MOESM2 of Alpha-synuclein-induced mitochondrial dysfunction is mediated via a sirtuin 3-dependent pathway

Additional file 2: Figure S2. (a) Representative cropped western blots showing αsyn in cytosolic and mitochondrial fractions from H4 WT-αsyn overexpressing cells. αSyn, GAPDH, and COXIV are all from same samples and immunoblot. (b) Western blot and quantification for SIRT3 in primary embryonic mouse neurons treated at DIV7 for 5 days with either AAV2/8 WT-αsyn or AAV2/8 venus control. Untagged αsyn overexpression shows a significant decrease in SIRT3 level compared to control. Error bars represent the mean ± SD. **p < 0.01 (n = 4)

MOESM1 of Alpha-synuclein-induced mitochondrial dysfunction is mediated via a sirtuin 3-dependent pathway

Additional file 1: Figure S1. (a) Luciferase activity in cytosolic fractions of H4 SL1&SL2 cells over time, n = 5 (b) Representative cropped western blots of denaturing SDS-page (left) and native-page (right) performed at 72 h to confirm the purity of the mitochondrial-enriched fractions, the lysates were probed for the golgi marker (GM130), inner mitochondrial membrane marker (COXIV), GAPDH and αsyn in cytosol and mitochondria from H4 SL1&SL2 cells. (c) Representative image of dot-blot assay of the mitochondrial fractions at 72 h probed for amyloid-specific A11 and αsyn disease-associated (5G4) antibodies. (d) SIRT3 expression was detected by immune-fluorescence in mitochondria. SIRT3 expression is decreased at 72 h in cells overexpressing αsyn. Representative image from 3 experiments. DAPI (nucleus; blue); αsyn (green); SIRT3 (mitochondria; orange); merged images (yellow). Scale bar = 10 μm. Error bars represent the mean ± SD (n = 3–5). **p < 0.01

MOESM3 of Alpha-synuclein-induced mitochondrial dysfunction is mediated via a sirtuin 3-dependent pathway

Additional file 3: Figure S3. (a) Representative cropped western blot showing DRP1 and p-DRP1 (Ser 616) in whole cells lysates from H4 SL1&SL2 cells over time (n = 4). Removal of tet leads to significantly increased p-DRP1 by 72 h. (b) Quantification of p-DRP1/DRP1 protein ratio in whole cell lysates. Error bars represent the mean ± SD. **p < 0.01

MOESM4 of Alpha-synuclein-induced mitochondrial dysfunction is mediated via a sirtuin 3-dependent pathway

Additional file 4: Figure S4. (a) Representative cropped western blot of lysates from dissected substantia nigra/midbrain (SN) of three rats injected unilaterally with control virus, AAV8-Hgluc (gaussia luciferase) demonstrates no change in SIRT3 protein levels when contralateral (C) uninjected SN is compared to ipsilateral (I) injected SN (b) Representative cropped western blot of AMPKα, p-AMPKα (Thr 172), CREB, and p-CREB (Ser 133) in SN lysate of AAV8-SL1&SL2 injected rat. αSyn expression leads to decreased p-AMPK and p-CREB protein levels in ipsilateral (I) injected SN compared to contralateral (C) uninjected SN, n = 5 rats total. Error bars represent the mean ± SD. *p < 0.05, **p < 0.01

Additional file 1 of A mutant methionyl-tRNA synthetase-based toolkit to assess induced-mesenchymal stromal cell secretome in mixed-culture disease models

Additional file 1: Fig. S1. Uncropped agarose gel from Figure 1B

Table_1_Honokiol decreases alpha-synuclein mRNA levels and reveals novel targets for modulating alpha-synuclein expression.docx

