Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Mar 12;14(3):257.
doi: 10.3390/ph14030257.

The Novel Alpha-2 Adrenoceptor Inhibitor Beditin Reduces Cytotoxicity and Huntingtin Aggregates in Cell Models of Huntington's Disease

Elisabeth Singer  1   2   3 , Lilit Hunanyan  4 , Magda M Melkonyan  4 , Jonasz J Weber  1   2   3 , Lusine Danielyan  5   6 , Huu Phuc Nguyen  3
Affiliations

The Novel Alpha-2 Adrenoceptor Inhibitor Beditin Reduces Cytotoxicity and Huntingtin Aggregates in Cell Models of Huntington's Disease

Elisabeth Singer et al. Pharmaceuticals (Basel). .

Abstract

Huntington's disease (HD) is a monogenetic neurodegenerative disorder characterized by the accumulation of polyglutamine-expanded huntingtin (mHTT). There is currently no cure, and therefore disease-slowing remedies are sought to alleviate symptoms of the multifaceted disorder. Encouraging findings in Alzheimer's and Parkinson's disease on alpha-2 adrenoceptor (α2-AR) inhibition have shown neuroprotective and aggregation-reducing effects in cell and animal models. Here, we analyzed the effect of beditin, a novel α2- adrenoceptor (AR) antagonist, on cell viability and mHTT protein levels in cell models of HD using Western blot, time-resolved Foerster resonance energy transfer (TR-FRET), lactate dehydrogenase (LDH) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) cytotoxicity assays. Beditin decreases cytotoxicity, as measured by TUNEL staining and LDH release, in a neuronal progenitor cell model (STHdh cells) of HD and decreases the aggregation propensity of HTT exon 1 fragments in an overexpression model using human embryonic kidney (HEK) 293T cells. α2-AR is a promising therapeutic target for further characterization in HD models. Our data allow us to suggest beditin as a valuable candidate for the pharmaceutical manipulation of α2-AR, as it is capable of modulating neuronal cell survival and the level of mHTT.

