A neurotoxic cryptic peptide arising from TDP-43-dependent cryptic splicing of PKN1

Domain: chemistry
Published: 2026-02-20
Editorial outcome: major_revision

Abstract
Dysfunction of transactive response DNA-binding protein 43 (TDP-43) drives neurodegeneration in amyotrophic lateral sclerosis (ALS) and Alzheimer’s disease (AD), in part through inducing aberrant RNA splicing. However, whether such mis-splicing yields stable, pathogenic proteins remains unclear. Here, we identify a TDP-43–repressed cryptic exon in Protein kinase N1 (PKN1), designated PKN1-5a1, which is activated in ALS patient brains and introduces a premature termination codon. This aberrant transcript escapes nonsense-mediated decay and is translated into a truncated peptide, PKN1-N207 (PKN207), detectable in AD brains with TDP-43 pathology. In mice, PKN207 impairs cognition, memory, and synaptic plasticity. Our findings demonstrate that TDP-43 loss–induced cryptic splicing can generate stable neurotoxic polypeptides, revealing a peptide-mediated mechanism in TDP-43 proteinopathies.

Introduction
Transactive response DNA-binding protein 43 (TDP-43) is a conserved RNA-binding protein that plays a key role in maintaining RNA splicing fidelity in the nervous system1. By recognizing UG-rich sequences in pre-mRNA, TDP-43 effectively suppresses aberrant insertion of unannotated cryptic exons, thereby ensuring normal expression of key neuronal genes2,3. Loss of TDP-43 function is widely recognized as a core pathological feature of amyotrophic lateral sclerosis (ALS)4, and is also commonly observed in frontotemporal dementia (FTD) and Alzheimer’s disease (AD). In FTD, TDP-43 pathology accounts for approximately 40–50% of cases, characterized by neuronal nuclear inclusions, cytoplasmic deposition, and concomitant gliopathy in the frontal and temporal lobes, and is closely associated with distinct clinical subtypes5,6. In AD, abnormal TDP-43 deposition is detected in about 50–70% of patients (with proportions varying across cohorts)1,4,7,8. Notably, limbic-predominant age-related TDP-43 encephalopathy (LATE), a special subtype of AD, is strongly linked to cognitive impairment in the elderly8–10.

In recent years, studies have gradually elucidated how loss of TDP-43 function disrupts neuronal gene expression through cryptic splicing and thereby mediates dysfunction1. For example, in STMN2 (Stathmin 2), TDP-43 loss activates an intronic aberrant event and introduces a premature polyadenylation site, causing transcription to terminate prematurely near exon 2 and preventing the production of functional STMN2 protein11,12; in UNC13A (unc-13 homolog A), TDP-43 loss induces insertion of a cryptic exon carrying a premature termination codon (PTC), triggering nonsense-mediated mRNA decay (NMD) and markedly downregulating functional UNC13A protein13,14. These mechanisms directly point to impairments in core processes such as axon growth and synaptic release, and are considered to constitute an important molecular basis of TDP-43 proteinopathies11,12,15,16. In addition, because of their specificity in ALS and early activation, cryptic splicing events are becoming potential biomarkers for disease subtyping and diagnosis17–21.

Notably, TDP-43–dependent cryptic splicing does not end solely in “loss of function”. Some events maintain open reading frame (ORF) continuity or retain key domains, thereby generating de novo proteins that participate in pathological processes. In the reported HDGFL2 (hepatoma-derived growth factor-related protein 2) event, the corresponding product can be detected in brain and cerebrospinal fluid (CSF) of ALS/FTD patients22; cryptic splicing of MYO18A (myosin XVIIIA) likewise produces an unannotated protein that retains functional domains and is hypothesized to be aggregation-prone23. These findings suggest that certain cryptic splicing events may exacerbate the progression of TDP-43 proteinopathies through gain-of-toxicity pathways.

Protein kinase N1 (PKN1) is a serine/threonine kinase involved in multiple processes, including cytoskeletal dynamics, axonal transport, intracellular signal transduction, and synaptic plasticity24,25. PKN1 can bind to neurofilament light chain (NEFL) and phosphorylate its head domain, thereby regulating neurofilament assembly and axonal structural stability26. Its activity is regulated by Rho-family small GTPase Rac1 and phosphoinositide-dependent protein kinase 1 (PDK1), and undergoes translocation or activation under hyperosmotic/stress conditions27. Under apoptotic stimulation, caspase-3 cleaves PKN1 to form a truncated species that is thought to have kinase activity distinct from the full-length conformation and potential toxic effects28.

In this work, we show that TDP-43 loss induces a cryptic splicing event in the PKN1 gene, designated PKN1-5a1, which inserts a 127-bp exon and introduces PTCs. This aberrant transcript partially escapes NMD and is translated into a stable truncated peptide, PKN1-N207 (PKN207). Using a specifically developed antibody, we detect PKN207 in AD brains with TDP-43 pathology. In vivo studies demonstrate that PKN207 expression impairs cognitive function and synaptic plasticity in mice. Proteomic profiling further reveals distinct alterations in neuronal pathways induced by PKN207. Our findings indicate that TDP-43 dysfunction not only causes loss-of-function effects but can also generate stable, neurotoxic polypeptides, revealing a peptide-level pathological mechanism in TDP-43 proteinopathies.

Results
TDP-43 knockdown induces PKN1 cryptic splicing and disrupts its protein expression

TDP-43 plays a pivotal role in maintaining RNA splicing fidelity, primarily by suppressing the aberrant insertion of cryptic exons. Loss of TDP-43 function leads to abnormal cryptic splicing in multiple crucial neuronal genes, resulting in a marked reduction of functional protein expression. Among these, STMN2 and UNC13A represent the most extensively studied targets, and their splicing dysregulation has been established as a key molecular event in neurodegenerative diseases1,11–14,29. Furthermore, other genes including PFKP (phosphofructokinase, Platelet), HDGFL2, and GAP43 (growth associated protein 43) have also been reported to be regulated through this mechanism. Notably, the aberrant splicing of STMN2 and GAP43 has been directly validated to impair axonal regeneration, linking these molecular defects to specific pathological phenotypes1,15,21,22,29. To discover additional TDP-43–regulated cryptic splicing events, we knocked down TDP-43 in neuroblastoma M17 cells and performed RNA-seq to systematically identify potential targets. Integrative Genomics Viewer (IGV) visualization showed that several known cryptic exon events, such as STMN2, PFKP, and HDGFL2, were all repeatedly detected under TDP-43 knockdown1,11,12,15, verifying the reliability of the sequencing data and the sensitivity of splicing-event detection (Supplementary Fig. 1a).

On this basis, we identified an aberrant splicing region between exons 4 and 5 of the PKN1 gene, in which two distinct splicing forms were detected: one involves insertion of a previously unannotated exon (127 bp), designated PKN1-5a1, with genomic coordinates hg38: chr19:14,450,088–14,450,214; the other is an alternative 3’ splice site (A3SS) of 230 bp with coordinates hg38: chr19:14,450,088–14,450,317, designated PKN1-A3SS (Fig. 1a). Among these, the PKN1-A3SS splicing form was previously reported in TDP-43–knockdown HeLa cells30; here, we characterize the PKN1-5a1 exon, a previously unannotated cryptic exon. We define its precise splice boundaries and sequence features, and elucidate its biological significance. Further analysis showed that the two splice sites of PKN1-5a1 are the canonical splice donor “GU” and acceptor “AG” (Fig. 1a), consistent with classical alternative splicing sequence features31; meanwhile, PKN1-5a1 contains multiple stop codons (Supplementary Fig. 1b). We calculated the PSI (percent-spliced-in) value of this region based on RNA-seq data and found that TDP-43 knockdown primarily activates insertion of the 5a1 exon, whereas the A3SS insertion event is relatively infrequent (Fig. 1b), suggesting that PKN1-5a1 may be the major splicing abnormality driven by TDP-43 loss.

We knocked down TDP-43 in multiple human cell types—including M17, HEK-293T, HeLa, and iNeurons (iPSC-derived cortical neurons)—and analyzed PKN1 splicing patterns. The iPSC-derived cortical neurons were obtained by differentiating human induced pluripotent stem cells (iPSC) and validated by MAP2 and NeuN immunostaining (Supplementary Fig. 1j), indicating successful differentiation into mature neurons. In M17 cells, we performed RT-PCR to detect splicing products in this region and observed that, in addition to the normally spliced product, two aberrant bands appeared after TDP-43 knockdown (Fig. 1c), corresponding to insertion of the PKN1-5a1 and A3SS splicing events (Fig. 1d), respectively, indicating that at least two forms of alternative splicing exist in this region. Subsequent qRT-PCR analysis showed that TDP-43 loss significantly upregulated insertion of PKN1-5a1 in M17, HEK-293T, HeLa, and iNeurons, whereas the A3SS event was detectable only in M17 cells (Fig. 1e and Supplementary Fig. 1c, d) and was expressed at a much lower level than 5a1. This suggests that TDP-43’s suppression of aberrant splicing of PKN1-5a1 is more conserved.

To directly visualize the expression of PKN1-5a1 splicing product at the cellular level, we introduced a dCas13a-GFP (green fluorescent protein) system for detection32,33. This system is based on a Cas13a variant from Leptotrichia wadei (dead LwaCas13a, dCas13a) whose nuclease activity is inactivated but which can still recognize target RNA under the guidance of a specific crRNA and form a fluorescent signal with GFP, thereby enabling spatial localization of target RNA molecules (Fig. 1f). We designed two crRNAs (designated crRNA-1 and crRNA-2) that recognize different regions of PKN1-5a1, with crRNA-1 targeting the junction between 5a1 and exon 4 and crRNA-2 targeting the junction between 5a1 and exon 5 (Fig. 1g). To ensure accessibility for binding, we used MFold34 to predict the secondary structure of the RNA sequence of the PKN1-5a1 splicing product. The results showed that the sequences targeted by crRNA-1 and crRNA-2 were both located in single-stranded loop/bulge regions exposed on the RNA surface, displaying good accessibility suitable for dCas13a binding (Fig. 1g). We then co-transfected the dCas13a-GFP system with either crRNA-1 or crRNA-2 into TDP-43–knockdown HEK-293T cells. After cell fixation, TDP-43 was detected by immunostaining using a TDP-43 antibody to assess knockdown efficiency, and the dCas13a-GFP signal was visualized with a GFP antibody. We found that magenta fluorescence was markedly reduced in the TDP-43–knockdown group, indicating a clear decrease in its expression. Meanwhile, distinct green fluorescent puncta were observed predominantly in the nuclei of TDP-43–deficient cells, whereas no obvious signal was detected in the control group (Fig. 1h and Supplementary Fig. 1e). Quantification of green puncta per cell further showed a significant increase in the number of dCas13a-GFP puncta in the shTDP-43 group (Fig. 1i and Supplementary Fig. 1f), suggesting that this cryptic splicing product is activated under TDP-43 deficiency and can be specifically recognized and visualized.

More importantly, we found that TDP-43 knockdown significantly reduced PKN1 protein level (Fig. 1j and Supplementary Fig. 1g-i). We therefore constructed a dual-fluorescence, splicing-dependent translation reporter system to verify the impact of PKN1-5a1 insertion on translation. In this system, EGFP expression serves as an internal control, whereas mCherry expression depends on whether splicing proceeds normally. Once the PKN1-5a1 exon is inserted, the introduction of premature termination codons blocks downstream mCherry translation (Fig. 1k). After co-transfection of this system with shTDP-43 or shCTR into HEK-293T cells, Western blots showed that mCherry protein was completely absent in the TDP-43 knockdown group (Supplementary Fig. 1k); flow cytometry further showed that before knockdown, double-positive (mCherry and EGFP) cells accounted for 57.75%, whereas after knockdown, this proportion was decreased to 11.37% (Fig. 1l). These results suggest that insertion of PKN1-5a1 interrupts downstream translation, thereby aborting PKN1 protein synthesis.

To further verify whether deletion of cryptic exon 5a1 could rescue the decline in PKN1 protein levels under TDP-43 deficiency, we constructed two reporter plasmids: PK-intron (containing exons 4 and 5, and the full intronic interval between them, including 5a1) and PK-Δ5a1 (in the same background but with the 5a1 region specifically removed) (Fig. 1m). After co-transfecting these two plasmids with shTDP-43 into HEK-293T cells and performing Western blots analysis, we found that under TDP-43 knockdown, PKN1 protein expression was markedly reduced only in the PK-intron group, whereas remained essentially unchanged in the PK-Δ5a1 group (Fig. 1n). Interestingly, a 25–33 kDa FLAG band was appeared under TDP-43 knockdown in the PK-intron group, but no such band in the PK-Δ5a1 group (Fig. 1n). Together, these results indicate that insertion of PKN1-5a1 is the direct cause of the downregulation of PKN1 expression in the context of TDP-43 loss.

TDP-43 knockdown reduces PKN1 protein expression in mice but does not activate the PKN1-5a1 cryptic splicing

Given that the PKN1-5a1 splicing event is markedly activated under TDP-43 deficiency, we further investigated the evolutionary conservation of this exon and its expression characteristics in mouse models. The results showed that PKN1-5a1 is highly conserved in primates but lacks a corresponding conserved sequence in mice (Supplementary Fig. 2a, b). This feature is similar to previously reported cryptic splicing events in STMN2 and UNC13A13,15, which likewise are conserved in humans or primates but absent or not spliced in mice, suggesting that such TDP-43–dependent cryptic splicing regulation exhibits pronounced species specificity during evolution.

