Journal: bioRxiv

Article Title: Telomerase mRNA-Lipid nanoparticles attenuate neuroinflammation after traumatic brain injury in mice

doi: 10.64898/2025.12.18.694748

Figure Lengend Snippet: (a,d) Encapsulation efficiency (%EE) of luciferase mRNA-loaded LNPs (Luc-LNP) and mouse TERT mRNA-loaded LNPs (mTERT-LNP) across three independently prepared batches. (b,e) Hydrodynamic diameter (bars, left y-axis) and polydispersity index (PDI; circles, right y-axis) of Luc-LNP (b) and mTERT-LNP (e) batches, showing uniform particle size (∼100 nm) and low PDI. (c,f) Zeta potential measurements of Luc-LNP (c) and mTERT-LNP (f) for three independent batches, indicating slightly negative surface charge. (g–j) In vitro cytocompatibility of Luc-LNP (g,i) and mTERT-LNP (h,j) in cultured cells at the indicated mRNA concentrations (0.5–2 µg/ml) compared with untreated control, assessed at 24 h (g,h) and 48 h (i,j). No appreciable reduction in cell viability is observed at any dose or time point. (k) mTERT-LNP RNA concentration after storage, measured on days 1, 8, and 14, demonstrating preserved RNA content over time. Data are presented as mean ± SEM of independent preparations/experiments. LNP, lipid nanoparticle; Luc, mRNA encoding luciferase; mTERT, mRNA encoding mouse telomerase reverse transcriptase; PDI, polydispersity index.

Article Snippet: The next step was an overnight incubation at 4°C using a solution of 3% NGS in PBS-T, containing primary antibodies: anti-rabbit Iba-1 (Wako, cat. #019-19741) at 1:500 to label microglia and macrophages, and anti-mouse TERT (Novus, cat. #NB100-317) at 1:500.

Techniques: Encapsulation, Luciferase, Zeta Potential Analyzer, In Vitro, Cell Culture, Control, Concentration Assay, Reverse Transcription

Journal: bioRxiv

Article Title: Telomerase mRNA-Lipid nanoparticles attenuate neuroinflammation after traumatic brain injury in mice

doi: 10.64898/2025.12.18.694748

Figure Lengend Snippet: (a) Schematic of the TERT mRNA-LNP system and in vivo study design. TERT or luciferase mRNA is encapsulated in Cy5.5-labeled LNPs and administered intravenously to 6-month-old male and female C57BL/6 mice subjected to controlled cortical impact TBI (day 0). Mice undergo IVIS imaging for mRNA-LNP biodistribution and protein expression on day 1 and neuropathologic and blood analyses on day 3. (b) Endogenous brain TERT mRNA levels were assessed by qPCR at baseline (control), 3 days post-injury (dpi), and 14 dpi, demonstrating an early decrease in TERT expression with partial recovery over time. (c) The telomere length (measured by the telomere-to-single-copy gene (T/S) ratio) decreased at 3 dpi and showed partial recovery by 14 dpi. (d) Representative RNAscope images of Tert mRNA (red) with DAPI nuclear counterstain (blue) in peri-contusional cortex from male and female mice at control and 3 dpi, illustrating reduced Tert signal after TBI. Scale bar, 50 µm. (e) Quantification of RNAscope Tert mRNA signal (% area) in peri-contusional cortex from male and female mice treated with luciferase control LNPs (Luc) or TERT mRNA-LNPs (Tert) and analyzed at 3 dpi, showing robust enhancement of Tert mRNA with TERT mRNA-LNP treatment. (f) qPCR analysis of cortical Tert mRNA levels (fold change) in Luc versus Tert groups at 3 dpi, confirming increased Tert transcript levels after TERT mRNA-LNP delivery. (g) Representative RNAscope images of Tert mRNA (red) with DAPI (blue) in peri-contusional cortex of male and female mice receiving Luc or mTERT LNPs. Scale bar, 50 µm. (h) Telomere length in peri-contusional cortex measured by qPCR (T/S ratio) at 3 dpi, showing partial restoration of telomere length in Tert-treated animals relative to Luc controls, in both sexes. (i) Quantification of TERT protein+ cells per field in peri-contusional cortex by immunohistochemistry (IHC), demonstrating increased TERT protein expression in Tert versus Luc groups at 3 dpi. (j) Representative IHC images of TERT protein (red) with DAPI (blue) in cortex from male and female Luc- and Tert-treated mice. Scale bar=50 µm. Data points represent individual mice; bars show mean ± SEM. Statistical significance is indicated by *p<0.05, **p<0.01, ****p<0.0001.

