Misfolded SOD1 forms high-density molecular complexes with synaptic molecules in mutant SOD1-linked familial amyotrophic lateral sclerosis cases.

Mutations in the superoxide dismutase 1 (sod1) gene cause familial amyotrophic lateral sclerosis (FALS), likely due to the toxic properties of misfolded mutant SOD1 protein. Here we report identification of various synaptic molecules forming molecular complexes with misfolded SOD1 in mutant SOD1-associated FALS patient tissues as well as in cellular FALS models. In the FALS cellular model system, we found that membrane depolarization that mimics synaptic hyperactivation/excitotoxicity could cause misfolding of mutant SOD, as well as acceleration of misfolded SOD1-synaptic protein complex formation. These results suggest that inhibition of synaptic release mechanism by association of misfolded SOD1 with synaptic molecules plays a role in the dysfunction of FALS.

Induction of protective immunity by vaccination with wild-type apo superoxide dismutase 1 in mutant SOD1 transgenic mice.

Vaccinations targeting extracellular superoxide dismutase 1 (SOD1) mutants are beneficial in mouse models of amyotrophic lateral sclerosis (ALS). Because of its misfolded nature, wild-type nonmetallated SOD1 protein (WT-apo) may have therapeutic application for vaccination of various SOD1 mutants. We compared the effects of WT-apo to those of a G93A SOD1 vaccine in low-copy G93A SOD1 transgenic mice. Both SOD1 vaccines induced antibody against G93A SOD1 and significantly delayed disease onset compared with saline/adjuvant controls. WT-apo SOD1 significantly extended the life span of vaccinated mice. The vaccines potentiated TH2 deviation in the spinal cord as determined by the ratio of interleukin-4 to interferon-γ (IFNγ) or tumor necrosis factor and induced C1q deposition around motor neurons. Transgenic mice had abundant microglial expression of signal transducers and activators of transcription 4, an activator of transcription of IFNγ, in the spinal cord implicating IFNγ in the pathogenesis. On the other hand, the sera from G93A SOD1-vaccinated mice showed higher IFNγ or tumor necrosis factor and yielded a lower IgG1/IgG2c ratio than the sera from WT-apo-vaccinated mice. These results indicate that the TH1/TH2 milieu is affected by specific vaccinations and that antigenicity might counteract beneficial effects by enhancing TH1 immunity. Thus, because of its lower TH1 induction, WT-apo may be a therapeutic option and have broader application in ALS associated with diverse SOD1 mutations.

Familial amyotrophic lateral sclerosis-linked mutant SOD1 aberrantly interacts with tubulin.

Mutations in the Cu,Zn-superoxide dismutase (SOD1) gene cause 20-25% of familial amyotrophic lateral sclerosis (ALS). Mutant SOD1 causes motor neuron degeneration through toxic gain-of-function(s). However, the direct molecular targets of mutant SOD1, underlying its toxicity, are not fully understood. In this study, we found that alpha/beta-tubulin is one of the major mutant SOD1-interacting proteins, but that wild-type SOD1 does not interact with it. The interaction between tubulin and mutant SOD1 was detected in the spinal cords of mutant G93A SOD1 transgenic mice before the onset of symptoms. Tubulin interacted with amino acid residues 1-23 and 116-153 of SOD1. Overexpression of mutant SOD1 resulted in the accumulation of tubulin in detergent-insoluble fractions. In a cell-free system, mutant SOD1 modulated tubulin polymerization, while wild-type SOD1 did not. Since tightly regulated microtubule dynamics is essential for neurons to remain viable, alpha/beta-tubulin could be an important direct target of mutant SOD1.

Mutant SOD1 impairs axonal transport of choline acetyltransferase and acetylcholine release by sequestering KAP3.

Mutations in the superoxide dismutase 1 (sod1) gene cause familial amyotrophic lateral sclerosis (FALS), likely due to the toxic properties of misfolded mutant SOD1 protein. Here we demonstrated that, starting from the pre-onset stage of FALS, misfolded SOD1 species associates specifically with kinesin-associated protein 3 (KAP3) in the ventral white matter of SOD1(G93A)-transgenic mouse spinal cord. KAP3 is a kinesin-2 subunit responsible for binding to cargos including choline acetyltransferase (ChAT). Motor axons in SOD1(G93A)-Tg mice also showed a reduction in ChAT transport from the pre-onset stage. By employing a novel FALS modeling system using NG108-15 cells, we showed that microtubule-dependent release of acetylcholine was significantly impaired by misfolded SOD1 species. Furthermore, such impairment was able to be normalized by KAP3 overexpression. KAP3 was incorporated into SOD1 aggregates in human FALS cases as well. These results suggest that KAP3 sequestration by misfolded SOD1 species and the resultant inhibition of ChAT transport play a role in the dysfunction of ALS.

Calcium-permeable AMPA receptors promote misfolding of mutant SOD1 protein and development of amyotrophic lateral sclerosis in a transgenic mouse model.

