Primary Neurons and Differentiated NSC-34 Cells Are More Susceptible to Arginine-Rich ALS Dipeptide Repeat Protein-Associated Toxicity than Non-Differentiated NSC-34 and CHO Cells.

A repeat expansion mutation in the C9orf72 gene is the most common known genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). In this study, using multiple cell-based assay systems, we reveal both increased dipeptide repeat protein (DRP) toxicity in primary neurons and in differentiated neuronal cell lines. Using flow cytometry and confocal laser scanning microscopy of cells treated with fluorescein isothiocyanate (FITC)-labeled DRPs, we confirm that poly-glycine-arginine (GR) and poly-proline-arginine (PR) DRPs entered cells more readily than poly-glycine-proline (GP) and poly-proline-alanine (PA) DRPs. Our findings suggest that the toxicity of C9-DRPs may be influenced by properties associated with differentiated and aging motor neurons. Further, our findings provide sensitive cell-based assay systems to test phenotypic rescue ability of potential interventions.

SOD1-positive aggregate accumulation in the CNS predicts slower disease progression and increased longevity in a mutant SOD1 mouse model of ALS.

Non-natively folded variants of superoxide dismutase 1 (SOD1) are thought to contribute to the pathogenesis of familial amyotrophic lateral sclerosis (ALS), however the relative toxicities of these variants are controversial. Here, we aimed to decipher the relationships between the different SOD1 variants (aggregated, soluble misfolded, soluble total) and the clinical presentation of ALS in the SOD1G93A mouse. Using a multi-approach strategy, we found that the CNS regions least affected by disease had the most aggregated SOD1. We also found that the levels of aggregated SOD1 in the spinal cord were inversely correlated with the disease progression. Conversely, in the most affected regions, we observed that there was a high soluble misfolded/soluble total SOD1 ratio. Taken together, these findings suggest that soluble misfolded SOD1 may be the disease driver in ALS, whereas aggregated SOD1 may serve to sequester the toxic species acting in a neuroprotective fashion.

Guanabenz Treatment Accelerates Disease in a Mutant SOD1 Mouse Model of ALS.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by loss of motor neurons. The mechanisms leading to motor neuron degeneration in ALS are unclear. However, there is evidence for involvement of endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) in ALS, notably in mutant SOD1 mediated models of ALS. Stress induced phosphorylation of the eIF2 alpha subunit by eukaryotic translation initiation factor 2-alpha kinase 3 Perk activates the UPR. Guanabenz is a centrally acting alpha2 adrenergic receptor agonist shown to interact with a regulatory subunit of the protein phosphatase, Pp1/Gadd34, and selectively disrupt the dephosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eif2alpha). Here we demonstrate that guanabenz is protective in fibroblasts expressing G93A mutant SOD1 when they are exposed to tunicamycin mediated ER stress. However, in contrast to other reports, guanabenz treatment accelerated ALS-like disease progression in a strain of mutant SOD1 transgenic ALS mice. This study highlights challenges of pharmacological interventions of cellular stress responses in whole animal models of ALS.

From transcriptome analysis to therapeutic anti-CD40L treatment in the SOD1 model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive loss of motor neurons. Using unbiased transcript profiling in an ALS mouse model, we identified a role for the co-stimulatory pathway, a key regulator of immune responses. Furthermore, we observed that this pathway is upregulated in the blood of 56% of human patients with ALS. A therapy using a monoclonal antibody to CD40L was developed that slows weight loss, delays paralysis and extends survival in an ALS mouse model. This work demonstrates that unbiased transcript profiling can identify cellular pathways responsive to therapeutic intervention in a preclinical model of human disease.

A novel role for tumor necrosis factor-like weak inducer of apoptosis (TWEAK) in the development of cardiac dysfunction and failure.

BACKGROUND: Tumor necrosis factor-like weak inducer of apoptosis (TWEAK), a member of the tumor necrosis factor superfamily, is a multifunctional cytokine known to regulate cellular functions in contexts of injury and disease through its receptor, fibroblast growth factor-inducible molecule 14 (Fn14). Although many of the processes and downstream signals regulated by the TWEAK/Fn14 pathway have been implicated in the development of cardiac dysfunction, the role of TWEAK in the cardiovascular system is completely unknown. METHODS AND RESULTS: Herein, we demonstrate that mouse and human cardiomyocytes express the TWEAK receptor Fn14. Furthermore, we determine that elevated circulating levels of TWEAK, induced via transgenic or adenoviral-mediated gene expression in mice, result in dilated cardiomyopathy with subsequent severe cardiac dysfunction. This phenotype was mediated exclusively by the Fn14 receptor, independent of tumor necrosis factor-alpha, and was associated with cardiomyocyte elongation and cardiac fibrosis but not cardiomyocyte apoptosis. Moreover, we find that circulating TWEAK levels were differentially upregulated in patients with idiopathic dilated cardiomyopathy compared with other forms of heart disease and normal control subjects. CONCLUSIONS: Our data suggest that TWEAK/Fn14 may be important in regulating myocardial structural remodeling and function and may play a role in the pathogenesis of dilated cardiomyopathy.

