Inducible Nitric Oxide Synthase Up-Regulation in a Transgenic Mouse Model of Familial Amyotrophic Lateral Sclerosis

Mutations in copper/zinc superoxide dismutase (SOD1) are associated with a familial form of amyotrophic lateral sclerosis (ALS), and their expression in transgenic mice produces an ALS-like syndrome. Here we show that, during the course of the disease, the spinal cord of transgenic mice expressing mutant SOD1 (mSOD1) is the site not only of a progressive loss of motor neurons, but also of a dramatic gliosis characterized by reactive astrocytes and activated microglial cells. These changes are absent from the spinal cord of age-matched transgenic mice expressing normal SOD1 and of wild-type mice. We also demonstrate that, during the course of the disease, the expression of inducible nitric oxide synthase (iNOS) increases. In both early symptomatic and end-stage transgenic mSOD1 mice, numerous cells with the appearance of glial cells are strongly iNOS-immunoreactive. In addition, iNOS mRNA level and catalytic activity are increased significantly in the spinal cord of these transgenic mSOD1 mice. None of these alterations are seen in the cerebellum of these animals, a region unaffected by mSOD1. Similarly, no up-regulation of iNOS is detected in the spinal cord of age-matched transgenic mice expressing normal SOD1 or of wild-type mice. The time course of the spinal cord gliosis and iNOS up-regulation parallels that of motor neuronal loss in transgenic mSOD1 mice. Neuronal nitric oxide synthase expression is only seen in neurons in the spinal cord of transgenic mSOD1 mice, regardless of the stage of the disease, and of age-matched transgenic mice expressing normal SOD1 and wild-type mice. Collectively, these data suggest that the observed alterations do not initiate the death of motor neurons, but may contribute to the propagation of the neurodegenerative process. Furthermore, the up-regulation of iNOS, which in turn may stimulate the production of nitric oxide, provides further support to the presumed deleterious role of nitric oxide in the pathogenesis of ALS. This observation also suggests that iNOS may represent a valuable target for the development of new therapeutic avenues for ALS.     

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.     

Apoptosis of human fibrosarcoma HT-1080 cells by epigallocatechin-3-<Emphasis Type="Italic">O</Emphasis>-gallate via induction of p53 and caspases as well as suppression of Bcl-2 and phosphorylated nuclear factor-κB

Animal tumor bioassays and in vitro cell culture systems have demonstrated that epigallocatechin-3-O-gallate (EGCG), the predominant catechin in green tea, possesses anti-proliferative and pro-apoptotic effects on various cancer cells and tumors. In this study, we investigated the effects of EGCG on cell growth, cell cycle progression, and apoptosis in human fibrosarcoma HT-1080 cells. The involvement of p53, Bcl-2, Bax, caspases, and nuclear factor-κB (NF-κB) was examined as a mechanism for the anti-cancer activity of EGCG. Time-dependent intracellular trafficking of EGCG was also determined using fluorescein isothiocyanate (FITC)-conjugated EGCG (FITC-EGCG). Our data show that EGCG treatment caused dose-dependent cell growth inhibition, cell cycle arrest at the G₀/G₁ phase, and DNA fragmentation suggesting the induction of apoptosis in HT-1080 cells. Immunoblot analysis revealed that the expression of p53, caspase-7 and -9 as well as the ratio of Bax/Bcl-2 protein increased significantly with higher EGCG concentrations and longer incubation times. Moreover, expression of phosphorylated NF-κB/p65 in HT-1080 cells was inhibited by EGCG treatment in a dose-dependent manner, while that of unphosphorylated NF-κB/p65 remained unaffected. Here we also reveal time-dependent internalization of FITC-EGCG into the cytosol of HT-1080 cells and its subsequent nuclear translocation. These results suggest that EGCG may interrupt exogenous signals directed towards genes involved in proliferation and cell cycle progression. Taken together, our data indicate that HT-1080 apoptosis may be mediated through the induction of p53 and caspases by the pro-oxidant activity of internalized EGCG, as well as suppression of Bcl-2 and phosphorylated NF-κB by the antioxidant activity of EGCG.     

