**8. Therapeutic strategy for ALS**

was evident and has been associated with mutations in mitochondrial DNA [175-176]. Although SOD1 is mainly localized in cytosols, it is also resilient in other subcellular com‐ partments such as the mitochondria [45,177-178] and even the endoplasmic reticulum [182]. The aggregates of mutant SOD1 were shown within the mitochondria of the spinal cord of SOD1G93A mice before the onset of symptoms [46-47] and were implicated in increased oxidative damage, decreased respiratory activity of mitochondria [48], and the appearance of mitochondrial swelling and vacuolization [47]. Dissociated cytochrome c from the interaction of mitochondria with mutant SOD1 activates apoptosis [44]. Mitochondria function as reservoirs of intracellular Ca2+, as ER. Once overloaded in cytosol, the accumulated Ca2+ in the mitochondria prepares the organelle to undergo permeability transition, and then swells and ruptures in their outermembrane, which in turn produces free radicals from them and oxidizes their lipids and DNA [179-180]. Ca2+-induced mitochondrial damage can also result in mitochondrial release of cytotoxic substances such as cytochrome c [181] and can affect caspase cascade. The homeostasis at the intracellular Ca2+ level was also disturbed in motor neurons of SOD1G93A mice [182]. Moreover, increased Ca2+ uptake into the mitochondria of motor neurons easily occurred after exposure to the glutamate agonist AMPA or kinate, and triggered increased ROS generation [183]. ALS-linked SOD1 has been shown to slow down fast axonal transport of mitochondria. The axonal mitochondria transport was primarily reduced in the anterograde direction, which suggests that the energy supply in the presynaptic terminals of the motor endplates is compromised [184]. Multiple functions of the mitochondria over cellular injury and the apearance of mitochondrial dysfunction in the presymptomatic stage may contribute to various routes of neuronal death in ALS. More recently, in mice that expressed human TDP-43 only in neurons that included motor neurons, massive accumulation of mitochondria in TDP-43-negative cytoplasmic inclusions in the motor neurons were reported and the lack of mitochondria in the motor axon terminal was observed [185]. In addition, the transgenic mice that overexpressed human TDP-43 driven by the mouse prion promoter demonstrated motor deficits, early mortality, and mitochondrial aggregation [186]. These results imply that TDP-43 is indirectly involved in mitochondrial dysfunction in neurodege‐

Autophagy is a degradative mechanism that is involved in the recycling and turnover of longlife proteins and organelles [187]. Autophagy is basically induced by lack of nutrients and energy or by various toxicants. Although its primary role is adaptation to scarcity, this degradative process is also critical for the normal turnover of cytoplasmic contents that include neurons. Genetic ablation of autophagy-related genes provokes neurodeneration even with lack of disease-like mutant proteins [188]. Recent studies verified the importance of the autophagy pathway in various pathological conditions that include neurodegenerative diseases [189]. Interestingly, the catabolic process is both beneficial and detrimental to cells, depending on its context and specific stimuli. The lethality of mutated SOD1 is the result of abnormal protein aggregates, which impair the degradation machinery such as the ubiqutin-

nerative diseases such as ALS.

46 Current Advances in Amyotrophic Lateral Sclerosis

**7. Autophagy in ALS**

#### **8.1. Separate routes of motor neuron degeneration in ALS**

The parallel pathway of oxidative stress and Fas-mediated apoptosis in motor neuron death in SOD1G93A mice was previously focused on [96]. This study provided the first evidence that combination therapy that targets oxidative stress and apoptosis together also delays the onset and progression of motor dysfunction and extends the survival time of ALS transgenic mice. Evidence was accumulated that shows that oxidative stress and apoptotic insults cause neuronal death through distinctive pathways and with unique morphological changes. The neurotrophins' nerve growth factor, the brain-derived neurotrophic factor (BDNF), neurotro‐ phin 3 (NT-3), and NT-4/5, and the insulin-like growth factors IGF-I and IGF-II, promote neuronal survival by preventing programmed cell death or apoptosis, but they significantly enhance necrotic degeneration of neurons exposed to oxidative stress or deprived of oxygen and glucose [199-200]. Neurotrophins can induce oxidative stress by upregulating NADPH oxidase, which leads to neuronal cell necrosis [201]. Surprisingly, the insulin-like growth factor 1 (IGF-1) prevented neuronal cell apoptosis and protected spinal motor neurons in ALS mice [199,202], but markedly potentiated neuronal cell necrosis induced by hydroxyl radicals or glutathione depletion [203]. Given that oxidative stress and apoptosis play a central role in motor neuron degeneration and can contribute to neuronal death through distinctive routes in ALS, it was hypothesized that a therapeutic approach that targets both oxidative stress and apoptosis would have additive effects on neuronal survival and the motor function. To pharmacologically prevent oxidative stress and apoptosis, Neu2000, a novel anti-oxidant, and Li+ , a well-known anti-apoptosis agent, were used. The former, a chemical derivative of aspirin and sulfasalazine, was developed to protect neurons from oxidative stress with greater potency and safety, and has been shown to be a potent and secure anti-oxidant in vitro and in animal models of hypoxic ischemia [204]. Li+ has been shown to prevent apoptosis through mecha‐

