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Figure 2. A53T a-Syn mutant regulates mTORC1 and then autophagy by regulating p300 localization (A) Activated mTORC1 and autophagy inhibition in A53T a-Syn mDA neurons. Blots are representative of three biologically independent experiments (n = 3). Two- tailed paired t test. *p < 0.05, **p < 0.01 vs. control iPSC-derived neurons. (B) mTORC1 activation and autophagy inhibition by A53T a-Syn in SH-SY5Y cells induced for 48 h. n = 4 independent experiments, one-way ANOVA with post hoc Tukey test. **p < 0.01, ***p < 0.001 vs. control cells; #p < 0.05, ##p < 0.01 vs. starved control cells; qqq p < 0.001 vs. Dox-treated cells. (C) Activation of mTORC1 signaling (phosphorylated S6) by inducing A53T a-Syn induced for 48 h. n = 4 independent experiments, R30 cells scored per condition per experiment. One-way ANOVA with post hoc Tukey test. *p < 0.05, ****p < 0.0001 vs. control cells; ##p < 0.01 vs. starved control cells. Scale bars, 5 and 2 mm (enlarged images).

Journal: Neuron

Article Title: Alpha-synuclein mutations mislocalize cytoplasmic p300 compromising autophagy, which is rescued by ACLY inhibition.

doi: 10.1016/j.neuron.2025.03.028

Figure Lengend Snippet: Figure 2. A53T a-Syn mutant regulates mTORC1 and then autophagy by regulating p300 localization (A) Activated mTORC1 and autophagy inhibition in A53T a-Syn mDA neurons. Blots are representative of three biologically independent experiments (n = 3). Two- tailed paired t test. *p < 0.05, **p < 0.01 vs. control iPSC-derived neurons. (B) mTORC1 activation and autophagy inhibition by A53T a-Syn in SH-SY5Y cells induced for 48 h. n = 4 independent experiments, one-way ANOVA with post hoc Tukey test. **p < 0.01, ***p < 0.001 vs. control cells; #p < 0.05, ##p < 0.01 vs. starved control cells; qqq p < 0.001 vs. Dox-treated cells. (C) Activation of mTORC1 signaling (phosphorylated S6) by inducing A53T a-Syn induced for 48 h. n = 4 independent experiments, R30 cells scored per condition per experiment. One-way ANOVA with post hoc Tukey test. *p < 0.05, ****p < 0.0001 vs. control cells; ##p < 0.01 vs. starved control cells. Scale bars, 5 and 2 mm (enlarged images).

Article Snippet: The following DNA or siRNA/shRNA constructs were also used: empty pEGFP from Clontech; pcDNA3.1-myc-6XHis from Invitrogen; pcDNA3.1-p300 (#23252), pcDNA3.1-p300 dominantnegative (DN;HAT-) (#23254), pSG5-HA-p300 (#89094), pRK5-HA-YFP-raptor (#73385), pRK5-HA-raptor (#8513), pcDNA3-AMPKa2 WT (#15991), pcDNA3-AMPKa2 K45R (#15992), pcDNA3-FLAG-LKB1 (#8590), pHM6-HTT(Q74)-HA (#40264), pEGFP-C1-a-Syn wt (#40822), pEGFP-C1-a-Syn-A53T (#40823) from Addgene. pCMV6-Myc-DDK-ACLY (#RC200508) from Origene.

Techniques: Mutagenesis, Inhibition, Two Tailed Test, Control, Derivative Assay, Activation Assay

Figure 3. AMPK inhibition by A53T a-Syn ACLY regulates p300-mediated mTORC1 activity and autophagic flux (A and B) Reduced AMPK activity (phosphorylation of AMPK) by A53T a-Syn in SH-SY5Y cells induced with Dox for 48 h (A) or A53T a-Syn mDA neurons (B). Blots are representative of at least three independent experiments (n = 3). Two-tailed paired t test. *p < 0.05, **p < 0.01 vs. A53T a-Syn-inducible SH-SY5Y cells without Dox (A) or control neurons (B). (C and D) Dot blot assay showing inhibition of phosphorylated p300 levels at Ser89 by A53T a-Syn in SH-SY5Y cells induced with Dox for 48 h (C) or A53T a-Syn mDA neurons (D). n = 3 independent experiments, two-tailed paired t test. *p < 0.05, **p < 0.01 vs. A53T a-Syn-inducible SH-SY5Y cells without Dox (C) or control neurons (D).

