More than 25 years after the discovery of the first mutation leading to amyotrophic lateral sclerosis (ALS), its pathogenesis is still unclear and no cure available. Given that mutations in more than 20 genes of different function lead to ALS, it is puzzling how they converge to the same phenotype of devastating motor neuron degeneration. Moreover, as the majority of ALS patients are considered sporadic with no defined genetic background, environmental factors are considered to contribute to the development and rapid disease progression. One of the hypothesis is that similar to cancer neurodegeneration is an aftermath of multiple hits i.e. that multiple cumulative insults lead to breakdown of neuronal and/or surrounding cell homeostasis. Here we will generate and characterize multiple-hit in vitro and in vivo ALS model systems in which we will sequentially apply potential neurodegeneration triggers. The foundation for additional manipulation will be mouse models that carry mutations similar to ALS patients: optineurin insufficiency model (Optn470T) and mutation in TDP-43 (TDP-43A315T). TDP-43 accumulates in damaged neurons and glial cells in > 95% ALS patients, and optineurin is a multifunctional adapter that regulates several key cellular processes such as inflammation, cell death and autophagy. Since our preliminary results suggest accumulation of TDP-43 in Optn470T model, we plan to investigate the causes of TDP-43 proteinopathy first in neuronal and microglial cell lines by removing optineurin by CRISPR-Cas9 technology, and then in primary brain cultures or Optn470T and TDP-43A315T model. To this end we will analyze the accumulation of proteins, secretion of exosomes and cell death upon manipulation of autophagy and inflammation. The latter will be triggered by LPS, and MCMV and F. novicida infections. To validate the relevance of this findings in vivo, we will make a complex genetic model of ALS by crossing the Optn470T and TDP-43A315T mice.