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- Bu səhifədəki naviqasiya:
- Protein instability and SOD1 aggregation.
- Models of mutant SOD1-mediated toxicity.
- NEUROSCIENCE
- Mutant SOD1 impairs multiple cellular functions.
- Mitochondrial dysfunction in ALS.
- The mitochondrion as a target of mutant SOD1.
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Aberrant redox chemistry Protein toxicity could also enhance the release of copper and zinc, and trigger copper and/or zinc-mediated neurotoxicity. The concept that mutant SOD1 provokes aberrant oxyradical reactions has been controversial. Some of the key findings have not been consistently detected; for example, increased levels of oxidative markers are evi- dent in transgenic ALS G93A-SOD1 mice and patients with SALS 58,59
but not in transgenic G37R mice 60 . Another inconsistency is the impact of forced expression of wild-type SOD1 jointly with mutant SOD1 transgenes. In G85R mice, genetic manipulations of levels of wild- type SOD1 (elimination or forced expression at high levels) do not affect the age of onset, the survival or the rate of progression; these findings could be used to argue against a direct role of heightened oxygen-mediated tox- icity in the degeneration of motor neurons 61 . By contrast, the forced expression of high levels of wild-type SOD1 in G93A, L126Z and A4V-SOD1 mice accelerates the onset of the disease 62,63
. This effect has been attributed to the enhanced formation of mitochondrial aggregates containing both mutant and wild-type SOD1 (REF. 63) . Also of note is evidence that transgenic mice that lack a protein required to load copper into SOD1 possess only minute levels of SOD1-bound copper and have drasti- cally reduced dismutation activity but still develop motor neuron disease 64 . Moreover, mice that harbour mutations in the histidines that bind copper in the SOD1 active site, which leads to loss of dismutase activity, also develop motor neuron disease 65 . Similarly, the G86R mutated murine form of SOD1 causes motor neuron disease in mice, even though a possible alternative copper-loading site, Cys111, is absent 66 . The most plausible interpreta- tion is that copper-mediated oxyradical chemistry is not required for the motor neuron pathology provoked by mutant SOD1; a caveat is that the mutant SOD1 might exert aberrant catalysis through copper loaded in a copper chaperone for SOD1 (CCS)-independent fashion on the surface of the protein 67 .
set of hypotheses propose that the conformational instability of mutant SOD1 induces the formation of harmful aggregates. In transgenic rodents with SOD1- mediated ALS (including copper-binding-site-null mice) and in some human ALS cases, aggregates that are immunoreactive for SOD1 are detected in motor neurons, the neuropil and astrocytes 61,68
. In these trans- genic mice, these aggregates become evident by the time of disease onset 69,70
, and increase in abundance with disease progression 61,69,70 . In vitro studies have shown that, in contrast to stable, dimeric wild-type SOD1, the mutant proteins oligomerize over time to form small pore-like structures that are similar to some forms of β-amyloid protein 71,72 .
to structures in human ALS neural tissue in vivo is not known. In the transgenic animal models, the formation of non-native, sub-microscopic, detergent-insoluble SOD1 species is a common feature of all mutant SOD1 proteins. Because this is not the case for the large, intra- cellular aggregates, it could be that the less evident microscopic aggregates are the vital aggregated protein component in mutant SOD1-mediated ALS. Are SOD1-associated protein inclusions toxic? If so, why are they toxic? As in other neurodegenerative disorders, it remains unclear whether protein aggregates that are detected in the CNS are harmful. As in Huntington’s disease