**Figure 1.** Multiple pathways of motor neuron degeneration and their therapeutic drugs in ALS: (1) increased Ca2+ in the motor neuron: dysfunction or downregulation of glutamate transporters such as GLT1 on the astrocytes, elevation of the Ca2+ permeable AMPA receptor via downregulation of or a deficit in the post-transcriptional edition of GluR2 sub-units, and mitochondrial dysfunction; (2) oxidative damage of the motor neuron: increased intracellular Ca2+ con‐ tents, high levels of mitochondria due to high energy demand, and increase in free metal ions such as copper and iron; (3) apoptosis in the motor neuron: activation of the Fas-mediated pathway, alteration of Bcl-2 family proteins via mitochondrial interaction with mSOD1, and initiation, propagation, or execution of caspase cascade; (4) inflamma‐ tion: non-cell-autonomous motor neuron death (the disease progression is coordinated by mSOD1 expression in all neuronal and non-neuronal cells) and concurrent activation of the innate immune system and systemic inflammation (BBB breakdown may induce a vicious cycle of inflammation); and (5) autophagy: increased autophagosome forma‐ tion.Current therapeutic drugs were developed basically against a specific route of ALS disease progression.

and neuronal survival in ALS and possibly other neurological diseases such as stroke, Alzheimer's disease, and Parkinson's disease. The authors' hypothesis was supported by other experiments in which a cocktail of neuroprotective drugs with different modes of action more significantly improved survival and the motor function than did monotherapy in transgenic

**Ceftriaxone 200 mg/kg ip Antiglutamatergic 10% Rothstein JD et al., 2005 [219] L-Arginine 6% drinking water Antiglutamatergic 20% Lee J et al., 2009 [220]**

**acidic dipetidase 30 mg/kg po Antiglutamatergic 15% Ghadge GD et al., [221]**

**route Hypothetical mechanism Survival Reference**

Multiple Routes of Motor Neuron Degeneration in ALS

http://dx.doi.org/10.5772/56625

49

**Lentiviral vecor Antiglutamatergic 30% Azzouz M et al., 2004 [218]**

**Creatine 1% diet Antioxidant 9% Klivenyi P et al., 1999 [78] 2% diet Antioxidant 17% Creatine 2% diet Antioxidant 20% Klivenyi P et al., 2004 [169] creatine 2% diet Antioxidant 12% Zhang W et al., 2003 [117] Vitamin E 200 IU chow Antioxidant No effect Gurney ME et al., 1996 [125] Edaravone 5 mg/kg ip Antioxidant 12.4% Ito H et al., 2008 [81] 15 mg/kg ip 17% AEOL-10150 2.5 mg/kg ip Antioxidant 26% Crow JP et al., 2005 [80] 2.5 mg/kg sc 22% N-acetylcysteine 2 mg/kg/d drinking water Antioxidant 7% Andreassen OA et al., 2000 [79] TRO19622 (Olesoxime) 3 mg/kg sc Antioxidant 10% Bordet T et al., 2007 [209] 30 mg/kg sc 8% Ammonium tetrathiomolybdate 5 mg/kg not described Antioxidant 25% Tokuda E et al., 2008 [210] Neu2000 30 mg/kg diet Antioxidant 15% Shin et al., 2007 [96] zVAD Antiapoptotic 22% Li et al., 2000 [111] Cyclosporin A 18mg/kg intrathecal Antiapoptotic 12% Keep M et al., 2001 [211] Minocycline 25 mg/kg ip Antiapoptotic/Anti-inflammatory 10% Van Den Bosch L et al., 2002 [116] 50 mg/kg ip 15.8% Minocycline 11 mg/kg Antiapoptotic/Anti-inflammatory 9% Zhu S et al., 2002 [115] Minocycline 22mg/kg/d ip Antiapoptotic/Anti-inflammatory 13% Zhang W et al., 2003 [117] Lithium 1 mEq/kg ip Antiapoptotic/Autophagy inducer 36% Fornai F et al., 2007 [192] Lithium 60 mg/kg ip Antiapoptotic/Autophagy inducer 8% Feng H et al., 2008 [212] Lithium 2% diet Antiapoptotic/Autophagy inducer 10% Shin et al., 2007 [96] Celecoxib 1500ppm chow Anti-inflammatory 25% Drachman DB et al., 2002 [168] Celecoxib 0.012% diet Anti-inflammatory 21% Klivenyi P et al., 2004 [169] Thalidomide 50 mg/kg Anti-inflammatory 12% Kiaei M et al., 2006 [213] 100 mg/kg 16% Glatiramer acetate 7ug/0.1 ml PBS immunization Anti-inflammatory 1.4% Banerjee R et al., 2008 [171]**

