Yulia Sidorova

Heparan sulfate proteoglycan syndecan-3 is a novel receptor for GDNF, neurturin, and artemin.

Syndecan-3 may act alone or as a coreceptor with RET to promote cell spreading, neurite outgrowth, and migration of cortical neurons by GNDF, NRTN, and ARTN.

The Structure of the Glial Cell Line-derived Neurotrophic Factor-Coreceptor Complex INSIGHTS INTO RET SIGNALING AND HEPARIN BINDING

Glial cell line-derived neurotrophic factor (GDNF), a neuronal survival factor, binds its co-receptor GDNF family receptor α1 (GFRα1) in a 2:2 ratio and signals through the receptor tyrosine kinase RET. We have solved the GDNF2·GFRα12 complex structure at 2.35 Å resolution in the presence of a heparin mimic, sucrose octasulfate. The structure of our GDNF2·GFRα12 complex and the previously published artemin2·GFRα32 complex are unlike in three ways. First, we have experimentally identified residues that differ in the ligand-GFRα interface between the two structures, in particular ones that buttress the key conserved ArgGFRα-Gluligand-ArgGFRα interaction. Second, the flexible GDNF ligand “finger” loops fit differently into the GFRαs, which are rigid. Third, and we believe most importantly, the quaternary structure of the two tetramers is dissimilar, because the angle between the two GDNF monomers is different. This suggests that the RET-RET interaction differs in different ligand2-co-receptor2-RET2 heterohexamer complexes. Consistent with this, we showed that GDNF2·GFRα12 and artemin2·GFRα32 signal differently in a mitogen-activated protein kinase assay. Furthermore, we have shown by mutagenesis and enzyme-linked immunosorbent assays of RET phosphorylation that RET probably interacts with GFRα1 residues Arg-190, Lys-194, Arg-197, Gln-198, Lys-202, Arg-257, Arg-259, Glu-323, and Asp-324 upon both domains 2 and 3. Interestingly, in our structure, sucrose octasulfate also binds to the Arg190-Lys202 region in GFRα1 domain 2. This may explain how GDNF·GFRα1 can mediate cell adhesion and how heparin might inhibit GDNF signaling through RET.

Heparin-binding determinants of GDNF reduce its tissue distribution but are beneficial for the protection of nigral dopaminergic neurons.

Glial cell line-derived neurotrophic factor (GDNF) protects and repairs dopamine neurons. It binds to GDNF family receptor alpha1 (GFRalpha1) and activates receptor tyrosine kinase. Heparan sulphate proteoglycans (HSPGs) also participate in the signalling of GDNF, though binding to HS may hinder the diffusion of infused GDNF. We assessed the importance of heparin-binding determinants in the neuroprotective effects of GDNF in the 6-OHDA rat model of Parkinsons disease. We utilized a truncated, non-heparin-binding Delta38N-GDNF or combined wtGDNF with heparin-binding growth-associated molecule (HB-GAM, pleiotrophin). Tissue diffusion of wtGDNF+/-HB-GAM and Delta38N-GDNF was also compared. A protective effect against ipsilateral d-amphetamine-induced turning was seen with 10 microg wtGDNF, 17 microg HB-GAM+10 microg wtGDNF or 10 microg Delta38N-GDNF at 8 weeks post lesion. This effect was most pronounced with wtGDNF alone. HB-GAM (17 or 50 microg) also reduced rotational behaviour, but did not protect dopaminergic cells. Otherwise, the survival of TH-positive cells in the substantia nigra correlated with the behavioural data. Although Delta38N-GDNF was more widely distributed than wtGDNF (irrespective of its origin), stable in a brain extract, and potent in mitogen-activated kinase assay, it was inferior in vivo. The results imply that GDNF binding to HSs is needed for the optimum neuroprotective effect.

Persephin signaling through GFRα1: The potential for the treatment of Parkinson's disease

Neurotrophic factors promote survival, proliferation and differentiation of neurons inducing intracellular signaling via specific receptors. The conventional biochemical methods often fail to reveal full repertoire of neurotrophic factor-receptor interactions because of their limited sensitivity. We evaluated several approaches to study signaling of Glial cell line-Derived Neurotrophic Factor (GDNF) family ligands and found that reporter-gene systems possess exceptionally high sensitivity and a heuristic power to identify novel biologically relevant growth factor-receptor interactions. We identified persephin, a GDNF family member, as a novel ligand for GFRalpha1/RET receptor complex. We confirmed this finding by several independent methods, including neurite outgrowth assay from the explants of sympathetic ganglia expressing Gfralpha1 and Ret mRNA but not persephins conventional receptor GFRalpha4. As the activation of GFRalpha1/RET was shown to rescue dopaminergic neurons, our results suggest the potential of persephin for the treatment of Parkinsons disease.

