Theophylline as a Potential Therapeutic Candidate for TDP-43 Proteinopathies

doi:10.21203/rs.3.rs-4862053/v1

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Theophylline as a Potential Therapeutic Candidate for TDP-43 Proteinopathies

https://doi.org/10.21203/rs.3.rs-4862053/v1

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The cytoplasmic aggregates of the TAR DNA-binding protein 43 (TDP-43) are a pathogenic hallmark of some neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and limbic-predominant age-related TDP-43 encephalopathy (LATE). Overexpression of TDP-43 induces cytoplasmic accumulation of TDP-43, leading to mitochondrial dysfunction and cell death. Theophylline treatment significantly decreased accumulation of cytoplasmic TDP-43 and cell death in the TDP-43 overexpressing human neuronal cell lines SH-SY5Y. TDP-43-induced eIF2α phosphorylation is also attenuated by theophylline treatment. Additionally, theophylline alleviated mitochondrial dysfunction caused by TDP-43 overexpression, restoring key mitochondrial respiration parameters such as basal and maximal respiration rates and ATP production. Moreover, Dietary supplementation with theophylline substantially improved both longevity and motility in Drosophila models expressing TDP-43 in neurons. Taken together, these findings suggest that theophylline may be a potential therapeutic candidate for TDP-43 proteinopathies.

Theophylline

TDP-43

neuroprotective effects

eIF2α

amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) frontotemporal dementia (FTD), and limbic predominant age-related TDP-43 encephalopathy (LATE) are debilitating neurodegenerative disorders characterized by progressive neuronal loss. A common pathological feature of these diseases is the aggregation of TAR DNA-binding protein 43 (TDP-43) in affected neurons and glia, an RNA-binding protein involved in various aspects of RNA metabolism [1]. Disease-associated mutations in TDP-43 lead to its abnormal accumulation in the cytoplasm and mitochondria, where it binds to mitochondria-transcribed messenger RNAs (mRNAs) encoding essential respiratory complex I subunits, such as ND3 and ND6 [2]. This binding disrupts mitochondrial function, resulting in increased production of reactive oxygen species (ROS), endoplasmic reticulum (ER) stress, and ultimately neuronal death [3–5]. The pathological role of TDP-43 in neurodegenerative diseases underscores the need for effective therapeutic strategies to mitigate its toxic effects [6]. Previous studies have shown that targeting TDP-43 localization and function can reduce its neurotoxicity [6]. Therefore, there is a critical need for pharmacological interventions that can modulate these processes in vivo.

Recent studies have highlighted the therapeutic potential of targeting TDP-43 to mitigate its neurotoxic effects. Theophylline, a methylxanthine drug commonly used for its bronchodilator properties [7], has emerged as a promising candidate for neuroprotection [8]. Theophylline is known to modulate various cellular pathways [7], and its potential effects on TDP-43-induced cytotoxicity warrant further investigation. Importantly, similar pharmacologic actions to caffeine and is one of its hepatic metabolites [9, 10]. Caffeine has been shown to elevate mitochondrial function, reduce ER stress, and protect against neuronal damage [11–13], which provides a strong rationale for exploring theophylline's therapeutic potential in TDP-43-associated neurodegenerative disorders.

In this study, we aim to investigate the effects of theophylline on TDP-43-induced cytotoxicity in neuronal cells and in a Drosophila model of TDP-43 proteinopathies. We hypothesize that theophylline treatment will reduce TDP-43-induced cellular and mitochondrial stress, thereby enhancing cell survival and improving neurodegenerative outcomes. Our findings will contribute to the understanding of theophylline’s potential as a therapeutic agent for TDP-43-associated neurodegenerative disorders, such as ALS, FTD and LATE.

Reagents and Antibodies

The following materials were obtained from the indicated vendors: dimethyl sulfoxide (DMSO; D8418, MilliporeSigma, Burlington, MA, USA), MG-132 (474791, MilliporeSigma), and theophylline (T1633-50G, MilliporeSigma). The following antibodies were obtained from the indicated vendors: TDP-43 (10782-2-AP, Proteintech, Rosemont, IL, USA), phospho-eIF2α (9721, Cell Signaling Technology, Danvers, MA, USA), eIF2α (9722, Cell Signaling Technology), α-tubulin (9099, Cell Signaling Technology), GAPDH (5174, Cell Signaling Technology) and Lamin A/C (05-714, MilliporeSigma).

