Amyotrophic Lateral Sclerosis Part 10 docx

342 Amyotrophic Lateral Sclerosis the aggregation is significantly retarded at pH < 5.5 While SOD1 exists as a dimer, the oxidized SOD1 dissociates into monomers and then forms non-amyloid aggregates with amorphous and fibrillar morphologies The oxidation-induced aggregation does not occur when SOD1 is in a holo state Zinc-binding affinity of SOD1 has been known to decrease with fALS mutations (Hayward et al., 2002); therefore, mutant SOD1 is more susceptible to aggregation through the metal-catalyzed oxidation than the wild-type protein Incubation time will be another key factor to induce the aggregation of a mature SOD1 (i.e a fully metallated SOD1 with an intramolecular disulfide bond) Usually, the aggregation kinetics of proteins has been monitored for at most 3 – 5 days, where either fully mature or even partially mature SOD1 does not aggregate in a physiological buffer without any chaotropic reagents Nonetheless, Hwang et al have extended the incubation time up to more than 300 hours (> 10 days) and found the fibrillar aggregation of fully mature SOD1 (with C6A/C111S mutations) under physiological conditions (~300 M proteins, pH 7.8, 37 oC) (Hwang et al., 2010) The SOD1 aggregates after a prolonged incubation did not show apple-green birefringence upon binding Congo Red nor strong enhancement of ThT fluorescence, consistent with properties of inclusions in SOD1-related fALS patients (Kato et al., 2000) It remains unknown if SOD1 retains metal ions even in the aggregated state, it is possible that such a long incubation of SOD1 proteins somehow leads to the partial loss and/or the altered binding geometries of metal ions In summary, SOD1 can adopt theoretically 44 types of modified states when metal binding, disulfide formation and dimerization are taken into account (Furukawa & O'Halloran, 2006) Many papers point out the strengths of the SOD1 aggregation model for ALS; however, as mentioned above, there is still no consensus on which state of SOD1 is responsible for aggregation observed in fALS cases Researchers including myself have thus continuingly pursued a mechanism describing why more than 100 ALS-causing mutations in SOD1 commonly facilitate the SOD1 aggregation process 2.2 TDP-43-positive inclusions in ALS patients TDP-43 is a DNA/RNA binding protein with 414 amino acids and contains two RNA recognition motifs (RRM1 and RRM2) and a C-terminal auxiliary region (Ayala et al., 2005) As of now, more than 40 mutations have been identified in the TDP-43 gene as being pathogenic, and most of the mutations are localized in the C-terminal region (http://alsod.iop.kcl.ac.uk) One of physiological functions of TDP-43 is to regulate an alternative splicing of several gene transcripts (Ayala et al., 2008a; Buratti & Baralle, 2001); usually, TDP-43 is localized at the nucleus but is also known to shuttle between nucleus and cytoplasm (Ayala et al., 2008b) Under pathological conditions, in contrast, TDP-43 is cleared from the nucleus and is mislocalized at the cytoplasm, where the ubiquitin- and TDP-43positive inclusions are observed (Arai et al., 2006; Neumann et al., 2006) Formation of TDP43 inclusions has been confirmed in sALS and SOD1-negative fALS but not in SOD1-linked fALS (Mackenzie et al., 2007) Actually, before identification of pathogenic mutations in the TDP-43 gene, proteomic analysis of ubiquitin-positive inclusions in sALS patients has revealed TDP-43 as a major component of inclusions (Arai et al., 2006; Neumann et al., 2006) TDP-43 immunoreactive inclusions have also been observed in many other neurodegenerative diseases such as frontotemporal lobar degeneration (FTLD), Huntington disease, and Alzheimer disease, which recently leads to a new disease category called TDP43 proteinopathies (Geser et al., 2009) Protein Aggregates in Pathological Inclusions of Amyotrophic Lateral Sclerosis 343 In pathological inclusions, TDP-43 is abnormally hyper-phosphorylated and cleaved to generate C-terminal fragments (Arai et al., 2006; Neumann et al., 2006) Pathological TDP-43 is also distinct from its normal counterpart because it exhibits decreased solubility in a buffer containing a detergent, Sarkosyl Ultrastructurally, inclusions observed in TDP-43 proteinopathies are characterized by bundles of straight fibrils with 10 – 20 nm diameter that are immunostained by anti-TDP-43 antibodies (Lin & Dickson, 2008) Similar to SOD1positive inclusions, however, TDP-43 inclusions are also not stained by Thioflavin S and Congo Red (Kerman et al., 2010), implying less amyloid characters Interestingly, the Cterminal fragments are enriched in the cytoplasmic inclusions in brain of ALS patients, but in the spinal cord, inclusions are composed of full-length TDP-43 (Igaz et al., 2008) Furthermore, Hasegawa et al have found the immunoblot distinction of TDP-43 among different TDP-43 