Samuel S. Newton

Phosphorylation is a key post-translational modification that can impact the function of a protein and the outcome of cell signaling pathways. Using phosphoproteomics to characterize the phosphorylation changes downstream of trophic factor signaling is important for better understanding the pleiotropic actions of this class of molecules. Insulin-like growth factor-1 (IGF-1) is a trophic factor that can influence cellular growth and differentiation, and IGF-1 signaling in the brain has been linked to cognitive processes. While its main signaling molecules are well characterized, we sought to perform a more in-depth and unbiased analysis of the IGF-1 phosphoproteome in neuronal cells. To obtain insight into the IGF-1 signaling pathway in neuronal cells, we performed a quantitative mass spectrometric phosphoproteomics analysis in rat pheochromocytoma (PC-12) cells. Our results illustrate a diverse phosphoproteome downstream of IGF-1, with insight into novel phosphoprotein sites, such as Plcβ3 (Ser¹¹⁰⁵), that likely influence IGF-1 signaling in neuronal cells. We also identify a robust upregulation of the Rho GTPase cycle and an activation of synaptogenesis signaling downstream of IGF-1. These results pave the way for more targeted studies on specific phosphoprotein sites, which will facilitate a better understanding of how these phosphorylation events impact IGF-1-related signaling outcomes in neuronal cells.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder that affects over 2% of the population worldwide and is characterized by repetitive behaviors, restricted areas of interest, deficits in social communication, and high levels of anxiety. Currently, there are no known effective treatments for the core features of ASD. The previous literature has established erythropoietin (EPO) as a promising antidepressant, working as a potent neurogenic and neurotrophic agent with hematopoietic side effects. Carbamoylated erythropoietin (CEPO), a chemically engineered non-hematopoietic derivative of EPO, appears to retain the neuroprotective factors of EPO without the hematologic properties. Recent evidence shows that CEPO corrects stress-related depressive behaviors in BALB/cJ (BALB) mice, which also have face validity as an ASD mouse model. We investigated whether CEPO can recover deficient social and anxiety-related behavioral deficits compared to C57BL/6J controls. After administering CEPO (40 μg/kg in phosphate-buffered saline) or vehicle over 21 days, we analyzed the mice’s performance in the three-chamber social approach, the open field, the elevated plus maze, and the Porsolt’s forced swim tasks. CEPO appeared to correct sociability in the three-chamber social approach task to C57 levels, increasing the amount of time the mice interacted with novel, social mice overall rather than altering the overall amount of exploratory activity in the maze. Consistent with this finding, there was no concomitant increase in the distance traveled in the open field, nor were there any alterations in the anxiety-related measures in the task. On the other hand, CEPO administration improved exploratory behavior in the elevated plus maze. This study marks the first demonstration of the benefits of a non-erythropoietic EPO derivative for social behavior in a mouse model of autism and merits further investigation into the mechanisms by which this action occurs.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder that affects over 2% of the population worldwide and is characterized by repetitive behaviors, restricted areas of interest, deficits in social communication, and high levels of anxiety. Currently, there are no known effective treatments for the core features of ASD. Previous literature has established erythropoietin (EPO) as a promising antidepressant, working as a potent neurogenic and neurotrophic agent with hematopoietic side effects. Carbamoylated erythropoietin (CEPO), a chemically engineered non-hematopoietic derivative of EPO, appears to retain the neuroprotective factors of EPO without the hematologic properties. Recent evidence shows that CEPO corrects stress-related depressive behaviors in BALB/cJ (BALB) mice, which also have face validity as an ASD mouse model. We investigated whether CEPO can recover deficient social and anxiety-related behavioral deficits, as compared to C57BL/6J controls. After administering CEPO (40 μg/kg in phosphate-buffered saline) or vehicle over 21 days, we analyzed performance in the three-chamber social approach, the open field, the elevated plus maze, and the Porsolt’s forced swim tasks. CEPO appears to correct sociability in the three-chamber social approach task to C57 levels, increasing the amount of time mice interacted with novel, social mice overall rather than altering the overall amount of exploratory activity in the maze. Consistent with this finding, there was no concomitant increase in distance traveled in the open field, nor was there an alteration of anxiety-related measures in the task. On the other hand, CEPO administration improved exploratory behavior in the elevated-plus maze. This study marks the first demonstration of the benefits of a non-erythropoietic EPO derivative for social behavior in a mouse model of autism and merits further investigation of the mechanisms by which this action occurs.

