Possible therapeutic targets identified for ALS and frontotemporal dementia

Prof. Ludo Van Den Bosch and his group at the VIB-KU Leuven Center for Brain & Disease Research investigate ALS and frontotemporal dementia (FTD). These devastating neurodegenerative diseases affect thousands in Belgium and have no effective treatment. The most common genetic cause of ALS and FTD is a mutation in the C9orf72 gene, but how this mutation leads to these conditions remains elusive. Now, the researchers identified key proteins, NEK6, HNRNPK and RRM2, that contribute to C9-related ALS and FTD. This opens up new therapeutic avenues for these disorders. Their results are published in high-impact journals Alzheimer's and Dementia:The Journal of the Alzheimer's Association and Acta Neuropathologica.

Van Den Bosch (VIB-KU Leuven): "From two completely different angles, we discovered that interfering with DNA damage counteracts toxicity leading to the major genetic forms of ALS and FTD. By identifying the proteins involved, our research does not only contribute to unraveling the disease mechanisms of ALS and FTD, but, hopefully, also to a cure for these terrible diseases."

Key points

• ALS and FTD are incurable, neurodegenerative diseases affecting over five thousand people in Belgium

• ALS and FTD have extensive clinical, pathological, and genetic overlaps

• A mutation in the C9orf72 gene is the most common genetic cause of ALS and FTD

• Now, three proteins, NEK6, HNRNPK and RRM2, were found to influence the DNA damage caused by the C9orf72 mutation.

• The discovered proteins are candidates for new therapeutic strategies for C9-related ALS and FTD.

FTD is the second most common form of dementia after Alzheimer's disease in patients younger than 65. Due to neuronal loss, people with FTD experience personality and behavioral changes as well as impairment of language skills. ALS is the most common degenerative motor neuron disease in adults. The disorder is characterized by a selective loss of motor neurons, resulting in progressive muscle weakness and paralysis, as well as swallowing and speech difficulties. Patients usually succumb to the disease within 2 to 5 years after diagnosis.

Patients with FTD can experience symptoms typically attributed to ALS and the other way around. An explanation for this overlap emerged with the discovery of a genetic link between the disorders, namely a mutation in the C9orf72 gene. This mutation is the most frequent genetic cause of both ALS and FTD and consists of an expanded repetition of a short DNA sequence which can be repeated in patients up to a thousand times. However, how exactly these repeats lead to ALS and FTD remains unclear. Prof. Ludo Van Den Bosch and his group study the disease mechanisms of this neurodegeneration using various models. Now, this led to the identification of a common pathway and key proteins that contribute to C9 ALS and FTD.

A world-first model identifies a novel target: NEK6

In ALS and FTD patients with the C9orf72 mutation, the RNA produced from the repeat DNA expansion can be translated into dipeptide repeat (DPR) proteins, which can be very toxic and has been identified as a major pathological change in C9-related patients. Dr. Wenting Guo explored the mechanisms of this DPR toxicity to find ways to inhibit it. With her team, Guo found that suppressing or inhibiting the NEK6 kinase reversed DNA damage in patient-derived neurons and counteracted the toxic effects of DPR proteins. Alzheimer's and Dementia: The journal of the American Alzheimer's society now published these results.

The discoveries were made using a world-first CRISPR/Cas9 knock-out screen in human-induced pluripotent stem cell (hiPSC) derived neurons. The study was performed in the Stem Cell Institute Leuven (SCIL).

Prof. Catherine Verfaillie, head of the SCIL, elaborates: "By using human iPSC-derived cortical neurons, we have a human model available for the first time to study the mechanisms underlying neuronal cell death. The CRISPR/Cas9 technology allows us to identify novel targets reversing the disease-causing processes."

Guo explains: "We are proud to present this brand-new discovery after five years of dedication. Our study offers a complete preclinical roadmap to discover and validate new therapeutic targets for C9-related ALS and FTD. Besides a featured research article in a top journal in this research field, we also filed a new patent with high applicational potential. We also provide a solid foundation for further drug development and hopefully will pave the way to clinical applications."

Van Den Bosch: "The intense collaboration with SCIL also benefitted from a successful worldwide collaboration and could also count on the expert services of different VIB Core Facilities. Both the VIB nucleomics and proteomics core were indispensable."

A model to study RNA toxicity identifies identifies novel targets: HNRNPK and RRM2

Along with DPR protein toxicity, RNA toxicity is another proposed mechanism for toxicity in C9-related ALS and FTD. Here, the repeat RNA produced from the C9-mutation can cause toxicity by binding to proteins that are then halted from executing their normal function. PhD student Elke Braems assessed the relevance of this less explored pathway using the in-house developed zebrafish model. Braems and colleagues discovered the importance of the HNRNPK and RRM2 protein in repeat RNA toxicity in ALS and FTD. The high-impact journal Acta Neuropathologica has now published their findings.

The individual contribution of DPR toxicity and RNA toxicity to C9-related disorders remains elusive, and, at present, there is no consensus on whether they could be non-mutually exclusive. One of the significant challenges in studying C9-related ALS and FTD is to separate RNA toxicity from DPR toxicity. Until recently, no model could investigate RNA toxicity without DPR-proteins being present. A solution was provided by the Van Den Bosch lab, which developed a unique zebrafish model in which the researchers could specifically look at the proteins involved in the pathway of RNA toxicity without the confounding factor of DPRs.

Now, this zebrafish model showed that increased levels of the HNRNPK protein can reverse DNA damage in neurons caused by repeat RNA toxicity. The researchers also identified the downstream target that underlies this mechanism, RRM2. The evidence suggests that the RRM2 protein is essential for maintaining an effective DNA damage response. However, it is impaired in C9 ALS and therefore represents a promising druggable target.

Braems explains: "During my master thesis, we used this zebrafish model as a screening tool and revealed HNRNPK as a modifier of RNA toxicity. This was the foundation for my current PhD project in which we successfully unraveled the exact mechanism of the HNRNPK protein in C9-related ALS."

In both studies, there was a very close collaboration with the university hospital in Leuven, where the modifiers were confirmed in patient material. Professor Philip Van Damme, neurologist at UZ Leuven, was fundamental to this cooperation.

"We don't understand the disease mechanisms of C9orf72 mutations enough, and therefore the modifiers identified in these studies provide hope for novel preclinical and clinical programs to find new treatment approaches for ALS and FTD."

While these results are promising, Van Den Bosch will continue researching the role of DNA damage in C9 ALS/FTD, saying: "NEK6, HNRNPK, and RRM2 may become new therapeutic targets for ALS and FTD, but further research into their complete mechanisms and the development of reliable NEK6-inhibitors and HNRNPK/RRM2 modifiers are certainly required."