**4. References**

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

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.

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

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,

modified/truncated in inclusions.

**2.3.2 FUS aggregates** *in vitro*

kinetics of full-length FUS proteins.

**3. Conclusion** 

**2.3.1 FUS aggregates in a rat model** 


Protein Aggregates in Pathological Inclusions of Amyotrophic Lateral Sclerosis 349

Culotta, V.C.; Klomp, L.W.J.; Strain, J.; Casareno, R.L.B.; Krems, B. & Gitlin, J.D. (1997). The

Da Cruz, S. & Cleveland, D.W. (2011). Understanding the role of TDP-43 and FUS/TLS in

de Belleroche, J.; Orrell, R.W. & Virgo, L. (1996). Amyotrophic lateral sclerosis: recent

Deng, H.X.; Hentati, A.; Tainer, J.A.; Iqbal, Z.; Cayabyab, A.; Hung, W.Y.; Getzoff, E.D.; Hu,

Deng, H.X.; Bigio, E.H.; Zhai, H.; Fecto, F.; Ajroud, K.; Shi, Y.; Yan, J.; Mishra, M.; Ajroud-

Deng, H.X.; Zhai, H.; Bigio, E.H.; Yan, J.; Fecto, F.; Ajroud, K.; Mishra, M.; Ajroud-Driss, S.;

Dobson, C.M. (2003). Protein folding and misfolding. *Nature*, Vol.426, No.6968, pp. 884-890, Doi, H.; Okamura, K.; Bauer, P.O.; Furukawa, Y.; Shimizu, H.; Kurosawa, M.; Machida, Y.;

Doi, H.; Koyano, S.; Suzuki, Y.; Nukina, N. & Kuroiwa, Y. (2010). The RNA-binding protein

Dormann, D.; Rodde, R.; Edbauer, D.; Bentmann, E.; Fischer, I.; Hruscha, A.; Than, M.E.;

Field, L.S.; Furukawa, Y.; O'Halloran, T.V. & Culotta, V.C. (2003). Factors controlling the

Forman, H.J. & Fridovich, I. (1973). On the stability of bovine superoxide dismutase. The

Forsberg, K.; Jonsson, P.A.; Andersen, P.M.; Bergemalm, D.; Graffmo, K.S.; Hultdin, M.;

Furukawa, Y.; Torres, A.S. & O'Halloran, T.V. (2004). Oxygen-induced maturation of SOD1:

*Neurosci Res*, Vol.66, No.1, pp. 131-133, ISSN 1872-8111

import. *EMBO J*, Vol.29, No.16, pp. 2841-2857, ISSN 1460-2075

effects of metals. *J Biol Chem*, Vol.248, No.8, pp. 2645-2649,

*PLoS One*, Vol.5, No.7, pp. e11552, ISSN 1932-6203

ALS and beyond. *Curr Opin Neurobiol*, pp. ISSN 1873-6882

No.7, pp. 747-757, ISSN 0022-3069

23472,

1047-1051,

1531-8249

6500, ISSN 0021-9258

Vol.278, pp. 28052-28059,

pp. 2872-2881,

copper chaperone for superoxide dismutase. *J Biol Chem*, Vol.272, No.38, pp. 23469-

advances in understanding disease mechanisms. *J Neuropathol Exp Neurol*, Vol.55,

P.; Herzfeldt, B.; Roos, R.P. & et al. (1993). Amyotrophic lateral sclerosis and structural defects in Cu,Zn superoxide dismutase. *Science*, Vol.261, No.5124, pp.

Driss, S.; Heller, S.; Sufit, R.; Siddique, N.; Mugnaini, E. & Siddique, T. (2011a). Differential Involvement of Optineurin in Amyotrophic Lateral Sclerosis With or Without SOD1 Mutations. *Arch Neurol*, Vol.68, No.8, pp. 1057-1061, ISSN 1538-3687

Heller, S.; Sufit, R.; Siddique, N.; Mugnaini, E. & Siddique, T. (2011b). FUSimmunoreactive inclusions are a common feature in sporadic and non-SOD1 familial amyotrophic lateral sclerosis. *Ann Neurol*, Vol.67, No.6, pp. 739-748, ISSN

Miyazaki, H.; Mitsui, K.; Kuroiwa, Y. & Nukina, N. (2008). RNA-binding protein TLS is a major nuclear aggregate-interacting protein in huntingtin exon 1 with expanded polyglutamine-expressing cells. *J Biol Chem*, Vol.283, No.10, pp. 6489-

FUS/TLS is a common aggregate-interacting protein in polyglutamine diseases.

Mackenzie, I.R.; Capell, A.; Schmid, B.; Neumann, M. & Haass, C. (2010). ALSassociated fused in sarcoma (FUS) mutations disrupt Transportin-mediated nuclear

uptake of yeast copper/zinc superoxide dismutase into mitochondria. *J Biol Chem*,

Jacobsson, J.; Rosquist, R.; Marklund, S.L. & Brannstrom, T. (2010). Novel antibodies reveal inclusions containing non-native SOD1 in sporadic ALS patients.

a key role for disulfide formation by the copper chaperone CCS. *EMBO J*, Vol.23,


Baumer, D.; Hilton, D.; Paine, S.M.; Turner, M.R.; Lowe, J.; Talbot, K. & Ansorge, O. (2010).

