There’s new hope for the future treatment of some leukodystrophies, neurodegenerative diseases in young children that progressively affect their quality of life, often leading to death before adulthood.
The development stems from the work of Benoit Coulombe, director of the Translational Proteomics Laboratory at the Clinical Research Institute of Montreal (IRCM) and a professor of biochemistry and molecular medicine in the Faculty of Medicine of Université de Montréal.
Published in the journal Molecular Brain, the new research shows that the drug Riluzole, approved by the U.S. Food and Drug Administration to treat certain forms of amyotrophic lateral sclerosis, can at least partially correct the molecular cause of some leukodystrophies.
“Indeed, we have shown that the causative mutations of some leukodystrophies affect the subunits of an important cellular enzyme, RNA polymerase III, preventing its normal assembly – it turned out that Riluzole can counteract this assembly defect,” said Maxime Pinard, the researcher responsible for the project in Coulombe’s lab.
“For diseases as serious and debilitating for patients and their families as leukodystrophies, learning about such advances in knowledge carries a great deal of hope, which IRCM warmly welcomes,” added Dr. Jean-François Côté, the IRCM’s president and scientific director.
Leukodystrophies are rare and almost exclusively genetic diseases characterized by a process of demyelination (damage to the myelin sheath) of the central and peripheral nervous system. The process is primitive in appearance and non-inflammatory and leads to cerebral sclerosis.
“More work is needed to evaluate the effect of Riluzole on patients in order to advance the development of therapeutic avenues for these diseases,” cautioned Marjolaine Verville, co-founder of the Leukodystrophy Foundation.
But already, she added, “the research from Dr. Coulombe’s laboratory is generating a lot of interest and hope in the community.” Her husband and Foundation co-founder, Éric Tailleur, agreed: “It clearly suggests that Riluzole could be used as a drug to treat this disease.”
POLR3-related leukodystrophy is a rare neurodegenerative disease and for which there is no cure. It affects previously healthy children and lead to progressive neurological symptoms, including motor and cognitive disturbances. Here, our approach has been to target the cause of this specific leukodystrophy. POLR3-related leukodystrophy is caused by biallelic pathogenic variants in genes encoding subunits of Pol III, i.e. POLR3A, POLR3B, POLR1C and POLR3K [25, 27,28,29,30, 37, 38].
AP-MS experiments revealed that some substitutions lead to subunits that cannot interact with their cognate partners to form a complete and functional Pol III enzyme [28, 30]. For example, POLR3B R103H causes a severe defect in complex assembly which makes it an interesting model to study Pol III assembly in cultured cells.
In addition to its use as a model to study the molecular mechanism leading to leukodystrophy-associated assembly defects, it can be used as an assay for the discovery of drugs that act early, and directly on the cause of the disease. In this manuscript, we show that riluzole, an FDA-approved drug for the treatment of ALS, partially corrects Pol III assembly defects induced by the leukodystrophy-causative amino acid substitution R103H in POLR3B.
Assembly of Pol III was studied in yeast [39, 40] but to our knowledge, there is no report of its study in mammalian cells. Looking at various time points after transient expression of either FLAG-tagged WT POLR3A or POLR3B, we were able to purify and quantify the bait protein interacting with a number of other Pol III subunits and co-factors in a statistically significant manner. The models shown in Fig. 2C and Fig. 3 C summarize our data on the dynamics of Pol III assembly which shows striking similarities with Pol II complex formation as reviewed by Wild and Cramer . In keeping with the Pol II model, we show that Pol III complex assembly follows a precise stepwise order that involves the formation of two distinct subcomplexes, each including one of the two largest Pol III subunits, POLR3A and POLR3B, and 9 other Pol III subunits.
However, our approach cannot distinguish between the contribution of the various assembly stages that could exist at each time point. The importance of the different subunits in this stepwise process was further confirmed in POLR3B-subcomplex assembly when the variant POLR3B R103H was pulled-down. In this instance, the data show that the mutation affects complex formation early and that the lack of POLR3B-POLR1C interaction may be involved in this defect. This defect could be related to the POLR3B R103 amino acid environment. Pol III crystallography has shown that R103 is found in a negatively charged pocket close to the POLR2K and POLR2L interaction interface . Histidine substitution would change the charge within the pocket and thus, cause protein instability affecting POLR2K, POLR2L and potentially POLR1C interaction with POLR3B. Other amino acid substitutions in this pocket region, namely L104F and R442C, were shown to be associated with POLR3-related leukodystrophy .
