Premature polyadenylation-mediated loss of stathmin-2 is a hallmark of TDP-43-dependent neurodegeneration

TDP-43 depletion or disease-causing mutation suppresses stathmin-2

Two independent strategies were undertaken to manipulate TDP-43 function in a human neuronal cell line: 1) depletion of TDP-43 by siRNA and 2) genome editing with CRISPR-Cas9 to introduce an ALS-causing mutation into both endogenous TDP-43 gene loci. The human neuronal cell line SH-SY5Y was chosen as a model as it has been shown to maintain a diploid karyotype and acquire substantial neuronal character when differentiated ²⁴. siRNA treatment reduced TDP-43 mRNA levels by >80% when compared to cells treated with a control siRNA (Fig. 1a). The consequences of lowering TDP-43 were then examined on a genome-wide basis. Libraries of RNAs were prepared, sequenced, and mapped to the human genome. This approach confirmed reduction of TDP-43 to 1/4^th of its initial level (Fig. 1b). Amid 299 and 219 mRNAs that were down or up regulated, respectively (with fold changes >1.5, false discovery rate [FDR] <0.05) (Supplementary Table 1), the most affected mRNA (with >85% reduction - Fig. 1b and Supplementary Fig. 1a) was the one encoding the neuronal growth-associated protein stathmin-2 ^16,18. Reduction in stathmin-2 mRNA upon TDP-43 depletion was confirmed by quantitative PCR (qPCR) (Fig. 1c) and an 8-fold reduction of the 22 kD stathmin-2 protein was identified by immunoblotting (Fig. 1d and Supplementary Fig. 1b).

Using a CRISPR-Cas9 site-selective nuclease, we next genetically engineered SH-SY5Y cells to express a familial ALS-causing mutation (asparagine substituted to serine at amino acid 352 – TDP-43^N352S) from both endogenous TDP-43 alleles (Supplementary Fig. 2a). Immunofluorescence imaging confirmed that mutant TDP-43 protein remained mostly nuclear, similar to wild type TDP-43 expressed in the original SH-SY5Y line (Fig. 1e and Supplementary Fig. 2b). Transcriptional profiling (i.e., RNA-seq) of the wild type and mutant lines revealed that introduction of the disease-causing TDP-43 mutation led to altered expression of 950 mRNAs (451 downregulated and 499 upregulated; fold change >1.5, FDR <0.05) (Supplementary Table 2), including moderate reduction in TDP-43 (Fig. 1f). In addition, previously described alternative splicing changes linked to TDP-43 loss of function were observed in TDP-43 mutant-expressing cells (Supplementary Fig. 2c), consistent with both loss of normal TDP-43 function and a gain of aberrant function, as seen previously in mice ^25–27.

Analysis of RNAs from isogenic wild type and mutant lines (using qPCR) confirmed a 1.7 fold reduction of stathmin-2 mRNA (Fig. 1g), while immunoblotting revealed more than two fold reduction of stathmin-2 protein in TDP-43^N352S mutant cells (Supplementary Fig. 2d). A search for gene expression changes that overlapped between cells with TDP-43 loss of function (Fig. 1b) and TDP-43^N352S mutant cells (Fig. 1f) identified stathmin-2 mRNA (and 18 additional RNAs) to be downregulated, together with another 23 that were upregulated (Fig. 1h).

Reduced stathmin-2 in TDP-43 mutant human neurons

We then tested whether stathmin-2 expression was reduced in human neurons directly converted from ALS patients’ fibroblasts. To this end, we used eight fibroblast lines obtained from an extended family that included four carriers heterozygous for the ALS-linked mutant TDP-43^N352S and four individuals without the mutation (Supplementary Fig. 3a and Supplementary Table 3). Overall stathmin-2 mRNA levels were already reduced (by 50%) (Supplementary Fig. 3b) in the mutant fibroblast lines relative to controls despite primarily nuclear localization of TDP-43 protein both in control and ALS patient fibroblasts (Supplementary Fig. 3c-d). Fibroblasts from all eight lines were directly induced into neurons (herein referred to as iNeurons) ²⁸ by expression of the neuronal-specific transcription activator Brn2 and reduction of the RNA-binding protein PTB (Fig. 2a).

