Nemo-like kinase disrupts nuclear import and drives TDP43 mislocalization in ALS

Probably not a substantial claim: may not pass S3 (load-bearing).

Exogenous NLK, like endogenous NLK, is both cytoplasmic and nuclear in distribution.

“Exogenous NLK, like endogenous NLK, is both cytoplasmic and nuclear in distribution (19).”

• cites [19] sourcePartly supported

  The Results (page unavailable, Fig. 1C) state that transfected NLK was detected in both cytoplasmic and nuclear compartments, supporting the claim about exogenous NLK. The passage does not establish that endogenous NLK has the same distribution. (LLM, conf=0.87)

  Experimental — accuracy not yet measured

May combine two claims.

TDP43 is primarily nuclear in healthy cells, in contrast to nuclear exclusion and cytosolic deposition characteristic of ALS.

“TDP43 is primarily nuclear in healthy cells, in contrast to the nuclear exclusion and cytosolic deposition of TDP43 that are characteristic of ALS (3).”

• cites [3] sourcePartly supported

  The Introduction states that TDP-43 is primarily nuclear and that pathological cytoplasmic inclusions containing it are characteristic of ALS. It does not state that nuclear exclusion is characteristic of ALS; a search for “nuclear exclusion” found no supporting passage. No page number is available in our copy. (LLM, conf=0.88)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

NLK overexpression reduces toxicity in Huntington's disease models, indicating context-dependent effects of NLK in neurodegeneration.

“Conversely, NLK overexpression reduces toxicity in Huntington’s disease models, indicating context-dependent effects of NLK in neurodegeneration (18).”

• cites [18] sourceCould not verify

  The Introduction mentions Huntington’s disease only as another polyglutamine disorder. The Results report that NLK overexpression exacerbates toxicity in an SBMA model, but our copy does not address whether it reduces toxicity in Huntington’s disease models (searched: Huntington, NLK overexpression and toxicity). (LLM, conf=0.76)

  Experimental — accuracy not yet measured

May combine two claims.

NLK depletion mitigates disease phenotypes in Drosophila and murine models of ALS, murine models of spinobulbar muscular atrophy, and murine models of spinocerebellar ataxia.

“NLK depletion mitigates disease phenotypes not just in Drosophila and murine models of ALS, but also murine models of spinobulbar muscular atrophy, and murine models of spinocerebellar ataxia (15–17).”

• cites [15] sourceNot checked — no full text of the source
• cites [16] sourcePartly supported

  The Results section reports that Nlk reduction improved survival and disease phenotypes in TDP-43 mouse models. The Introduction reports earlier alleviation of disease phenotypes in SCA1 and SBMA animal models. Our copy does not report a Drosophila ALS model, so that part of the claim is unsupported by this source. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites [17] sourcePartly supported

  The Results and Discussion report that reduced NLK expression mitigated disease phenotypes in Drosophila and mouse models of spinocerebellar ataxia type 1. Our copy does not report NLK-depletion outcomes in ALS or spinobulbar muscular atrophy models, so it supports only the SCA1 portion of the cited sentence. (LLM, conf=0.90)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing).

NLK is a proline-directed serine/threonine kinase.

“Nemo-like kinase (NLK)—a proline-directed serine/threonine kinase—promotes the mislocalization of TDP43 and other RNA-binding proteins by disrupting nuclear import.”

• Also stated in: § Introduction · plain

  “nemo-like kinase (NLK), a proline directed serine-threonine kinase that regulates cell differentiation (12), proliferation, and apoptosis (13, 14).”

• cites [12] sourceCould not verify

  The passages on nuclear import discuss karyopherins, MTR10 and C9orf72 dipeptide-repeat toxicity, but do not identify NLK or describe its kinase activity (Abstract; page unavailable). Searches for “Nemo-like kinase NLK proline-directed serine/threonine kinase” and “NLK TDP43 nuclear import kinase” found no passage addressing the attributed description. (LLM, conf=0.75)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing).

NLK interacts with several proteins associated with neurodegenerative diseases, including poly-Q expanded androgen receptor in spinobulbar muscular atrophy (SBMA) and ataxin-1 in spinocerebellar ataxia 1 (SCA1).

“NLK interacts with several proteins associated with neurodegenerative diseases, including poly-Q expanded androgen receptor in spinobulbar muscular atrophy (SBMA) and ataxin-1 in spinocerebellar ataxia 1 (SCA1) (16, 17); accumulations of these proteins may also contribute to changes in NLK expression and/or activity.”