Antisense peptide nucleic acid-mediated knockdown of the p75 neurotrophin receptor delays motor neuron disease in mutant SOD1 transgenic mice

Re-expression of the death-signalling p75 neurotrophin receptor (p75NTR) is associated with injury and neurodegeneration in the adult nervous system. The induction of p75NTR expression in mature degenerating spinal motor neurons of humans and transgenic mice with amyotrophic lateral sclerosis (ALS) suggests a role of p75NTR in the progression of motor neuron disease (MND). In this study, we designed, synthesized and evaluated novel antisense peptide nucleic acid (PNA) constructs targeting p75NTR as a potential gene knockdown therapeutic strategy for ALS. An 11-mer antisense PNA directed at the initiation codon, but not downstream gene sequences, dose-dependently inhibited p75NTR expression and death-signalling by nerve growth factor (NGF) in Schwann cell cultures. Antisense phosphorothioate oligonucleotide (PS-ODN) sequences used for comparison failed to confer such inhibitory activity. Systemic intraperitoneal administration of this antisense PNA to mutant superoxide dismutase 1 (SOD1G93A) transgenic mice significantly delayed locomotor impairment and mortality compared with mice injected with nonsense or scrambled PNA sequences. Reductions in p75NTR expression and subsequent caspase-3 activation in spinal cords were consistent with increased survival in antisense PNA-treated mice. The uptake of fluorescent-labelled antisense PNA in the nervous system of transgenic mice was also confirmed. This study suggests that p75NTR may be a promising antisense target in the treatment of ALS.     

Early decrease of survival signal-related proteins in spinal motor neurons of presymptomatic transgenic mice with a mutant SOD1 gene

The mechanisms of motor neuronal death in amyotrophic lateral sclerosis (ALS) remain to be unclear. Phosphatidy-linositol 3-kinase (PI3-K) and its main downstream effector, Akt/protein kinase B (PKB) have been shown to play a central role in neuronal survival against apoptosis supported by neurotrophic factors. In order to investigate a possible impairment of survival signaling, we examined expressions of PI3-K and Akt in the spinal cord of the transgenic mice overexpressing a mutant Cu/Zn superoxide dismutase (SOD1) gene, a valuable model for human ALS. Immunoblotting and immunohistochemical analyses showed that the majority of spinal motor neurons lost the immunoreactivities for both PI3-K and Akt in the early and presymptomatic stage that preceded significant loss of the neurons. The present results suggest that an early decrease of survival signal proteins in the spinal motor neurons may account for the subsequent motor neuronal loss in this animal model of ALS.     

Apoptotic death of neurons exhibiting peripherin aggregates is mediated by the proinflammatory cytokine tumor necrosis factor-alpha

Peripherin, a neuronal intermediate filament protein associated with axonal spheroids in amyotrophic lateral sclerosis (ALS), induces the selective degeneration of motor neurons when overexpressed in transgenic mice. To further clarify the selectivity and mechanism of peripherin-induced neuronal death, we analyzed the effects of peripherin overexpression in primary neuronal cultures. Peripherin overexpression led to the formation of cytoplasmic protein aggregates and caused the death not only of motor neurons, but also of dorsal root ganglion (DRG) neurons that were cultured from dissociated spinal cords of peripherin transgenic embryos. Apoptosis of DRG neurons containing peripherin aggregates was dependent on the proinflammatory central nervous system environment of spinal cultures, rich in activated microglia, and required TNF-alpha. This synergistic proapoptotic effect may contribute to neuronal selectivity in ALS.     

XIAP decreases caspase-12 cleavage and calpain activity in spinal cord of ALS transgenic mice

Amyotrophic lateral sclerosis (ALS) is characterized by the selective degeneration of motor neurons. The cause for nerve cell demise is not clear but involves activation of the caspase family of cysteine proteases. We have shown that ER stress and caspase-12 activation occur in ALS transgenic mice carrying the mutant copper/zinc superoxide dismutase (SOD1) gene. In these mice, we found that the antiapoptotic proteins, X-linked Inhibitor of Apoptosis Protein (XIAP) and the related protein, MIAP2 were decreased. To study the role of this, we generated double transgenic mice expressing XIAP in ALS spinal cord neurons using the Thy1 promoter. Overexpression of XIAP inhibited caspase-12 cleavage and reduced calpain activity in the ALS mice. XIAP also reduced the breakdown of calpastatin that is an inhibitor of calpain. In the double transgenic mice, life span was increased by about 12%. These data support the view that XIAP has beneficial effects in ALS and extends survival. The neuroprotective effect of XIAP involves inhibition of caspases and the stabilization of the calpastatin/calpain system that is altered in the ALS mice.     

