
Background: ErbB4 was identified as a causative gene for a rare familial ALS designated as ALS19 (1). We previously reported that ErbB4 expression decreases in the spinal motor neurons of sporadic ALS patients. A recent study also showed decreased ErbB4 expression in the motor neurons of SOD1 transgenic mice (2). Furthermore, accumulating evidence suggests that ErbB4 signaling insufficiency is related to other neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease (3)(4)(5).
Objectives: This study investigated the relevance of ErbB4 to frontotemporal dementia (FTD).
Methods: We immunohistochemically analyzed the brains of FTD patients (including Pick's disease (n=1), corticobasal degeneration (n=4), progressive supranuclear palsy (n=3), argyrophilic grain disease (n=2), and FTLD17 (n=2)) by using a polyclonal anti-ErbB4 antibody raised against the intracellular C-terminal region of ErbB4 to evaluate the expression of ErbB4 in affected brain regions. This is an ongoing research study.
Results: ErbB4 accumulates in the pathological hallmarks of FTLD-TAU. These include Pick bodies in Pick's disease, ballooned neurons and coiled bodies in corticobasal degeneration, globose neurofibrillary tangles and coiled bodies in progressive supranuclear palsy, grains and coiled bodies in argyrophilic grain disease and various hyperphosphorylated tau inclusions in FTLD17. In contrast, astrocytic tau inclusions were not stained with anti-ErbB4 antibody.
Discussion and conclusions: This study showed that ErbB4 localizes to neuron-and oligodendroglia-derived pathological hallmarks in FTLD-TAU. Based on these findings, we hypothesize that ErbB4 colocalizes with abnormally phosphorylated tau causing impaired ErbB4 signaling. An alternative hypothesis is that the increased expression of ErbB4 and upregulation of its signaling cascade might accelerate the phosphorylation of tau. In summary, ErbB4, a causative gene product of familial ALS, might be involved in the pathophysiology of FTLD-TAU.
Background: A number of studies propose that bulbaronset ALS (B-ALS), compared with limb-onset ALS (L-ALS), may present with a unique neurodegenerative profile that is associated with specific and concomitant cognitive/language impairments (1). Other studies report no such association (2). Theories about propagation of neuropathological changes in ALS progression are also debated. Two predominant hypotheses postulate that: (1) disease propagates from initial lesion site to proximate neuroanatomical regions (3); or (2) propagation occurs within neuronal networks that form functional and structural connectomes (4). Recent literature supporting the connectome hypothesis predominates (5).
Objectives/Design: To investigate regional histopathological features of cases with B-ALS vs. L-ALS in order to investigate the connectome hypothesis in the context of bulbar disease. We hypothesized that brain structures associated with bulbar motor/speech dysfunction such as Broca area, Wernicke area, primary auditory cortex, bulbar motor cortex, and subcortical white matter may demonstrate greater histopathology disease burden in B-ALS than L-ALS.
Methods/Analysis: Brain tissue samples from 5 B-ALS, 5 L-ALS, and 5 sex-and age-matched controls, all righthanded, were examined. Both ALS groups were matched by disease duration and premorbidly well characterized clinically with respect to bulbar motor and extramotor involvement and genetic workup. Histopathological evaluation included a central pathology review of left hemispheric regions of interest (ROIs): Broca and Wernicke areas, primary motor, premotor, supplementary motor cortices, and primary auditory cortex, as well as brainstem motor nuclei, cerebellum, and white matter tracts (specifically arcuate and uncinate fasciculi, corticospinal, and corticobulbar tracts). Currently, all ROIs for ALS cases have been sampled and pathologic analysis is underway using semi-quantitative assessment of routine and immunohistochemical stains to determine neuronal loss (NeuN), gliosis (GFAP), macrophage/microglial density (CD163), proteinopathy (TDP43), axonal density (NF), and myelin pallor (HE/LFB).
Discussion: This ongoing work will result in better understanding of bulbar ALS from its clinical presentation to its neuropathology. Findings will shed light on mechanisms of pathology propagation in the disease, aid in accurate patient subtyping, and help predict disease progression, all paramount for discovery of the etiology and treatment for this currently incurable disease. Objectives: To identify protein-protein interactomes for ALS, HSP/PLS, and SMA and to reveal canonical pathways, proteins, and cellular functions critical for selective vulnerability of CSMN and SMN, and to further delineate commonalities and differences among each motor neuron disease.

