
==== Front
Sci Rep
Sci Rep
Scientific Reports
2045-2322
Nature Publishing Group UK London

39294194
70822
10.1038/s41598-024-70822-8
Article
Location and function of TDP-43 in platelets, alterations in neurodegenerative diseases and arising considerations for current plasma biobank protocols
Luthi-Carter Ruth ruth.luthi-carter@acimmune.com

1
Cappelli Sara 2
Le Roux-Bourdieu Morgan 1
Tentillier Noemie 1
Quinn James P. 34511
Petrozziello Tiziana 6
Gopalakrishnan Lathika 7
Sethi Purva 8
Choudhary Himanshi 2
Bartolini Giorgia 1
Gebara Elias 1
Stuani Cristiana 2
Font Laure 1
An Jiyan 7
Ortega Vanessa 7
Sage Jessica 812
Kosa Edina 8
Trombetta Bianca A. 34
Simeone Roberto 9
Seredenina Tamara 1
Afroz Tariq 1
Berry James D. 3610
Arnold Steven E. 3456
Carlyle Becky C. 3413
Adolfsson Oskar 1
Sadri-Vakili Ghazaleh 356
Buratti Emanuele 2
Bowser Robert 7
Agbas Abdulbaki 8
1 https://ror.org/00e8cky09 grid.476060.3 0000 0004 7702 9629 AC Immune, SA (ACIU), EPFL Innovation Park Building B, 1015 Lausanne, Switzerland
2 https://ror.org/043bgf219 grid.425196.d 0000 0004 1759 4810 International Centre for Genetic Engineering and Biotechnology, Padriciano 99, 34149 Trieste, Italy
3 https://ror.org/002pd6e78 grid.32224.35 0000 0004 0386 9924 Massachusetts General Hospital Department of Neurology, 114 16th Street, Charlestown, MA 02129 USA
4 Massachusetts Alzheimer’s Disease Research Center (ADRC), 114 16th Street, Charlestown, MA 02129 USA
5 MassGeneral Institute for Neurodegenerative Disease, 114 16th Street, Charlestown, MA 02129 USA
6 https://ror.org/002pd6e78 grid.32224.35 0000 0004 0386 9924 Sean M. Healey and AMG Center for ALS at MassGeneral, Massachusetts General Hospital, 165 Cambridge Street, Boston, MA 02114 USA
7 https://ror.org/01fwrsq33 grid.427785.b 0000 0001 0664 3531 Department of Translational Neuroscience, Barrow Neurological Institute, 350 W. Thomas Road, Phoenix, AZ 85013 USA
8 https://ror.org/052em3f88 grid.258405.e 0000 0004 0539 5056 Kansas City University, 1750 Independence Ave, Kansas City, MO 64106 USA
9 Dipartimento di Medicina Trasfusionale Giuliano-Isontina, Azienda Sanitaria Universitaria Giuliano Isontina (ASUGI), Trieste, Italy
10 Neurological Clinical Research Institute, 165 Cambridge Street, Boston, MA 02114 USA
11 grid.418767.b 0000 0004 0599 8842 Present Address: Eisai US, 35 Cambridgepark Drive, Cambridge, MA 02140 USA
12 grid.418412.a 0000 0001 1312 9717 Present Address: Boehringer Ingelheim Vetmedica, St Joseph, MO 64503 USA
13 https://ror.org/052gg0110 grid.4991.5 0000 0004 1936 8948 Present Address: Department of Physiology, Anatomy and Genetics and Kavli Institute for Nanoscience Discovery, University of Oxford, Oxford, OX13QU UK
18 9 2024
18 9 2024
2024
14 2183724 10 2023
21 8 2024
© The Author(s) 2024
2024
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The TAR DNA Binding Protein 43 (TDP-43) has been implicated in the pathogenesis of human neurodegenerative diseases and exhibits hallmark neuropathology in amyotrophic lateral sclerosis (ALS). Here, we explore its tractability as a plasma biomarker of disease and describe its localization and possible functions in the cytosol of platelets. Novel TDP-43 immunoassays were developed on three different technical platforms and qualified for specificity, signal-to-noise ratio, detection range, variation, spike recovery and dilution linearity in human plasma samples. Surprisingly, implementation of these assays demonstrated that biobank-archived plasma samples yielded considerable heterogeneity in TDP-43 levels. Importantly, subsequent investigation attributed these differences to variable platelet recovery. Fractionations of fresh blood revealed that ≥ 95% of the TDP-43 in platelet-containing plasma was compartmentalized within the platelet cytosol. We reasoned that this highly concentrated source of TDP-43 comprised an interesting substrate for biochemical analyses. Additional characterization of platelets revealed the presence of the disease-associated phosphoserine 409/410 TDP-43 proteoform and many neuron- and astrocyte-expressed TDP-43 mRNA targets. Considering these striking similarities, we propose that TDP-43 may serve analogous functional roles in platelets and synapses, and that the study of platelet TDP-43 might provide a window into disease-related TDP-43 dyshomeostasis in the central nervous system.

Subject terms

Drug discovery
Neuroscience
Physiology
Biomarkers
Neurology
Pathogenesis
Target ALS FoundationBB-2022-C5 Industry-Led Consortium Project Grant NOSRESCUEALSalsfindingacureissue-copyright-statement© Springer Nature Limited 2024
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pmcIntroduction

Increasing evidence has implicated TDP-43 in the pathogenesis of multiple neurodegenerative diseases1–5. These studies have revealed TDP-43 to be a rational target for novel therapies and biomarkers for diseases including ALS, frontotemporal degeneration (FTD), Alzheimer’s disease (AD) and Limbic-predominant Age-related TDP-43 Encephalopathy (LATE). Protein dyshomeostasis and aggregation are key etiolological features of most neurodegenerative diseases6 and TDP-43 is among the most common proteins to be detected in pathology within affected brain or spinal cord regions at autopsy7.

TDP-43 exists in multiple proteoforms in the central nervous system (CNS), where it undergoes post-translational modifications (including phosphorylation, ubiquitination, SUMOylation, cysteine sulfonation, and acetylation3,8–10), is subject to proteolysis2,11, and exists as variants arising from a number of TARDBP splice isoforms12–14. Evidentiary support links some of these proteoforms with disease and disease progression3,10,15.

TDP-43 shuttles between the nucleus and cytosol to fulfil its normal functions16. Histopathology studies of TDP-43-associated diseases indicate that misfolded TDP-43 accumulates in extranuclear cellular compartments, often with reduced nuclear levels of protein17. Reduced levels of nuclear TDP-43 demonstrate corresponding losses of TDP-43 function17,18.

TDP-43-targeted therapeutic agents are being developed to slow or reverse the process of TDP-43 aggregation, to prevent pathological TDP-43 spreading and/or to recover TDP-43 function19,20. Blood-based biomarkers to detect and evaluate TDP-43 pathology are urgently needed for differential diagnosis, evaluation of disease progression and to assess target engagement for such therapies21–25.

In the process of developing new TDP-43-targeting bioassays, we discovered that TDP-43 levels are low in the non-cellular phase of blood plasma but that TDP-43 is highly concentrated in platelets. Here we describe our analyses and consider the potential biological significance of this localization. Although many cellular functions of TDP-43 occur in the nucleus26, others, such as RNA binding, RNA transport, regulation of mRNA translation, miRNA processing, the formation of intracellular RNA/stress granules and the transport of macromolecules in and out of mitochondria, are known to take place in extranuclear compartments17,26. We propose that the latter TDP-43 functions also occur in anucleate platelets, and we further postulate that the study of platelet TDP-43 may provide insights into synaptic TDP-43 functions within the CNS, including its etiologic role(s) in neurologic diseases.

Our findings not only comprise an important consideration for the analysis of TDP-43 as a biomarker, but also highlight a potential confound in plasma biobanking protocols.

Results

Novel high-sensitivity assays for TDP-43 detection

We tested a variety of commercially available and ACIU proprietary antibodies (see “Methods” section) to develop novel immunoassays for TDP-43 quantification in human biofluids. The current study utilizes two immunoassay platforms, Single-Molecule Bead Array (SIMOA™) and Meso Scale Discovery (MSD™), for the detection of C-terminal and N-terminal (or full-length) TDP-43 in biofluids, respectively (Fig. 1A). We also use previously developed capillary electrophoresis immunoassays (CEIs) for detecting TDP-43 phosphorylated at Serine 409 and/or Serine 410 (pSer(409/410)TDP-43, pTDP-43) and total TDP-43 in human biofluids (Fig. 1A). These assays were developed independently in three different laboratories and used here to collectively address the molecular characteristics and distribution of plasma TDP-43.Fig. 1 Assessment of the performance of novel TDP-43 assays in plasma. (A) A schematic diagram of TDP-43 showing epitopes targeted in the C-terminal (B–D, and Fig. 2C), N-terminal (E and Fig. 2C) and p409/p410 (Fig. 2C) immunoassays; see further details in “Methods” section and Suppl. Fig. 6. (B,E) The calibration curves of the novel SIMOA and MSD assays. (C,D) Assay dilution linearity and parallelism for the C-terminal SIMOA assay. For SIMOA, each data point is the mean ± SD of two technical replicates in a single experiment. For MSD, each data point represents the mean ± SD of technical triplicates in a single experiment. In both cases, the error bars are either shown or fall within the limits of the data symbols.

