Scientific Research & Diligence for Drug Discovery

Tundra reads the full-text literature, patents, clinical trial records, and more. Then grounds every claim in a source. Built for biotech investors, BD, and translational science.

May 27

Now let me pull full texts from the most experimentally rich papers. I'll fetch several in parallel batches.

Now let me pull a few of the patents for their experimental content.

I now have enough full-text data to compile a comprehensive, source-grounded list. Here it is:

TDP-43 / ALS — In Vitro Preclinical Experiment Menu

Organized by experimental category. Each entry includes a verbatim source quotation from the bookshelf literature, with paper title, PMCID, and context. Patent-derived experiments are flagged separately at the end.

Category 1: TDP-43 Overexpression & Mutant Expression Models

1. Inducible WT/mutant TDP-43 expression in mouse neuronal cell line (NSC-34) for innate immune pathway activation

Doxycycline-inducible lentiviral TDP-43 in NSC-34 cells, measuring NF-κB and type I IFN upregulation with WT vs. Q331K/A315T mutants. Entry point for drug screening.

"To examine inflammation triggered by TDP-43, we employed inducible expression of empty vector (control [Ctrl]), wild-type (WT), or ALS mutant (Q331K) TDP-43 in a mouse neuronal cell line, which confirmed upregulation of NF-κB and type I IFN pathways."

— TDP-43 Triggers Mitochondrial DNA Release via mPTP to Activate cGAS/STING in ALS (PMC7599077, Section: Inflammatory Signaling from TDP-43 Is Dependent on cGAS/STING)

2. Inducible TDP-43 overexpression in mouse embryonic fibroblasts (MEFs) with genetic panel knockouts (cGAS, STING, MAVS, PKR, Bak/Bax) for innate immune sensor mapping

A chemical genetic dissection of the upstream sensor triggering TDP-43-driven inflammation.

"To identify the innate immune sensor regulating this response, we repeated the model in mouse embryonic fibroblasts (MEFs) genetically deficient for a panel of candidates that are known to regulate NF-κB and type I IFN production."

— TDP-43 Triggers Mitochondrial DNA Release via mPTP to Activate cGAS/STING in ALS (PMC7599077, Section: Inflammatory Signaling from TDP-43 Is Dependent on cGAS/STING)

3. TDP-43-EGFP dose-response toxicity in primary rodent cortical neurons with longitudinal automated fluorescence microscopy (AFM)

Gold-standard live-cell survival assay; measures hazard ratio as a function of TDP-43 expression level. Can be run with WT, A315T, M337V, or other mutants.

"We fused WT and ALS-associated mutant (A315T) TDP43 to enhanced green fluorescent protein (EGFP) and transfected primary rodent cortical neurons with different amounts of construct and mApple, a survival and morphology marker."

— Autophagy induction enhances TDP43 turnover and survival in neuronal ALS models (PMC4106236, Section: TDP43 toxicity is dose-dependent)

4. Overexpression of WT and ALS-mutant TDP-43 in HEK293T cells for subcellular fractionation and mitochondrial localization

Biochemical framing experiment; establishes that disease-associated mutations (G298S, A315T, A382T) increase mitochondrial TDP-43 levels.

"Whereas all fibroblasts displayed comparable expression of total TDP-43, G298S and A382T fibroblasts demonstrated significantly higher levels of mitochondrial TDP-43 than NHFs."

— The inhibition of TDP-43 mitochondrial localization blocks its neuronal toxicity (PMC4974139, Section: ALS-mutations increase TDP-43 mitochondrial localization)

Category 2: iPSC-Derived Motor Neuron (hMN) Models

5. iPSC-derived motor neurons from ALS patients (familial TDP-43 mutations) — cGAS/STING pathway activation + cGAMP quantification

Human disease-relevant model; measures cGAMP production, IFN/cytokine gene expression, and motor neuron viability over 28 days in culture.

"We also confirmed activation of the cGAS/STING pathway in induced pluripotent stem cell (iPSC)-derived motor neurons (MNs) from ALS patients carrying familial mutations in TDP-43."