BackgroundIntracytoplasmic inclusions comprised of aggregated alpha-synuclein (αsyn) represent a key histopathological feature of neurological disorders collectively termed “synucleinopathies,” which includes Parkinson’s disease (PD). Mutations and multiplications in the SNCA gene encoding αsyn cause familial forms of PD and a large body of evidence indicate a correlation between αsyn accumulation and disease. Decreasing αsyn expression is recognized as a valid target for PD therapeutics, with down-regulation of SNCA expression potentially attenuating downstream cascades of pathologic events. Here, we evaluated if Honokiol (HKL), a polyphenolic compound derived from magnolia tree bark with demonstrated neuroprotective properties, can modulate αsyn levels in multiple experimental models.MethodsHuman neuroglioma cells stably overexpressing αsyn, mouse primary neurons, and human iPSC-derived neurons were exposed to HKL and αsyn protein and SNCA messenger RNA levels were assessed. The effect of HKL on rotenone-induced overexpression of αsyn levels was further assessed and transcriptional profiling of mouse cortical neurons treated with HKL was performed to identify potential targets of HKL.ResultsWe demonstrate that HKL can successfully reduce αsyn protein levels and SNCA expression in multiple in vitro models of PD with our data supporting a mechanism whereby HKL acts by post-transcriptional modulation of SNCA rather than modulating αsyn protein degradation. Transcriptional profiling of mouse cortical neurons treated with HKL identifies several differentially expressed genes (DEG) as potential targets to modulate SNCA expression.ConclusionThis study supports a HKL-mediated downregulation of SNCA as a viable strategy to modify disease progression in PD and other synucleinopathies. HKL has potential as a powerful tool for investigating SNCA gene modulation and its downstream effects.</p

Table_2_Honokiol decreases alpha-synuclein mRNA levels and reveals novel targets for modulating alpha-synuclein expression.DOCX

BackgroundIntracytoplasmic inclusions comprised of aggregated alpha-synuclein (αsyn) represent a key histopathological feature of neurological disorders collectively termed “synucleinopathies,” which includes Parkinson’s disease (PD). Mutations and multiplications in the SNCA gene encoding αsyn cause familial forms of PD and a large body of evidence indicate a correlation between αsyn accumulation and disease. Decreasing αsyn expression is recognized as a valid target for PD therapeutics, with down-regulation of SNCA expression potentially attenuating downstream cascades of pathologic events. Here, we evaluated if Honokiol (HKL), a polyphenolic compound derived from magnolia tree bark with demonstrated neuroprotective properties, can modulate αsyn levels in multiple experimental models.MethodsHuman neuroglioma cells stably overexpressing αsyn, mouse primary neurons, and human iPSC-derived neurons were exposed to HKL and αsyn protein and SNCA messenger RNA levels were assessed. The effect of HKL on rotenone-induced overexpression of αsyn levels was further assessed and transcriptional profiling of mouse cortical neurons treated with HKL was performed to identify potential targets of HKL.ResultsWe demonstrate that HKL can successfully reduce αsyn protein levels and SNCA expression in multiple in vitro models of PD with our data supporting a mechanism whereby HKL acts by post-transcriptional modulation of SNCA rather than modulating αsyn protein degradation. Transcriptional profiling of mouse cortical neurons treated with HKL identifies several differentially expressed genes (DEG) as potential targets to modulate SNCA expression.ConclusionThis study supports a HKL-mediated downregulation of SNCA as a viable strategy to modify disease progression in PD and other synucleinopathies. HKL has potential as a powerful tool for investigating SNCA gene modulation and its downstream effects.</p

Image_2_Honokiol decreases alpha-synuclein mRNA levels and reveals novel targets for modulating alpha-synuclein expression.JPEG

BackgroundIntracytoplasmic inclusions comprised of aggregated alpha-synuclein (αsyn) represent a key histopathological feature of neurological disorders collectively termed “synucleinopathies,” which includes Parkinson’s disease (PD). Mutations and multiplications in the SNCA gene encoding αsyn cause familial forms of PD and a large body of evidence indicate a correlation between αsyn accumulation and disease. Decreasing αsyn expression is recognized as a valid target for PD therapeutics, with down-regulation of SNCA expression potentially attenuating downstream cascades of pathologic events. Here, we evaluated if Honokiol (HKL), a polyphenolic compound derived from magnolia tree bark with demonstrated neuroprotective properties, can modulate αsyn levels in multiple experimental models.MethodsHuman neuroglioma cells stably overexpressing αsyn, mouse primary neurons, and human iPSC-derived neurons were exposed to HKL and αsyn protein and SNCA messenger RNA levels were assessed. The effect of HKL on rotenone-induced overexpression of αsyn levels was further assessed and transcriptional profiling of mouse cortical neurons treated with HKL was performed to identify potential targets of HKL.ResultsWe demonstrate that HKL can successfully reduce αsyn protein levels and SNCA expression in multiple in vitro models of PD with our data supporting a mechanism whereby HKL acts by post-transcriptional modulation of SNCA rather than modulating αsyn protein degradation. Transcriptional profiling of mouse cortical neurons treated with HKL identifies several differentially expressed genes (DEG) as potential targets to modulate SNCA expression.ConclusionThis study supports a HKL-mediated downregulation of SNCA as a viable strategy to modify disease progression in PD and other synucleinopathies. HKL has potential as a powerful tool for investigating SNCA gene modulation and its downstream effects.</p