Nevertheless, by immunofluorescence staining and Western blots, we observed a significant decrease in PKN1 protein levels in the hippocampus of TDP-43–knockdown mice (Supplementary Fig. 2c, d). However, IGV analysis and RT-PCR in TDP-43–knockdown N2a cells did not reveal obvious changes in PKN1 splicing patterns or mRNA levels (Supplementary Fig. 2e). We speculate that TDP-43 knockdown may affect PKN1 protein stability in mouse cells. To test this hypothesis, after knocking down TDP-43 in N2a cells, we used cycloheximide (CHX) to inhibit protein translation and collected samples at 0, 4, 8, and 12 hours, assessing PKN1 expression by Western blots. We found that PKN1 protein was already decreased at 0 hours in shTDP-43 N2a cells, and its degradation rate accelerated significantly over time (Supplementary Fig. 2f). Although the rat genome harbors splice sites in this region that conform to the GU–AG rule, the overall sequence is not conserved relative to the human PKN1-5a1 region. Using non–exon-spanning primers for RT-PCR in TDP-43–knockdown rat B104 cells, we likewise did not observe bands other than the constitutively spliced product (Supplementary Fig. 2g). These results suggest that the presence of GU–AG splice signals alone is insufficient to drive cryptic exon splicing; the occurrence of the event may depend on higher-order cis-regulatory information.

In summary, although PKN1-5a1 lacks splicing activity in rodents, TDP-43 knockdown still induces a reduction of PKN1 protein expression in mouse brain tissue; this regulatory mechanism may involve protein stability rather than changes at the transcriptional or splicing level. These results suggest that TDP-43 regulation of PKN1 is markedly species-specific, and the physiological and pathological significance of its cryptic splicing remains to be further validated in primate models.

PKN1 cryptic exon in ALS neurons

In recent years, cryptic splicing events have been increasingly recognized as potentially disease-specific and important molecular markers in neurodegenerative disorders17–19,21,22,29. Accordingly, we analyzed the publicly available ALS brain RNA-seq dataset (GSE153960) from the New York Genome Center (NYGC), and investigated the activation of PKN1 cryptic exon 5a1 to assess its clinical relevance as a candidate biomarker of ALS.

First, using RNA-seq reads covering all known PKN1 exons, we estimated full-length transcript expression with TPM (transcripts per million). Given that PKN1 has multiple splicing isoforms and sequencing depth varies across samples, TPM—being length-normalized—reflects within-sample abundance and is suitable for cross-sample comparisons, making it preferable for full-length transcript–level analyses. PKN1 expression was reduced not only in regions classically affected by TDP-43 pathology4,35,36, e.g., frontal cortex, temporal cortex, motor cortex, and cervical/lumbar spinal cord; but, interestingly, also showed a similar trend in the cerebellum, which is typically considered less impacted by TDP-43 (Fig. 2a). This suggests that downregulation may not be solely attributable to loss of TDP-43 function. Given that PKN1 is a phosphor-protein involved in neuronal cytoskeletal regulation24,25, we speculated that reduced PKN1 expression in the cerebellum may reflect broader neurodegenerative processes such as neuronal loss. We also noted varying degrees of PKN1 reduction in some “Other” group samples, although statistical differences were comparatively modest.

To delineate the regional distribution of PKN1 splicing abnormalities, we quantified PKN1-5a1 expression. Specifically, for the hg38 interval chr19:14,450,088–14,450,214, we calculated RPM (reads per million) as a relative measure of the cryptic exon. Compared with length-dependent metrics (e.g., TPM/FPKM), RPM is more appropriate for fixed loci such as cryptic exons, effectively controlling for between-sample depth differences and widely used for exon- or event-level quantification. Preliminary analyses detected reads over this interval in a subset of healthy controls (a read was counted toward PKN1-5a1 if ≥75% of its aligned length overlapped the 127-bp 5a1 region). However, junction-level visualization revealed that these signals largely arose from a low-abundance A3SS event (chr19:14,450,088–14,450,317) that shares the same 5′ donor with 5a1 (Fig. 1a), causing some junction reads to be misassigned to PKN1-5a1 and generating false positives in RPM analysis. To avoid A3SS contamination, we computed an endpoint-strict CE-pair RPM from RegTools/LeafCutter counts—counting only Exon4→CE and CE→Exon5 and discarding A3SS-exclusive junctions—which strengthened specificity and the reliability of between-region comparisons (see ‘Methods’).

After correction, PKN1-5a1 was significantly upregulated across multiple CNS regions in ALS, with the most pronounced changes in canonical disease sites—frontal cortex, temporal cortex, motor cortex, and cervical and lumbar spinal cord—whereas no clear changes were observed in the cerebellum (Fig. 2b). By contrast, the cryptic exon was virtually absent in healthy controls, supporting strong disease specificity. In the “Other” group, a subset of individuals also showed elevated PKN1-5a1, particularly in cortical regions, with levels intermediate between ALS and normal control (NC). This may indicate TDP-43 dysfunction in certain atypical cases, but this hypothesis requires further histological or molecular-pathological validation. Overall, PKN1-5a1 expression is concentrated in cortex and spinal cord—regions heavily affected by TDP-43 pathology4,35,36—and remains low in the typically less affected cerebellum. This spatial pattern aligns with the regional selectivity of TDP-43 pathology in ALS, and further supports PKN1-5a1 as a candidate biomarker of TDP-43 proteinopathies–associated splicing abnormalities.

TDP-43 suppresses cryptic splicing of PKN1 through direct binding to conserved UG-rich elements in intron 5

As an aberrant splicing event abundantly detected in ALS patients (Fig. 2b), PKN1-5a1 has potential value as a target for intervention. Therefore, we further focused on the regulatory role of TDP-43 in the splice selection of PKN1, exploring how it finely regulates the generation of this cryptic exon through binding to pre-mRNA, with the aim of providing a theoretical basis for mechanistic understanding and the development of subsequent therapeutic strategies.

To test this hypothesis, we analyzed TDP-43 CLIP-seq data in the POSTAR3 database37. The results showed multiple high-confidence TDP-43 binding sites in the intronic region between exons 4 and 5 of PKN1, these sites are enriched for UG-repeat sequences and were ranked from high to low according to the crosslinking-induced truncation site (CITS) score (Fig. 3a). We further performed RNA immunoprecipitation (RIP) in M17 cells. Compared with the IgG control, TDP-43 specifically enriched PKN1 transcripts containing the 5a1 region (Fig. 3b), supporting the existence of direct binding between TDP-43 and PKN1 pre-mRNA in cells.

To clarify the functional significance of these TDP-43 binding sites in regulating PKN1 cryptic splicing, we constructed a minigene containing exon 4, intron 5, and exon 5 of PKN1 (PKmini), and co-transfected with shTDP-43 or shCTR into HEK-293T cells. RT-PCR analysis showed that under shTDP-43 condition, in addition to the normal splicing product, the cryptic splicing product PKN1-5a1 was detected, whereas only normal splice form was produced in the shCTR group (Fig. 3c). This result not only validated the repressive role of TDP-43 in this cryptic splicing event, but also indicated that the PKmini system can be served as an important tool for analyzing downstream splicing regulatory mechanisms.

To further characterize TDP-43 regulatory sites, we predicted six conserved binding sites based on the CLIP-seq results (site1–site6), among which sites 1–5 are located upstream of the cryptic exon PKN1-5a1 and site 6 downstream (Fig. 3a). Based on this structural feature, we prioritized constructing mutant minigenes lacking site1–5 (PKmini^D1–5) (Fig. 3d) or lacking site 6 (PKmini^D6) (Fig. 3h). RT-PCR results showed that PKmini^D1–5 markedly induced the cryptic splicing product PKN1-5a1 (Fig. 3e), whereas PKmini^D6 primarily produced an A3SS product (Fig. 3i). Sanger sequencing further validated the splice junctions of these aberrant events (Supplementary Fig. 3a), indicating that deletion of distinct TDP-43 binding sites can regulate PKN1 splice selection through specific mechanisms. To further verify the functional role of TDP-43 in the above splicing regulation, we performed TDP-43 overexpression and knockdown on the background of PKmini^D1–5 or PKmini^D6, respectively. The results showed that TDP-43 overexpesssion significantly suppressed aberrant splicing in both mutants and enhanced normal splicing (Fig. 3g, k); conversely, TDP-43 knockdown further increased aberrant splicing (Fig. 3f, j). Notably, when querying human SNP databases (e.g., dbSNP), we found that although these variants are not associated with specific diseases, multiple SNPs annotated as nonpathogenic are located in the site 6 region (Supplementary Fig. 3b). To further assess their functional impact, we constructed mutant minigenes containing the above SNPs, and these variants likewise induced aberrant splicing events in the minigene (Supplementary Fig. 3c), further supporting site 6 as one of the key cis-acting elements regulating PKN1 splicing.

To clarify the cis-acting mechanism of the site 1–6 regions in TDP-43–dependent splicing regulation, we constructed a mutant simultaneously lacking all six sites (PKmini^D1–6) (Fig. 3l). In this construct, the A3SS event could still occur, but the responsiveness to TDP-43 expression level was completely lost (Fig. 3m), i.e., neither overexpression nor knockdown of TDP-43 significantly altered its splicing pattern. By contrast, on the PKmini^D6 background lacking only site 6, TDP-43 expression still effectively regulated A3SS splicing levels (Fig. 3j, k). Given that TDP-43 could still regulate A3SS splicing when only site 6 was deleted, whereas this regulation was completely lost when sites 1–6 were deleted, these results suggest that TDP-43 regulation of A3SS splicing may depend on cis-acting elements in the site 1–5 region, whereas site 6 is more likely involved in selection of the A3SS acceptor site, thereby influencing the final splicing outcome. To further pinpoint key cis elements, we generated multiple combinatorial deletion mutants (e.g., PKmini^D1+6, PKmini^D2+6, etc.) (Fig. 3l) and systematically evaluated their effects on the A3SS event. PSI analysis showed that among all the combinations, deletion of site 3 or site 5 led to a more pronounced reduction in TDP-43’s ability to suppress aberrant splicing (Fig. 3m), suggesting that these two sites may be key cis elements mediating TDP-43 splicing regulation.

Finally, to determine whether TDP-43 directly binds specific sites on the pre-mRNA of PKN1, we designed biotin-labeled RNA probes covering the site 1–site 6 regions (Fig. 3n) and performed electrophoretic mobility shift assay (EMSA). We incubated the probes with recombinant TDP-43 protein. In native polyacrylamide gels, we observed distinct nucleic acid–protein complex bands for TDP-43 with site 1, site 2, site 3, site 5, and site 6, indicating binding capability at these sites (Fig. 3o), whereas site 4 showed no complex formation, suggesting no obvious affinity for this region. In addition, to further verify the binding specificity and affinity between TDP-43 and the candidate cis elements, we performed EMSA assay for each probe under different TDP-43 concentrations. We found that as TDP-43 concentration was increased, the intensity of complex bands formed with the probes of site 1, site 2, site 3, site 5, and site 6 were increased significantly in a typical dose-dependent manner (Supplementary Fig. 3d), indicating stable and specific binding. Conversely, site 4 showed no complex band even at high TDP-43 concentrations, further indicating a lack of binding to TDP-43. Together, these results indicate that TDP-43 precisely regulates PKN1 splicing by directly binding multiple conserved sites on the pre-mRNA of PKN1, thereby suppressing production of cryptic exon 5a1 and maintaining the correct splicing of PKN1 mRNA (Fig. 3p).

The RRM1 domain and dimerization capacity jointly maintain the fidelity of TDP-43 regulation on PKN1 splicing

To delineate the structural basis by which TDP-43 regulates PKN1 cryptic splicing, we systematically evaluated the functional roles of its dimerization capacity and RNA-recognition domains in this process. Although previous studies have indicated that the dimeric state of TDP-43 influences its RNA splicing function, the mechanistic underpinnings in specific cryptic splicing events remain to be clarified29,38. To test whether this structural basis controls PKN1 splicing, we overexpressed dimerization-impaired mutant, TDP-43^90–414 (90-414), and TDP-43^N6A (N6A), a dimerization-defective N-terminal mutant carrying six alanine substitutions38–40 in HEK-293T cells. Cells were then treated with the cell-permeable dimer crosslinker disuccinimidyl glutarate (DSG) and analyzed by Western blots. We found that full-length TDP-43 (TDP-43^FL) formed stable dimers and higher-ordered oligomers, whereas 90–414 and N6A existed predominantly as monomers, with virtually no crosslinked bands (Fig. 4a), confirming markedly impaired dimerization for both mutants. Functionally, when 90–414 or N6A was co-transfected with the splicing reporter PKmini into HEK-293T cells without endogenous TDP-43 knockdown, neither mutant induced insertion of PKN1-5a1 (Fig. 4b). However, upon TDP-43 knockdown, both mutants failed to effectively suppress insertion of the cryptic exon PKN1-5a1 (Fig. 4c), whereas TDP-43^FL significantly suppressed this cryptic splicing event under the same conditions, indicating that loss of dimerization weakens the splicing regulatory function of TDP-43.