— TDP-43 Triggers Mitochondrial DNA Release via mPTP to Activate cGAS/STING in ALS (PMC7599077, Section: Inflammatory Signaling from TDP-43 Is Dependent on cGAS/STING)

6. iPSC-derived motor neuron survival assay with STING inhibitor (H-151) over 28-day terminal differentiation

Tests whether pharmacological STING blockade rescues the intrinsic cell-death excess observed in ALS patient MNs. Establishes cell-intrinsic role of cGAS/STING.

"Interestingly, we observed that ALS patient iPSC-derived motor neurons had impaired survival 28 days after terminal differentiation, whereas control motor neurons were still predominantly viable. Addition of the STING inhibitor H-151 during the final 28 days of culture prevented excess motor neuron death, suggesting a cell-intrinsic role of the cGAS/STING pathway."

— TDP-43 Triggers Mitochondrial DNA Release via mPTP to Activate cGAS/STING in ALS (PMC7599077, Section: In Vitro In Vivo STING Inhibition Ameliorates Neurodegeneration and)

7. siRNA TDP-43 knockdown in iPSC-derived human motor neurons (hMNs) + transcriptome-wide RNA-Seq

Identifies TDP-43-regulated transcripts in the motor-neuron-specific context; led to discovery of STMN2 as a key downstream target. Can be adapted to profile any candidate target.

"We then used qRT-PCR to validate that TDP-43 mRNA levels had been depleted in hMNs treated with siTDP43s, but not in those exposed to a scrambled siRNAs. We further confirmed TDP-43 depletion at the protein level by immunoblot, with siTDP43-treated hMNs showing a 54–65% reduction in TDP-43 levels."

— ALS-implicated protein TDP-43 sustains levels of STMN2, a mediator of motor neuron growth and repair (PMC7153761, Section: Motor neuron RNAs regulated by TDP-43)

8. Autophagy inducer treatment (fluphenazine, methotrimeprazine) in iPSC-derived motor neurons and astrocytes carrying TDP-43 M337V mutation — longitudinal AFM survival

Tests whether neuronal autophagy induction reduces ALS-mutant TDP-43 toxicity in human disease-relevant cells.

"MTM and FPZ reduced the risk of death for M337V-TDP43 MNs, without affecting the survival of WT TDP43 MNs."

— Autophagy induction enhances TDP43 turnover and survival in neuronal ALS models (PMC4106236, Section: Autophagy induction prevents TDP43-mediated cell death)

9. iPSC-derived neurons from ALS patient fibroblasts (TDP-43 A382T) — mitochondrial fragmentation and dysfunction assay (TMRM, Seahorse OCR, ATP)

Establishes phenotypic consequences of endogenous mutant TDP-43 on bioenergetics in patient-derived human neurons.

"Both control and A382T human neurons were indistinguishable in TDP-43 nuclear localization, neuronal morphology and viability. However, compared with control human neurons, A382T human neurons exhibited significant mitochondrial fragmentation/dysfunction and increased vulnerability to oxidative stress, which could be alleviated by PM1 treatment."

— The inhibition of TDP-43 mitochondrial localization blocks its neuronal toxicity (PMC4974139, Section: Mitochondrial localization is critical for TDP-43 toxicity)

Category 3: Protein Clearance & Degradation Pathway Assays

10. Optical Pulse Labeling (OPL) for single-cell TDP-43 half-life measurement in primary neurons (TDP-43–Dendra2)

A uniquely powerful assay for directly measuring TDP-43 clearance kinetics at single-cell resolution, uncoupled from toxicity and aggregation confounds.

"Optical pulse labeling (OPL) uses photoconvertible proteins, such as Dendra2, that irreversibly switch their spectral properties when exposed to short-wavelength light. We tagged WT TDP43 with Dendra2 and expressed the construct in rodent primary cortical neurons, then photoconverted with 405-nm light."

— Autophagy induction enhances TDP43 turnover and survival in neuronal ALS models (PMC4106236, Section: Measuring TDP43 clearance)

11. LC3-Dendra2 autophagic flux assay in primary cortical neurons — single-cell measurement of autophagic throughput

Enables live-cell, quantitative measurement of autophagic flux in neurons; validated against Atg7 knockdown. Designed for small-molecule screening.