Article Snippet: The next step was an overnight incubation at 4°C using a solution of 3% NGS in PBS-T, containing primary antibodies: anti-rabbit Iba-1 (Wako, cat. #019-19741) at 1:500 to label microglia and macrophages, and anti-mouse TERT (Novus, cat. #NB100-317) at 1:500.

Techniques: In Vivo, Luciferase, Labeling, Imaging, Expressing, Control, RNAscope, Immunohistochemistry

Journal: bioRxiv

Article Title: Telomerase mRNA-Lipid nanoparticles attenuate neuroinflammation after traumatic brain injury in mice

doi: 10.64898/2025.12.18.694748

Figure Lengend Snippet: (a,b) Quantification of microglial activation in peri-contusional cortex at 3 days post-injury (dpi) in male and female mice treated with luciferase control LNPs (Luc) or TERT mRNA-LNPs (Tert). Iba-1+ cells per field (a) and Iba-1+ area (%) (b) were measured by immunohistochemistry, showing a significant reduction in Iba-1+ cell number with Tert treatment in both sexes, with a trend toward reduced Iba-1+ area. (c) Representative confocal images of Iba-1 immunofluorescence (green) with DAPI nuclear counterstain (blue) in peri-contusional cortex from male and female Luc- and Tert-treated mice at 3 dpi. Top panels show low-magnification views; bottom panels show higher-magnification insets highlighting microglial morphology. (d-i) qPCR analysis of cortical cytokine mRNA expression at 3 dpi comparing Luc and Tert groups: IL-1β (d), TNF-α (e), IL-6 (f), TGF-β (g), IL-18 (h), and IL-10 (i). Tert treatment significantly reduced IL-1β, TNF-α, and IL-6 transcripts, with no significant changes in TGF-β, IL-18, or IL-10. Data points represent individual mice; bars indicate mean ± SEM. Statistical significance is denoted as *p< 0.05, **p< 0.01.

Article Snippet: The next step was an overnight incubation at 4°C using a solution of 3% NGS in PBS-T, containing primary antibodies: anti-rabbit Iba-1 (Wako, cat. #019-19741) at 1:500 to label microglia and macrophages, and anti-mouse TERT (Novus, cat. #NB100-317) at 1:500.

Techniques: Activation Assay, Luciferase, Control, Immunohistochemistry, Immunofluorescence, Expressing

Journal: bioRxiv

Article Title: Telomerase mRNA-Lipid nanoparticles attenuate neuroinflammation after traumatic brain injury in mice

doi: 10.64898/2025.12.18.694748

Figure Lengend Snippet: (a) Representative Western blots of serum C-reactive protein (CRP) from male and female mice at 3 days post-injury (dpi) treated with luciferase control LNPs (Luc) or TERT mRNA-LNPs (Tert). Stain-free total protein is shown as a loading control. (b) Densitometric quantification of CRP levels (arbitrary units) normalized to total protein demonstrates a significant reduction in circulating CRP in Tert-versus Luc-treated mice, particularly in males. (c) Serum malondialdehyde (MDA) concentrations, a marker of lipid peroxidation and oxidative stress, showing decreased MDA in Tert-treated animals at 3 dpi. (d–h) Representative hematoxylin and eosin (H&E)-stained sections of spleen (d), kidney (e), lung (f), heart (g), and liver (h) from male and female Luc- and Tert-treated mice at 3 dpi. No treatment-related histopathologic abnormalities are observed in any organ, indicating that TERT mRNA-LNP administration is well tolerated systemically. Scale bar in (h), 100 µm (applies to all histological panels). Data points represent individual mice; bars indicate mean ± SEM. Statistical significance: *p< 0.05, ***p< 0.001.