Mutant Cu/Zn-superoxide dismutase (SOD1) protein aggregation has been suggested as responsible for amyotrophic lateral sclerosis (ALS), although the operative mediating factors are as yet unestablished. To evaluate the contribution of motoneuronal Ca2+-permeable (GluR2 subunit-lacking) alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-type glutamate receptors to SOD1-related motoneuronal death, we generated chat-GluR2 transgenic mice with significantly reduced Ca2+-permeability of these receptors in spinal motoneurons. Crossbreeding of the hSOD1G93A transgenic mouse model of ALS with chat-GluR2 mice led to marked delay of disease onset (19.5%), mortality (14.3%) and the pathological hallmarks such as release of cytochrome c from mitochondria, induction of cox2 and astrogliosis. Subcellular fractionation analysis revealed that unusual SOD1 species first accumulated in two fractions dense with neurofilaments/glial fibrillary acidic protein/nuclei and mitochondria long time before disease onset, and then concentrated into the former fraction by disease onset. All these processes for unusual SOD1 accumulation were considerably delayed by GluR2 overexpression. Ca2+-influx through atypical motoneuronal AMPA receptors thus promotes a misfolding of mutant SOD1 protein and eventual death of these neurons.

CHIP promotes proteasomal degradation of familial ALS-linked mutant SOD1 by ubiquitinating Hsp/Hsc70.

Over 100 mutants in superoxide dismutase 1 (SOD1) are reported in familial amyotrophic lateral sclerosis (ALS). However, the precise mechanism by which they are degraded through a ubiquitin-proteasomal pathway (UPP) remains unclear. Here, we report that heat-shock protein (Hsp) or heat-shock cognate (Hsc)70, and the carboxyl terminus of the Hsc70-interacting protein (CHIP), are involved in proteasomal degradation of mutant SOD1. Only mutant SOD1 interacted with Hsp/Hsc70 in vivo, and in vitro experiments revealed that Hsp/Hsc70 preferentially interacted with apo-SOD1 or dithiothreitol (DTT)-treated holo-SOD1, compared with metallated or oxidized forms. CHIP, a binding partner of Hsp/Hsc70, interacted only with mutant SOD1 and promoted its degradation. Both Hsp70 and CHIP promoted polyubiquitination of mutant SOD1-associated molecules, but not of mutant SOD1, indicating that mutant SOD1 is not a substrate of CHIP. Moreover, mutant SOD1-associated Hsp/Hsc70, a known substrate of CHIP, was polyubiquitinated in vivo, and polyubiquitinated Hsc70 by CHIP interacted with the S5a subunit of the 26S proteasome in vitro. Furthermore, CHIP was predominantly expressed in spinal neurons, and ubiquitinated inclusions in the spinal motor neurons of hSOD1(G93A) transgenic mice were CHIP-immunoreactive. Taken together, we propose a novel pathway in which ubiquitinated Hsp/Hsc70 might deliver mutant SOD1 to, and facilitate its degradation, at the proteasome.

The crucial role of caspase-9 in the disease progression of a transgenic ALS mouse model.

Mutant copper/zinc superoxide dismutase (SOD1)-overexpressing transgenic mice, a mouse model for familial amyotrophic lateral sclerosis (ALS), provides an excellent resource for developing novel therapies for ALS. Several observations suggest that mitochondria-dependent apoptotic signaling, including caspase-9 activation, may play an important role in mutant SOD1-related neurodegeneration. To elucidate the role of caspase-9 in ALS, we examined the effects of an inhibitor of X chromosome-linked inhibitor of apoptosis (XIAP), a mammalian inhibitor of caspase-3, -7 and -9, and p35, a baculoviral broad caspase inhibitor that does not inhibit caspase-9. When expressed in spinal motor neurons of mutant SOD1 mice using transgenic techniques, XIAP attenuated disease progression without delaying onset. In contrast, p35 delayed onset without slowing disease progression. Moreover, caspase-9 was activated in spinal motor neurons of human ALS subjects. These data strongly suggest that caspase-9 plays a crucial role in disease progression of ALS and constitutes a promising therapeutic target.

VAChT-Cre. Fast and VAChT-Cre.Slow: postnatal expression of Cre recombinase in somatomotor neurons with different onset.

The cholinergic gene locus (CGL) consists of the genes encoding the choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter (VAChT). To establish a cholinergic-specific Cre-expressing mouse, we constructed a transgene expression vector (VAChT-Cre) with 11.3 kb human CGL in which a Cre-IRES-EGFP unit was inserted in the VAChT open reading frame. The activity of Cre, whose expression was driven by the VAChT promoter, was examined by crossing a reporter mouse (CAG-CAT-Z) in which expression of LacZ is activated upon Cre-mediated recombination. Transgenic lines with the VAChT-Cre construct displayed the restricted Cre expression in a subset of cholinergic neurons in the somatomotor nuclei and medial habenular nucleus, but absent in visceromotor and other central and peripheral cholinergic neurons. Cre expression was first observed at postnatal day 7 and later detected in approximately 40-60% of somatomotor neurons. Based on the onset of Cre expression, we generated two mouse lines (two alleles; VAChT-Cre. Fast and VAChT-Cre.Slow) in which Cre expression reaches maximal levels fast and slow, respectively. The use of VAChT-Cre mice should allow us to deliver Cre to a subset of postnatal motor neurons, thereby bypassing lethality and facilitating analysis of gene function in adult motor neurons.