"To accurately measure flux, the rate of protein breakdown must be measured directly for an autophagy substrate. We therefore developed an in situ assay of autophagy flux by fusing LC3 to Dendra2, expressing LC3-Dendra2 in primary rodent cortical neurons and measuring the turnover of LC3-Dendra2 through OPL."

— Autophagy induction enhances TDP43 turnover and survival in neuronal ALS models (PMC4106236, Section: Induction of autophagy increases TDP43 clearance)

12. Proteasome inhibition (MG-132) pulse-chase as a model of TDP-43 nuclear depletion + cryptic exon inclusion in hMNs

Creates an acute pharmacological model of TDP-43 mislocalization; directly links proteostasis dysfunction to downstream STMN2 deregulation and splicing.

"Strikingly, using the Pearson's coefficient analysis and nuclear to cytoplasmic ratio, we observed that TDP-43 staining in the nucleus was greatly diminished after 24 hours. Following MG-132 washout, we found that TDP-43 staining became indistinguishable from that in unchallenged neurons after 4 days."

— ALS-implicated protein TDP-43 sustains levels of STMN2, a mediator of motor neuron growth and repair (PMC7153761, Section: STMN2 levels tuned by TDP-43 levels and localization)

Category 4: Mitochondrial Biology Assays

13. Cell-free mitochondrial import assay — recombinant WT and ALS-mutant TDP-43 with isolated mouse brain mitochondria

Biochemically characterizes how TDP-43 enters the inner mitochondrial membrane; enables testing of import inhibitors without confounds from whole-cell biology.

"We then employed a cell-free mitochondrial import assay using Flag-tagged recombinant WT/mutant human TDP-43 protein (rTDP-43) and isolated mouse brain mitochondria."

— The inhibition of TDP-43 mitochondrial localization blocks its neuronal toxicity (PMC4974139, Section: ALS-mutations increase TDP-43 mitochondrial localization)

14. TAT-peptide competitive inhibitors of TDP-43 mitochondrial import (PM1/PM3) in primary cortical neurons — mitochondrial function + neuronal viability readouts

Directly tests the therapeutic concept of blocking TDP-43 entry to the IMM; measures mΔψ, OCR, ATP, and SYTOX-green cell death.

"PM1 and PM3, but not control peptides cPMs, suppressed rTDP-43 import, and reduced mitochondrial TDP-43 in HEK293 cells and primary neurons."

— The inhibition of TDP-43 mitochondrial localization blocks its neuronal toxicity (PMC4974139, Section: TDP-43 mitochondrial localization depends on internal motifs)

15. Mitochondrial translation assay (AHA pulse-labeling with emetine block) for ND3/ND6 synthesis in HEK293 cells overexpressing TDP-43

Directly tests whether excess mitochondrial TDP-43 inhibits translation of specific OXPHOS mRNAs; can be applied to primary neurons or patient fibroblasts.

"We investigated the effect of TDP-43 on mitochondrial translation in HEK293 cells by the azidohomoalanine (AHA) incorporation assay in the presence of emetine to block cytosolic translation. The synthesis of ND3/6 was significantly reduced by WT TDP-43 overexpression."

— The inhibition of TDP-43 mitochondrial localization blocks its neuronal toxicity (PMC4974139, Section: Mitochondrial TDP-43 inhibits translation of ND3/6 mRNAs)

16. Complex I activity measurement + Blue Native PAGE supercomplex analysis in isolated mitochondria from cells/fibroblasts

Quantifies TDP-43-driven disassembly of respiratory Complex I; applicable to ALS patient fibroblasts as a functional biomarker assay.

"Consistent with the specific inhibitory effect of TDP-43 on the expression of complex I subunits ND3/6, WT TDP-43 overexpression caused a significant reduction of complex I expression and function, as well as a slight disassembly of complex I-comprised supercomplexes."

— The inhibition of TDP-43 mitochondrial localization blocks its neuronal toxicity (PMC4974139, Section: Mitochondrial TDP-43 impairs OXPHOS complex I)

17. mtDNA depletion (ρ⁰ cells) experiment in THP-1 cells with inducible TDP-43 — test whether inflammatory signaling requires mitochondrial DNA

Genetically/pharmacologically depletes mtDNA with ethidium bromide to establish whether mtDNA is the ligand activating cGAS downstream of TDP-43.