• cites [16] sourceSupported
• cites [17] sourceSupported

Probably not a substantial claim: may not pass S3 (load-bearing).

TGF-ß is upregulated in ALS.

“Notably, TGF-ß is upregulated in ALS (55), and aberrant Wnt signaling is associated with TDP43 mislocalization in cellular and animal models (56).”

• cites [55] sourceCould not verify

  The Axis I discussion addresses ALS biomarkers such as neurofilaments, tau, and TDP-43, but does not report TGF-β upregulation. Searches for TGF-β and transforming growth factor beta in our copy found no passage addressing that claim; the copy is not marked as truncated. (LLM, conf=0.73)

  Experimental — accuracy not yet measured

May combine two claims.

Disruption of the Ran gradient, mislocalization of nuclear pore components, and disturbance of nuclear pore architecture have likewise been described in ALS/FTLD-TDP samples and disease models.

“Disruption of the Ran gradient, mislocalization of nuclear pore components, and disturbance of nuclear pore architecture — all of which we observed upon NLK overexpression — have likewise been described in ALS/FTLD-TDP samples and disease models (9–11, 50).”

• cites [9] sourcePartly supported

  The Results report Ran GTPase mislocalization after POM121 knockdown, and the Discussion describes earlier reports of nuclear pore protein mislocalization in disease models. But this paper's electron-microscopy Results find no overt change in nuclear pore architecture in C9orf72 patient-derived neurons; it also reports reduced nuclear nucleoporins without corresponding cytoplasmic mislocalization. The claim therefore overstates what this source shows, particularly for pore architecture. (LLM, conf=0.88)

  Experimental — accuracy not yet measured
• cites [10] sourcePartly supported

  The Results section on the Ran gradient reports a reduced nuclear-to-cytoplasmic Ran ratio in C9-ALS iPS neurons, while the nuclear-pore-pathology Results and Extended Data describe abnormal RanGAP1, Nup205, and Nup107 localization in C9-ALS patient tissue and models. The paper does not establish a disturbance of nuclear pore architecture as distinct from component pathology, or document all three findings across ALS/FTLD-TDP samples and models generally. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [11] sourcePartly supported

  The paper’s results (Fig. 3a–c; page unavailable in our copy) describe mislocalized Nup107 and abnormal nuclear-envelope architecture in a C9ORF72 repeat-expansion fly model. Its patient-derived neurons show altered RNA distribution, but the copy does not describe a disrupted Ran gradient or establish these structural findings across ALS/FTLD-TDP samples generally. (LLM, conf=0.86)

  Experimental — accuracy not yet measured
• cites [50] sourceCould not verify

  The Introduction and Discussion describe TDP43 mislocalization and hUPF1-mediated protection in ALS models, but do not address the Ran gradient, nuclear pore components, or pore architecture. Searches for those terms and nuclear transport found no passage in our copy supporting the attributed claim; page numbers were not available. (LLM, conf=0.75)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing).

Approximately 50% of individuals with frontotemporal lobar degeneration (FTLD) show TDP43 mislocalization as in ALS.

“Approximately 50% of individuals with frontotemporal lobar degeneration (FTLD) show TDP43 mislocalization as in ALS(3).”

• cites [3] sourcePartly supported

  The Introduction (page unavailable) describes pathological cytoplasmic TDP-43 inclusions in both FTLD and ALS, supporting the comparison. It does not give a prevalence figure for FTLD, so the claim that approximately 50% of affected individuals show mislocalization is unsupported by this copy. (LLM, conf=0.87)

  Experimental — accuracy not yet measured

May combine two claims.

At baseline, NLK is highly expressed in the CNS, and its expression is unregulated by oxidative and osmotic stress.

“At baseline, NLK is highly expressed in the CNS, and its expression is unregulated by oxidative and osmotic stress (52, 53), conditions associated with the cytosolic accumulation of TDP43 and other, predominantly nuclear, RNA binding proteins.”

• cites [52] sourceCould not verify

  The passages on axon degeneration discuss DLK and JNK, not NLK. Searches for NLK, Nemo-like kinase, CNS expression, and oxidative or osmotic stress found no passage in our copy addressing the attributed claim. (LLM, conf=0.76)

  Experimental — accuracy not yet measured
• cites [53] sourceCould not verify

  The Results section on neuronal stress examines DLK levels after nerve growth factor withdrawal, not NLK expression. Searches for NLK, CNS baseline expression, and oxidative or osmotic stress found no passage addressing the attributed claim. Page numbers are unavailable in our copy. (LLM, conf=0.77)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Up to half of people with ALS demonstrate cognitive impairment reminiscent of FTLD, while ~1/3 of those with FTLD show motor neuron disease that is indistinguishable from ALS.