The choroid plexus (CP) is an understudied tissue in the central nervous system and is primarily implicated in cerebrospinal fluid (CSF) production. CP also produces numerous neurotrophic factors (NTF) which circulate to different brain regions. Regulation of NTFs in the CP during natural aging is largely unknown. Here, we investigated the age and gender-specific transcription of NTFs along with the changes in the tight junctional proteins (TJPs) and the water channel protein Aquaporin (AQP1). Male and female mice were used for our study. Age-related transcriptional changes were analyzed using quantitative PCR at three different time points: mature adult, middle-aged, and aged. Transcriptional changes during aging were further confirmed with digital droplet PCR. Additionally, we used immunohistochemical analysis (IHC) for the evaluation of in vivo protein expression. We further investigated the cellular phenotype of these NTFS, TJP, and water channel proteins in the mouse CP by co-labeling them with the classical vascular marker, Isolectin B4, and epithelial cell marker, Plectin. Aging significantly altered NTF gene expression in the CP. Brain-derived neurotrophic factor (BDNF), Midkine (MDK), VGF, Insulin-like growth factor (IGF1), IGF2, Klotho (KL), Erythropoietin (EPO), and its receptor (EPOR) were reduced in the aged CP of males and females. Vascular endothelial growth factor (VEGF) transcription was gender-specific; in males, gene expression was unchanged in the aged CP, while females showed an age-dependent reduction. Age-dependent changes in VEGF localization were evident, from vasculature to epithelial cells. IGF2 and klotho localized in the basolateral membrane of the CP and showed an age-dependent reduction in epithelial cells. Water channel protein AQP1 localized in the tip of epithelial cells and showed an age-related reduction in mRNA and protein levels. TJP’s JAM, CLAUDIN1, CLAUDIN2 and CLAUDIN5 were reduced in aged mice. Our study highlights transcriptional level changes in the CP during aging. The age-related transcriptional changes exhibit similarities as well as gene-specific differences in the CP of males and females. Altered transcription of the water channel protein AQP1 and TJPs could be involved in reduced CSF production during aging. Importantly, reduction in the neurotrophic factors and longevity factor Klotho can play a role in regulating brain aging.

Trophic factors are secreted proteins that can modulate neuronal integrity, structure, and function. Previous preclinical studies have shown synergistic effects on decreasing apoptosis and improving behavioral performance after stroke when combining two such trophic factors, erythropoietin (EPO) and insulin-like growth factor-1 (IGF-1). However, EPO can elevate the hematocrit level, which can be life-threatening for non-anemic individuals. A chemically engineered derivative of EPO, carbamoylated EPO (CEPO), does not impact hematological parameters but retains neurotrophic effects similar to EPO. To obtain insight into CEPO and IGF-1 combination signaling, we examined immediate early gene (IEG) expression after treatment with CEPO, IGF-1, or CEPO + IGF-1 in rat pheochromocytoma (PC-12) cells and found that combining CEPO and IGF-1 produced a synergistic increase in IEG expression. An in vivo increase in the protein expression of Npas4 and Nptx2 was also observed in the rat hippocampus. We also examined which kinase signaling pathways might be mediating these effects and found that while AKT inhibition did not alter the pattern of IEG expression, both ERK and JAK2 inhibition significantly decreased IEG expression. These results begin to define the molecular effects of combining CEPO and IGF-1 and indicate the potential for these trophic factors to produce positive, synergistic effects.