proteinopathies (Hasegawa et al., 2008); for example, Sarkosyl-insoluble fractions of ALS and FTLD brains exhibit different electrophoretic band patterns of the Cterminal fragments of phosphorylated TDP-43 in the Western blots Depending upon the clinicopathological subtypes of TDP-43 proteinopathies, multiple pathways can thus be considered for the formation of TDP-43 inclusions; however, molecular mechanisms of truncation and phosphorylation in TDP-43 remain unknown 2.2.1 TDP-43 aggregates in mouse models Homozygous disruption of the TDP-43 gene is embryonic lethal in mice (Kraemer et al., 2010), and post-natal deletion of the TDP-43 gene by utilizing a Cre recombinase also produces lethality albeit without any ALS-like symptoms (Chiang et al., 2010) Expression of wild-type human TDP-43 has also been reported to be toxic in mice in a dose-dependent manner; indeed, TDP-43 transgenic mice exhibit a wide variety of motor dysfunctions, which appears to depend upon the promoter regulating the expression of the transgene (Da Cruz & Cleveland, 2011) More toxic effects of ALS-causing mutations (A315T and M337V examined so far) in the TDP-43 transgene has not been established yet Surprisingly, any of the transgenic mice expressing wild-type and mutant TDP-43 have not reproduced the formation of ubiquitin- and TDP-43-positive inclusions When human TDP-43 with A315T mutation is expressed in mice under the control of mouse prion promoter (Wegorzewska et al., 2009), the mice develop gait abnormality with an average survival of about 150 days, and ubiquitin-positive inclusions are observed in specific neuronal populations including spinal motor neurons Despite this, those ubiquitin-positive inclusions are not immunostained with anti-TDP-43 antibodies, and very limited amounts of C-terminally truncated TDP-43 are confirmed Furthermore, mutant TDP-43 exhibits similar solubility in a Sarkosylcontaining buffer to that of mouse endogenous wild-type TDP-43 Although truncation as well as insolubilization of TDP-43 characterizes the TDP-43 proteinopathies, both of these pathological processes may hence not be required for neurodegeneration In contrast, Wils et al have constructed a mouse expressing wild-type human TDP-43 under the control of a neuronal murine Thy-1 promoter and found a dose-dependent degeneration of cortical and spinal motor neurons (Wils et al., 2010) Immunohistochemical analysis has further confirmed the formation of ubiquitin-positive inclusions, which are stained by an anti-TDP-43 antibody and also an antibody recognizing Ser409/410-phosphorylated TDP43 Abnormal phosphorylation on TDP-43 is thus reproduced in this model mouse; furthermore, the C-terminal truncation of human TDP-43 is observed albeit much less amounts than that in ALS patients Despite this, human TDP-43 in the affected mice remains 344 Amyotrophic Lateral Sclerosis soluble in a Sarkosyl-containing buffer, showing that the pathological processes of TDP-43 are not completely reproduced in the transgenic mouse model Transgenic rats expressing human wild-type and mutant (M337V) TDP-43 have also been made (Zhou et al., 2010) Soon after the birth, TDP-43M337V transgenic rats become paralyzed at 20 - 30 days and die at postnatal ages; in contrast, TDP-43WT transgenic rats exhibit no paralysis by the age of 200 days Mutation-specific toxicity of TDP-43 has thus been reproduced in these rat transgenic models, but TDP-43 inclusions are rarely detected and present only in the cortex of paralyzed TDP-43M337V transgenic rats A very faint amount of truncated TDP-43 is detected, and phosphorylated TDP-43 is accumulated at the cytoplasm of spinal motor neurons These molecular changes of TDP-43 are, however, confirmed in both TDP-43WT and TDP-43M337V transgenic rats, implying little roles of truncation and phosphorylation in expressing the mutant-specific toxicity of TDP-43 Accordingly, it still remains to be established in the rodent models how mutant TDP-43 exerts its toxicity and is involved in the inclusion formation under pathological conditions 2.2.2 TDP-43 aggregates in vitro Bacterially expressed TDP-43 normally forms insoluble inclusion bodies, which hampers biochemical characterization of TDP-43 proteins Johnson et al have nonetheless succeeded to obtain soluble full-length 6 x His-tagged TDP-43 by using a cold shock expression system in E.coli (Johnson et al., 2009) Agitation of 3 M full-length TDP-43 in 40 mM HEPES/150 mM KCl/20 mM MgCl2/1 mM DTT, pH 7.4 at 25 oC increases solution turbidity within an hour, supporting the high aggregation propensities of TDP-43 A TDP-43 truncate that is devoid of the C-terminal auxiliary domain does not increase its solution turbidity, suggesting an important role of the C-terminal domain in the aggregation in vitro Aggregates of full-length TDP-43 exhibit both filament-like and thread-like morphologies but did not react with