Bosco, D.A.; Morfini, G.; Karabacak, N.M.; Song, Y.; Gros-Louis, F.; Pasinelli, P.; Goolsby,

Bruijn, L.I.; Miller, T.M. & Cleveland, D.W. (2004). Unraveling the Mechanisms Involved in Motor Neuron Degeneration in ALS. *Annu Rev Neurosci*, Vol.27, pp. 723-749, Brundin, P.; Melki, R. & Kopito, R. (2010). Prion-like transmission of protein aggregates in

Buck, M. (1998). Trifluoroethanol and colleagues: cosolvents come of age. Recent studies

Buratti, E. & Baralle, F.E. (2001). Characterization and functional implications of the RNA

Chattopadhyay, M.; Durazo, A.; Sohn, S.H.; Strong, C.D.; Gralla, E.B.; Whitelegge, J.P. &

Chen, A.K.; Lin, R.Y.; Hsieh, E.Z.; Tu, P.H.; Chen, R.P.; Liao, T.Y.; Chen, W.; Wang, C.H. &

Chia, R.; Tattum, M.H.; Jones, S.; Collinge, J.; Fisher, E.M. & Jackson, G.S. (2010). Superoxide

Chiang, P.M.; Ling, J.; Jeong, Y.H.; Price, D.L.; Aja, S.M. & Wong, P.C. (2010). Deletion of

Clavaguera, F.; Bolmont, T.; Crowther, R.A.; Abramowski, D.; Frank, S.; Probst, A.; Fraser,

Crozat, A.; Aman, P.; Mandahl, N. & Ron, D. (1993). Fusion of CHOP to a novel RNA-

*Cell Biol*, Vol.11, No.7, pp. 909-913, ISSN 1476-4679

exon 9. *J Biol Chem*, Vol.276, No.39, pp. 36337-36343, ISSN 0021-9258 Charcot, J.M. & Joffroy, A. (1869). Deux cas d'atrophie musculaire progressive avec lésions

mutations. *Neurology*, Vol.75, No.7, pp. 611-618, ISSN 1526-632X

Vol.13, No.11, pp. 1396-1403, ISSN 1546-1726

*Physiol. Norm. Pathol.*, Vol.6, pp. 744-760,

1471-0080

ISSN 1091-6490

ISSN 1520-5126

6490

e10627, ISSN 1932-6203

644, ISSN 0028-0836

5835

Juvenile ALS with basophilic inclusions is a FUS proteinopathy with FUS

H.; Fontaine, B.A.; Lemay, N.; McKenna-Yasek, D.; Frosch, M.P.; Agar, J.N.; Julien, J.P.; Brady, S.T. & Brown, R.H., Jr. (2010). Wild-type and mutant SOD1 share an aberrant conformation and a common pathogenic pathway in ALS. *Nat Neurosci*,

neurodegenerative diseases. *Nat Rev Mol Cell Biol*, Vol.11, No.4, pp. 301-307, ISSN

with peptides and proteins. *Q Rev Biophys*, Vol.31, No.3, pp. 297-355, ISSN 0033-

binding properties of nuclear factor TDP-43, a novel splicing regulator of CFTR

de la substance grise et des faisceaux antérolatéraux de la moelle épinière. *Arch.* 

Valentine, J.S. (2008). Initiation and elongation in fibrillation of ALS-linked superoxide dismutase. *Proc Natl Acad Sci U S A*, Vol.105, No.48, pp. 18663-18668,

Huang, J.J. (2010). Induction of amyloid fibrils by the C-terminal fragments of TDP-43 in amyotrophic lateral sclerosis. *J Am Chem Soc*, Vol.132, No.4, pp. 1186-1187,

dismutase 1 and tgSOD1 mouse spinal cord seed fibrils, suggesting a propagative cell death mechanism in amyotrophic lateral sclerosis. *PLoS One*, Vol.5, No.5, pp.

TDP-43 down-regulates Tbc1d1, a gene linked to obesity, and alters body fat metabolism. *Proc Natl Acad Sci U S A*, Vol.107, No.37, pp. 16320-16324, ISSN 1091-

G.; Stalder, A.K.; Beibel, M.; Staufenbiel, M.; Jucker, M.; Goedert, M. & Tolnay, M. (2009). Transmission and spreading of tauopathy in transgenic mouse brain. *Nat* 

binding protein in human myxoid liposarcoma. *Nature*, Vol.363, No.6430, pp. 640-


Protein Aggregates in Pathological Inclusions of Amyotrophic Lateral Sclerosis 351

Huang, C.; Zhou, H.; Tong, J.; Chen, H.; Liu, Y.J.; Wang, D.; Wei, X. & Xia, X.G. (2011). FUS

Hwang, Y.M.; Stathopulos, P.B.; Dimmick, K.; Yang, H.; Badiei, H.R.; Tong, M.S.; Rumfeldt,