Riluzole, the only therapeutic drug approved for ALS, extends life expectancy to up to 3 months in human patients. The symptomatic drug potentially targets gluatamate- or oxidativestress-induced neurodegeneration with marginal apoptosis effects [25]. As mentioned,

mouse ALS models [117,207].

**Table 1.** List of drugs tested with ALS mice

**N-acetylated a-linked**

**8.2. Current treatment and new approach of ALS medications**

**AM1241 1 mg/kg ip Anti-inflammatory 3%**

**Celastrol 8 mg/kg diet Anti-inflammatory 13% Kiaei M et al., 2005 [213] 2 mg/kg 9.4% Nordihydroguaiaretic acid 2500ppm po Anti-inflammatory 10% West M et al., 2004 [214] RO-28-2653 100 mg/kg po Anti-inflammatory 11% Lorenzl S et al., 2006 [215] Riluzole 100ug/ml drinking water Antiglutamatergic 10% Gurney ME et al., 1996 [117] Riluzol 30 mg/kg drinking water Antiglutamatergic 11% Waibel et al., 2004 [224] Gabapentin 3% chow Antiglutamatergic 5% Gurney ME et al., 1996 [117] Memantine 10 mg/kg subcutaneus Antiglutamatergic 7% Wang et al., 2005 [216] Memantine 30 mg/kg drinking water Antiglutamatergic 5% Joo IS et al., 2007 [26] 90 mg/kg drinking water 1% vegf 1.0 ug/kg ip Antiglutamatergic 8% Zheng C et al., 2004 [217]**

**Compound Dose Administration**

nisms that involve Bcl-2 upregulation, glycogen synthase kinase-3 beta inhibition, and activation of phosphatidylinositol 3-kinase that activates serine/threonine kinase Akt-1 and phospholipase C gamma [205-206]. An additional benefit of Li+ was recently demonstrated the induction of an autophagy pathway at a low dose, clears altered mitochondria and protein aggregates [192]. In the results of this study, the concurrent administration of Neu2000 and Li + , which block free-radical-mediated necrosis and Fas-mediated apoptosis, respectively, significantly delayed the onset and progression of motor neuron degeneration and motor function deficits. Thus, targeting both oxidative stress and the Fas apoptosis pathway with concurrent treatment with Neu2000 and Li+ may further improve the neurological function


**Table 1.** List of drugs tested with ALS mice

nisms that involve Bcl-2 upregulation, glycogen synthase kinase-3 beta inhibition, and activation of phosphatidylinositol 3-kinase that activates serine/threonine kinase Akt-1 and phospholipase C gamma [205-206]. An additional benefit of Li+ was recently demonstrated the induction of an autophagy pathway at a low dose, clears altered mitochondria and protein aggregates [192]. In the results of this study, the concurrent administration of Neu2000 and Li

tion.Current therapeutic drugs were developed basically against a specific route of ALS disease progression.

**Figure 1.** Multiple pathways of motor neuron degeneration and their therapeutic drugs in ALS: (1) increased Ca2+ in the motor neuron: dysfunction or downregulation of glutamate transporters such as GLT1 on the astrocytes, elevation of the Ca2+ permeable AMPA receptor via downregulation of or a deficit in the post-transcriptional edition of GluR2 sub-units, and mitochondrial dysfunction; (2) oxidative damage of the motor neuron: increased intracellular Ca2+ con‐ tents, high levels of mitochondria due to high energy demand, and increase in free metal ions such as copper and iron; (3) apoptosis in the motor neuron: activation of the Fas-mediated pathway, alteration of Bcl-2 family proteins via mitochondrial interaction with mSOD1, and initiation, propagation, or execution of caspase cascade; (4) inflamma‐ tion: non-cell-autonomous motor neuron death (the disease progression is coordinated by mSOD1 expression in all neuronal and non-neuronal cells) and concurrent activation of the innate immune system and systemic inflammation (BBB breakdown may induce a vicious cycle of inflammation); and (5) autophagy: increased autophagosome forma‐

, which block free-radical-mediated necrosis and Fas-mediated apoptosis, respectively, significantly delayed the onset and progression of motor neuron degeneration and motor function deficits. Thus, targeting both oxidative stress and the Fas apoptosis pathway with

may further improve the neurological function

+

concurrent treatment with Neu2000 and Li+

48 Current Advances in Amyotrophic Lateral Sclerosis

and neuronal survival in ALS and possibly other neurological diseases such as stroke, Alzheimer's disease, and Parkinson's disease. The authors' hypothesis was supported by other experiments in which a cocktail of neuroprotective drugs with different modes of action more significantly improved survival and the motor function than did monotherapy in transgenic mouse ALS models [117,207].