, it might be that protein inclusions are favour- able, perhaps because they sequester the mutant protein. By contrast, studies have reported a correlation between protein aggregation and a clinical phenotype of trans- genic SOD1 mice: mutations that cause a more severe disease (gauged by shortened survival) have a shorter half-life and the protein products are more likely to form aggregates 73,74 . There is considerable speculation about the potential toxicity of inclusions. It has been proposed that inclusions could both mediate oxyradical chemistry (see above) and overwhelm the proteasome. The latter is predicted to impair protein degradation and recycling (not only of mutant SOD1 but also of other proteins normally processed by proteasomes) and sequester proteins that are crucial for cellular processes, such as heat-shock proteins (HSPs). Mutant SOD1 directly interacts with HSPs, such as HSP70, HSP40, HSP27 and Figure 1 | Models of mutant SOD1-mediated toxicity. The instability of the mutant protein contributes to its toxicity, sometimes enhanced by the release of Zn. In the aberrant redox chemistry model, mutant superoxide dismutase 1 (SOD1) is unstable and the active channel opens, permitting aberrant chemistry through promiscuous interaction with non-conventional substrates. Hydrogen peroxide (H 2 O
) or nitronium ion (ONOO – ) can react with reduced SOD1 (SOD1 – Cu + ). Molecular oxygen (O 2 ) can react aberrantly with Zn-deficient SOD1 to generate an excess of superoxide anion (O 2 .- ). The unstable protein could also release free copper and/or zinc, which might be toxic. In the protein toxicity model, unstable, conformationally altered mutant SOD1 could form toxic, proteinaceous deposits. Aggregated SOD1 could inhibit chaperone and/or proteasome activity, with subsequent misfolding and insufficient clearance of numerous proteins. Alternatively, these aggregates could sequester, inactivate or enhance the toxicity of other proteins crucial for cellular processes. R E V I E W S NATURE REVIEWS |
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Microglia Glutamate Caspase activation Caspase activation EAAT2 Cytokines (for example, TNF α) Nucleus Proteasome stress
Mitochondrial stress
ER stress Golgi
Cell body Microtubule Neurofilaments Axon
Synapse Cell death Δ
metabolism ATP
Ca ++ CytC Δ
Vesicle trafficking Mutant SOD1 Toxicity Glutamate receptor Synaptic vesicle Altered axonal transport Axonal transport motor
Mutant SOD1
Mutant SOD1 Presynaptic neuron terminal BCL2
The founding member of a family of apoptosis-regulating proteins. Many BCL2 family members regulate mitochondria-dependent steps in cell death pathways, with some suppressing and others promoting the release of apoptogenic proteins from these organelles. Apoptosis A mode of cell death in which the cell triggers its own destruction by activating pre- programmed intracellular suicide machinery. Caspases A family of intracellular cysteine endopeptidases that have a key role in mammalian apoptosis. They cleave proteins at specific aspartate residues. Astrogliosis Proliferation and ramification of glial cells in response to brain damage. Microgliosis Proliferation and activation of microglial cells, which are the primary immune effector cells in the brain. αβ-crystallin 75 , perhaps impairing the chaperone and/or the anti-apoptotic function of these proteins. It is likely that mutant SOD1 also sequesters the anti-apoptotic protein BCL2
at the surface of mitochondria 76 . Apoptosis in ALS. Biochemical markers of apoptosis can be detected in the terminal stages of human and trans- genic ALS 77–85
. Early reports suggested that SOD1 muta- tions transform SOD1 from an anti- to a pro-apoptotic protein. Cultured neuronal cells either transfected or microinjected with mutant SOD1 cDNAs die by apop- tosis 78,79
. Mutant SOD1 protein is also pro-apoptotic in an ALS transgenic mouse model 79–83 . Additional evidence linking SOD1 to apoptosis comes from the impairment of the association of cytochrome c (an intermediate in the apoptosis pathway) with the inner membrane of the mitochondrion in ALS transgenic mice, and from the finding that a gradual reduction in intra-mitochondrial cytochrome c correlates with disease progression 84 . Activation of caspase 1 ( CASP1
) is the earliest molec- ular abnormality in the SOD1-G85R mice, occurring months before CASP3
activation, motor neuron death and clinical onset 79–89 . In the spinal cord, activated CASP3 is found in both motor neurons and astrocytes 80 , where it cleaves the astroglial excitatory amino acid transporter 2 ( EAAT2 ) 85 . In the G93A-SOD1 mice at least, the activa- tion of another downstream caspase ( CASP7
) also coincides with disease onset 86 . In agreement with the hierarchical order of caspase activation, in the G93A- SOD1 mice, cytochrome c translocation to the cytosol and concomitant CASP9