TDP-43 contains two conserved RNA recognition motifs (RRMs), among which RRM1 is considered central to RNA binding and splicing regulation41. To validate the role of RRMs in PKN1 splicing, we co-transfected TDP-43^FL, loss-of-function mutant of RRM1 (F147/149L) or RRM2 (F227/229L), deletion mutant of RRM1 (ΔRRM1) or RRM2 (ΔRRM2) with PKmini into HEK-293T cells, and analyzed by RT-PCR. Interestingly, we found that F147/149L and ΔRRM1 both markedly induced aberrant insertion of PKN1-5a1, with the effects resembling TDP-43 knockdown, particularly pronounced for ΔRRM1 (Fig. 4b), suggesting a dominant-negative effect of RRM1 deletion. Similarly, upon a TDP-43 knockdown background (Supplementary Fig. 4a), co-transfection of these mutants with PKmini into HEK-293T cells revealed that TF147/149L and ΔRRM1 failed to suppress PKN1-5a1 insertion, consistent with the loss-of-function phenotype (Fig. 4c). Moreover, ΔRRM1 not only failed to suppress the cryptic event but further enhanced PKN1-5a1 insertion. Notably, even without PKmini transfection, overexpression of ΔRRM1 in HEK-293T cells significantly induced insertion of 5a1 in the endogenous PKN1 transcript (Fig. 4d), further indicating that the ΔRRM1 mutant interferes with normal regulation of endogenous splice sites through a dominant-negative mechanism. In addition, we observed that F147/149L and ΔRRM1 formed aberrant nuclear condensates and colocalized with TDP-43^FL in HEK-293T cells, as previously describe42–44, whereas other constructs (e.g., TDP-43^FL, F227/229L, or ΔRRM2) did not form nuclear condensates (Supplementary Fig. 4b).

Previous studies have shown that dominant-negative mutants can heterodimerize with the wild-type counterparts, thereby impairing normal function45. Together with the prior finding that the N terminus of TDP-43 has dimerization capacity38–40, we further hypothesized that ΔRRM1 might exert dominant-negative effects by forming heterodimers with endogenous wild-type TDP-43, thereby disrupting recognition and binding of RNA targets. To test this hypothesis, we generated a double mutant lacking both dimerization capacity and RRM1 (ΔRRM1^N6A), and co-transfected with PKmini into HEK-293T cells, then analyzed by RT-PCR. We found that, unlike ΔRRM1—which markedly induced PKN1-5a1 insertion—ΔRRM1^N6A did not elicit obvious cryptic exon insertion, indicating a lack of dominant-negative activity (Fig. 4e). To further determine whether these mutants retain the ability to associate with wild-type TDP-43, we employed the bimolecular fluorescence complementation (BiFC) system. TDP-43 and each mutant were fused to Venus N-terminal fragment 173 (VN173) or Venus C-terminal fragment 155 (VC155) fragments of the BiFC system, and co-expressed in HEK-293T cells. If dimerization occurs, the VN and VC fragments will produce a GFP signal. We found that TDP-43-VN173 and TDP-43-VC155 generated strong nuclear GFP signals, confirming homodimerization; ΔRRM1-VC155 also formed heterodimers with TDP-43-VN173; by contrast, N6A-VC155 and ΔRRM1^N6A-VC155 failed to induce GFP signals, indicating complete loss of dimerization capacity and further indicating that RRM1 forms heterodimers with wild-type TDP-43 via the N terminus (Supplementary Fig. 4c).

We had previously observed that ΔRRM1 formed pronounced nuclear condensates and colocalized with wild-type TDP-43 (WT), and, together with the ability of ΔRRM1 to form heterodimers with WT, we hypothesized that ΔRRM1 might recruit WT into the aberrant nuclear condensates formed via dimerization. To test this, we designed two key conditions: (1) whether ΔRRM1 condensates recruit TDP-43-VN173/ΔRRM1-VC155 heterodimers efficiently; and (2) whether these condensates recruit TDP-43-VN173/TDP-43-VC155 homodimers. We co-transfected TDP-43-VN173 with ΔRRM1-VC155 or TDP-43-VC155, and ΔRRM1 mutant into HEK-293T cells. Fluorescence imaging of living cells showed robust BiFC punctate signals in the TDP-43-VN173/ΔRRM1-VC155 and ΔRRM1 group, whereas only weak BiFC punctate signals were observed in the TDP-43-VN173/TDP-43-VC155 and ΔRRM1 group (Fig. 4f). Given that the fluorescences in BiFC system are arised from stabilized protein–protein interactions, these results suggest that ΔRRM1 condensates preferentially recruit TDP-43/ΔRRM1 heterodimers rather than TDP-43 homodimers (Fig. 4g). To determine whether ΔRRM1 is specific for this BiFC punctate signals, we performed parallel control experiments by co-expressing wild-type TDP-43 and TDP-43-VN173 with either ΔRRM1-VC155 or TDP-43-VC155; no similar nuclear puncta were observed (Supplementary Fig. 4d), further supporting ΔRRM1 as the key driver of the aberrant aggregation of TDP-43. Whether such condensates functionally “sequester” wild-type TDP-43 and thereby impair its binding to PKN1 pre-mRNA remain to be further validated using the MS2–MCP system46.

Taken together, these results support our hypothesis that TDP-43^ΔRRM1 can form heterodimers with endogenous TDP-43 via its retained N-terminal region, thereby disrupting normal recognition and binding to RNA targets and inducing aberrant splicing through a dominant-negative mechanism, whereas N-terminal dimerization–deficient mutations effectively block this process (Fig. 4h).

TDP-43 knockdown generates and accumulates the cryptic peptide PKN1-N207

Previously, we knocked down TDP-43 in HEK-293T cells expressing PK-intron, and observed a ~25–33 kDa FLAG band (Fig. 1n), suggesting that an aberrant transcript arising from a cryptic splicing event might be translated into a truncated cryptic peptide. Interestingly, this splicing product contains a PTC (Supplementary Fig. 1a) and should be subject to NMD47. However, the stable presence of this band implies that it may partially escape NMD.

To test this hypothesis, we treated TDP-43–knockdown HEK-293T cells with the translation inhibitor CHX (2 μg/mL) to indirectly inhibit NMD activity and performed to qRT-PCR analysis. We found that PKN1-5a1 expression was increased ~1.4-fold in the CHX group, compared with the DMSO group (Supplementary Fig. 5a). Furthermore, we knocked down up-frameshift protein 1 (UPF1), a key effector of NMD48 (Fig. 5a), to specifically block this pathway. We found that UPF1 knockdown alone did not induce aberrant splicing of PKN1-5a1, but in the context of TDP-43 deficiency, combined UPF1 knockdown further upregulated its expression by ~1.6-fold (Fig. 5a), supporting that PKN1-5a1 transcript can be partially recognized and degraded by NMD. Notably, either by CHX treatment or UPF1 knockdown, the magnitude of PKN1-5a1 upregulation was limited; together with our previous observation of the “FLAG band”. These suggest that PKN1-5a1 may not be a typical NMD target and may escape degradation by being translated into a truncated peptide. This, of course, required validation at the endogenous level. Therefore, we generated stable TDP-43–knockdown HEK-293T and M17 cell lines. Specifically, cells were infected with lenti/shTDP-43, treated with puromycin (2 μg/mL) for 4 days, and then cultured without antibiotics for an additional 6 days. It should be noted that TDP-43 knockdown markedly suppressed the proliferative capacity of HEK-293T and M17 cells, making long-term stable passaging difficult. To assess proliferation, we performed an EdU incorporation assay on HEK-293T cells at day 10 post-infection. We found a clear decrease in cell density and a significant reduction in EdU-positive cells in the shTDP-43 group (Supplementary Fig. 5b), indicating impaired proliferation, consistent with previous studies49. We then collected the selected HEK-293T and M17 cells for Western blots analysis, using a specific antibody against the N terminus of PKN1 to monitor the expression of full-length PKN1 and the truncated peptide. We found that TDP-43 knockdown led to a marked decrease in full-length PKN1, accompanied by a pronounced increase in a truncated peptide within the 25~33 kDa range (Fig. 5b and Supplementary Fig. 5c). Based on the splice junctions and reading frame, this band corresponds to the first 207 amino acids of PKN1; we therefore designated the cryptic peptide PKN207. To further analyze its accumulation at different time points, we set a time course of TDP-43 knockdown at 48, 72, 96, and 120 hours, and performed to Western blots. We found that full-length PKN1 was gradually decreased from 48 hours onward, but the PKN207 band was clearly detected only after 120 hours of sustained TDP-43 deficiency (Fig. 5c), suggesting that this truncated peptide must gradually accumulate under continuous loss of TDP-43 to reach a detectable level.

To further evaluate the stability of this cryptic peptide PKN207 in HEK-293T cells, we constructed a Tet-on inducible expression system to drive expression of full-length PKN1 (Tet-PKFL) and the N207 cryptic peptide (Tet-PKN207), respectively (Fig. 5d). After induction with doxycycline (Dox, 1 μg/mL) for 24 hours, the medium was replaced with Dox-free medium, then treated with CHX (2 μg/mL); cells were collected at 0, 4, 8, and 12 hours for immunoblotting. We found that with prolonged CHX treatment, full-length PKN1 levels were gradually declined, while PKN207 levels remained stable throughout (Fig. 5d), indicating that PKN207 cryptic peptide has high intracellular stability and may continuously accumulate in HEK-293T cells (Fig. 4g). Furthermore, we simultaneously knocked down TDP-43 and PKN1 in HEK-293T cells to examine changes in PKN207 expression. We observed that TDP-43 knockdown markedly enhanced PKN207 expression, whereas concurrent PKN1 knockdown clearly reduced the truncated peptide (Fig. 5e), further demonstrating that it originates from aberrant splicing of the PKN1 gene. These results indicate that the TDP-43–knockdown–induced cryptic splicing of PKN1 can, after prolonged accumulation, generate and express the cryptic peptide PKN207, and that this peptide exhibits high stability, suggesting that it may persistently exert functions in cells.

A specific antibody reveals that the PKN207 cryptic peptide is detectable in AD brain with TDP-43 pathology

Given that the PKN1-5a1 cryptic exon has been validated in RNA-seq data of ALS cohort from NYGC, we further hypothesized that whether this cryptic splicing event truly occurs in patients, its translation product—the PKN207 cryptic peptide—might also be detectable in patients with TDP-43 pathology. To test this hypothesis, we attempted to generate antibodies capable of specifically recognizing PKN207. Sequence alignment showed that the C terminus of PKN207 protein contains a unique amino acid sequence “LAL”, which replaces the “SPD” sequence present in full-length PKN1, resulting in a distinct C-terminal structure (Supplementary Fig. 5d). To maximize antibody specificity, we selected an eight–amino acid peptide, “DDTQGLAL”, spanning this differential region, as the immunogen. Structural predictions indicated that this region is surface-exposed and exhibits favorable antigenicity, hydrophilicity, and immunogenicity (Supplementary Fig. 5e). On this basis, we immunized rabbits intraperitoneally with the peptide and prepared four polyclonal anti-PKN207 antibodies (E29426–E29429) (Supplementary Fig. 5f).

To validate the specificity of the antibodies for PKN207, we overexpressed either full-length PKN1 (PKFL) or the PKN207 cryptic peptide in HEK-293T cells and performed to Western blots analysis. All four antibodies showed a specific band of approximately 27–33 kDa in the PKN207 group, while no signal in the PKFL group, indicating that these antibodies do not cross-react with full-length PKN1 (Fig. 5f). We then further validated their recognition in TDP-43–knockdown or control HEK-293T cells. All four antibodies showed a band of approximately 27 kDa in the shTDP-43 group, no obvious signal in the control group (Fig. 5g); among them, E29426 exhibited the lowest background and best specificity (Fig. 5g). These results indicate that this antibody series specifically recognize PKN207 but not PKFL. To further assess the cellular localization of PKN207, we overexpressed PKN1^FL or PKN1^N207 in HEK-293T cells, and performed to nuclear–cytoplasmic fractionation and Western blots. We found that PKN1 localized mainly in the cytoplasm with a portion in the nucleus, whereas PKN207 localized exclusively in the cytoplasm (Supplementary Fig. 5g). We next evaluated the applicability of these antibodies in immunofluorescence staining. We transfected PKN207-EGFP into HEK-293T cells and performed immunostaining with the four antibodies (red fluorescence). We observed strong diffuse cytoplasmic red signals regardless of EGFP transfection (Supplementary Fig. 5h–k), indicating notable nonspecific background of these polyclonal antibodies in immunofluorescence.