Zebrafish GDNF and its co-receptor GFRα1 activate the human RET receptor and promote the survival of dopaminergic neurons in vitro

Glial cell line-derived neurotrophic factor (GDNF) is a ligand that activates, through co-receptor GDNF family receptor alpha-1 (GFRα1) and receptor tyrosine kinase “RET”, several signaling pathways crucial in the development and sustainment of multiple neuronal populations. We decided to study whether non-mammalian orthologs of these three proteins have conserved their function: can they activate the human counterparts? Using the baculovirus expression system, we expressed and purified Danio rerio RET, and its binding partners GFRα1 and GDNF, and Drosophila melanogaster RET and two isoforms of co-receptor GDNF receptor-like. Our results report high-level insect cell expression of post-translationally modified and dimerized zebrafish RET and its binding partners. We also found that zebrafish GFRα1 and GDNF are comparably active as mammalian cell-produced ones. We also report the first measurements of the affinity of the complex to RET in solution: at least for zebrafish, the Kd for GFRα1-GDNF binding RET is 5.9 μM. Surprisingly, we also found that zebrafish GDNF as well as zebrafish GFRα1 robustly activated human RET signaling and promoted the survival of cultured mouse dopaminergic neurons with comparable efficiency to mammalian GDNF, unlike E. coli-produced human proteins. These results contradict previous studies suggesting that mammalian GFRα1 and GDNF cannot bind and activate non-mammalian RET and vice versa.

Menadione Suppresses Benzo(α)pyrene-Induced Activation of Cytochromes P450 1A: Insights into a Possible Molecular Mechanism

Oxidative reactions that are catalyzed by cytochromes P450 1A (CYP1A) lead to formation of carcinogenic derivatives of arylamines and polycyclic aromatic hydrocarbons (PAHs), such as the widespread environmental pollutant benzo(α)pyrene (BP). These compounds upregulate CYP1A at the transcriptional level via an arylhydrocarbon receptor (AhR)-dependent signaling pathway. Because of the involvement of AhR-dependent genes in chemically induced carcinogenesis, suppression of this signaling pathway could prevent tumor formation and/or progression. Here we show that menadione (a water-soluble analog of vitamin K3) inhibits BP-induced expression and enzymatic activity of both CYP1A1 and CYP1A2 in vivo (in the rat liver) and BP-induced activity of CYP1A1 in vitro. Coadministration of BP and menadione reduced DNA-binding activity of AhR and increased DNA-binding activity of transcription factors Oct-1 and CCAAT/enhancer binding protein (C/EBP), which are known to be involved in negative regulation of AhR-dependent genes, in vivo. Expression of another factor involved in downregulation of CYP1A—pAhR repressor (AhRR)—was lower in the liver of the rats treated with BP and menadione, indicating that the inhibitory effect of menadione on CYP1A is not mediated by this protein. Furthermore, menadione was well tolerated by the animals: no signs of acute toxicity were detected by visual examination or by assessment of weight gain dynamics or liver function. Taken together, our results suggest that menadione can be used in further studies on animal models of chemically induced carcinogenesis because menadione may suppress tumor formation and possibly progression.

Differential Spinal and Supraspinal Activation of Glia in a Rat Model of Morphine Tolerance

Development of tolerance is a well known pharmacological characteristic of opioids and a major clinical problem. In addition to the known neuronal mechanisms of opioid tolerance, activation of glia has emerged as a potentially significant new mechanism. We studied activation of microglia and astrocytes in morphine tolerance and opioid-induced hyperalgesia in rats using immunohistochemistry, flow cytometry and RNA sequencing in spinal- and supraspinal regions. Chronic morphine treatment that induced tolerance and hyperalgesia also increased immunoreactivity of spinal microglia in the dorsal and ventral horns. Flow cytometry demonstrated that morphine treatment increased the proportion of M2-polarized spinal microglia, but failed to impact the number or the proportion of M1-polarized microglia. In the transcriptome of microglial cells isolated from the spinal cord (SC), morphine treatment increased transcripts related to cell activation and defense response. In the studied brain regions, no activation of microglia or astrocytes was detected by immunohistochemistry, except for a decrease in the number of microglial cells in the substantia nigra. In flow cytometry, morphine caused a decrease in the number of microglial cells in the medulla, but otherwise no change was detected for the count or the proportion of M1- and M2-polarized microglia in the medulla or sensory cortex. No evidence for the activation of glia in the brain was seen. Our results suggest that glial activation associated with opioid tolerance and opioid-induced hyperalgesia occurs mainly at the spinal level. The transcriptome data suggest that the microglial activation pattern after chronic morphine treatment has similarities with that of neuropathic pain.