activation follow CASP1 and precede CASP3 and CASP7 activation 86 . Because the activation of CASP1 has been involved in both apoptosis and inflammation, it is possible that prolonged activa- tion is a consequence of (and exacerbates) chronic neural inflammation ( astrogliosis and
microgliosis ) in these mice. Alternatively, in contrast to developmental apoptosis in which cell death proceeds rapidly, mutant SOD1- mediated early caspase activation might initiate a slowly Figure 2 | Mutant SOD1 impairs multiple cellular functions. The toxicity of mutant superoxide dismutase 1 (SOD1) is multi-factorial, operating through diverse, interrelated pathways. Within the motor neuron, mutant SOD1 adversely affects DNA/RNA metabolism, mitochondria (diminishing ATP production and calcium buffering, increasing the release of free calcium), neurofilaments and axonal transport, and the function of the endoplasmic reticulum (ER), the Golgi apparatus and proteasomes. In turn, ER stress and apoptotic cascades are activated. Through an interaction with chromogranin (a secretory glycoprotein), mutant SOD1 could be secreted from the motor neuron. The pathological process also involves the activation and dysfunction of astrocytes and microglia. Astroglial uptake of glutamate is reduced, contributing to excessive extracellular glutamate and excitotoxicity. Microglia secrete cytokines such as tumour necrosis factor-
α (TNFα) that could be toxic and cause cellular damage. So, the death process in motor neurons reflects a complex interplay between intrinsic (autonomous) and extrinsic (non-cell autonomous) phenomena. CytC, cytochrome c; EAAT2, excitatory amino acid transporter 2. Modified, with permission, from REF. 187
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Cytoplasm b Mutant SOD1 Toxicity Matrix
Outer membrane PTP
BCL2 TOM
Activation of apoptotic pathway Altered mitochondrial permeability CytC
Inhibition of protein import Intermembrane space Toxic interaction with BCL2 Inner membrane Spinal cord Liver
CytC release ATP depletion BCL2 WT
Mutant SOD1
Aggregates of Mutant SOD1 Toxic mitochondrial SOD1/BCL2 aggregates Cell death progressive cell death process that kills motor neurons and the surrounding cells over months and years. Is this slow apoptotic process directly triggered by mutant SOD1? The gradual activation of an apoptotic pathway in ALS, and the anti-apoptotic influence of wild-type SOD1 (REF. 87) , indicate that mutant SOD1 is directly involved in the initiation of caspase activation and cell death. The observations that both wild-type and mutant SOD1 bind the anti-apoptotic BCL2 and, more importantly, in so doing trap BCL2 into detergent resist- ant aggregates, are consistent with this view 76
. When entrapped in inclusions, BCL2 could be directly rendered non-functional. To the extent that BCL2 functions by binding other pro- and anti-apoptotic proteins, such segregation might also indirectly block BCL2 function. Yet another possibility is that, on binding to mutant SOD1, BCL2 undergoes conformational modification and becomes toxic. Entrapment and depletion of BCL2 is supported by studies that show reduced levels of BCL2 in SOD1 mice and patients with ALS. However, this is only pertinent to mutant SOD1 and does not explain the observed changes in BCL2 expression and function in non-SOD1 ALS cases. Once the pro-apoptotic death signal has been gen- erated, secondary events in the spinal cord amplify the disease process. These include the activation of microglia and T cells and the release of inflammatory factors and cytokines such as interleukin 1 β, COX2 and tumour necrosis factor- α (TNFα) 88–91
. TNF α, which activates CASP8 in late stages of the disease in ALS transgenic mice 92,93
, and its receptor are part of a superfamily of proteins that includes Fas. Cultured embryonic motor neurons are selectively sensitive to Fas-induced apop- tosis, which suggests that these inflammatory/pro- apoptotic molecules mediate a motor neuron-specific apoptotic pathway and thereby selectively intoxicate motor neurons in ALS 92 . Another intriguing mechanism signalling neuroinflammation and toxicity is predicated on the accumulation of mutant but not wild-type SOD1 in the endoplasmic reticulum 94 . Results from recent stud- ies show that mutant SOD1 interacts with proteins such as chromogranin in the endoplasmic reticulum and is thereby retained within that compartment, to be traf- ficked to the cell surface and secreted extracellularly 95 .
