Therapeutic Effect of Berberine on Huntington's Disease Transgenic Mouse Model.

Huntington disease (HD) represents a family of neurodegenerative diseases that are caused by misfolded proteins. The misfolded proteins accumulate in the affected brain regions in an age-dependent manner to cause late-onset neurodegeneration. Transgenic mouse models expressing the HD protein, huntingtin, have been widely used to identify therapeutics that may retard disease progression. Here we report that Berberine (BBR), an organic small molecule isolated from plants, has protective effects on transgenic HD (N171-82Q) mice. We found that BBR can reduce the accumulation of mutant huntingtin in cultured cells. More importantly, when given orally, BBR could effectively alleviate motor dysfunction and prolong the survival of transgenic N171-82Q HD mice. We found that BBR could promote the degradation of mutant huntingtin by enhancing autophagic function. Since BBR is an orally-taken drug that has been safely used to treat a number of diseases, our findings suggest that BBR can be tested on different HD animal models and HD patients to further evaluate its therapeutic effects

Large Polyglutamine Repeats Cause Muscle Degeneration in SCA17 Mice

SummaryIn polyglutamine (polyQ) diseases, large polyQ repeats cause juvenile cases with different symptoms than those of adult-onset patients, who carry smaller expanded polyQ repeats. The mechanisms behind the differential pathology mediated by different polyQ repeat lengths remain unknown. By studying knockin mouse models of spinal cerebellar ataxia-17 (SCA17), we found that a large polyQ (105 glutamines) in the TATA-box-binding protein (TBP) preferentially causes muscle degeneration and reduces the expression of muscle-specific genes. Direct expression of TBP with different polyQ repeats in mouse muscle revealed that muscle degeneration is mediated only by the large polyQ repeats. Different polyQ repeats differentially alter TBP’s interaction with neuronal and muscle-specific transcription factors. As a result, the large polyQ repeat decreases the association of MyoD with TBP and DNA promoters. Our findings suggest that specific alterations in protein interactions by large polyQ repeats may account for the unique pathology in juvenile polyQ diseases

Oral administration of BBR reduced mutant Htt aggregation and increased autophagy in transgenic N171-82Q mice.

<p><b>(A)</b> Fluorescent immunohistostaining of mouse brain slices from transgenic N171-82Q HD mice and WT siblings, orally gavaged with 40 mg/kg BBR or vehicle daily. <b>(B)</b> Western blotting of the cortex samples of 3 mice per group (HD+BBR vs. HD+vehicle) showing mutant Htt aggregates in the stacking gel and P62. Non-specific immunoreactive bands are also shown on the blots. Actin was used to show the internal control protein on western blot. <b>(C)</b> Densitometry analysis of above Western blots. The data are mean±SE (n = 3). <b>(D)</b> Quantitative RT-PCR analysis of transgenic mutant Htt mRNA in the cortex of the BBR-treated or untreated N171-82Q mice. n = 3 mice per group. Data are presented as mean CT±SEM. <b>(E)</b> Western blotting of WT mouse cortex samples (3-mouse pool per group) after the mice had been orally gavaged with 40 mg/kg BBR or vehicle daily for 24 weeks and sacrificed 4 h after the last BBR administration. <b>(F)</b> Densitometry analysis of Western blots in (E) showing the ratio of P62 to actin. The quantitative data are presented as mean±SE.</p

Oral administration of BBR ameliorates neurological symptoms in transgenic N171-82Q mice.

<p><b>(A, B)</b> Rotarod test of motor coordination of N171-82Q transgenic mice and WT siblings, orally gavaged with 40 mg/kg BBR or vehicle daily. <b>(C, D)</b> Grip strength test on transgenic N171-82Q mice and WT siblings treated with BBR or vehicle. <b>(E)</b> Balance beam test on 18-week-old N171-82Q and WT mice that had been treated with BBR or vehicle. <b>(F)</b> Survival curve for N171-82Q mice and WT siblings treated with BBR or vehicle. (<b>G</b>) The BBR-treated N171-82Q group survived 15 days longer than their untreated HD siblings. <b>(H)</b> Body weight of N171-82Q mice and WT siblings treated with or without BBR. The quantitative data are presented as mean±SE. *p<0.05. **p<0.01. ***p<0.001, n = 7 mice per group.</p

BBR increases autophagic activity in cultured cells.

<p><b>(A)</b> Western blotting of LC3B in HEK293 cells transfected with or without Htt and treated with BBR (0, 5, 25, 50, or 100 μM). <b>(B)</b> Densitometry analysis of the ratios of LC3-I to LC3-II in above Western blots. <b>(C)</b> Western blotting of Htt-transfected HEK293 cells treated with or without 50 μM BBR or bafilomycin (BFA), an autophagy inhibitor. Antibody to P62, which decreases when autophagy activates, was used to confirm the altered activity of autophagy. <b>(D)</b> Densitometry analysis of the ratios of aggregated Htt or P62 to actin in Western blots in (C). The quantitative data are presented as mean±SE.</p

BBR reduced Htt aggregation <i>in vitro</i> in a dose and time-dependent manner.

<p><b>(A)</b> Immunocytostaining images (10 X) of Htt-120Q- or Htt-20Q-transfected HEK293 cells that were treated with different concentrations of BBR (0, 5, 25, 50, 100 μM). <b>(B)</b> Western blot analysis of Htt-transfected cells treated with or without BBR at different concentrations. Aggregated Htt in the stacking gel was detected by mEM48 antibody. Soluble mutant Htt and its potential degraded products are also shown. <b>(C)</b> Densitometry analysis of the ratios of aggregated Htt or Htt-20Q to β-actin on western blots in (B). <b>(D)</b> Fluorescent microscopic images of Htt-120Q-transfected HEK293 cells that were treated with 50 μM BBR at 0 h, 12 h or 24 h post-transfection. <b>(E)</b> Cell-counting analysis of images obtained in (D) showing the percentage of aggregates relative to the total cells revealed by DAPI nuclear staining. <b>(F)</b> Western blotting of Htt-120Q transfected HEK293 cells treated with 50 μm BBR for different times showing aggregated Htt in the stacking gel. <b>(G)</b> Densitometry analysis of the ratios of mutant Htt to β-actin on western blots in (F). The quantitative data are presented as mean±SE.</p

Age-dependent neurological phenotypes of N-terminal HTT KI mice.

<p>(<b>A</b>) The survival curve of N160Q-KI and wild type mice. (<b>B</b>) Body weight of N160Q KI mice. (<b>C</b>) Rotarod tests showing that motor deficits of N160Q KI mice increase with age. (<b>D</b>) Balance beam test showing a motor deficit of N160Q KI mice at 12 months of age. Age-matched WT mice (n = 19) and KI mice (n = 22) were examined. The data are presented as mean±SE. * P<0.05 compared with WT.</p