the amyloid-diagnostic dyes, Congo Red and ThT A subset of fALSlinked mutations (M337V, Q331K) slightly facilitates the aggregation kinetics of full-length TDP-43 A high propensity for fibrillation has been also shown for the synthetic peptide fragment of a TDP-43 C-terminal region (Gly 287- Met 322) (Chen et al., 2010) Fibrillar aggregates of the C-terminal peptide did not increase the intensity of Thioflavin T fluorescence Interestingly, an ALS-causing mutation, G294A, but not A315T renders the fibrillar aggregates ThT-positive While fibrils of all C-terminal peptides (wild-type, A315T, G294A) possess -sheet rich structures, ALS mutations would affect the biochemical/structural properties of TDP-43 aggregates I have recently reported that bacterially expressed full-length TDP-43 is resolubilized, purified in the presence of GdnHCl, and then refolded by dilution of GdnHCl (Furukawa et al., 2011) Such refolded TDP-43 proteins retain the physiological DNA binding function but forms fibrillar aggregates by agitation at 37 oC in 100 mM Na-Pi/100 mM NaCl/5 mM EDTA/5 mM DTT/10 % glycerol, pH 8.0 A C-terminal half of TDP-43 assumes a core in the fibrillar aggregates and reproduces the fibrillation propensities of full-length TDP-43 proteins These in vitro TDP-43 fibrils are insoluble in a Sarkosyl-containing buffer, which is a consistent feature with the pathological inclusions A seeding activity is also a notable feature of TDP-43 fibrils in vitro, where pre-formed fibrils (or called “seeds”) function as a structural template to facilitate the recruitment of soluble proteins into insoluble fibrils This seeding reaction has been found to also occur inside the cultured cells by transducing the cells with in vitro TDP-43 fibrils; thereby, the formation of Sarkosyl-insoluble and ubiquitinated TDP-43 inclusions is well Protein Aggregates in Pathological Inclusions of Amyotrophic Lateral Sclerosis 345 reproduced in the cell This is notable because simple overexpression of TDP-43 in the cultured cells has never generated the Sarkosyl-insoluble inclusions It remains controversial whether the aggregation of TDP-43 is a cause or a result of the disease; however, as recently proposed in the other neurodegenerative diseases (Aguzzi & Rajendran, 2009; Brundin et al., 2010), a seeding activity of TDP-43 proteins may contribute to the propagation of pathological changes with the progression of diseases All recent in vitro studies on TDP-43 proteins have revealed its high propensities for aggregation, which are provided by the C-terminal auxiliary domain Given that most of the fALS-causing mutations are located at this domain, the mutational alteration in the aggregation propensities of TDP-43 might be a part of the ALS pathomechanism More in vitro experiments will, however, be required to reveal if the aggregation reactions of TDP-43 are affected by mutation, truncation, and phosphorylation 2.3 FUS-positive inclusions in ALS patients FUS was initially identified as the N-terminus of FUS-CHOP (CCAAT/enhancer binding protein homologous protein), a fusion oncoprotein expressed in human myxoid liposarcoma with the t(12;16) chromosomal translocation (Crozat et al., 1993) Like TDP-43, FUS is a DNA/RNA binding protein with 526 amino acids and comprised of multiple domains as follows (from N-terminal to C-terminal); a Q/G/S/Y-rich domain, a G-rich domain, an RNA-recognition motif (RRM), an R/G-rich domain, a Zn-finger motif, and a region containing a nuclear localization signal (NLS) (Dormann et al., 2010; Iko et al., 2004) Under physiological conditions, FUS has been proposed to be involved in transcription regulation (Uranishi et al., 2001), RNA splicing (Yang et al., 1998), and RNA transport including nucleo-cytoplasmic shuttling (Zinszner et al., 1997) Late in 2007, which was before identification of pathological mutations in the FUS gene, FUS protein was found as one of major proteins recruited into neuronal intranuclear inclusions in patients of Huntington disease (Doi et al., 2008) In this neurodegenerative disease, a polyglutamine tract in a protein called huntingtin (HTT) is abnormally expanded, leading to fibrillar aggregation of mutant HTT in affected neurons (Zoghbi & Orr, 2000) FUS is sequestered by fibrillar HTT aggregates and then becomes insoluble and possibly dysfunctional (Doi et al., 2008) Loss of physiological functions of FUS would, therefore, contribute to neuronal cell death in Huntington’s disease (Doi et al., 2008) as well as other polyglutamine diseases (Doi et al., 2010) Then, fALS-causing mutations in the FUS gene have been identified in 2009 (Kwiatkowski et al., 2009; Vance et al., 2009), and, as of now, at least 40 mutations have been reported, most of which are localized at a G-rich domain and a C-terminal NLS-containing region (http://alsod.iop.kcl.ac.uk) Although neuropathological analysis of fALS patients with