Limits...
Tricyclic pyrone compounds prevent aggregation and reverse cellular phenotypes caused by expression of mutant huntingtin protein in striatal neurons.

Trushina E, Rana S, McMurray CT, Hua DH - BMC Neurosci (2009)

Bottom Line: Most data suggest that polyglutamine-mediated aggregation associated with expression of mutant huntingtin protein (mhtt) contributes to the pathology.Suppression of aggregation is accepted as one of the markers of successful therapeutic approaches.TP compounds effectively inhibit aggregation caused by mhtt in neurons and glial cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Chemistry, CBC Building, Kansas State University, Manhattan, KS 66506, USA. trushina.eugenia@mayo.edu

ABSTRACT

Background: Huntington's disease (HD) is a progressive neurodegenerative disorder caused by a CAG repeat expansion mutation in the coding region of a novel gene. The mechanism of HD is unknown. Most data suggest that polyglutamine-mediated aggregation associated with expression of mutant huntingtin protein (mhtt) contributes to the pathology. However, recent studies have identified early cellular dysfunctions that preclude aggregate formation. Suppression of aggregation is accepted as one of the markers of successful therapeutic approaches. Previously, we demonstrated that tricyclic pyrone (TP) compounds efficiently inhibited formation of amyloid-beta (Abeta) aggregates in cell and mouse models representing Alzheimer's Disease (AD). In the present study, we aimed to determine whether TP compounds could prevent aggregation and restore early cellular defects in primary embryonic striatal neurons from animal model representing HD.

Results: TP compounds effectively inhibit aggregation caused by mhtt in neurons and glial cells. Treatment with TP compounds also alleviated cholesterol accumulation and restored clathrin-independent endocytosis in HD neurons.

Conclusion: We have found that TP compounds not only blocked mhtt-induced aggregation, but also alleviated early cellular dysfunctions that preclude aggregate formation. Our data suggest TP molecules may be used as lead compounds for prevention or treatment of multiple neurodegenerative diseases including HD and AD.

Show MeSH

Related in: MedlinePlus

Treatment with CP2 restores caveolin-related endocytosis inhibited by mhtt expression. A. Treatment with CP2 does not affect internalization of fluorescently labelled Tfn (red) in control or HD neurons. Images represent internalization of Tfn in live neurons 10 min after addition to the cells. Tfn internalization and localization were indistinguishable in untreated and CP2-pretreated control and HD cells. Data for 2 μM CP2 treatment is shown. Scale bar, 5 μm. B. Internalization of LacCer (green) is inhibited in striatal HD72 neurons (HD72, middle panel) comparing to control neurons. Treatment with 2 μM CP2 restores internalization of LacCer in HD72 neurons (HD72+2 μM CP2). Images of live cells in A and B were acquired using LSM 510 confocal microscope with 100× oil DIC lens (1.4 na). Scale bar, 5 μm. C. Quantification of Tfn and LacCer internalization in control and HD72 striatal neurons with and without CP2 treatment acquired in experiments shown in A and B. At least 10 cells were taken into analysis in each 3 independent experiments. *, p < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2719645&req=5

Figure 4: Treatment with CP2 restores caveolin-related endocytosis inhibited by mhtt expression. A. Treatment with CP2 does not affect internalization of fluorescently labelled Tfn (red) in control or HD neurons. Images represent internalization of Tfn in live neurons 10 min after addition to the cells. Tfn internalization and localization were indistinguishable in untreated and CP2-pretreated control and HD cells. Data for 2 μM CP2 treatment is shown. Scale bar, 5 μm. B. Internalization of LacCer (green) is inhibited in striatal HD72 neurons (HD72, middle panel) comparing to control neurons. Treatment with 2 μM CP2 restores internalization of LacCer in HD72 neurons (HD72+2 μM CP2). Images of live cells in A and B were acquired using LSM 510 confocal microscope with 100× oil DIC lens (1.4 na). Scale bar, 5 μm. C. Quantification of Tfn and LacCer internalization in control and HD72 striatal neurons with and without CP2 treatment acquired in experiments shown in A and B. At least 10 cells were taken into analysis in each 3 independent experiments. *, p < 0.001.

Mentions: Internalization of essential extracellular components in neurons occurs through multiple endocytic pathways including clathrin-dependent and clathrin-independent caveolin-related endocytosis [23]. In order to evaluate whether CP2 treatment could restore defective endocytosis in HD72 neurons, we monitored internalization of fluorescently labelled cargoes specific for each pathway. Alexa Fluor 594-labeled transferrin, Tfn, was used as a marker for clathrin-mediated endocytosis [24], and BODIPY-lactosylceramide, LacCer, as a marker for clathrin-independent caveolin-related endocytosis [25]. Consistent with previous observations, expression of mhtt had no effect on Tfn internalization in HD72 neurons comparing to control cells (Figure 4A, C). Treatment with either 2 or 5 μM of CP2 also did not affect Tfn internalization or intracellular localization in HD72 neurons comparing to control or untreated HD72 cells (Figure 4A, C). These data suggest that neither CP2 treatment nor mhtt expression affected clathrin-mediated endocytosis in striatal neurons. In contrast, the uptake of LacCer was inhibited by 70% in HD neurons relative to control cells (Figure 4B, C). However, pre-treatment with 2 μM of CP2 restored the uptake of LacCer in HD cells to approximately 60% of that in control neurons (Figure 4B, C). Thus, CP2 partially restores lipid trafficking defect caused by expression of mhtt, and the beneficial effect of CP2 is evident prior to visible formation of aggregates.


