7-methyl-5-(1-((3-(trifluoromethyl)phenyl)acetyl)-2-3-dihydro-1h-indol-5-yl)-7h-pyrrolo(2-3-d)pyrimidin-4-amine and Amyotrophic-Lateral-Sclerosis

Insoluble, hyperubiquitylated TAR DNA-binding protein of 43 kDa (TDP-43) in the central nervous system characterizes frontotemporal dementia and ALS in many individuals with these neurodegenerative diseases. The causes for neuropathological TDP-43 aggregation are unknown, but it has been suggested that stress granule (SG) formation is important in this process. Indeed, in human embryonic kidney HEK293E cells, various SG-forming conditions induced very strong TDP-43 ubiquitylation, insolubility, and reduced splicing activity. Osmotic stress-induced SG formation and TDP-43 ubiquitylation occurred rapidly and coincided with colocalization of TDP-43 and SG markers. Washout experiments confirmed the rapid dissolution of SGs, accompanied by normalization of TDP-43 ubiquitylation and solubility. Surprisingly, interference with the SG process using a protein kinase R-like endoplasmic reticulum kinase inhibitor (GSK2606414) or the translation blocker emetine did not prevent TDP-43 ubiquitylation and insolubility. Thus, parallel pathways may lead to pathological TDP-43 modifications independent of SG formation. Using a panel of kinase inhibitors targeting signaling pathways of the osmotic shock inducer sorbitol, we could largely rule out the stress-activated and extracellular signal-regulated protein kinase modules and glycogen synthase kinase 3β. For arsenite, but not for sorbitol, quenching oxidative stress with

Topics: Acetylcysteine; Adenine; Amyotrophic Lateral Sclerosis; DNA-Binding Proteins; Frontotemporal Dementia; Glycogen Synthase Kinase 3 beta; Heat-Shock Proteins; HEK293 Cells; Humans; Indoles; Mutation; Osmotic Pressure; Oxidative Stress; Protein Aggregation, Pathological; Protein Transport; Signal Transduction; Solubility; Sorbitol; Ubiquitination

Loss of protein folding homeostasis features many of the most prevalent neurodegenerative disorders. As coping mechanism to folding stress within the endoplasmic reticulum (ER), the unfolded protein response (UPR) comprises a set of signaling mechanisms that initiate a gene expression program to restore proteostasis, or when stress is chronic or overwhelming promote neuronal death. This fate-defining capacity of the UPR has been proposed to play a key role in amyotrophic lateral sclerosis (ALS). However, the several genetic or pharmacological attempts to explore the therapeutic potential of UPR modulation have produced conflicting observations. In order to establish the precise relationship between UPR signaling and neuronal death in ALS, we have developed a neuronal model where the toxicity of a familial ALS-causing allele (mutant G93A SOD1) and UPR activation can be longitudinally monitored in single neurons over the process of neurodegeneration by automated microscopy. Using fluorescent UPR reporters we established the temporal and causal relationship between UPR and neuronal death by Cox regression models. Pharmacological inhibition of discrete UPR processes allowed us to establish the contribution of PERK (PKR-like ER kinase) and IRE1 (inositol-requiring enzyme-1) mechanisms to neuronal fate. Importantly, inhibition of PERK signaling with its downstream inhibitor ISRIB, but not with the direct PERK kinase inhibitor GSK2606414, significantly enhanced the survival of G93A SOD1-expressing neurons. Characterization of the inhibitory properties of both drugs under ER stress revealed that in neurons (but not in glial cells) ISRIB overruled only part of the translational program imposed by PERK, relieving the general inhibition of translation, but maintaining the privileged translation of ATF4 (activating transcription factor 4) messenger RNA. Surprisingly, the fine-tuning of the PERK output in G93A SOD1-expressing neurons led to a reduction of IRE1-dependent signaling. Together, our findings identify ISRIB-mediated translational reprogramming as a new potential ALS therapy.

Topics: Acetamides; Adenine; Amyotrophic Lateral Sclerosis; Animals; Cell Survival; Cells, Cultured; Cerebral Cortex; Cyclohexylamines; eIF-2 Kinase; Endoplasmic Reticulum Stress; HEK293 Cells; HeLa Cells; Humans; Indoles; Mice; Models, Biological; Mutation; Neurons; Rats, Sprague-Dawley; Signal Transduction; Superoxide Dismutase; Survival Analysis; Unfolded Protein Response

Amyotrophic lateral sclerosis (ALS) is a fatal, late-onset neurodegenerative disease primarily affecting motor neurons. A unifying feature of many proteins associated with ALS, including TDP-43 and ataxin-2, is that they localize to stress granules. Unexpectedly, we found that genes that modulate stress granules are strong modifiers of TDP-43 toxicity in Saccharomyces cerevisiae and Drosophila melanogaster. eIF2α phosphorylation is upregulated by TDP-43 toxicity in flies, and TDP-43 interacts with a central stress granule component, polyA-binding protein (PABP). In human ALS spinal cord neurons, PABP accumulates abnormally, suggesting that prolonged stress granule dysfunction may contribute to pathogenesis. We investigated the efficacy of a small molecule inhibitor of eIF2α phosphorylation in ALS models. Treatment with this inhibitor mitigated TDP-43 toxicity in flies and mammalian neurons. These findings indicate that the dysfunction induced by prolonged stress granule formation might contribute directly to ALS and that compounds that mitigate this process may represent a novel therapeutic approach.

Topics: Adenine; Amyotrophic Lateral Sclerosis; Analysis of Variance; Animals; Ataxins; DNA-Binding Proteins; Drosophila melanogaster; Eukaryotic Initiation Factor-2; Gene Ontology; High-Throughput Screening Assays; Humans; Immunoblotting; Immunohistochemistry; Indoles; Luminescent Proteins; Motor Neurons; Nerve Tissue Proteins; Phosphorylation; Poly(A)-Binding Proteins; Red Fluorescent Protein; Retina; RNA Interference; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Small Molecule Libraries; Spinal Cord