The SIMOA-based C-terminal immunoassay (see “Methods” section) targets the RRM2 domain and a C-terminal to amino acid 360. Comparison of its results to those of the N-terminal domain assay for a given sample can provide an estimate of the presence of N-terminal versus C-terminal sequences present and can thereby dissect possible TDP-43 splice isoform and proteolytic variations2,3,8–15. The SIMOA assay showed good specificity, signal-to-noise ratio, detection range, variation, spike recovery and dilution linearity in human plasma samples (Fig. 1B–D). Its quantification range in plasma was 90 fM–21 pM using the digital mode of detection only (Fig. 1B, see also “Methods” section).

An MSD-based immunoassay was used to detect full-length or N-terminal fragments of TDP-43; the capture antibody epitope lies between amino acids 203–209 and the detection antibody epitope resides near the N-terminus (Fig. 1E). The assay conditions were optimized to achieve accurate and sensitive measurements of TDP-43 in human biofluids, with a lower limit of quantification of 0.1 pM in plasma (Fig. 1E).

A CEI-based immunoassay was also used to examine total TDP-43 and phosphorylated (S409/410) TDP-43 derivatives and elucidate potential differences in their apparent mass.

Independent analyses and technical platforms demonstrate that plasma TDP-43 is predominantly contained in platelets

In the process of qualifying the C-terminal TDP-43 assay, it was noted that different sources of human plasma yielded a wide range of TDP-43 concentrations. Further investigation showed that TDP-43 levels were inversely correlated with the centrifugation speed used to process the whole blood and recover the plasma (Suppl. Fig. 1). Subsequent studies with the MSD-based N-terminal assay revealed that centrifugation of a fresh blood sample at 1750 × g for 10 min created a vertical gradient of TDP-43 levels with maximal levels in the plasma near the buffy coat (Fig. 2A). These results suggested the possibility that TDP-43 was present in a sedimentable plasma compartment, with platelets being the most likely candidate.Fig. 2 Fractionation of fresh blood samples demonstrates that TDP-43 is compartmentalized in the platelet cytosol. (A) Diagram at left illustrates a typical biobank protocol for plasma collection, followed by the removal of serial samples from the top surface of the plasma. Diagram at right depicts serial collection of 50 µl samples from the top surface of a fresh plasma was followed by TDP-43 quantitation of the fourfold diluted samples using an N-terminally directed TDP-43 MSD assay (as in Fig. 1E). The graph shows the relative amounts of TDP-43 in each fraction, demonstrating that TDP-43 is unevenly distributed, with increasing concentrations in deeper fractions. (B) Schematic diagram of blood processing by differential centrifugation to achieve separation of platelets and PC. (For additional details, see “Methods” section and Suppl. Fig. 1). (C) Results from C-terminally targeted SIMOA assay (as in Fig. 1B-D). Each point represents the mean of technical duplicate samples; bar plot represents group mean ± SD. (D) Results of N-terminally directed TDP-43 MSD assay (as in Fig. 1E). (E) Results from p(409/410) TDP-43 CEI assay (see also Fig. 4). PRP and PC fractions demonstrate significantly higher TDP-43 levels than PPP and PM fractions as indicated (*); data in (C,D) were analyzed by one-way ANOVA followed by the Tukey multiple comparison test; data in E were analyzed by a two-way Student t-test. Data in (C–E) are presented as mean ± SD. Points represent measures from individual healthy control blood donors; n = 5 (C), n = 8 (D), n = 6 (E).

We subsequently investigated this hypothesis by isolating platelets from additional fresh blood samples. We performed this using a multi-step differential centrifugation protocol similar to previously published methods for platelet isolation (see Fig. 2B and “Methods” section). The first, low-speed spin was used to remove erythrocytes and nucleated blood cells, with the supernatant comprising platelet-rich-plasma (PRP). The platelet-rich plasma underwent an additional centrifugation to pellet the platelets, leaving exosomes and lipoprotein complexes in the supernatant (platelet poor plasma, PPP). This yielded specific (> 95–98% pure) recovery of resting platelets, as revealed by microscopic and fluorescence-activated cell sorting analyses for size, shape and P Selectin (CD62P) negativity (Suppl. Fig. 2A,B). Platelet pellets were resuspended in a small volume of buffer and lysed, after which the platelet membranes (PM) were separated from the platelet cytosol (PC) by an additional centrifugation. The plasma and platelet fractions were verified biochemically using western blotting (Suppl. Fig. 2C,D). All fractions were subsequently analyzed for TDP-43 levels.

TDP-43 quantitations by both the MSD-based N-terminal and the SIMOA-based C-terminal assays in different fractions of human blood detected comparable levels of TDP-43 in PRP and platelet cytosol, indicating that platelets largely account for the TDP-43 content in PRP. In contrast, PPP, which consists of soluble plasma proteins, lipoproteins and extracellular vesicles, contained ˂5% of TDP-43 compared to PRP. Additionally, the TDP-43 concentration in platelet cytosol versus platelet membranes was assessed, revealing that platelet TDP-43 is localized within the cytoplasmic compartment (Fig. 2C,D). These results are also consistent with our data using CEI assays, which demonstrated compartmentalization of pTDP-43 in the platelet cytosol, with a very low amount in the PPP (Fig. 2E).

Qualitative analysis of ALS-associated TDP-43 proteoforms in plasma/platelets

Next, we sought to examine disease-associated post-translational modifications and splice variants of TDP-43 in plasma from ALS patients and healthy controls (Fig. 3). Multiple attempts were made to detect TDP-43 variants and modifications by immunoprecipitation (IP) from ALS plasma followed by LC–MS/MS proteomic analyses (Fig. 3B,C). The recovery of TDP-43 from plasma by IP was > 95% (Suppl. Fig. 3), but the total collection amount was small due to the low starting concentration. Trypsin digestion alone was insufficient to recover C-terminal peptide signals in the MS, which we initially attributed to the absence of C-terminal cleavage sites from which peptides could be generated (Fig. 3B). However, a combination of trypsin and chymotrypsin digestion, which contained theoretical C-terminal cleavage sites, also exhibited a limited recovery of C-terminal peptides and no post-translational modifications or splice isoforms (Fig. 3C). In contrast, when the same procedure was applied to SH-SY5Y cell lysates, we obtained 97% sequence coverage of the TDP-43 amino acid sequence, although still without detecting post-translational modifications, endogenous proteolysis or splice isoform translation products (Fig. 3D) (with the exception of methionine oxidation of Met85, which could represent a processing artifact). There are a number of possible explanations for these findings, including that C-terminally variant proteoforms are unstable, of low abundance or highly heterogeneous in plasma, and/or that such species are underrepresented in SH-SY5Y cells compared to the predominant presence of unmodified, full-length TDP-43.Fig. 3 LC–MS/MS analysis of TDP-43 proteoforms in ALS plasma. (A) Schematic diagram of TDP-43 illustrating known sites of post-translational modification according to previous literature (see text). (B) Illustration of peptide mapping results from an LC–MS/MS study of a TDP-43-positive fraction of human plasma obtained by immunoprecipitation. The immunoprecipitated material was separated by SDS-PAGE and a Coomassie-stained gel band of 43–48 kDa was digested with Trypsin prior to LC–MS/MS analysis. (C) Illustration of mapped peptides from a second IP LC–MS/MS experiment from human ALS plasma in which the Coomassie-stained band was digested with trypsin and chymotrypsin before undergoing LC–MS/MS. (D) Results from a parallel LC–MS/MS experiment in which the starting material comprised Coomassie-stained SDS-PAGE bands from SH-SY5Y cell lysates digested with trypsin and chymotrypsin, which resulted in 97% peptide coverage. All peptides mapped to the Uniprot Q13148 reference sequence, with no post-translational modifications detected except from an oxidized methionine at position 85.

