Khashayar Vakili, United States of America
Healey Center for ALS, Massachusetts General Hospital NeurologyAuthor Of 2 Presentations
ALTERATIONS IN WWOX LEVELS LEAD TO MITOCHONDRIAL DYSFUNCTION IN AMYOTROPHIC LATERAL SCLEROSIS.
Abstract
Aims
Understanding the pathogenic mechanisms leading to amyotrophic lateral sclerosis (ALS) is crucial for the development of new therapies. Here, we sought to determine whether alterations in the WW domain-containing oxidoreductase (WWOX), a protein involved in DNA damage response, oxidative stress, and neurodegeneration, may contribute to ALS pathogenesis.
Methods
Genetic data from ALS patients were obtained from Project MinE data browser. Western blots were used to assess alterations in WWOX levels as well as in the levels of proteins involved in the mitochondrial electron transport chain (mtETC) in ALS and control post-mortem motor cortex (mCTX). Interactions between WWOX and mtETC proteins were assessed by co-immunoprecipitation (co-IP). SH-SY5Y cells were used to assess cell viability, mtETC proteins levels, and mitochondrial morphology following treatment with wild-type and mutant recombinant WWOX proteins (rWWOXWT, rWWOXSTOP261E and rWWOOXSTOP353Q).
Results
WWOX levels were decreased in ALS mCTX. Furthermore, there were rare, genetic variants in WWOX in 4,366 ALS samples from Project MinE. Two mutations (WWOXSTOP261E and WWOXSTOP353Q) were identified in the short-chain alcohol dehydrogenases (SDR) domain involved in regulating the mtETC. Furthermore, our data demonstrated a significant decrease in ATP5A and COXII levels in ALS mCTX and revealed that WWOX interact with ATP5A. To link mutations in WWOX to mitochondrial dysfunction, SH-SY5Y cells were treated with rWWOXSTOP261E and rWWOXSTOP353Q. Both rWWOXSTOP261E and rWWOXSTOP353Q induced mitochondrial dysfunctions and decreased mtETC proteins levels. Lastly, rWWOXSTOP353Q reduced mitochondrial length in treated SH cells.
Conclusions
Together, our results suggest that mutations in WWOX may contribute to mitochondrial dysfunction in ALS.
DRP1 AND TAU INCREASE MITOCHONDRIAL FISSION IN AMYOTROPHIC LATERAL SCLEROSIS
Abstract
Aims
Although mitochondrial dysfunction has been widely described in amyotrophic lateral sclerosis (ALS), the mechanisms underlying these alterations remain to be elucidated. Here, we sought to verify whether the accumulation of the microtubule binding protein tau, and dynamin-related protein 1 (DRP1), the GTPase catalyzing mitochondrial fission, cause mitochondrial fragmentation in ALS.
Methods
Synaptoneurosomes (SNs) were prepared from ALS and control post-mortem motor cortex (mCTX). Western blots, and immunohistochemistry were used to assess tau, and DRP1 levels. Mitochondrial number and axonal degeneration were assessed by electron microscopy. DRP1 and tau interactions were studied by co-immunoprecipitation (co-IP). Mitochondrial morphology was assessed in SH-SY5Y cells following treatment with tau, ALS or control SNs in the absence or presence of DRP1 silencing (siDRP1) and a selective tau degrader (QC-01-175).
Results
Our results indicate that there was a decrease in the number of mitochondria in ALS together with an increase in DRP1. Furthermore, tau fibrils were observed in the white matter from ALS mCTX together with an increase in axonal degeneration. Additionally, hyperphosphorylated tau mis-localized to the synapses, and was shown to interact with DRP1 in ALS. Lastly, treatment of SHSY5Y cells with ALS SNs, enriched in both pTau and DRP1, significantly increased mitochondrial fission by reducing mitochondrial length and volume. Importantly, knocking down DRP1 or reducing tau levels significantly mitigated alterations in mitochondrial length and volume induced by ALS SNs.
Conclusions
Together, our findings suggest that increases in DRP1 and pTau cause mitochondrial fragmentation in ALS and importantly targeting this molecular pathway mitigates these alterations.