“In addition, up to half of people with ALS demonstrate cognitive impairment reminiscent of FTLD, while ~1/3 of those with FTLD show motor neuron disease that is indistinguishable from ALS (3, 45).”

• cites [3] sourceCould not verify

  The Introduction says TDP-43 is associated with both FTLD and ALS, but it does not address how often people with either condition have symptoms of the other. Searches for cognitive impairment, motor neuron disease, and the claimed proportions found no supporting passage in our copy; pages were not provided. (LLM, conf=0.76)

  Experimental — accuracy not yet measured
• cites [45] sourcePartly supported

  The Introduction (page unavailable) notes that MATR3 mutations have been found in individuals with both ALS and frontotemporal dementia, supporting a general clinical overlap. It does not report cognitive impairment in up to half of people with ALS, motor neuron disease in about one-third of people with FTLD, or the claimed clinical equivalence. (LLM, conf=0.78)

  Experimental — accuracy not yet measured

May combine two claims.

NLK is an essential kinase - homozygous Nlk deletion results in death in utero or postnatally, depending on the mouse strain.

“NLK is an essential kinase — homozygous Nlk deletion results in death in utero or postnatally, depending on the mouse strain (57, 58).”

• cites [57] sourceCould not verify

  The Introduction and Results discuss GRN loss of function in human brain organoids, not NLK deletion in mice. Searches for NLK, Nlk deletion, and strain-dependent embryonic or postnatal death found no passage addressing the claim in our copy. (LLM, conf=0.76)

  Experimental — accuracy not yet measured
• cites [58] sourceCould not verify

  The Introduction and Discussion passages concern TDP-43 pathology, and the Methods describe TDP-43 experiments; none addresses Nlk deletion. Searches for NLK/Nlk, homozygous deletion, and embryonic or postnatal lethality found no passage about the claimed mouse-strain-dependent outcome in our copy. (LLM, conf=0.76)

  Experimental — accuracy not yet measured

Classified as probably not a substantial claim. May combine two claims.

Cell lines 1021 and 793 are control lines, and 883 and 321 are C9ORF lines.

“The cell lines 1021 and 793 (controls) and 883 and 321 (C9ORF) were generated and characterized as previously described (7).”

• cites [7] sourceCould not verify

  The ‘Nucleocytoplasmic transport alterations’ section discusses C9orf72 iPSC-derived neurons, but does not identify cell lines 1021, 793, 883, or 321. Searches for those identifiers and for the lines’ generation and characterization found no confirming passage in our copy. (LLM, conf=0.75)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing).

The human iPSC line WTC11 was used in isogenic GRN+/+ and GRN−/− forms.

“Isogenic human iPSC line WTC11 (GRN+/+ and GRN−/−) were generated by Dr. Bruce R. Conklin, as previously described (61).”

• cites [61] sourceCould not verify

  The Methods sections on iPSC reprogramming and CRISPR/Cas9 genome editing describe patient-derived iPSCs and C9ORF72-edited cells, respectively. Searches for WTC11, GRN isogenic lines, and Bruce R. Conklin found no passage in our copy identifying the claimed GRN+/+ and GRN−/− WTC11 lines or their origin. (LLM, conf=0.78)

  Experimental — accuracy not yet measured

Probably not a substantial claim: may not pass S3 (load-bearing).

In automated primary neuron survival analyses, cell death was scored by criteria including rounding of the soma, loss of fluorescence, and degeneration of neuritic processes.

“Custom Python scripts were used to track ROIs over time, and cell death marked a set of criteria that include rounding of the soma, loss of fluorescence and degeneration of neuritic processes (63, 64).”

• cites [63] sourceCould not verify

  The Material & Methods sections on microscopy and cell culture describe fixed-cell imaging and HeLa and SH-SY5Y cultures, but do not describe automated primary-neuron survival analysis. Searches for soma rounding, fluorescence loss, neurite degeneration, and Python-based cell tracking found no cell-death scoring criteria in our copy; page numbers were unavailable. (LLM, conf=0.73)

  Experimental — accuracy not yet measured
• cites [64] sourceNot checked — no full text of the source

May combine two claims.