"mtDNA depletion from THP-1 cells containing inducible WT and mutant TDP-43 was achieved in culture with EtBr for 3 weeks, as judged by qPCR of mitochondrial gene expression. We then treated the cells with doxycycline (Dox) to induce TDP-43 (WT or Q331K), followed by quantification of inflammatory cytokine expression."

— TDP-43 Triggers Mitochondrial DNA Release via mPTP to Activate cGAS/STING in ALS (PMC7599077, Section: TDP-43 Triggers mtDNA Release into the Cytoplasm)

18. mPTP inhibition (cyclosporin A) or PPID/cyclophilin D genetic deletion in MEFs — block TDP-43-induced cytosolic mtDNA release

Tests whether the mPTP is the escape route for mtDNA; pharmacological angle provides direct therapeutic target validation.

"Pharmacological inactivation of the mPTP using cyclosporin A (CsA) prevented TDP-43(Q331K)-mediated mtDNA leakage into the cytoplasm, thus attenuating IFNB1 expression."

— TDP-43 Triggers Mitochondrial DNA Release via mPTP to Activate cGAS/STING in ALS (PMC7599077, Section: TDP-43 Triggers mtDNA Release into the Cytoplasm via the mPTP)

Category 5: TDP-43 Target RNA / Splicing Assays

19. Formaldehyde RNA immunoprecipitation (fRIP) for TDP-43–STMN2 mRNA binding confirmation in hMNs

Biochemically validates direct TDP-43 binding to STMN2 transcript; can be used to test whether candidate therapeutic interventions disrupt or preserve this interaction.

"We next asked whether additional transcripts influenced by TDP-43 knockdown, housekeeping transcripts, or a MN-specific transcript were enriched. Of these, we observed strong and significant enrichment for STMN2 transcripts."

— ALS-implicated protein TDP-43 sustains levels of STMN2, a mediator of motor neuron growth and repair (PMC7153761, Section: TDP-43 regulates STMN2 levels)

20. RT-PCR detection of STMN2 cryptic exon inclusion in hMNs under TDP-43 depletion or proteasome inhibition

Functional readout of TDP-43 splicing activity; the cryptic exon inclusion in STMN2 (confirmed by sequencing) provides a clean molecular biomarker of TDP-43 loss of nuclear function.

"Although hMNs growing under normal conditions showed no detectable evidence for this cryptic event, following proteasome inhibition, it could be readily observed."

— ALS-implicated protein TDP-43 sustains levels of STMN2, a mediator of motor neuron growth and repair (PMC7153761, Section: STMN2 levels tuned by TDP-43 levels and localization)

Category 6: Cytoskeletal & Axonal Biology Assays

21. Sholl analysis of neurite complexity in hMNs after TDP-43 siRNA knockdown or STMN2 CRISPR knockout

Quantifies the structural consequences of TDP-43 loss of function on motor neuron morphology; uses automated neurite tracing in fixed cultures.

"Sholl analysis, which quantifies the number of neurite branches at a given interval from the center of the soma, revealed significantly reduced neurite complexity in neurons treated with siTDP-43."

— ALS-implicated protein TDP-43 sustains levels of STMN2, a mediator of motor neuron growth and repair (PMC7153761, Section: TDP-43 depletion inhibits neurite and axon growth)

22. Microfluidic axotomy and axon regeneration assay in hMNs (soma + axon compartment devices)

Physically severs axons and quantifies regrowth; directly models the nerve repair deficit relevant to ALS disease progression.

"Neurons cultured for 7 days in the soma compartment of the device extended axons through the microchannels into the axon chamber. We severed axons without disturbing cell bodies in the soma compartment and then measured axon extension from the microchannel across a time course to assess regrowth after injury."

— ALS-implicated protein TDP-43 sustains levels of STMN2, a mediator of motor neuron growth and repair (PMC7153761, Section: TDP-43 depletion inhibits neurite and axon growth)

23. JNK inhibition (SP600125) as a post-translational STMN2 rescue strategy — STMN2 protein quantification + Sholl/axon regeneration readouts

Tests whether boosting STMN2 stability pharmacologically can restore motor neuron structural phenotypes caused by TDP-43 dysfunction.

