CD38/cADPR/Ca2+ Pathway Promotes Cell Proliferation and Delays Nerve Growth Factor-induced Differentiation in PC12 Cells

Intracellular Ca²⁺ mobilization plays an important role in a wide variety of cellular processes, and multiple second messengers are responsible for mediating intracellular Ca²⁺ changes. Here we explored the role of one endogenous Ca²⁺-mobilizing nucleotide, cyclic adenosine diphosphoribose (cADPR), in the proliferation and differentiation of neurosecretory PC12 cells. We found that cADPR induced Ca²⁺ release in PC12 cells and that CD38 is the main ADP-ribosyl cyclase responsible for the acetylcholine (ACh)-induced cADPR production in PC12 cells. In addition, the CD38/cADPR signaling pathway is shown to be required for the ACh-induced Ca²⁺ increase and cell proliferation. Inhibition of the pathway, on the other hand, accelerated nerve growth factor (NGF)-induced neuronal differentiation in PC12 cells. Conversely, overexpression of CD38 increased cell proliferation but delayed NGF-induced differentiation. Our data indicate that cADPR plays a dichotomic role in regulating proliferation and neuronal differentiation of PC12 cells.Mobilization of intracellular Ca²⁺ stores is involved in diverse cell functions, including fertilization, cell proliferation, and differentiation (1–4). At least three endogenous Ca²⁺-mobilizing messengers have been identified, including inositol trisphosphate (IP₃),³ nicotinic adenine acid dinucleotide phosphate (NAADP), and cyclic adenosine diphosphoribose (cADPR). Similar to IP₃, cADPR can mobilize calcium release in a wide variety of cell types and species, from protozoa to animals. The cADPR-mediated Ca²⁺ signaling has been indicated in a variety of cellular processes (5–7), from abscisic acid signaling and regulation of the circadian clock in plants, to mediating long-term synaptic depression in hippocampus.Ample evidence shows that the ryanodine receptors are the main intracellular targets for cADPR (1, 2, 8). Ryanodine receptors (RyRs) are intracellular Ca²⁺ channels widely expressed in various cells and tissues, including muscles and neurons. It is the major cellular mediator of Ca²⁺-induced Ca²⁺ release (CICR) in cells. There are three isoforms of ryanodine receptors: RyR1, RyR2, and RyR3, all of which have been implicated in the cADPR signaling (1, 2, 8). However, evidence regarding cADPR acting directly on the receptors is lacking (9). It has been suggested that accessory proteins, such as calmodulin and FK506-binding protein (FKBP), may be involved instead (10–15).cADPR is formed from nicotinamide adenine dinucleotide (NAD) by ADP-ribosyl cyclases. Six ADP-ribosyl cyclases have been identified so far: Aplysia ADP-ribosyl cyclase, three sea urchin homologues (16, 17), and two mammalian homologues, CD38 and CD157 (18). CD38 is a membrane-bound protein and the main mammalian ADP-ribosyl cyclase. As a novel multifunctional enzyme, CD38 catalyzes the synthesis and hydrolysis of both cADPR and NAADP, two structurally and functionally distinct Ca²⁺ messengers. Virtually all mammalian tissues ever examined have been shown to express CD38. CD38 knock-out mice exhibit multiple physiological defects, ranging from impaired immune responses, metabolic disturbances, to behavioral modifications (1, 6, 18).CD38 was originally identified as a lymphocyte differentiation antigen (18). Indeed, CD38/cADPR has been linked to cell differentiation (5). For example, in human HL-60 cells, CD38 expression and the consequential accumulation of cADPR play a causal role in mediating granulocytic differentiation (19). In addition, expression of CD38 in HeLa and 3T3 cells not only increased intracellular Ca²⁺ concentration but also induced cell proliferation by significantly reducing the S phase duration, leading to shortened cell doubling time (20). The ability of cADPR to increase cell proliferation has also been observed in human T cells (21), human hemopoietic progenitors (22), human peripheral blood mononuclear cells (23), human mesenchymal stem cells (24), and murine mesangial cells (25).The PC12 cell line was derived from rat adrenal medulla and has been used extensively as a neuronal model, since it exhibits many of the functions observed in primary neuronal cultures (26). Most importantly, PC12 cells can be induced by nerve growth factor (NGF) to differentiate into cells with extensive neurite outgrowths, resembling neuronal dendritic trees (26, 27). In contrast to NGF, numerous growth factors and neurotransmitters can induce the proliferation of PC12 cells instead (26). Both IP₃ receptor- and ryanodine receptor-mediated Ca²⁺ stores have been shown to be present in PC12 cells (28–31). The type 2 ryanodine receptor is expressed in PC12 cells and activation of the NO/cGMP pathway in PC12 cells results in calcium mobilization, which is mediated by cADPR and similar to that seen in sea urchin eggs (32). It has been demonstrated that NAADP, another Ca²⁺-mobilizing messenger, is also a potent neuronal differentiation inducer in PC12 cells, while IP₃ exhibits no such role (33, 34). Whether cADPR is involved in the proliferation and differentiation of PC12 cells is unknown.Here we show that activation of the CD38/cADPR/Ca²⁺ signaling is required for the ACh-induced proliferation in PC12 cells, while inhibition of the pathway accelerates NGF-induced neuronal differentiation. Our data indicate that cADPR is important in regulating cell proliferation and neuronal differentiation in PC12 cells.     

