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Human mutant huntingtin disrupts vocal learning in transgenic songbirds

Nature Neuroscience volume 18, pages 1617–1622 (2015)Cite this article

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Abstract

Speech and vocal impairments characterize many neurological disorders. However, the neurogenetic mechanisms of these disorders are not well understood, and current animal models do not have the necessary circuitry to recapitulate vocal learning deficits. We developed germline transgenic songbirds, zebra finches (Taneiopygia guttata) expressing human mutant huntingtin (mHTT), a protein responsible for the progressive deterioration of motor and cognitive function in Huntington's disease (HD). Although generally healthy, the mutant songbirds had severe vocal disorders, including poor vocal imitation, stuttering, and progressive syntax and syllable degradation. Their song abnormalities were associated with HD-related neuropathology and dysfunction of the cortical–basal ganglia (CBG) song circuit. These transgenics are, to the best of our knowledge, the first experimentally created, functional mutant songbirds. Their progressive and quantifiable vocal disorder, combined with circuit dysfunction in the CBG song system, offers a model for genetic manipulation and the development of therapeutic strategies for CBG-related vocal and motor disorders.

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Figure 1: Genotyping of the transgenic zebra finch.
Figure 2: Vocal learning disorder in mutant transgenics.
Figure 3: Progressive song change in adult mutants.
Figure 4: Neuropathology of mutant transgenics.

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Acknowledgements

We thank F. Nottebohm for his support and critical reading of the manuscript, and N. Wexler for organizing a songbird symposium to support this project. We are grateful to R. Agate for his advice on embryo injection, and A. Reiner, who provided advice on IHC. Rockefeller University Bio-Imaging Resource Center for help and advice relating to stereology methods and confocal microscopy. This study was supported by CHDI funding, an Irma T. Hirschl research award, and a Herbert and Nell Singer scholarship, with additional support from The Rockefeller University to W.L.

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Authors and Affiliations

  1. Laboratory of Animal Behavior, The Rockefeller University, New York, New York, USA

    Wan-chun Liu, Jessica Kohn, Sarah K Szwed, Eben Pariser, Sharon Sepe & Bhagwattie Haripal

  2. Laboratory of Mammalian Cell Biology and Development, The Rockefeller University, New York, New York, USA

    Naoki Oshimori

  3. Neurodegeneration Laboratory, University of California, San Diego, La Jolla, California, USA

    Martin Marsala

  4. Vector Development Core Lab, UCSD School of Medicine, La Jolla, California, USA

    Atsushi Miyanohara

  5. CHDI Management Inc., New York, New York, USA

    Ramee Lee

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  1. Wan-chun Liu
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Contributions

W.L. and R.L. designed the study. W.L., J.K., S.K.S., E.P., B.H., N.O. and S.S. performed the experiments. A.M. and M.M. designed and produced the constructs. W.L., J.K., S.K.S. and E.P. analyzed the data. W.L., J.K., S.K.S. and R.L. wrote the manuscript.

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Correspondence to Wan-chun Liu.

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Integrated supplementary information

Supplementary Figure 1 Breeding and pedigrees of mHTT (145Q) transgenic zebra finches.

Five F0 finches were selectively bred based on the presence of song abnormality and transgene verification with PCR using gDNA extracted from blood samples. These five transgenic lines were bred up to the second (F2), third (F3), or fourth (F4) generations. Listed in each box is the number of 145Q transgenic offspring compared to total progeny.

Supplementary Figure 2 Song development in mHTT transgenics.

a, An example of abnormal song development in mutant transgenics. A juvenile mutant, W208, at 65 dph produced only a few unlearned syllables immediately followed by a “silent” note (light green bar), characterized by a clear singing gesture and beak movement with no audible sound. The silent note is later replaced by a simple, repetitive call note (syllable J) when song is crystallized. b, An example of juvenile song learning with later degradation as an adult. Mutant bird W201 imitates a few song syllables (E and F) from its tutor, but the imitated syllables are later deleted and replaced by simpler, repetitive call notes (H and J) by 95-100 dph. c, Due to the progressive degradation of song syllables, some of the 145Q mutants precisely copy tutor syllables during the sensitive period of song learning (i.e., first two months of age) but these copied syllables are later deleted or replaced by improvised syllables. Therefore, the ratio of copied syllables/ total syllables decreases from 2 months to 3 months to 12 months of age (n=15 mutant bird, 13 wild-type birds; Kruskal-Wallis one way ANOVA, F(1,27)=1.98; P=0.014). d, The similarity score to tutor song gradually declines from 3 to 9 months of age due to progressive song degradation, but remains relatively stable afterwards (n=13 males per group). e, The adult, crystallized song of transgenic mutant males (145Q; n=20 males) is significantly different from the adult song of wild-type normal males (WT; n=18 males; Mann-Whitney U test, P<0.05) in several acoustic features: continuity in time, frequency modulation (FM), and pitch goodness. f, In mutant 145Q adults that only sing improvised syllables (n=6 males), the song syntax (number of syllable transitions) remains variable without stabilization. This instability is sustained after 15 months of age.

