Journal: bioRxiv

Article Title: ATF4 activates a transcriptional program that chronically suppresses mTOR activity promoting neurodegeneration in Parkinson’s disease models

doi: 10.1101/2025.06.09.658667

Figure Lengend Snippet: A. Cortical neurons were transduced with Ad-ATF4 or Ad-GFP (50-MOI) for 24 hours and mRNA levels of Sesn2, Trib3 and Ddit4 were determined by qRT-PCR. Expression is reported as fold increase over untransduced neurons from the same culture (n=5; t-test, *p<0.05). B. ATF4+/+ and ATF4-/- cortical neurons were treated with MPP+ (50µM) or 6-OHDA (10µM) for 16 hours and Sesn2, Trib3 and Ddit4 mRNA levels was determined by qRT-PCR. Expression is reported as fold increase over vehicle treated neurons from the same culture (n=3-4; t-test, *p<0.05). C. ATF4+/+ and ATF4-/- cortical neurons were treated with MPP+ (50μM) or 6-OHDA (10μM) for the indicated times and SESN2 and DDIT4 protein levels were assessed by western blot and quantified by densitometry and normalized to the loading control Actin or Cyclophilin B (n=3; 2-way-ANOVA *p<0.05). D. Mesencephalic neurons derived from ATF4+/+ and ATF4-/- mice were treated with MPP+ (25μM) or 6-OHDA (5μM) for 16 hours and Sesn2, Trib3 and Ddit4 mRNA levels were assessed in dopaminergic neurons using multiplex RNAScope fluorescence in situ hybridization combined with tyrosine hydroxylase immunostaining. Representative images showing PD neurotoxin induced expression of Sesn2, Trib3 and Ddit4 mRNA fluorescence puncta in wildtype and ATF4-deficient dopaminergic (TH+) neurons (left scale bar=20uM, right scale bar=5uM). Data represent the mean ± SEM number of transcripts per DA neuron (n=3, 2-way-ANOVA, *p<0.05). E. Wildtype and ATF4-deficient mesencephalic neurons were seeded with α-Syn PFFs (5μg/ml) for 9 days and Sesn2, Trib3 and Ddit4 mRNA levels were assessed in dopaminergic neurons using multiplex RNAScope fluorescence in situ hybridization combined with tyrosine hydroxylase immunostaining. Data represent the mean ± SEM number of transcripts per DA neuron (n=3, t-test, *p<0.05). F-H. RNA was isolated from N2 animals or atfs-1 LoF animals treated with MPP+ [100uM] or 6-OHDA [100uM] and a-syn or a- syn::atfs-1LoF animals at indicated time points. Heat maps represent fold change in gene expression over time as calculated by RT-qPCR. Values were normalized to act-1 housekeeping mRNA and fold change was calculated based on normalized mRNA levels in untreated or wildtype animals at respective time points (n=3, 2way-ANOVA, *p<0.05).

Article Snippet: Shuttle plasmids for the generation of recombinant adenoviral vectors expressing Trib3, DDIT4 and SESN2 were constructed from pcDNA-Trb3 (Addgene #131157), pCMS-eGFP- RTP801 (Addgene #65057) and pGEX-Sesn2 (Addgene #61873) respectively in a two-stage cloning process.

Techniques: Transduction, Quantitative RT-PCR, Expressing, Western Blot, Control, Derivative Assay, Multiplex Assay, RNAscope, Fluorescence, In Situ Hybridization, Immunostaining, Isolation, Gene Expression