P155 HUMAN MUTATIONS HELP CONNECT GENES TO PATHWAYS AND NETWORKS, AND OFFER
Methods: We first compiled a published list of mutations causative or associated with ALS, HSP, PLS, and SMA, then determined the binding partners of each mutated gene product via curated and published information using large-data management tool boxes, such as ingenuity pathway analysis. After protein interactome for each disease was determined, we investigated the common and unique proteins, interaction domains, important canonical pathways, signaling molecules, upstream regulators, and protein-protein network interactions that are associated with each disease.
Results: To date, causative and associated genes have been identified in ALS (n=23; n=28), HSP/PLS (n=36; n=29), and SMA (n=4; n=17). ALS (n=1139), HSP/PLS (n=611) and SMA (n=672) binding partners were identified and among these binding partners common and unique proteins, proteins with most frequent interactions with mutated gene products were identified. We further identified canonical pathways, networks and individual proteins within these networks exhibiting the greatest number of interactions for all ALS, all HSP/PLS, and all SMA binding partners as well as disease exclusive and overlapping partners for each motor neuron disease. Our ongoing studies focus on the secreted molecules that are common and unique among diseases.

Conclusion:
We conclude that human mutations inform us on the basis of selective motor neuron vulnerability. We find that it is not the individual gene, but the proteinprotein network associated with each gene product that is responsible for the emergence of selective vulnerability and progressive degeneration in the context of ALS, SMA, and HSP/PLS. The information we generated has begun to reveal the most important cellular events that are perturbed and become dysfunctional in the presence of mutations. Identification of common and unique pathways and protein interaction dynamics important for distinct motor neuron population will help build effective treatment strategies in the near future.  2). Furthermore, such selective vulnerability seen in SMA is irrelevant to any morphological characteristics (1). We put forward the hypothesis that intrinsic molecular differences between motor neurones could modulate the disease severity.
Objectives: Our aim is to identify the intrinsic disease modifiers between motor neurones with different vulnerability.
Methods: Based on previous results (1), the motor neurones innervating extensor digital longus (EDL, resistant), gastrocnemius (GS, intermediate), and anterior tibialis (TA, vulnerable) were selected for this study. The motor neurones were retrogradely labeled in normal FVB mice (2 weeks age) with WGA-HRP and isolated with laser capture dissection (LCM) for transcriptome microarray analysis. Data mining of probe expression values using DAVID and GSEA created a comprehensive interpretation of the differences between the motor neuron groups. The potential disease modifying factors were subjected to in vivo and in vitro functionality validation.
Results: In this study, we directly accessed the transcriptional profiles of different motor neurones innervating different muscles by using microarray combined with laser capture microdissection. It enabled us to uncover subtle but important differences that may differentiate the vulnerabilities among these three groups of motor neurones. Additionally, wild-type mice were used to eliminate the false-positive result that may be secondary to the general disease response. Overall, we find resistant motor neurones display higher activities of cellular metabolisms such as oxidative phosphorylation, protein synthesis, and degradation.
Discussion and conclusions: Motor neurones cope with the disease differently. As such, it is believed that the understanding of intrinsic differences between more and less vulnerable motor neurones is the key to identifying protective pathways that could be therapeutic targets. Introduction: A connection between Alzheimer's disease and Down syndrome has been described extensively in the literature. The higher risk for Alzheimer's disease in people with Down syndrome has been attributed to the increased production of amyloid beta due to an extra copy of chromosome 21, but other genes on chromosome 21 may also play a role, such as superoxide dismutase (SOD1). Little is known about the consequences of trisomy 21 for other neurodegenerative diseases.
Case study: We present the case of a 49-year-old male diagnosed with trisomy 21 at an early age, who developed severe intellectual deficiency and progressive muscle weakness of the upper limbs at the age of 47. He also complained of pain in the shoulders, and became unable to move his right arm within months. The symptoms worsened over the following year. He progressively stopped walking, developed dysphagia, further impairment of the lower limbs and eventually died.
