α-Synuclein (a-Syn), a protein implicated in Parkinson disease, contributes significantly to dopamine metabolism. a-Syn binding inhibits the activity of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis. Phosphorylation of TH stimulates its activity, an effect that is reversed by protein phosphatase 2A (PP2A). In cells, a-Syn overexpression activates PP2A. Here we demonstrate that a-Syn significantly inhibited TH activity in vitro and in vivo and that phosphorylation of a-Syn serine 129 (Ser-129) modulated this effect. In MN9D cells, a-Syn overexpression reduced TH serine 19 phosphorylation (Ser(P)-19). In dopaminergic tissues from mice overexpressing human a-Syn in catecholamine neurons only, TH-Ser-19 and TH-Ser-40 phosphorylation and activity were also reduced, whereas PP2A was more active. Cerebellum, which lacks excess a-Syn, had PP2A activity identical to controls. Conversely, a-Syn knock-out mice had elevated TH-Ser-19 phosphorylation and activity and less active PP2A in dopaminergic tissues. Using an a-Syn Ser-129 dephosphorylation mimic, with serine mutated to alanine, TH was more inhibited, whereas PP2A was more active in vitro and in vivo. Phosphorylation of a-Syn Ser-129 by Polo-like-kinase 2 in vitro reduced the ability of a-Syn to inhibit TH or activate PP2A, identifying a novel regulatory role for Ser-129 on a-Syn. These findings extend our understanding of normal a-Syn biology and have implications for the dopamine dysfunction of Parkinson disease.

Fragile X mental retardation syndrome, the most common form of inherited mental retardation, is caused by the absence of the fragile X mental retardation protein (FMRP). FMRP has been shown to use its arginine-glycine-glycine (RGG) box to bind to a subset of RNA targets that form a G quadruplex structure. We performed a detailed analysis of the interactions between the FMRP RGG box and the microtubule associated protein 1B (MAP1B) mRNA, a relevant in vivo FMRP target. We show that MAP1B RNA forms an intramolecular G quadruplex structure, which is bound with high affinity and specificity by the FMRP RGG box. We determined that hydrophobic interactions are important in the FMRP RGG box-MAP1B RNA association, with minor contributions from electrostatic interactions. Our findings that at low protein:RNA ratios the RNA G quadruplex structure is slightly stabilized, whereas at high ratios is unfolded, suggest a mechanism by which the FMRP concentration variation in response to a neurotransmitter stimulation event could act as a regulatory switch for the protein function, from translation repressor to translation activator.Keywords: FMRP; fragile X syndrome; G quadruplex; MAP1B RNA; RGG boxThe lack of fragile X mental retardation protein (FMRP) results in the fragile X syndrome, the most prevalent inherited mental disorder (Crawford et al. 2001). The absence of FMRP is due to the transcriptional inactivation of the fmr1 gene, caused by an unstable expansion of a CGG trinucleotide repeat in its 59-untranslated region (UTR) (Jin and Warren 2000;O'Donnell and Warren 2002). The function of FMRP has been extensively studied, however its cellular role and how its loss causes mental retardation is still not fully understood. It is believed that FMRP is a translational regulator for specific messenger RNAs (mRNAs), and it has been shown that it is associated with actively translating ribosomes (Antar and Bassell 2003;Jin and Warren 2003;Massimiliano et al. 2004). Nucleic acid chaperone properties have also been attributed to this protein (Gabus et al. 2004). This 632 amino acid protein has two types of RNA binding domains: two K-homology (KH) domains and one arginine-glycine-glycine (RGG) box, suggesting that FMRP exerts its function through RNA binding (Siomi et al. 1993).Several studies reported that FMRP uses its RGG box to bind with high affinity to mRNAs rich in G content that could adopt more complex G quadruplex structures Darnell et al. 2001;Schaeffer et al. 2001;Ramos et al. 2003). Formed from four guanine residues, a G quartet has a planar conformation stabilized by Hoogsteen base pairs (Fig. 1A). Several such planar structures can stack to form G quadruplexes, which are stabilized by potassium or sodium cations, but are disrupted in the presence of lithium cations (Davis 2004;Hazel et al. 2004;Mergny et al. 2005). It has also been shown that FMRP uses its KH2 domain to bind to a synthetic RNA that harbors a loop-loop kissing complex (Darnell et al. 2005). However, despite these extensive efforts, the in vivo...

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...