Tricyclic pyrone compounds prevent aggregation and reverse cellular phenotypes caused by expression of mutant huntingtin protein in striatal neurons.

Trushina E, Rana S, McMurray CT, Hua DH - BMC Neurosci (2009)

Treatment with CP2 restores caveolin-related endocytosis inhibited by mhtt expression. A. Treatment with CP2 does not affect internalization of fluorescently labelled Tfn (red) in control or HD neurons. Images represent internalization of Tfn in live neurons 10 min after addition to the cells. Tfn internalization and localization were indistinguishable in untreated and CP2-pretreated control and HD cells. Data for 2 μM CP2 treatment is shown. Scale bar, 5 μm. B. Internalization of LacCer (green) is inhibited in striatal HD72 neurons (HD72, middle panel) comparing to control neurons. Treatment with 2 μM CP2 restores internalization of LacCer in HD72 neurons (HD72+2 μM CP2). Images of live cells in A and B were acquired using LSM 510 confocal microscope with 100× oil DIC lens (1.4 na). Scale bar, 5 μm. C. Quantification of Tfn and LacCer internalization in control and HD72 striatal neurons with and without CP2 treatment acquired in experiments shown in A and B. At least 10 cells were taken into analysis in each 3 independent experiments. *, p < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2719645&req=5

Figure 4: Treatment with CP2 restores caveolin-related endocytosis inhibited by mhtt expression. A. Treatment with CP2 does not affect internalization of fluorescently labelled Tfn (red) in control or HD neurons. Images represent internalization of Tfn in live neurons 10 min after addition to the cells. Tfn internalization and localization were indistinguishable in untreated and CP2-pretreated control and HD cells. Data for 2 μM CP2 treatment is shown. Scale bar, 5 μm. B. Internalization of LacCer (green) is inhibited in striatal HD72 neurons (HD72, middle panel) comparing to control neurons. Treatment with 2 μM CP2 restores internalization of LacCer in HD72 neurons (HD72+2 μM CP2). Images of live cells in A and B were acquired using LSM 510 confocal microscope with 100× oil DIC lens (1.4 na). Scale bar, 5 μm. C. Quantification of Tfn and LacCer internalization in control and HD72 striatal neurons with and without CP2 treatment acquired in experiments shown in A and B. At least 10 cells were taken into analysis in each 3 independent experiments. *, p < 0.001.
Mentions: Internalization of essential extracellular components in neurons occurs through multiple endocytic pathways including clathrin-dependent and clathrin-independent caveolin-related endocytosis [23]. In order to evaluate whether CP2 treatment could restore defective endocytosis in HD72 neurons, we monitored internalization of fluorescently labelled cargoes specific for each pathway. Alexa Fluor 594-labeled transferrin, Tfn, was used as a marker for clathrin-mediated endocytosis [24], and BODIPY-lactosylceramide, LacCer, as a marker for clathrin-independent caveolin-related endocytosis [25]. Consistent with previous observations, expression of mhtt had no effect on Tfn internalization in HD72 neurons comparing to control cells (Figure 4A, C). Treatment with either 2 or 5 μM of CP2 also did not affect Tfn internalization or intracellular localization in HD72 neurons comparing to control or untreated HD72 cells (Figure 4A, C). These data suggest that neither CP2 treatment nor mhtt expression affected clathrin-mediated endocytosis in striatal neurons. In contrast, the uptake of LacCer was inhibited by 70% in HD neurons relative to control cells (Figure 4B, C). However, pre-treatment with 2 μM of CP2 restored the uptake of LacCer in HD cells to approximately 60% of that in control neurons (Figure 4B, C). Thus, CP2 partially restores lipid trafficking defect caused by expression of mhtt, and the beneficial effect of CP2 is evident prior to visible formation of aggregates.

Bottom Line: Most data suggest that polyglutamine-mediated aggregation associated with expression of mutant huntingtin protein (mhtt) contributes to the pathology.Suppression of aggregation is accepted as one of the markers of successful therapeutic approaches.TP compounds effectively inhibit aggregation caused by mhtt in neurons and glial cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Chemistry, CBC Building, Kansas State University, Manhattan, KS 66506, USA. trushina.eugenia@mayo.edu

ABSTRACT

Background: Huntington's disease (HD) is a progressive neurodegenerative disorder caused by a CAG repeat expansion mutation in the coding region of a novel gene. The mechanism of HD is unknown. Most data suggest that polyglutamine-mediated aggregation associated with expression of mutant huntingtin protein (mhtt) contributes to the pathology. However, recent studies have identified early cellular dysfunctions that preclude aggregate formation. Suppression of aggregation is accepted as one of the markers of successful therapeutic approaches. Previously, we demonstrated that tricyclic pyrone (TP) compounds efficiently inhibited formation of amyloid-beta (Abeta) aggregates in cell and mouse models representing Alzheimer's Disease (AD). In the present study, we aimed to determine whether TP compounds could prevent aggregation and restore early cellular defects in primary embryonic striatal neurons from animal model representing HD.

Results: TP compounds effectively inhibit aggregation caused by mhtt in neurons and glial cells. Treatment with TP compounds also alleviated cholesterol accumulation and restored clathrin-independent endocytosis in HD neurons.

Conclusion: We have found that TP compounds not only blocked mhtt-induced aggregation, but also alleviated early cellular dysfunctions that preclude aggregate formation. Our data suggest TP molecules may be used as lead compounds for prevention or treatment of multiple neurodegenerative diseases including HD and AD.

Show MeSH
Related in: MedlinePlus