FUS mutations has been still limited, cytoplasmic mislocalization of nuclear FUS protein in motor neurons is a major pathological hallmark Indeed, as shown by a recent study (Dormann et al., 2010), fALS-causing mutations at the C-terminal region of FUS result in the functional impairment of the NLS, facilitating the cytoplasmic mislocalization of mutant FUS In FUS-related fALS, FUS-immunoreactive cytoplasmic inclusions are observed, which have been recently found to exhibit mutation-dependent heterogeneity (Mackenzie et al., 2011) For example, P525L mutation in FUS associates with a relatively early onset (twenties) of ALS, where round FUS-immunoreactive neuronal cytoplasmic inclusions are found In contrast, late-onset (forties to sixties) ALS cases are linked to R521C mutation in FUS and 346 Amyotrophic Lateral Sclerosis have tangle-like FUS-immunoreative neuronal and glial cytoplasmic inclusions Furthermore, it has been reported that FUS-immunoreactive inclusions are observed in spinall spinal anterior horn neurons in all sporadic and familial ALS cases tested, except for those with SOD1 mutations (Deng et al., 2011b) Although mutations in FUS account for only a small fraction of fALS and sALS cases, FUS proteins may be a common component of cytosolic inclusions in non-SOD1 ALS In motor neurons of patients with juvenile ALS, FUS has been shown to form filamentous aggregates with a diameter of 15 – 20 nm, which are often associated with small granules (Baumer et al., 2010; Huang et al., 2011) Staining with Thioflavin T/S and Congo Red has not, however, been performed yet on the inclusions of FUS-linked ALS It also remains unknown if pathological FUS decreases its solubility or is modified/truncated in inclusions 2.3.1 FUS aggregates in a rat model As of now, there is no mouse model of FUS-linked ALS, but a transgenic rat expressing wild-type or ALS-causing mutant (R521C) FUS has been published (Huang et al., 2011) Only the mutant FUS transgenic rats developed paralysis at an early age (1 – 2 mo) with a significant loss of neurons in the frontal cortex and dentate gyrus These pathological changes are not observed in age-matched wild-type FUS transgenic rats, although, at the advanced age (> 1 yr), wild-type FUS transgenic rats display a deficit in spatial learning and memory with a moderate loss of neurons in the frontal cortex and dentate gyrus Immunohistochemical analysis of the cortex and spinal cord has shown the appearance of ubiquitin-positive inclusions at the paralysis stages of both wild-type and mutant FUS rats; however, the inclusions are not immunostained with anti-FUS antibodies Given that several different anti-FUS antibodies show distinct immunoreactivities toward FUS-containing inclusions in sALS cases (Deng et al., 2011b), more detailed investigations will be necessary to characterize the possible aggregation of FUS forming pathological inclusions 2.3.2 FUS aggregates in vitro There is only one paper published on the aggregation reaction of purified FUS proteins (Sun et al., 2011); Sun et al have prepared GST-fused FUS proteins intervened with a TEV protease site and found that the cleavage of 2.5 – 5 M GST-FUS with a TEV protease produces full-length FUS in 100 mM Tris/200 mM trehalose/0.5 mM EDTA/20 mM glutathione, pH 8.0, and starts aggregation without a lag-time at 22° C in the absence of agitation The resultant in vitro aggregates of FUS do not increase the intensity of ThT fluorescence and are completely soluble in an SDS-containing buffer They have further examined the aggregation reactions of several truncated FUS proteins and shown that the N-terminal region of FUS (1 – 422) is enough to reproduce the aggregation behavior of fulllength FUS Aggregates of both full-length FUS and truncated FUS (1 – 422) are fibrillar in the morphologies, which resemble to the FUS inclusions in the ALS patients No effects of fALS-causing mutations (H517Q, R521H, R521C) are observed on the in vitro fibrillation kinetics of full-length FUS proteins 3 Conclusion In this chapter, recent progress has been reviewed on aggregation mechanisms of ALS pathogenic proteins, SOD1, TDP-43 and FUS Common to all these three proteins, Protein Aggregates in Pathological Inclusions of Amyotrophic Lateral Sclerosis 347 structural/biochemical characters of aggregates in vitro are much dependent upon experimental conditions, and it remains obscure which of aggregates in vitro reproduces the pathological inclusions in patients In particular, post-translational processes such as metallation, disulfide formation, phosphorylation, and truncation appear to affect the aggregation pathway(s) of the pathogenic proteins In future, therefore, it will become more important to correlate any abnormalities in these post-translational modifications with pathogenicity of ALS Very recently, mutations in another gene, optineurin (OPTN), have been