An Integrated Approach of Differential Mass Spectrometry and Gene Ontology Analysis Identified Novel Proteins Regulating Neuronal Differentiation and Survival

MS-based quantitative proteomics is widely used for large scale identification of proteins. However, an integrated approach that offers comprehensive proteome coverage, a tool for the quick categorization of the identified proteins, and a standardized biological study method is needed for helping the researcher focus on investigating the proteins with biologically important functions. In this study, we utilized isobaric tagging for relative and absolute quantification (iTRAQ)-based quantitative differential LC/MS/MS, functional annotation with a proprietary gene ontology tool (Molecular Annotation by Gene Ontology (MANGO)), and standard biochemical methods to identify proteins related to neuronal differentiation in nerve growth factor-treated rat pheochromocytoma (PC12) cells, which serve as a representative model system for studying neuronal biological processes. We performed MS analysis by using both nano-LC-MALDI-MS/MS and nano-LC-ESI-MS/MS for maximal proteome coverage. Of 1,482 non-redundant proteins semiquantitatively identified, 72 were differentially expressed with 39 up- and 33 down-regulated, including 64 novel nerve growth factor-responsive PC12 proteins. Gene ontology analysis of the differentially expressed proteins by MANGO indicated with statistical significance that the up-regulated proteins were mostly related to the biological processes of cell morphogenesis, apoptosis/survival, and cell differentiation. Some of the up-regulated proteins of unknown function, such as PAIRBP1, translationally controlled tumor protein, prothymosin α, and MAGED1, were further analyzed to validate their significant functions in neuronal differentiation by immunoblotting and immunocytochemistry using each antibody combined with a specific short interfering RNA technique. Knockdown of these proteins caused abnormal cell morphological changes, inhibition of neurite formation, and cell death during each course of the differentiation, confirming their important roles in neurite formation and survival of PC12 cells. These results show that our iTRAQ-MANGO-biological analysis framework, which integrates a number of standard proteomics strategies, is effective for targeting and elucidating the functions of proteins involved in the cellular biological process being studied.MS-based quantitative proteomics strategies such as iTRAQ¹ (1) and stable isotope labeling with amino acids in cell culture (2) are powerfully effective for the comprehensive characterization of biological phenomena (1–5). Although these methods have been applied for cancer biomarker (6, 7) and drug target (8) discovery, their use in the elucidation of biological and functional processes has been limited because of certain technical problems that arise when attempting to meaningfully process the immense amount of data obtained from such experiments. The following four main issues are typically the sources of such difficulties. 1) Quantitative identification by one type of MS system may fail to cover the total proteome because of ionization efficiency differences, such as those between ESI and MALDI, for certain peptides, leading to theoretical limitations in proteome coverage. 2) The public protein databases are often insufficient for searching non-human species because of the limited available genomic information. 3) The identification of the functions and biological processes of thousands of proteins is a formidable task because of the lack of simple and user-friendly software to automate gene ontology (GO) annotation. Furthermore it is difficult to convert large lists of taxonomically diverse proteins into their human orthologs to obtain the richest GO information available. 4) Lastly biological validation strategies for identified proteins have not been standardized. Therefore, we believe an analysis framework that provides (a) comprehensive proteome data; (b) a simple and quick tool for organizing, enriching, and sorting those data to reveal candidate molecules for relation to certain processes; and (c) a standardized biological validation technique would greatly benefit this field. We therefore designed a concise, three-step, sequential proteomics strategy that addresses the above concerns and utilized it successfully in studying the mechanism of neuronal differentiation in PC12 cells.PC12 cells (9) have been widely used as a model of neurons because of their unique advantages, such as