We ultimately selected the E29426 antibody to determine whether it could be used to detect PKN207 expression in patients with TDP-43 pathology. Given that ALS brain tissue was unavailable and TDP-43 pathology is present in ~20–50% of AD patients7,50,51, we examined hippocampal tissue from TDP-43-positive AD cases. First, we assessed the presence of phosphorylated TDP-43 (pTDP-43) in the RIPA-insoluble fraction of AD samples by Western blots. We found clear pTDP-43 signals in AD1 and AD5, indicating typical TDP-43 proteinopathy (Fig. 5h). We then examined PKN207 expression in the AD samples. Although there was no significant difference in PKFL between the AD and control groups (Fig. 5i), we detected a distinct ~25–33 kDa band in AD1 and AD5 using the E29426 antibody, whereas no such band was observed in the control group or in TDP-43-negative AD cases (Fig. 5h, j). Further RT-PCR analysis also showed that the PKN1-5a1 cryptic exon was detected in AD1, AD2, and AD5 (Fig. 5k). Notably, PKN207 expression mirrored pTDP-43 pathology, being present in AD1 and AD5 (which showed marked aggregation) but absent in AD2 and controls (which lacked corresponding pathology) (Fig. 5j). In contrast, the PKN1-5a1 cryptic exon transcript was detected at low levels in AD2 as well (Fig. 5k), yet no corresponding PKN207 protein was detected, demonstrating that cryptic splicing does not always produce a stable protein. We speculate that even in the absence of classic aggregated pTDP-43, functional abnormalities of TDP-43 (e.g., mild nuclear loss or weakened splicing regulatory capacity) may still occur in AD2, thereby inducing activation of specific cryptic splicing. This splicing abnormality may represent an early molecular feature of TDP-43 loss of function that has not yet progressed to visible aggregate pathology. In addition, the splicing product may not yet have accumulated to a detectable protein level in vivo, leading to failure to detect the corresponding PKN207 band. In summary, compared with the splicing event itself, the expression of PKN207 cryptic peptide may more directly reflect TDP-43 pathology, and the E29426 antibody holds promise as a more specific biomarker for TDP-43 proteinopathies.

Expression of the PKN207 cryptic peptide induces cognitive impairment in mice

We detected the expression of PKN207 cryptic peptide in TDP-43-positive AD brains, whereas PKFL showed no obvious change (Fig. 5h), suggesting that PKN207 may directly mediate neuronal dysfunction without altering full-length protein expression, and may even participate in the development of cognitive impairment. Given the limited number of AD samples in the immunoblot analysis, we further analyzed RNA-seq data from the parahippocampal gyrus (PHG) in the Mount Sinai Brain Bank (MSBB) AD transcriptome database to expand the sample size and validate the finding at the transcriptional level. We found that the PKN1-5a1 cryptic exon was stably detected in AD patients, and nearly absent in healthy controls (Fig. 6a), supporting its activation in disease and potential pathogenicity. Meanwhile, full-length PKN1 mRNA expression was slightly increased, which may explain the lack of a significant change at the protein level. Notably, activation of this cryptic splicing event was observed even at Braak stage 1, an early stage of AD, suggesting that it may be induced at the early stage of the disease process.

To test the potential neurotoxicity of PKN207, we injected three neuron-specific promoter adeno-associated viruses (AAV-hSyn) into the hippocampus bilaterally of 2-month-old C57BL/6J mice: AAV-PKFL (encoding full-length PKN1), AAV-PKN207 (encoding the N207 cryptic peptide), and an empty control virus AAV-CTR (Fig. 6b). We also validated the expression of AAV-PKFL and AAV-PKN207 in the mouse hippocampus by Western blots, and confirmed robust expression of both PKFL and the PKN207 peptide (Supplementary Fig. 6e). Four months later, Morris water maze (MWM) testing showed significant cognitive deficits in both the AAV-PKFL and AAV-PKN207 groups (Fig. 6c), indicating that overexpression of either protein may adversely affect learning and memory.

Considering that cognitive impairment is often accompanied by structural damage to neuronal axons, we further measured NEFL level in CSF 4 months post virus injection. CSF-NEFL was significantly higher in the PKFL and N207 groups than in their respective control groups (Fig. 6d), suggesting compromised axonal integrity accompanied by increased neurofilament release. This change in a fluid biomarker reflects structural pathological alterations at the central nervous system level52–55.

To further verify whether PKN207 is cytotoxic, we overexpressed PKFL or PKN207 in primary mouse hippocampal neurons and assessed toxicity by lactate dehydrogenase (LDH) release. Cytotoxicity was significantly induced in both groups, with PKFL showing slightly higher toxicity than PKN207 (Fig. 6e). Nevertheless, as a splicing product specific to TDP-43 loss, PKN207 has a unique pathological context, suggesting that it may possess disease-state–specific pathogenic mechanisms.

Methods
Ethical statement
This study was conducted in accordance with the ethical principles of the Declaration of Helsinki. The use of human post-mortem brain tissue and associated clinicopathological data was approved by the Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology (Approval No. [2019] IEC(S775)). All tissue samples were obtained from the National Health and Human Brain Tissue Resource Center (China). Informed consent for brain donation and the use of tissue for research purposes was obtained from all donors ante-mortem. The brain bank operates under strict ethical guidelines to ensure donor anonymity and confidentiality.

All animal procedures were approved by the Animal Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology (IACUC4696). The study was performed in accordance with the relevant guidelines and regulations.

Human brain tissue
Human brain autopsy specimens were obtained from the National Health and Disease Human Brain Tissue Resource Center (Table 1). Each specimen carried a unique Brain Bank identifier, with donor ID, sampled brain region, sex, age at death, brain wet weight, and Braak neuropathological stage recorded. Clinical and neuropathological diagnoses were made according to established criteria, referencing the NIA–AA guidelines for the neuropathologic assessment of AD57, Braak staging followed the original method58. Inclusion criteria were based on ante-mortem clinical presentation and postmortem histological evaluation. The primary clinical diagnosis among included cases was AD. Detailed donor information is provided in the accompanying table (including Brain Bank ID, sampled brain region, age at death, sex, Braak stage, and brain wet weight). Normal group refers to a healthy population with no history of neurological diseases.

Plasmid Information and Construction
Detailed information of the plasmids used in this study and their construction procedures is provided in Supplementary Table 2.

Animals and stereotaxic injection
Two-month-old C57BL/6J mice were randomly assigned to three groups: AAV-Con (n=15), AAV-PKN1 (n=25), and AAV-PKN207 (n=25). Operators were blinded to group allocation. AAV9 vectors (SYN promoter; working titer 1×10¹² vg/mL) were provided by Obio Co., Ltd. (Shanghai). Mice were anesthetized with isoflurane (induction 3–4%, maintenance 1.5–2.0%; RWDR510-22-10) and positioned in a stereotaxic frame (RWD 69100). Using bregma as the reference, bilateral hippocampal injections were made at AP −2.0 mm, ML ±1.5 mm, and DV −1.8 mm. A Hamilton syringe (LFGJ-80383) connected to a micro-injection pump was used to deliver virus at 100 nL/min; 0.5 μL was infused per side (≈5×10⁸ vg), for a total dose of ≈1×10⁹ vg per mouse. The needle was left in place for 5 min after infusion and then withdrawn slowly to minimize reflux. A heating pad was used intraoperatively; postoperative analgesia was provided with meloxicam (2 mg/kg, subcutaneous, once daily for 2 days) and recovery was monitored. Mice were housed at 22±2°C with 40–60% humidity under a 12-h light/dark cycle, with food and water available ad libitum. Surgical and stereotaxic procedures followed established methods, and injection coordinates were referenced to Paxinos & Franklin, The Mouse Brain in Stereotaxic Coordinates, 4th ed.

Behavioral testing—Morris water maze (MWM)
At 4 months after viral injection, the MWM was used to assess spatial learning and memory. The apparatus consisted of a circular pool (diameter 120 cm) filled with opaque water maintained at 22±1°C. A hidden platform (diameter 10 cm) was fixed in the same quadrant and submerged ≈1 cm below the water surface. Training lasted 4 days with 4 trials per day, with mice released from different quadrants; the maximum swim time per trial was 90 s. The escape latency was recorded upon reaching the platform; trials in which the platform was not reached were assigned 90 s. After each trial, mice were allowed to remain on the platform for 15 s. Behavior was automatically recorded and analyzed using the EthoVision XT video-tracking system (Noldus). Learning ability was evaluated by the mean escape latency during the training period.

Cisterna magna CSF collection and NEFL measurement
Mice received AAV injection at 2 months of age, and CSF was collected 4 months later for analysis. Groups were: AAV-CTR (n=10), AAV-PKFL (n=20), and AAV-PKN207 (n=20). Procedures were performed under aseptic conditions with continuous isoflurane anesthesia (1–2%). With the mouse placed prone and the neck flexed to expose the cisterna magna, the area was disinfected with 75% ethanol; a midline skin incision was made, muscles were separated, and the transparent dura mater was exposed. Under a stereomicroscope, a pulled glass capillary (outer diameter ≈0.5 mm) was inserted vertically into the cisterna magna, and CSF was slowly aspirated; approximately 5–15 μL of clear fluid was obtained per mouse. Blood contamination was strictly avoided; any blood-tinged sample was discarded. CSF was immediately transferred to RNase-free microcentrifuge tubes, kept on ice, and stored at −80°C. NEFL levels were measured using a mouse NEFL ELISA kit (Jonln, JL51267) according to the manufacturer’s instructions.

LDH release assay in mouse hippocampal neurons
To evaluate the effects of differential PKN1 expression on cell membrane integrity, LDH levels in the culture supernatant of primary hippocampal neurons were measured using an LDH Cytotoxicity Assay Kit (Beyotime, C0017). Three groups were included: control virus (Null), PKN1 full-length lentivirus (PKFL), and N207 truncated lentivirus (PKN207). Cells were seeded at equal density in 24-well plates and, after stable adherence, subjected to the indicated treatments. Supernatants were collected 48 h later. According to the manufacturer’s instructions, supernatants were mixed with the working reagent and incubated at room temperature for 30 min protected from light; after addition of the stop solution, absorbance was read at 490 nm (OD₄₉₀). Cytotoxicity was assessed by comparing LDH release in each experimental group relative to the control group. Four technical replicates were included per group, and background values from blank wells were subtracted prior to analysis.

fEPSP recordings
Acute hippocampal slice electrophysiology was performed on 6-month-old C57BL/6J mice at 4 months after AAV injection. Mice were anesthetized with isoflurane and rapidly decapitated; the brain was removed, rinsed with ice-cold artificial CSF (aCSF), and mounted on a vibratome. Coronal cortico-hippocampal slices (350 μm) were prepared in aCSF at 4°C equilibrated with 95% O₂/5% CO₂, then transferred to room-temperature aCSF (20.5±1°C) to recover for ≥1 h. Recordings were completed within 8 h of tissue collection. The aCSF contained: 118 mM NaCl, 10 mM glucose, 20 mM sucrose, 25 mM NaHCO₃, 3.0 mM KCl, 1.5 mM CaCl₂, 1.0 mM NaH₂PO₄, and 1.0 mM MgCl₂; osmolality 305–315 mOsm, pH 7.42±0.02.

Recordings were performed under continuous superfusion with aCSF (31.0±2°C, 95% O₂/5% CO₂). A bipolar stimulating electrode (CBARC75, FHC Inc.) was placed in the Schaffer collateral pathway, and a glass recording electrode (1–2 MΩ) was positioned in the CA1 stratum radiatum. Stimuli (100–400 μA, 0.1–0.2 ms) were delivered at the lowest intensity that evoked 50% of the maximal fEPSP amplitude. After establishing a stable baseline for ≥10 min, long-term potentiation (LTP) was induced with theta-burst stimulation: 4 trains, each comprising 10 bursts at 5 Hz, with 4 pulses per burst at 100 Hz. Post-tetanus responses were recorded for 60 min, and fEPSP slopes were normalized to baseline. Signals were acquired using an AM Systems differential amplifier.

RNA sequencing
M17 cells were infected with lentiviruses (Obio Co., Ltd., Shanghai, China): control (Lenti-shCTR) and a TARDBP–targeting knockdown virus (Lenti-shTDP-43). 96 h later, all samples were lysed in TRIzol (Takara, 9109), immediately stored at −80°C, and subsequently submitted to BGI Genomics (Shenzhen, China) for RNA sequencing. Each group included three biological replicates. Libraries were prepared using poly(A)+ mRNA enrichment and sequenced with paired-end 150-bp reads (PE150), yielding approximately 10 Gb per sample. Reads were aligned to the NCBI human reference genome (hg38) and used for downstream gene-expression and splicing analyses. The raw sequencing data (FASTQ) have been deposited in the NCBI SRA database.

DIA mass spectrometry (Orbitrap Astral platform)
Hippocampus stored at −80°C from AAV-CTR, AAV-PKN207, and AAV-PKFL mice, with three biological replicates per group, following protein extraction and trypsin digestion, peptides were combined in equal amounts and submitted to Shanghai Bioprofile Biotechnology Co., Ltd. for data-independent acquisition (DIA) using the Orbitrap Astral platform. Chromatographic separation was performed on a Vanquish Neo UHPLC (Thermo Scientific). Mobile phase A was 0.1% formic acid in water; mobile phase B was 0.1% formic acid in 80% acetonitrile. The column was pre-equilibrated with 96% A. Samples were enriched on a trap column (PepMap Neo 5 μm C18, 300 μm × 5 mm; Thermo Scientific), then subjected to gradient separation on a μPAC Neo High Throughput analytical column (Thermo Scientific), followed by DIA acquisition on an Orbitrap Astral mass spectrometer (Thermo Scientific). The total analysis time was 8 min.

