New avenue for treatment of ALS and frontotemporal dementia? A canonical m6A reader is a potential new target for ALS and frontotemporal dementia

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Author: Michael McMillan PhD, Scientist I at QurAlis. Lead author of “RNA methylation influences TDP43 binding and disease pathogenesis in models of amyotrophic lateral sclerosis and frontotemporal dementia” published in Molecular Cell. 

Proteintech has always supported and been highly interested in ALS research. Dr Michael McMillan, a Proteintech customer, used our TDP43 (10782-2-AP) and YTHDF2 (24744-1-AP) antibodies in a recent Molecular Cell paper (PMID: 36634675), for which he is the first author. This highly detailed and insightful paper demonstrates how TDP43 RNA binding capabilities are modified by YTHDF2, a canonical m6A reader, thus highlighting a potential therapeutic target.

Dr McMillan performed this work while at the University of Michigan, under the supervision of Dr Sami Barmada. Now, Dr McMillan works as a Scientist I at QurAlis, and kindly wrote a summary of his fascinating paper.

Graphical abstract from McMillan et al., 2023 Cell.

Could RNA methylation play a role in ALS?

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that, despite recent advancements, still largely remains a mystery mechanistically and has limited therapeutic options. ALS patients can present with different clinical, biochemical, and genetic features, further complicating treatment of the disease. However, in roughly 95% of ALS cases, cytoplasmic inclusions of an RNA binding protein TDP43 (TAR-binding protein of 43 kDa) occur. TDP43 acts as a critical protein for RNA splicing, transport, translation, and stability. Previous data from our lab suggests that TDP43 specifically destabilizes transcripts encoding ribosomal proteins and oxidative phosphorylation enzymes (PMID: 30030424). These same transcripts were also shown to be upregulated when an RNA methyltransferase enzyme related to the RNA modification, N6-methyladenosine (m6A), is knocked down. Evidence supports that TDP43 interacts with other components of the m6A pathway, leading us to hypothesize that TDP43 may be recognizing m6A on RNA transcripts to target certain transcripts.

TDP43 recognizes m6A methylation

Using bioinformatics, we found a slight but significant increase in TDP43 binding motifs upstream of m6A sites, suggesting that TDP43 may be influenced by the presence of m6A modifications. To investigate if TDP43 binds m6A methylated transcripts, we performed DART-seq, a method which detects m6A sites  independent of m6A antibodies, in cells where we endogenously tagged TDP43 with HaloTag to pulldown bound RNA transcripts. The results showed that m6A was enriched in TDP43-bound transcripts compared to controls,and within the transcript that encodes TDP43, TARDBP, we detected m6A sites within specific defined binding regions. Follow up experiments using minigene reporter demonstrated that m6A is crucial for TDP43 RNA substrate recognition.

YTHDF2 reduction mitigates RNA hypermethylation observed in ALS spinal cord

With this result and our previous evidence implicating m6A in TDP43 binding, we questioned whether m6A was dysregulated in sporadic ALS (sALS). Using sALS and control spinal cord we performed an m6A array to measure m6A levels in the two groups, finding significant hypermethylation in ALS samples. After finding substantial RNA methylation in ALS spinal cord, we wondered whether RNA methylation contributes to TDP43 mediated toxicity. To measure the impact that RNA methylation had on TDP43 toxicity, we designed a CRISPR-Cas9 platform to modulate readers, writers, and erasers of m6A and monitored neuronal survival in a TDP43 based model. Knocking out writers, erasers, and readers of m6A had varying effects on survival, but only knockout of the m6A reader Ythdf2 reduced TDP43 toxicity. This effect was also observed in human induced pluripotent stem cell (iPSCs) models of ALS, where knockdown of YTHDF2 extended neuronal survival. Conversely, overexpression of YTHDF2 in neurons decreased neuronal survival, and immunostaining in ALS spinal cord displayed an increase in YTHDF2 protein compared to control.