Role of Wnt1 and Fzd1 in the spinal cord pathogenesis of amyotrophic lateral sclerosis-transgenic mice

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by chronic progressive degeneration of motor neurons resulting in muscular atrophy, paralysis, and ultimately death. We have investigated the expression of Wnt1 and Fzd1 in the spinal cords of SOD1G93A ALS transgenic mice, SOD1G93A-transfected N2a cells, and primary cultured astrocytes from SOD1G93A transgenic mice. In addition, we provided further insight into the role of Wnt1 and Fzd1 in the pathogenesis of ALS transgenic mice and discuss the mechanisms underlying the Wnt signal pathway which may be useful in the treatment of ALS. The results indicate the involvement of Wnt1 and Fzd1 in the pathogenesis and development of ALS.     

Nitric Oxide Potentiates TNF-α-Induced Neurotoxicity Through Suppression of NF-κB

Modulation of enzyme activity through nitrosylation has recently been identified as a new physiological activity of nitric oxide (NO). We hypothesized that NO enhances the TNF-α-induced death of retinal neurons through a suppression of nuclear factor-κB (NF-κB) by nitrosylation. In this study, cells from the RGC-5 line were exposed to different concentrations (2.0, 10, and 50 ng/ml) of TNF-α, and the degree of TNF-α-induced cell death was determined by the WST-8 assay and by flow cytometric measurements of the externalization of phosphatidylserine. The effects of etanercept, a soluble TNFR-Fc fusion protein, and S-nitroso-N-penicillamine (SNAP), an NO donor, on the toxicity were determined. Experiments were also performed to determine whether nitric oxide synthase (NOS) was associated with the toxicity of TNF-α. The activation of NF-κB was determined by the detection of the p65 subunit in the nuclear extracts. Our results showed that exposure of RGC-5 cells to different concentrations of TNF-α significantly decreased the number of living cells in a dose-dependent way. The death was partially due to apoptosis with an externalization of phosphatidylserine, and the death was suppressed by etanercept. Exposure to TNF-α increased the activation of NF-κB and the expression of iNOS. Although NF-κB inhibitors suppressed the increase of iNOS, they also potentiated the TNF-α-induced death. Both L-NAME and aminoguanidine, both NOS inhibitors, rescued the cells from death. In contrast, addition of SNAP caused nitrosylation of the inhibitory κB kinase, and suppressed the NF-κB activation and potentiated the TNF-α-induced neurotoxicity. These results indicate that NO potentiates the neurotoxicity of TNF-α by suppressing NF-κB.     

ER Stress and Unfolded Protein Response in Amyotrophic Lateral Sclerosis

Several theories on the pathomechanism of amyotrophic lateral sclerosis (ALS) have been proposed: misfolded protein aggregates, mitochondrial dysfunction, increased glutamate toxicity, increased oxidative stress, disturbance of intracellular trafficking, and so on. In parallel, a number of drugs that have been developed to alleviate the putative key pathomechanism of ALS have been under clinical trials. Unfortunately, however, almost all studies have finished unsuccessfully. This fact indicates that the key ALS pathomechanism still remains a tough enigma. Recent studies with autopsied ALS patients and studies using mutant SOD1 (mSOD1) transgenic mice have suggested that endoplasmic reticulum (ER) stress-related toxicity may be a relevant ALS pathomechanism. Levels of ER stress-related proteins were upregulated in motor neurons in the spinal cords of ALS patients. It was also shown that mSOD1, translocated to the ER, caused ER stress in neurons in the spinal cord of mSOD1 transgenic mice. We recently reported that the newly identified ALS-causative gene, vesicle-associated membrane protein-associated protein B (VAPB), plays a pivotal role in unfolded protein response (UPR), a physiological reaction against ER stress. The ALS-linked P56S mutation in VAPB nullifies the function of VAPB, resulting in motoneuronal vulnerability to ER stress. In this review, we summarize recent advances in research on the ALS pathomechanism especially addressing the putative involvement of ER stress and UPR dysfunction.     