linked to fALS cases, and hyaline inclusions in the anterior horn cells of spinal cord were immunoreactive for OPTN in patients with OPTN mutation (E478G) (Maruyama et al., 2010) Furthermore, albeit controversial, skein-like inclusions in all the sALS and non-SOD1 fALS have been reported to be immunostained with an anti-OPTN antibody (Deng et al., 2011a) Aggregation of an OPTN protein would thus be of relevance to describe the pathomechanism of both sporadic and familial ALS In spite of recent efforts to identify the causative genes for fALS, most of the cases are still genetically unidentified (Da Cruz & Cleveland, 2011) Given that the skein-like inclusions in the spinal anterior horn cells are characteristic of ALS, proteomic analysis of those inclusions will help to identify as-yet-unknown proteins pathogenic for ALS In addition, the component analysis of skein-like inclusions will help to describe the common pathomechanism of sporadic and familial ALS cases 4 References Aguzzi, A & Rajendran, L (2009) The transcellular spread of cytosolic amyloids, prions, and prionoids Neuron, Vol.64, No.6, pp 783-790, ISSN 1097-4199 Arai, T.; Hasegawa, M.; Akiyama, H.; Ikeda, K.; Nonaka, T.; Mori, H.; Mann, D.; Tsuchiya, K.; Yoshida, M.; Hashizume, Y & Oda, T (2006) TDP-43 is a component of ubiquitin-positive tau-negative inclusions 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1531-8249 Hayward, L.J.; Rodriguez, J.A.; Kim, J.W.; Tiwari, A.; Goto, J.J.; Cabelli, D.E.; Valentine, J.S & Brown, R.H., Jr (2002) Decreased metallation and activity in subsets of mutant 366 Amyotrophic Lateral Sclerosis 4.8 Clioquinol Clioquinol (5-chloro-7-iodo-8-hydroxyquinoline) is a quinoline metal chelator that binds selectively to zinc and copper ions (Cherny et al 2001) Having a hydrophobic nature, it crosses the BBB easily Recent research with clioquinol in neurological disorders contributed by an imbalance in metal ions has led to promising results, presenting the possibility of a new therapeutic strategy In AD transgenic mice, treatment with clioquinol resulted in the dissolution of aberrant neocortex beta amyloid (Aβ) aggregates, which are enriched with copper and zinc ions (Cherny et al 2001) In a pilot phase II clinical trial, the drug was well tolerated and led to a significant decrease in Aβ plasma levels in AD patients, providing support for future trials (Ritchie et al 2003) In PD, elevated levels of iron in the substantia nigra, the brain region affected in PD, has been reported In mice, oral administration of clioquinol antagonized the action of the Parkinson’s inducing agent 1-methyl-4-phenyl1,2,3,6-tetra-pyridine (MPTP) (Kaur et al 2003) In HD, where iron, copper and zinc have been implicated, clioquinol improved the symptoms and lifespan of transgenic HD mice (Nguyen et al 2005) A second generation 8-hydroxyquinoline, PBT2, has been developed to improve the safety and efficacy of clioquinol and also its pharmaceutical properties, such as solubility and bioavailability In preclinical in vivo and in vitro trials on transgenic AD mice, PBT2 was more effective in lowering plaque formation and reducing plaque toxicity More importantly, it may also improve cognition 5 Conclusion The current consensus is that ALS is a multifactorial disease However, an explanation for the initiation of the putative causative mechanism of ALS remains elusive, and there lacks a hypothesis that can link all the mechanisms together In recent years, the implication of the kynurenine pathway in multiple diseases, particularly neurodegenerative diseases, has led to an increase in assessing the efficacy of drugs targeting the kynurenine pathway in ameliorating disease symptoms and/or retarding disease progression The kynurenine pathway has been demonstrated to be involved in ALS and this provides an important link that ties together some of the major hypotheses underlying the pathogenesis of ALS, namely glutamate excitotoxicity, oxidative stress, non-cell-autonomous mechanism and apoptosis, which are also the major mechanisms via which QUIN exerts its neurotoxicity effects Due to the multiple pathways involved in the pathogenesis and progression of ALS, it may be speculated that a combination therapy could be more efficacious Hence, by targeting the kynurenine pathway, it is hoped that more effective therapeutic agents, acting in synergy with other agents, may uncover a better treatment for ALS 6 Appendix 3HAA3-hydroxyanthranilic acid 4-HNE4-hydroxynonenal Aβ Beta amyloid ADAlzheimer’s disease ALSAmyotrophic lateral sclerosis BBBBlood-brain barrier The Kynurenine Pathway 367 CNSCentral nervous system CSFCerebrospinal fluid D-1-MTD-1-methyl-tryptophan EAEExperimental autoimmune encephalomyelitis GCN2General control non-derepressible-2 kinase GTPGuanosine triphosphate HDHuntington’s disease HIVHuman immunodeficiency virus IDOIndoleamine 2,3-dioxygenase