IGV visualization
RNA-seq reads were aligned to the NCBI GRCh38 reference using STAR v2.7.10a to generate BAM files. To display expression intensity, BAM files were converted to BigWig format with bamCoverage from deepTools v3.5.1. Tracks were then visualized in Integrative Genomics Viewer (IGV).

RNA immunoprecipitation (RIP)
RIP was performed with RIP RNA-binding protein immunoprecipitation kit (GENE CREATE, JKR23003) according to the manufacturer’s instructions. Endogenous TDP-43 was immunoprecipitated from M17 cell lysates using the anti–TDP-43 antibody (ProteinTech, 10782-2-AP). RNA was extracted from the immunoprecipitates and reverse-transcribed to cDNA using a reverse transcription kit (ABclonal, RK20429). The resulting cDNA was used as template for RT-PCR to detect transcripts containing PKN1 cryptic exon 5a1. A rabbit IgG isotype control (GENECREATE, JKR23003) served as the negative control. Primers: Rip-F, 5′-TTTCCCTAGGACTGGCCCTGT-3′; Rip-R, 5′-GACAAACCATCATGAAGATGCATCACG-3′. PCR cycling conditions: 95°C for 5 min; 35 cycles of 95°C for 30 s, 60°C for 10 s, and 72°C for 10 s; followed by 72°C for 5 min. PCR products were resolved by agarose gel electrophoresis. PCR reactions were performed with OneTaq 2× Master Mix (standard buffer) (Vazyme).

RNA electrophoretic mobility shift assay (RNA EMSA)
Experiments were performed with Chemiluminescent RNA EMSA Kit (Beyotime, GS606) according to the manufacturer’s instructions. 5′-biotin–labeled RNA probes were synthesized by Sangon Biotech and used at a final concentration of 150 pmol/L, adding 1 μL per reaction. Recombinant TDP-43 protein was purchased from MCE (HY-P71788); 2 μg was added to each binding reaction, and a concentration series (0.4, 0.8, 1.2, 1.6, 2.0 μg) was used to evaluate the affinity of different probes for TDP-43. Probe sequences (5' to 3') were: probe 1, CUGUGACUGUGUUUCUCUGCCC; probe 2, UGUAGUAUGCUGUGGUUGUCCA; probe 3, GAAUGAGGUGUGUCAACACGGG; probe 4, GACUUAGAGUCUCACUCUGUCA; probe 5, GUGUGCCUGUGUGUCAUUGCUC; probe 6, CUCACUGCCCUGUAUUCCACGC. Samples were separated on 6% EMSA precast gels (Beyotime, GS306S) at constant voltage (100 V) for ≈1.5 h in pre-chilled 0.5× TBE (Tris-Borate-EDTA buffer). Proteins were then wet-transferred to nylon membrane (Beyotime, FFN10) in pre-chilled 0.5× TBE at 360 mA for 1 h; membranes were pre-soaked in 0.5× TBE for 30 min prior to transfer. After transfer, membranes were UV-crosslinked for 10 min at ≈8 cm from the light source. Following blocking, membranes were incubated with streptavidin–HRP for 20 min, washed according to the kit instructions, and developed with an ultrasensitive ECL substrate. Images were acquired using a ChemiScope 6100 imaging system (Clinx).

Cell culture and transfection
Human embryonic kidney 293T cell line (HEK-293T), human cervix epithelial cell line (HeLa), mouse neuroblastoma Neuro-2a cell line (N2a), and rat neuroblastoma B104 cell line were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, C11995500BT) supplemented with 15% fetal bovine serum (FBS) (Vazyme, F101-01), 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco, 15140122); and human neuroblastoma M17 cells were cultured in DMEM/F12 (Gibco, C11330500BT) with 10% FBS, penicillin/streptomycin (Gibco), and incubated in a humidified atmosphere containing 5% CO₂ at 37°C. Cells were seeded to culture plates (Sigma, 27964-99-4), and all transfections were performed with Lipofectamine® 2000 (Invitrogen) according to the manufacturer’s instructions. Empty vectors were used as controls for the corresponding transfection.

Primary cortical neurons were prepared from E18.5 C57BL6/J embryos. After decapitation of the pregnant dam, embryos were placed in ice-cold Ca²⁺/Mg²⁺-free PBS. Under a stereomicroscope, the cerebral cortex was dissected and meninges removed. Cortex and hippocampus (with superficial vascular membranes removed) were separately incubated in 0.25% trypsin-EDTA (Gibco, 25200072) at 37°C for 15 min, with gentle inversion every 5 min. Tissues were gently triturated using a 5 mL pipette tip to obtain single-cell suspensions, passed through a 40 μm cell strainer, and centrifuged at 500 g for 5 min. Pelleted cells were resuspended in high-glucose DMEM and plated onto poly-L-lysine–coated coverslips or culture plates (Sigma). After incubation at 37°C for 4 h, the medium was replaced with Neurobasal (Gibco, 21103049) supplemented with B27 (Gibco), GlutaMAX (Gibco, 35050061), and penicillin/streptomycin (Gibco) for maintenance culture; half of the medium was replaced every 3 days. Plasmid transfection was performed with Lipofectamine® 2000 (Invitrogen).

shRNA lentiviral treatment timelines: (1) Cell lines: Day 0, seeding; Day 2, addition of shRNA lentiviruses; Day 3, medium change; Day 6, harvest for RT-qPCR or immunoblotting. (2) Primary hippocampal neurons: Day 0, seeding; Day 6, infection; Day 7, medium change; Day 12, sampling for LDH cytotoxicity assay. (3) iPSC-derived cortical neurons: Day 0, seeding; Day 14, upon appearance of typical neuronal morphology, switch to maturation medium (Neurobasal + B27 + GlutaMAX) and add shRNA lentiviruses; Day 16, medium change; Day 22, harvest for RT-qPCR or immunoblotting.

iPSC source and cortical neuron differentiation
Human iPSCs were derived from normal human dermal fibroblasts and maintained on Matrigel-coated 6-well plates (Corning, 354234) in xeno-free mTeSR1 medium (STEMCELL Technologies, 85850) at 37°C in 5% CO₂. Medium was changed daily, and cultures were passaged every 4–5 days using ReLeSR (STEMCELL, 100-0484). Typical colonies were round with smooth edges and high refractivity.

Cortical neuron differentiation followed a dual-SMAD inhibition strategy. On Day 0, healthy iPSCs were dissociated to single cells and plated on Matrigel; neural induction was performed in basal medium (DMEM/F12 + N2 + B27 minus vitamin A) supplemented with 10 μM SB431542 and 100 nM LDN-193189 for 7 days. Beginning on Day 7, inhibitors were gradually withdrawn, and 20 ng/mL FGF2 was added to expand neural progenitor cells (NPCs). Neuron-like morphology emerged by Day 14; cultures were then switched to a maturation medium (Neurobasal + B27 + GlutaMAX) supplemented with 10 ng/mL BDNF, 10 ng/mL GDNF, 200 μM ascorbic acid, and 1 μM cAMP to support differentiation and maturation. By approximately Day 30, cortical neurons stably expressing NeuN and MAP2 were obtained and, after confirmation by immunofluorescence, used for splicing analyses and functional assays.

Protein extraction and immunoblotting (Western blots)
Lysis and quantification: Cells were lysed in RIPA buffer (Beyotime, P0013B) supplemented with EDTA-free protease inhibitor (MCE, HY-K0010) and phosphatase inhibitor (MCE, HY-K0021). Lysates were centrifuged at 12,000 g for 15 min at 4°C, and supernatants were collected. Protein concentrations were determined by the BCA assay (Beyotime, P0012). Equal amounts of protein were mixed with SDS loading buffer and denatured at 95°C for 10 min.

Electrophoresis and transfer: Proteins were separated by 10% SDS–PAGE and transferred to PVDF membranes (Millipore Sigma, IPVH00010). Membranes were blocked in 5% nonfat milk in Tris-buffered saline (TBS) for 1 h at room temperature, incubated with primary antibodies (diluted in blocking buffer) overnight at 4°C, washed in TBST (TBS + 0.1% Tween-20, 3 × 5 min), and then incubated with HRP-conjugated secondary antibodies (ABclonal, AS014 or AS003; 1:5,000) for 1 h at room temperature.

Detection and quantification: Signals were developed by enhanced chemiluminescence (ECL) and imaged using a ChemiScope 6100 system (Clinx). Band intensities were quantified with Fuji (ImageJ). Details of all primary antibodies are provided in Supplementary Table 1.

Extraction of RIPA-insoluble proteins from AD hippocampi
Approximately 2 μg of parahippocampal gyrus tissue from AD patients was homogenized on ice for 30 min in 300 μL prechilled RIPA buffer (50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 1% Triton X-100; 0.1% SDS; 2 mM EDTA; supplemented with protease and phosphatase inhibitors). The homogenate was centrifuged at 500 g for 5 min at 4°C to remove debris; the resulting supernatant was collected and further centrifuged at 16,000 g for 15 min at 4°C. The final supernatant was designated the RIPA-soluble fraction, and the pellet the RIPA-insoluble fraction.

The pellet was washed once with prechilled RIPA buffer, resuspended in 10% SDS lysis buffer (10% SDS; 250 mM Tris-HCl, pH 6.8), and subjected to water-bath sonication until fully solubilized. Protein concentrations of both fractions were determined by the BCA assay (Beyotime). The RIPA-insoluble fraction was used for subsequent immunoblot analysis of pTDP-43 (S409/410).

Immunofluorescence staining
HEK-293T, M17, and HeLa cells were seeded on glass coverslips in 24-well plates and transfected/treated as described above. Cells were fixed at room temperature with 4% paraformaldehyde in PBS for 15 min, then permeabilized with 0.5% Triton X-100/PBS (Biosharp, BS084) for 15–30 min. Blocking was performed with 3% goat serum in PBST (0.1% Triton X-100/PBS) for 1 h at room temperature.

Primary and secondary antibodies were diluted in blocking buffer and incubated either overnight at 4°C (up to 48 h) or for 1–2 h at room temperature, as required. After staining, samples were washed in PBST (3 × 5 min). Coverslips were mounted using an aqueous mounting medium containing DAPI (ZSGB-Bio, ZLI9557). Images were acquired and analyzed using a Zeiss LSM800 confocal microscope or an OLYMPUS IX81 inverted fluorescence microscope. Details of all primary antibodies are provided in Supplementary Table 1.

Protein crosslinking
To stabilize native intracellular interactions, cells were crosslinked with disuccinimidyl glutarate (DSG; MCE, HY-114697). Cells at ≈80% confluence grown in 10-cm dishes were washed once with PBS and the medium was replaced with PBS containing an EDTA-free protease inhibitor (MCE, HY-K0010). DSG was added to a final concentration of 400 μM, and cells were gently rotated at room temperature for 30 min. The reaction was quenched with 20 mM Tris (pH 7.4) at room temperature for an additional 15 min. Cells were then collected by centrifugation at 1500 g for 5 min.

EdU cell proliferation assay
During the generation of a stable TDP-43 knockdown HEK-293T cell line, we observed markedly reduced proliferation. To verify this, DNA synthesis was assessed using an EdU-555 Cell Proliferation Kit (Beyotime, C0075S). Cells were infected with lentiviruses (shCTR and shTDP-43) and cultured for 10 days; on Day 10, cells were seeded at equal density onto sterile round coverslips placed in 6-well plates. After attachment and entry into logarithmic growth, EdU working solution was added (final concentration 10 μM) and cells were incubated for 2 h at 37°C in 5% CO₂.

Following incubation, cells were washed twice with PBS and processed according to the manufacturer’s protocol: fixation (30 min), permeabilization, Click-reaction fluorescent labeling, and DAPI nuclear staining, all performed at room temperature protected from light. Images were acquired with an Olympus SLIDEVIEW VS200 fluorescence microscope. EdU⁺ cells exhibited red fluorescence, whereas DAPI stained nuclei blue to determine total cell counts. The fraction of EdU⁺ cells (EdU⁺/total cells) was quantified with ImageJ and used to evaluate DNA synthesis and proliferative capacity.

NMD blockade
To assess whether aberrantly spliced transcripts induced by TDP-43 knockdown are regulated by nonsense-mediated mRNA decay (NMD), NMD was inhibited in M17 cells by pharmacological and genetic approaches.

Pharmacological inhibition: Cycloheximide (CHX; MCE, HY-12320) was added to shTDP-43 and shCTR cells at 200 μg/mL. After 6 h of treatment, samples were collected for RT–qPCR.

Genetic interference: shRNAs targeting UPF1 (shUPF1#1/#2) were constructed and co-infected with shTDP-43 into M17 cells. Total RNA was extracted after 96 h.

Regulation by NMD was inferred by comparing the expression of aberrant splicing transcripts (e.g., PKN1-5a1) before and after CHX treatment or UPF1 knockdown.

