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The choroid plexus (ChP) is the blood-cerebrospinal fluid (CSF) barrier and the primary source of CSF. Acquired hydrocephalus, caused by brain infection or hemorrhage, lacks drug treatments due to obscure pathobiology. Our integrated, multi-omic investigation of post-infectious hydrocephalus (PIH) and post-hemorrhagic hydrocephalus (PHH) models revealed that lipopolysaccharide and blood breakdown products trigger highly similar TLR4-dependent immune responses at the ChP-CSF interface. The resulting CSF "cytokine storm", elicited from peripherally derived and border-associated ChP macrophages, causes increased CSF production from ChP epithelial cells via phospho-activation of the TNF-receptor-associated kinase SPAK, which serves as a regulatory scaffold of a multi-ion transporter protein complex. Genetic or pharmacological immunomodulation prevents PIH and PHH by antagonizing SPAK-dependent CSF hypersecretion. These results reveal the ChP as a dynamic, cellularly heterogeneous tissue with highly regulated immune-secretory capacity, expand our understanding of ChP immune-epithelial cell cross talk, and reframe PIH and PHH as related neuroimmune disorders vulnerable to small molecule pharmacotherapy.

Exosomes are synthesized and secreted by different cell types and contain proteins, lipids, metabolites and RNA species that reflect the physiological status of the cell of origin. As such, exosomes are increasingly being used as a novel reservoir for disease biomarker discovery. However, isolation of exosomes can be challenging due to their nonuniformity of shape and variable tissue of origin. Moreover, various analytical techniques used for protein detection and quantitation remain insensitive to the low amounts of protein isolated from exosomes. Despite these challenges, techniques to improve proteomic yield and increase protein dynamic range continue to improve at a rapid rate. In this review, we highlight the importance of exosome proteomics in neurodegenerative and neuropsychiatric disorders and the associated technical difficulties. Furthermore, current progress and technological advancements in exosome proteomics research are discussed with an emphasis on disease-associated protein biomarkers.

Many neurological disorders and diseases including drug addiction are associated with specific neuronal cell types in the brain. The striatum, a region that plays a critically important role in the development of addictive drug-related behavior, provides a good example of the cellular heterogeneity challenges associated with analyses of specific neuronal cell types. Such studies are needed to identify the adaptive changes in neuroproteomic signaling that occur in response to diseases such as addiction. The striatum contains two major cell types, D1 and D2 type dopaminoceptive medium spiny neurons (MSNs), whose cell bodies and processes are intermingled throughout this region. Since little is known about the proteomes of these two neuronal cell populations, we have begun to address this challenge by using fluorescence-activated nuclear sorting (FANS) to isolate nuclei-containing fractions from striatum from D1 and D2 &ldquo, Translating Ribosome Affinity Purification&rdquo, (TRAP) mice. This approach enabled us to devise and implement a robust and reproducible workflow for preparing samples from specific MSN cell types for mass spectrometry analyses. These analyses quantified at least 685 proteins in each of four biological replicates of 50 K sorted nuclei from two D1 mice/replicate and from each of four biological replicates of 50 K sorted nuclei from two D2 mice/replicate. Proteome analyses identified 87 proteins that were differentially expressed in D1 versus D2 MSN nuclei and principal component analysis (PCA) of these proteins separated the 8 biological replicates into specific cell types. Central network analysis of the 87 differentially expressed proteins identified Hnrnpd and Hnmpa2b1 in D1 and Cct2 and Cct7 in D2 as potential central interactors. This workflow can now be used to improve our understanding of many neurological diseases including characterizing the short and long-term impact of drugs of abuse on the proteomes of these two dopaminoceptive neuronal populations.

Despite its importance for γ-aminobutyric acid (GABA) inhibition and involvement in neurodevelopmental disease, the regulatory mechanisms of the K(+)/Cl(−) co-transporter KCC2 (encoded by SLC12A5) during maturation of the central nervous system (CNS) are not entirely understood. Here, we applied quantitative phosphoproteomics to systematically map sites of KCC2 phosphorylation during CNS development in the mouse. KCC2 phosphorylation at Thr(906) and Thr(1007), which inhibits KCC2 activity, underwent dephosphorylation in parallel with the GABA excitatory-inhibitory sequence in vivo. Knock-in mice expressing the homozygous phospho-mimetic KCC2 mutations T906E/T1007E (Kcc2(E/E)), which prevented the normal developmentally regulated dephosphorylation of these sites, exhibited early postnatal death from respiratory arrest and a marked absence of cervical spinal neuron respiratory discharges. Kcc2(E/E) mice also displayed disrupted lumbar spinal neuron locomotor rhythmogenesis and touch-evoked status epilepticus associated with markedly impaired KCC2-dependent Cl(−) extrusion. These data identify a previously unknown phosphorylation-dependent KCC2 regulatory mechanism during CNS development that is essential for dynamic GABA-mediated inhibition and survival.