IFN-γInterferon gamma ILInterleukin KMOKynurenine 3-monooxygenase KYNKynurenine KYNAKynurenic acid MCPMonocyte chemoattractant protein MIPMacrophage inflammatory protein MPTPMethyl-4-phenyl-1,2,3,6-tetra-pyridine mRNAMessenger ribonucleic acid NF-κBNuclear factor kappa-light-chain-enhancer of activated B cells NMDAN-methyl D-aspartate NRNMDA receptor PICPicolinic acid QUINQuinolinic acid ROSReactive oxygen species SOD1Superoxide dismutase 1 TDOTryptophan 2,3-dioxygenase 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Contribution in ALS: Inflammation and Excitotoxicity Kim Staats1,2 and Ludo Van Den Bosch1,2 2VIB 1University of Leuven, Vesalius Research Center, Belgium 1 Introduction Amyotrophic Lateral Sclerosis (ALS) is a devastating progressive neurodegenerative disease, due to the loss of motor neurons and denervation of muscle fibres, resulting in increasing muscle weakness and paralysis The disease has an incidence of 2.7 cases per 100,000 people in Europe (Longroscino et al., 2010) It is diagnosed from teen years onward, but is more prevalent in the later years of life In lack of a medical cure, average life expectancy post diagnosis is between 2 and 5 years, though 10% of all patients live longer than 10 years Patients mainly succumb to the disease by respiratory insufficiency or may opt for euthanasia where legislature permits (Maessen et al., 2010) Although ALS is characterised by degeneration of central nervous system tissue, mental functions remain largely unaffected resulting in a locked-in state (Kotchoubey et al., 2003) At current, there is but one medicine to treat the disease, riluzole, slowing disease progression moderately (Miller et al., 2007) 1.1 Basic genetics of ALS Mutations in the ubiquitously expressed Cu/Zn superoxide dismutase 1 (SOD1) gene can cause ALS SOD1 detoxifies cell damaging free radicals and its mutations account for 20% of the ALS patients suffering from the disease by familial origin (fALS) worldwide The remaining 90% of ALS patients suffer from the disease by unknown sporadic causes (sALS), though a common mechanism is predicted as fALS and sALS patients display indistinguishable clinical phenotypes Overexpression of mutant forms of human SOD1 causes the ALS phenotype of transgenic SOD1 mice, accounting for an invaluable contribution to ALS research (Gurney et al., 1994) Many hallmarks of the disease are shared between patients and this rodent model, including specific motor neuron loss, aggregate formation, astrogliosis, microgliosis and progressive paralysis As the genetic ablation of SOD1 does not produce an ALS-like phenotype in mice (Reaume et al., 1996; Shefner et al., 1999) the pathogenic mechanism of mutant SOD1 is a toxic gain of function This gain of function may be exerted by protein misfolding, aggregation, impaired proteasome functioning, impaired retrograde transport, excitotoxic cell death or other mechanisms (reviewed in Bruijn et al., 2004) Mutations in other genes also cause familial ALS, including mutations in vesicle-associated membrane proteinassociated protein B (VAPB), TAR DNA binding protein (TDP-43), fused in 378 Amyotrophic Lateral Sclerosis sarcoma/translocated in liposarcoma (FUS/TLS), optineurin and valsolin containing protein (VCP) (Johnson et al.; Maruyama et al.; Rutherford et al., 2008; Van Deerlin et al., 2008; Del Bo et al., 2009; Kwiatkowski et al., 2009; Vance et al., 2009) Unfortunately, the discovery of these mutant genes has not yet progressed into useful ALS model organisms, so most of the work described below was conducted with mutant SOD1based ALS models 1.2 Non-cell autonomous ALS Multiple cell types contribute to the pathology making ALS a non-cell autonomous disease (Boillee et al., 2006a) By addition or deletion of mutant SOD1 in specific cell types, it is known that mutant SOD1 influences the disease depending on the cell type, including astrocytes (Yamanaka et al., 2008; Wang et al., 2011a), microglia (Boillee et al., 2006b), Schwann cells (Lobsiger et al., 2009) and motor neurons (Jaarsma et al., 2008) Additionally, ablation of T-cells (Beers et al., 2008; Chiu et al., 2008), B-cells (Naor et al., 2009), CD4+ and CD8+ cells (Beers et al., 2008) decrease survival of ALS mice, demonstrating the role of immune cells in disease progression Although ALS is a non-cell autonomous disease, mutant SOD1 expressed solely in motor neurons is sufficient to initiate the disease, albeit with a slower disease progression (Jaarsma et al., 2008) Motor neurons in the motor cortex, brainstem and spinal cord undergo cell death selectively in patients A number of hypotheses attempt to explain this cell type selectivity, including the long axons of the motor neurons (Fischer and Glass, 2007), their poor intracellular calcium buffering capacity (Grosskreutz et al., 2010) and motor neuron specific cell death pathways (Raoul et al., 2002; Raoul et al., 