stability, homogeneity, strong nerve growth factor (NGF) responsiveness, high differentiation potential, and a wealth of accessible background material, which help to facilitate their manipulation (10). This cell line has also been used for studying the mechanisms of neuronal disorders such as Alzheimer (11), Huntington (12), and Parkinson diseases (13) and neurofibromatosis type 1 (14–16). Here we used PC12 cells as a model for characterizing the mechanisms of neuronal differentiation and neurodegenerative disorders by means of MS-based quantitative proteomics.NGF is one member of a family of structurally and functionally related dimeric polypeptides, neurotrophins, that are essential for the development and maintenance of distinct neuronal populations in the central and peripheral nervous systems (17). The initial signaling cascades in the neuronal cells right after NGF stimulation have been subjected to thorough investigation and characterization by using PC12 cells. After binding of extracellular NGF to the cell membrane-localized tropomyosin-related kinase A (TrkA) receptor, TrkA receptors dimerize and subsequently autophosphorylate each other. Then the phosphorylated receptors recruit a complex of signaling molecules and induce a number of intracellular signaling cascades involving the signaling molecules, such as phosphoinositide 3-kinase, phospholipase C-γ, and Ras (18). The posttranslational modifications, such as phosphorylation cascades, triggered by NGF stimulation play important roles in PC12 cell differentiation. However, knowledge of the precise dynamic molecular events of protein expression in response to NGF signaling in PC12 cells after an interval that allows the stimulation to take full effect and produce morphological changes remains far from complete.Several reported studies have applied such methods as expressed sequence tag (19), restriction landmark cDNA scanning (20), targeted display (21), serial analysis of gene expression (22), and cDNA microarray (23) to survey the global change of differentially expressed genes in PC12 cells before and after NGF treatment (19–23). However, the genes and underlying mechanisms associated with the acquisition of a neuronal phenotype in these cells have not been clarified. Also a few proteomics approaches have been used for identifying the proteins related to NGF-inducible neurite formation in PC12 cells. For example, 2-D electrophoresis was applied in whole-cell extract separation to study the NGF modulation of protein synthesis (24); however, only two peptides were identified (25). Even currently available PC12 cell 2-D databases include merely a few proteins related to NGF stimulation (26–29). There is thus a paucity of functional proteomic information related to PC12 cell biological processes that may be attributed to technical limitations such as those listed above.In this study, we performed the first proteomics survey of proteins differentially expressed in PC12 cells during NGF treatment by using a semiquantitative differential LC shotgun method, namely isobaric tagging for relative and absolute quantitation (iTRAQ) coupled with concurrent use of two tandem MS/MS systems, namely nano-LC-MALDI-TOF-TOF and nano-LC-ESI-Quadrupole/quadrupole/time-of-flight mass spectrometers. The total list of proteins identified was converted into a new file linked to the GO database by our proprietary GO analysis tool for proteomes (MANGO) and categorized by biological process and function using specific classification methods. Thereafter we classified the subset of proteins that were up- or down-regulated during neurite formation into specific molecular categories by combining the differential data obtained by iTRAQ with the proteomic GO analysis results. We then attempted to characterize the functional mechanism of NGF-induced PC12 cell neuronal differentiation. Interestingly the specific up-regulated groups classified in this study were related to apoptosis/cell survival in addition to cell motility, differentiation, stress stimulation, and morphogenesis. To investigate the molecular functions of the up-regulated proteins in relation to both PC12 cell differentiation and apoptosis/survival during neurite formation, some of them were further analyzed with a biochemical and cellular biological strategy using a combined antibody and siRNA technique. Lastly we demonstrated the advantages that our concise, sequential proteomics strategy offers for studying the molecular mechanisms of cellular biological events such as cell differentiation and survival/apoptosis.     