Image_1_Honokiol decreases alpha-synuclein mRNA levels and reveals novel targets for modulating alpha-synuclein expression.JPEG

BackgroundIntracytoplasmic inclusions comprised of aggregated alpha-synuclein (αsyn) represent a key histopathological feature of neurological disorders collectively termed “synucleinopathies,” which includes Parkinson’s disease (PD). Mutations and multiplications in the SNCA gene encoding αsyn cause familial forms of PD and a large body of evidence indicate a correlation between αsyn accumulation and disease. Decreasing αsyn expression is recognized as a valid target for PD therapeutics, with down-regulation of SNCA expression potentially attenuating downstream cascades of pathologic events. Here, we evaluated if Honokiol (HKL), a polyphenolic compound derived from magnolia tree bark with demonstrated neuroprotective properties, can modulate αsyn levels in multiple experimental models.MethodsHuman neuroglioma cells stably overexpressing αsyn, mouse primary neurons, and human iPSC-derived neurons were exposed to HKL and αsyn protein and SNCA messenger RNA levels were assessed. The effect of HKL on rotenone-induced overexpression of αsyn levels was further assessed and transcriptional profiling of mouse cortical neurons treated with HKL was performed to identify potential targets of HKL.ResultsWe demonstrate that HKL can successfully reduce αsyn protein levels and SNCA expression in multiple in vitro models of PD with our data supporting a mechanism whereby HKL acts by post-transcriptional modulation of SNCA rather than modulating αsyn protein degradation. Transcriptional profiling of mouse cortical neurons treated with HKL identifies several differentially expressed genes (DEG) as potential targets to modulate SNCA expression.ConclusionThis study supports a HKL-mediated downregulation of SNCA as a viable strategy to modify disease progression in PD and other synucleinopathies. HKL has potential as a powerful tool for investigating SNCA gene modulation and its downstream effects.</p

Image_3_Honokiol decreases alpha-synuclein mRNA levels and reveals novel targets for modulating alpha-synuclein expression.JPEG

BackgroundIntracytoplasmic inclusions comprised of aggregated alpha-synuclein (αsyn) represent a key histopathological feature of neurological disorders collectively termed “synucleinopathies,” which includes Parkinson’s disease (PD). Mutations and multiplications in the SNCA gene encoding αsyn cause familial forms of PD and a large body of evidence indicate a correlation between αsyn accumulation and disease. Decreasing αsyn expression is recognized as a valid target for PD therapeutics, with down-regulation of SNCA expression potentially attenuating downstream cascades of pathologic events. Here, we evaluated if Honokiol (HKL), a polyphenolic compound derived from magnolia tree bark with demonstrated neuroprotective properties, can modulate αsyn levels in multiple experimental models.MethodsHuman neuroglioma cells stably overexpressing αsyn, mouse primary neurons, and human iPSC-derived neurons were exposed to HKL and αsyn protein and SNCA messenger RNA levels were assessed. The effect of HKL on rotenone-induced overexpression of αsyn levels was further assessed and transcriptional profiling of mouse cortical neurons treated with HKL was performed to identify potential targets of HKL.ResultsWe demonstrate that HKL can successfully reduce αsyn protein levels and SNCA expression in multiple in vitro models of PD with our data supporting a mechanism whereby HKL acts by post-transcriptional modulation of SNCA rather than modulating αsyn protein degradation. Transcriptional profiling of mouse cortical neurons treated with HKL identifies several differentially expressed genes (DEG) as potential targets to modulate SNCA expression.ConclusionThis study supports a HKL-mediated downregulation of SNCA as a viable strategy to modify disease progression in PD and other synucleinopathies. HKL has potential as a powerful tool for investigating SNCA gene modulation and its downstream effects.</p