Cell Culture

The SH-SY5Y human neuroblastoma cell line (HTB-11, ATCC, Manassas, VA, USA) was grown in Dulbecco's modified Eagle's medium (DMEM; 11995-073, Gibco, Waltham, MA, USA), which was supplemented with 10% heat-inactivated fetal bovine serum (FBS; 16000-044, Gibco) and 50 µg/mL penicillin-streptomycin (15140-122, Gibco).

Lentiviral Transduction of SH-SY5Y cells

Third-generation lentiviral constructs, specifically pLenti-C-mGFP-P2A-Puro (vector) and human wild-type (WT) TDP-43, were utilized to generate lentiviruses following established protocols [14–16]. The lentivirus was produced by Koma Biotech (Seoul, Korea) based on the previously mentioned protocol. The prepared viral particles were divided into aliquots and preserved at − 80°C for later use SH-SY5Y cells were infected with lentivirus encoding either GFP alone or GFP-tagged wild-type human TDP-43 at a MOI of 25, with the addition of 4 µg/mL polybrene (TR-1003, MilliporeSigma). The cells with viral particles were centrifuged at 1,000 × g and 32°C for 80 minutes, followed by overnight incubation in a 37°C CO₂ incubator. The culture medium was then replaced with DMEM containing 10% FBS to reduce potential viral cytotoxicity. Cells were collected for analysis two days after viral transduction.

Cytotoxicity Test

To measure the ratio of cells undergoing necrosis and apoptosis, we employed flow cytometry using an Annexin V-FITC apoptosis detection kit (556547, BD Biosciences, San Jose, CA, USA). The analysis was performed on cells stained with Annexin V-FITC and propidium iodide (PI), following the instructions provided by the manufacturer. SH-SY5Y cells overexpressing TDP-43 (1 × 10⁶ cells/well) were treated with or without theophylline prior to experimentation. The cellular samples were resuspended in 500 µL of binding solution (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl₂, pH 7.4) and then treated with 5 µL of Annexin V labeled with FITC. Following 15 minutes incubation at 37°C in the dark, 4 µL of propidium iodide was added to identify necrotic cells. Within 60 minutes of sample preparation, we conducted flow cytometric measurements using a MoFlo Astrios system (Beckman Coulter, Brea, CA, USA).

Immunoblot Analysis

For Immuno blot analysis, we extracted total proteins using Cell lysis buffer (9803, Cell Signaling Technology) enhanced with freshly added protease and phosphatase inhibitor cocktail (Roche, Basel, Switzerland). We then separated equal amounts of protein samples on Bolt™ 4–12% Bis-Tris gels (NW04120BOX, Thermo Fisher Scientific, Waltham, MA, USA) and transferred them to polyvinylidene difluoride (PVDF; IB24001, Thermo Fisher Scientific) membranes. The membranes were blocked for 1 hour using Tris Buffered Saline (TBS; M1304, mentos) containing 0.05% TWEEN20 (P2287, MilliporeSigma) and either 5% skim milk (232100, BD Difco) or 2% bovine serum albumin (BSA; A3059, MilliporeSigma). Following blocking, the membranes were incubated with primary antibodies at 4°C for 12 hours. We visualized the target proteins using an ECL™ Prime Western Blotting System (45-002-401, Amersham, Buckinghamshire, UK). The relative protein expression levels were quantified from three independent experiments using a Fusion-FX instrument (Viber Lourmat, Collegien, France).

Preparation of Nuclear and Cytoplasmic Extracts

For subcellular fractionation, cells were lysed using the NE-PER nuclear and cytoplasmic extraction reagents kit (78833, Thermo Fisher Scientific) following the manufacturer's protocol. Briefly, cellular fractions were separated into nuclear and cytoplasmic components using ice-cold CER I and CER II buffers through centrifugation at 16,000 × g for 5 minutes at 4°C. The cytosolic supernatants were carefully collected and stored frozen. The pellet containing the nuclei was resuspended by vortexing in ice-cold NER buffer, followed by another centrifugation step at 16,000 × g for 10 minutes at 4°C to obtain the nuclear extract supernatant.

