AAV-Mediated Expression of Dominant-Negative ULK1 Increases Neuronal Survival and Enhances Motor Performance in the MPTP Mouse Model of Parkinson’s Disease

Abstract

Loss of nigrostriatal projections by axonal degeneration is a key early event in Parkinson’s disease (PD) pathophysiology, being accountable for the lack of dopamine in the nigrostriatal system and resulting in motor symptoms such as bradykinesia, rigidity, and tremor. Since autophagy is an important mechanism contributing to axonal degeneration, we aimed to evaluate the effects of competitive autophagy inhibition in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD in vivo. Adeno-associated viral vector (AAV)–mediated overexpression of a dominant-negative form of the unc-51 like autophagy-initiating kinase (ULK1.DN) in the substantia nigra was induced 3 weeks before MPTP treatment. Analysis of motor behavior demonstrated a significant improvement of ULK1.DN expressing mice after MPTP treatment. Immunohistochemical analyses of dopaminergic nigral neurons and nigrostriatal projections revealed a significant protection from MPTP-induced neurotoxicity after ULK1.DN expression. Western blot analysis linked these findings to an activation of mTOR signaling. Taken together, our results indicate that expression of ULK1.DN can attenuate MPTP-induced axonal neurodegeneration, suggesting that ULK1 could be a promising novel target in the treatment of PD.

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Abbreviations

AAD:

Acute axonal degeneration

AAV:

Adeno-associated virus

AMPK:

AMP-activated protein kinase

AP:

Anterior posterior

ATG:

Autophagy related

DA:

Dopamine

DOPAC:

Dihydroxyphenylacetic acid

DV:

Dorsoventral

FIP200:

FAK family interacting protein of 200 kDa

HPLC:

High-performance liquid chromatography

HVA:

Homovanillic acid

LC3:

Microtubule-associated proteins 1A/1B light chain 3B

MPP +:

1-Methyl-4-phenylpyridinium.

ML:

Mediolateral

MPTP:

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine

mTOR:

Mechanistic target of rapamycin kinase

p62:

Sequestosome 1/p62

PD:

Parkinson’s disease

SN:

Substantia nigra

SNpc:

Substantia nigra pars compacta

TH:

Tyrosine hydroxylase

ULK1:

Unc-51 like autophagy activating kinase

ULK1.DN:

Dominant-negative ULK1.

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Acknowledgments

The authors thank Elisabeth Barski, Sabine Ceramella, and Barbara Müller for excellent technical support. We thank Sharon A. Tooze (Francis Crick Institute, London, UK) for providing the ULK1.DN plasmid.

Funding

D.B. and B.F.V. were supported by a scholarship from the Department of Neurology, University Medical Center Göttingen. V.T.R. was a fellow of the National Council for Scientific and Technological Development (CNPq), Brazil. L.T., V.D., M.B. and P.L. received funding from the Cluster of Excellence and DFG Research Center Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB), Göttingen.

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Correspondence to Paul Lingor.

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All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All procedures performed in this study involving animals were in accordance with the ethical standards and followed the regulations of the animal research council at the University Medical Center Göttingen and legislation of the State of Lower Saxony, Germany (33.9-42502-04-16/2239).

This article does not contain any studies with human participants performed by any of the authors.

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Fig. S1
figure7

Injection of AAV.ULK1.DN into the substantia nigra pars compacta (SNpc). Adeno-associated viral vectors (AAV) were injected into the SNpc relative to Bregma at anterior posterior: −3.0 mm, mediolateral: −1.2 mm and dorsoventral: −4.5 mm (a). Representative overview of mCherry expression after AAV injection into the SNpc of the mouse brain. Cell nuclei were counterstained with DAPI to visualize brain structures. Scale bar: 1 mm (b). Representative photomicrograph of the mouse midbrain region visualizing the co-localization of mCherry expression and tyrosine hydroxylase (TH) expression in the SNpc after AAV injection. Cell nuclei were counterstained with DAPI. Scale bar: 500 μm (c). (PNG 1426 kb)

Fig. S2
figure8

Immunohistochemical analysis of p62 in the substantia nigra pars compacta (SNpc). Representative photomicrographs of the SNpc in the treatment groups CTRL PBS, ULK1.DN PBS, CTRL MPTP and ULK1.DN MPTP show an increase of p62 protein after ULK1.DN expression. White arrowheads indicate p62 signal. Scale bar: 50 μm. (PNG 1120 kb)

Fig. S3
figure9

Immunohistochemical analysis of mTOR in the substantia nigra pars compacta (SNpc). Representative photomicrographs of the SNpc in the treatment groups CTRL PBS, ULK1.DN PBS, CTRL MPTP and ULK1.DN MPTP show an increase of mTOR protein after ULK1.DN expression. Scale bar: 50 μm. (PNG 1471 kb)

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Balke, D., Tatenhorst, L., Dambeck, V. et al. AAV-Mediated Expression of Dominant-Negative ULK1 Increases Neuronal Survival and Enhances Motor Performance in the MPTP Mouse Model of Parkinson’s Disease. Mol Neurobiol 57, 685–697 (2020). https://doi.org/10.1007/s12035-019-01744-0

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Keywords

  • ULK1
  • Autophagy
  • Axonal degeneration
  • MPTP mouse model
  • Parkinson’s disease