Igaz, L.M.; Kwong, L.K.; Xu, Y.; Truax, A.C.; Uryu, K.; Neumann, M.; Clark, C.M.; Elman,

Iko, Y.; Kodama, T.S.; Kasai, N.; Oyama, T.; Morita, E.H.; Muto, T.; Okumura, M.; Fujii, R.;

Jensen, L.T. & Culotta, V.C. (2005). Activation of CuZn superoxide dismutases from

Johnson, B.S.; Snead, D.; Lee, J.J.; McCaffery, J.M.; Shorter, J. & Gitler, A.D. (2009). TDP-43 is

Kabashi, E.; Valdmanis, P.N.; Dion, P.; Spiegelman, D.; McConkey, B.J.; Vande Velde, C.;

Karch, C.M.; Prudencio, M.; Winkler, D.D.; Hart, P.J. & Borchelt, D.R. (2009). Role of mutant

Kato, S.; Takikawa, M.; Nakashima, K.; Hirano, A.; Cleveland, D.W.; Kusaka, H.; Shibata, N.;

Kawamata, H. & Manfredi, G. (2008). Different regulation of wild-type and mutant Cu,Zn

*Natl Acad Sci U S A*, Vol.106, No.19, pp. 7774-7779, ISSN 1091-6490

*Chem*, Vol.277, No.18, pp. 15923-15931,

1553-7404

1083-351X

194, ISSN 1525-2191

44834-44840, ISSN 0021-9258

20339, ISSN 0021-9258

184, ISSN 1466-0822

Vol.280, No.50, pp. 41373-41379, ISSN 0021-9258

Vol.40, No.5, pp. 572-4, ISSN 1546-1718

Vol.17, No.21, pp. 3303-3317, ISSN 1460-2083

superoxide dismutases associated with familial amyotrophic lateral sclerosis. *J Biol* 

transgenic rats develop the phenotypes of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. *PLoS Genet*, Vol.7, No.3, pp. e1002011, ISSN

J.A.; Chen, P.; Karanassios, V. & Meiering, E.M. (2010). Nonamyloid aggregates arising from mature copper/zinc superoxide dismutases resemble those observed in amyotrophic lateral sclerosis. *J Biol Chem*, Vol.285, No.53, pp. 41701-41711, ISSN

L.B.; Miller, B.L.; Grossman, M.; McCluskey, L.F.; Trojanowski, J.Q. & Lee, V.M. (2008). Enrichment of C-terminal fragments in TAR DNA-binding protein-43 cytoplasmic inclusions in brain but not in spinal cord of frontotemporal lobar degeneration and amyotrophic lateral sclerosis. *Am J Pathol*, Vol.173, No.1, pp. 182-

Takumi, T.; Tate, S. & Morikawa, K. (2004). Domain architectures and characterization of an RNA-binding protein, TLS. *J Biol Chem*, Vol.279, No.43, pp.

Caenorhabditis elegans does not require the copper chaperone CCS. *J Biol Chem*,

intrinsically aggregation-prone, and amyotrophic lateral sclerosis-linked mutations accelerate aggregation and increase toxicity. *J Biol Chem*, Vol.284, No.30, pp. 20329-

Bouchard, J.P.; Lacomblez, L.; Pochigaeva, K.; Salachas, F.; Pradat, P.F.; Camu, W.; Meininger, V.; Dupre, N. & Rouleau, G.A. (2008). TARDBP mutations in individuals with sporadic and familial amyotrophic lateral sclerosis. *Nat Genet*,

SOD1 disulfide oxidation and aggregation in the pathogenesis of familial ALS. *Proc* 

Kato, M.; Nakano, I. & Ohama, E. (2000). New consensus research on neuropathological aspects of familial amyotrophic lateral sclerosis with superoxide dismutase 1 (SOD1) gene mutations: inclusions containing SOD1 in neurons and astrocytes. *Amyotroph Lateral Scler Other Motor Neuron Disord*, Vol.1, No.3, pp. 163-

superoxide dismutase localization in mammalian mitochondria. *Hum Mol Genet*,


Furukawa, Y. & O'Halloran, T.V. (2005). ALS mutations have the greatest destabilizing effect

Furukawa, Y.; Fu, R.; Deng, H.X.; Siddique, T. & O'Halloran, T.V. (2006). Disulfide cross-

Furukawa, Y. & O'Halloran, T.V. (2006). Posttranslational modifications in Cu,Zn-

Furukawa, Y.; Kaneko, K.; Yamanaka, K.; O'Halloran, T.V. & Nukina, N. (2008). Complete

Furukawa, Y.; Kaneko, K.; Watanabe, S.; Yamanaka, K. & Nukina, N. (2011). A seeding

Geser, F.; Martinez-Lage, M.; Kwong, L.K.; Lee, V.M. & Trojanowski, J.Q. (2009).

