α-Synuclein aggregation alters tyrosine hydroxylase phosphorylation and immunoreactivity: Lessons from viral transduction of knockout mice

doi:10.1016/j.neulet.2008.02.014

Neuroscience Letters

Volume 435, Issue 1, 11 April 2008, Pages 24-29

https://doi.org/10.1016/j.neulet.2008.02.014 Get rights and content

Abstract

Tyrosine hydroxylase (TH), the rate limiting enzyme in catecholamine synthesis, is frequently used as a marker of dopaminergic neuronal loss in animal models of Parkinson's disease (PD). We have been exploring the normal function of the PD-related protein α-synuclein (α-Syn) with regard to dopamine synthesis. TH is activated by the phosphorylation of key seryl residues in the TH regulatory domain. Using in vitro models, our laboratory discovered that α-Syn inhibits TH by acting to reduce TH phosphorylation, which then reduces dopamine synthesis [X.-M. Peng, R. Tehranian, P. Dietrich, L. Stefanis, R.G. Perez, Alpha-synuclein activation of protein phosphatase 2A reduces tyrosine hydroxylase phosphorylation in dopaminergic cells, J. Cell. Sci. 118 (2005) 3523–3530; R.G. Perez, J.C. Waymire, E. Lin, J.J. Liu, F. Guo, M.J. Zigmond, A role for alpha-synuclein in the regulation of dopamine biosynthesis, J. Neurosci. 22 (2002) 3090–3099]. We recently began exploring the impact of α-Syn on TH in vivo, by transducing dopaminergic neurons in α-Syn knockout mouse (ASKO) olfactory bulb using wild type human α-Syn lentivirus. At 3.5–21 days after viral delivery, α-Syn expression was transduced primarily in periglomerular dopaminergic neurons. Cells with modest levels of α-Syn consistently co-labeled for Total-TH. However, cells bearing aggregated α-Syn, as revealed by proteinase K or Thioflavin-S treatment had significantly reduced Total-TH immunoreactivity, but high phosphoserine-TH labeling. On immunoblots, we noted that Total-TH immunoreactivity was equivalent in all conditions, although tissues with α-Syn aggregates again had higher phosphoserine-TH levels. This suggests that aggregated α-Syn is no longer able to inhibit TH. Although the reason(s) underlying reduced Total-TH immunoreactivity on tissue sections await(s) confirmation, the dopaminergic phenotype was easily verified using phosphorylation-state-specific TH antibodies. These findings have implications not only for normal α-Syn function in TH regulation, but also for measuring cell loss that is associated with synucleinopathy.

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Acknowledgements

We thank NINDS (NS42094) and the Fox Foundation for support, D. Trono and Y.J. Liu for plasmids, H.M. Na, S.M. Reid and C.J. Pedersen for technical assistance, and J. Wang for critical reading of the manuscript. We dedicate our work to M.J. Fox, R. Beyer, and J. Cordy, and in memory of L. “Rusty” Lanelli.

References (41)

A. Abeliovich et al.
Mice lacking alpha-synuclein display functional deficits in the nigrostriatal dopamine system
Neuron
(2000)
C. Alves da Costa et al.
Alpha-synuclein lowers p53-dependent apoptotic response of neuronal cells. Abolishment by 6-hydroxydopamine and implication for Parkinson's disease
J. Biol. Chem.
(2002)
A.L. Biere et al.
Parkinson's disease-associated alpha-synuclein is more fibrillogenic than beta- and gamma-synuclein and cannot cross-seed its homologs
J. Biol. Chem.
(2000)
D.G. Campbell et al.
Identification of four phosphorylation sites in the N-terminal region of tyrosine hydroxylase
J. Biol. Chem.
(1986)
M.-C. Chartier-Harlin et al.
alpha-synuclein locus duplication as a cause of familial Parkinson's disease
Lancet
(2004)
Y. Chu et al.
Age-associated increases of alpha-synuclein in monkeys and humans are associated with nigrostriatal dopamine depletion: is this the target for Parkinson's disease?
Neurobiol. Dis.
(2007)
N. Golts et al.
Magnesium inhibits spontaneous and iron-induced aggregation of alpha-synuclein
J. Biol. Chem.
(2002)
M. Hashimoto et al.
Human recombinant NACP/alpha-synuclein is aggregated and fibrillated in vitro: relevance for Lewy body disease
Brain Res.
(1998)
J.W. Haycock
Phosphorylation of tyrosine hydroxylase in situ at serine 8, 19, 31, and 40
J. Biol. Chem.
(1990)
P. Ibanez et al.
Causal relation between alpha-synuclein gene duplication and familial Parkinson's disease
Lancet
(2004)