A targeted approach to detect TDP-43 and pTDP-43 in platelets by CEI proved more successful (Fig. 4A). The pSer(409/410) post-translational modification was chosen due to extensive findings that it is detected in TDP-43 neuropathology27–30. Interestingly, both TDP-43 and pTDP43 electropherograms showed different patterns in platelets from ALS samples compared to those from healthy control samples (Fig. 4A,B). A high-molecular weight (~ 230 kDa) species was detected by both anti-pTDP-43 and anti-TDP-43 (pan) antibodies in ALS platelet cytosol samples, whereas it was completely absent in samples from healthy controls. According to the literature, this species could represent oligomerized, misfolded, oxidized and/or post-translationally modified proteoforms31–33.Fig. 4 Further characterization of platelet TDP-43 species. (A,B) Differences in relative abundance and electrophoretic migration of various TDP-43 proteoforms in ALS and control platelet cytosol observed in the CEI assay. The figure shows representative results from (A) control samples and (B) ALS samples. The electropherograms depict an overlapping view of the CEI signals from 14–3-3 γ (loading control, green-filled), TDP-43 (pink-filled), and pSer(409/410)TDP-43 (labeled as pTDP-43, orange-filled). Additional higher apparent molecular weight TDP-43-positive species are indicated by blue and purple shading. The two line traces (blue or green) indicate separate CEI runs to detect total TDP-43 or pTDP-43 (together with the 14–3-3 γ loading control). In (A), the control sample shows a TDP-43 peak at ~ 44 kDa (pink shading), a pTDP-43 peak at ~ 48–49 kDa (orange shading), and TDP-43 and pTDP-43 peaks at ~ 92 kDa (blue, potentially representing TDP-43 dimerization). An additional TDP-43-positive peak is observed at ~ 59 kDa. In (B), the ALS sample shows a signal equivalent to control for 14–3-3 γ (green) and a pTDP-43-positive band at ~ 48–49 kDa (orange). The first total TDP-43 peak, however, runs more slowly in ALS samples than in control samples, with an apparent MW of ~ 54 kDa (pink). In addition, unique TDP-43-positive and pTDP-43-positive species were detected at ~ 228–249 kDa in the ALS sample, whereas the species at ~ 44, ~ 59 and ~ 92 kDa observed in controls were not detected. These phenomena were repeatedly observed in ALS and control samples (n = 10,10). (C) Super-resolution microscopy images of TDP-43 in platelets. Immunofluorescence to visualize total TDP-43 (pan-TDP-43), TDP-43 pS409/410, pS369, and pS375 in platelets is shown in green. The intensities and distributions of TDP-43 immunoreactivity were compared to that of Tubulin (shown in red). Bar = 1 µm.

Conversely, in healthy control samples, a unique TDP-43 peak was detected at ~ 92 kDa that was absent from ALS samples. This peak potentially represents functionally intact TDP-43 dimers34. Recent work has postulated that decreased TDP-43 dimerization is a key step to inducing TDP-43 pathology31; therefore, the apparent loss of dimeric TDP-43 in ALS suggests that platelets may replicate the pathologic process observed in the CNS.

Finally, the third change in the ALS TDP-43 CEI profile is the slower apparent migration of the total TDP-43 peak relative to the pTDP-43 peak, with an estimated mass of ~ 54–58 kDa in the ALS samples instead of the ~ 43–44 kDa species seen in the control samples. Further work is required to fully characterize the post-translational modifications and/or oligomerization that account for these observations. These results nonetheless indicate that platelet derived TDP-43 warrants further consideration for developing biomarkers for CNS-related diseases.

Imaging TDP-43 in platelets

In order to obtain visual images of TDP-43 in platelets we conducted immunocytochemistry for the unmodified TDP-43 protein and various phosphorylated forms that have been described to occur in TDP-43 pathology (pS409/410, pS375, pS369) and examined healthy control platelet samples using super-resolution microscopy. As shown in Fig. 4C (compared to the positive control, tubulin), the immunostaining for total TDP-43 shows a strong signal that labels large puncta in the body of platelets. Interestingly, pS409/410 TDP-43 immunolabeling of platelets from healthy subjects also revealed readily observable puncta, despite that pS409/410 TDP-43 is typically only seen in degenerating neurons in ALS or other neurodegenerative disease autopsy specimens. Likewise, a strong signal was also detected for pS369, in contrast with immunostaining for pS375, which gave a very weak signal. Taken together, these results further confirm that TDP-43 is highly expressed in platelets and that it can undergo at least some of the post-translational modifications that occur in human neuropathology.

TDP-43-regulated, CNS-expressed transcripts are detected in human platelets

Since TDP-43 functions as an RNA binding protein, we conducted an unbiased evaluation of platelet RNAs by RNA sequencing (RNAseq) and queried the presence of known TDP-43 targets, including those in common with neurons and astroglia. As we have previously shown that TDP-43 activity can be profoundly affected by cell-type35, we compared the platelet RNA profile with those of neuron-like SH-SY5Y and astrocyte-like U87 human cell lines (Figs. 5, 6). As expected, a principal component analysis readily separates the transcriptomes of these three sample types (Fig. 5A). This did not preclude the analysis of common transcriptomic features, however.Fig. 5 mRNA sequencing and differential expression analysis of platelets versus neural cell lines. (A) A principal component analysis was performed on normalized “rlog” gene counts obtained from three biological replicates of U87 (red), SH-SY5Y (green) and platelet (light blue) samples. The first (PC1) and second (PC2) dimension are plotted on the x-axis and y-axis, respectively. (B) Volcano plot representations of RNAseq data obtained from platelets versus SH-SY5Y (on the left) and platelets versus U87 (on the right). Each mRNA is represented by a single dot, with statistically differentially expressed mRNAs being color-coded with respect to platelet expression (lower expression indicated in green and higher expression indicated in red, respectively, with non-differentially expressed mRNAs represented in grey. The threshold for statistical significance is indicated by the horizontal dashed grey line (FDR adj. p = 0.05). The top 10 down- and upregulated mRNAs (by adj. p value) are indicated in blue text. TARDBP (encoding TDP-43) is indicated in black. (C) Over-representation of differentially expressed genes among Gene Ontology (GO) categories for Platelets versus SH-SY5Y (on the left) and Platelets versus U87 (on the right). Top 10 GO categories (BP: biological process) are presented as horizontal bars with the relative number of identified mRNAs labeled as its Count). Categories related to brain disorders are designated with filled blue bars; these show significant enrichment in both neuron-like and astrocyte-like cells.

Fig. 6 Comparison of Platelet mRNA expression and mRNAs regulated by TDP-43 depletion in neural cell lines. (A) RNA-sequencing-based differential expression analysis was conducted for SH-SY5Y cells with and without TDP-43 depletion. The pie chart displays the number of non-differentially expressed genes (in grey) and significantly differentially expressed genes (DEGs, in blue). Expanded bars report the number of upregulated (in red) and downregulated (in green) RNAs (DEGs) in TDP-43-depleted samples. For DEGs, the number of genes detected as expressed in platelets (using the criterion of DESeq2 normalized counts (normCount_Platelets) greater than 0) are also reported. For each analysis, the percentage (%) is calculated relative to the total number of genes interrogated (33,117). The level of expression (normalized counts) of genes detected in platelets and differentially expressed in TDP-43-depleted SH-SY5Y versus SH-SY5Y cells is reported in the bar chart at the right. (B) RNA-sequencing-based differential expression analysis was conducted for U87 cells with and without TDP-43 depletion. The pie chart displays the number of non-differentially expressed genes (in grey) and DEGs (in blue). Expanded bars report the number of upregulated (in red) and downregulated (in green) DEGs in TDP-43-depleted samples. For DEGs, the number of genes detected as expressed in platelets (using the criterion of DESeq2 normalized counts (normCount_Platelets) greater than 0) are also reported. For each analysis, the percentage (%) is calculated relative to the total number of genes interrogated (33,117). The level of expression (normalized counts) of genes detected in platelets and differentially expressed in TDP-43-depleted U87 versus U87 cells is reported in the bar chart at the right. (C) Cross-comparison of TDP-43-responsive RNAs in neural cells with RNA expression levels in platelets. The pie chart displays the number of DEGs in common to TDP-43-depleted SH-SY5Y and U87 cells (orange) compared to all genes analyzed (red). Expanded bars to the right of the pie chart report the number of platelet-expressed genes (normCount_Platelets > 0, in acid green) and non-platelet-expressed genes (normCount_Platelets = 0, in light green). The bar chart at the right reports representative TDP-43-responsive genes detected at levels of normCount_Platelets > 10. TARDBP (encoding TDP-43) is highlighted as a red bar. Statistical analyses of top up- and down-regulated genes are shown in Suppl. Fig. 6.