#### **8.2. Current treatment and new approach of ALS medications**

Riluzole, the only therapeutic drug approved for ALS, extends life expectancy to up to 3 months in human patients. The symptomatic drug potentially targets gluatamate- or oxidativestress-induced neurodegeneration with marginal apoptosis effects [25]. As mentioned,


AAD-2004, which has a dual mode of action as an anti-oxidant and an mPGES-1 inhibitor, had better efficacy on the motor function and survival than those of riluzole and ibuprofen.

Multiple Routes of Motor Neuron Degeneration in ALS

http://dx.doi.org/10.5772/56625

51

In support of such a notion, a phase II clinical trial was recently conducted, which showed that

In ALS, knowledge of the contribution of multiple pathways to the degeneration of motor neurons has expanded greatly and has challenged clinical trials of drugs that target the processes. Better understanding of the detrimental processes that cause neurodegeneration will help define its medical importance and clarify the therapeutic potential of interfering with

[1] Rosen, D.R. Sapp, P. O'Regan, J. McKenna-Yasek, D. Schlumpf, K.S. Haines, J.L. Gu‐ sella, J.F. Horvitz, H.R. & Brown, R.H. Jr. Genetic linkage analysis of familial amyo‐ trophic lateral sclerosis using human chromosome 21 microsatellite DNA markers.

[2] Gurney, M.E. Transgenic-mouse model of amyotrophic lateral sclerosis. N Engl J

[3] Won SJ, Kim DY, Gwag BJ. Cellular and molecular pathways of ischemic neuronal

[4] Nicholls D, Attwell D. The release and uptake of excitatory amino acids. Trends

[5] Barbour B, Brew H, Attwell D. Electrogenic glutamate uptake in glial cells is activat‐

ed by intracellular potassium. Nature. 1988 Sep 29;335(6189):433-435.

the suggested strategy may be feasible and efficient.

**9. Conclusion**

**Author details**

**References**

Jin Hee Shin1\* and Jae Keun Lee2

1 GNT Pharma, South Korea

\*Address all correspondence to: ppzini@hanmail.net

Am J Med Genet. 1994 May;15(51): 61-69.

death. J Biochem Mol Biol. 2002 Jan 31;35(1):67-86

Med. 1994 Dec 22;331(25):1721-1722.

Pharmacol Sci. 1990 Nov;11(11):462-468.

2 School of Life Science and Biotechnology, Korea University, South Korea

them.

#### **Table 2.** Additive effect of combination therapy in ALS mice

therapeutic strategies and drugs developed based on them, as shown in Figure 1, explain the multiple-disease-causing process of ALS. As shown in Table 1, many drugs were evaluated in mice that expressed mutant SOD1. Most of the drugs were beneficial to the motor function and survival in the tests with the mice. Several drugs (such as creatine, celecoxib, gabapentin, topiramate, lamotrigine, minocycline, thalidomide, valproate, vitamin E, and even lithium) showed beneficial effects in animal ALS models, but none of them significantly prolonged the survival or improved the quality of life of human ALS patients. The therapeutic effects on the animal models and the human patients significantly differed due to the following translational mismatch issues: first, the methological inappropriateness of the drug screening with the use of animals that had biological confounding variables such as sex and differences in the treatment initiation time point; second, the lack of correct pharmacokinetics, which were considered in a dose-ranging study of safety/toxicity and BBB penetration; and finally, the methodological pitfall of ALS clinical trials due to the insufficiency of the number of patients, the inclusion of heterogeneous populations, the short duration of the trial, and the inadequate analysis of the efficacy. It should be noted that the combination of creatine and celecoxib improved the motor function in a randomized clinical phase II trial of ALS patients and SOD1G93A mice, although single treatment with either creatine or celecoxib failed to show beneficial effects in human ALS trials [208], which suggests the greater efficacy of combined anti-oxidant and NSAID therapy than those of monotherapy. Several pieces of evidence support the notion that therapeutic combinations are more effective than individual agents in animal ALS models (Table2). More recently, the authors reported that a single agent named AAD-2004, which has a dual mode of action as an anti-oxidant and an mPGES-1 inhibitor, had better efficacy on the motor function and survival than those of riluzole and ibuprofen.

In support of such a notion, a phase II clinical trial was recently conducted, which showed that the suggested strategy may be feasible and efficient.