"Subsequent immunoblot analysis showed STMN2 protein levels were significantly increased after JNK inhibition. Notably, the reduced levels of STMN2 observed after siTDP43 treatment could be boosted to levels greater than those observed in control hMNs."

— ALS-implicated protein TDP-43 sustains levels of STMN2, a mediator of motor neuron growth and repair (PMC7153761, Section: JNK inhibition rescues STMN2 and neurite defects)

Category 7: Stress Granule & Translational Stress Assays

24. Primary rat cortical neurons + TDP-43-EGFP overexpression + PERK inhibitor (GSK2606414) — longitudinal AFM survival at pharmacological doses

Therapeutic rescue experiment in primary neurons; quantifies whether reduction of eIF2α phosphorylation (stress granule attenuation) mitigates TDP-43 toxicity.

"We assessed the ability of GSK2606414 to mitigate TDP-43 toxicity, we first performed a dose-response to the compound, testing a range of compound concentrations. These studies showed that treatment of primary neurons with PERKi GSK2606414 mitigated TDP-43 toxicity at 250 and 500 nM."

— Therapeutic modulation of eIF2α phosphorylation rescues TDP-43 toxicity in amyotrophic lateral sclerosis disease models (PMC3934366, Section: Pharmacologic rescue of TDP-43 toxicity)

25. cGAS/STING pharmacological blockade screen (RU.521 for cGAS, H-151 for STING) in THP-1 cells with TDP-43 overexpression — cytokine gene expression + signaling Western blot

Validates druggability of the cGAS/STING pathway in human myeloid cells; entry point for compound optimization.

"Indeed, these drugs prevented expression of STING and IFNB1 and TNF in response to overexpressed WT and mutant TDP-43."

— TDP-43 Triggers Mitochondrial DNA Release via mPTP to Activate cGAS/STING in ALS (PMC7599077, Section: Inflammatory Signaling from TDP-43 Is Dependent on cGAS/STING)

Patent-Referenced Experiments (from bookshelf patents — abstracts reviewed)

P1. Conformational antibody binding assay for misfolded TDP-43 detection

Antibodies targeting W68 in the DAGWGNL epitope context; in vitro binding assays (ELISA, immunoprecipitation) for detecting pathological TDP-43 conformations.

Patent: US-12559548-B2 — "Antibodies to misfolded TDP-43 and methods of use" (Cashman/Kaplan, UBC / Promis Neurosciences)

P2. Cellular reporter assay for STMN2 expression restoration

iPSC-hMN-based screening platform for identifying candidate agents that restore full-length STMN2 RNA expression (vs. cryptic exon inclusion).

Patent: US-12496327-B2 — "Methods and compositions for restoring STMN2 levels" (Eggan et al., Harvard)

P3. ASO dose-response knockdown assay in cell culture (TDP-43 mRNA targeting)

Cellular testing of antisense oligonucleotides against defined target regions in TARDBP mRNA; evaluates TDP-43 protein knockdown efficiency and selectivity.

Patent: US-2026085310-A1 — "Antisense oligonucleotide targeting tdp-43 mRNA or pre-mRNA" (Nippon Shinyaku)

P4. TDP-43 aggregation resistance assay with PLD-mutant variants (aromatic spacing engineering)

Expresses Regeneron's aggregation-resistant TDP-43 prion-like domain variants; assesses aggregation propensity in cellular models.

Patent: US-2026035420-A1 — "Aggregation-resistant variants of tdp-43" (Regeneron)

P5. Peptide treatment in cell-based TDP-43 pathology model

Celosia Therapeutics' peptide (SEQ ID NO:1 optionally with destabilization domain) administered to cells; measures rescue of TDP-43-associated pathological markers.

Patent: US-12544418-B2 — "Compositions and methods for treatment" (Ittner/Ke, Celosia Therapeutics)

This gives 25 literature-grounded experiments and 5 patent-anchored concepts — a menu of 30 modular options across mechanistic validation, target engagement, functional rescue, and drug screening paradigms. Let me know which you'd like to prioritize for the development plan and I can help you think through sequencing, assay tiering, or what each experiment would prove.

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