TDP-43 was almost exclusively nuclear in wild type and mutant fibroblasts (Supplementary Fig. 3c-d). When converted into iNeurons, a higher proportion of wild type TDP-43 accumulated in the cytoplasm and this relocalization was enhanced in iNeurons expressing the N352S mutation (Fig. 2b and Supplementary Fig. 3e). Importantly, stathmin-2 mRNA was significantly downregulated (more than 3 fold) in all four sets of TDP-43^N352S iNeurons relative to the four iNeuron lines derived from fibroblasts of healthy family members (p<0.01, n=4) (Fig. 2c).

We next tested iNeurons generated from familial ALS patient fibroblasts carrying three other mutations: glycine to serine at position 298 (TDP-43^G298S), alanine to threonine at position 382 (TDP-43^A382T), and asparagine to serine at position 390 (TDP-43^N390S). Stathmin-2 mRNA was reduced (relative to iNeurons from non-ALS individuals) in all of these familial ALS iNeurons (Fig. 2c-d). These data demonstrate that, similar to reduction of TDP-43, ALS-linked mutations in TDP-43 suppress stathmin-2 expression level in human iNeurons.

TDP-43 represses premature polyadenylation in stathmin-2 pre-mRNA

The stathmin-2 gene is annotated to contain 5 constitutive exons (Refseq ID: NM_001199214.1) plus a proposed alternative exon between exons 4 and 5 whose use was undetectable (using RNA-seq) under any condition in our SH-SY5Y cells (Fig. 3a). Reduction or mutation in TDP-43, however, induced a new spliced exon, with RNA-seq reads mapping within intron one (Fig. 3a, red arrow). This new exon, herein called “exon 2a”, was absent in wild type cells, but appeared either when TDP-43 was depleted or when endogenous TDP-43 was edited to carry the N352S mutation. Prominent utilization of this new exon was observed when TDP-43 was reduced, in line with >6 fold suppression of RNAs containing exons 2 through 5 (Fig. 3a, upper panel). This was accompanied by the corresponding loss of stathmin-2 protein (Fig. 1d and Supplementary Fig. 1b).

Altered splicing and ligation of exon 1 to exon 2a in SH-SY5Y cells after TDP-43 depletion or in the presence of a TDP-43 mutation was then confirmed by RT-PCR and qPCR (Fig. 3b). However, while fusion between exon 1 and exon 2a was consistently observed (Fig. 3b), no RNAs containing exon 2a ligated to the downstream exon 2 were identified using primers targeting the flanking exons 1 and 2 (Supplementary Fig. 4a-c). To test the possibility that incorporation of exon 2a drives alternative polyadenylation within what is normally intron one (Fig. 3c), we used anchored oligo(dT) primers for reverse transcription followed by PCR and sequencing. This analysis confirmed polyadenylation of the stathmin-2 RNA containing exon 2a as the terminal exon (Fig. 3d-e).

A polyadenylation signal “AUUAAA” ²⁹ was identified 24 nucleotides upstream to the polyadenylation site (Fig. 3d). This cryptic polyadenylation sequence in the human stathmin-2 gene is conserved among most primates, but is notably absent in mouse or rat (Supplementary Fig. 4d). qPCR analysis on nascent stathmin-2 pre-mRNAs identified reduction in full-length stathmin-2 transcripts in SH-SY5Y cells expressing mutant TDP-43 (Supplementary Fig. 4e), consistent with increased levels of truncated exon 2a-containing RNAs (Fig. 3b).

The prematurely polyadenylated RNA includes 227 nucleotides originating from exon 2a (hg38; chr8:79,616,822–79,617,048) with its predicted 16 amino acid translation product initiating at the normal AUG codon in exon 1 and ending at 11 codons into exon 2a (Fig. 3c-d). Three “GUGUGU” hexamers marking potential binding sites of TDP-43 (as we and others have previously described ^8,9) were identified in a region spanning 30 nucleotides downstream of the 3’ splice site that produces exon 2a (Fig. 3d). Indeed, analysis of ultraviolet cross-linking and immuno-precipitation (iCLIP) data for TDP-43 in human SH-SY5Y cells ⁹, confirmed TDP-43 binding (shown by mapped reads) to the stathmin-2 pre-mRNA at a single region containing the three GUGUGU sequences (Fig. 3f), thus confirming physical interaction with TDP-43 protein. Altogether, we conclude that reduction in TDP-43 causes de-repression and efficient use of a cryptic polyadenylation site in what normally is intron 1, which results in truncation of the stathmin-2 pre-mRNA and suppression of functional stathmin-2 expression.