2006; Genestine et al., 2011) The contribution of mutant SOD1 expressing astrocytes in the non-cell autonomous character of ALS has been studied by excising mutant SOD1 from astrocytes which increases survival in two different mutant SOD1 mouse models (Yamanaka et al., 2008; Wang et al., 2011a) These results denote the toxic character of mutant SOD1 in astrocytes that accelerate disease progression significantly by mechanisms such as, but not exclusively, the below described mechanisms of neuroinflammation and excitotoxicity This is schematically presented in figure 1 1.3 Neuroinflammation observed in ALS Neuroinflammation occurs in a number of neurodegenative diseases, including ALS (reviewed in Papadimitriou et al., 2010 and Philips and Robberecht, 2011), and entails the reactive state of astrocytes (astrogliosis) and microglia (microgliosis) and the infiltration of lymphocytes Initially perceived as a bystander effect, neuroinflammation is currently seen as beneficial at first, removing damaged cells and secreting supportive factors, and potentially detrimental thereafter by excessive release of cytokines (Beers et al., 2011a) Evidence of inflammation is detected in post mortem tissue (Schiffer et al., 1996; Anneser et al., 2004; Casula et al., 2011; Sta et al., 2011; Wang et al., 2011b), in cerebrospinal fluid (CSF) (Baron et al., 2005; Tateishi et al., 2010) and in blood samples of ALS patients (Poloni et al., 2000) In accordance, similar parameters of neuroinflammation are detected in ALS rodent models (among many others in Kiaei et al., 2006; Keller et al., 2009; Beers et al., 2011b) Inflammation is generally perceived as hazardous in ALS, as increasing inflammation in ALS models exacerbates disease progression and diminishes survival (Nguyen et al., 2004; Gowing et al., 2009) Fittingly, therapeutic strategies targeting inflammation are often advantageous in ALS rodent models (see below) The Astrocytic Contribution in ALS: Inflammation and Excitotoxicity 379 1.4 Excitotoxicity in ALS An additional detrimental mechanism in ALS is excitotoxicity; an overstimulation of neurons causing neurodegeneration Glutamate binds to the N-methyl D-aspartate (NMDA) or α-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA) receptors, allowing extracellular sodium and calcium to enter motor neurons Increased levels of intracellular calcium consequently cause neuronal cell death The importance of excitotoxicity in ALS is demonstrated by the beneficial effects obtained by treating patients with riluzole Although the precise mechanism of this drug is not yet known, it blocks NMDA receptors, enhances re-uptake of glutamate from the synaptic cleft and inhibits glutamate release by blocking voltage-gated sodium channels (Siniscalhi et al., 1999), thus preventing motor neuron cell death Riluzole treatment increases predicted lifespan with a significant 12% in ALS mice (Bensimon et al., 1994; Lacomblez et al., 1996) and increases the probability of one year survival in patients by 9% (Miller et al., 2007) Unfortunately, it does not halt disease progression An overview of the current knowledge of the astrocytic contribution in ALS will be addressed in this chapter separately for the mechanisms inflammation and excitotoxicity 2 Astrocytes in inflammation Despite that microglia are the main immune cells of the central nervous system (reviewed in Ransohoff, 2010), astrocytes can also become reactive and contribute to neuroinflammation and are the focus of this chapter, with microglial inflammatory effects residing beyond the scope of this chapter During neuroinjury or neurodegeneration the production of cytokines induce astrogliosis in which astrocytes increase glial fibrillary acidic protein (GFAP) and vimentin expression as well as an array of other genes This response increases neuronal survival and includes both supportive factors (e.g growth factors and glutamate transporters) and cytokines to sustain/promote neuroinflammation Interestingly, during neuroinflammation the number of astrocytes increases by the differentiation of chondroitin sulfate proteoglycan, NG2, positive cells to astrocytes and not by astrocytic proliferation (Gowing et al., 2008) 2.1 Increasing inflammation in ALS To assess the effect of inflammation in ALS and thus to discover whether boosting the inherent inflammation would be beneficial, lipopolysaccharide (LPS) was daily administered to ALS mice (Nguyen et al., 2004) The effect of this treatment was a clear decrease in lifespan, implying that an increase of inflammation is detrimental in ALS (Nguyen et al., 2004) Another study, initially intended to decrease inflammation, administered macrophage colony stimulating factor (M-CSF) to ALS mice and observed an unexpected increase of microgliosis also leading to a decreased survival (Gowing et al., 2009) Although not directed specifically at astrocytes, this work has