Phospho-TDP-43 (Ser409/410) assay principle

The Phospho-TDP-43 (Ser409/410) assay measures TDP-43 when phosphorylated at Ser409/410. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Phospho-TDP-43 (Ser409/410) assay uses 2 antibodies, one labeled with a donor fluorophore and the other with an acceptor. The first antibody was selected for its specific binding to the phosphorylated motif on the protein, the second for its ability to recognize the protein independent of its phosphorylation state. Protein phosphorylation enables an immune-complex formation involving both labeled antibodies and which brings the donor fluorophore into close proximity to the acceptor, thereby generating a FRET signal. Its intensity is directly proportional to the concentration of phosphorylated protein present in the sample and provides a means of assessing the protein’s phosphorylation state under a no-wash assay format.

Phospho-TDP-43 (Ser409/410) two-plate assay protocol

The two-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates to a 384-well low volume detection plate before adding Phospho-TDP-43 (Ser409/410) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Phospho-TDP-43 (Ser409/410) one-plate assay protocol

Detection of Phosphorylated TDP-43 (Ser409/410) with HTRF reagents can be performed in a single plate used for culturing, stimulation, and lysis. No washing steps are required. This HTS designed protocol enables miniaturization while maintaining robust HTRF quality.

Phospho-TDP-43 (Ser409/410) modulation using Calyculin A

Neuro2a cells were cultured in a 96-well plate (50,000 cells/well) for 24h, and then treated for 30 min with increasing concentrations of Calyculin A (protein phosphatase 1 and 2A inhibitor). After treatment, cells were lysed with 10 µL of supplemented lysis buffer #1 (4X) for 30 min at room temperature under gentle shaking (as described in the suspension cell protocol).

After lysis, 16 µL of lysates were transferred into a 384-well low volume white microplate, and 4 µL of the HTRF Phospho-TDP-43 (Ser409/410) or Total TDP-43 detection antibodies were added. The HTRF signal was recorded after an overnight incubation at room temperature.

As expected, Calyculin A triggered a dose-dependent accumulation of phosphorylated TDP-43 at Ser409/410 through inhibition of PP1/2, while the expression level of the protein was not modulated by the treatment.

Inhibition of Phospho-TDP-43 (Ser409/410) with CK1 inhibitors

Neuro2a cells were cultured in a 96-well plate (50,000 cells/well) for 24h, and then treated for 1h30 with the Casein Kinase 1 (CK1) inhibitors IC 261 or PF 670462 (100 µM) followed by Calyculin A activation (30 min, 100 nM).

After cell lysis, 16 µL of lysates were transferred into a 384-well low volume white microplate and 4 µL of the HTRF Phospho-TDP-43 (Ser409/410) or Total TDP-43 detection antibodies were added. The HTRF signal was recorded after an overnight incubation.

As expected, the results obtained show an inhibition of TDP-43 Ser409/410 triggered by Calyculin A after CK1 inhibitor treatment, while the expression level of the protein was not modulated by the treatment.

Detection of accurate level of phosphorylated TDP-43 in an aggregation model induced by staurosporine

HeLa cells were cultured in a 96-well plate (50,000 cells/well) for 24h and then treated for 6h with 1 µM of staurosporine (a well-established caspase activator, cleaving TDP-43 to induce aggregation of C-terminal fragments). After treatment, the cell culture medium was removed and cells were lysed with 50 µL of supplemented lysis buffer #1 (1X) for 30 min at RT under gentle shaking.

Following lysis, three wells with the same condition were pooled, and then 2x60 µL of each condition were treated with Protein Disaggregation kit (Revvity #64DAGGRPET) as per the instructions described in the protocol.

After checking of the disaggregation steps, 16 µL of each condition were transferred into a 384 well low volume white microplate before the addition of 4µL of the HTRF phospho-TDP-43 (Ser409/410) detection reagents. The HTRF signal was recorded after an overnight incubation.

As expected, the staurosporine-induced TDP-43 aggregation provoked an increase in the phosphorylation level of TDP-43 at Ser409/410, detectable only after disaggregation.

Besides demonstrating the compatibility of the Protein Disaggregation kit with HTRF® phospho assay, these results suggest the need to use this process to better detect the accurate level of phosphorylation on aggregated TDP-43.

Assessement of Phospho-TDP-43 (Ser409/410) levels in various cell lines

Adherent  human & mouse cells Neuro 2A, HeLa, and SH-SY5Y were seeded at 50,000 cells/well in a 96-well microplate. After a 24h incubation, the cells were treated 30 min with 100 nM of Calyculin A. Following treatment, cells were lysed with 10 µL of supplemented lysis buffer #1 (4X) for 30 min at room temperature under gentle shaking, (as per the suspension cell protocol).

After lysis, 16 µL of lysate were transferred into a 384-well low volume white microplate before the addition of 4 µL of the HTRF phospho TDP-43 (Ser409/410) detection reagents. The HTRF signal was recorded after an overnight incubation.

The HTRF phospho TDP-43 (Ser409/410) assay efficiently detected phospho TDP-43 (Ser409/410) in human and mouse cellular models with different phosphorylation levels after the same treatment.

HTRF TDP-43 phospho-Ser409/410 assay compared to Western Blot

HeLa cells were cultured in a T175 flask in complete culture medium at 37°C, 5% CO2. After a 48h incubation, the cells were treated with Calyculin A (30 min, 100 nM), then  lysed with 3 mL of supplemented lysis buffer #1 (1X) for 30 minutes at RT under gentle shaking.

Serial dilutions of the cell lysate were performed using supplemented lysis buffer, and 16 µL of each dilution were transferred into a low volume white microplate before the addition of 4 µL of HTRF phospho-TDP-43 (Ser409/410) detection reagents. Equal amounts of lysates were used for a side-by-side comparison between HTRF and Western Blot.

Using the HTRF phospho-TDP-43 (Ser409/410) assay, 4000 cells/well were enough to detect a significant signal, while 16,000 cells were needed to obtain a minimal chemiluminescent signal using Western Blot. Therefore in these conditions, the HTRF phospho-TDP-43 (Ser409/410) assay was 4 times more sensitive than the Western Blot technique.

TDP-43 (TAR DNA binding protein 43) is a DNA and RNA-binding protein which plays a crucial role in RNA metabolism. Mainly located in the nucleus, TDP-43 shuttles between the nucleus and the cytoplasm.

The phosphorylation of TDP-43 is regulated, even if it can be an aberrant process in pathological conditions. Several kinases, including Casein Kinases (CK1 & 2), Tau Tubulin Kinases (TTBK 1 & 2), and cell division cycle 7 (CDC7), are known to promote TDP-43 phosphorylation, whereas phosphatases (PP1 &2) and Calcineurin catalyze its dephosphorylation. Dysregulation of these processes by some mutations, oxidative stress, etc. may lead to an increase in TDP-43 phosphorylation. The phosphorylation of TDP-43 impacts cell functions such as RNA binding or alternative splicing, and can induce a mislocalization and accumulation in the cytoplasm, triggering aggregate formation

In pathological conditions, phospho-TDP-43 Ser409/410 is a hallmark of proteinopathies such as Amyotrophic lateral sclerosis (ALS),  FrontoTemporal Dementia (FTD), or Alzheimer’s Disease (AD). Aberrant phosphorylation, cytoplasmic accumulation, and aggregation of TDP-43 impair the clearance through the proteasome and autophagy mechanisms, leading to neuron cell toxicity.

TDP-43 phosphorylation and its regulation may provide new therapeutic directions to treat neurodegenenerative diseases.