P36. 4-hydroxy(phenyl) retinamide (4HPR) worsens the pathological features of ALS-related TBK1-mutant motor neurons by further disrupting the autophagic flux at the elongation phase

Abstract

TANK-binding kinase 1 (TBK1) is a protein playing a critical role in the activation of pro-survival pathways, by phosphorylating Akt, and in autophagy, as its phosphorylation and activation of the autophagic receptors p62 and optineurin is fundamental for the correct cargo recruitment at the forming autophagosome. Moreover, mutations in TBK1 gene have been recently linked to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). In our study we aimed to uncover the molecular pathological mechanisms characterizing ALS patients carrying mutations in TBK1 gene. For this scope, we used as a model human induced Pluripotent Stem Cells (hiPSCs), and differentiated them into spinal motor neurons (MNs). TBK1-mutant MNs display reduced TBK1 protein levels up to 50%, supporting the theory that haploinsufficiency might be the pathogenic mechanism leading to ALS in these specific cases. Interestingly, as observed in post-mortem tissues, TBK1 MNs are characterized by decreased viability during long-term culture, and accumulate aberrant cytosolic aggresomes. In order to elucidate the impairment affecting the degrative pathway leading to aggresomes accumulation, and eventually circumvent the pathological phenotype, we performed a small molecule screening, which identified 4-hydroxy(phenyl) retinamide (4HPR) as a potent autophagy modifier. This synthetic retinoid failed in reverting the pathological phenotype in mutant motor neurons:in fact, TBK1-mutant cells accumulated immature phagophores when treated with 4HPR. This highlighted an impaired elongation phase in autophagy, further confirmed by the down regulation of several genes involved in this specific step of the degradative pathway. Notably, over expression of Atg10, a protein directly involved in the autophagic elongation phase, rescued the detrimental effect exerted by the retinoid. Finally, we demonstrated that 4HPR leads to a strong and faster accumulation of disease markers also in human C9ORF72-mutant motor neurons and pre-synthomatic SOD1G93A mice. Our results bring novel mechanistic insights into the autophagy impairment occurring in ALS, confirming that induction of the degratative pathway worsens the disease progression if the levels of the autophagy interplayers are not properly re-established.