Keywords: Huntington’s disease; alpha-2 adrenoceptor; autophagy; beditin; huntingtin; neurodegeneration; neuronal cell survival.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure A1
Figure A1
α2A-AR levels in STHdh cells and R6/2 mice. (a) Western blot of α2A-AR in R6/2 cortex homogenates, Jurkat homogenates, and MEFQ111/Q111 cells. Monomeric form indicated by unfilled arrowhead, dimeric form by grey arrowhead. (b) Quantification of immunoblots shown in Figure 2a with normalization to β- actin. ** p = 0.0021 (dimer) and ** p = 0.004 (dimer/monomer) (c) Western blot and quantification of α2A-AR levels in cortex homogenates of 11\-week-old R6/2 mice. Unpaired t-test with Welch’s correction p = 0.06, n = 3.
Figure A2
Figure A2
Beditin induces autophagy independent of mTOR. (a) Representative images of LC3B–mCherry–EGFP overexpression in HEK293T cells treated with beditin, rapamycin, or PBS (control). (b) Western blot and quantification of p62 levels after 24 h of beditin treatment in STHdhQ111/Q111 and MEFQ111/Q111 cells.
Figure 1
Figure 1
Chemical structure of 2-(2-amino-4-thiazolyl)-1,4-benzodioxane hydrochloride (beditin), a new benzodioxane derivative.
Figure 2
Figure 2
α2A- adrenoceptor (AR) levels in STHdh cells and R6/2 mice. The dimeric form of the α2A-AR is increased in STHdhQ111/Q111 cells and R6/2 mice. (a) Western blot of α2A-AR in STHdhQ7/Q7 and STHdhQ111/Q111 cells and quantification of α2A-AR signal of the band representing the dimeric form of the protein (top panel, grey shapes) and the dimer/monomer ratio (bottom panel, outlined shapes). Unpaired t-test with Welch’s correction. * p = 0.01; n = 3, *** p = 0.0007. (b) Western blot analysis of α2A-AR levels in striatum homogenates of 11-week-old R6/2 males vs wildtype (WT). Quantification of the α2A-AR signal of the band representing the dimeric form of the protein (top panel, grey shapes) and the monomeric form (bottom panel, outlined shapes). Unpaired t-test with Welch’s correction. * p = 0.016; n = 4–5; ** p = 0.0067; n = 4–5.
Figure 3
Figure 3
Beditin reduces cytotoxicity in STHdh cells. Cytotoxicity is reduced by treating STHdhQ111/Q111 cells with 10 µM of beditin. (a) Brightfield microscopic images of STHdhQ111/Q111 cells 24 h after seeding (0 h treatment) and control vs. 10 µM beditin (bed.) after 48 h of treatment. Scale bar: 20 µM. (b) Representative images of fluorescent terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining (white arrows) in STHdhQ111/Q111 cells. (c) Quantification of TUNEL-positive cells. Unpaired t-test with Welch’s correction. ** p = 0.0075; n = 4. (d) Lactate dehydrogenase (LDH) cytotoxicity assay in STHdhQ111/Q111 cells after 48 h of treatment. Unpaired t-test with Welch’s correction. ** p = 0.003; n = 3.
Figure 4
Figure 4
Beditin induces autophagy independent of mechanistic target of rapamycin (mTOR). Beditin induces autophagy in STHdh cells. − = control; + = indicated substance present (a) Western blot images of the autophagic flux assay in STHdhQ111/Q111 cells and respective quantification of LC3-II and p62 levels. Two-way ANOVA with Dunnett’s multiple comparison to control without bafilomycin A1 (baf.A1). p62: p = 0.053 (control + baf.A1), * p = 0.046 (beditin‒baf.A1), ** p = 0.002 (beditin + baf.A1), n = 3. (b) Assessment of LC3-II levels by Western blot and quantification of LC3-II levels in the presence of baf.A1. STHdh cells were treated for 4 h with beditin (10 µM). Unpaired t-test with Welch’s correction. * p = 0.048; n = 6. (c) Evaluation of mTOR signaling upon beditin treatment (10 µM) in comparison to rapamycin (rapam.) treatment (400 nM) by S6RP phosphorylation at Ser235/236 (P-S6RP).
Figure 5
Figure 5
Levels of soluble mutant HTT (mHTT) in STHdhQ111/Q111 cells. Beditin (bed.) does not reduce total soluble mHTT levels in STHdhQ111/Q111 cells, treated for 48 h. (a) Western blot of HTT in STHdhQ111/Q111 cells by polyQ (1C2) and HTT (D7F7) detection and (b) respective quantification of total 1C2 signal (top panel, grey shapes) and D7F7 (lower panel). In the lower panel, quantification of the full-length band is shown in grey and of a lower fragment signal is shown in red. Unpaired t-test with Welch’s correction. *** p = 0.0001; n = 4. (c) time-resolved Foerster resonance energy transfer (TR-FRET) analysis of soluble mHTT levels in lysates of STHdhQ111/Q111 cells treated for 24 h; n = 4. HA-tagged HTT (HA-HTT) of different polyQ lengths serve as negative (15Q) and positive (128Q) controls for mHTT detection. (d) Immunocytochemistry (ICC) of mHTT (D7F7) in STHdhQ111/Q111 cells after 48 h of treatment and quantification of fluorescent signal; scale bar: 50 µM. Unpaired t-test with Welch’s correction. ** p = 0.0014; n = 4.
Figure 6
Figure 6
Beditin reduces mHTT exon 1 aggregation in a transient overexpression model. (a) Fluorescent imaging of HTT exon 1 49Q overexpression in human embryonic kidney (HEK) 293T cells for 24 h, with beditin (bed.) 5 µM and 10 µM treatment at 10× magnification (scale bar: 100 µm) for determination of aggregate count and 63× (scale bar: 20 µm) for determination of aggregate size. Aggregates at 10× magnification are highlighted by white arrow heads. (b) Quantification of aggregate count per cells in field of view (FOV) (top panel) and quantification of aggregate size (bottom panel). One-way ANOVA with multiple comparisons against control and Dunnett’s multiple comparison correction. Aggregate number: * p = 0.031 (5 µM) and * p = 0.018 (10 µM); n = 4. Aggregate size: * p = 0.044 (5 µM) and * p = 0.048 (10 µM); n = 4. (c) Filter trap analysis of HTT exon 1 49Q overexpression in HEK293T cells for 24 h with beditin 5 µM and 10 µM treatment. A representative image of filter trap immunodetection is shown above the respective quantification. One-way ANOVA with multiple comparisons against control and Dunnett’s multiple comparison correction. * p = 0.027 (10 µM); n = 5.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. The Huntington’s Disease Collaborative Research Group A novel gene containing a trinucleotide repeat that is expanded and unstable on huntington’s disease chromosomes. The huntington’s disease collaborative research group. Cell. 1993;72:971–983. doi: 10.1016/0092-8674(93)90585-E. - DOI - PubMed
    1. Wild E.J., Tabrizi S.J. Therapies targeting DNA and rna in huntington’s disease. Lancet Neurol. 2017;16:837–847. doi: 10.1016/S1474-4422(17)30280-6. - DOI - PMC - PubMed
    1. Zuccato C., Valenza M., Cattaneo E. Molecular mechanisms and potential therapeutical targets in huntington’s disease. Physiol. Rev. 2010;90:905. doi: 10.1152/physrev.00041.2009. - DOI - PubMed
    1. Ferrante R.J., Kowall N.W., Beal M.F., Martin J.B., Bird E.D., Richardson E.P., Jr. Morphologic and histochemical characteristics of a spared subset of striatal neurons in huntington’s disease. J. Neuropathol. Exp. Neurol. 1987;46:12–27. doi: 10.1097/00005072-198701000-00002. - DOI - PubMed
    1. MacDonald E., Kobilka B.K., Scheinin M. Gene targeting—Homing in on α2-adrenoceptor-subtype function. Trends Pharmacol. Sci. 1997;18:211–219. doi: 10.1016/S0165-6147(97)01063-8. - DOI - PubMed

LinkOut - more resources