Gitcho, M.A.; Baloh, R.H.; Chakraverty, S.; Mayo, K.; Norton, J.B.; Levitch, D.; Hatanpaa,

Gurney, M.E.; Pu, H.; Chiu, A.Y.; Dal Canto, M.C.; Polchow, C.Y.; Alexander, D.D.;

Hadano, S.; Hand, C.K.; Osuga, H.; Yanagisawa, Y.; Otomo, A.; Devon, R.S.; Miyamoto, N.;

Hasegawa, M.; Arai, T.; Nonaka, T.; Kametani, F.; Yoshida, M.; Hashizume, Y.; Beach, T.G.;

diseases. *J Neurol*, Vol.256, No.8, pp. 1205-1214, ISSN 1432-1459

*Antioxid Redox Signal*, Vol.8, No.5-6, pp. 847-867, ISSN 1523-0864

*J Biol Chem*, Vol.280, pp. 17266-17274,

No.21, pp. 18664-18672, ISSN 1083-351X

*Science*, Vol.264, No.5166, pp. 1772-1775,

No.2, pp. 166-173, ISSN 1061-4036

1083-351X

ISSN 1531-8249

*U S A*, Vol.103, No.18, pp. 7148-7153, ISSN 0027-8424

on the apo, reduced form of SOD1, leading to unfolding and oxidative aggregation.

linked protein represents a significant fraction of ALS-associated Cu, Znsuperoxide dismutase aggregates in spinal cords of model mice. *Proc Natl Acad Sci* 

superoxide dismutase and mutations associated with amyotrophic lateral sclerosis.

loss of post-translational modifications triggers fibrillar aggregation of SOD1 in familial form of ALS. *J Biol Chem*, Vol.283, No.35, pp. 24167-24176, ISSN 0021-9258 Furukawa, Y.; Kaneko, K.; Yamanaka, K. & Nukina, N. (2010). Mutation-dependent

polymorphism of Cu,Zn-superoxide dismutase aggregates in the familial form of amyotrophic lateral sclerosis. *J Biol Chem*, Vol.285, No.29, pp. 22221-22231, ISSN

reaction recapitulates intracellular formation of Sarkosyl-insoluble transactivation response element (TAR) DNA-binding protein-43 inclusions. *J Biol Chem*, Vol.286,

Amyotrophic lateral sclerosis, frontotemporal dementia and beyond: the TDP-43

K.J.; White, C.L., 3rd; Bigio, E.H.; Caselli, R.; Baker, M.; Al-Lozi, M.T.; Morris, J.C.; Pestronk, A.; Rademakers, R.; Goate, A.M. & Cairns, N.J. (2008). TDP-43 A315T mutation in familial motor neuron disease. *Ann Neurol*, Vol.63, No.4, pp. 535-538,

Caliendo, J.; Hentati, A.; Kwon, Y.W.; Deng, H.X. & et al. (1994). Motor neuron degeneration in mice that express a human Cu,Zn superoxide dismutase mutation.

Showguchi-Miyata, J.; Okada, Y.; Singaraja, R.; Figlewicz, D.A.; Kwiatkowski, T.; Hosler, B.A.; Sagie, T.; Skaug, J.; Nasir, J.; Brown, R.H., Jr.; Scherer, S.W.; Rouleau, G.A.; Hayden, M.R. & Ikeda, J.E. (2001). A gene encoding a putative GTPase regulator is mutated in familial amyotrophic lateral sclerosis 2. *Nat Genet*, Vol.29,

Buratti, E.; Baralle, F.; Morita, M.; Nakano, I.; Oda, T.; Tsuchiya, K. & Akiyama, H. (2008). Phosphorylated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. *Ann Neurol*, Vol.64, No.1, pp. 60-70, ISSN 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

superoxide dismutases associated with familial amyotrophic lateral sclerosis. *J Biol Chem*, Vol.277, No.18, pp. 15923-15931,


Protein Aggregates in Pathological Inclusions of Amyotrophic Lateral Sclerosis 353

Mackenzie, I.R.; Ansorge, O.; Strong, M.; Bilbao, J.; Zinman, L.; Ang, L.C.; Baker, M.;

Maruyama, H.; Morino, H.; Ito, H.; Izumi, Y.; Kato, H.; Watanabe, Y.; Kinoshita, Y.; Kamada,

McCord, J.M. & Fridovich, I. (1969). Superoxide dismutase. An enzymic function for erythrocuprein (hemocuprein). *J Biol Chem*, Vol.244, No.22, pp. 6049-6055, Millecamps, S.; Salachas, F.; Cazeneuve, C.; Gordon, P.; Bricka, B.; Camuzat, A.; Guillot-

correlations. *J Med Genet*, Vol.47, No.8, pp. 554-560, ISSN 1468-6244

Munch, C.; O'Brien, J. & Bertolotti, A. (2011). Prion-like propagation of mutant superoxide

Neumann, M.; Sampathu, D.M.; Kwong, L.K.; Truax, A.C.; Micsenyi, M.C.; Chou, T.T.;

Okamoto, K.; Mizuno, Y. & Fujita, Y. (2008). Bunina bodies in amyotrophic lateral sclerosis.