Protein stability assays
DOX-inducible assessment of PKN1 and PKN207 stability in HEK-293T cells: HEK-293T cells were transfected with Tet-PKFL or Tet-PKN207 constructs. After 24 h, doxycycline (DOX; 1 μg/mL; MCE, HY-N0565) was added for 24 h to induce expression, after which induction was terminated by replacing the medium with DOX-free high-glucose DMEM. CHX (200 μg/mL) was then added to inhibit de novo protein synthesis, and cells were harvested at 0, 4, 8, and 12 h. Total proteins were analyzed by immunoblotting, and band intensities across time points were compared to evaluate the degradation rates of PKN1 and PKN207.

PKN1 stability under TDP-43 knockdown in N2a cells: In N2a cells, a CHX chase was initiated on Day 5 after establishing TDP-43 knockdown. Cells were switched to fresh serum-free medium, then CHX (200 μg/mL) was added, and samples were collected at 0, 4, 8, and 12 h. Total proteins were subjected to immunoblotting, and degradation rates of PKN1 were compared between knockdown and control groups to assess the effect of TDP-43 on PKN1 stability.

RNA extraction and cryptic exon detection
Cultured cells and neurons were directly lysed in TRIzol (Takara, 9109) to extract total RNA; human brain tissue was mechanically homogenized and likewise extracted with TRIzol. cDNA was synthesized using a reverse transcription kit (ABclonal, RK20429). RT–PCR was performed with OneTaq 2× Master Mix with Standard Buffer (Vazyme, P222-03-AA) under the following program: 95°C for 5 min; 35 cycles of 95°C for 15 s, 60°C for 15 s, and 72°C for 15 s; followed by 72°C for 5 min.

Detection of the PKN1 cryptic exon 5a1 used primers: forward (ceF1), 5′-CAGCACCAAGGACCGGAAGCTG-3′; reverse (ceR1), 5′-TCAGGGCCAGTCCTTGGGTGT-3′. Detection of the alternative 3′ splice site (A3SS) used primers: forward (ceF1, as above); reverse (ceR2), 5′-GTCAGGACTCCCTGTGAGCGTGGAAATA-3′. Amplification of total PKN1 transcripts (non–junction-spanning) used: F1, 5′-ATGTTGCAGGACAGTAAGACCAAGA-3′; R1, 5′-CGCAGTACGTTCTTGGCACCC-3′.

All PKN1 minigene constructs and their mutants were also assayed by RT–PCR with the exon-spanning primers F1/R1 to simultaneously detect the 5a1 and A3SS events. PCR products were resolved on 2–3% TAE (Tris-acetate-EDTA) agarose gel and imaged with the SH-510 bioimaging system (Hangzhou Shenhua Technology Co., Ltd.). Band intensities were quantified in ImageJ.

RT–qPCR
RNA extraction and cDNA synthesis were performed as described above; primers were identical to those used for RT–PCR. Reactions employed a SYBR Green kit (ABclonal, RK21203) on a real-time PCR system (Bio-Rad, CFX2000). The 20 μL reaction contained: 10 μL 2× SYBR Mix, 0.5 μL forward primer (10 μM), 0.5 μL reverse primer (10 μM), 50 ng cDNA, and ddH₂O to volume. Cycling conditions were: 95°C for 5 min; 35 cycles of 95°C for 15 s and 60°C for 10 s; followed by a melt curve to verify specificity. Relative expression was calculated using the ΔΔCt method.

RNA-Seq analysis of ALS patient samples
Data source and preprocessing. Public human brain RNA-seq data were obtained from GSE153960 (NYGC ALS Consortium)17. The project applied unified QC, including verification of tissue type, brain region, and sex using >250 marker genes. For samples included in this study, the median sequencing depth was 4.2× 10⁷ read pairs (range: 1.6 × 10⁷–1.67× 10⁸). Raw reads were processed with the RAPiD-nf (Nextflow) pipeline: adapter trimming with Trimmomatic v0.36; alignment to GRCh38 (hg38.primary_assembly) using STAR v2.7.2a with GENCODE v30 annotation; and gene-level quantification with RSEM v1.3.1. Unless otherwise specified, downstream analyses used only uniquely mapped reads (STAR criteria: NH=1 and MAPQ ≥255). When library strandedness was not reported, it was inferred with RSeQC infer_experiment.py, and strand corrections were applied accordingly in adapter annotation and sequence motif checks.

PKN1 full-length expression. Based on the GENCODE v30 PKN1 locus (chr19:14,433,305–14,471,895; GRCh38), RSEM outputs were summarized and TPM was computed: transcript counts were length-normalized to RPK, then normalized by the sum of RPKs across all transcripts within each sample to enable cross-sample comparison.

Splice-junction extraction and clustering. To obtain splicing evidence across the PKN1 region, splice junctions were extracted from BAM files with RegTools v0.5.1 (minimum anchor length 8 bp; maximum intron size 500 kb) and clustered with LeafCutter v0.2.9 (min_reads_per_cluster=30, min_cluster_fraction=1e-4) to generate a sample-by-junction count matrix. Junctions mapping ambiguously, of low complexity, or overlapping genomic blacklist regions were excluded. For stability, only junctions with ≥5 uniquely mapped reads in at least one group were carried forward.

Definition, coordinates, and deconvolution of PKN1-5a1. The cryptic exon of interest, PKN1-5a1, lies within the intron between exons 4 and 5 at chr19:14,445,088–14,445,214 (127 bp, GRCh38). A3SS event is present in the same region (chr19:14,445,088–14,445,317). To prevent A3SS contamination in quantification, we applied endpoint-strict matching and event typing: (1) Paired cryptic-exon junctions (CE-pair): Exon4 → CE boundary (14,445,088) and CE boundary (14,445,214) → Exon5. Both junctions must be supported by uniquely mapped reads within the same sample called as “PKN1-5a1 detected”. (2) A3SS-exclusive junctions: Exon4 → A3SS site (14,445,317) or A3SS site → Exon5 were labeled as A3SS and not counted toward the CE-pair. (3) The CE segment had to reside entirely within a single annotated intron, not overlap any annotated exon, and, after strand correction, both splice sites had to conform to the GT–AG rule (CT–AC on the negative strand). (4) N_mapped denotes the total number of uniquely mapped reads in a sample (primary, unique-mapped; counted by read end), excluding unmapped, secondary, or supplementary alignments; for paired-end data, each read end was counted separately.

RPM metric and definition of “positive” samples. For each sample, the relative abundance of the CE-pair was quantified by RPM (reads per million mapped reads): RPM up→CE = C up→CE / N mapped ×10⁶, RPM CE→down = C CE→down / N mapped ×10⁶, and the total CE-pair abundance was reported as: RPM pair = RPM up→CE + RPM CE→down. To mitigate depth effects, N mapped was the sample’s total uniquely mapped reads. A sample was called positive only if both up→CE and CE→down junctions each had ≥1 uniquely mapped read and their endpoints strictly matched the CE boundaries. For cross-sample summaries/plots, only samples in which at least one CE junction had ≥5 uniquely mapped reads in any group were displayed.

Between-group comparisons and statistics. Group differences (e.g., ALS vs. control or across brain regions) were evaluated using RPM pair as the primary metric and the Wilcoxon rank-sum test, with Benjamini–Hochberg correction (FDR <0.05 as the significance threshold). Robustness was additionally assessed by comparing positive-sample proportions with Fisher’s exact test (BH-FDR correction). To reduce false positives, we imposed a consistency filter: in cases, both CE-junction RPMs had to increase concordantly, and in controls the median RPM pair was approximately 0.

Visualization and software. Data processing was performed in R v4.2.2. Plots were generated with ggplot2, ggpubr, and patchwork. Distributions of RPM pair were shown as violin plots overlaid with boxplots and individual sample points.

Mount Sinai AD transcriptome analysis
Bulk RNA-seq data from the MSBB project (brain area BA36; AMP-AD; Synapse ID: syn3159438)19 were analyzed. Adapters were trimmed with Trimmomatic v0.36; reads were aligned to GRCh38 using STAR v2.7.2a with GENCODE v30 annotation; TPM was quantified with RSEM v1.3.1. Downstream analyses used only uniquely mapped reads (primary, unique-mapped; counted by read end).

Splice junctions were extracted with RegTools v0.5.1 (minimum anchor 8 bp; maximum intron 500 kb) and clustered with LeafCutter v0.2.9; only junctions with ≥5 uniquely mapped reads in at least one group were retained for comparison. PKN1-5a1 was defined as the concurrent presence of the paired junctions Exon4 → 14,445,088 and 14,445,214 → Exon5 (endpoints strictly matched; GT–AG/CT–AC after strand correction). Junctions for the co-located A3SS event (14,445,317) were labeled separately and excluded from the CE count. For each sample, RPM was computed as: RPM up→CE = C up→CE / N mapped ×10⁶, RPM CE→down = C CE→down / N mapped ×10⁶, and the total paired CE abundance was: RPM pair = RPM up→CE + RPM CE→down, where N mapped denotes the sample’s total uniquely mapped reads.

Group comparisons were performed between Braak 0 and Braak 1–6, using log₂(RPM+1) and log₂(TPM+1) as inputs; statistical testing used the Wilcoxon rank-sum test.