TWEAK induces liver progenitor cell proliferation.

Progenitor ("oval") cell expansion accompanies many forms of liver injury, including alcohol toxicity and submassive parenchymal necrosis as well as experimental injury models featuring blocked hepatocyte replication. Oval cells can potentially become either hepatocytes or biliary epithelial cells and may be critical to liver regeneration, particularly when hepatocyte replication is impaired. The regulation of oval cell proliferation is incompletely understood. Herein we present evidence that a TNF family member called TWEAK (TNF-like weak inducer of apoptosis) stimulates oval cell proliferation in mouse liver through its receptor Fn14. TWEAK has no effect on mature hepatocytes and thus appears to be selective for oval cells. Transgenic mice overexpressing TWEAK in hepatocytes exhibit periportal oval cell hyperplasia. A similar phenotype was obtained in adult wild-type mice, but not Fn14-null mice, by administering TWEAK-expressing adenovirus. Oval cell expansion induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) was significantly reduced in Fn14-null mice as well as in adult wild-type mice with a blocking anti-TWEAK mAb. Importantly, TWEAK stimulated the proliferation of an oval cell culture model. Finally, we show increased Fn14 expression in chronic hepatitis C and other human liver diseases relative to its expression in normal liver, which suggests a role for the TWEAK/Fn14 pathway in human liver injury. We conclude that TWEAK has a selective mitogenic effect for liver oval cells that distinguishes it from other previously described growth factors.

Tweak induces mammary epithelial branching morphogenesis.

Members of the tumor necrosis factor (TNF) superfamily regulate cell survival and proliferation and have been implicated in cancer. Tweak (TNF-related weak inducer of apoptosis) has pleiotropic biological functions including proapoptotic, proangiogenic and proinflammatory activities. We explored a role for Tweak in mammary gland transformation using a three-dimensional model culture system. Tweak stimulates a branching morphogenic phenotype, similar to that induced by pro-oncogenic factors, in Eph4 mammary epithelial cells cultured in matrigel. Increased proliferation and invasiveness are observed, with a concomitant inhibition of functional differentiation. Levels of matrix metalloproteinase-9 (MMP-9) are significantly increased following Tweak treatment. Notably, MMP inhibitors are sufficient to block the branching phenotype induced by Tweak. The capacity to promote proliferation, inhibit differentiation and induce invasion suggests a role for Tweak in mammary gland tumorigenesis. Consistent with this, we have observed elevated protein levels of the Tweak receptor, Fn14, in human breast tumor cell lines and xenograft models as well as in primary human breast tumors. Together, our results suggest that the Tweak/Fn14 pathway may be protumorigenic in human breast cancer.

Tumor necrosis factor-like weak inducer of apoptosis-induced neurodegeneration.

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is a member of the tumor necrosis factor (TNF) family of cytokines. It has proangiogenic and proinflammatory properties in vivo and induces cell death in tumor cell lines. TWEAK effects are mediated by the membrane receptor Fn14. In a systematic search for genes regulated in a murine stroke model with the tag-sequencing technique massively parallel signature sequencing, we have identified TWEAK as an induced gene. After 24 hr of focal cerebral ischemia in vivo or oxygen glucose deprivation in primary cortical neurons, both TWEAK and its receptor Fn14 were significantly upregulated. TWEAK induced cell death in primary neurons. Transfection of a nuclear factor (NF)-kappaB-luciferase fusion gene demonstrated that TWEAK stimulated transcriptional activity of NF-kappaB through Fn14 and the IkappaB kinase. Inhibition of NF-kappaB reduced TWEAK-stimulated neuronal cell death, suggesting that NF-kappaB mediates TWEAK-induced neurodegeneration at least in part. Intraperitoneal injection of a neutralizing anti-TWEAK antibody significantly reduced the infarct size after 48 hr of permanent cerebral ischemia. In summary, our data show that TWEAK induces neuronal cell death and is involved in neurodegeneration in vivo.