Results: At the autopsy, the patient presented characteristic craniofacial morphological features of Down syndrome. Macroscopic examination of the brain showed atrophy of the superior temporal gyrus and the frontoparietal cortex, a small hippocampus and dilatation of the lateral ventricles. Microscopic examination of the brain showed typical features of Alzheimer's disease with amyloid deposits in the cerebral cortex, basal ganglia, and cerebellar cortex. Gallyas staining showed the presence of numerous neuritic plaques and widespread neurofibrillary degeneration at the level of the hippocampus. Immunohistological stains for alpha-synuclein did not reveal the presence of Lewy bodies. The spinal cord examination showed atrophy of the corticospinal tract. CD68 immunohistochemistry revealed abundant macrophages in the medullary pyramids and lateral columns and an associated microglial reaction. TDP-43 immunohistochemistry showed a filamentous staining in the cytoplasm and a loss of nuclear staining within motor neurons. Ubiquitin immunohistochemistry showed weak staining of some spinal nerve roots. Interestingly, SOD1 immunohistochemistry showed a staining in the cytoplasm colocalizing with TDP-43.
Conclusion: The present case shows that neurodegenerative disease in Down syndrome patients can take other forms besides Alzheimer's disease, including amyotrophic lateral sclerosis. In familial ALS (FALS), any one of the reported SOD1 inherited mutations in the SOD1 gene can lead to misfolding of the protein exerting its toxic gain of function. As cytoplasmic TDP-43 inclusions in mutant SOD1-related FALS cases are relatively rare, the presence of cytoplasmic misfolded SOD1 colocalizing with TDP43 suggests that the present case is sporadic in nature and that non-native conformers of SOD1-linked ALS and SALS might converge on a common pathogenic pathway.
DOI: 10.1080/21678421.2016.1232060/0029 Background: The high incidence of ALS-Parkinsonism-Dementia complex reported on Guam in the 1950s was linked to exposure to cyanobacterial toxins. Epidemiological studies in USA, France, and Sweden support the view that exposure to cyanobacterial toxins increases the risk of developing ALS. Biomagnification of the cyanobacterial toxin b-methylamino-L-alanine (BMAA) through human food chains (molluscs and fish) has also been reported. Most cyanobacteria synthesize both of the non-protein amino acids (NPAAs) BMAA and 2,4 diaminobutyric acid (DAB). Little is known, however, about the toxicity of DAB and the toxicity of BMAA and DAB in combination. NPAAs can be neurotoxic due to their ability to mimic/replace protein amino acids in metabolic pathways, become mistakenly incorporated into proteins or to exert excitotoxic effects on glutamate receptors. Although multiple mechanisms of toxicity have been proposed for BMAA no one has carried out systematic analysis to determine how BMAA affects the whole proteome.
Objectives: (i) To examine the ability of DAB to enter the human food chain; (ii) To use proteomic approaches to examine the range of mechanisms by which BMAA and DAB could initiate neurodegenerative disease.
Methods: We carried out proteomic analysis on human neuroblastoma cells treated with BMAA (500mM) and DAB (500mM), and BMAA and DAB in combination to determine which cellular pathways were altered.
Results: Among the metals analyzed in serum, as concentration resulted significantly lower in patients than in controls (p=0.007); Mn and Hg showed lower levels in patients. Auto-CM analysis linked closely high concentrations of Al and Se to the ALS group. Levels of metals in whole blood have been correlated with levels in serum. Our proteomics data show that some proteins related to Acute Phase Response (APR) and lipid homeostasis are decreased in patients (APOA1, APOA2, TTR, RET4, and SAP) while only ANT3 results increased. For some of these proteins we can describe a drastic reduction in the first 5 years of disease. Apo"4 allele is more represented in the patient's group than in controls.
Discussion: Impaired metal homeostasis, attributable to environmental exposure, could lead to mineral overload. Waters of the creek of the narrow valley, where these subjects are located, are reported to be strongly polluted due to acid mine drainage. Besides promoting oxidative stress, metals can compete for the binding sites of metalcontaining proteins, such as those containing iron-sulfur clusters (3). The different expression of the APR proteins reported could be a reflection of the disease status of the subjects analyzed, possibly linking ALS to a chronic inflammation status. Enrichment in Apo"4 allele frequency in patients may provide a link between neurodegeneration and lipid metabolism disturbances. It is important to highlight the fact that all the proteins found differentially expressed in our study have already been described in other studies. This strengthens our methodological approach, based on a small number of patients but with a common environmental exposure.