Alpha-synuclein aggregation alters tyrosine hydroxylase phosphorylation and immunoreactivity: lessons from viral transduction of knockout mice.
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 alpha-synuclein (alpha-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 alpha-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 alpha-Syn on TH in vivo, by transducing dopaminergic neurons in alpha-Syn knockout mouse (ASKO) olfactory bulb using wild type human alpha-Syn lentivirus. At 3.5-21 days after viral delivery, alpha-Syn expression was transduced primarily in periglomerular dopaminergic neurons. Cells with modest levels of alpha-Syn consistently co-labeled for Total-TH. However, cells bearing aggregated alpha-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 alpha-Syn aggregates again had higher phosphoserine-TH levels. This suggests that aggregated alpha-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 alpha-Syn function in TH regulation, but also for measuring cell loss that is associated with synucleinopathy.
Alerte
Tshianda N M
TN
Department of Neurology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15260, United States.
Akinfolarin
Akinwande A
AA
Friedrich
Emily E
EE
Mader
Samantha A
SA
Hong
Chang-Sook
CS
Perez
Ruth G
RG
D000818:Animals; D002478:Cells, Cultured; D004195:Disease Models, Animal; D004298:Dopamine / Q000096:biosynthesis*; D015536:Down-Regulation / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007150:Immunohistochemistry; D002479:Inclusion Bodies / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D016086:Lentivirus / Q000235:genetics; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D009410:Nerve Degeneration / Q000235:genetics / Q000378:metabolism / Q000503:physiopathology; D009474:Neurons / Q000378:metabolism* / Q000473:pathology; D009830:Olfactory Bulb / Q000166:cytology / Q000378:metabolism; D010300:Parkinson Disease / Q000235:genetics / Q000378:metabolism* / Q000503:physiopathology; D010766:Phosphorylation; D013378:Substantia Nigra / Q000378:metabolism* / Q000473:pathology / Q000503:physiopathology; D014161:Transduction, Genetic / Q000379:methods; D014162:Transfection / Q000379:methods; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism*; D051844:alpha-Synuclein / Q000378:metabolism*
D051844:alpha-Synuclein; D014446:Tyrosine 3-Monooxygenase; D004298:Dopamine