Oztug Durer, Z.A.; Cohlberg, J.A.; Dinh, P.; Padua, S.; Ehrenclou, K.; Downes, S.; Tan, J.K.;

Piao, Y.S.; Wakabayashi, K.; Kakita, A.; Yamada, M.; Hayashi, S.; Morita, T.; Ikuta, F.;

Rae, T.D.; Schmidt, P.J.; Pufahl, R.A.; Culotta, V.C. & O'Halloran, T.V. (1999). Undetectable

Rakhit, R.; Cunningham, P.; Furtos-Matei, A.; Dahan, S.; Qi, X.F.; Crow, J.P.; Cashman, N.R.;

*Neuropathology*, Vol.28, No.2, pp. 109-115, ISSN 0919-6544

and 2000. *Brain Pathol*, Vol.13, No.1, pp. 10-22, ISSN 1015-6305

dismutase. *Science*, Vol.284, No.5415, pp. 805-808,

No.1, pp. 87-98, ISSN 1432-0533

pp. 223-226, ISSN 1476-4687

pp. 3548-3553, ISSN 1091-6490

133, ISSN 1095-9203

e5004, ISSN 1932-6203

Stewart, H.; Eisen, A.; Rademakers, R. & Neumann, M. (2011). Pathological heterogeneity in amyotrophic lateral sclerosis with FUS mutations: two distinct patterns correlating with disease severity and mutation. *Acta Neuropathol*, Vol.122,

M.; Nodera, H.; Suzuki, H.; Komure, O.; Matsuura, S.; Kobatake, K.; Morimoto, N.; Abe, K.; Suzuki, N.; Aoki, M.; Kawata, A.; Hirai, T.; Kato, T.; Ogasawara, K.; Hirano, A.; Takumi, T.; Kusaka, H.; Hagiwara, K.; Kaji, R. & Kawakami, H. (2010). Mutations of optineurin in amyotrophic lateral sclerosis. *Nature*, Vol.465, No.7295,

Noel, L.; Russaouen, O.; Bruneteau, G.; Pradat, P.F.; Le Forestier, N.; Vandenberghe, N.; Danel-Brunaud, V.; Guy, N.; Thauvin-Robinet, C.; Lacomblez, L.; Couratier, P.; Hannequin, D.; Seilhean, D.; Le Ber, I.; Corcia, P.; Camu, W.; Brice, A.; Rouleau, G.; LeGuern, E. & Meininger, V. (2010). SOD1, ANG, VAPB, TARDBP, and FUS mutations in familial amyotrophic lateral sclerosis: genotype-phenotype

dismutase-1 misfolding in neuronal cells. *Proc Natl Acad Sci U S A*, Vol.108, No.9,

Bruce, J.; Schuck, T.; Grossman, M.; Clark, C.M.; McCluskey, L.F.; Miller, B.L.; Masliah, E.; Mackenzie, I.R.; Feldman, H.; Feiden, W.; Kretzschmar, H.A.; Trojanowski, J.Q. & Lee, V.M. (2006). Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. *Science*, Vol.314, No.5796, pp. 130-

Nakano, Y.; Bowman, C.J.; Hoskins, J.L.; Kwon, C.; Mason, A.Z.; Rodriguez, J.A.; Doucette, P.A.; Shaw, B.F. & Selverstone Valentine, J. (2009). Loss of metal ions, disulfide reduction and mutations related to familial ALS promote formation of amyloid-like aggregates from superoxide dismutase. *PLoS One*, Vol.4, No.3, pp.

Oyanagi, K. & Takahashi, H. (2003). Neuropathology with clinical correlations of sporadic amyotrophic lateral sclerosis: 102 autopsy cases examined between 1962

intracellular free copper: The requirement of a copper chaperone for superoxide

Kondejewski, L.H. & Chakrabartty, A. (2002). Oxidation-induced misfolding and


Kerman, A.; Liu, H.N.; Croul, S.; Bilbao, J.; Rogaeva, E.; Zinman, L.; Robertson, J. &

Klunk, W.E.; Jacob, R.F. & Mason, R.P. (1999). Quantifying amyloid by congo red spectral

Kraemer, B.C.; Schuck, T.; Wheeler, J.M.; Robinson, L.C.; Trojanowski, J.Q.; Lee, V.M. &

Kwiatkowski, T.J., Jr.; Bosco, D.A.; Leclerc, A.L.; Tamrazian, E.; Vanderburg, C.R.; Russ, C.;

Lagier-Tourenne, C.; Polymenidou, M. & Cleveland, D.W. (2010). TDP-43 and FUS/TLS:

Leitch, J.M.; Jensen, L.T.; Bouldin, S.D.; Outten, C.E.; Hart, P.J. & Culotta, V.C. (2009).