Journal: Neuron

Article Title: Alpha-synuclein mutations mislocalize cytoplasmic p300 compromising autophagy, which is rescued by ACLY inhibition.

doi: 10.1016/j.neuron.2025.03.028

Figure Lengend Snippet: Figure 3. AMPK inhibition by A53T a-Syn ACLY regulates p300-mediated mTORC1 activity and autophagic flux (A and B) Reduced AMPK activity (phosphorylation of AMPK) by A53T a-Syn in SH-SY5Y cells induced with Dox for 48 h (A) or A53T a-Syn mDA neurons (B). Blots are representative of at least three independent experiments (n = 3). Two-tailed paired t test. *p < 0.05, **p < 0.01 vs. A53T a-Syn-inducible SH-SY5Y cells without Dox (A) or control neurons (B). (C and D) Dot blot assay showing inhibition of phosphorylated p300 levels at Ser89 by A53T a-Syn in SH-SY5Y cells induced with Dox for 48 h (C) or A53T a-Syn mDA neurons (D). n = 3 independent experiments, two-tailed paired t test. *p < 0.05, **p < 0.01 vs. A53T a-Syn-inducible SH-SY5Y cells without Dox (C) or control neurons (D).

Article Snippet: The following DNA or siRNA/shRNA constructs were also used: empty pEGFP from Clontech; pcDNA3.1-myc-6XHis from Invitrogen; pcDNA3.1-p300 (#23252), pcDNA3.1-p300 dominantnegative (DN;HAT-) (#23254), pSG5-HA-p300 (#89094), pRK5-HA-YFP-raptor (#73385), pRK5-HA-raptor (#8513), pcDNA3-AMPKa2 WT (#15991), pcDNA3-AMPKa2 K45R (#15992), pcDNA3-FLAG-LKB1 (#8590), pHM6-HTT(Q74)-HA (#40264), pEGFP-C1-a-Syn wt (#40822), pEGFP-C1-a-Syn-A53T (#40823) from Addgene. pCMV6-Myc-DDK-ACLY (#RC200508) from Origene.

Techniques: Inhibition, Activity Assay, Phospho-proteomics, Two Tailed Test, Control, Dot Blot

Figure 4. ACLY regulates p300-mediated mTORC1 activity and autophagic flux (A) Nuclear translocation of p300 with 10 mM HC treatment. Scale bar, 5 mm. n = 4 independent experiments, R30 cells scored per condition per experiment. Two-tailed paired t test. *p < 0.05 vs. control cells. Arrows indicate p300 signals in the cytoplasm. (B) Reduced raptor acetylation after inhibiting ACLY using 10 mM HC for 24 h (left) or by transfecting siRNA (right). n = 3 independent experiments. Two-tailed paired t test. *p < 0.05, **p < 0.01 vs. vehicle-treated or control siRNA transfected cells.

Journal: Neuron

Article Title: Alpha-synuclein mutations mislocalize cytoplasmic p300 compromising autophagy, which is rescued by ACLY inhibition.

doi: 10.1016/j.neuron.2025.03.028

Figure Lengend Snippet: Figure 4. ACLY regulates p300-mediated mTORC1 activity and autophagic flux (A) Nuclear translocation of p300 with 10 mM HC treatment. Scale bar, 5 mm. n = 4 independent experiments, R30 cells scored per condition per experiment. Two-tailed paired t test. *p < 0.05 vs. control cells. Arrows indicate p300 signals in the cytoplasm. (B) Reduced raptor acetylation after inhibiting ACLY using 10 mM HC for 24 h (left) or by transfecting siRNA (right). n = 3 independent experiments. Two-tailed paired t test. *p < 0.05, **p < 0.01 vs. vehicle-treated or control siRNA transfected cells.

Article Snippet: The following DNA or siRNA/shRNA constructs were also used: empty pEGFP from Clontech; pcDNA3.1-myc-6XHis from Invitrogen; pcDNA3.1-p300 (#23252), pcDNA3.1-p300 dominantnegative (DN;HAT-) (#23254), pSG5-HA-p300 (#89094), pRK5-HA-YFP-raptor (#73385), pRK5-HA-raptor (#8513), pcDNA3-AMPKa2 WT (#15991), pcDNA3-AMPKa2 K45R (#15992), pcDNA3-FLAG-LKB1 (#8590), pHM6-HTT(Q74)-HA (#40264), pEGFP-C1-a-Syn wt (#40822), pEGFP-C1-a-Syn-A53T (#40823) from Addgene. pCMV6-Myc-DDK-ACLY (#RC200508) from Origene.

Techniques: Activity Assay, Translocation Assay, Two Tailed Test, Control, Transfection

Figure 5. Inhibition of ACLY restores altered autophagy regulation to reduce pathological a-Syn aggregates and rescues pathological phe- notypes induced by a-Syn (A) Rescue of p300 localization in cytoplasm by A53T a-Syn after HC treatment. Blots are representative of at least three independent experiments (n = 3). One- way ANOVA with post hoc Tukey test. *p < 0.05 vs. A53T a-Syn-inducible SH-SY5Y cells without Dox; ##p < 0.01 vs. Dox-treated cells. (B) Rescue of increased p300 activity in A53T a-Syn-expressing cells by 10 mM HC treatment. n = 4 independent experiments, one-way ANOVA with post hoc Tukey test. *p < 0.05 vs. A53T a-Syn-inducible SH-SY5Y cells without Dox; ##p < 0.01 vs. Dox-treated cells.