Stathmin-2 mRNA is prominently expressed in motor neurons

Comparison of published evidence of the abundance of stathmin-2 mRNA in ribosome-bound RNAs across specific cell types of the murine central nervous system ³⁰ revealed 25 and 15 fold enrichment of translated stathmin-2 mRNAs in motor neurons relative to astrocytes and oligodendrocytes, respectively (Supplementary Fig. 5a). Remarkably, further analysis of these data ³¹ identified stathmin-2 to be among the 25 most abundant actively translated mRNAs isolated from adult motor neurons in mice. Similarly, our analysis of RNA sequencing from laser capture microdissected (LCM) human lumbar spinal motor neurons from seven healthy controls ³¹ determined that in human spinal motor neurons stathmin-2 is the 20th most abundant mRNA, only slightly less abundant that the mRNAs encoding the three neurofilament subunits NF-M, NF-L and NF-H (Supplementary Fig. 5b). Further, of the four stathmin genes, only stathmin-2 was enriched within human motor neurons (Supplementary Fig. 5c).

Suppression of stathmin-2 by premature polyadenylation is a hallmark of ALS

Analysis of the RNA sequencing reads mapping to the stathmin-2 gene from lumbar motor neurons captured by laser microdissection revealed a clear segregation between controls and sporadic ALS cases. RNAs from all analyzed sporadic ALS patients (n=13) contained inclusion of exon 2a (Fig. 4a and Supplementary Fig. 6). In contrast, RNAs including exon 2a were absent in mRNA from all tested (n=7) non-ALS individuals. Importantly, just as we had determined in TDP-43 mutant expressing SH-SY5Y cells and patient-derived iNeurons (Fig. 1 and 2), overall stathmin-2 mRNA levels in sporadic ALS patients were significantly decreased relative to those in motor neurons from non-ALS individuals (Fig. 4b). By contrast, stathmin-1 mRNA expression was low in both healthy and ALS spinal motor neurons (Fig. 4b). Additionally, analysis of RNAs extracted from anterior horns of thoracic spinal cord from 13 sporadic ALS patients and three familial ALS patients carrying GGGGCC expansion in C9orf72 revealed aberrant processing of stathmin-2 mRNA in all, but not in RNAs from eight healthy individuals or three familial ALS patients with SOD1 mutations (Fig. 4c and Supplementary Fig. 7a). Consistent with the latter, mice developing fatal motor neuron disease from expressing an ALS-linked mutation in SOD1 retained normal stathmin-2 mRNA levels despite apparent motor neuron damage ³⁰, demonstrating that stathmin-2 expression is not affected by motor neuron degeneration per se (Supplementary Fig. 5a). Recognizing that TDP-43 pathology is found in sporadic and familial ALS linked to C9orf72 expansion ³² but not SOD1-mediated ALS (Supplementary Fig. 8) ³³, these results are consistent with premature polyadenylation of stathmin-2 mRNA being triggered by TDP-43 dysfunction and the corresponding suppression of stathmin-2 expression in both thoracic (Fig. 4c and Supplementary Fig. 7a) and lumbar (Fig. 4a) motor neurons.

Extension of similar analyses to the motor cortex identified altered processing of stathmin-2 mRNA to include exon 2a in 2/2 C9orf72 samples and 6/9 sporadic ALS samples (Fig. 4d). For ALS cases that were also diagnosed with frontotemporal dementia (FTD), premature polyadenylation was seen in frontal cortex-derived RNAs in 3 of 4 sporadic ALS/FTD patients, and one of one C9orf72 expansion patient (Supplementary Fig. 7b).

Lastly, chromogenic in situ hybridization (CISH) was used to test on a cell by cell basis for stable accumulation of exon 2a-containing stathmin-2 RNAs in spinal cord and motor cortex samples from five control individuals and four sporadic ALS patients (Fig. 4e-g and Supplementary Fig. 7c,d). In agreement with prior analyses of RNAs derived by laser microdissection (Fig. 4a) or from bulk tissues (Fig. 4c,d), RNA probes hybridizing to stathmin-2 exon 2a were increased in both nuclear and cytoplasmic compartments of spinal cord neurons (97% of sporadic ALS motor neurons versus 0% of control; n=69 and 41, respectively) and in neurons from layers 3 and 5 of motor cortex from all ALS-derived samples, with 100% of scored sporadic ALS cortical neurons staining positive for exon 2a (n=50) versus 0% of control cortical neurons (n=50). Low basal levels of hybridization (presumably corresponding to the newly transcribed stathmin-2 pre-mRNA) were found in tissues from non-ALS individuals, with an intensity only slightly above that of a scrambled sequence control probe.