LeVine, H., 3rd (1999). Quantification of beta-sheet amyloid fibril structures with thioflavin

Lin, W.L. & Dickson, D.W. (2008). Ultrastructural localization of TDP-43 in filamentous

Liu, H.N.; Sanelli, T.; Horne, P.; Pioro, E.P.; Strong, M.J.; Rogaeva, E.; Bilbao, J.; Zinman, L. &

Luk, K.C.; Song, C.; O'Brien, P.; Stieber, A.; Branch, J.R.; Brunden, K.R.; Trojanowski, J.Q. &

Mackenzie, I.R.; Bigio, E.H.; Ince, P.G.; Geser, F.; Neumann, M.; Cairns, N.J.; Kwong, L.K.;

T. *Methods Enzymol*, Vol.309, pp. 274-284, ISSN 0076-6879

Vol.116, No.2, pp. 205-213, ISSN 0001-6322

No.47, pp. 20051-20056, ISSN 1091-6490

*Neurol*, Vol.61, No.5, pp. 427-434, ISSN 0364-5134

75-80, ISSN 1531-8249

chaperone CCS. *J Biol Chem*, Vol.284, No.33, pp. 21863-21871, 0021-9258 Lelie, H.L.; Liba, A.; Bourassa, M.W.; Chattopadhyay, M.; Chan, P.K.; Gralla, E.B.; Miller,

shift assay. *Methods Enzymol*, Vol.309, pp. 285-305, ISSN 0076-6879

*Neuropathol*, Vol.119, No.3, pp. 335-344, ISSN 1432-0533

419, ISSN 1432-0533

No.5918, pp. 1205-1208, ISSN 1095-9203

pp. R46-R64, ISSN 1460-2083

Chakrabartty, A. (2010). Amyotrophic lateral sclerosis is a non-amyloid disease in which extensive misfolding of SOD1 is unique to the familial form. *Acta* 

Schellenberg, G.D. (2010). Loss of murine TDP-43 disrupts motor function and plays an essential role in embryogenesis. *Acta Neuropathol*, Vol.119, No.4, pp. 409-

Davis, A.; Gilchrist, J.; Kasarskis, E.J.; Munsat, T.; Valdmanis, P.; Rouleau, G.A.; Hosler, B.A.; Cortelli, P.; de Jong, P.J.; Yoshinaga, Y.; Haines, J.L.; Pericak-Vance, M.A.; Yan, J.; Ticozzi, N.; Siddique, T.; McKenna-Yasek, D.; Sapp, P.C.; Horvitz, H.R.; Landers, J.E. & Brown, R.H., Jr. (2009). Mutations in the FUS/TLS gene on chromosome 16 cause familial amyotrophic lateral sclerosis. *Science*, Vol.323,

emerging roles in RNA processing and neurodegeneration. *Hum Mol Genet*, Vol.19,

Activation of Cu,Zn-superoxide dismutase in the absence of oxygen and the copper

L.M.; Borchelt, D.R.; Valentine, J.S. & Whitelegge, J.P. (2011). Copper and zinc metallation status of copper-zinc superoxide dismutase from amyotrophic lateral sclerosis transgenic mice. *J Biol Chem*, Vol.286, No.4, pp. 2795-2806, ISSN 1083-351X

neuronal inclusions in various neurodegenerative diseases. *Acta Neuropathol*,

Robertson, J. (2009). Lack of evidence of monomer/misfolded superoxide dismutase-1 in sporadic amyotrophic lateral sclerosis. *Ann Neurol*, Vol.66, No.1, pp.

Lee, V.M. (2009). Exogenous alpha-synuclein fibrils seed the formation of Lewy body-like intracellular inclusions in cultured cells. *Proc Natl Acad Sci U S A*, Vol.106,

Forman, M.S.; Ravits, J.; Stewart, H.; Eisen, A.; McClusky, L.; Kretzschmar, H.A.; Monoranu, C.M.; Highley, J.R.; Kirby, J.; Siddique, T.; Shaw, P.J.; Lee, V.M. & Trojanowski, J.Q. (2007). Pathological TDP-43 distinguishes sporadic amyotrophic lateral sclerosis from amyotrophic lateral sclerosis with SOD1 mutations. *Ann* 


Protein Aggregates in Pathological Inclusions of Amyotrophic Lateral Sclerosis 355

Siddique, T.; Figlewicz, D.A.; Pericak-Vance, M.A.; Haines, J.L.; Rouleau, G.; Jeffers, A.J.;

Son, M.; Puttaparthi, K.; Kawamata, H.; Rajendran, B.; Boyer, P.J.; Manfredi, G. & Elliott, J.L.

Soto, C. (2003). Unfolding the role of protein misfolding in neurodegenerative diseases. *Nat* 

Sreedharan, J.; Blair, I.P.; Tripathi, V.B.; Hu, X.; Vance, C.; Rogelj, B.; Ackerley, S.; Durnall,

Stathopulos, P.B.; Rumfeldt, J.A.; Scholz, G.A.; Irani, R.A.; Frey, H.E.; Hallewell, R.A.;

aggregates in vitro. *Proc Natl Acad Sci USA*, Vol.100, No.12, pp. 7021-7026, Subramaniam, J.R.; Lyons, W.E.; Liu, J.; Bartnikas, T.B.; Rothstein, J.; Price, D.L.; Cleveland,

Sun, Z.; Diaz, Z.; Fang, X.; Hart, M.P.; Chesi, A.; Shorter, J. & Gitler, A.D. (2011). Molecular

Turner, B.J. & Talbot, K. (2008). Transgenics, toxicity and therapeutics in rodent models of

Uranishi, H.; Tetsuka, T.; Yamashita, M.; Asamitsu, K.; Shimizu, M.; Itoh, M. & Okamoto, T.