Supplementary Figure 3 Changes in adult mutant song after LMAN and Area X lesions.

a, Prior to LMAN lesioning, a 145Q mutant adult (bird R30, 120 dph) shows crystallized song syllables but multiple song types. Post-lesion, the song syntax crystallizes and only one song type can be identified. Additionally, one syllable (syllable D) becomes highly repetitive. b, Area X lesions induce immediate change in only one of two song types and syntax order (ABCD to ACD) in a mutant 145Q adult (bird Y37, 122 dph). Right panel: the syllable transition A→B disappears after X-lesion, and A→C predominates. The total number of song types remains unchanged. c, After LMAN lesion (n=4 mutants and 4 controls), syllable structure crystallizes (similarity of post-lesioned to pre-lesioned song; Mann-Whitney U test, U=16, * P<0.05) and syntax variation is reduced (post-lesioning change in syllable transitions, Mann-Whitney U test, P=0.016). In post-Area X lesioned mutants (n=4), the syllable structure remained unchanged (P>0.05) but the number of syntax variations either increases or decreases (Error bars, mean±s.d.).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–3 and Supplementary Table 1 (PDF 3091 kb)

Supplementary Methods Checklist

Supplementary information of statistical analysis and experimental methods. (PDF 386 kb)

Rigidity and body tremor of a juvenile mutant zebra finch.

An example showing a juvenile zebra finch at around 40 days post hatching has body rigidity and tremor while the eyes are closed. (MOV 1090 kb)

A normal, resting, wild type, juvenile zebra finch.

An example showing the resting behavior of a normal, wild type, juvenile zebra finch at around 40 days post hatching. (MOV 399 kb)

Head dystonia of a mutant juvenile canary

An example of head dystonia observed in a mutant juvenile canary at around 45 days post hatching, the bird also had no fear toward human caretaker and perched on human fingers. (MOV 1275 kb)

A normal, resting, wild type, juvenile canary.

An example showing the resting behaviour of a normal, wild type, juvenile canary. (MOV 566 kb)

Supplementary Audio 1: An example of highly repetitive, stuttering-like mutant adult song.

A mutant (145Q) adult zebra finch produces highly repetitive, stuttering-like song. (WAV 507 kb)

Supplementary Audio 2: An example of abnormal, repetitive song produced by a mutant adult finch.

A mutant (145Q) adult zebra finch produces highly abnormal song that consists of multi-syllable repetition. (WAV 461 kb)

Supplementary Audio 3: An example of normal, wild type, adult finch song.

A typical song of a wild-type, adult zebra finch, which consists of multiple syllables with no or few serial repetition. (WAV 430 kb)

Supplementary Audio 4: An example of progressive song deterioration in an adult mutant finch at age of 100 days old.

The song produced by an adult mutant zebra finch (LG20) at 100 days of age. (WAV 221 kb)

Supplementary Audio 5: An example of progressive song deterioration in an adult mutant finch at age of 150 days old.

The song produced by mutant finch (LG20) shows syllable replacement at age of 150 days of age. (WAV 373 kb)

Supplementary Audio 6: An example of progressive song deterioration in an adult mutant finch at age of 250 days old.

The song of a mutant finch (LG20) continues to change and the syllables are replaced by unlearned notes. (WAV 234 kb)

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Liu, Wc., Kohn, J., Szwed, S. et al. Human mutant huntingtin disrupts vocal learning in transgenic songbirds. Nat Neurosci 18, 1617–1622 (2015). https://doi.org/10.1038/nn.4133

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