Exploring the molecular mechanisms of the human vgf-derived antidepressant neuropeptide tlqp-62 on neurodifferentiation

Resumen

Neuropsychiatric disorders, as major depression, schizophrenia or bipolar disorder, affect about 10% of world population, having a huge impact on society and a bigger stigma. Knowledge from a molecular and biological view is still poor, and diagnostic and treatment are lacking effectiveness, leading to a high suicide index. These disorders have a genetic and environmental component, and some genes have been pointed out as risk factors. Moreover, although diagnosed is performed through selfreporting of patient in a clinical interview and behaviour observation, there are evidence from patients suffering from these disorders of hippocampal abnormality with reduced volume and decreased cell proliferation which affect cognitive functions and memory. Changes can be reversed by treatment with antidepressants, antipsychotics or physical exercise, by having an impact on neurogenesis. Neurogenesis is the generation of new neurons from neural stem cells, occurring mainly during the embryonic brain development, but it continues during life in some areas of the brain, mainly being in the subgranular zone of dentate granule cells in the dentate gyrusof the hippocampus and subventricular zone of interneurons in the olfactory bulb. This process generates new cells that suffer migration, differentiation with axon and dendrite outgrowth, and synapse formation to integrate the pre-existing neuronal circuit. This confers plasticity to this region and understanding the molecular mechanisms underlying adult neurogenesis may give critical insight for successful treatment for these disorders. The BDNF/TrkB/CREB signalling pathway has been shown to be implicated in hippocampal neurogenesis, together with some other proteins, as DISC1 and VGF. DISC1 was found mutated, leading to its dysregulation, in individuals with schizophrenia, schizoaffective disorder, bipolar disorder or major depression. This protein is linked to neurogenesis and it functions as a scaffold protein, interacting with several other proteins important for neurodevelopment and synaptic function. Our group has reported that DISC1 knockdown produces a significant downregulation of VGF, a nerve growth factor responsive gene, in SH-SY5Y cells. DISC1 apparently does not interact directly with VGF but seems to regulate this gene expression through the PI3K/AKT/CREB pathway. VGF is a peptide precursor for TLQP-62, an antidepressant neuropeptide that promotes hippocampal neurogenesis, with an effect on memory and learning, through a BDNFdependent mechanism, increasing synaptic activity and dendritic branching. Thus, this neuropeptide is a very attractive target for further search and investigation of its role and molecular mechanisms in neurogenesis and neurodifferentiation. Also, the identification of TLQP-62 receptor(s) and mediated signalling pathways are crucial for a better understanding of these neuropeptide molecular mechanisms, as well as the threedimensional structures, for further investigation for agonists to be used as a treatment for chronic mental disorders. The human derived cell line SH-SY5Y reproduces biochemical and morphological properties of neurons, being often used as an in vitro model for human. Those cells can be induced to differentiate into a more neuron-like phenotype, through retinoic acid induction, and express high levels of VGF. Therefore, can be properly used as a model for neurogenesis concerning the neurodifferentiation step. In the present study the effect of TLQP-62 on SH-S5Y5 was evaluated, concerning its ability to induce proliferation and/or neurodifferentiation with neuritogenesis and dendritogenesis. Morphological and proteomic evaluation were performed. TLQP-62 is capable alone of induce neurodifferentiation on SH-SY5Y cells, rather than proliferation, promoting more and longer dendrites with occasional connection between cells. Also, in a proteomic analysis, several proteins involved in neurodevelopment and synaptic plasticity, as cell growth and communication, biosynthesis and metabolism, fatty acid and glucose metabolism, and immune an inflammatory response are increased by TLQP-62. These processes are known to be implicated in neurogenesis and in neurological disorders. Moreover, TLQP-62 has already been described as having a role in glucose homeostasis. To better understand the molecular mechanisms of TLQP-62 in neurogenesis and neurodifferentiation processes, as dendritogenesis, it is crucial to know the TLQP-62 receptor(s), and for that an avidin affinity chromatography assay was performed to membrane fraction of SH-SY5Y cells incubated with biotin-TLQP-62 crosslinked with sulfo-EMCS. Olfactory receptor 5P3 was isolated, a GPCR, apparently classified as an olfactory receptor based on homology and with no known ligands. TLQP-62 is capable of increase cAMP levels in a cell model overexpressing OR5P3, proving it is a ligand for this receptor and may act through a Gs protein signalling pathway. Also HSPA8 and HSPD1 were isolated. HSPA8 have been previously described as a TLQP-21 binding protein. HSPA8 was proved to interact with TLQP-62 by label-free dynamic mass redistribution analysis. The structure of TLQP-62 was also explored in order to understand its interaction with OR5P3 and HSPA8. TLQP-62 acts as a random coil in solution transitory with an α-helix, which might stabilized upon binding with HSPA8 or with its receptor OR5P3. Or HSPA8OR5P3-TLQP-62 might form a complex to activate a signal transduction pathway. Further structural insights into this putative complex can help developing a pharmacological drug against of this receptor to enhance neurogenesis and be used as a treatment of some neuropsychiatric and other neurological disorders. Thus, all data taken together help to better understand TLQP-62 mechanism of action in neurogenesis and neurodifferentiation, and also in mental and neurological disorders.