Live Cell Imaging

Images were acquired and processed to assess cell survival using the IncuCyte® ZOOM Live-Cell Imaging system (Sartorius, Göttingen, Germany), following established methods for monitoring the dynamics of cytotoxicity [15, 16]. SH-SY5Y cells were cultured into 96-well plates at a density of 8 × 10³ cells per well, followed by transduction with either GFP-tagged or GFP-tagged human WT TDP-43. Cells treated with theophylline (20, 40 µM) or vehicle (DMSO) were transferred to the IncuCyte® ZOOM instrument and incubated for 92 hours with image capture every 4 hours. IncuCyte® Cytotox Red dye (4632, Sartorius) was used to mark dead cells. Subsequently, all images were analyzed to quantify the percentage of cells infected with GFP and stained with IncuCyte® Cytotox Red dye.

Measurement of Oxygen Consumption rate in SH-SY5Y cells

The oxygen consumption rate (OCR) was assessed in SH-SY5Y cells transfected with TDP-43 using a Seahorse XF24 flux analyzer (Agilent, Santa Clara, CA, USA). We replaced the medium with 200 µL of XF assay medium (103680-100, Agilent technologies), which contained 5 mM glucose, 1 mM pyruvate, and 4 mM glutamine, pre-warmed to 37°C. The cells were then degassed for 1 hour in a non- CO₂ incubator before starting the assay. We first recorded baseline oxygen consumption rate (OCR) measurements. Subsequently, we used the Cell Mito Stress Test kit (103015-100, Agilent technologies) to assess mitochondrial function by sequentially adding 1 µM oligomycin, 2 µM FCCP, and a combination of 0.5 µM antimycin A and 0.5 µM rotenone. To calculate the final OCR values, we subtracted the non-mitochondrial oxygen consumption (measured after antimycin A + rotenone addition) from all readings. We excluded any technical replicates that fell outside one standard deviation from the mean. In SH-SY5Y cells, we normalized OCR values to the mean protein content of each well, which we determined using a BCA protein assay kit (23225, Thermo Fisher Scientific).

Fly Strains

Drosophila were cultured on standard cornmeal agar media at 24°C, except where specified otherwise. The UAS-TDP-43 fly line was previously characterized [3], and all fly stocks were acquired from The Bloomington Drosophila Stock Center (Indiana University, IN, USA).

Climbing and Lifespan Assays

Adult male flies overexpressing TDP-43 (aged 0 to 1 day) were randomly assigned to individual vials containing either RU-486 (Mifepristone, M8046, MilliporeSigma, 70 µg/ml) supplemented fly food or ethanol (1.00983.2511, MilliporeSigma) only as control, with each group consisting of more than 100 flies. The flies were transferred to fresh media mixed with or without theophylline (10 µM) every two days, and deceased flies were counted during each transfer. The climbing ability of adult flies was assessed using a method previously outlined [3].

Statistical Analysis

All experiments were conducted in triplicate, and the results are expressed as mean ± standard deviation. Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparison test followed (GraphPad Prism Software, San Diego, CA, USA). Statistical significance was defined as p < 0.05 and denoted as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 and n.s indicating not significant.

Theophylline attenuates TDP-43-induced neuronal cell death

Abnormal TDP-43 aggregates are a hallmark of the overlapping neurodegenerative disorders ALS, FTD and LATE [19–22]. TDP-43 is a main causative protein of degenerative brain diseases that elevates ER and oxidative stress by disrupting the mitochondrial electron transport system [23–25]. In both an SH-SY5Y cellular stress model and frontotemporal dementia (FTD) brain samples, cytoplasmic TDP-43 is found to associate with polyribosomes [26]. To assess the potential cytotoxicity of TDP-43 in SH-SY5Y cells, we used a lentiviral system to express either GFP alone or GFP-tagged human TDP-43. At 2 to 3 days post-transduction, we confirmed the expression of TDP-43 protein in SH-SY5Y cells by fluorescence microscopy (Fig. 1a), and flow cytometry analysis showed that the transduction efficiency of GFP was about 70% and that of TDP-43 was about 60% (data not shown). To assess how theophylline impacts TDP-43-induced neuronal toxicity, we employed a real-time cell viability monitoring system using live-cell imaging technology. We cultured SH-SY5Y cells in 96-well plates and infected them with lentiviral particles carrying either GFP-tagged TDP-43 or GFP alone. We then marked dead cells using Incucyte® Cytotox Red dye and captured cell images at 4 hours intervals. Theophylline was administered the day after lentivirus transduction, and cell death induced by TDP-43 was observed during co-administration of theophylline for up to 92 hours post-transduction. TDP-43-induced neurotoxicity was observed until 92 hours of transduction, and theophylline treatment significantly alleviated pattern TDP-43-induced cytotoxicity in SH-SY5Y cells (Fig. 1b). Theophylline did not show any changes in cytotoxicity in GFP-expressing cells (Fig. 1b, Supplementary data 1). The flow cytometry analysis also showed that TDP-43-induced cell death was substantially mitigated by approximately 4% on average by theophylline treatment (Fig. 1c). These results indicate that theophylline exerts a protective effect on TDP-43-induced neurotoxicity.