The change in TDP43 NC ratio was driven by both a reduction in nuclear TDP43 and a concordant increase in cytoplasmic TDP43.

“The change in TDP43 NC ratio was driven by both a reduction in nuclear TDP43 as well as a concordant increase in cytoplasmic TDP43, consistent with nuclear-cytoplasmic TDP43 mislocalization (Figure 1C–D).”

May combine two claims.

WT NLK overexpression reduced the NC ratio of Ran, driven by both a decrease in nuclear Ran and an increase in cytoplasmic Ran.

“WT NLK overexpression also reduced the NC ratio of Ran, driven by both a decrease in nuclear Ran and an increase in cytoplasmic Ran (Figure 4J, K).”

May combine two claims.

Our work revealed NLK upregulation solely within affected neurons displaying TDP43 pathology, in association with evidence of disrupted nucleocytoplasmic trafficking in the same cells.

“In contrast, our work revealed NLK upregulation solely within affected neurons displaying TDP43 pathology, in association with evidence of disrupted nucleocytoplasmic trafficking in the same cells, suggesting that NLK expression changes are restricted to cells with TDP43 redistribution.”

May combine two claims.

Elevated NLK expression selectively in neurons with TDP-43 pathology both at the RNA level (in FTLD-TDP frontal cortex) and at the protein level (in ALS spinal cord).

“Notably, we uncovered elevated NLK expression selectively in neurons with TDP-43 pathology both at the RNA level (in FTLD-TDP frontal cortex) and at the protein level (in ALS spinal cord).”

Probably not a substantial claim: may not pass S3 (load-bearing).

The concordance index was 0.587, indicating a moderate ability of the model to discriminate between different hazard levels.

“The concordance index was 0.587, indicating a moderate ability of the model to discriminate between different hazard levels.”

May combine two claims.

NLK overexpression prevented RanGAP1 sumoylation and reduced the amount of RanGAP1 in complex with RanBP2.

“NLK overexpression prevented RanGAP1 sumoylation (Figure 4A) and reduced the amount of RanGAP1 in complex with RanBP2 (Figure 4C).”

May combine two claims.

WT NLK expression drives mislocalization of YFP-NLSMATR3, but not endogenous Matrin-3.

“Notably, WT NLK expression drives mislocalization of YFP-NLSMATR3, but not endogenous Matrin-3 (Figure 1J, K), potentially due to the relatively large size of Matrin-3 (95kDa, vs 27kDa for YFP-NLSMATR3) (23, 24).”

May combine two claims.

WT NLK overexpression resulted in a more diffuse and evenly distributed nuclear signal, and minimal changes in the polyA mRNA NC ratio.

“While untransfected and KN NLK-expressing cells displayed a punctate pattern of polyA mRNA within the nucleus, WT NLK overexpression resulted in a more diffuse and evenly distributed nuclear signal, and minimal changes in the polyA mRNA NC ratio (Figure 5C and S4C).”

May combine two claims.

Overexpression of WT NLK significantly increased the NC ratio of both KPNA2 and KPNB1, driven by a significant increase in their nuclear fractions without a corresponding decrease in their cytoplasmic abundance.

“Overexpression of WT NLK significantly increased the NC ratio of both KPNA2 and KPNB11, driven by a significant increase in their nuclear fractions without a corresponding decrease in their cytoplasmic abundance (Figure 3A–D, S1A–B).”

May combine two claims.

Partial NLK knockdown in our investigations was associated with extended survival in C9ORF72 and TARDBP mutant human iPSC-derived neurons and was safe in control neurons.

“Similarly, partial NLK knockdown in our investigations was associated with extended survival in C9ORF72 and TARDBP mutant human iPSC-derived neurons and was safe in control neurons.”

May combine two claims.

NLK overexpression is toxic, while NLK reduction promotes neuronal survival in models of ALS and FTLD-TDP.

“Collectively, these data imply that NLK overexpression is toxic, while NLK reduction promotes neuronal survival in models of ALS and FTLD-TDP.”

May combine two claims.

NLK-induced disruption of the Ran gradient correlates with disruption of the RanGAP1-RanBP2 complex and impaired nucleocytoplasmic transport.

“As such, NLK-induced disruption of the Ran gradient correlates with disruption of the RanGAP1-RanBP2 complex and impaired nucleocytoplasmic transport.”

May combine two claims.