We first examined the expression of the TARDBP RNA that encodes TDP-43. TARDP RNA was readily detected in all three cell types, including platelets, which had a normalized read count of > 1000. This confirmed that TDP-43 protein expression in platelets is further ensured by a resident TARDBP RNA pool. Looking at the differential expression of 33,117 detected transcripts, we identified 8275 differentially expressed genes (DEGs) between platelets and SH-SY5Y cells. Specifically, 7386 and 889 mRNAs were found down- and upregulated, respectively (Fig. 5B, left panel). On the other hand, 7709 mRNAs were differentially expressed in platelets compared to U87 cells. Out of these mRNAs, 6368 and 1341 were found to be down- and upregulated, respectively (Fig. 5B, right panel). In this analysis, the selection of DEGs was made using the following cut-offs: p adj. < 0.05 and fold change < 1.3 for upregulated genes and p adj. < 0.05 and fold change < 0.7 for downregulated genes. A general overview of the differential expression results was obtained by performing gene set enrichment analysis (GSEA) (Fig. 5C and Suppl. Figs. 4, 5). As expected, mRNAs belonging to platelet-related Gene Ontology (GO) categories, such as “platelet aggregation” and “platelet alpha granule” were found enriched in platelets compared to the neural cell lines, whereas brain-related categories, such as “axonogenesis”, “axon development”, “synapse organization” and “developmental regulation of neuron projection”, were significantly suppressed in platelets compared to SH-SY5Y and U87 cell lines. In fact, a large group of 5966 transcripts were found to be similarly differentially expressed in both pairwise comparisons. This is consistent with the fact that SH-SY5Y and U87 share expression of sets of genes associated with brain function and development, and thus distinct from those whose expression is characteristic of platelets. Examples of these commonly regulated transcripts are reported in the volcano plots (Fig. 5B), such as CA2, CAV2, HBB, CXCL5 (higher expression in platelets) and RBFOX2, MAST2, LAMB1, ISOC2, POMT2, EXTL3, GPC1, ECHDC1, AGK, PVR, PXN, FAT1, GOPC, TMEM98, FOXC1 and ZDHHC8 (higher expression in neural cells).

Conversely, we also evaluated whether neuronal and glial TDP-43 target RNAs were also contained in platelets. The purpose of this exercise was to identify transcripts expressed in platelets that could be misregulated by the presence of misfolded or non-functional TDP-43. To identify TDP-43 target RNAs, we performed a transcriptome analysis of SH-SY5Y and U87 cells after siRNA knockdown of TDP-43 (Fig. 6). Diminution of TDP-43 by ~ 80% (Suppl. Fig. 6A) altered the levels of 3717 mRNAs in SH-SY5Y cells (Fig. 6A, Suppl. Fig. 7A), and diminution of TDP-43 by ~ 80% (Supp. Fig. 6B) altered the levels of 8245 mRNAs in U87 cells (Fig. 6B, Suppl. 7B). Interestingly, among these DEGs, platelets expressed 1986 of the mRNAs differentially expressed in SH-SY5Y cells and 5023 of the mRNAs differentially expressed in U87 cells by the criterion of normalized RNAseq counts greater than zero. These results indicate that platelets express a set of mRNAs potentially affected by TDP-43 dysregulation. We also considered the overlap between TDP-43-responsive genes in the two neural cell types. This analysis identified 1210 mRNAs coordinately regulated by TDP-43 deficiency, of which 266 were also expressed in platelets (normalized RNAseq counts greater than zero (Fig. 6C)). This result further supports our hypothesis that similarities in the TDP-43-mediated regulation of RNAs expressed in both neural cells and platelets may drive and/or reflect the effects of TDP-43 proteinopathies. Representative TDP-43-responsive neural DEGs with normalized RNAseq counts > 10 in platelets are shown in Fig. 6C (bar graph). These included YWHAZ, NRGN, HPCAL1, DYNT3, GLUL, MBNL1, LIMK and SNX3.

Taken together, these data indicate that, despite being anucleate, platelets contain high levels of TDP-43 protein and many of its known RNA targets, thereby suggesting a role for TDP-43 in platelet RNA homeostasis and protein translation. The presence of the same TDP-43 target RNAs in both neural cell types and platelets may allow elucidation of shared functions of TDP-43. Moreover, this unique compartmentalization raises the possibility that disease-related changes in TDP-43 function also occur in platelets. If confirmed, this might provide a novel approach for detecting disease-associated events in accessible biofluids that reflect central events in neurodegenerative diseases. The insights gained here can now be translated into the appropriate collection of platelet samples to address this question.

Discussion

Using three immunoassays on distinct assay platforms, we determined that the majority of TDP-43 collected within blood plasma fractions is contained in platelets. Our results confirm and extend recent studies suggesting altered levels of TDP-43 in platelets in neurodegenerative diseases36,37. Variations of centrifugation speeds during plasma separation and variability in extracting plasma close to the buffy coat likely are responsible for the variable levels of TDP-43 detected in plasma across various studies38. Our results highlight the importance of using well-defined and carefully followed biobanking procedures in order to properly quantify biomarker analytes such as TDP-43.

Platelets and synapses surprisingly share several commonalities in terms of structural organization and protein composition that have intriguing implications for understanding the possible role of platelets in neurologic diseases or their use as surrogate biomarker substrates (Fig. 7). These commonalities include their small size, lacking a nucleus, expression of neurotransmitter receptor ion channels (e.g. for glutamate, serotonin, acetylcholine and ATP) and requirement for rapid, local, signal-dependent translation. Given that the brain is a highly vascularized structure with regulated permeability, there may also be opportunities for the exchange of molecular components and signaling to platelets via the blood-CNS barrier.Fig. 7 Potentially similar roles of TDP-43 in synapses and platelets. Upper text highlights features shared by platelets and synapses. Drawings depict the roles that TDP-43 may fulfil in both synapses and platelets, including regulating pre-mRNA splicing, RNA transport, RNA translation, and mitochondrial homeostasis. Line and lariat structures depict RNA; black points represent translation-capable ribosomes attached to the rough endoplasmic reticulum; beige ovoids depict mitochondria; grey circles represent secretory vesicles; features at the perimeter represent receptors and ion channels at the plasma membrane. Green arrows point to descriptions of how TDP-43 dysfunction might hypothetically manifest in both synapses and platelets, based on its known pathological effects in neurons. The question mark indicates the speculative nature of these features with respect to platelets.

What are the potential functions of TDP-43 in platelets? Possible similarities in TDP-43’s extranuclear roles in platelets and synapses, in both healthy and disease conditions, are summarized in Fig. 7 and described further below.

Many of the loss-of-function effects of disease-related TDP-43 dyshomeostasis are thought to involve its essential role(s) in the nucleus, most notably the regulation of pre-mRNA splicing39. Since platelets are anucleate in nature, we can consider how the splicing of relevant genes encoding platelet components might be regulated in their megakaryocyte precursors. Most requisite platelet proteins, including TDP-43, are expressed by megakaryocytes during endomitosis and packaged into their proplatelet extensions prior to the release of platelets into the bloodstream40. It is therefore plausible that some of the nuclear functions of TDP-43, such as regulation of RNA splicing, take place at the level of platelet-related gene transcription in the megakaryocyte. The platelet contents can provide a window into these events.

In addition, cytoplasmic intron-containing RNA transcripts exist in both platelets and synapses41–43. It has recently come to light that platelets can splice pre-mRNAs in a signal-dependent manner despite being anucleate. For example, platelets contain unspliced IL1B pre-mRNA that can be processed in a signal-dependent manner into a translatable, intronless IL1B transcript44. Moreover, a more extensive search for splicing events in platelets showed that treatment with COLL/TRAP agonists induces splicing in 65 pre-mRNAs that includes intron removal in CLTC, BANK1, and TM9SF2 transcripts, leading to upregulation in their translation45.

TDP-43 also has multiple known functions as an mRNA-binding protein, and many of these remain relevant for platelet function after release from the proplatelet extension. In fact, these functions may more closely parallel TDP-43’s role in neuronal synapses, where signal-dependent regulation of the synthesis of specific proteins is required locally. TDP-43-dependent regulation of its bound mRNAs can occur at multiple downstream checkpoints, including mRNA transport, mRNA sequestration, mRNA degradation and/or recruitment into translationally active ribosomal complexes46–48. TDP-43’s regulation of local protein synthesis in synapses is largely restricted to components needed for synaptic plasticity and remodeling (Fig. 7). It will be interesting to discover which mRNAs may be directly regulated by TDP-43 in platelets and whether there are perturbations to this regulation in neurodegenerative diseases.