Theophylline mitigates cytoplasmic mislocalization of TDP-43 in SH-SY5Y cells

We utilized immunoblotting to examine the effect of theophylline on TDP-43 protein levels. The level of total exogenous TDP-43 protein was not affected by theophylline treatment (Fig. 2a). Next, we investigated the effect of theophylline on the cytoplasmic mislocalization of TDP-43 in SH-SY5Y cells. Interestingly, theophylline treatment affects the TDP-43 cytoplasmic/nuclear ratio and significantly reduces the portion of cytoplasmic TDP-43 (Fig. 2b). Previous studies indicated that cytoplasmic mislocalization of TDP-43 induces an increase in eIF2α phosphorylation through ER stress [25, 26]. This increase in phosphorylation is an important factor in TDP-43-induced neurotoxicity [3, 27]. Thus, we examined whether the reduction of cytoplasmic TDP-43 by theophylline in SH-SY5Y cells affects downstream cellular stress responses such as ER stress. Theophylline treatment markedly reduced the levels of p-eIF2α (Ser51) in TDP-43-over expressing cells (Fig. 2c).

Theophylline attenuates TDP-43-induced mitochondrial dysfunction.

Recent studies indicate that preventing TDP-43 from localizing to mitochondria blocks both mitochondrial dysfunction and TDP-43-induced neurotoxicity [2]. Thus, we also investigated whether theophylline treatment restored mitochondrial dysfunction. We performed bioenergetics measurements of mitochondria to assess whether this TDP-43-induced mitochondrial dysfunction is restored after theophylline treatment. Overexpression of TDP-43 resulted in significant functional reductions in basal respiration rate, ATP production, and maximal respiration rate (Fig. 3a, b). Theophylline treatment significantly restored mitochondrial respirate parameters like basal respiration rate, ATP production, and maximal respiration rate in TDP-43-expressing cells (Fig. 3a, b). These results demonstrate that theophylline improves cell survival through the attenuation of TDP-43-mediated mitochondrial dysfunction in SH-SY5Y cells.

Theophylline alleviates neuronal toxicity of TDP-43 in a Drosophila model.

To assess the in vivo efficacy of theophylline against TDP-43-induced neurotoxicity, we employed a Drosophila model expressing human TDP-43 throughout the whole nervous system [30]. In our previous studies, we established a Drosophila model of TDP-43 proteinopathies and demonstrated that overexpression of TDP-43 in the fly neurons leads to neurodegeneration [28, 29]. We examined the effects of theophylline on the shortened lifespan and locomotor deficits induced by TDP-43 expression. Flies with neuron-specific TDP-43 expression exhibited significant deficits in lifespan and climbing ability (Fig. 4a, b). Theophylline treatment extended the lifespan of flies with neuron-specific TDP-43 expression (Fig. 4a). Moreover, TDP-43-expressing flies demonstrated significantly diminished climbing ability compared to control flies. This TDP-43-induced climbing deficit was significantly mitigated by theophylline treatment (Fig. 4b). Our results collectively demonstrate that theophylline effectively protects against TDP-43-induced neuronal damage in vivo, highlighting its potential as a therapeutic agent for TDP-43 proteinopathies.

This study provides evidence that theophylline exerts protective effects against TDP-43-induced neurotoxicity, which is a common feature of many neurodegenerative disorders.