A probe hybridizing to exon five of the stathmin-2 mRNA revealed an apparent reduction in affected regions of sporadic ALS, with only 35% of sALS lumbar motor neurons showing weakly positive staining for STMN2 exon five versus 70% of control patient motor neurons (n=65 and 61, respectively). The reduction of exon five staining was even more substantial in the motor cortex where 0% of scored neurons showed a positive signal, compared with a strong signal in 100% of non-ALS cortical neurons (Fig. 4e,f and Supplementary Fig. 7c,d).

Impaired axonal regeneration upon TDP-43 loss in human iPSC-derived motor neurons is alleviated by restoring stathmin-2

To determine the functional consequence of TDP-43-mediated reduction in stathmin-2, induced pluripotent stem cells (iPSC) were generated using a non-integrating approach, validated to retain a normal karyotype (Supplementary Fig. 9a) and expression of pluripotent stem cell markers (Supplementary Fig. 9b-d), and differentiated into motor neurons (Supplementary Fig. 9e). Motor neuron precursors (expressing Hb9) appeared within 21 days (Supplementary Fig. 9f) and by 28 days developed Map2 positive dendrites and long axons accumulating neurofilaments (Supplementary Fig. 9g-h). At day 29, motor neurons were treated with antisense oligonucleotides (ASOs) that direct catalytic degradation of TDP-43 mRNAs through the action of endogenous RNase H. TDP-43 mRNA levels were reduced in a dose-dependent manner (Fig. 5a).

ASO-induced reduction in TDP-43 mRNA (up to 55%) was consistently accompanied by a greater (up to 12 fold) dose-dependent reduction in stathmin-2 mRNAs, coupled with a corresponding increase in RNAs containing exon 1 ligated to exon 2a (Fig. 5b). Dose-dependent suppression (up to 15 fold) of stathmin-2 expression in iPSC-derived motor neurons (without affecting TDP-43 levels) was also achieved with an ASO targeted to the 3’ UTR of full length stathmin-2 mRNA (Fig. 5c). Immunoblotting confirmed that within 12 days of treatment with ASOs targeting either TDP-43 or stathmin-2 mRNAs there was nearly complete loss of the 22kD stathmin-2 protein (Fig. 5d). Motor neuronal viability was not affected under these conditions (Supplementary Fig. 9i).

To assess the functional consequences of stathmin-2 depletion in the axonal regeneration process (Fig. 5e), motor neuron precursors were seeded into the proximal somatic compartment of a microfluidic device. Over a nine days maturation period, axons extended through microgroove-embedded channels (830 μm in length) that exclude neuronal cell bodies but allow axonal extension into the distal compartment. Matured motor neurons were treated for 20 days with ASOs added to the somatic compartment to reduce synthesis and accumulation of either TDP-43 or stathmin-2 (Fig. 5f). Axons were maintained after loss of either protein. After axons in the distal compartment were mechanically axotomized, axonal regrowth of motor neurons treated with non-targeting, control ASOs initiated within 24 hours (visualized by NF-H immunostaining in red - Fig. 5g-h). Stathmin-2 appeared in a punctate pattern along these regenerating axons and accumulated in the growth cones (Fig. 5g), consistent with a role in promoting axonal regrowth. Indeed, following ASO-mediated reduction in stathmin-2 (Fig. 5i,j) or TDP-43 (Fig. 5k,l), axonal regeneration after axotomy was almost completely suppressed, with 10% (Fig. 5j) and 13% (Fig. 5l) recovery rates relative to those treated with control ASOs, respectively. Together, these data demonstrate increased vulnerability of motor neurons upon reduced accumulation of the neuronal-growth factor stathmin-2.

We next tested if the failure to regenerate after axotomy of iPSC-derived human motor neurons following depletion of either stathmin-2 or TDP-43 could be rescued by restoration of stathmin-2 expression. To do this, after a 16 day ASO-mediated suppression of stathmin-2 or TDP-43, iPSC-derived motor neurons were transduced with a lentivirus carrying a stathmin-2-encoding gene whose RNA did not contain the cryptic polyadenylation site found in the endogenously encoded stathmin-2 gene. Despite a sustained reduction in stathmin-2, subsequent restoration of stathmin-2 expression almost completely rescued regeneration after axotomy (Fig. 5m,n). More remarkably, although TDP-43 affects the levels or splicing of many RNAs (Fig. 1b and ref. ^8,9), restoration of stathmin-2 alone was sufficient to rescue regeneration after axotomy of TDP-43 depleted motor neurons (Fig. 5o,p).