Statistical analysis
Statistical analyses were performed using GraphPad Prism 8.0 (GraphPad Software Inc., San Diego, CA, USA). For comparisons between two groups, two-sided unpaired Student’s t-test or two-sided Wilcoxon test was used as indicated in the figure legends. For comparisons among multiple groups, one-way ANOVA was applied where appropriate. For large-scale transcriptomic datasets, including ALS and AD patient cohorts, statistical comparisons were performed using a two-sided Wilcoxon rank-sum test. For analyses involving comparison of regression slopes, analysis of covariance (ANCOVA) was used. Correlation analyses were performed using Pearson correlation, and correlation matrices were visualized as Pearson correlation heatmaps. A P value ≤0.05 was considered statistically significant. Data are presented as mean ± SEM unless otherwise stated.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript, Yang, Wang, Yan, Luo and colleagues focus on the relationship between PKN1 missplicing and the RNA binding protein TDP-43 in the development of neurodegenerative disease. The manuscript bridges bioinformatics, cell biology, animal modeling, and examinations in human post-mortem material to define the conservation of PKN1 missplicing and the potential toxicity of PKN1 cryptic peptides that emerge upon TDP-43 loss of function.\nStrengths of the study include the broad and diverse methods used to examine the influence of TDP-43 on PKN1 splicing, as well as the resulting impact of PKN1 5a1 cryptic peptide expression. The mechanistic studies demonstrating how TDP-43 regulates PKN1 splicing is particularly strong. Further, inclusion of patient samples helps ground the work and indicates some level of translatability.\nEven so, several issues limit my enthusiasm for the manuscript. Conceptually, it is unclear how a pan-regulated transcript such as PKN1 could be responsible for cell type-specific effects of TDP-43 dysregulation in disease. Second, although the authors attempt to demonstrate PKN1 missplicing in Alzheimer’s disease tissue, there is no exploration of similar relationships in ALS or FTLD-TDP, conditions that frequently referenced by the authors themselves for their association with TDP-43 proteinopathy. Third, and perhaps most importantly, the authors make several conclusions that are not fully supported by the data.\nAbstract:\nThe abstract fails to demonstrate the relevance of the work for those who are not familiar with TDP-43. The abstract also leaves out crucial information about PKN1 and why readers should be interested in this.\nIntroduction:\nLine 49: TDP-43 pathology in AD is now recognized as LATE.\nThe authors leave out a more balanced discussion of other cryptic splicing targets of TDP-43, such as those described in PMID: 38277467 and 38278991\nLine 77: pan-neuronal reduction in PKN1 contradicts the vulnerability of select populations (i.e. motor neurons) to degeneration in ALS\nLine 90: the findings suggest that PKN1 cryptic splicing is sufficient for toxicity, but NOT that PKN1 cryptic splicing is critical - this would require additional experiments, asking whether prevention of PKN1 cryptic splicing in ALS models can reverse phenotypes.\nResults:\nFigure 1: The rationale behind cross-referencing data from human cortex, M17 cels and HeLa cells is unclear - cell type-specific differences in TDP-43 splicing are likely important for the disease itself. Otherwise, ALS would be a systemic disease with multiple cell types affected. As it stands, it is unclear whether generalized splicing deficiencies from TDP-43 loss would be relevant to a motor neuron disease such as ALS.\n1A: this panel is very crowded, making it difficult to determine relevant expression changes.\n1D: as it stands, it is unclear whether the targets increased in the ‘Hela rescue’ condition actually went down in the TDP43 KO condition compared to WT cells.\n1F: this panel requires additional explanation. The legend leaves out critical information, including where the data came from and how the genes were identified.\n1K: these images are not very convincing, as 1/2 of the ds-Red positive cells (including the one that is assessed by line scanning in 1L) look like they might be dead\nLine 112-113: the findings show that PKN1 is regulated by TDP-43, but say nothing about its importance (i.e. whether it is a ‘key downregulated gene’)\nFigure 2J: please show effects of CHX at baseline (l.e. in controls or cells without TDP-43 shRNA)\n2M: these data are interesting, but without quantification it is difficult to determine the magnitude and reproducibility of the effect\nSup Figure 2: without additional experiments, the authors cannot conclude whether TDP43 KD-induced changes in PKN1 are ‘a compensatory response rather than a direct effect’ as stated in line 189. Data in Fig. S2D show PKN1 reduction in primary neurons with TDP43 KD, contrary to stable PKN1 levels in N2a cells (Fig. S2G). This suggests that the regulation by TDP43 is cell type-dependent, rather than indirect.\nSup Fig 6: How do the authors account for the observation that TDP43(F147/149L) induced cryptic splicing of PKN1, while also repressing PKN1-5a1 expression in the background of TDP-43 KD. Both TDP43(F147/149L) and TDP43(dRRM1) exhibit reduced RNA binding activity, and may result in dominant-negative loss of function due to sequestration of endogenous TDP-43. Therefore, both would be expected to behave similarly.\nIn addition, the authors should explain in more detail the rationale behind TDP43(90-414) and TDP43(N6A) variants. To conclude that these variants disrupt PKN1 splicing by preventing TDP43 dimerization, the authors need to show disrupted dimerization; alternatively, these mutations may affect TDP43 localization (by impacting the NLS or other means, as in PMID: 37431963)\nSup Fig. 7: how do the authors account for the selective effect of the M3337V mutation in splicing activity of TDP43? This is inconsistent with the notion that PKN1 is a conserved missplicing event in ALS.\nFigure 4: if cryptic spliced PKN1 is degraded by NMD, why is the N207 cryptic peptide so abundant? Does the abundance change when using inhibitors of NMD that do not affect protein translation (unlike CHX)?\n4D: data in this panel could be misleading. It appears that the overall expression of the 5a1-containing construct is much lower than the WT version, and the nuclear localization is due primarily to the low level expression in the cytoplasm. The low levels by IF is also in direct contrast to the experiments in Fig. 2 G showing elevated protein levels by WB.\nFigure 5D: data in this panel are not convincing for an upregulation of antibody reactivity with TDP43 KD. First, the negative control should include transfection with a plasmid expressing a NT shRN and a similar marker such as DsRed. Second, the antibody signal in DsRed (+) cells in the bottom panel (shTDP-43) appears no different from signal in DsRed (-) cells. Third, there is no quantification of results.\nFigure 6: immunofluorescence images in panel 6G are not very convincing for specific E29426 reactivity. It appears that the signal closely overlaps pTDP43, and at this magnification lipofuscin staining is a concern.\nFigure 7: ‘cortex’ is misspelled in panel A. It is also important to show normal (full length) PKN1 mRNA in all brain regions as well.\n7A: it is unclear how PKN1 could be regulated by TDP43 in multiple cell types (Fig. 1), regions of the brain (Fig. 7) and even serum, but not in cerebellum.\n7A: in addition, please show distributions via violin plot or other, similar, plot\n7C: there appear to be 2 separate regressions between TARDBP and PKN1 expression, in both brain and blood. Can the authors account for this?\n7D: please show RT-PCR for flPKN1 mRNA as well.\n7E: no loading control is shown for these studies\nFigure 8: it is difficult to place these findings in perspective without comparison to PKN1 overexpression or KD, particularly the latter if the authors believe N207 has dominant negative effects by dimerizing with WT PKN1.\nFigure 9: it is unusual that changes in NeuN were noted, but not synaptophysin or PSD95. Often changes in synaptic markers precede frank neuron loss (i.e. PMID: 36970154)\n9H: some AD patients have elevated NEFH/FM, whereas others have elevated NEFL in comparison to controls, with variable overlap with N207 abundance. How do the authors account for this?\nDiscussion:\nLine 520: please reference prior studies that observed similar phenomena, including PMID: 32649883, 31018129\nLine 533: the statement regarding ‘molecular explanation for the clinically observed NEFL upregulation’ could be misleading. Neuronal injury results in cleavage and secretion of NEFL fragments that can be detected in CSF — this is not unique to ALS or other TDP-43 related diseases, and instead can be seen with almost all forms of neuronal injury.\nLine 540 and 543: while this reviewer appreciates the condensed and clear discussion, many of the conclusions are not fully supported by the data. As discussed above, the conservation of PKN1 missplicing by TDP43 is interesting, but does not fit with the selectivity of ALS for neurons, and in particular motor neurons. The fact that the PKN1 5a1 cryptic exon can be directed in SOME but not all patients is not promising for a diagnostic assay, making a negative result difficult to interpret. Further, the sensitivity of the N207 antibodies remains unclear without additional studies, as this was not specifically tested in the current manuscript.\nOf note, while TDP-43 pathology in AD is common, this is now referred to as comorbid limbic predominant age-related TDP43 encephalopathy (LATE).", "reviewer_2": "In the manuscript by Yang M. et al, the authors describe a novel cryptic peptide generated by a TDP-43 regulated cryptic exon in PKN1 upon loss of TDP-43. After biochemical and molecular characterisation and generation of antibodies against the cryptic peptide, the authors investigate the cryptic peptide in AD brains with TDP-43 pathology as well as tissue and blood from ALS patients. They also investigate the impact of PKN1-N207 in mouse models describing neuronal dysfunction and cognitive impairment upon its expression as well as upregulation of NEFL in the hippocampus. The manuscript suggests direct involvement of this cryptic splicing event in disease, also providing a potential new biomarker and will be of high interest to the field. The amount of work the authors have invested in this study is impressive but there are several aspects that need to be addressed before publication.\n## Major points.\n* Point 1. The authors are inconsistent in the n numbers shown in western blots, RT-PCRs etc. across cell lines.\nIn Figure 1G-J, the authors present RNAi of TDP-43 in different cellular systems, showing triplicates for M17 cells (Fig. 1G), unicates for iNeurons (Fig. 1H), duplicates for Hela (Fig. 1I), and 293T cells (Fig. 1J). The authors should present triplicates in all these panels.\nIn Fig. 2C-D and 2F-J the number of shown replicates vary. Please make them triplicates.\nIn Fig. 3G, please show triplicates.\nIn Fig. 4A,B,G the replicate number varies from 3-4. Please make it triplicates.\n* Point 2. In Fig. 2J and NMD experiments: Translation inhibition is fairly indirect. It is an insufficient way of assessing whether a transcript is a NMD target in the absence of additional evidence, given the pleotropic effects of cycloheximide treatment and cascading effects of general translation inhibition. The authors should include either complementary UPF1 and SMG1 knockdown or inhibition of NMD using inhibitors specific to the pathway, e.g. iSmg1 (PMID: 23021994). The small stabilization of the PNK1-5a1 mRNA by roughly 2-fold upon cycloheximide treatment further suggests that it would be a poor NMD target. Quantitative RT-PCR would be preferred detection method.\nAlso, the steady accumulation of the cryptic PNK1(N207) peptide originating from PKN1-5a1 is contradictory to PKN1-5a1 being an efficient NMD target (that would prevent the production of the faulty truncated protein).\n* Point 3. The use of the dCAS13a-GFP fluorescence reporter is a nice idea but no images are shown in Fig. 2M under control knockdown conditions nor is any quantitation given, e.g. # punctae per nucleus, that would justify statement on lines 177-178 of the manuscript regarding nuclear accumulation. Please show images -/+ TDP-43 knockdown for both crRNA1 and crRNA2 and quantify the fluorescence signal.\n* Point 4. In Fig. 3C, unless I am missing something, the primer pairs Rip-F1 + RipR1 and Rip-F3 + Rip-R3 are not used here. Please clarify the situation and/or modify the figure accordingly.\n* Point 5. The EMSA data in Fig. 3R provides evidence of TDP-43 binding. However, for statements regarding different affinities (as in the figure legend), the authors should perform serial dilutions of TDP-43 protein against the RNA for each construct, which would be necessary do measure the affinity. Alternatively, the authors can use another quantitative assay, e.g. thermophoresis.\n* Point 6. Overexpression experiments in Fig. S6 indicate a dominant negative effect of the TDP-43 deltaRRM1. These experiments were also performed under depletion of endogenous TDP-43 and rescue with presumably RNAi-resistant mutant TDP-43 (please clarify) in Fig. S6F. However, no proof of the depletion of endogenous TDP-43 is shown neither was the splicing reporter quantified. Also n numbers seem to be missing. As shown, under TDP/43 depletion, the F147/149L mutant appears to rescue the PNK1 ex4-ex5 splicing but not the deltaRRM1 mutant or the F227/229L mutant. This suggests rather that RRM2 is required for proper PKN1 splicing and that deltaRRM1 is a dominant negative mutant altogether?\nFurthermore, the immunofluorescence should include TDP-43 staining to assess whether endogenous TDP-43 is sequestered from other regions into the nuclear punctae together with the ectopic HA-tagged mutant TDP-43.\n* Point 7. Throughout the manuscript the authors use structural modeling, but they do not seem to provide details for the modeling in the methods section nor figure legends. Without quality indicators or confidence metrics for the alphafold 3 models, these cannot be assessed. The authors should provide these and update the methods accordingly.\n*Point 8. The materials and methods section is lacking essential information which needs to be corrected 1) For the RNA-Sequencing and Astral-DIA experiments there is not sufficient information to replicate the experiments as apart from which company has done it, no information is given 2) Identity or culture conditions for iPSCs on lines 747-749. 3) I cannot find any details on how iPSC-derived neurons were differentiated from them for use in Fig. 1G-J referenced on lines 115-116, in Fig. 2F referenced on lines 149-150, and Fig. 2G. Confusingly, in the figure panels they are labelled as iNeurons whereas the figure legends calls them just “iPSC”.\n4) how were primary cortical neurons isolated from E18.5 mouse embryos (lines 698-701)?\n* Point 9. Given that cycloheximide treatment induces the PKN1-5a1 transcript generating the cryptic peptide (as shown in Fig. 2J), it is not suitable to assess the stability of the cryptic peptide using it in Fig. 4G. Indeed, it seems impossible to explain based on protein stability alone that the PKN1(N207) peptide accumulates under a translation elongation inhibition regime. Instead, the expression and stability of the cryptic PKN1(N207) peptide vs. the normal PKN1 should be verified by proteasome inhibition (e.g. MG132), inhibition of translation initiation (e.g. edeine or pactamycin), and another translation elongation inhibitor (e.g. emetine). Ideally, these experiments would be also repeated with cDNA constructs that will not be subject to alternative splicing and differential transcript expression to assess the protein stability independently.\n* Point 10. In the testing of the PKN1(N207)-specific antibody E29426 in Fig. 5H, the authors use a fluorescent dsRed maker for the TDP-43 shRNA expressing vector, but this does not assess the TDP-43 depletion or else the author need to describe the system more clearly. Irrespective of the dsRed expression (and supposedly TDP-43 depletion), many cells in the shown image still stain strongly with E29426. Therefore, the authors need to include a TDP-43 staining to assess the TDP-43 knockdown and perform a quantitative analysis. In this way, they could also correlate the E29426 signal with TDP-43 expression in individual cells, which would be more convincing evidence for the antibody’s specificity in the immunofluorescence staining. As present, it does not seem convincing.\n* Point 11: In Fig. 9H, the E29426 western blot signal is not always visible in full. Please present a wider panel or a non-cropped image here.\n* Point 12: Regarding the AD diagnosis in donor tissues, the Materials and methods state “Clinical and neuropathological diagnoses were made as described previously. Subjects were screened based on both clinical and neuropathological assessments.” on lines 674-676. However, the authors do not provide these references nor what assessments or disease staging were used in support of the diagnosis. Were standardized assessments used as inclusion criteria and, if so, which? Likewise, the use of standardized pathological classification is required to place some of the results in the right context, e.g. Braak staging. The authors should clarify these points.