The protozoan parasite Entamoeba histolytica causes amoebiasis, a major public health problem in developing countries. Motility of E. histolytica is important for its pathogenesis. Blebbing is an essential process contributing to cellular motility in many systems. In mammalian cells, formation of plasma membrane blebs is regulated by Rho-GTPases through its effectors, such as Rho kinase, mDia1, and acto-myosin proteins. In this study, we have illuminated the role of EhRho1 in bleb formation and motility of E. histolytica. EhRho1 was found at the site of bleb formation in plasma membrane of trophozoites. Overexpression of mutant EhRho1 defective for Guanosine triphosphate (GTP)-binding or down-regulating EhRho1 by antisense RNA resulted in reduced blebbing and motility. Moreover, serum-starvation reduced blebbing that was restored on serum-replenishment. Lysophosphatidic acid treatment induced bleb formation, whereas wortmannin inhibited the process. In all these cases, concentration of GTP-EhRho1 (active) and Phosphatidylinositol 4,5-bisphosphate (PIP2) inversely correlated with the level of plasma membrane blebbing. Our study suggests the role of EhRho1 in blebbing and bleb-based motility through PI3 kinase pathway in E. histolytica.

The choroid plexus epithelium (CPE) secretes higher volumes of fluid (cerebrospinal fluid, CSF) than any other epithelium and simultaneously functions as the blood-CSF barrier to gate immune cell entry into the central nervous system. Posthemorrhagic hydrocephalus (PHH), an expansion of the cerebral ventricles due to CSF accumulation following intraventricular hemorrhage (IVH), is a common disease usually treated by suboptimal CSF shunting techniques. PHH is classically attributed to primary impairments in CSF reabsorption, but little experimental evidence supports this concept. In contrast, the potential contribution of CSF secretion to PHH has received little attention. In a rat model of PHH, we demonstrate that IVH causes a Toll-like receptor 4 (TLR4)- and NF-κB-dependent inflammatory response in the CPE that is associated with a ∼3-fold increase in bumetanide-sensitive CSF secretion. IVH-induced hypersecretion of CSF is mediated by TLR4-dependent activation of the Ste20-type stress kinase SPAK, which binds, phosphorylates, and stimulates the NKCC1 co-transporter at the CPE apical membrane. Genetic depletion of TLR4 or SPAK normalizes hyperactive CSF secretion rates and reduces PHH symptoms, as does treatment with drugs that antagonize TLR4-NF-κB signaling or the SPAK-NKCC1 co-transporter complex. These data uncover a previously unrecognized contribution of CSF hypersecretion to the pathogenesis of PHH, demonstrate a new role for TLRs in regulation of the internal brain milieu, and identify a kinase-regulated mechanism of CSF secretion that could be targeted by repurposed US Food and Drug Administration (FDA)-approved drugs to treat hydrocephalus.

The parasite Entamoeba histolytica is the etiological agent of amoebiasis and phagocytosis plays a key role in virulence of this organism. Signaling pathways involved in activation of cytoskeletal dynamics required for phagocytosis remain to be elucidated. Phagocytosis is initiated with sequential recruitment of EhC2PK, EhCaBP1, EhCaBP3 and an atypical kinase EhAK1 after particle attachment. Here we show that EhARPC1, an essential subunit of the actin branching complex Arp 2/3 is recruited to the phagocytic initiation sites by EhAK1. Imaging, expression knockdown of different molecules and pull down experiments suggest that EhARPC1 interacts with EhAK1 and that it is required during initiation of phagocytosis and phagosome formation. Moreover, recruitment of EhARPC2 at the phagocytosis initiation by EhAK1 is also observed, indicating that the Arp 2/3 complex is recruited. In conclusion, these results suggests a novel mechanism of recruitment of Arp 2/3 complex during phagocytosis in E. histolytica., Author Summary E. histolytica is the causative agent of amoebiasis and leads to morbidity and mortality in developing countries. It is known to phagocytose immune and non-immune cells, epithelial tissue, erythrocytes and commensal bacteria. The high rate of phagocytosis in this protist parasite provides a unique system to study the signaling cascade that is activated after attachment of the particle to the cell surface. The major objective of the signaling pathway is to generate force for uptake of the particle and this is done through stimulating cytoskeleton to form appropriate structures. However, the molecular mechanism of the same is still largely unknown in E. histolytica, though this pathway has been characterized in many other systems. We have been investigating this pathway by using red blood cells as a particle and have identified different molecules required during the initial stages of phagocytosis. In this study we demonstrate the mechanism by which actin cytoskeleton branching complex EhARP2/3 is recruited at the site of erythrophagocytosis and show that the recruitment is through an atypical alpha kinase EhAK1. A number of different approaches, such as pull down assay, conditional suppression of EhAK1 expression and imaging were used to decipher this pathway. Therefore this study provides a mechanism by which actin dynamics couples to the initial signaling system, activated on attachment of RBC to the cell receptors.