led to the understanding of the hazardous character of neuroinflammation in ALS 2.2 Astrogliosis in ALS Reactive astrocytes alter gene expression including an upregulation of the intermediate filaments GFAP and vimentin that allow for visualisation of astrogliosis by increased immunoreactivity of these filaments in patient and ALS model tissue Post mortem spinal 380 Amyotrophic Lateral Sclerosis cord tissue from fALS and sALS patients display astrogliosis (Schiffer et al., 1996), implying that reactivity of astrocytes is not limited to the familial form of ALS Interestingly, astrogliosis levels are similar between long surviving and short surviving ALS patients, although this is not the case for microglial activation and the amount of dendritic cells (Sta et al., 2011) An extra facet of astrogliosis in ALS is an increased immunoreactivity of tolllike receptor 4 in astrocytes of sALS patients (Casula et al., 2011) Astrogliosis in ALS mice is present at symptomatic stages preceeding microgliosis (Kiaei et al., 2006; Keller et al., 2009; Yang et al., 2011) Interestingly, GFAP is not necessary for astrogliosis as GFAP deficient astrocytes can still become reactive and do not affect survival of ALS mice (Yoshii et al., 2011) 2.3 Mutant SOD1 affects astrocytic inflammatory behaviour The expression of mutant SOD1 in astrocytes alters their function in vivo and in vitro To begin, deletion of mutant SOD1 in astrocytes in two distinct ALS models demonstrates the detrimental effect of mutant SOD1 in astrocytes mainly post onset, as deletion increased lifespan of ALS mice (Yamanaka et al., 2008; Wang et al., 2011a) Intriguingly, astrogliosis was unaltered, implying that the negative effect of mutant SOD1 in astrocytes is not due to altered levels of astrogliosis (Yamanaka et al., 2008), but potentially by astrocytes inducing microgliosis (Yamanaka et al., 2008; Wang et al., 2011a) An alternative approach arrives from the field of transplantation in which non-transgenic mesenchymal stem cells are transplanted into the spinal cord of ALS rats and differentiate into astrocytes, thus diluting the mutant SOD1 positive astrocytes in the spinal cord (Boucherie et al., 2009) This approach also shows unaltered astrogliosis, but also decreased microgliosis and cyclooxygenase 2 (COX2) expression, and extends murine ALS life span (Boucherie et al., 2009) The processes explaining this hazardous effect of mutant SOD1 in astrocytes has been investigated in vitro To begin, an interesting approach of transducing human astrocytes with wild-type SOD1 or mutant SOD1 increases inflammation in mutant SOD1 cultures (Marchetto et al., 2008) In addition, the mutant SOD1 transduced astrocytes provide a less viable environment for human embryonic stem cell derived motor neurons (Marchetto et al., 2008) The latter was rescued by using a NADPH oxidase 2 (NOX2) inhibitor, apocynin (Marchetto et al., 2008) Other studies concur that mutant SOD1 primary astrocytes exhibit a higher gene expression of cytokines on baseline and when stimulated by interferon γ (IFNγ) or tumor necrosis factor α (TNFα) (Hensley et al., 2006), implying once again that mutant SOD1 expression may affect the threshold of astrocytes to produce proinflammatory cytokines Accordingly, the expression of interferon simulated genes is detected in astrocytes of presymptomatic ALS mice (Wang et al., 2011b) and genetic ablation and knockdown of the interferon alpha receptor type 1 (IFNAR1) increase ALS mouse survival by 5% and 10%, respectively (Wang et al., 2011b) Intriguingly, Aebischer et al stress the importance of interferon signalling in mutant SOD1 astrocytes by demonstrating that mutant SOD1 astrocytes trigger the selective death of motor neurons mediated by IFNγ (Aebischer et al., 2011) This mechanism is dependent on the activation of the lymphotoxin-β receptor by LIGHT (TNFSF14) and genetic ablation of LIGHT extends survival of ALS mice by 13%, but does not postpone disease onset (Aebischer et al., 2011) Although this is a large increase in disease survival, clearly other mechanisms remain to play a role The above described altered functioning of mutant SOD1 expressing astrocytes is induced by an overexpression of multiple copies of mutant SOD1 It is unclear whether these effects ... Insoluble mutant SOD1 is partly oligoubiquitinated in amyotrophic lateral sclerosis mice J Biol Chem, Vol.281, No.44, pp 33325-33335, ISSN 0021-9258 348 Amyotrophic Lateral Sclerosis Baumer, D.;... ISSN 1460-2083 352 Amyotrophic Lateral Sclerosis Kerman, A.; Liu, H.N.; Croul, S.; Bilbao, J.; Rogaeva, E.; Zinman, L.; Robertson, J & Chakrabartty, A (2 010) Amyotrophic lateral sclerosis is a non-amyloid... sporadic amyotrophic lateral sclerosis from amyotrophic lateral sclerosis with SOD1 mutations Ann Neurol, Vol.61, No.5, pp 427-434, ISSN 0364-5134 Protein Aggregates in Pathological Inclusions of Amyotrophic