Analysis of Non-canonical Fibroblast Growth Factor Receptor 1 (FGFR1) Interaction Reveals Regulatory and Activating Domains of Neurofascin

Fibroblast growth factor receptors (FGFRs) are important for many different mechanisms, including cell migration, proliferation, differentiation, and survival. Here, we show a new link between FGFR1 and the cell adhesion molecule neurofascin, which is important for neurite outgrowth. After overexpression in HEK293 cells, embryonal neurofascin isoform NF166 was able to associate with FGFR1, whereas the adult isoform NF186, differing from NF166 in additional extracellular sequences, was deficient. Pharmacological inhibitors and overexpression of dominant negative components of the FGFR signaling pathway pointed to the activation of FGFR1 after association with neurofascin in neurite outgrowth assays in chick tectal neurons and rat PC12-E2 cells. Both extra- and intracellular domains of embryonal neurofascin isoform NF166 were able to form complexes with FGFR1 independently. However, the cytosolic domain was both necessary and sufficient for the activation of FGFR1. Cytosolic serine residues 56 and 100 were shown to be essential for the neurite outgrowth-promoting activity of neurofascin, whereas both amino acid residues were dispensable for FGFR1 association. In conclusion, the data suggest a neurofascin intracellular domain, which activates FGFR1 for neurite outgrowth, whereas the extracellular domain functions as an additional, regulatory FGFR1 interaction domain in the course of development.The four known fibroblast growth factor receptors (FGFRs),² which are targeted by a large family of 22 fibroblast growth factor ligands, represent a highly diverse signaling system important for migration, proliferation, differentiation, and survival of many different cell types (1, 2). fibroblast growth factor activation of FGFR leads to the activation of mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K), and phospholipase Cγ (PLCγ), depending on the cellular system under study. Non-canonical FGFR interactions with NCAM, cadherins, and syndecan via extracellular domains were also described (1). However, the contribution of intracellular interactions of FGFR1 with further membrane co-receptors is poorly understood. Only cytosolic interaction between FGFRs and EphA4 have been described that are involved in mutual transphosphorylation (3).The cell adhesion molecule neurofascin is important for cell-cell communication in the nervous system (4, 5). Neurofascin regulates many different functions in the brain, suggesting that it functions as a key regulator for both developing and differentiated neural cells. Different alternatively spliced neurofascin isoforms are expressed in different cells and at different times of development (6). Embryonal neurofascin NF166 is important for neurite outgrowth and guidance (7, 8). Recently, a role for neurofascin NF166 for early processes of inhibitory synaptogenesis at the axon hillock and for the positioning of inhibitory synapses at the axon initial segment has been proven (9, 10).In the more developed nervous system, NF166 is replaced by NF186, which is inhibitory for neurite outgrowth (11). NF186 is linked to the cortical actin cytoskeleton via ankyrin_G (12). Clustering of voltage-gated sodium channels both at axon initial segments and at the nodes of Ranvier is conferred by neurofascin NF186 (13, 14). A further cytosolic interaction partner is the PDZ molecule syntenin-1 (15).Despite the well known functional importance of neurofascin in the nervous system, corresponding signaling pathways have not been investigated. In contrast, signaling by the related molecules NCAM and L1 have been studied with regard to the induction of neurite outgrowth in greater detail (for a review, see Refs. 16–18). Both NCAM and L1 induce neurite outgrowth through activation of FGFR1 (19–23). NCAM may further undergo lateral interactions with PrP (prion precursor protein) or GFRα, which is part of the glia-derived neurotrophic factor receptor (24, 25). In addition to FGFR1 interaction, both L1 and NCAM are connected to non-receptor tyrosine kinases. However, whereas NCAM employs the non-receptor kinase c-Fyn as an upstream component, L1 is linked to c-Src (26, 27). L1 converges with NCAM signaling upstream of the MAPK pathway at the level of Raf (18, 21, 28, 29). NCAM may induce alternative signaling pathways, including protein kinase A-dependent signaling or G-proteins (18, 30). NCAM signaling to the nucleus may include activation of CREB and c-Fos or NF-κB (29, 31, 32).Here, we elucidate the molecular mechanisms of neurofascin-FGFR1 interaction for neurite outgrowth. We show that both cytosolic and the extracellular domains are important for the association of FGFR1 with neurofascin. Although the cytosolic domain represents a critical determinant for FGFR1 activation, the extracellular sequences of neurofascin act as a regulator for FGFR1-dependent signal transduction in the course of development.     

Activin A Suppresses Osteoblast Mineralization Capacity by Altering Extracellular Matrix (ECM) Composition and Impairing Matrix Vesicle (MV) Production