Article Snippet: The next step was an overnight incubation at 4°C using a solution of 3% NGS in PBS-T, containing primary antibodies: anti-rabbit Iba-1 (Wako, cat. #019-19741) at 1:500 to label microglia and macrophages, and anti-mouse TERT (Novus, cat. #NB100-317) at 1:500.

Techniques: Western Blot, Luciferase, Control, Staining, Marker

Journal: bioRxiv

Article Title: A non-canonical AKT1-TERT pathway coordinates autophagy and ERphagy

doi: 10.1101/2025.11.24.690135

Figure Lengend Snippet: a) Schematic representation of the experimental design for AKT2 inhibition (CCT128930, 6 nM) in iPSC RPE and hFRPE cells. b) Western blot analysis demonstrates a significant increase in phospho-TERT at Ser824 in the nuclear fraction following AKT2 inhibition in iPSC RPE CFH Y402H cells. The AKT2 inhibition treatment also shows an increase in phospho-AKT1 at Ser473 in the nucleus. This also marked a reduction in nuclear localization of AKT2 upon AKT2 inhibition. A total of 25 µg of protein was loaded in each well. Vinculin was utilized as a control for the cytoplasmic fraction, while Lamin B1 served as a nuclear loading control. c) Densitometry data indicate elevated nuclear phospho-TERT expression following AKT2 inhibition in iPSC RPE CFH Y402H cells. d) Immunofluorescence imaging reveals increased nuclear localization of phospho-TERT after AKT2 inhibition in hFRPE cells. Phalloidin stains the actin cytoskeleton and DAPI staining for the nucleus. (scale bar = 10 µm; zoomed inset = 5 µm). e) Quantification of phospho-TERT intensity in the nuclei of 400 cells (n = 4, with 100 nuclei counted per sample). f) Western blot analysis showing the expression levels of phospho-Src, phospho-Foxo3a, phospho-Foxo1, and PRAS40 in the RPE of 15-month-old Akt2 FL, Akt2 KI, and Akt2 cKO mice (n=3). Elevated Src phosphorylation, indicative of active Src and increased phosphorylation of Foxo3a, which indicates its inactive state in cytoplasm, was observed in the RPE of Akt2 KI mice compared to Akt2 cKO and wild-type controls. A total of 10 µg of protein was loaded in each well, and Vinculin was used as the loading control. g) Western blot analysis shows the subcellular localization of phospho-FOXO3a and total FOXO3a in iPSC RPE CFH Y402H cells. In DMSO-treated controls, phospho-FOXO3a is primarily nuclear, indicating its degradation, while dephosphorylated FOXO3a suggests an active form. Total FOXO3a levels increase in the nucleus after AKT2 inhibition. A total of 25 µg of protein was loaded in each well. β-tubulin was utilized as a control for the cytoplasmic fraction, while H3 served as a nuclear loading control. h) Immunofluorescence imaging reveals increased nuclear localization of FOXO3a after AKT2 inhibition in hFRPE cells. Phalloidin stains the actin cytoskeleton and DAPI staining for the nucleus (scale bar = 10 µm; zoomed inset = 5 µm). i) Quantification of FOXO3a intensity in the nuclei of 400 cells (n = 4, with 100 nuclei counted per sample). Values represent mean ± s.d. (n = 4). Statistical analysis was performed using Student’s t -test or one-way ANOVA followed by Tukey’s post hoc test for multiple comparisons. ** P < 0.01, *** P < 0.001, **** P < 0.001. ns= no significance.

Article Snippet: The primary antibodies Akt1 (2938S), Akt2 (3063S), mTOR (2983S), RPTOR (2280S), RICTOR (2114S), PERK (5683T), p-PERK (3179S), eIF2α (9722S), p-eIF2α (9721S), ATF4 (11815S), BiP (3177T), CHOP (2895T), FOXO3a (12829S), p-FOXO3a (9464S), β-actin (4970S), Vinculin (13901S) and c-MYC (5605T) were purchased from Cell Signaling Technology, Inc. TERT (NB100-317) was purchased from Novus Biologicals.

