Alpha-synuclein aggregation alters tyrosine hydroxylase phosphorylation and immunoreactivity: lessons from viral transduction of knockout mice

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 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.

Figure 1. α-Syn is co-expressed with Total-TH in periglomerular olfactory neurons of lentivirus transduced α-Syn knockout mice

At 7 days after stereotaxic delivery of α-Syn lentivirus (A) DAPI stained nuclei reveal normal olfactory bulb morphology. Arrows point to periglomerular regions. (B) Many periglomerular neurons were labeled for Total-TH, confirming their dopaminergic phenotype. Arrowhead points to a group of four TH-ir neurons at the edge of a glomerulus. (C) Many neurons express wild type human α-Syn after transduction. Arrowhead points to the same four neurons as in B. (D) At high magnification the merged Total-TH-ir and α-Syn-ir appears yellow, confirming α-Syn expression in DA neurons (arrowhead). (E) Non-transduced (Ntd) olfactory bulb of a PBS-injected ASKO mouse lacks α-Syn-ir. Total-TH antibody used was TH-S. Size bars, 100 μm (A–C), 25 μm (D, E).

Figure 2. α-Syn transduction, but not GFP transduction, reduces Total-TH immunoreactivity in dopaminergic neurons

(A) Untransduced periglomerular dopaminergic neurons in ASKO mice at 3.5 days post-surgery have no α-Syn-ir, abundant Total-TH-ir that overlaps with TH PSer19-ir. B) Many α-Syn transduced neurons contain Total-TH-ir, confirming efficient α-Syn transduction of dopaminergic neurons. Cells with strong α-Syn-ir, also have strong TH-PSer19-ir but Total-TH-ir is reduced, which appear purple in the merged image (at arrowheads). (C) Intensity profiles of cells 1, 2, and 3, from Figure 2B reveal that cell 1 has low α-Syn-ir, low PSer19-ir, and high Total-TH-ir; cell 2 has moderate levels of α-Syn-ir, PSer19-ir, and Total-TH-ir; while cell 3 has high α-Syn-ir, high Pser19-ir, but low Total-TH-ir. (D) GFP-transduced neurons were immunostained for GFP using CY-3 immunofluorescence at 14 days post-surgery (red). The circled GFP-ir neuron has strong Total-TH-ir (green circled cell) and low levels of PSer19-ir (blue circled cell). GFP-transduced dopaminergic neurons appear yellow in the merged image including the circled cell and other cells in the same microscopic field. (E) Tissues from α-Syn-transduced olfactory bulbs have equivalent Total-TH levels but elevated PSer19 compared to GFP-transduced tissues at 3.5 and 7 days post-transduction (post-TDX). Actin serves as a loading control. Total-TH antibodies used were TH-S in A and B, AB1542 in D, and MAB318 in E. Size bars, 20 μm.

Figure 3. α-Syn aggregation, confirmed by proteinase K or Thioflavin-S labeling, correlates with TH PSer19 immunoreactivity

(A) Proteinase K-resistant α-Syn-ir in isolated neurons (red) that lack Total-TH-ir (green) but have high levels of PSer19-ir (blue), which appear purple in the merged image of cells at 7 days post-transduction. (B) Aggregated α-Syn (red) colocalizes with Thioflavin-S (green) in neurons at 14 days post-transduction. Total-TH-ir is reduced in these cells (arrowhead) compared to untransduced dopaminergic neurons at the edge of the injection, which lack α-Syn and have strong Total-TH-ir (arrow). Merged signal appears yellow due to strong α-Syn-ir and Thioflavin-S signal. Total-TH antibodies used were TH-S in A, and P40101-0 in B. Size bars, 25 μm.