R.O. Kuljis et al.

Lewy bodies in tyrosine hydroxylase-synthesizing neurons of the human cerebral cortex

Neurosci. Lett.

(1989)

A.B. Manning-Bog et al.

The herbicide paraquat causes up-regulation and aggregation of alpha-synuclein in mice: paraquat and alpha-synuclein

J. Biol. Chem.

(2002)

S. Nakashima et al.

Tyrosine hydroxylase protein in Lewy bodies of parkinsonian and senile brains

J. Neurol. Sci.

(1984)

H.J. Rideout et al.

Alpha-Synuclein is required for the fibrillar nature of ubiquitinated inclusions induced by proteasomal inhibition in primary neurons

J. Biol. Chem.

(2004)

L. Bobrovskaya et al.

Phosphorylation of Ser19 increases both Ser40 phosphorylation and enzyme activity of tyrosine hydroxylase in intact cells

J. Neurochem.

(2004)

R. Cappai et al.

Dopamine promotes alpha-synuclein aggregation into SDS-resistant soluble oligomers via a distinct folding pathway

FASEB J.

(2005)

K.A. Conway et al.

Accelerated in vitro fibril formation by a mutant alpha-synuclein linked to early-onset Parkinson disease

Nat. Med.

(1998)

K.A. Conway et al.

Accelerated oligomerization by Parkinson's disease linked alpha-synuclein mutants

Ann. NY Acad. Sci.

(2000)

R.J. George et al.

In vitro phosphorylation of bovine adrenal chromaffin cell tyrosine hydroxylase by endogenous protein kinases

J. Neurochem.

(1989)

J.W. Haycock et al.

Role of serine-19 phosphorylation in regulating tyrosine hydroxylase studied with site- and phosphospecific antibodies and site-directed mutagenesis

J. Neurochem.

(1998)

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α-Synuclein aggregation alters tyrosine hydroxylase phosphorylation and immunoreactivity: Lessons from viral transduction of knockout mice

Abstract

Section snippets

Acknowledgements

Neuron

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Lancet

Neurobiol. Dis.

J. Biol. Chem.

Brain Res.

J. Biol. Chem.

Lancet

Neurosci. Lett.

J. Biol. Chem.

J. Neurol. Sci.

J. Biol. Chem.

Phosphorylation of Ser19 increases both Ser40 phosphorylation and enzyme activity of tyrosine hydroxylase in intact cells

J. Neurochem.

Dopamine promotes alpha-synuclein aggregation into SDS-resistant soluble oligomers via a distinct folding pathway

FASEB J.

Accelerated in vitro fibril formation by a mutant alpha-synuclein linked to early-onset Parkinson disease

Nat. Med.

Accelerated oligomerization by Parkinson's disease linked alpha-synuclein mutants

Ann. NY Acad. Sci.

In vitro phosphorylation of bovine adrenal chromaffin cell tyrosine hydroxylase by endogenous protein kinases

J. Neurochem.

Role of serine-19 phosphorylation in regulating tyrosine hydroxylase studied with site- and phosphospecific antibodies and site-directed mutagenesis

J. Neurochem.