Techniques: Inhibition, Western Blot, Control, Expressing, Immunofluorescence, Imaging, Staining, Phospho-proteomics

Journal: bioRxiv

Article Title: A non-canonical AKT1-TERT pathway coordinates autophagy and ERphagy

doi: 10.1101/2025.11.24.690135

Figure Lengend Snippet: a) Co-immunoprecipitation (Co-IP) with anti-TERT antibody shows increased interaction between TERT and c-MYC following AKT2 inhibition; no interaction with FOXO3a was detected. A schematic illustrates TERT’s interaction with c-MYC and the lack of interaction with FOXO3a. b) Co-immunoprecipitation with anti-FOXO3A antibody demonstrates an enhanced interaction between FOXO3a and c-MYC after AKT2 inhibition; no interaction between FOXO3a and TERT was observed. A schematic depicts the interaction of FOXO3a with c-MYC, highlighting the absence of interaction with TERT. c) Western blot showing UPR arm of PERK through eIF2α and ATF4 and ER chaperone BiP in the cytoplasmic fraction upon AKT2 inhibition. PERK-eIF2α increases ATF4 expression upon AKT2 inhibition. A total of 25 µg of protein was loaded in each well. Vinculin was utilized as a control for the cytoplasmic fraction, while H3 served as a nuclear loading control. d) Co-IP of TERT showing interaction with ATF4 and not PERK upon AKT2 inhibition. e) Co-IP of FOXO3a showing exclusive interaction with PERK and ATF4 upon AKT2 inhibition. f) Western blot analysis indicates elevated expression at both 6 hours and 24 hours of phospho-AMPKα and AMPK-regulated phospho-ULK1 Ser555 following AKT2 inhibition at both 6 and 24 hours, while mTORC1-regulated phospho-ULK1 Ser757 was reduced at 6 hours. g) Western blot results show increased expression of autophagy induction genes, including ATG3, ATG4B, ATG7, and ATG13, after AKT2 inhibition at both 6 and 24 hours. h) Immunofluorescence analysis using a tandem adenoviral GFP-RFP-LC3 construct demonstrates autophagy flux in hFRPE cells infected with the tandem construct overnight, labeling autophagosomes (yellow) and autolysosomes (red). Post DMSO or AKT2i treatment for 24 hours, a significant increase in red puncta (autolysosomes) was observed in AKT2 inhibition condition compared to DMSO controls, indicating enhanced autophagy flux. This was further amplified upon autophagy blockade with bafilomycin (100nM) for 4 hours. Scale bar = 10 µm i) Quantification of autophagosome (yellow puncta) and autolysosomes (red puncta) in DMSO or AKT2i treatment with or without bafilomycin A (100nM for 4 hours). j) LC3 flux ratio analysis in iPSC RPE CFH Y402H cells treated with DMSO or AKT2i for 24 hours, followed by treatment with or without bafilomycin (100 nM for 4 hours). The ratio of LC3-II to LC3-I illustrates autophagic flux, with AKT2i treatment resulting in a significant increase in the LC3-II/LC3-I ratio, indicating enhanced autophagy. In the presence of bafilomycin, the LC3-II/LC3-I ratio further increases due to the accumulation of LC3-II, while p62 levels also rise, suggesting an inhibition of autophagic degradation and highlighting impaired flux despite increased autophagosome formation. k) Quantification of LC3 flux ratio. l) Quantification of percent accumulation of p62 with bafilomycin A treatment (100nM for 4 hours). Values represent mean ± s.d. (n = 4). Statistical analysis was performed using Student’s t -test or one-way ANOVA followed by Tukey’s post hoc test for multiple comparisons. * P < 0.05, ** P < 0.01, **** P < 0.001.

Article Snippet: The primary antibodies Akt1 (2938S), Akt2 (3063S), mTOR (2983S), RPTOR (2280S), RICTOR (2114S), PERK (5683T), p-PERK (3179S), eIF2α (9722S), p-eIF2α (9721S), ATF4 (11815S), BiP (3177T), CHOP (2895T), FOXO3a (12829S), p-FOXO3a (9464S), β-actin (4970S), Vinculin (13901S) and c-MYC (5605T) were purchased from Cell Signaling Technology, Inc. TERT (NB100-317) was purchased from Novus Biologicals.

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Inhibition, Western Blot, Expressing, Control, Immunofluorescence, Construct, Infection, Labeling, Amplification