During bone formation, osteoblasts deposit an extracellular matrix (ECM) that is mineralized via a process involving production and secretion of highly specialized matrix vesicles (MVs). Activin A, a transforming growth factor-β (TGF-β) superfamily member, was previously shown to have inhibitory effects in human bone formation models through unclear mechanisms. We investigated these mechanisms elicited by activin A during in vitro osteogenic differentiation of human mesenchymal stem cells (hMSC). Activin A inhibition of ECM mineralization coincided with a strong decline in alkaline phosphatase (ALP¹) activity in extracellular compartments, ECM and matrix vesicles. SILAC-based quantitative proteomics disclosed intricate protein composition alterations in the activin A ECM, including changed expression of collagen XII, osteonectin and several cytoskeleton-binding proteins. Moreover, in activin A osteoblasts matrix vesicle production was deficient containing very low expression of annexin proteins. ECM enhanced human mesenchymal stem cell osteogenic development and mineralization. This osteogenic enhancement was significantly decreased when human mesenchymal stem cells were cultured on ECM produced under activin A treatment. These findings demonstrate that activin A targets the ECM maturation phase of osteoblast differentiation resulting ultimately in the inhibition of mineralization. ECM proteins modulated by activin A are not only determinant for bone mineralization but also possess osteoinductive properties that are relevant for bone tissue regeneration.The quality of bone tissue is determined by the balanced action of the anabolic bone cells, the osteoblasts, and their catabolic counterparts, the osteoclasts. This process of bone remodeling occurs throughout life and can be influenced by a wide variety of molecules, having ultimately an impact on the quality of bone (1, 2). Activins and inhibins are members of the TGF-β superfamily with predominant antagonistic effects in their classically known target tissues, such as in gonadotropin producing cells in the pituitary and their role in reproduction (3, 4). Like other TGF-β member, activins elicit biological responses by binding to type I and II serine/threonine kinase receptors at the cell surface. Upon ligand binding, signaling is further transduced in the cytoplasm by phosphorylated Smad protein complexes that once in the nucleus regulate gene expression. This signaling pathway is highly complex because of crosstalk between different ligands (Activins, BMPs, TGF-β) binding to multiple serine/threonine kinase receptors that activate different Smad proteins signaling to the nucleus. Activin is known to signal using type II receptors ACVR2A or ACVR2B and the type I receptor ACVRIB (shared with BMPs) activating Smad2 and 3 proteins (shared with TGF-β). Inhibins exert their inhibitory effects on activin by competitive binding to the activin receptors in the presence of betaglycan. This signaling regulates a wide array of biological activities from cell proliferation, differentiation to tumor development and endocrine signaling (5, 6) in many cell lineages like hematopoietic (7, 8) and monocyte/macrophage (9, 10). Several consequences of these reproductive hormones, especially those of activin A, are also described in relation to bone metabolism. Activin A is present in bone tissue (11, 12) affecting both osteoclasts and osteoblasts. While having a consistent pro-osteoclastogenic effect (9, 13), the activin A impact on osteoblast differentiation is more controversial (see (14) for review) Several reports support a stimulatory effect of activin A on osteoblast differentiation and mineralization in vitro and in vivo (9, 15, 16). On the other hand, two different studies, using rat and human bone formation models, have demonstrated that activin A treatment has a coherent inhibitory influence on osteogenesis leading to significant reduction of the mineralization capacity (11, 17). These opposing effects of activin A on osteoblastogenesis may simply reflect species differences, however, it may be also driven by heterogeneity of the used cell model or the stage of osteoblast differentiation (14). Nevertheless, a negative role of activin A in bone formation is also supported by other in vivo studies in mice and primates in which blockage of activin signaling resulted in increased bone mass (18, 19). Moreover, transgenic mice overexpressing human inhibin A showed increased bone formation (20).The extracellular compartment is crucial for bone because it determines most of the bone quality properties (21, 22), including its strength, stability, and integrity. Interestingly, a mature extracellular matrix (ECM) is characterized by the capacity to mineralize even in the absence of further osteoblast activity (11, 23). This biomineralization process is complex and not fully elucidated but it is thought to be started within MVs (24). Osteoblasts in bone and other cells in mineralization competent tissues, such as cartilage (25), tendon (26), teeth (27), and calcifying vasculature (28) produce and release from their plasma membrane these vesicles with diameters ranging between 50 and 200 nm. It is inside these membrane-enclosed particles that first crystals of mineral are formed and grow, before the vesicle membrane is permeated and the mineral crystallization advances into the ECM (29, 30). In this context, proteins that can mobilize calcium and inorganic phosphate (Pi), the backbone of the hydroxyapatite crystals present in bone, are of utmost importance. P_i donor proteins found in MVs include alkaline phosphatase (ALP) and inorganic pyrophosphatases (31) whereas the annexin family of proteins is postulated to be crucial for calcium influx into the vesicles (32–34).In this study we investigated the inhibitory effect of activin A on human mesenchymal stem cells (hMSC) derived osteoblast differentiation and mineralization. We have previously shown that in human osteoblast cultures activin A influences the expression of many ECM genes altering ECM maturity (11). Thus, we focused our analysis on extracellular environment changes, namely the ECM and matrix vesicles (MVs). The characterization of these compartments was done using the state-of-the-art quantitative proteomics tools including SILAC metabolic labeling and mass spectrometry. Furthermore, the importance of ECM composition for osteoblast differentiation was also determined.     

Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

High-definition De Novo Sequencing of Crustacean Hyperglycemic Hormone (CHH)-family Neuropeptides