Journal: Neuron

Article Title: Alpha-synuclein mutations mislocalize cytoplasmic p300 compromising autophagy, which is rescued by ACLY inhibition.

doi: 10.1016/j.neuron.2025.03.028

Figure Lengend Snippet: Figure 5. Inhibition of ACLY restores altered autophagy regulation to reduce pathological a-Syn aggregates and rescues pathological phe- notypes induced by a-Syn (A) Rescue of p300 localization in cytoplasm by A53T a-Syn after HC treatment. Blots are representative of at least three independent experiments (n = 3). One- way ANOVA with post hoc Tukey test. *p < 0.05 vs. A53T a-Syn-inducible SH-SY5Y cells without Dox; ##p < 0.01 vs. Dox-treated cells. (B) Rescue of increased p300 activity in A53T a-Syn-expressing cells by 10 mM HC treatment. n = 4 independent experiments, one-way ANOVA with post hoc Tukey test. *p < 0.05 vs. A53T a-Syn-inducible SH-SY5Y cells without Dox; ##p < 0.01 vs. Dox-treated cells.

Article Snippet: The following DNA or siRNA/shRNA constructs were also used: empty pEGFP from Clontech; pcDNA3.1-myc-6XHis from Invitrogen; pcDNA3.1-p300 (#23252), pcDNA3.1-p300 dominantnegative (DN;HAT-) (#23254), pSG5-HA-p300 (#89094), pRK5-HA-YFP-raptor (#73385), pRK5-HA-raptor (#8513), pcDNA3-AMPKa2 WT (#15991), pcDNA3-AMPKa2 K45R (#15992), pcDNA3-FLAG-LKB1 (#8590), pHM6-HTT(Q74)-HA (#40264), pEGFP-C1-a-Syn wt (#40822), pEGFP-C1-a-Syn-A53T (#40823) from Addgene. pCMV6-Myc-DDK-ACLY (#RC200508) from Origene.

Techniques: Inhibition, Activity Assay, Expressing

Figure 6. ACLY inhibition rescues a-Syn-induced pathology in SNCA zebrafish model (A) Failure of nuclear translocation of p300 by A53T a-Syn expression. Representative images of antibody staining for a-Syn, p300, and DAPI were performed on cryosections across the spinal cord of transgenic Dendra-a-SYN_WT and A53T fish with mosaic expression of the transgene at 2 d.p.f. n = 14 fish per group, two- tailed unpaired t test. *p < 0.05 vs. Dendra-a-SYN _WT. Scale bar, 5 mm. (B) Enhanced mTORC1 activity in fish expressing human A53T a-Syn mutation. Representative images and quantification of S6K1 and p-S6K1 western blots to measure mTORC1 activity in fish with pan-neuronal expression of Dendra-a-SYN_WT and A53T at 2 d.p.f. n = 12 independent clutches, two-tailed unpaired t test. *p < 0.05 vs. Dendra-a-SYN_WT. (C) Inhibition of autophagic flux in fish carrying the A53T mutation. Representative images and quantification of western blots for the levels of LC3-II in fish with pan-neuronal expression of Dendra-a-SYN_WT or A53T at 2 d.p.f. in basal conditions and after treatment with 10 mM NH4Cl for 4 h. Graph represents the ratios of LC3-II vs. actin normalized to a single negative sibling control run in all blots. The same sample from a negative sibling was run in parallel on every gel and used for

Journal: Neuron

Article Title: Alpha-synuclein mutations mislocalize cytoplasmic p300 compromising autophagy, which is rescued by ACLY inhibition.

doi: 10.1016/j.neuron.2025.03.028

Figure Lengend Snippet: Figure 6. ACLY inhibition rescues a-Syn-induced pathology in SNCA zebrafish model (A) Failure of nuclear translocation of p300 by A53T a-Syn expression. Representative images of antibody staining for a-Syn, p300, and DAPI were performed on cryosections across the spinal cord of transgenic Dendra-a-SYN_WT and A53T fish with mosaic expression of the transgene at 2 d.p.f. n = 14 fish per group, two- tailed unpaired t test. *p < 0.05 vs. Dendra-a-SYN _WT. Scale bar, 5 mm. (B) Enhanced mTORC1 activity in fish expressing human A53T a-Syn mutation. Representative images and quantification of S6K1 and p-S6K1 western blots to measure mTORC1 activity in fish with pan-neuronal expression of Dendra-a-SYN_WT and A53T at 2 d.p.f. n = 12 independent clutches, two-tailed unpaired t test. *p < 0.05 vs. Dendra-a-SYN_WT. (C) Inhibition of autophagic flux in fish carrying the A53T mutation. Representative images and quantification of western blots for the levels of LC3-II in fish with pan-neuronal expression of Dendra-a-SYN_WT or A53T at 2 d.p.f. in basal conditions and after treatment with 10 mM NH4Cl for 4 h. Graph represents the ratios of LC3-II vs. actin normalized to a single negative sibling control run in all blots. The same sample from a negative sibling was run in parallel on every gel and used for