A Novel Small Molecule GDNF Receptor RET Agonist, BT13, Promotes Neurite Growth from Sensory Neurons in Vitro and Attenuates Experimental Neuropathy in the Rat

Neuropathic pain caused by nerve damage is a common and severe class of chronic pain. Disease-modifying clinical therapies are needed as current treatments typically provide only symptomatic relief; show varying clinical efficacy; and most have significant adverse effects. One approach is targeting either neurotrophic factors or their receptors that normalize sensory neuron function and stimulate regeneration after nerve damage. Two candidate targets are glial cell line-derived neurotrophic factor (GDNF) and artemin (ARTN), as these GDNF family ligands (GFLs) show efficacy in animal models of neuropathic pain (Boucher et al., 2000; Gardell et al., 2003; Wang et al., 2008, 2014). As these protein ligands have poor drug-like properties and are expensive to produce for clinical use, we screened 18,400 drug-like compounds to develop small molecules that act similarly to GFLs (GDNF mimetics). This screening identified BT13 as a compound that selectively targeted GFL receptor RET to activate downstream signaling cascades. BT13 was similar to NGF and ARTN in selectively promoting neurite outgrowth from the peptidergic class of adult sensory neurons in culture, but was opposite to ARTN in causing neurite elongation without affecting initiation. When administered after spinal nerve ligation in a rat model of neuropathic pain, 20 and 25 mg/kg of BT13 decreased mechanical hypersensitivity and normalized expression of sensory neuron markers in dorsal root ganglia. In control rats, BT13 had no effect on baseline mechanical or thermal sensitivity, motor coordination, or weight gain. Thus, small molecule BT13 selectively activates RET and offers opportunities for developing novel disease-modifying medications to treat neuropathic pain.

A deep convolutional neural network approach for astrocyte detection

Astrocytes are involved in various brain pathologies including trauma, stroke, neurodegenerative disorders such as Alzheimer’s and Parkinson’s diseases, or chronic pain. Determining cell density in a complex tissue environment in microscopy images and elucidating the temporal characteristics of morphological and biochemical changes is essential to understand the role of astrocytes in physiological and pathological conditions. Nowadays, manual stereological cell counting or semi-automatic segmentation techniques are widely used for the quantitative analysis of microscopy images. Detecting astrocytes automatically is a highly challenging computational task, for which we currently lack efficient image analysis tools. We have developed a fast and fully automated software that assesses the number of astrocytes using Deep Convolutional Neural Networks (DCNN). The method highly outperforms state-of-the-art image analysis and machine learning methods and provides precision comparable to those of human experts. Additionally, the runtime of cell detection is significantly less than that of other three computational methods analysed, and it is faster than human observers by orders of magnitude. We applied our DCNN-based method to examine the number of astrocytes in different brain regions of rats with opioid-induced hyperalgesia/tolerance (OIH/OIT), as morphine tolerance is believed to activate glia. We have demonstrated a strong positive correlation between manual and DCNN-based quantification of astrocytes in rat brain.

Small-Molecule Ligands as Potential GDNF Family Receptor Agonists

To find out potential GDNF family receptor α1 (GFRα1) agonists, small molecules were built up by molecular fragments according to the structure-based drug design approach. Molecular docking was used to identify their binding modes to the biological target GFRα1 in GDNF-binding pocket. Thereafter, commercially available compounds based on the best predicted structures were searched from ZINC and MolPort databases (similarity ≥ 80%). Five compounds from the ZINC library were tested in phosphorylation and luciferase assays to study their ability to activate GFRα1–RET. A bidental compound with two carboxyl groups showed the highest activity in molecular modeling and biological studies. However, the relative position of these groups was important. The meta-substituted structure otherwise identical to the most active compound 2-[4-(5-carboxy-1H-1,3-benzodiazol-2-yl)phenyl]-1H-1,3-benzodiazole-5-carboxylic acid was inactive. A weaker activity was detected for a compound with a single carboxyl group, that is, 4-(1,3-benzoxazol-2-yl)benzoic acid. The substitution of the carboxyl group by the amino or acetamido group also led to the loss of the activity.