mutant SOD1 activates microglial and astrocytic cells to provoke a neuroinflammatory response around the motor neuron 95 . Mitochondrial dysfunction in ALS. Numerous studies have focused on the role of the mitochon- drion in the pathogenesis of neurodegenerative diseases like ALS. Evidence of mitochondrial dysfunc- tion in ALS patients includes clusters of abnormal mito- chondria and morphological defects identified within mitochondria in skeletal muscles and intramuscular nerves in human SALS cases 96,97 . In such cases biochemical analyses have delineated defects in the respiratory chain complexes I and IV in muscle 98 and elevated levels of mitochondrial calcium 99 in muscle and spinal cord. In ALS mice, the main morphological evidence for mitochondrial pathology is the presence of vacuolated mitochondria (for example, in the G93A and G37R trans- genic lines). In the G93A-SOD1 mice, mitochondrial Figure 3 | The mitochondrion as a target of mutant SOD1. Long considered a cytoplasmic protein, superoxide dismutase 1 (SOD1) also localizes to the mitochondrion; its precise localization (outer membrane, intermembrane space or matrix) is not fully defined. a | Mutant SOD1 forms insoluble aggregates that could directly damage the mitochondrion through: swelling, with expansion and increased permeability of the outer membrane and intermembrane space, leading to release of cytochrome c (CytC) and caspase activation; inhibition of the translocator outer membrane (TOM) complex, preventing mitochondrial protein import; and aberrant interactions with mitochondrial proteins such as BCL2. b | Aggregates of mutant SOD1 and BCL2 are found specifically in spinal cord but not liver mitochondria, a finding that might relate to the motor neuronal specificity of mutant SOD1 phenotypes. Codeposition of mutant SOD1 with BCL2 might eliminate BCL2 function, disrupt the mitochondrial membrane, deplete energy, deregulate mitochondrial bioenergetics and activate the mitochondrial apoptotic pathway. WT, wild- type. Modified, with permission, from REF. 76
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ER Vesicle
Axon Microtubules Mitochondrion Neurofilaments Anterograde cargo
Synthesis and trafficking of vesicles and proteins Cell body Dynein–dynactin complex (retrograde motor) Kinesin
(anterograde motor) Retrograde transport Anterograde transport Synapse
Uptake Release
Neurotrophic factors
Retrograde cargo
Kinesins A class of motor proteins that attach to microtubules and transport vesicles along the tubule. vacuoles derived from a detachment between the inner and the outer membrane 100
are evident early, and drastically increase in both number and volume as the disease progresses 101–103
. In vitro and in vivo studies further define mitochon- drial defects in ALS mice. The expression of mutant SOD1 in neuronal cell lines or in cultured primary motor neurons depolarizes mitochondria 104,105
, impairs calcium homeostasis 106 and reduces ATP production 107 . Similarly, G93A-SOD1 mice show reduced respiratory chain activity and reduced ATP synthesis; these increase in severity as disease progresses. Whether mitochondrial dysfunction and pathology represent primary or secondary pathological events is unknown, as mitochondrial abnormalities can both result from and cause oxidative toxicity. In either circumstance, the pathological mitochondria can mediate cell death by releasing calcium into the cytoplasm, producing inadequate levels of ATP and triggering apoptosis (FIG. 3b) . It is likely that mitochon- drial function modifies the course of motor neuron degeneration. In ALS mice, inhibition of manganese super- oxide dismutase (a mitochondrial dismutase) accelerates disease progression. Conversely, the disease is slowed by interventions that improve mitochondrial function — for example, creatine, which inhibits opening of the mitochondrial transition pore, and minocycline, which blocks the egress of cytochrome c 108,109 .
indicated that mutant SOD1 could directly damage mito- chondria. It is evident that a fraction of SOD1 is localized in the mitochondrion 76,110–113 .
targeted SOD1 in vitro reveal that mutant SOD1 more potently triggers cell death when localized to mitochon- dria and, once so targeted, forms intra-mitochondrial protein aggregates 114 . Several interlocking mechanisms explain how mutant SOD1 impairs mitochondria from within
(FIG. 3a) . In G93A-SOD1 mice, mutant SOD1 colocalizes with cytochrome c and the peroxisomal membranes associated with the vacuoles 100 . Therefore, mutant SOD1 could be involved in fusing the peroxi- somes and the outer mitochondrial membrane, a process that could form pores in the mitochondrial membrane allowing the release of cytochrome c and the initiation of an apoptotic cascade. A second hypothesis is that mutant SOD1 is selectively recruited to the outer mitochondrial membrane, where it forms aggregates that slowly disrupt the protein translocation machinery (the translocator outer membrane (TOM) complex) of the mitochon- drion and limits the import of functional proteins into the mitochondrion 112
. Two studies suggest that mutant SOD1 is selectively recruited to mitochondria only in affected tissues 76,112
. It has therefore been proposed that the selective loss of TOM function in spinal cord mito- chondria might form the basis for the tissue specificity of ALS. Finally, mutant SOD1 aggregates could damage the mitochondrion through abnormal interaction with other mitochondrial proteins such as BCL2 (REF. 76) . A recent paper challenged the notion of a toxic mito- chondrial mutant SOD1, suggesting that mitochondrial load ing of mutant SOD1 is simply the result of over- expression of the mutant SOD1 that normally does not associate with mitochondria 115 .
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