A complete understanding of the biological functions of large signaling peptides (>4 kDa) requires comprehensive characterization of their amino acid sequences and post-translational modifications, which presents significant analytical challenges. In the past decade, there has been great success with mass spectrometry-based de novo sequencing of small neuropeptides. However, these approaches are less applicable to larger neuropeptides because of the inefficient fragmentation of peptides larger than 4 kDa and their lower endogenous abundance. The conventional proteomics approach focuses on large-scale determination of protein identities via database searching, lacking the ability for in-depth elucidation of individual amino acid residues. Here, we present a multifaceted MS approach for identification and characterization of large crustacean hyperglycemic hormone (CHH)-family neuropeptides, a class of peptide hormones that play central roles in the regulation of many important physiological processes of crustaceans. Six crustacean CHH-family neuropeptides (8–9.5 kDa), including two novel peptides with extensive disulfide linkages and PTMs, were fully sequenced without reference to genomic databases. High-definition de novo sequencing was achieved by a combination of bottom-up, off-line top-down, and on-line top-down tandem MS methods. Statistical evaluation indicated that these methods provided complementary information for sequence interpretation and increased the local identification confidence of each amino acid. Further investigations by MALDI imaging MS mapped the spatial distribution and colocalization patterns of various CHH-family neuropeptides in the neuroendocrine organs, revealing that two CHH-subfamilies are involved in distinct signaling pathways.Neuropeptides and hormones comprise a diverse class of signaling molecules involved in numerous essential physiological processes, including analgesia, reward, food intake, learning and memory (1). Disorders of the neurosecretory and neuroendocrine systems influence many pathological processes. For example, obesity results from failure of energy homeostasis in association with endocrine alterations (2, 3). Previous work from our lab used crustaceans as model organisms found that multiple neuropeptides were implicated in control of food intake, including RFamides, tachykinin related peptides, RYamides, and pyrokinins (4–6).Crustacean hyperglycemic hormone (CHH)¹ family neuropeptides play a central role in energy homeostasis of crustaceans (7–17). Hyperglycemic response of the CHHs was first reported after injection of crude eyestalk extract in crustaceans. Based on their preprohormone organization, the CHH family can be grouped into two sub-families: subfamily-I containing CHH, and subfamily-II containing molt-inhibiting hormone (MIH) and mandibular organ-inhibiting hormone (MOIH). The preprohormones of the subfamily-I have a CHH precursor related peptide (CPRP) that is cleaved off during processing; and preprohormones of the subfamily-II lack the CPRP (9). Uncovering their physiological functions will provide new insights into neuroendocrine regulation of energy homeostasis.Characterization of CHH-family neuropeptides is challenging. They are comprised of more than 70 amino acids and often contain multiple post-translational modifications (PTMs) and complex disulfide bridge connections (7). In addition, physiological concentrations of these peptide hormones are typically below picomolar level, and most crustacean species do not have available genome and proteome databases to assist MS-based sequencing.MS-based neuropeptidomics provides a powerful tool for rapid discovery and analysis of a large number of endogenous peptides from the brain and the central nervous system. Our group and others have greatly expanded the peptidomes of many model organisms (3, 18–33). For example, we have discovered more than 200 neuropeptides with several neuropeptide families consisting of as many as 20–40 members in a simple crustacean model system (5, 6, 25–31, 34). However, a majority of these neuropeptides are small peptides with 5–15 amino acid residues long, leaving a gap of identifying larger signaling peptides from organisms without sequenced genome. The observed lack of larger size peptide hormones can be attributed to the lack of effective de novo sequencing strategies for neuropeptides larger than 4 kDa, which are inherently more difficult to fragment using conventional techniques (34–37). Although classical proteomics studies examine larger proteins, these tools are limited to identification based on database searching with one or more peptides matching without complete amino acid sequence coverage (36, 38).Large populations of neuropeptides from 4–10 kDa exist in the nervous systems of both vertebrates and invertebrates (9, 39, 40). Understanding their functional roles requires sufficient molecular knowledge and a unique analytical approach. Therefore, developing effective and reliable methods for de novo sequencing of large neuropeptides at the individual amino acid residue level is an urgent gap to fill in neurobiology. In this study, we present a multifaceted MS strategy aimed at high-definition de novo sequencing and comprehensive characterization of the CHH-family neuropeptides in crustacean central nervous system. The high-definition de novo sequencing was achieved by a combination of three methods: (1) enzymatic digestion and LC-tandem mass spectrometry (MS/MS) bottom-up analysis to generate detailed sequences of proteolytic peptides; (2) off-line LC fractionation and subsequent top-down MS/MS to obtain high-quality fragmentation maps of intact peptides; and (3) on-line LC coupled to top-down MS/MS to allow rapid sequence analysis of low abundance peptides. Combining the three methods overcomes the limitations of each, and thus offers complementary and high-confidence determination of amino acid residues. We report the complete sequence analysis of six CHH-family neuropeptides including the discovery of two novel peptides. With the accurate molecular information, MALDI imaging and ion mobility MS were conducted for the first time to explore their anatomical distribution and biochemical properties.     

Proinsulin and the Genetics of Diabetes Mellitus

Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.     

Analysis of Proteins That Rapidly Change Upon Mechanistic/Mammalian Target of Rapamycin Complex 1 (mTORC1) Repression Identifies Parkinson Protein 7 (PARK7) as a Novel Protein Aberrantly Expressed in Tuberous Sclerosis Complex (TSC)