Another shared requirement of platelets and synapses is a local energy source, which is fulfilled by resident mitochondria. Multiple reports have described the localization of TDP-43 to mitochondria, via the TIM22 complex1–5. Moreover, Yu et al. have shown that TDP-43 accumulation can trigger the opening of the mitochondrial permeability transition pore (mPTP) and mitochondrial DNA release via the voltage-dependent anion channel 1 (VDAC1)5. TDP-43 is also known to associate with the Optic atrophy-1 protein (OPA1)-mitofilin complex10, which is critical for mitochondria fusion, cristae integrity, and mtDNA maintenance6–8. These data suggest additional platelet-related mechanisms by which TDP-43 neurotoxicity may be conveyed through oxidative stress and inflammation47–53. We anticipate that TDP-43-related disease pathways involving mitochondria might also be accessible for study in platelets49–53.

The above discussion primarily considers disease-related changes in TDP-43 accumulation and function as potentially being mirror images of each other in separate body compartments, i.e. as parallel but independent events. On the other hand, might changes in platelet TDP-43 be directly connected to TDP-43 protein dyshomeostasis and/or pathology in the nervous system?

We previously described evidence of platelet activation in the ALS choroid plexus blood-CSF barrier54. These included apparent disruptions to epithelial tight junctions and vascular integrity. Changes were also noted in the choroid plexus per se, with losses in pericyte markers and increases in macrophage infiltration. Others have also observed that the blood-spinal cord barrier is damaged in ALS patients55. Moreover, a recent literature review conducted by Kopeikina and Ponomarev56 put forward the interesting notion that platelets might enter the CNS parenchyma and interact with neuronal cells. In their own studies, Kopeikina et al. highlighted a role of platelets in the inflammation-related development of epileptic seizures57. Another paper from the same group provided evidence that platelet derived serotonin and platelet activating factor can also play key roles in the protective neuroinflammation58. These various known and proposed interactions between the blood platelets, cerebrovasculature, choroid plexus and CNS could provide a means for CNS-associated proteoforms of TDP-43 to contact or enter platelets or activate cell surface receptors on platelets to induce TDP-43 post-translational modifications similar to those occurring in the CNS. Moreover, ALS-associated effects on platelets could drive (further) changes to the choroid plexus and its associated vasculature and immune components.

It is also intriguing to consider whether TDP-43 dysfunction contributes to primary microvessel-related pathways to neurodegeneration. Perturbations in the regulation of platelet aggregation per se would likely lead to a pathological change in hemostasis. Moreover, this could be further compounded by a potential capillary endothelium-related etiology. Interestingly, VEGFA mRNA, whose protein product is both a neurotropic factor and a regulator of angiogenesis, is one of the first elucidated TDP-43 targets59. In addition, vascular accumulation and aggregation of TDP-43 is seen in ALS spinal cord and FTLD frontal cortex60. Intriguingly, changes in serotonin levels and platelet morphometric and mechanical properties have also been observed previously in ALS61,62.

An increasing number of studies have suggested that platelets be considered as a potential peripheral model to study the dyshomeostasis of neurodegeneration-associated proteins and their downstream consequences. These include the metabolism of amyloid precursor protein (APP) and the pathophysiology of the onset of Alzheimer’s disease63, the accumulation of alpha-synuclein in Parkinson’s disease6, and the differential accumulation of phosphorylated TDP-4336. We are undertaking further studies of platelets to elucidate their potential as a biomarker for TDP-43 proteinopathies.

The variable levels of TDP-43 reported in biobanked plasma samples that may be prepared using different protocols highlights an important issue that requires further consideration. Although blood comprises a readily accessible tissue for biomarker development, the disposition of neurodegeneration-related proteins across blood subcompartments is not uniformly taken into account. Our data highlight the importance of making these considerations a uniform step in sample processing and assay development. There is not likely to be a simple and uniform solution to this important issue, however, because different proteins are almost certainly distributed differentially in blood components. For TDP-43, the collection of PRP or serum would be the simplest biobanking approaches, although processing of PRP into platelet cytosol may be required to detect specific TDP-43 proteoforms, due to their lower abundance compared to total TDP-43.

In summary, we report that TDP-43 is highly enriched in platelets and therefore represents a significant reservoir of TDP-43 in the bloodstream. This highlights interesting and potentially important facets of TDP-43 biology and biochemistry that may help clarify its relationship(s) to neurodegeneration. The present findings also have implications for more carefully defining how blood samples are processed for biomarker assay development. Further work is being undertaken by our team to create protocols and assays that may assist the diagnosis and monitoring of TDP-43 proteinopathies.

More broadly, our study highlights new and intriguing commonalities between platelets and synapses that reinforce the possibility of using platelets as a peripheral model to study neurodegenerative diseases. Platelets are readily accessible for sampling, making them a valuable resource for biomarker discovery, investigation of disease mechanisms and potentially monitoring impact of drug treatments.

Methods

Antibodies

Anti-phospho (S409/S410)TDP-43 (#8007-1-RR), and pan anti-TDP-43 (# 10782-2-AP) antibodies were obtained from Proteintech. An additional anti-TDP-43 antibody (#MAB77782-100) was obtained from R&D Systems. A sulfo-tag conjugated anti-mouse secondary antibody (#R32AC-1) was obtained from MSD. Novel mouse monoclonal antibodies against human TDP-43 were generated by AC Immune. The SIMOA assay reported here utilized ACI-mAb-1 that binds to the RRM2 domain (Suppl. Fig. 7) and ACI-mAb-2 that binds to a C-terminal epitope.

Ethics

Research involving human research participants was performed in accordance with the Declaration of Helsinki and all relevant guidelines and regulations as described below. Informed consent was obtained from all participants and/or their legal guardians. Blood samples for CEI assays were collected under the Kansas City University Institutional Review Board (IRB)-approved protocol 299644 (Agbas). Blood samples for MSD assays were collected from healthy donors (n = 8) with informed consent and approved by the St. Joseph’s Hospital and Medical Center IRB (#PHX-21-500-101-70-09). Blood was drawn into acid citrate dextrose anticoagulant tubes and processed for the isolation of platelets within two h of collection. For SIMOA assays, whole blood samples were obtained from the Interregional Blood Transfusion Service of the Swiss Red Cross (Lausanne, Switzerland). Blood was drawn into K+/EDTA coated tubes and processed for isolation of the different blood fractions within four h post collection. Plasma samples for SIMOA and IP experiments were obtained from the Northeast ALS Sample Repository (https://neals.org/als-researchers/neals-sample-repository). Platelet samples for RNA sequencing analysis were collected from human platelet concentrates of 5 healthy donors with informed consent and approved by “Dipartimento di Medicina Trasfusionale Giuliano-Isontina, Azienda Sanitaria Universitaria Giuliano Isontina (ASUGI), Trieste, Italy”. Human post-mortem motor cortex (mCTX) tissues from ALS and control cases were obtained from the Massachusetts ADRC with approval from the Mass General Brigham IRB. Post-mortem consent was obtained from the appropriate representative (next of kin or health care proxy) prior to autopsy. Anonymized details regarding the cases are provided in Suppl. Table 1.

Platelet cytosol preparation

8–9 ml of blood was collected in an ACD-containing vacutainer (yellow-top) tube and centrifuged at 200 × g for 20 min at room temperature (RT). The supernatant (PRP) was collected carefully by pipetting down to 0.5 cm above the buffy coat. The platelet activating factor inhibitor PGI2 was added to the PRP at a concentration of 1 µg/ml, mixed gently, and subjected to centrifugation at 1200 × g for 15 min at RT. The supernatant (PPP) was collected in a separate tube, aliquoted, and stored at − 80 °C for future use. The platelet pellet was gently washed without creating foam with 1 ml of citrate wash buffer (11 mM glucose, 128 mM NaCl, 4.3 mM NaH2PO4, 7.5 mM Na2HPO4, 4.8 mM Na-Citrate, 2.4 mM citric acid, pH 6.5, warmed to RT prior to use). The platelet suspension was subsequently centrifuged at 1200 × g for 15 min at RT. The supernatant was discarded and the platelet pellet was resuspended in 0.6 ml of rupture buffer (250 mM sucrose, 1 mM EDTA, 10 mM Tris–HCl, pH 7.4) including protease (Calbiochem Cocktail Set III) and phosphatase (Calbiochem Cocktail Set II) inhibitors. The platelet suspension was incubated in an ice-bath for 20 min and sonicated for 20 s at setting 3 in continuous mode without causing frothing. The platelet lysate was subjected to centrifugation at 20,000 × g for 30 min at 4 °C to remove membranous debris. The clear platelet cytosol fraction was aliquoted and stored at − 80 °C. In some experiments, the platelet membrane fraction was also saved for further analysis. The BCA assay was used for total protein quantification. Slight variations to this protocol were made for SIMOA experiments (see subsequent methods section).

