Pharmacological Intervention Targeting FAF1 Restores Autophagic Flux for α-Synuclein Degradation in the Brain of a Parkinson's Disease Mouse Model.

α-Synuclein accumulation is implicated in the pathogenesis of neurodegenerative diseases, including Parkinson's disease (PD). Previously, we reported that Fas-associated factor 1 (FAF1), which plays a role in PD pathogenesis, potentiates α-synuclein accumulation through autophagy impairment in dopaminergic neurons. In this study, we show that KM-819, a FAF1-targeting compound, which has completed phase I clinical trials, interferes with α-synuclein accumulation in the mouse brain, as well as in human neuronal cells (SH-SY5Ys). KM-819 suppressed the accumulation of monomeric, oligomeric, and aggregated forms of α-synuclein in neuronal cells. Furthermore, KM-819 restored the turnover rate of α-synuclein in FAF1-overexpressing SH-SY5Y cells, implicating KM-819-mediated reconstitution of the α-synuclein degradative pathway. In addition, KM-819 reconstituted autophagic flux in FAF1-transfected SH-SY5Y cells, also suppressing α-synuclein-induced mitochondrial dysfunction. Moreover, oral administration of KM-819 also interfered with α-synuclein accumulation in the midbrain of mice overexpressing FAF1 via an adeno-associated virus system. Consistently, KM-819 reduced α-synuclein accumulation in both the hippocampus and the midbrain of human A53T α-synuclein transgenic mice. Collectively, these data imply that KM-819 may have therapeutic potential for patients with PD.

Fas-associated factor 1 induces the accumulation of α-synuclein through autophagic suppression in dopaminergic neurons.

Impairment of protein clearance mechanisms leads to α-synuclein accumulation in dopaminergic neurons, contributing to the pathogenesis of Parkinson's disease (PD). Based on the finding that Fas-associated factor 1 (FAF1), a positive modulator of PD, colocalizes with α-synuclein in PD patient brains, we investigated the existence of pathological interplay between FAF1 and α-synuclein. Monomeric and high-molecular-weight forms of α-synuclein were increased in FAF1-overexpressing SH-SY5Y cells. In particular, α-synuclein turnover was accelerated by genetic depletion of FAF1 in SH-SY5Y cells. Therefore, we questioned whether FAF1 is involved in the α-synuclein clearance process. Autophagy inhibitors, but not proteasome inhibitors, restored concurrent attenuation of α-synuclein expression by FAF1 depletion in SH-SY5Y cells. Moreover, we found alterations in autophagy markers in SH-SY5Y cells caused by FAF1 overexpression, indicating that FAF1 disturbed α-synuclein clearance through the autophagy-lysosome pathway. Indeed, FAF1 activated the mammalian target of rapamycin (mTOR) pathway, subsequently suppressing autophagosome formation. Consistently, α-synuclein-mediated mitochondrial dysfunction was observed in FAF1-overexpressing SH-SY5Y cells. Furthermore, FAF1 overexpression using stereotaxic injection of adeno-associated virus led to α-synuclein accumulation and autophagy dysregulation in the PD model mice. Taken together, our results reveal a novel role for FAF1: that of a negative regulator of autophagic α-synuclein clearance.

A novel function of FAF1, which induces dopaminergic neuronal death through cell-to-cell transmission.

BACKGROUND: Fas-associated factor 1 (FAF1) has been implicated in Parkinson's disease (PD) and activates the cell death machinery in the cytosol. However, the presence of extracellular FAF1 has not been studied. METHODS: Serum-free conditioned medium (CM) from FAF1-transfected SH-SY5Y cells was concentrated and analyzed by western blotting. Exosomes were isolated from CM by ultracentrifugation and analyzed by western blotting, electron microscopy and nanoparticle tracking analysis. Soluble FAF1 from CM was immunodepleted using anti-FAF1 antibody. Transmission of secreted FAF1 was examined by transwell assay under a confocal microscope. CM-induced cell death was determined by measuring propidium iodide (PI) uptake using a flow cytometer. RESULTS: FAF1 was secreted from SH-SY5Y cells via exocytosis and brefeldin A (BFA)-resistant secretory pathways. Furthermore, FAF1 was secreted as a vesicle-free form and a genuine exosome cargo in the lumen of exosomes. In addition, FAF1 increased the number of exosomes, suggesting a regulatory role in exosome biogenesis. Extracellular FAF1 was transmitted via endocytosis to neighboring cells, where it induced cell death through apoptotic and necrotic pathways. CONCLUSIONS: This study presents a novel route by which FAF1 induces neuronal death through cell-to-cell transmission. Video Abstract.

FAF1 mediates necrosis through JNK1-mediated mitochondrial dysfunction leading to retinal degeneration in the ganglion cell layer upon ischemic insult.

BACKGROUND: Aberrant cell death induced by ischemic stress is implicated in the pathogenesis of ischemic diseases. Fas-associated factor 1 (FAF1) has been identified as a death-promoting protein. This study demonstrates that FAF1 functions in death signaling triggered by ischemic insult. METHODS: The expression changes of FAF1 and phophorylated JNK1 were detected by Western blotting. Immunoprecipitation was employed to investigate protein-protein interaction. We determined the cell death using flow cytometry and lactate dehydrogenase release measurement. To validate the death-promoting role of FAF1 in the retina, we generated conditional retinal FAF1 knockout mice. We used hematoxylin and eosin staining to detect retinal cell death in retinal ganglion cell layer. RESULTS: FAF1 was found to function upstream of c-Jun N-terminal kinase 1 (JNK1), followed by mitochondrial dysregulation and necrotic cell death processes upon ischemic insult. We investigated whether FAF1 is involved in the pathogenesis of ischemic diseases using a retinal ischemia model. Indeed, FAF1 potentiated necrosis through JNK1 activation upon ischemic stress in retinal cells demonstrating retinal ganglion-like character. Conditional FAF1 depletion attenuated JNK1 activation in the retinas of Dkk3-Cre;Faf1flox/flox mice and ameliorated death of retinal cells due to elevated intraocular pressure (IOP). CONCLUSIONS: Our results show that FAF1 plays a key role in ischemic retinal damage and may be implicated in the pathogenesis of retinal ischemic disease.

FAF1 mediates regulated necrosis through PARP1 activation upon oxidative stress leading to dopaminergic neurodegeneration.

Cumulative damage caused by oxidative stress results in diverse pathological conditions. Therefore, elucidating the molecular mechanisms underlying cell death following oxidative stress is important. Here, we describe a novel role for Fas-associated factor 1 (FAF1) as a crucial regulator of necrotic cell death elicited by hydrogen peroxide. Upon oxidative insult, FAF1 translocated from the cytoplasm to the nucleus and promoted the catalytic activation of poly(ADP-ribose) polymerase 1 (PARP1) through physical interaction. Moreover, FAF1 depletion prevented PARP1-linked downstream events involved in the triggering of cell death, including energetic collapse, mitochondrial depolarization and nuclear translocation of apoptosis-inducing factor (AIF), implying that FAF1 has a key role in PARP1-dependent necrosis in response to oxidative stress. We further investigated whether FAF1 might contribute to the pathogenesis of Parkinson's disease through excessive PARP1 activation. Indeed, the overexpression of FAF1 using a recombinant adeno-associated virus system in the mouse ventral midbrain promoted PARP1 activation and dopaminergic neurodegeneration in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease. Collectively, our data demonstrate the presence of an FAF1-PARP1 axis that is involved in oxidative stress-induced necrosis and in the pathology of Parkinson's disease.