Many biological processes involve the mechanistic/mammalian target of rapamycin complex 1 (mTORC1). Thus, the challenge of deciphering mTORC1-mediated functions during normal and pathological states in the central nervous system is challenging. Because mTORC1 is at the core of translation, we have investigated mTORC1 function in global and regional protein expression. Activation of mTORC1 has been generally regarded to promote translation. Few but recent works have shown that suppression of mTORC1 can also promote local protein synthesis. Moreover, excessive mTORC1 activation during diseased states represses basal and activity-induced protein synthesis. To determine the role of mTORC1 activation in protein expression, we have used an unbiased, large-scale proteomic approach. We provide evidence that a brief repression of mTORC1 activity in vivo by rapamycin has little effect globally, yet leads to a significant remodeling of synaptic proteins, in particular those proteins that reside in the postsynaptic density. We have also found that curtailing the activity of mTORC1 bidirectionally alters the expression of proteins associated with epilepsy, Alzheimer''s disease, and autism spectrum disorder—neurological disorders that exhibit elevated mTORC1 activity. Through a protein–protein interaction network analysis, we have identified common proteins shared among these mTORC1-related diseases. One such protein is Parkinson protein 7, which has been implicated in Parkinson''s disease, yet not associated with epilepsy, Alzheimers disease, or autism spectrum disorder. To verify our finding, we provide evidence that the protein expression of Parkinson protein 7, including new protein synthesis, is sensitive to mTORC1 inhibition. Using a mouse model of tuberous sclerosis complex, a disease that displays both epilepsy and autism spectrum disorder phenotypes and has overactive mTORC1 signaling, we show that Parkinson protein 7 protein is elevated in the dendrites and colocalizes with the postsynaptic marker postsynaptic density-95. Our work offers a comprehensive view of mTORC1 and its role in regulating regional protein expression in normal and diseased states.The mechanistic/mammalian target of rapamycin complex 1 (mTORC1)¹ is a serine/threonine protein kinase that is highly expressed in many cell types (1). In the brain, mTORC1 tightly coordinates different synaptic plasticities — long-term potentiation (LTP) and long-term depression (LTD) — the molecular correlates of learning and memory (2–5). Because mTORC1 is at the core of many synaptic signaling pathways downstream of glutamate and neurotrophin receptors, many hypothesize that dysregulated mTORC1 signaling underlies cognitive deficits observed in several neurodegenerative diseases (3, 6–17). For example, mTORC1 and its downstream targets are hyperactive in human brains diagnosed with Alzheimer''s disease (AD) (18–20). Additionally in animal models of autism spectrum disorder (ASD), altered mTORC1 signaling contributes to the observed synaptic dysfunction and aberrant network connectivity (13, 15, 21–27). Furthermore, epilepsy, which is common in AD and ASD, has enhanced mTORC1 activity (28–32).Phosphorylation of mTORC1, considered the active form, is generally regarded to promote protein synthesis (33). Thus, many theorize that diseases with overactive mTORC1 arise from excessive protein synthesis (14). Emerging data, however, show that suppressing mTORC1 activation can trigger local translation in neurons (34, 35). Pharmacological antagonism of N-methyl-d-aspartate (NMDA) receptors, a subtype of glutamate receptors that lies upstream of mTOR activation, promotes the synthesis of the voltage-gated potassium channel, Kv1.1, in dendrites (34, 35). Consistent with these results, in models of temporal lobe epilepsy there is a reduction in the expression of voltage-gated ion channels including Kv1.1 (30, 31, 36). Interestingly in a model of focal neocortical epilepsy, overexpression of Kv1.1 blocked seizure activity (37). Because both active and inactive mTORC1 permit protein synthesis, we sought to determine the proteins whose expression is altered when mTORC1 phosphorylation is reduced in vivo.Rapamycin is an FDA-approved, immunosuppressive drug that inhibits mTORC1 activity (38). We capitalized on the ability of rapamycin to reduce mTORC1 activity in vivo and the unbiased approach of mass spectrometry to identify changes in protein expression. Herein, we provide evidence that mTORC1 activation bidirectionally regulates protein expression, especially in the PSD where roughly an equal distribution of proteins dynamically appear and disappear. Remarkably, using protein–protein interaction networks facilitated the novel discovery that PARK7, a protein thus far only implicated in Parkinson''s disease, (1) is up-regulated by increased mTORC1 activity, (2) resides in the PSD only when mTORC1 is active, and (3) is aberrantly expressed in a rodent model of TSC, an mTORC1-related disease that has symptoms of epilepsy and autism. Collectively, these data provide the first comprehensive list of proteins whose abundance or subcellular distributions are altered with acute changes in mTORC1 activity in vivo.     

Phosphorylated Tau Interacts with c-Jun N-terminal Kinase-interacting Protein 1 (JIP1) in Alzheimer Disease