MSD

The capture antibody comprised a polyclonal mouse anti-TDP43 antibody (Proteintech, # 10782-2-AP). The antibody was diluted in 1X PBS to a final concentration of 1.5 µg/ml. Microtiter plate wells were coated with the diluted capture antibody and incubated overnight at 4 °C with no shaking. After incubation, the wells were washed with wash buffer (1X PBS containing 0.5% Tween-20) to remove unbound capture antibody. The coated microtiter plate wells were blocked with blocking buffer (1% BSA, 1% casein TBS) at 4 °C overnight without shaking, to minimize non-specific binding. The wells were washed and the plasma fractions were combined 1:4 with sample diluent (nVector) and added to the wells in triplicate. The sealed plates were incubated at 4 °C overnight on a plate shaker. The wells were washed to remove unbound analyte and other components. An anti-TDP43 mouse monoclonal detection antibody (mAb) (R&D Systems, Cat #MAB77782-100) was incubated with the washed plates at 37 °C for 2 h on a shaker. The plates were then washed and incubated with MSD SULFO-TAG™ labeled anti-mouse antibody (1:1000) diluted in 1% casein TBS (0.5 µg/ml sulfo-tag in 1% Casein/TBS) at 37 °C for 1 h with shaking. The microplates were washed and incubated with MSD Read Buffer A (MSD, Cat #R92TG-2) for 5 min at room temperature. The resulting signal was measured using a MESO QuickPlex SQ 120 instrument. A standard curve was generated by plotting the signal intensities of the known concentrations of recombinant TDP-43 protein (Origene, Cat #TP710010). The concentrations of TDP-43 in the samples were interpolated from the standard curve using the corresponding signal intensities. Further description of assay development and analytical validation will be provided in a separate publication.

SIMOA

For SIMOA experiments on fresh blood plasma fractions, 9 ml of blood was collected in a K+/EDTA sample tube and centrifuged for 10 min at 200 × g. The supernatant (PRP) was carefully collected without disturbing the buffy coat interface, and an aliquot of PRP was collected for analysis. The remainder of the PRP was subsequently centrifuged at 5000 × g for 20 min, and the supernatant was collected carefully so as not to disturb the platelet pellet. An aliquot of this supernatant (PPP) was also saved for analysis. The platelet pellet was then resuspended in RIPA buffer and sonicated five times, 10 s each, on ice. A final centrifugation was performed at 20,000 × g for 10 min at 4 °C, after which the supernatant (PC) was separated from the platelet membrane pellet.

Assay development was conducted with blood plasma prepared by centrifugation of whole blood (collected in K+/EDTA tubes) at 1750 × g for 15 min. A C-terminal TDP-43 assay was developed using the three-step HomeBrew protocol as described in the Quanterix manual. An antibody targeting the RRM2 region (ACI-mAb 1) was conjugated to Simoa Singleplex beads (Quanterix, Cat #103612) for analyte capture; a biotinylated C-terminal antibody (ACI-mAb 2) was used for detection. Bead conjugation and biotinylation were performed according to Quanterix HomeBrew guidelines. Samples were incubated for 30 min with the capture beads, 10 min with the detection antibody and 10 min with streptavidin ß‐galactosidase (SBG, Quanterix, Cat #103397). All incubation steps were performed at 25 °C, in the Simoa® Microplate Shaker (Quanterix) at 800 rpm; the plate was washed in-between the incubations and read with the SR-X instrument (Quanterix). On the day of the assay, a freshly thawed aliquot of the recombinant human TDP-43 was used to prepare the calibrator curve and quality controls. Calibrators, controls, samples and detection antibody were diluted in PBS (D1408, Sigma, Cat #D1408) supplemented with NaCl 300 mM (Sigma, Cat #S7653), CHAPS 0.6% (Sigma, Cat #C3023), BSA 2% (Sigma, Cat #A3294) and 50 µg/ml of an heterophilic blocker (Tru BLOCK ULTRA, MilanAnalytica, Cat #8000). SBG was diluted in its corresponding diluent (Quanterix, Cat #100376). The calibration curve was obtained by plotting the Digital Average Enzyme per Bead (AEBDigital) against the logarithm of calibrator concentrations using the software GraphPad Prism (version 8.4.3). AEBDigital was calculated according to the fraction of active beads measured by the SR-X, using to the formula: − ln[1 − fon]62. Samples were quantified using a Four Parameter Logistics (4-PL) fit of calibrators, with a 1/Y2 weighting. All measurements were performed in duplicate.

CEI

The assay was conducted according to the manufacturer’s protocol with some modifications. The detailed step-by-step protocol is published in65.

Primary antibodies, TDP-43 pan antibody and p(S409/410) TDP-43 antibody were titrated for achieving antibody saturation point where the signal-to-noise ratio was high. In this setting, 1/200 and 1/10 titration was used for anti-TDP-43 pan (Proteintech #10782-2-AP) and anti-p(S409/410)TDP-43 (Proteintech #80007-1-RR) antibodies, respectively. Platelet cytosol protein concentration was determined as 0.6 mg/ml where the highest signal-to-noise ratio was achieved. Anti-14-3-3-gamma antibody (1/10 titration) (Novus Bio #NBP22-27202) was included in assay system as protein loading control66. The assay procedure was fully automated and assay results were analyzed by Compass software (Compass for SW, v.6.1.0).

IP of TDP-43 from human ALS plasma

Recombinant AC Immune anti-TDP-43 or control IgG2a monoclonal antibodies were coupled to M-270 Epoxy Dynabeads® (Invitrogen) with rotation for 24 h at 4 °C. After antibody coupling, the supernatant was removed using the magnet and Dynabeads® were washed in 1 ml phosphate buffered saline (PBS) containing 0.6% (v/v) CHAPS detergent (Sigma-Aldrich). 0.6% CHAPS was then added to washed, pooled human ALS plasma and incubated with anti-TDP-43-conjugated (or control) Dynabeads® with rotation for 1 h at 4 °C. The Dynabeads® were captured using a magnetic stand and the plasma supernatant was removed and retained to be used in a second IP. The conjugated Dynabeads® were then removed from the magnet, resuspended in 100 μl PBS + 0.6% CHAPS and transferred to a fresh tube. The Dynabeads® were magnetically re-captured and the supernatant was removed. The beads were subsequently washed 3 times with 1 ml PBS + 0.6% CHAPS in a similar manner. The washed beads were then resuspended in elution buffer followed by removal of the eluate, from which proteins were precipitated by addition of cold ethanol. The supernatant reserved from the first IP was then sequentially re-immunoprecipitated in the same manner using a second and third batch of mAb-conjugated beads, from which the subsequent TDP-43-enriched eluate was also ethanol precipitated. The eluted, precipitated protein pellets were resuspended in Laemmli buffer and pooled. 1/10th of the total IP eluate was separated by SDS-PAGE in a 4–12% Bis–Tris gel alongside the control IP sample and transferred to a nitrocellulose membrane. Immunoblotting was then used to verify that the TDP-43-positive bands at 43 kDa and 47 kDa were clearly distinguishable from the heavy and light chain IgG species (revealed by prior staining of the nitrocellulose membrane for total protein using REVERT™ 700 stain (LI-COR Biosciences)). A C-term SIMOA assay (Fig. 1) of proteins collected from the final IP supernatant showed that the serial precipitation procedure captured > 97% of endogenous plasma TDP-43 (Suppl. Fig. 3). The remainder of the TDP-43 immunoprecipitate was separated by SDS-PAGE in a 4–12% Bis–Tris gel and subsequently stained with Coomassie Blue 250. 13 gel samples were excised from the TDP-43 IP lane corresponding to potential protein bands with apparent MW’s of ~ 47 to ~ 15 k Da and sent for LC–MS/MS analyses (see below). Only the ~ 47 kDa and ~ 43 kDa bands yielded detectable TDP-43-derived peptides, however.