Journal: bioRxiv

Article Title: ATF4 activates a transcriptional program that chronically suppresses mTOR activity promoting neurodegeneration in Parkinson’s disease models

doi: 10.1101/2025.06.09.658667

Figure Lengend Snippet: A. Cortical neurons were transduced with lenti-shRNA vectors targeting either Sesn2, Trib3, or Ddit4. After 5 days, neurons were treated with MPP+ (50μM) or 6-OHDA (10μM) for 24 hours or seeded with α-Syn PFFs (5μg/ml) for an additional 9 days. The levels of p-mTOR (S2448), p-S6 (S235/236), p-AKT (S473) and p- FOXO3a (S253) and corresponding total protein was assessed by western blot and quantified by densitometry. The ratio of phosphorylated protein/total protein is reported relative to untreated control levels for each shRNA vector (n=5; 2-way-ANOVA, *p<0.05). B. Mesencephalic neuron cultures were transduced with lenti-shRNA and after 5 days treated with MPP+ (25μM) or 6-OHDA (5μM) for 48 hours or seeded with α-Syn PFFs (5μg/ml) for an additional 12 days. The number of TH+ neurons was quantified and is reported relative to TH+ counts in vehicle treated neurons from the same culture. Data represents the mean ± SEM and statistical differences were determined by ANOVA (n=4; *p<0.05).

Article Snippet: Shuttle plasmids for the generation of recombinant adenoviral vectors expressing Trib3, DDIT4 and SESN2 were constructed from pcDNA-Trb3 (Addgene #131157), pCMS-eGFP- RTP801 (Addgene #65057) and pGEX-Sesn2 (Addgene #61873) respectively in a two-stage cloning process.

Techniques: Transduction, shRNA, Western Blot, Control, Plasmid Preparation

Journal: bioRxiv

Article Title: ATF4 activates a transcriptional program that chronically suppresses mTOR activity promoting neurodegeneration in Parkinson’s disease models

doi: 10.1101/2025.06.09.658667

Figure Lengend Snippet: A. Cortical or neurons were transduced with recombinant adenoviral vectors (50-MOI) expressing GFP alone or co-expressing GFP and either SESN2, DDIT4 or Trib3 alone or indicated combinations for 24 hours and p-mTOR(S2448)/total mTOR was assessed by western blot and quantified by densitometry (n=3; ANOVA, *p<0.05). B. PUMA+/+ and PUMA-/- cortical neurons were co-transduced with Ad-Trib3/Ad- SESN2/Ad-DDIT4 or Ad-EGFP (3X) for 24 hours and p-mTOR (S2448), p-S6 (S235/236), p-AKT (S473) and p-Foxo3a (S253) levels were determined by western blot and quantified by densitometry (n=3; 2-way ANOVA *p<0.05). C. Mesencephalic neuron cultures were transduced with indicated vectors and dopamine neuron survival was assessed by TH+ counts at 48 hours. Survival is reported relative to untransduced neurons from the same culture (n=3; ANOVA *p<0.05). D. Midbrain neurons were treated with the dual mTOR inhibitor OSI-027 (5μM) for 24 hours and then co-immunostained for tyrosine hydroxylase (TH) and PUMA. Confocal images were acquired and the fluorescence intensity of PUMA was quantified in dopaminergic (TH+) neurons from 3 independent experiments (n=17-23 cells per group; t- test, *p<0.05). E. PUMA+/+ and PUMA-/- cortical neurons were treated with OSI-027 (5μM) and PUMA protein levels were assessed by western blot and quantified by densitometry and normalized to the loading control Actin (n=3; ANOVA *p<0.05). F. Midbrain neurons derived from PUMA+/+ and PUMA-/- mice were treated with OSI-027 for 48 hours and the number of TH+ cells was counted and reported relative to TH+ counts in vehicle treated neurons from the same culture. Data represents the mean ± SEM and statistical differences were determined by 2-way-ANOVA (n=4; *p<0.05). G. PUMA+/+ and PUMA-/- midbrain neuron cultures were transduced with Ad-Trib3/Ad-SESN2/Ad-DDIT4 or Ad-EGFP (3X) and DA neuron survival was assessed by TH+ cell counts after 48 hours (n=3; ANOVA *p<0.05).

Article Snippet: Shuttle plasmids for the generation of recombinant adenoviral vectors expressing Trib3, DDIT4 and SESN2 were constructed from pcDNA-Trb3 (Addgene #131157), pCMS-eGFP- RTP801 (Addgene #65057) and pGEX-Sesn2 (Addgene #61873) respectively in a two-stage cloning process.