Article Snippet: The following DNA or siRNA/shRNA constructs were also used: empty pEGFP from Clontech; pcDNA3.1-myc-6XHis from Invitrogen; pcDNA3.1-p300 (#23252), pcDNA3.1-p300 dominantnegative (DN;HAT-) (#23254), pSG5-HA-p300 (#89094), pRK5-HA-YFP-raptor (#73385), pRK5-HA-raptor (#8513), pcDNA3-AMPKa2 WT (#15991), pcDNA3-AMPKa2 K45R (#15992), pcDNA3-FLAG-LKB1 (#8590), pHM6-HTT(Q74)-HA (#40264), pEGFP-C1-a-Syn wt (#40822), pEGFP-C1-a-Syn-A53T (#40823) from Addgene. pCMV6-Myc-DDK-ACLY (#RC200508) from Origene.

Techniques: Inhibition, Translocation Assay, Expressing, Staining, Transgenic Assay, Two Tailed Test, Activity Assay, Mutagenesis, Western Blot, Control

Figure 7. ACLY inhibition rescues a-Syn-induced pathology in SNCA mouse model (A) Schematic of the study paradigm, including the intracerebroventricular (ICV) infusion by minipump and designated collection time points. (B) p300 localization, mTORC1, and autophagy levels in the brains of SNCA mice after injection with 100 mM HC for 28 days. n = 6 each group, two-tailed unpaired t test. *p < 0.05, **p < 0.01, ***p < 0.001 vs. vehicle-injected SNCA mice. (C) Rescue of AMPK activity after injection with 100 mM HC for 28 days. n = 6 each group, two-tailed unpaired t test. *p < 0.05 vs. vehicle-injected SNCA mice. (D) Decreased total human a-Syn level after injection with 100 mM HC for 28 days. n = 8 for HC-treated and n = 9 for vehicle control group, two-tailed unpaired t test. **p < 0.01 vs. vehicle-injected SNCA mice. Scale bar, 20 and 10 mm (enlarged images). Arrowheads indicate human a-Syn signals.

Journal: Neuron

Article Title: Alpha-synuclein mutations mislocalize cytoplasmic p300 compromising autophagy, which is rescued by ACLY inhibition.

doi: 10.1016/j.neuron.2025.03.028

Figure Lengend Snippet: Figure 7. ACLY inhibition rescues a-Syn-induced pathology in SNCA mouse model (A) Schematic of the study paradigm, including the intracerebroventricular (ICV) infusion by minipump and designated collection time points. (B) p300 localization, mTORC1, and autophagy levels in the brains of SNCA mice after injection with 100 mM HC for 28 days. n = 6 each group, two-tailed unpaired t test. *p < 0.05, **p < 0.01, ***p < 0.001 vs. vehicle-injected SNCA mice. (C) Rescue of AMPK activity after injection with 100 mM HC for 28 days. n = 6 each group, two-tailed unpaired t test. *p < 0.05 vs. vehicle-injected SNCA mice. (D) Decreased total human a-Syn level after injection with 100 mM HC for 28 days. n = 8 for HC-treated and n = 9 for vehicle control group, two-tailed unpaired t test. **p < 0.01 vs. vehicle-injected SNCA mice. Scale bar, 20 and 10 mm (enlarged images). Arrowheads indicate human a-Syn signals.

Article Snippet: The following DNA or siRNA/shRNA constructs were also used: empty pEGFP from Clontech; pcDNA3.1-myc-6XHis from Invitrogen; pcDNA3.1-p300 (#23252), pcDNA3.1-p300 dominantnegative (DN;HAT-) (#23254), pSG5-HA-p300 (#89094), pRK5-HA-YFP-raptor (#73385), pRK5-HA-raptor (#8513), pcDNA3-AMPKa2 WT (#15991), pcDNA3-AMPKa2 K45R (#15992), pcDNA3-FLAG-LKB1 (#8590), pHM6-HTT(Q74)-HA (#40264), pEGFP-C1-a-Syn wt (#40822), pEGFP-C1-a-Syn-A53T (#40823) from Addgene. pCMV6-Myc-DDK-ACLY (#RC200508) from Origene.

Techniques: Inhibition, Injection, Two Tailed Test, Activity Assay, Control