In the absence of sumoylation, RanGAP1 is unable to bind RanBP2 or localize to the nuclear envelope.

“In the absence of sumoylation, RanGAP1 is unable to bind RanBP2 or localize to the nuclear envelope (Figure 4C, (31–33).”

• cites [31] sourceNot checked — no full text of the source
• cites [32] sourceNot checked — no full text of the source
• cites [33] sourceNot checked — no full text of the source

May combine two claims.

Overexpression of NLK significantly impairs the mechanisms that regulate nucleocytoplasmic transport, leading to the mislocalization of pertinent RNA-binding proteins and ultimately neuron death.

“Collectively, our data indicate that overexpression of NLK significantly impairs the mechanisms that regulate nucleocytoplasmic transport, leading to the mislocalization of pertinent RNA-binding proteins and ultimately neuron death.”

May combine two claims.

These effects correlate with the mislocalization of the RanBP2-RanGAP1 complex and collapse of the Ran gradient.

“These effects correlate with the mislocalization of the RanBP2-RanGAP1 complex and collapse of the Ran gradient, both of which are crucial for functional nucleocytoplasmic transport.”

May combine two claims.

NLK may contribute to the pathology of ALS-FTLD-TDP by impairing nucleocytoplasmic transport and promoting neurotoxicity.

“These results suggest that NLK may contribute to the pathology of ALS-FTLD-TDP by impairing nucleocytoplasmic transport and promoting neurotoxicity.”

May combine two claims.

NLK is upregulated in human patients and in disease models featuring TDP43 pathology.

“Together, these data show that NLK is upregulated in human patients and in disease models featuring TDP43 pathology, in accord with our data demonstrating TDP43 mislocalization upon NLK overexpression.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Only RNA samples with an RNA integrity number (RIN) of at least 9.4 were used for bulk RNA sequencing.

“Only samples with an RNA integrity number (RIN) ≥ 9.4 were used to perform bulk RNA sequencing.”

Probably not a substantial claim: may not pass S3 (load-bearing).

NLK is also likely to act on distinct substrates and disease-related pathways, including the integrated stress response and autophagy.

“RanBP2 and RanGAP1 are two potential targets for NLK with clear connections to nucleocytoplasmic transport, but NLK is also likely to act on distinct substrates and disease-related pathways, including the integrated stress response and autophagy (15).”

• cites [15] sourceNot checked — no full text of the source

May combine two claims.

RanBP2 and RanGAP1 are two potential targets for NLK with clear connections to nucleocytoplasmic transport.

“RanBP2 and RanGAP1 are two potential targets for NLK with clear connections to nucleocytoplasmic transport, but NLK is also likely to act on distinct substrates and disease-related pathways, including the integrated stress response and autophagy (15).”

Probably not a substantial claim: may not pass S3 (load-bearing).

Survival analysis of iNeurons used a Cox proportional hazards model stratified by biological replicate.

“Cox proportional hazards model was applied to the datasets, stratifying by biological replicates, and the corresponding hazard plots were generated in R.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Aberrant Wnt signaling is associated with TDP43 mislocalization in cellular and animal models.

“Notably, TGF-ß is upregulated in ALS (55), and aberrant Wnt signaling is associated with TDP43 mislocalization in cellular and animal models (56).”

• cites [56] sourceNot checked — no full text of the source

Probably not a substantial claim: may not pass S3 (load-bearing).

All plasmids used were verified by Sanger sequencing.

“All plasmids were verified by Sanger sequencing.”

Probably not a substantial claim: may not pass S3 (load-bearing).

At least three biological replicates were used per experiment.

“At least three biological replicates were used per experiment.”

Probably not a substantial claim: may not pass S3 (load-bearing).

For data from Liu et al., DESeq2 P-values were corrected for multiple testing using the Benjamini-Hochberg method.

“Data from Liu et al. was analyzed with assistance from the TDP43 loss of function database (69); DESeq2 P-values were calculated using Wald tests and corrected for multiple testing using the Benjamini–Hochberg method.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Our study examined tissue from both male and female patients, and similar findings are reported for both sexes.

“Our study examined tissue from both male and female patients, and similar findings are reported for both sexes.”

Probably not a substantial claim: may not pass S3 (load-bearing).

Manual analysis of iNeuron survival used programmatic de-identification to achieve blinded analysis.

“For manual analysis of iNeuron survival, image time-series were processed by flat-field correction and image registration, followed by programmatic de identification for blinded analysis.”