Caffeine, which is metabolically interchangeable with theophylline and shares similar biological activity, is also known to have neuroprotective effects [9, 10]. Studies have shown that caffeine enhances mitochondrial function, reduces ER stress, and protects neurons from oxidative damage [11, 30, 31]. These effects of caffeine align with the observed benefits of theophylline, suggesting that its similarities to caffeine contributes to its neuroprotective property.

Theophylline and caffeine act primarily as antagonists on A1, A2A, and A2B adenosine receptors [34–36]. Previous studies have shown that A2AR regulates TDP-43 mislocalization and neurotoxicity through modulation of the AMP-activated protein kinase pathway in the NSC-34 cell line and a TDP-43-overexpressing transgenic mouse model [35, 36]. Since A2AR is also expressed in the human spinal cord, future studies should investigate the effects of theophylline on A2AR and its potential to inhibit TDP-43-mediated toxicity [37].

This study reveals a neuroprotective effect of theophylline against TDP-43-induced neurotoxicity. Theophylline treatment significantly reduces cytoplasmic TDP-43 accumulation, ameliorates mitochondrial dysfunction, and downregulates eIF2α phosphorylation. These findings, further validated by in vivo studies using a Drosophila model, suggest the therapeutic potential of theophylline for TDP-43 proteinopathies. Specifically exploring how theophylline affects TDP-43 localization, mitochondrial function, and cellular stress response could be crucial for developing effective pharmacological interventions to mitigate TDP-43 toxicity and improve patient outcomes in TDP-43 proteinopathies.

ALS, amyotrophic lateral sclerosis

FTD, frontotemporal dementia

LATE, limbic predominant age-related TDP-43 encephalopathy

OCR, oxygen consumption rate

TDP-43, TAR DNA-binding protein 43

ROS, reactive oxygen species

FUNDING

This work was supported by KBRI basic research program through Korea Brain Research Institute funded by Ministry of Science and ICT 24-BR-02-04, the National Research Foundation of Korea (NRF) grant funded by Korean government (MSIT; 2021R1C1C1008688, RS-2024-00343239) and a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number : HI14C1135)

AUTHORS’ CONTRIBUTIONS

Conceptualization, methodology, validation, Y-MJ, HKK, YK and H-JK; data curation, formal analysis, visualization, Y-MJ, HKK, YK and H-JK; writing—original draft preparation, HKK; writing—review and editing, Y-MJ, HKK, YK, SL, SK, MJ, JW and H-JK; supervision, H-JK; project administration, MJ and HJK; funding acquisition, H-JK. All authors have read and agreed to the published version of the manuscript.

Ethics approval and consent to participate

Not applicable

Consent for publication

Not applicable

Availability of data and materials

Kindly reach out to the author regarding requests for data and materials.

Competing interests

The authors declare no conflict of interest.

Acknowledgements

Not applicable

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Suppl.Figure1.tif
Supplementary data 1. Cytotoxic effects of theophylline. GFP expressing SH-SY5Y cells were stained with Cytotox Red, a marker of dead cells. Images were captured every 4 hours using an IncuCyte live-cell imaging system. The number of cells exhibiting both green fluorescence (live cells) and red fluorescence (dead cells) was quantified. Theophylline (THE, 20 or 40 μM) did not show cytotoxicity in GFP-expressing SH-SY5Y cells. n.s., not significant (one-way ANOVA with Tukey’s multiple comparison test).
Theophyllinewesternblotrawdata.xlsx

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Theophylline as a Potential Therapeutic Candidate for TDP-43 Proteinopathies

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Version 1

Abstract

Figures

INTRODUCTION

MATERIALS AND METHODS

Reagents and Antibodies

Cell Culture

Lentiviral Transduction of SH-SY5Y cells

Cytotoxicity Test

Immunoblot Analysis

Preparation of Nuclear and Cytoplasmic Extracts

Live Cell Imaging

Measurement of Oxygen Consumption rate in SH-SY5Y cells

Fly Strains

Climbing and Lifespan Assays

Statistical Analysis

RESULTS

Theophylline attenuates TDP-43-induced neuronal cell death

Theophylline mitigates cytoplasmic mislocalization of TDP-43 in SH-SY5Y cells

DISCUSSION

Abbreviations

Declarations

References

Supplementary Files

Status:

Version 1