Decreased Phosphorylation Levels of TrkB Neurotrophin Receptor in the Spinal Cords from Patients with Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is characterized by the selective degeneration of specific populations of cranial and spinal motor neurons. In this study, we examined the expression of the high affinity functional receptor for BDNF, TrkB, and assessed the functional state of TrkB by examining the level of phosphorylation on tyrosine residues in ALS spinal cords. The data showed that TrkB-immunoprecipitates prepared from cell-free lysates of ALS spinal cords by use of an anti-TrkB antibody contained much more TrkB protein than from controls. These TrkB proteins expressed in ALS spinal cords, however, are much less phosphorylated on tyrosine residues than those of controls. Moreover, RT-PCR analysis of TrkB mRNA in ALS spinal cords demonstrated that the expression of Trk B mRNA is also upregulated in ALS spinal cords compared with those of controls. These data strongly suggest that there exists an abnormality in TrkB-mediated intracellular signaling in ALS spinal cords and shed a light on the possibility of the therapeutic intervention by normalizing this intracellular signaling.     

Calcium signal-initiated early activation of NF-κB in neurons is a neuroprotective event in response to kainic acid-induced excitotoxicity

We demonstrate that activation of nuclear factor κB (NF-κB) in neurons is neuroprotective in response to kainic acid (KA)-induced excitotoxicity. Combination of Western blotting, immunocytochemistry, and electrophoresis mobility shift assay showed that KA exposure induced a fast but transient nuclear translocation of the NF-κB p65 subunit and increased DNA-binding activity of NF-κB in primary cultured cortical neurons. The transient NF-κB activity was associated with upregulation of antiapoptotic Bcl-xL and XIAP gene products revealed by real-time PCR. Knockdown of p65 decreased neuronal viability and antiapoptotic gene expression. In addition, we showed that KA-stimulated DNA-binding activity of NF-κB was associated with reactive oxygen species and calcium signals, using AMPA/KA receptor antagonist, calcium chelator, and antioxidant. These results suggest that the fast and transient activation of NF-κB initiated by calcium signals is one of the important proximal events in response to KA-induced excitotoxicity, which has neuroprotective effect against KA-induced apoptosis.     

In Vivo Gene Electroporation of Glial Cell Line-Derived Neurotrophic Factor (GDNF) into Skeletal Muscle of SOD1 Mutant Mice

Motor neurons degenerate with intracellular vacuolar change and eventually disappear in spinal cords of SOD1 mutant mice, resembling human amyotrophic lateral sclerosis (ALS). The GDNF gene was electroporatically transferred into the leg muscles of SOD1 mutant mice and expressed in muscle cells. This gene therapy with GDNF delayed the deterioration of motor performance, being retrogradely transported into spinal motor neurons. However, the number of the motor neurons and survival of the mutant mice were not improved by GDNF treatment. These results indicate that in vivo gene electroporation of GDNF into muscles could be an appropriate therapeutic approach to ameliorate an early dysfunction of motor neurons in SOD1 mutant mice, but further improvement is needed to use this gene transfer as an effective treatment of ALS.     

Death receptor 6 (DR6) antagonist antibody is neuroprotective in the mouse SOD1G93A model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the death of motor neurons, axon degeneration, and denervation of neuromuscular junctions (NMJ). Here we show that death receptor 6 (DR6) levels are elevated in spinal cords from post-mortem samples of human ALS and from SOD1^G93A transgenic mice, and DR6 promotes motor neuron death through activation of the caspase 3 signaling pathway. Blocking DR6 with antagonist antibody 5D10 promotes motor neuron survival in vitro via activation of Akt phosphorylation and inhibition of the caspase 3 signaling pathway, after growth factor withdrawal, sodium arsenite treatment or co-culture with SOD1^G93A astrocytes. Treatment of SOD1^G93A mice at an asymptomatic stage starting on the age of 42 days with 5D10 protects NMJ from denervation, decreases gliosis, increases survival of motor neurons and CC1⁺ oligodendrocytes in spinal cord, decreases phosphorylated neurofilament heavy chain (pNfH) levels in serum, and promotes motor functional improvement assessed by increased grip strength. The combined data provide clear evidence for neuroprotective effects of 5D10. Blocking DR6 function represents a new approach for the treatment of neurodegenerative disorders involving motor neuron death and axon degeneration, such as ALS.     