LC–MS/MS analyses of TDP-43-derived peptides

Gel bands appearing selectively in the anti-TDP-43 IP samples were reduced, alkylated with iodoacetamide, and digested with trypsin (Fig. 3B) or trypsin and chymotrypsin (Fig. 3C) in situ, as described previously67. Peptides were then subjected to microcapillary liquid chromatography tandem mass spectrometry (LC–MS/MS) on an Orbitrap Eclipse mass spectrometer (Thermo Scientific). Full MS spectra were acquired using the Orbitrap analyzer and MS/MS (MS2) spectra were acquired after Collision-induced dissociation fragmentation using the linear ion trap. Data were acquired in a data-dependent mode using the full MS spectrum as a master scan based on which the most intense peptide ions were selected for MS2. MS2 spectra were assigned using a SEQUEST68 proteomics analysis platform requiring cysteine residues to be carbamidomethylated (mass increment of 57.02146 Da), while methionine residues were allowed to be oxidized (15.99492 Da). Peptide assignments were made based on human protein sequences in the Uniprot database (downloaded on 03/27/2020) and expected experimental contaminants such as trypsin fragments. Based on the target-decoy database search strategy69 and employing linear discriminant analysis and posterior error histogram sorting, peptide and protein assignments were filtered to a false discovery rate (FDR) of ˂ 1%70. The identified fragments are illustrated in Fig. 3B,C. The double digestion resulted in a higher peptide sequence coverage of approximately 20% and included a higher proportion of N-terminal sequences. Identification of C-terminal peptides was not achieved. Given that we used a C-terminally-targeted mAb in our immunopurification strategy, we do not attribute this to the immunopurification per se.

To further investigate potential biases in our LC–MS/MS procedure, we immunopurified TDP-43 from human SH-SY5Y cells using the same mAbs and subjected the resultant protein bands to the same analysis. In contrast to the analyses from plasma, the cell line experiment yielded outstanding (97%) peptide coverage of TDP-43, albeit still without the identification of TDP-43 PTMs (Fig. 3D). This indicates that (a) only a small proportion of TDP-43 may be post-translationally modified and (b) the recovery of sequences of the C-terminal region are achievable if using sufficient input material.

Platelet RNA extraction from human platelet concentrate

RNA extraction was performed from 10 ml of human platelet concentrate (3 × 1011 platelets obtained from 5 healthy donors). Briefly, 10 ml of concentrate was centrifuged at 1200 × g for 15 min at room temperature. The platelet pellet (~ 7.2 × 109 platelets) was then washed twice with citrate buffer (11 mM glucose, 128 mM NaCl, 4.3 mM NaH2PO4, 7.5 mM Na2HPO4, 4.8 mM sodium citrate, 2.4 mM citric acid, pH 6.5), each time centrifuged at 1200 × g for 15 min at room temperature. RNA was then extracted as follows. 1 ml of QIAzol Lysis Reagent (Qiagen) was added to each sample and left to incubate for 5 min at room temperature. Then, 200 µl of chloroform were added to the mixture. After 5 min incubation at room temperature, samples were centrifuged at 12,000 × g for 15 min at 4 °C. The supernatant was transferred into a fresh microcentrifuge tube containing 500 µl of chloroform, carefully mixed and centrifugated at 12,000 × g for 15 min at 4 °C. Then, 1 µl of 20 µg/µl glycogen (Thermo Scientific, #R0551) and 500 µl of pre-chilled isopropanol were added to the supernatant. The mixture was carefully mixed and incubated for 2 h at − 70 °C. After this, samples were thawed on ice and centrifuged at 12,000 × g for 15 min at 4 °C. The pellet was washed twice with 70%-80% EtOH, respectively, and each time centrifuged at 7500 × g for 10 min at 4 °C. Finally, RNA was resuspended in 40 µl of DNase-free water and quantified using an Eppendorf BioPhotometer D30 (Eppendorf).

Human neuroblastoma (SH-SY5Y) cell line: maintenance and siRNA treatment

Human neuroblastoma (SH-SY5Y) cell line (ECACC) was cultured up to 20 passages in Dulbecco’s modified Eagle’s medium Nutrient Mixture F-12 Ham (Sigma-Aldrich), supplemented with 15% fetal bovine serum (FBS) (Life-Technologies), 1% MEM Non-essential Amino Acid Solution (100X) (Sigma-Aldrich), and 1% Antibiotic–Antimycotic-stabilized suspension (Sigma-Aldrich) at 37 °C with humidified atmosphere of 5% CO2. RNA interference treatments of SH-SY5Y cells were performed using Lipofectamine RNAiMAX (Life-Technologies) and 80 nM siRNA concentration, according to the manufacturer’s instructions. TDP-43 (TARDBP) depletion was achieved using siRNA against the following sequence: 5’-gcaaagccaagaugagccu-3’. siRNA against firefly luciferase (siLUC) was used as a control: 5′-uaaggcuaugaagagauac-3′. Briefly, 8 × 105 cells were seeded in 6-well plates in the presence of a transfection mixture composed of: 150 μl Opti-MEM (Life-Technologies), 3 μl of 40 μM gene-specific siRNA (siTDP-43) or control siRNA (siLUC) and 9 μl of Lipofectamine RNAiMAX reagent. After 48 h, cells were collected and prepared for RNA extraction. This procedure diminished cellular TDP-43 protein levels by ~ 80% (Suppl. Fig. 6A).

Human glioblastoma/astrocytoma (U87) cell line: maintenance and siRNA treatment

The human glioblastoma/astrocytoma (U87) cell line (kindly provided by Marco Baralle, RNA biology group, ICGEB, Trieste, Italy) was cultured up to 20 passages in DMEM (1X), high glucose, GlutaMAX Supplement, pyruvate (Life-Technologies) supplemented with 10% fetal bovine serum (FBS from Life-Technologies) and 1% Antibiotic–Antimycotic-stabilized suspension (Sigma-Aldrich), at 37 °C with humidified atmosphere of 5% CO2. RNA interference treatments of U87 cells were performed using Lipofectamine RNAiMAX reagent (Life-Technologies) and 80 nM siRNA concentration, according to the manufacturer’s instructions. TDP-43 (TARDBP) depletion was achieved using siRNA against the following sequence: 5′-gcaaagccaagaugagccu-3′. siRNA against fire-fly luciferase (siLUC) was used as a control: 5′-uaaggcuaugaagagauac-3′. At day zero, 6 × 105 cells were seeded in a 60 mm tissue culture dish to reach 70–80% confluence after 24 h. Two rounds of silencing were performed at day 1 and day 2 by incubating cells with a mixture composed of: 300 μl Opti-MEM (Life-Technologies), 6 μl of 40 μM gene-specific siRNA (siTDP-43) or control siRNA (siLUC) and 18 μl of Lipofectamine RNAiMAX reagent. Cells were then collected and prepared for RNA extraction on day 3 (after 24 h from the last round of gene knockdown). This procedure diminished cellular TDP-43 protein levels by ~ 80% (Suppl. Fig. 6B).

RNA extraction and RNA-sequencing analysis

Total RNA was extracted from human cell lines (SH-SY5Y and U87) using the miRNeasy Kit (Qiagen), according to the manufacturer’s instructions. RNA extraction from platelet samples was carried out as described above. Library preparation and RNA sequencing were performed by Novogene (https://en.novogene.com/) using Illumina HiSeq NovaSeq 600 instrument, as three independent experiments. Reads were mapped to the GRChg38 human genome assembly and batch effects were removed using ComBat-Seq. Principal component analysis (PCA) and differential gene expression analysis were carried out on total count matrix using DEseq2 (v1.38.3) and vsn R packages (v.3.66.0), respectively. Low count genes (rowSums < 10) were pre-filtered out before to carrying out differential gene expression analysis. Factor levels were assigned using the “contrast” argument and independent filtering was performed by setting the “alpha” argument to 0.05. The overall distribution of differentially expressed genes (DEGs) were evaluated using the following cut-off: up-regulated genes Fold Change (FC) > 1.3 and padj < 0.05; down-regulated genes FC < 0.7 and padj < 0.05. ClusterProfiler package (v4.6.2) from R was also used for gene set enrichment (GSE) and over-representation gene ontology (GO) analysis of RNA sequencing data; ClusterProfiler uses a hypergeometric model to identify class overrepresentation and possible associations between bioinformatically curated ontology groupings and a given gene list. Graphical representation of RNA sequencing data was realized using ggplot2 R package (v3.4.2).

Western blot analysis of cell lines

Total protein samples were extracted by sonicating SH-SY5Y or U87 cells for 10 min at high power with a BioRuptor UCD-200 (Diagenode) in a mild lysis buffer composed of 1X Phosphate Saline Buffer (PBS) and 1X Complete Protease Inhibitor Cocktail (Roche Diagnostics). 10 μg of resultant protein extract from each sample was resuspended in 1X NuPage LDS Sample Buffer (Invitrogen) prepared with 2.5% beta-mercaptoethanol and boiled at 95 ℃ for 5 min. Samples were loaded on a precast NuPage 10% Bis–Tris gel (Invitrogen) and run with 1X NuPage MOPS SDS Running Buffer (Invitrogen). The gel was then electroblotted onto a Nitrocellulose Power Blotter Select Transfer Stack (Invitrogen) by using a Power Blotter System (Invitrogen). The membrane was developed using a Purity Western Blot Detection System (UVItec Ltd.) with the following antibodies: polyclonal rabbit anti-TDP-43 1:1000 (Proteintech) and polyclonal mouse anti-Tubulin 1:10,00071. The chemiluminescence was detected with ECL Luminata Classico Western HRP substrate (Merck Millipore) and the images were acquired and analyzed using Alliance 9.7 Western Blot Imaging System (UVItec Ltd.). Tubulin was used as total protein loading control.

