α-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 α-synuclein (α-Syn) with regard to dopamine synthesis. TH is activated by the phosphorylation of key seryl residues in the THregulatory-domain. Using in vitro models, our laboratory discovered that α-Syn inhibits TH by acting to reduce TH phosphorylation, which then reduces dopamine synthesis [31,33]. 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 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. KeywordsParkinson's disease; lentivirus; knockout mice; transduction Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Short term regulation of TH depends on the phosphorylation of seryl residues, Ser19, Ser31, and Ser40, in the TH regulatory domain [5,14]. We previously demonstrated significant reduction in TH phosphorylation in dopaminergic cells that overexpress α-Syn [31,33]. The serine that regulates 14-3-3 binding to TH is phospho-Ser19 (PSer19), a site that is highly phosphorylated in dopaminergic neurons throughout the brain [36]. To evaluate the impact of α-Syn on TH in vivo, in the absence of endogenous α-Syn, we obtained ASKO mice [1] and generated wild type human α-Syn lentivirus using established methodologies [16]. Herein, we share our novel findings revealing that when α-Syn becomes aggregated, immunoreactivity (ir) for Total-TH appears to be reduced in dopaminergic...

Loss of MicroRNA-7 Regulation Leads to α-Synuclein Accumulation and Dopaminergic Neuronal Loss In Vivo

“…38 Interestingly aggregated human α-synuclein has also been shown to reduce TH activity in dopaminergic neurons. 42 As miRs have many targets it is impossible to rule out that miR-7 may be affecting TH expression and dopaminergic neuronal health via another method other than upregulation of α-synuclein. The absence of strong binding sites for miR-7 in the TH 3'-UTR (data not shown) suggests that miR-7 does not affect TH expression directly but an interesting future experiment would be to look at miR-7T in an α-synuclein knock out animal.…”

Expression and Subcellular Location of Alpha-Synuclein During Mouse-Embryonic Development

“…However, expression was found as early as E9.5 in the midbrain/hindbrain junction, suggesting that a-SYN may play a yet undefined role in early brain development. a-SYN is reported to regulate DA synthesis by binding to and inhibiting tyrosine hydroxylase (TH) (Peng et al 2005;Alerte et al 2008). Previous studies show that the first appearance of TH-positive cells is in the midbrain at E9.0-9.5 of mouse development (Di Porzio et al 1990;PerroneCapano and Di Porzio 2000).…”

Molecular and Neurochemical Mechanisms in PD Pathogenesis

“…Alphasynyuclein inhibits tyrosine hydroxylase by reducing the enzyme phosphorylation, which then reduces dopamine synthesis in vitro (Peng et al 2005;Perez et al 2002;Alerte et al 2008). Alpha-synuclein phosphorylation at serine 129 is characteristic of Parkinson disease and this increases the conformational flexibility of alpha-synuclein and inhibits its fibrillogenesis in vitro but does not perturb its membrane-bound conformation (Paleologou et al 2008).…”