Van Deerlin, V.M.; Leverenz, J.B.; Bekris, L.M.; Bird, T.D.; Yuan, W.; Elman, L.B.; Clay, D.;

protein FUS/TLS. *PLoS Biol*, Vol.9, No.4, pp. e1000614, ISSN 1545-7885 Tiwari, A. & Hayward, L.J. (2003). Familial amyotrophic lateral sclerosis mutants of

Vol.104, No.14, pp. 6072-6077, ISSN 0027-8424

No.5870, pp. 1668-1672, ISSN 1095-9203

*Rev Neurosci*, Vol.4, No.1, pp. 49-60, ISSN 1471-003X

*Chem*, Vol.278, No.8, pp. 5984-5992, ISSN 0021-9258

Vol.276, No.16, pp. 13395-13401, ISSN 0021-9258

ISSN 0022-3069

0028-4793

No.4, pp. 301-307,

0301-0082

1474-4422

posterior column involvement. *J Neuropathol Exp Neurol*, Vol.55, No.4, pp. 481-490,

Sapp, P.; Hung, W.Y.; Bebout, J.; McKenna-Yasek, D. & et al. (1991). Linkage of a gene causing familial amyotrophic lateral sclerosis to chromosome 21 and evidence of genetic-locus heterogeneity. *N Engl J Med*, Vol.324, No.20, pp. 1381-1384, ISSN

(2007). Overexpression of CCS in G93A-SOD1 mice leads to accelerated neurological deficits with severe mitochondrial pathology. *Proc Natl Acad Sci U S A*,

J.C.; Williams, K.L.; Buratti, E.; Baralle, F.; de Belleroche, J.; Mitchell, J.D.; Leigh, P.N.; Al-Chalabi, A.; Miller, C.C.; Nicholson, G. & Shaw, C.E. (2008). TDP-43 mutations in familial and sporadic amyotrophic lateral sclerosis. *Science*, Vol.319,

Lepock, J.R. & Meiering, E.M. (2003). Cu/Zn superoxide dismutase mutants associated with amyotrophic lateral sclerosis show enhanced formation of

D.W.; Gitlin, J.D. & Wong, P.C. (2002). Mutant SOD1 causes motor neuron disease independent of copper chaperone-mediated copper loading. *Nat Neurosci*, Vol.5,

determinants and genetic modifiers of aggregation and toxicity for the ALS disease

copper/zinc superoxide dismutase are susceptible to disulfide reduction. *J Biol* 

mutant SOD1-mediated familial ALS. *Prog Neurobiol*, Vol.85, No.1, pp. 94-134, ISSN

(2001). Involvement of the pro-oncoprotein TLS (translocated in liposarcoma) in nuclear factor-kappa B p65-mediated transcription as a coactivator. *J Biol Chem*,

Wood, E.M.; Chen-Plotkin, A.S.; Martinez-Lage, M.; Steinbart, E.; McCluskey, L.; Grossman, M.; Neumann, M.; Wu, I.L.; Yang, W.S.; Kalb, R.; Galasko, D.R.; Montine, T.J.; Trojanowski, J.Q.; Lee, V.M.; Schellenberg, G.D. & Yu, C.E. (2008). TARDBP mutations in amyotrophic lateral sclerosis with TDP-43 neuropathology: a genetic and histopathological analysis. *Lancet Neurol*, Vol.7, No.5, pp. 409-416, ISSN

aggregation of superoxide dismutase and its implications for amyotrophic lateral sclerosis. *J Biol Chem*, Vol.277, No.49, pp. 47551-47556,


Rakhit, R.; Crow, J.P.; Lepock, J.R.; Kondejewski, L.H.; Cashman, N.R. & Chakrabartty, A.

Reaume, A.G.; Elliott, J.L.; Hoffman, E.K.; Kowall, N.W.; Ferrante, R.J.; Siwek, D.F.; Wilcox,

Ren, P.H.; Lauckner, J.E.; Kachirskaia, I.; Heuser, J.E.; Melki, R. & Kopito, R.R. (2009).