Techniques: Transduction, Recombinant, Expressing, Western Blot, Fluorescence, Control, Derivative Assay

Journal: Pharmaceuticals

Article Title: Brain-Wide Transgene Expression in Mice by Systemic Injection of Genetically Engineered Exosomes: CAP-Exosomes

doi: 10.3390/ph17030270

Figure Lengend Snippet: Generation and characterization of genetically engineered MSCs-derived exosomes. ( A ). The schematic diagram showing the various steps including isolation and growth of BM-MSCs derived-AAV-CAP domain-Lamp2b fusion protein expression plasmid recombination, generation of BM-MSC derived CAP-Exosomes and Lamp2b-exosomes. ( B ). Western blot analysis shows expression of fusion protein CAP-Lamp2b-HA in total protein isolated 48 h after electroporation of CAP-Lamp2b with BM-MSCs as both the Hemagglutinin HA-tag antibody specific and Lamp2b- specific positive signal shows in the 1st western blot run (( B ). Right panel in lane1) and after striping of 2nd western blot (( B ), Left panel, lane1) respectively, and neither in the protein isolated from pcDNA-hygro-Lamp2b electroporated BM-MSCs (( B ), Lane 2, Right and Left panel), nor in the total protein isolated from just BM-MSCs (( B ), Lane 3, Right and Left panel). ( C ). Western blot analysis of a total of 10 9 exosomal particles (average size of 100–150 nm) showed that both Lamp2b and HA specific signal are present in ( C ) (Lane 2) indicating the CAP-Lamp2b-HA fusion protein inclusion on the exosomes.

Article Snippet: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ph17030270/s1 , Figure S1: Graphical presentation of cloning strategy for AAV-CAP domain in pcDNA GNSTM-3-Flag-10-lamp2b-HA expression plasmid vector. (Addgene, USA, Plasmid no.71293).

Techniques: Derivative Assay, Isolation, Expressing, Plasmid Preparation, Western Blot, Electroporation

Journal: Pharmaceuticals

Article Title: Brain-Wide Transgene Expression in Mice by Systemic Injection of Genetically Engineered Exosomes: CAP-Exosomes

doi: 10.3390/ph17030270

Figure Lengend Snippet: Targeting efficiency and specificity of CAP-Exosomes and Lamp2b-exosomes after systemic delivery. ( A ) a schematic for the production of CAP-exosomes and Lamp-2b-exosomes. Presence of high numbers of DiI-labeled (red) CAP-exosomes ( B ) compared to Lamp2b-exosmes ( C ) in the frontal cortex region of respective mice. In the frontal cortex (FC) of the mice very high DiI-labeled exosomes were found (3D-confocal micrograph ( D ), red fluorescence) along with high efficiency GFP-transgene expression (( E ), green fluorescence). Note that visual inspection of merging (Red + Green) confocal micrograph ( F ) revealed a high percentage of only red fluorescence but not merged colors. NeuN stained micrograph of the hippocampus (( G ), white) with DiI labeled exosomes (( H ), red) and GFP- expression (( I ), green) indicate that systemically delivered CAP-Exosomes loaded GFP- transgene capable of brain wide and highly neuron specific delivery of genes and its expression in the mouse brain. Both the red ( J ) and green ( K ) fluorescence were low indicating CAP- domain expressing exosomes minimizing off-target GFP-gene expression. In a separate sets of animals IV injection of DiI labeled and GFP loaded hygro-Lamp2b exosomes to mice resulted in high exosomal uptake ( L ) and GFP-gene expression ( M ) in the animal’s liver.

Article Snippet: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ph17030270/s1 , Figure S1: Graphical presentation of cloning strategy for AAV-CAP domain in pcDNA GNSTM-3-Flag-10-lamp2b-HA expression plasmid vector. (Addgene, USA, Plasmid no.71293).

Techniques: Labeling, Fluorescence, Expressing, Staining, IV Injection