RNA extraction and cDNA synthesis

For RNA extraction from cultured cells and neurons, direct lysis was applied using Trizol reagent (Invitrogen) and RNA was purified according to the manufacturer’s instructions. Human tissue samples were first homogenized in Trizol, using a mechanical tissue homogenizer, and RNA was extracted accordingly.

RT-PCR and real-time PCR

For cDNA synthesis, 1 μg total RNA was reverse transcribed using the high-capacity reverse transcription kit (ABI) or superscript III (Invitrogen) according to the manufactures’ instructions. RT-PCR reactions were performed using Q5 High-Fidelity DNA polymerase (NEB) in a T100 thermocycler PCR machine (Bio-Rad). For splicing analyses, RT-PCR products were separated on 2% polyacrylamide gels and then incubated with SYBR gold (Invitrogen) for imaging and analysis. Quantification of alternative splicing band intensities were determined using ImageJ software and an average of 3 biological replicates was plotted. Quantitative real-time PCR was carried out in triplicates, using iTaq Universal SYBR green (Bio-Rad) in a CFX384 real-time PCR machine. mRNA expression of transferrin receptor protein-1 (TFRC) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as endogenous control genes, as indicated. All primers are listed in Supplementary Table 4.

RNA sequencing

Total RNA was extracted from SH-SY5Y cells as indicated above. Subsequently, 1 μg of total RNA was used for mRNA libraries preparation using the TruSeq RNA kit (Illumina). For the TDP-43 knockdown experiment, 3 biological replicates were sequenced per each condition (siControl/siTDP-43). For TDP-43 genome-editing experiments, 2 biological replicates were sequenced per each condition (TDP-43^WT/WT, TDP-43^N352S/N352S). Real-time qPCR validations were done with at least 3 replicates. RNA sequencing was carried out on an Illumina HiSeq 4000 platform with a median of 46M reads per sample. 50bp single-end FASTQ files were obtained using the Illumina demultiplexing pipeline. STAR ⁵⁴ and RSEM ⁵⁵ were used to align the reads to the human reference sequence HG38 and to calculate the raw counts and transcripts per million (TPM) values for genes, respectively. Genes differentially expressed between sample groups were identified by DESeq2 ⁵¹.

Genome editing

Both TDP-43 alleles were genetically modified in SH-SY5Y cells. A single guided RNA targeting TDP-43 was designed (Benchlin webtool) and cloned into pSpCas9–2A-GFP plasmid ⁵⁶ (px458-Addgene) using the BbSI restriction site. The sequence for guided RNA targeting TDP-43 is: GCGGGTAATAACCAAAACCA. To promote homologous recombination, pSpCas9–2A-GFP-gRNA plasmid was electroporated using the Amaxa Nucleofactor (assay A-023) along with a 180 long single-stranded donor oligonucleotides (IDT), containing the desired ALS-causing mutation and four synonymous single-nucleotide replacements to avoid DNA cleavage recurrence by Cas9. 48 hours following electroporation, cells were collected and dissociated using Accutase. GFP-positive cells were sorted and single-cell seeded into 96 wells plates using the SH800S Sony cell sorter. Individual clones were expanded and DNA was extracted for PCR amplification of TDP-43 genomic locus. Replacement of AAT to AGT (c.1055A>G) was confirmed by Sanger sequencing. The entire TDP-43 coding region and all exon-intron junctions (> 500 base-pairs length of each intronic region) were then sequenced to verify the absence of any additional DNA alteration. qPCR analysis was performed with a wild type isogenic SH-SY5Y cell line that had undergone the mutagenesis attempt but not acquired the TDP-43 mutation during the original screening progress.

Immunoblotting

Total-cells extracts were collected in radioimmunoprecipitation lysis buffer (RIPA buffer). Proteins concentrations were determined by Bradford assay (Bio-Rad) and equal amounts of total protein were boiled in SDS sample buffer for 10 minutes before running in 10% acrylamide gel. Proteins were transferred to PVDF, and blocked in 5% milk solution in tris-buffered saline and 0.1% Tween-20 (TBST) for 1 hour before overnight incubation with the following primary antibodies: anti-TDP-43 (1:1000) (ProteinTech, 10782–2-AP), anti-Stathmin-2 (1:2000) (nbp1–49461, NovusBiologicals), anti-Tubulin (1:10,000) (DM1A, Abcam). Immunoblots were washed in TBST and probed with HRP-conjugated secondary antibodies diluted 1:5000 for one hour in room temperature (GE Healthcare), before being exposed to films.