HSP90 and HSP70 have long been shown to participate in nascent protein folding, complex formation, and protein stability . Moreover, the involvement of HSP90 in Pol II assembly [7, 40] requires the presence of the PAQosome core subunit RPAP3 . Herein, we show that HSP70 and HSP90 also interact with POLR3A and POLR3B during Pol III assembly.
However, their interaction with POLR3A was observed at later steps during this process, after 8-10 h. This either suggests that HSP70 and HSP90 act later to help in proper protein folding during Pol III assembly or that another chaperone such as the PAQosome  could be involved. On the other hand, early interaction with HSP70 followed by HSP90 was observed when POLR3B was pulled-down, suggesting that these chaperones do participate in POLR3B folding and subcomplex assembly.
Similarly to what has been reported for other RNA polymerases , we found that PAQosome subunits showed statistically significant association with the Pol III complex when POLR3A was pulled-down. This observation suggests that the PAQosome is involved in Pol III complex assembly. Inhibition of various PAQosome subunits has been shown to disrupt other protein complex formation such as that of Pol II , snRNP U5  and PIKKs .
Our data further suggests that the interaction between POLR3A and the PAQosome occurs early, at the same time as POLR2E, a subunit that is common to both Pol III and PAQosome complexes. The strength of the interaction between POLR2E and URI shown in a previous study could explain this rapid recruitment of the PAQosome to POLR3A . Interestingly, only two PAQosome subunits, namely RUVBL1 and RUVBL2, showed statistically significant association with the Pol III complex when POLR3B was pulled-down.
Their interaction occurred early and was not affected by the leukodystrophy-associated substitution R103H in POLR3B. Further investigation is needed to determine the precise order of interactions between the POLR3A-subcomplex and PAQosome subunits, to determine its function in the proper assembly of Pol III complex and to increase our understanding of the exact role of the PAQosome in the process.
GPN1 and GPN3 were also found to significantly interact with the POLR3B subcomplex. These two co-factors are essential for the biogenesis of Pol II  and were shown to be required for nuclear translocation of Pol II and Pol III . Niesser et al. have shown that they can act as GTPase-driving chaperones .
Interestingly, GPN1 and GPN3 were recruited with the POLR3B subcomplex and these interactions were significantly reduced in the R103H variant, suggesting that POLR1C, and potentially other interacting subunits such as POLR1D and POLR2L, are required for GPNs’ recruitment at the subcomplex. This hypothesis is supported by the fact that GPN1 was significantly reduced in the POLR1C N74S variant as observed by Thiffault et al. . Moreover, the amino acid N74 was shown by Ramsay et al.  and Girbig et al.  to be located at an interface that mediates POLR1C interaction with POLR3B [4, 5]. Further investigation is required to understand the exact role of GPNs during Pol III assembly.
Proteostasis is a complex process involving various systems such as chaperones and the proteasomes that can be used as therapeutic targets [45, 46]. Various compounds targeting these systems have proven efficacious at improving folding of several key proteins associated with neurological diseases . One of these compounds, riluzole, has been shown to act as an antiglutamate drug that blocks excessive release of glutamate in motor neurons [48, 49], inhibiting Na+ channel  in ALS animal models, and has since been the subject of repurposing studies for various neurological diseases  and cancers [52, 53].
In our experiments, riluzole treatment resulted in a positive impact on Pol III assembly in the leukodystrophy-causative POLR3B R103H model. Specifically, riluzole was able to significantly increase the interaction level between POLR1C and POLR3B as well as other Pol III subunits. Even if the impact of the R103H variant on small RNAs expression level is not known, it was shown that complex assembly could affect expression level of some small RNA in other Pol III complex assembly defects, as we have previously shown with POLR1C variants . Further experiments would be required to investigate if riluzole could rescue R103H-expressing Pol III function.
The exact mechanism of action of riluzole in our model and whether it is beneficial for other leukodystrophy-causative mutations with assembly defects remains unknown. However, Yang et al. have shown that riluzole can affect the expression level of HSF1 in the cytoplasm  and its translocation stimulates the expression of proteostasis-associated molecules . Work in progress will precise the mechanism of action of riluzole, particularly if the PAQosome or its expression is the target of its action on Pol III assembly. But at this stage, this is purely speculative.
Original Research: Open access.
“Riluzole partially restores RNA polymerase III complex assembly in cells expressing the leukodystrophy-causative variant POLR3B R103H” by Maxime Pinard et al. Molecular Brain