Rodriguez, J.A.; Shaw, B.F.; Durazo, A.; Sohn, S.H.; Doucette, P.A.; Nersissian, A.M.; Faull,

Ross, C.A. & Poirier, M.A. (2004). Protein aggregation and neurodegenerative disease. *Nat* 

Serpell, L.C. & Smith, J.M. (2000). Direct visualisation of the beta-sheet structure of synthetic Alzheimer's amyloid. *J Mol Biol*, Vol.299, No.1, pp. 225-231, ISSN 0022-2836 Shaw, B.F.; Lelie, H.L.; Durazo, A.; Nersissian, A.M.; Xu, G.; Chan, P.K.; Gralla, E.B.; Tiwari,

Shibata, N.; Hirano, A.; Kobayashi, M.; Sasaki, S.; Kato, T.; Matsumoto, S.; Shiozawa, Z.;

Shibata, N.; Asayama, K.; Hirano, A. & Kobayashi, M. (1996a). Immunohistochemical study

Shibata, N.; Hirano, A.; Kobayashi, M.; Siddique, T.; Deng, H.X.; Hung, W.Y.; Kato, T. &

pathogenesis. *Proc Natl Acad Sci U S A*, Vol.102, No.30, pp. 10516-10521, Rosen, D.R.; Siddique, T.; Patterson, D.; Figlewicz, D.A.; Sapp, P.; Hentati, A.; Donaldson,

sclerosis. *J Biol Chem*, Vol.277, No.49, pp. 47551-47556,

sclerosis. *J. Biol. Chem.*, Vol.279, No.15, pp. 15499-15504,

dismutase. *J Biol Chem*, Vol.277, No.18, pp. 15932-15937,

sclerosis. *Nature*, Vol.362, No.6415, pp. 59-62,

*Med*, Vol.10 Suppl, pp. S10-S17, ISSN 1078-8956

*J Biol Chem*, Vol.283, No.13, pp. 8340-8350, ISSN 0021-9258

sclerosis. *Neurosci Lett*, Vol.179, No.1-2, pp. 149-152,

47,

5866

aggregation of superoxide dismutase and its implications for amyotrophic lateral

(2004). Monomeric Cu,Zn-superoxide dismutase is a common misfolding intermediate in the oxidation models of sporadic and familial amyotrophic lateral

H.M.; Flood, D.G.; Beal, M.F.; Brown, R.H., Jr.; Scott, R.W. & Snider, W.D. (1996). Motor neurons in Cu/Zn superoxide dismutase-deficient mice develop normally but exhibit enhanced cell death after axonal injury. *Nat Genet*, Vol.13, No.1, pp. 43-

Cytoplasmic penetration and persistent infection of mammalian cells by polyglutamine aggregates. *Nat Cell Biol*, Vol.11, No.2, pp. 219-225, ISSN 1476-4679 Rodriguez, J.A.; Valentine, J.S.; Eggers, D.K.; Roe, J.A.; Tiwari, A.; Brown, R.H. & Hayward,

L.J. (2002). Familial amyotrophic lateral sclerosis-associated mutations decrease the thermal stability of distinctly metallated species of human copper/zinc superoxide

K.F.; Eggers, D.K.; Tiwari, A.; Hayward, L.J. & Valentine, J.S. (2005). Destabilization of apoprotein is insufficient to explain Cu,Zn-superoxide dismutase-linked ALS

D.; Goto, J.; O'Regan, J.P.; Deng, H.X. & et al. (1993). Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral

A.; Hayward, L.J.; Borchelt, D.R.; Valentine, J.S. & Whitelegge, J.P. (2008). Detergent-insoluble aggregates associated with amyotrophic lateral sclerosis in transgenic mice contain primarily full-length, unmodified superoxide dismutase-1.

Komori, T.; Ikemoto, A.; Umahara, T. & et al. (1994). Cu/Zn superoxide dismutaselike immunoreactivity in Lewy body-like inclusions of sporadic amyotrophic lateral

on superoxide dismutases in spinal cords from autopsied patients with amyotrophic lateral sclerosis. *Dev Neurosci*, Vol.18, No.5-6, pp. 492-498, ISSN 0378-

Asayama, K. (1996b). Intense superoxide dismutase-1 immunoreactivity in intracytoplasmic hyaline inclusions of familial amyotrophic lateral sclerosis with posterior column involvement. *J Neuropathol Exp Neurol*, Vol.55, No.4, pp. 481-490, ISSN 0022-3069


**15** 

*Australia* 

**The Kynurenine Pathway** 

Yiquan Chen1 and Gilles Guillemin1,2

*University of New South Wales, Sydney,* 

*1Department of Pharmacology, School of Medical Sciences,* 

*2St Vincent's Centre for Applied Medical Research, St Vincent's Hospital, Sydney,* 

The kynurenine pathway represents a major route for the catabolism of tryptophan (TRP). In the body, TRP is transported around the periphery either bound to albumin (90%) or in free form (10%), the two states existing in equilibrium (McMenamy 1965). However, only free form TRP can be transported across the blood-brain barrier (BBB) by the competitive and nonspecific L-type amino acid transporter (Hargreaves and Pardridge 1988). Once in the central nervous system (CNS), TRP acts as a precursor to several metabolic pathways, such as for the synthesis of kynurenine (KYN), serotonin, melatonin and protein (Fig. 1) (Ruddick *et al.* 2006).

Fig. 1. TRP in the CNS. Only free TRP can cross the BBB and act as precursor for protein, serotonin, tryptamine, and kynurenine and kynuramine synthesis. The kynurenine pathway

is a major pathway for TRP catabolism. Adapted from (Ruddick *et al.* 2006).

**1. Introduction** 