Establishment of a cell model of ALS disease: Golgi apparatus disruption occurs independently from apoptosis

The Golgi apparatus (GA) appears disrupted in motor neurons of amyotrophic lateral sclerosis (ALS). Here, mouse motor neuron-like NSC-34 cell lines stably expressing human superoxide dismutase 1 (hSOD1)^wt and mutant hSOD1^G93A, as an ALS cell model, were constructed. The number of cells with disrupted GA increased from 14% to 34%. Furthermore, NSC-34/hSOD1^G93A cells showed lower levels of proliferation and differentiation. GA disruption was not caused by apoptosis as determined by several techniques including caspase-3 activation. Similarly, spinal cords from ALS patients did not show caspase-3 activation. Therefore, NSC-34/hSOD1^G93A cells are a suitable cell model to study GA dysfunction in ALS.     

Targeted Depletion of TDP-43 Expression in the Spinal Cord Motor Neurons Leads to the Development of Amyotrophic Lateral Sclerosis-like Phenotypes in Mice

ALS, or amyotrophic lateral sclerosis, is a progressive and fatal motor neuron disease with no effective medicine. Importantly, the majority of the ALS cases are with TDP-43 proteinopathies characterized with TDP-43-positive, ubiquitin-positive inclusions (UBIs) in the cytosol. However, the role of the mismetabolism of TDP-43 in the pathogenesis of ALS with TDP-43 proteinopathies is unclear. Using the conditional mouse gene targeting approach, we show that mice with inactivation of the Tardbp gene in the spinal cord motor neurons (HB9:Cre-Tardbp(lx/-)) exhibit progressive and male-dominant development of ALS-related phenotypes including kyphosis, motor dysfunctions, muscle weakness/atrophy, motor neuron loss, and astrocytosis in the spinal cord. Significantly, ubiquitinated proteins accumulate in the TDP-43-depleted motor neurons of the spinal cords of HB9:Cre-Tardbp(lx/-) mice with the ALS phenotypes. This study not only establishes an important role of TDP-43 in the long term survival and functioning of the mammalian spinal cord motor neurons, but also establishes that loss of TDP-43 function could be one major cause for neurodegeneration in ALS with TDP-43 proteinopathies.     

DREAM-Dependent Activation of Astrocytes in Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of unknown origin and characterized by a relentless loss of motor neurons that causes a progressive muscle weakness until death. Among the several pathogenic mechanisms that have been related to ALS, a dysregulation of calcium-buffering proteins in motor neurons of the brain and spinal cord can make these neurons more vulnerable to disease progression. Downstream regulatory element antagonist modulator (DREAM) is a neuronal calcium-binding protein that plays multiple roles in the nucleus and cytosol. The main aim of this study was focused on the characterization of DREAM and glial fibrillary acid protein (GFAP) in the brain and spinal cord tissues from transgenic SOD1^G93A mice and ALS patients to unravel its potential role under neurodegenerative conditions. The DREAM and GFAP levels in the spinal cord and different brain areas from transgenic SOD1^G93A mice and ALS patients were analyzed by Western blot and immunohistochemistry. Our findings suggest that the calcium-dependent excitotoxicity progressively enhanced in the CNS in ALS could modulate the multifunctional nature of DREAM, strengthening its apoptotic way of action in both motor neurons and astrocytes, which could act as an additional factor to increase neuronal damage. The direct crosstalk between astrocytes and motor neurons can become vulnerable under neurodegenerative conditions, and DREAM could act as an additional switch to enhance motor neuron loss. Together, these findings could pave the way to further study the molecular targets of DREAM to find novel therapeutic strategies to fight ALS.