POLDIP3 splicing analysis

Total RNA was extracted as described in the previous section. PCR analysis of POLDIP3 exon 3 splicing was performed using MiniAmp™ Plus Thermal Cycler (Applied Biosystem) with the following conditions: 94 °C for 2 min, 94 °C for 45 s, 50 °C for 45 s and 72 °C for 45 s for 35 cycles; and 72 °C for 10 min for the final extension. The sequence for the primers was: Forward: 5’-gcttaatgccagaccgggagttg-3’; Reverse: 5’-tcatcttcatccaggtcatataaatt-3’. The results were analyzed by capillary electrophoresis using the QIAxcel Advanced Instrument (QIAGEN).

Characterization of novel anti-TDP-43 mAbs

The ability for the antibodies to bind specifically to TDP-43 was validated in homogenates from SH-SY5Y cells treated with a control siRNA (siControl) or a siRNA targeting TDP-43 (siTDP-43). TDP-43 immunoblotting was also performed on samples from human post-mortem mCTX from control and ALS donors.

Samples were homogenized in RIPA buffer composed of 0.5% Triton X-100, 50 mM Tri-HCl, pH 7.4, 0.2% sodium dodecyl sulfate (SDS), 100 mM NaCl, 1 mM EGTA, 1 mM PMSF, 1 mM Na3VO4, and 1X Complete Protease Inhibitor Cocktail (Roche Diagnostics). Western blots were performed with 50 µg of either cell or brain homogenates were resuspended in SDS sample buffer and separated on a 4–12% Bis–Tris protein gel for 90 min at 120 V. Proteins were then transferred to a PVDF membrane in an iBlot Dry Blotting System (Thermo Fisher Scientific), and the membrane was blocked with 5% bovine serum albumin (BSA) in tris-buffered saline with Tween 20 (TBST) before immunodetection with ACI mAb-1 antibody (1:1200) and β-Actin (1:1000; Cell Signaling) overnight at 4 °C. Primary antibody incubation was followed by 4 washes in TBST before incubation with the secondary antibody (HRP-conjugated goat anti-rabbit IgG, and HRP-conjugated goat anti-mouse IgG; Jackson ImmunoResearch Laboratories) for 1 h. After four washes in TBST, proteins were visualized using the ECL detection system (Thermo Fisher Scientific).

Immunohistochemistry was performed on paraffin-embedded motor cortex sections from control and ALS brain tissues. Seven-µm-thick paraffin-embedded brain sections were immunostained for ACI mAb-1 (1:3000) using a Bond Rx autostainer (Leica Biosystems), according to the manufacturer’s instructions and as previously reported72,73. Briefly, slides were batch processed with the following settings: Bake and Dewax, immunohistochemistry protocol F 60 min, HIER 20 min with ER1. Slides were then transferred into water and dehydrated by 1-min incubation into baths of 70% ethanol, 95% ethanol, 100% ethanol, and xylene. Slides were then cover slipped using Permount Mounting Medium (Fisher Scientific) and left to dry overnight. Slides were scanned using a NanoZoomer Digital Pathology-HT scanner (C9600-12; Hamamatsu Photonics, Japan) at a magnification of 20X. Scanned slide images were visualized in NDP.view2 viewing software (Hamamatsu, Japan).

Results are presented in Suppl. Fig. 8.

Platelet immunocytochemistry and super-resolution microscopy

The freshly isolated platelet pellet (as above) was dissolved in Tyrode’s buffer (NaCl 134 mM, KCl 2.9 mM, MgCl2 1 mM, HEPES 10 mM, Glucose 5 mM, NaHCO3 12 mM, Na2HPO4 0.34 mM, BSA 0.3% w/v, pH 7.4) and the samples were prepared as previously described74. 50µL of platelets were incubated at 37 °C for 3 h on coverslip treated with 1:5 diluted Poly-L-Lysine. They were then permeabilized using 0.2% Trion x-100 for 5 min, proceeded with a blocking in 2%BSA/PBS for 25 min. Overnight incubation at 4 °C was performed with a dilution of 1:200 of the primary antibodies for Tubulin (Merck) TDP-43 (Proteintech), TDP-43 S409/410 (Cosmo Bio)75, pS369 (Eurogentec)76, and pS375 (Eurogentec)77,78. The primary antibodies were revealed by Alexa Fluor® 488/594 conjugated fluorescent secondary antibodies (Invitrogen) by incubation for 1 h at room temperature in dark. The coverslips were then mounted onto slides using ProLong™ Gold Antifade reagent (Invitrogen) and were dried for 24 h at room temperature before transferring them to 4 °C. Super-resolution imaging was performed using a z-stack captured on a Zeiss lattice SIM Elyra7 structured illumination microscope, using a 63 × oil objective with default lattice width for each laser line and 13 phases of illumination. The reconstruction of the z-stacks was acquired using default parameters for the SIM module. The z projection was obtained with maximal intensity.

Supplementary Information

Supplementary Information.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-70822-8.

Acknowledgements

We gratefully acknowledge funding for this work from the Target ALS Foundation (Industry-Led Consortium Project Grant to RLC, GSV, SEA; Grant BB-2022-C5 to RLC, RB, AA, EB). Additional support has been provided by NOSRESCUEALS (AriSLA, Italy) and alsfindingacure (USA) to EB. We also acknowledge the important roles of the NEALS Biorepository, St. Joseph’s Hospital and Medical Center (IRB # PHX-21-500-101-70-09) and University of Kansas Medical Centre ALS clinic for providing biofluids from ALS and healthy control volunteers for this study (IRB #140689+,147493). Sincere thanks also go to Wilhelm Haas and the Taplin Biological Mass Spectrometry Facility at Harvard Medical School for support with proteomic analyses and Silvano Piazza and Giulia Canarutto at ICGEB for support with bioinformatics analyses. We also acknowledge Clotilde Lagier-Tourenne for helpful discussions and Florian Udry and Bojana Portmann for the critical reading of the manuscript.

Author contributions

T.A. & T.S. designed the production and characterization of novel mAbs. T.P. & G.S.V. conducted immunohistochemistry and western blot experiments to characterize novel mAbs; R.L.C., M.L.R.B. and O.A. designed and interpreted SIMOA experiments, N.T., M.L.R.B., L.F., B.A.T. & E.G. conducted SIMOA and ELISA experiments and analyzed data, G.B. isolated platelets from human blood and conducted western blot experiments, A.A. designed CEI experiments and analyzed the data; P.S. prepared the pre-assay workflow, conducted CEI assays, and participated in data analysis E.K. and J.S. isolated platelets from human blood and conducted CEI assays; E.B., S.C., H.C., C.S., R.S. conducted platelet RNA experiments and associated bioinformatic analyses; J.P.Q., N.T., B.C.C., R.L.C., S.E.A. & J.D.B. provided immunopurified TDP-43 samples and conducted proteomic experiments; R.B., J.A., V.O. & L.G. designed and conducted MSD experiments and analyzed data; R.L.C. compiled the first draft of the manuscript; all authors reviewed and contributed to the manuscript.

Data availability

The RNAseq datasets generated during and analyzed during the current study are publicly available in the Gene Expression Omnibus repository (Series record GSE245303). The remaining data analyzed during this study are included in this published article (and its Supplementary Information files). Reasonable requests for other information regarding the study will also be granted if addressed to the authors via the Corresponding Author.

Competing interests

RLC, MLRB, LF, GB, OA, TS, TA, MKV and AP are employees of AC Immune and are entitled to stock options. NT and EG are former employees of AC Immune. RB and JA have stock options in nVector, Inc., a company developing biomarker assays for neurologic diseases. EB is a member of the Scientific Advisory Board of VectorY. SEA has served on scientific advisory panels or consulted with Allyx Therapeutics, BioVie, Daewoong Pharmaceutical, EIP Pharma, Quince Therapeutics, Jocasta, NeuroSense, Risen Pharmaceutical, Sage Therapeutics, and Vandria. All other authors declare that they have no competing interests.

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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