\n## Minor points\n* Fig. 1A is very busy and would benefit of fewer genes being highlighted. It would be also clearer if the NA samples would be filtered out in their respective tissue groups.\n* Fig. 1B: Upset plot might be clearer representation.\n* Fig. 1F: 'Cortex' is misspelt. The plot is a bit chaotic and the x-axis label is missing. I assume it is differential expression as log2(FC). The y-axis should be re-ordered by value.\n* Fig. 3I-P The presentation of the PKN1 minigene constructs and data is very busy. In particular, panel 3O might profit from the constructs being presented in a column with the data next to them.\n* Fig. 4G: The figure legend states that PKN1(N207) is unstable, however text states that it “exhibited markedly greater stability”\n* Fig. 4H: Which peptide is green, cyan, or grey, PKN1(FL) or PKN1(N207)? What is the inset showing? The inset does not match the indicated square in the full structure.\n* Fig. S7C-D. The figure legend text is poorly worded. I guess 'Blue color represents spatially interacting proteins and nucleic acids' means that the interacting residues are colored blue, not the whole protein (the rest of which is green) nor RNA (orange).\n* In Fig. 6D, Fig. 6F and Fig. 9H, please remove donor IDs from the top panels and use 'Control-1, Control-2, etc.' and 'AD-1, AD-2, etc.' or similar instead. Also explain what 'normal' means in this context.\n* Fig. 7A: 'Crotex' returns in all four panels! Please correct the spelling.\n* In the figure legend to Fig. 8E-F, the authors should clarify what ‘ARW’ abbreviates.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_4": "The authors have conducted a very large investigation into the role of PKN in TDP-43 diseases.\nThey describe a cryptic exon that leads to both RNA degradation and also cryptic peptide production. The associated development of a novel antibody targeting this cryptic peptide is a novel aspect of the study. The work goes on to identify this event in patient material and then test whether it is toxic in mice.\nThere is novelty in the work, however there are major points that need to be clarified.\nIn many instances the authors perform multiple analyses to show the same result – they could present some in supplementary in order to streamline the reading. On the other hand, several points need further experiments to be supported.\nOverall, this work provides insights into a novel cryptic peptide and its potential implications in disease, but more evidence needs to be provided to support the findings, methods are highly lacking and the discussion of the findings are limited.\nThe relevance of the initial human tissue panels of Figure 1 is of unclear relevance. Expression can be driven by cell type composition. The reduction at expression levels is very indirect – and indeed PKN is reduced also in the cerebellum, where there is no TDP-43 pathology. Similarly, it is unclear why HDGFL2 should be downregulated - this is likely caused by cell population differences. The authors should focus on the cell data from figure 1 splicing data from figure 2 to introduce the work.\nThe NMD and lack of RNA cytoplasmic localisation (Cas13 experiments showing nuclear localisation) are hard to reconcile with the detection of the peptide. Numerous points need to be therefore clarified. The authors need to perform PKN KD in WT and TDP-KD cells and show that both the FL and truncated cryptic peptide bands shown in figure 4B disappear. Further, given the wealth of other systems they have presented, it would be important to show the truncated protein in multiple lines. The authors should assess the change in localisation of the FL and peptide with cell fractionation and more reliable quantifications of ICC in figure 4.\nThe CHX experiments in Figure 4 need to be explained. Given the translation inhibition action of CHX, how do the authors reach any conclusions? CHX may increase the RNA t/2 inhibiting NMD, but why would it impact on the protein levels?\nThe localisation in human tissue presented in figure 6 is not very convincing. It is not necessary to use all the first part of the figure to show Tau and pTDP stainings. The authors need to instead present appropriate numbers and quantify co-stainings with the cryptic peptide in much greater detail. The current low magnification image presented is not convincing.\nDetection of the CE and peptide in the periphery is interesting. Authors should show results also when trying to detect both cryptic and non-cryptic bands in figure 7D. The work on n=2 in blood has to be significantly expanded. The difference between plasma and serum is unclear and the band seems to be smaller than in other blots.\nIn regards to the mouse experiments, can the authors confirm that the controls are the expression of FL-PKN, and not just empty GFP AAV? This is essential and should be made more clear. If so, a blot with anti-GFP needs to be performed to compare the protein expression levels. As well as RNA quantifications in order to control for overexpression levels.\nThe proposed link between NEFL and PKN1 is interesting but remains somewhat unclear. NF levels are typically thought to increase in patients due to neuronal loss. The suggestion that levels are high due to overexpression is novel, but needs to be demonstrated. Characterization of the induced overexpression and whether it happens through changes in translation, transcription, stability etc. should be investigated. The discussion could benefit from a more nuanced approach, either softening this claim or providing additional context to clarify the potential relationship.\nParts of the manuscript, such as the section on M337V are weak (e.g. the fact that m337V does not bind the RNA equally is not sufficiently supported; why does M337V have a different molecular weight in Fig. S7E?) and are not essential for the manuscript and could be worth removing unless significantly improved.\nSpecific points:\nText\nThe analysis of post-mortem for example, if it’s a neuronal gene the fact that it goes down in postmortem is unsurprising, given neurons die in disease.\nLine 49. It is not the majority of FTD cases, but around 45% (another 40+% of FTD is characterised by tau aggregation, and other cases are due to agregation of FUS or other/unknown genes). In general it is better to cite the original work reporting a data/concept. Riva et al, citation is not the appropriate one here.\nLine 52. Is it “deletion”/knockout or “reduction”/knockdown?\nLine 72. Manser et al work talk about an interesting mechanism by which PKN1 may disrupt neurofilament assembly, this should be brought up in the discussion section.\nLine 72. Xiao et al never talk about PKN\nLine 74. Gudi et al is a purification paper – what is the relevance?\nLine 129-130. Are these the same M17 cell lines? Or iNeurons? Or new ones?\nLine 132. Same CE can be found in Humphrey et al, BMC medical genomics 2017, so would cite this paper and remove the word “novel”.\nLine 135. “PKN1-5a1 CE” instead of “PKN1 gene”.\nLine 182. referencing for claims about UNC13A and STMN2.\nLine 398. Reference to original works, not to introduction of unrelated works.\nLine 435. Why would the tissue expression of the native proteins be the reason to find the mis-spliced events in those tissue?\nLines 441-443. This cohort needs a better characterization.\nMethods comments\nThe methods are extremely lacking and are not reproducible.\nMissing methods for CHX experiment.\nMissing methods for analysis of RNA-sequencing for post-mortem publicly available data. Missing for differential gene expression throughout, for both in-house RNA-sequencing and publicly used data,e.g. Ferguson HeLA analysis.\nUnclear how figure 1A is generated from methods, is this the downloaded RSEM files or the data produced by the referenced paper? How was differential expression assessed?\nNo information on how the RNA-seq from the m17 cells was analyzed either, what was used to align, what was used to call difference expression etc?\nAll supplementary figures revolve around Figure 3\nMissing primary antibodies\nMethods need referencing.\nFigure Comments:\nThere are two table 1 listed\nCrotex instead of cortex throughout figures.\nThe shTDP-43/Ds-red is used in figure 1, but Ds-red positivity meaning a TDP-43 KD is not mentioned until figure 5 this should be made clear in text.\nFigure 1A. Unclear why genes have been listed, figure is generally difficult to read, should perhaps highlight at max UNC13A, STMN2, PKN1.\nFigure 1A legend. Move the GSE number to methods.\nFigure 1A and 1B. Cerebellum shouldn’t be affected by TDP-43 pathology in ALS, so need to comment on why it is showing similar changes.\nFigure 1D. This comparison should probably also be made with either a line of text explaining what was done (e.g. we took genes which were upregulated after the Hela rescue in Ferguson with our own knockdown data) or a venn diagram, but generally given there are too many panels a line of text would do.\nFigure 1D legend. Move the GSE number to methods.\nFigure 1E. It should read “ALS intersection with M17”, not “VS”.\nFigure 1F. Why is it called “preALS”? Are the causative mutation for familial ALS known? They should be tested and reported.\nFigure 1G,H,I,J please add quantification for WB analysis. Also, supplementary materials should include full-sized, uncropped blots.\nFigure 1K. Please add other immunofluorescence replicates in supplementary materials.\nFigure 1L. what is a line profile?\nFig2\nFigure S1A,B,C. Center the IGV lines on the cryptic exon and highlight it.\nFigure 2A and 2B can be a single panel. Also, needs coherence between using U or T.\nFigure 2D is missing size (bp).\nI would move gels (2C,D,F,G,H) to supplementary and keep only quantifications in main figure.\nFigure 2I needs replicates.\nFig3\nFigure S2A and S2B. It looks to me that the splicing donor and acceptor sites are conserved between human and rat for example. Do you think it is possible for a cryptic exon (despite different) to be included in rat upon TDP-43 depletion? Comment on if the mouse neuronal lines also saw differential splicing? No methods given for this analyses so unclear what was done here. Line 189. Why should it be a “compensatory mechanism” and not a destabilisation at the protein level? Also, showing an IGV track is not sufficient to prove that RNA levels are not changed.\nFigure 3A is uninformative - would opt to remove entirely, alternatively showing the binding sites in a supplementary figure on IGV, or else wise,\nThis figure reports 131 bp but the CE is 127. Please elaborate.\nFigure 3B. It is unclear what the distribution of binding score refers to. No methods for this either.\nReference to Figure 3F is missing in main text.\nFigure 3H. elaborate on the GFP+/mCherry- cloud in the control condition?\nNaming of Figure 3I to 3P is confusionary.\nFig 3R - typo expoure\nFigure S4. Should read “Sanger” instead of “Snager”.\nFigure S5 methods. Also it would be interesting to see if people carrying these variants have ALS phenotype/NEFH elevation.\nLine 249. Need to write EMSA in full. Expand EMSA methods.\nFigure S6. Missing S6B in legend (should be with S6A).\nFigure S7A and B. please add the significant p values for clarity?\nFigure S7D should read V337 since V substitutes the M.\nFigure S7E. Please show quantification and add uncropped western blot.\nFigure S7F. I don’t understand if the quantification is coming from one or all three replicates.\nFig4\nLine 350. Should read “Figure 4C and D” instead of “Figure 4A and B”. Also, other replicates should be included in supplementary.\nFigure 4D. Which condition has 14 cells and which has 20?\nFigure 4G legend does not correspond to what is shown.\nLine 368. DSG should be written in full in the main text, not only in figure legend.\nFigure 4M. Why not using ACTB as a control as in all other figures?\nFig5\nLine 373. Only the last 3 aminoacid are different\nFigure 5B. Would be good to see the scores for the AF3 prediction, as usually the extremities of proteins are not well predicted.\nLine 379. Should be “5D”, not “5B”.\nLine 384. Should be “5E”, not “5C”.\nLine 387. Should be “5F”, not “5D”.\nMissing reference to Figure 5G in main text. I also don’t see the necessity of adding this panel to the main figure.\nLine 393. Should be “5H”, not “5E”. Also, include in supplementary the other replicates.\nFig6\nFigure 6B. I don’t see a big difference in the red fluorescence between AD and control. please quantify, better if adding an housekeeping protein to normalise?\nFigure 6D. Need coherent names between labels in figure and text.\nReference to Figure 6E is missing from the main text and figure legend. It would be nice to add colour/shape by sample to see if those with expression of PKN1-N207 are the same with reduced levels of PKN1. Also, is this an N-term or C-term antibody for PKN1?\nFigure 6F. There is a sixth AD case that is not described in supplementary table 1.\nFigure 6G. Show more cases.\nFigure S8. Why is the control positive for pTDP43 and PKN1-N207? Showing some quantification (and more cases) could help.\nFig7\nFigure 7A. “Cortex” instead of “Crotex”.\nFigure 7B. What is a sigmark? Also, I don’t see correlation values. Honestly this analysis between TDP-43 and PKN1 RNA expression is not needed and it does not lead in any logical way to the blood subsequent work - testing in blood is enough of a justification in itself given its a widely available biofluid, 7B and 7C don’t add to the message of the paper and should be removed.\nFig8\nCan you elaborate on the differences between males and females in the different experiments?\nFig9\nProteomics methods are missing.\nMethods for enrichment analysis are missing.\nFigure 9C. Is it enough to show only 4 genes in the heatmap? How does it compare with a full transcriptomics heatmap?\nFigure 9H. Please correlate this finding with those in Figure 6."}, "author_response": ""}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed many concerns, through additional experiments, removal of unverified data, and text editing. The revised manuscript is significantly more focused and stronger.\nI would suggest 2 additional edits revised manuscript:\n- the new Fig. 4 and Sup. Fig. 4 perform experiments with a \"NES\" deletion construct. I would strongly encourage the authors to remove these experiments, or the \"NES\" deletion conditions. Several manuscripts have provided convincing evidence showing that the \"NES\" is not a functional nuclear export sequence - see PMID: 29728608, 29728564, 29545601. These experiments will only raise doubt and confusion for readers, and are not necessary for the authors' conclusion that dRRM1 constructs induce a dominant-negative inhibition of TDP-43 splicing activity.\n- the new discussion states \"providing, to our knowledge, the first explicit link between toxicity mediated by a cryptic splicing product and the pathology of neurodegenerative diseases\" (line 768-770). I would suggest removing this or toning the language down, as there are now preprints and evidence that cryptic peptides elicit an immune response that could contribute to disease (PMID: 40667053).", "reviewer_2": "The authors have substantially revised their manuscript in response to our feedback and critical assessment, and most of the reviewers’ concerns have been adequately addressed.\nWe note that the revisions extend beyond the sections highlighted in yellow, and in several instances the changes are more extensive than what is immediately apparent.\nThere remain some points to address:\n* lane 169, \"thereby providing higher sequence specificity [than FISH]\", that is not true as a short gRNA will not necessarily have more specificity than an RNA-based FISH probe.\n* lanes 178-180, \"After cell fixation and immunostaining, TDP-43 was labeled with magenta fluorescence to verify knockdown efficiency.\" This is poorly phrased and thus an insufficent description of the experiment. Please clarify what antibodies were used for immunostaining. Is the GFP detected with an antibody after fixation?\n* Figure 1H. It is not clear what antibodies were used here. The TDP-43 antibodies in Supp Table 1 only list use for WB. Also, the dCas13-GFP is usually used in live cell imaging but the figure legend states immunofluorescence staining. Did the GFP signal survive fixation, or was this stained with a GFP antibody? Either way, there is still information missing.\n* In several images (Fig. 1H, DAPI; Supplementary Figs. 1E and 5H–K) we notice numerous small, extranuclear DAPI-positive puncta distributed in the cytoplasm. This pattern is suggestive of an active mycoplasma contamination, which can confound experimental outputs.\n* Figure 2. In the figure, \"cortex\" is still mispelled as \"crotex\".\n* lanes 584-587 \"Therefore, this antibody series are suitable for Western blots but not for immunofluorescence analysis. However, puzzlingly, PKN207-EGFP showed more nuclear localization, which is opposite to our results of nuclear–cytoplasmic fractionation.\" Since the antibody is not suitable for immunofluorescence, this discussion is not needed.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "1 August 20"}]

Decision letter