Cerebral hypoperfusion is associated with enhanced cognitive decline and increased risk of neuropsychiatric disorders. Erythropoietin (EPO) is a neurotrophic factor known to improve cognitive function in preclinical and clinical studies of neurodegenerative and psychiatric disorders. However, the clinical application of EPO is limited due to its erythropoietic activity that can adversely elevate hematocrit in non-anemic populations. Carbamoylated erythropoietin (CEPO), a chemically engineered non-erythropoietic derivative of EPO, does not alter hematocrit and maintains neurotrophic and behavioral effects comparable to EPO. Our study aimed to investigate the role of CEPO in cerebral hemodynamics. Magnetic resonance imaging (MRI) analysis indicated increased blood perfusion in the hippocampal and striatal region without altering tight junction integrity. In vitro and in vivo analyses indicated that hippocampal neurotransmission was unaltered and increased cerebral perfusion was likely due to EDRF, CGRP, and NOS-mediated vasodilation. In vitro analysis using human umbilical vein endothelial cells (HUVEC) and hippocampal vascular gene expression analysis showed CEPO to be a non-angiogenic agent which regulates the MEOX2 gene expression. The results from our study demonstrate a novel role of CEPO in modulating cerebral vasodilation and blood perfusion.

A significant body of research has demonstrated that antidepressants regulate neurotrophic factors and that neurotrophins themselves are capable of independently producing antidepressant-like effects. While brain derived neurotrophic factor (BDNF) remains the best studied molecule in this context, there are several structurally diverse trophic factors that have shown comparable behavioral effects, including basic fibroblast growth factor (FGF-2), insulin-like growth factor 1 (IGF-1) and vascular endothelial growth factor (VEGF). In this review we discuss the structural and biochemical signaling aspects of these neurotrophic factors with antidepressant activity. We also include a discussion on a cytokine molecule erythropoietin (EPO), widely known and prescribed as a hormone to treat anemia but has recently been shown to function as a neurotrophic factor in the central nervous system (CNS).

Cognitive deficits are widespread in psychiatric disorders, including major depression and schizophrenia. These deficits are known to contribute significantly to the accompanying functional impairment. Progress in the development of targeted treatments of cognitive deficits has been limited and there exists a major unmet need to develop more efficacious treatments. Erythropoietin (Epo) has shown promising procognitive effects in psychiatric disorders, providing support for a neurotrophic drug development approach. Several preclinical studies with non-erythropoietic derivatives have demonstrated that the modulation of behavior is independent of erythropoiesis. In this review, we examine the molecular, cellular and cognitive actions of Epo and non-erythropoietic molecular derivatives by focusing on their neurotrophic, synaptic, myelin plasticity, anti-inflammatory and neurogenic mechanisms in the brain. We also discuss the role of receptor signaling in Epo and non-erythropoietic EPO-mimetic molecules in their procognitive effects.

In recent years, erythropoietin (EPO) has emerged as a useful neuroprotective and neurotrophic molecule that produces antidepressant and cognitive-enhancing effects in psychiatric disorders. However, EPO robustly induces erythropoiesis and elevates red blood cell counts. Chronic administration is therefore likely to increase blood viscosity and produce adverse effects in non-anemic populations. Carbamoylated erythropoietin (CEPO), a chemically engineered modification of EPO, is non-erythropoietic but retains the neurotrophic and neurotrophic activity of EPO. Blood profile analysis after EPO and CEPO administration showed that CEPO has no effect on red blood cell or platelet counts. We conducted an unbiased, quantitative, mass spectrometry-based proteomics study to comparatively investigate EPO and CEPO-induced protein profiles in neuronal phenotype PC12 cells. Bioinformatics enrichment analysis of the protein expression profiles revealed the upregulation of protein functions related to memory formation such as synaptic plasticity, long term potentiation (LTP), neurotransmitter transport, synaptic vesicle priming, and dendritic spine development. The regulated proteins, with roles in LTP and synaptic plasticity, include calcium/calmodulin-dependent protein kinase type 1 (Camk1), Synaptosomal-Associated Protein, 25 kDa (SNAP-25), Sectretogranin-1 (Chgb), Cortactin (Cttn), Elongation initiation factor 3a (Eif3a) and 60S acidic ribosomal protein P2 (Rplp2). We examined the expression of a subset of regulated proteins, Cortactin, Grb2 and Pleiotrophin, by immunofluorescence analysis in the rat brain. Grb2 was increased in the dentate gyrus by EPO and CEPO. Cortactin was induced by CEPO in the molecular layer, and pleiotrophin was increased in the vasculature by EPO. The results of our study shed light on potential mechanisms whereby EPO and CEPO produce cognitive-enhancing effects in clinical and preclinical studies.