Conclusions and implications

These findings implicate m6A dysregulation in disease and potentially identify a source of the observed RNA instability in ALS, as the main influence of m6A deposition is to decrease RNA stability of that transcript. RNA destabilization. The increased YTHDF2 found in ALS patient spinal cord could be creating cellular toxicity in concordance with mislocalized TDP43, supported by data where YTHDF2 depletion extended neuronal lifespan in multiple models of ALS, and YTHDF2 overexpression increased risk of death. The ties to RNA degradation pathways and the data demonstrated in this paper make m6A an exciting new pathway implicated in ALS, and identifies YTHDF2 as a potential novel therapeutic target towards finding treatments for ALS.

Antibodies and related products for m6A modifications

m6A Monoclonal Antibody (68055-1-Ig)

m6A IHCeasy Kit
(KHC0143)

m6A Writers	m6A Readers	m6A Erasers
METTL3	YTHDC1	FTO
METTL14	YTHDC2	ALKBH5
METTL16	YTHDF1
WTAP	YTHDF2
KIAA1429	YTHDF3

Antibodies and products for TDP43

Proteintech’s TDP-43 polyclonal antibody 10782-2-AP played a seminal role in the discovery of this protein (Neuman et al. 2006). Our whole protein immunogen strategy enabled the visualisation of aggregated TDP-43 in more disease scenarios.

Proteintech TDP43 products have over 1500 citations to date, and we have developed a wide range of products to target TDP-43 in a variety of states such as the pathogenic c-terminal fragment and phosphorylated TDP-43.

Antigen Name	CatalogNo	Host and Clonality	Application
TDP-43	10782-2-AP	Rabbit Polyclonal	FC, IF, IHC, WB, ELISA
TDP-43 (C-terminal)	12892-1-AP	Rabbit Polyclonal	IF, IHC, IP, WB, ELISA
TDP-43	18280-1-AP	Rabbit Polyclonal	FC, IF, IHC, IP, WB, ELISA
Phospho-TDP43 (Ser409/410)	22309-1-AP	Rabbit Polyclonal	WB, ELISA
TDP-43 (human specific)	60019-2-Ig	Mouse Monoclonal	FC, IHC, IP, WB, ELISA
Phospho-TDP43 (Ser403/404)	66079-1-Ig	Mouse Monoclonal	FC, WB, ELISA
Phospho-TDP43 (Ser409/410)	66318-1-Ig	Mouse Monoclonal	WB, ELISA
TDP-43	66734-1-Ig	Mouse Monoclonal	FC, IHC, WB, ELISA
TDP-43 (C-terminal)	67345-1-Ig	Mouse Monoclonal	FC, IF, IHC, WB, ELISA
TDP-43	80001-1-RR	Rabbit Recombinant	FC, IHC, WB, ELISA
TDP-43 (for IF/FC)	80002-1-RR	Rabbit Recombinant	FC, IF, ELISA
Phospho-TDP43 (Ser409/410)	80007-1-RR	Rabbit Recombinant	IHC, WB, ELISA
TDP-43	CL488-10782	Rabbit Polyclonal	FC (Intra), IF
TDP-43 (C-terminal)	CL488-12892	Rabbit Polyclonal	FC (Intra), IF
TDP-43 (C-terminal)	CL488-67345	Mouse Monoclonal	IF
TDP-43 (for IF/FC)	CL488-80002	Rabbit Recombinant	FC (Intra), IF
TDP-43 (C-terminal)	CL594-12892	Rabbit Polyclonal	FC (Intra), IF
TDP-43 (C-terminal)	CL594-67345	Mouse Monoclonal	IF
TDP-43 (for IF/FC)	CL594-80002	Rabbit Recombinant	FC (Intra), IF
Human TDP-43 ELISA kit	KE00005	Mix	ELISA (plasma and serum)
Human TDP-43 ELISA kit	KE00009	Mix	ELISA (CSF and cell lysate)