Immunofluorescence and image acquisition

SH-SY5Y cells were grown on poly-D-lysine (Sigma) coated 4-wells glass bottom chambers (Thermo). iNeurons were grown on matrigel coated (Corning, 356230) 4-wells glass bottom chambers (Thermo). At end point, cells were washed one time with phosphate buffer (PBS) and fixed by 4% paraformaldehyde in phosphate buffer (PBS) for 30 minutes at room temperature. Two washes with PBS were followed by cell membrane permeabilization using 0.1% Triton X-100 (Sigma) for 15 minutes at room temperature. After one wash with PBS, cells were incubated for 30 minutes with blocking solution containing 3% bovine serum albumin (Sigma) + 3% donkey serum (Jackson ImmunoResearch) in PBS. Blocking solution was removed and replaced with primary TDP-43 antibody (Proteintech # 10782–2-AP) diluted 1:1000 in fresh blocking solution for overnight incubation at 4°C. Following overnight incubation and 3 washes with PBS, cells were incubated with fluorescently tagged secondary antibody conjugated to Alexa-488 (Thermo Fisher Scientific) diluted 1:500 in blocking solution. Finally, cells were washed 3 times in PBS, the second wash included 10 minutes incubation with DAPI solution (Thermo Fisher Scientific, 100ng/ml) for nuclear staining. The preparation was mounted with ProLong Gold antifade reagent (Thermo Fisher Scientific). Fluorescence images were acquired with Olympus FV1000 confocal microscope, at the microscopy core, the Ludwig institute, UCSD.

Fluorescence quantification from micrographs

Fluorescence quantifications were performed using FIJI image processing software (http://imagej.net). Freehand tool of the software was used to select the subcellular regions such as nuclei or cytoplasm for quantification. Data were plotted in GraphPad Prism software for illustration.

In Situ Hybridization of STMN2 RNA isoforms

Short 5’-Digoxigenin-labeled Locked Nucleic Acid (LNA) probes were designed by QIAGEN (Truncated Exon2A STMN2 RNA isoform probe sequence: TCACACAGAGAGCCAAATTCTT; Normal STMN2 mature mRNA probe sequence to the 3’UTR of STMN2: ATCCTGATATCGCATGATCCAT). For each tissue two serial FFPE tissue sections (7μm) were cut onto charged glass slides. Each of the two sections was hybridized with one of the STMN2 probes using standard ISH protocols and revealed with a commercial anti-DIG antibody and NBT developer kit. Slides were inspected under brightfield microscopy and serial sections of individual neurons were identified and imaged at 40x magnification on a Keyence BZ-X700 fluorescent microscope. For quantification, spinal motor neurons were identified by their large cytoplasm and/or nucleus, the presence of lipofuscin, and their position within Rexed lamina IX of the spinal cord. Neurons of the motor cortex were identified by their shape and relative size and position within cortical layering, with special attention to layers 3 and 5.

Immunohistochemistry for TDP-43 and phosphorylated TDP-43

Formalin-fixed paraffin-embedded sections with 6 µm thickness were deparaffinized with Citrisolv (Decon Lab #1601H) and hydrated with different dilutions of alcohol. Endogenous peroxidase activity was quenched with 0.06% H₂O₂ in methanol for 15 min. Antigen retrieval was performed in a high pH solution (Vector #H-3301) in a pressure cooker at 125 °C for 20 min. Sections were blocked with 2% FBS (Gibco #10438–026) and 0.2% Triton X-100 (Sigma #T8787) in PBS for 60 min. Following antigen retrieval and blocking, the sections were incubated with primary antibodies at 4 °C overnight as follows: TDP-43 (anti-Rabbit, Proteintech #10782–2-AP, 1:5,000) and phosphorylated TDP-43 (pTDP-43) (pS409/410, anti-Rabbit, Cosmobio #TIP-PTD-P02, 1:1,000). The secondary antibody (ImmPRESS HRP Reagent Kit, anti-Rabbit IgG, Vector #MP-7401) was incubated at room temperature for 60 min, and signals were detected using NovaRED peroxidase substrate kit (Vector #SK-4800) for 2 minutes. Counterstaining was performed with hematoxylin (Ricca chemical #3537–32). TDP-43 and pTDP-43 immunohistochemistry was performed using consecutive sections for each patient.

Cell culture and transfection