Congenital hydrocephalus (CH), featuring markedly enlarged brain ventricles, is thought to arise from failed cerebrospinal fluid (CSF) homeostasis and is treated with lifelong surgical CSF shunting with substantial morbidity. CH pathogenesis is poorly understood. Exome sequencing of 125 CH trios and 52 additional probands identified three genes with significant burden of rare damaging de novo or transmitted mutations: TRIM71 (p = 2.15 x 10(-7)), SMARCC1 (p = 8.15 x 10(-10)), and PTCH1 (p = 1.06 x 10(-6)). Additionally, two de novo duplications were identified at the SHH locus, encoding the PTCH1 ligand (p = 1.2 x 10(-4)). Together, these probands account for similar to 10\% of studied cases. Strikingly, all four genes are required for neural tube development and regulate ventricular zone neural stem cell fate. These results implicate impaired neurogenesis (rather than active CSF accumulation) in the pathogenesis of a subset of CH patients, with potential diagnostic, prognostic, and therapeutic ramifications.

Phagocytosis plays a key role in survival and pathogenicity of Entamoeba histolytica. We have recently demonstrated that an atypical kinase EhAK1 is involved in phagocytosis in this parasite. It is recruited to the phagocytic cups through interaction with EhCaBP1. EhAK1 manipulates actin dynamics by multiple mechanisms including phosphorylation of G-actin. Biochemical analysis showed that EhAK1 is a serine/threonine kinase with broad ion specificity and undergoes multiple trans-autophosphorylation. Three autophosphorylation sites were identified by mass spectrometry. Out of these Thr279 appears to be involved in both autophosphorylation as well as substrate phosphorylation. Over expression of the mutant Thr279A inhibited erythrophagocytosis showing dominant negative phenotype. Multiple alignments of different kinases including alpha kinases displayed conserved binding sites that are thought to be important for function of the protein. Mutation studies demonstrated the importance of some of these binding sites in kinase activity. Binding studies with fluorescent-ATP analogs supported our prediction regarding ATP binding site based on sequence alignment. In conclusion, EhAK1 has multiple regulatory features and enrichment of EhAK1 at the site of phagocytosis stimulates trans-autophosphorylation reaction that increases kinase activity resulting in enhanced actin dynamics and phagocytosis. Some of the properties of EhAK1 are similar to that seen in alpha kinases.

A variety of functions in eukaryotes, such as cell migration, contraction, secretion, proliferation, differentiation, and exocytosis, are initiated and sustained by signaling processes. Most of the signalling pathways have been described in detail for many eukaryotic systems, particularly for mammalian systems. Signaling pathways quite often consist of cell-surface receptors, intracellular components that function as adaptors and transducers including those that generate second messengers leading to either alteration in gene expression or cytoskeleton dynamics. We have rudimentary knowledge about the organization of signaling systems in Entamoeba histolytica and the mechanisms by which initiation is coupled with functional readout, in spite of identification of a number of molecules known to participate in these pathways in other organisms. In this chapter we provide a summary of our current understanding on calcium and G-protein signaling pathways of E. histolytica and their role in different biological processes.

EhCaBP1 is a well-characterized calcium binding protein from Entamoeba histolytica with four canonical EF-hand motifs. The crystal structure of EhCaBP1 reveals the trimeric organization of N-terminal domain. The solution structure obtained at pH 6.0 indicated its monomeric nature, similar to that of calmodulin. Recent domain-wise studies showed clearly that the N-terminal domain of EhCaBP1 is capable of performing most of the functions of the full-length protein. Additionally, the mode of target binding in the trimer is similar to that found in calmodulin. To study the dynamic nature of this protein and further validate the trimerization of N-terminal domain at physiological conditions, the crystal structure of N-terminal domain was determined at 2.5 Å resolution. The final structure consists of EF-1 and EF-2 motifs separated by a long straight helix as seen in the full-length protein. The spectroscopic and stability studies, like far and near-ultraviolet circular dichroism spectra, intrinsic and extrinsic fluorescence spectra, acrylamide quenching, thermal denaturation, and dynamic light scattering, provided clear evidence for a conversion from trimeric state to monomeric state. As the pH was lowered from the physiological pH, a dynamic trimer-monomer transition was observed. The trimeric state and monomeric state observed in spectroscopic studies may represent the x-ray and NMR structures of the EhCaBP1. At pH 6.0, the endogenous kinase activation function was almost lost, indicating that the monomeric state of the protein, where EF-hand motifs are far apart, is not a functional state.