Amyotrophic lateral sclerosis (ALS) is the most common adult-onset motor neuron disease. It causes degeneration of motor neurons in the primary motor cortex, brain stem, and spinal cord which subsequently leads to rapidly progressive paralysis of skeletal muscles and ultimately to death due to respiratory failure, usually within 3 to 5 years after disease onset.
The majority of ALS cases are acquired spontaneously (sporadic ALS; sALS), while only 10%–15% of ALS cases are inherited (familial ALS; fALS) [1
]. Recent breakthroughs in genetics have enlarged the number of known mutations causing fALS, among them mutations in genes coding for superoxide dismutase 1 (SOD1), TAR DNA-binding protein (TARDP), fused-in sarcoma/translocation in liposarcoma (FUS/TLS), and, most recently, a repeat expansion of C9orf72, the cause of chromosome 9-linked ALS and frontotemporal lobar dementia (FTLD) [2
]. Mutation in the gene for profilin1 (PFN1) was recently reported to be associated with several fALS families. Four mutations (C71G, M114T, E117G, and G118V) were identified in several families [3
]. The etiology of sALS is less clear and must be considered multifactorial and polygenic in the majority of cases.
Several interdependent and interacting mechanisms have been shown to induce motor neuron damage in both fALS and sALS: excitotoxicity, aberrant RNA processing, altered axonal transport, protein aggregation, mitochondrial dysfunction, toxicity of nonneuronal (glial) cells and oxidative stress [4
]. Even if it is unclear whether oxidative stress is a primary or a secondary cause of neurodegeneration in ALS, data from both human tissue and studies in transgenic animal models suggest that it is a major contributory factor leading to chronic motor neuron death. In mutant superoxide dismutase 1 (SOD1) ALS-mouse models [5
] as well as in human familial and sporadic ALS, markers of oxidative damage of proteins, lipids and DNA are elevated in brain and spinal cord [6
]. A larger number of nonspecific antioxidants (e.g., creatine, coenzyme Q 10, vitamine E, N-acetyl-cysteine, and others) have been tested in transgenic mouse models for ALS and were efficient regarding survival, disease progression and motor neuron loss in the spinal cord. Clinical trials in ALS patients have not yet been able to prove efficacy of antioxidant treatment in the clinical setting, but ongoing trials such as the dexpramipexole study still use compounds with antioxidant potential which underlines the importance and acceptance of this therapeutic strategy [10
Inflammation and mitochondrial dysfunction are considered as major pathomechanisms in motor neuron degeneration. These processes both have strong interconnections to oxidative stress cascades. These three pathways are overlapping and interconnected and appear to form a vicious cycle that each could be an initiator as well as a mediator of motor neuron death ().
Figure 1 Three major toxic pathways that contribute to neurodegeneration. Nrf2/ARE signaling activation via triterpenoids can reduce oxidative damage, lessen inflammation, and restore mitochondria resulting in more robust motor neurons able to defend themselves (more ...)
Mitochondrial injury is known to result in an excess of oxygen radicals. Depending on the cell type whose mitochondria are injured, that may trigger different sets of reactions. For instance, glial cells with injured mitochondria can produce proinflammatory molecules that could be toxic to neurons and other cells nearby. These types of inflammatory reactions are stressors for neurons and will be adding to oxidative stress that neurons are subject to. All these in turn will be damaging to mitochondria and other cellular organelles which may lead to further inflammatory reactions in neurons and other cells in CNS (see review by Sun et al. [11
A key molecule regulating the cellular antioxidant response is the basic region leucine-zipper transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2). In basal conditions, Nrf2 is bound to the endogenous inhibitor Kelch-like ECH associated protein1 (Keap1). Once activated, it translocates to the nucleus of the cell where it forms heterodimers with other transcription factors such as c-Jun and small Maf proteins (G/F/K), binds to the antioxidant response element (ARE), a regulatory enhancer region within gene promoters. c-Jun is then supposed to act mainly as transcriptional activator while the small Mafs as well as c-Myc inactivate gene transcription after Nrf2 binding [12
]. Nrf2-ARE binding regulates the expression of more than 200 genes involved in the cellular antioxidant and anti-inflammatory defense such as phase 2 detoxification enzymes (NAD(P)H quinone oxyreductase, glutathione), enzymes which are necessary for glutathione biosynthesis, extracellular superoxide dismutase, glutamate-6-phosphate-dehydrogenase, heat shock proteins and ferritin, furthermore pro- und anti-inflammatory enzymes such as cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and heme oxygenase-1 (HO-1) [13
]. Nrf2 has also been reported to regulate the expression of genes promoting mitochondrial biogenesis such as mitochondrial transcription factors (TFAM) and is therefore directly involved in mitochondrial preservation [16
Induction of Nrf2 by compounds of different chemical classes was shown to be directly correlated to the inhibition of proinflammatory responses (Cox-2 and iNOS expression), but the anti-inflammatory effects of these molecules are only partially Nrf2-dependent and the exact relation between Nrf2-induction and anti-inflammatory properties remains to be clarified [17
]. Probably by regulation of intracellular glutathione content, Nrf2 further has direct cytoprotective effects via the inhibition of Fas-mediated apoptotic pathways [15
The endogenous inhibitor of Nrf2 is the actin-bound cytoskeletal zinc metalloprotein Keap1. Several models of interaction of Keap1 and Nrf2 have been proposed [18
]. Modifications of cysteine residues of Keap1 apparently alter the interaction of Keap1 with Nrf2 and lead to its relocation to the cytoplasm where it is subsequently degraded by the ubiquitin-proteasome system [18
]. Decreased Keap-Nrf2-binding (via oxidation of sulfhydryl groups or phosphorylation) results in intranuclear shuttling of Nrf2 and subsequent transcription of ARE-driven genes [13
]. Keap1 and Nrf2 therefore constitute a cellular sensor for damage caused by free oxygen radicals [21
]. Some studies reported constant shuttling of Keap1 between the nucleus and the cytoplasm under physiological conditions. Karyopherin-6 (KPNA6) has been identified as a protein which facilitates nuclear import and attenuates Nrf2 signaling [26
]. Furthermore, KPNA6 accelerates the clearance of Nrf2 protein from the nucleus, and even promotes the restoration of the Nrf2 protein to basal levels. These findings suggest that KPNA6-mediated Keap1 nuclear import plays an essential role in modulating the Nrf2-dependent antioxidant response and maintaining cellular redox homeostasis.
In addition, it has also been shown that Nrf2 protein stability can be regulated in a Keap1-independent manner by phosphorylation via glycogen synthase kinase-3 (GSK-3β
] and that Nrf2 function can further be modified by regulation of its transcription [28
There are multiple factors that activate Nrf2 in any given cell, for example, environmental stressors such as cigarette smoke, infection, oxidative stress, or inflammation. Several reports have shown that disruption of Nrf2 impairs the induction of the Nrf2/ARE pathway leading to exacerbation of oxidative stress, inflammation, and mitochondrial dysfunction (reviewed by [40
]). Restorative effects of Nrf2 were reported in mice exposed to cigarette smoke [41