In Alzheimer disease (AD) and frontotemporal dementia the microtubule-associated protein Tau becomes progressively hyperphosphorylated, eventually forming aggregates. However, how Tau dysfunction is associated with functional impairment is only partly understood, especially at early stages when Tau is mislocalized but has not yet formed aggregates. Impaired axonal transport has been proposed as a potential pathomechanism, based on cellular Tau models and Tau transgenic mice. We recently reported K369I mutant Tau transgenic K3 mice with axonal transport defects that suggested a cargo-selective impairment of kinesin-driven anterograde transport by Tau. Here, we show that kinesin motor complex formation is disturbed in the K3 mice. We show that under pathological conditions hyperphosphorylated Tau interacts with c-Jun N-terminal kinase- interacting protein 1 (JIP1), which is associated with the kinesin motor protein complex. As a result, transport of JIP1 into the axon is impaired, causing JIP1 to accumulate in the cell body. Because we found trapping of JIP1 and a pathological Tau/JIP1 interaction also in AD brain, this may have pathomechanistic implications in diseases with a Tau pathology. This is supported by JIP1 sequestration in the cell body of Tau-transfected primary neuronal cultures. The pathological Tau/JIP1 interaction requires phosphorylation of Tau, and Tau competes with the physiological binding of JIP1 to kinesin light chain. Because JIP1 is involved in regulating cargo binding to kinesin motors, our findings may, at least in part, explain how hyperphosphorylated Tau mediates impaired axonal transport in AD and frontotemporal dementia.The microtubule-associated protein Tau is predominantly found in the axonal compartment of neurons, where it binds to microtubules (1). In human brain, six isoforms of Tau are expressed, due to alternative splicing of exons 2, 3 and 10 (2). Tau consists of an amino-terminal projection domain followed by 3 or 4 microtubule binding repeats (3R or 4R), due to splicing of exon 10, and a carboxyl-terminal tail region. In the AD³ and FTD brain, Tau forms filamentous inclusions (3). They are found in nerve cell bodies and apical dendrites as neurofibrillary tangles (NFTs), in distal dendrites as neuropil threads, and in the abnormal neurites that are associated with some amyloid plaques (neuritic plaques) (3). Hyperphosphorylation of Tau is thought to be an initiating step (4), as it detaches Tau from microtubules and makes it prone to form aggregates (1, 5). Whereas in AD no mutations have been identified in the MAPT gene encoding Tau, so far 42 intronic and exonic mutations have been found in familial forms of FTD (6). Their identification assisted in the generation of transgenic mouse models that reproduce NFT formation and memory impairment (7).The models were also instrumental in testing hypotheses that had been brought forward to link Tau pathology to functional impairment (8–10). In particular, defects in axonal transport have been implicated in neurodegenerative disorders (11, 12). Tau binding to microtubules affects axonal transport (13), and in cell culture overexpression of Tau was shown to lead to impaired transport of mitochondria and vesicles (14, 15). Axonal transport defects have also been reproduced in wild-type Tau transgenic mice (16) and in K369I mutant Tau K3 mice (17), whereas Tau expression failed to inhibit axonal transport in other systems (18, 19). This apparent discrepancy may depend on the type of cargos analyzed and, specifically, the experimental paradigm, e.g. using phosphorylated (16, 17, 20) versus non-phosphorylated Tau (18).To dissect Tau-mediated axonal transport defects at a molecular level, we used K3 mice that overexpress human Tau carrying the pathogenic FTD K369I mutation (17). We observed a pronounced hyperphosphorylation of transgenic Tau in many brain areas. Clinically, the mice present with an early onset motor phenotype that is, at least in part, caused by impairment of axonal transport in neurons of the substantia nigra. Interestingly, only selected aspects of anterograde axonal transport were impaired, in particular those of kinesin-I motor complex-driven vesicles and mitochondria. Our data suggest a selective impairment of axonal transport rather than a generalized, non-selective blockage of microtubules that has been established in cell culture systems, which fail to phosphorylate Tau at the high levels that are found in vivo even under physiological conditions. More importantly, in AD and FTD Tau is even more phosphorylated, i.e. hyperphosphorylated at physiological sites and de novo at pathological sites, preventing it from binding to microtubules (1).Based on our findings of an impaired kinesin-I-driven axonal transport in the K3 mice, we speculated that hyperphosphorylated Tau may impair anterograde transport by interfering directly with components of the kinesin-I motor complex rather than disrupting the binding of the kinesin heavy chain (see below) to microtubules. Axonal transport along microtubules is mediated by members of the kinesin superfamily (KIF) of motor proteins (21–23). The KIFs typically consist of an ATPase domain that interacts with microtubules and drives movement and a domain that links to cargos, either directly or indirectly, as in the case of KIF5, by assembling with the kinesin light chain (KLC) to form the kinesin-I (KIF5/KLC) motor complex (24). In addition, increasing evidence suggests that scaffolding proteins mediate and regulate the binding of cargos to KIFs (21, 25–27). These include the scaffold protein JNK-interacting protein (JIP) that is involved in the linkage of cargos to the kinesin-I motor complex via KLC (25, 28–33).Here, by using the K3 mouse model, we identified a novel interaction of Tau and JIP in neurons that causes a trapping of JNK interacting protein 1 (JIP1) in the cell body of K3 mice, cell culture systems, and human AD brain. We found that the pathological interaction of hyperphosphorylated Tau and JIP1 competes with the physiological binding of JIP1 to KLC.