An scFv intrabody against the nonamyloid component of alpha-synuclein reduces intracellular aggregation and toxicity

Prevention of abnormal misfolding and aggregation of α synuclein (syn) protein in vulnerable neurons should be viable therapeutic strategies for reducing pathogenesis in Parkinson's disease. The nonamyloid component (NAC) region of α-syn shows strong tendencies to form β-sheet structures, and deletion of this region has been shown to reduce aggregation and toxicity in vitro and in vivo. The binding of a molecular species to this region may mimic the effects of such deletions. Single-chain variable fragment (scFv) antibodies retain the binding specificity of antibodies and, when genetically manipulated to create high-diversity libraries, allow in vitro selection against peptides. Accordingly, we used a yeast surface display library of an entire naïve repertoire of human scFv antibodies to select for binding to a NAC peptide. Candidate scFv antibodies (after transfer to mammalian expression vectors) were screened for viability in a neuronal cell line by transient cotransfection with A53T mutant α-syn. This provided a ranking of the protective efficacies of the initial panel of intracellular antibodies (intrabodies). High steady-state expression levels and apparent conformational epitope binding appeared more important than in vitro affinity in these assays. None of the scFv antibodies selected matched the sequences of previously reported anti-α-syn scFv antibodies. A stable cell line expressing the most effective intrabody, NAC32, showed highly significant reductions in abnormal aggregation in two separate models. Recently, intrabodies have shown promising antiaggregation and neuroprotective effects against misfolded mutant huntingtin protein. The NAC32 study extends such work significantly by utilizing information about the pathogenic capacity of a specific α-syn region to offer a new generation of in vitro-derived antibody fragments, both for further engineering as direct therapeutics and as a tool for rational drug design for Parkinson's disease.     

Structural insights into the recognition of peroxisomal targeting signal 1 by Trypanosoma brucei peroxin 5

Glycosomes are peroxisome-like organelles essential for trypanosomatid parasites. Glycosome biogenesis is mediated by proteins called “peroxins,” which are considered to be promising drug targets in pathogenic Trypanosomatidae. The first step during protein translocation across the glycosomal membrane of peroxisomal targeting signal 1 (PTS1)-harboring proteins is signal recognition by the cytosolic receptor peroxin 5 (PEX5). The C-terminal PTS1 motifs interact with the PTS1 binding domain (P1BD) of PEX5, which is made up of seven tetratricopeptide repeats. Obtaining diffraction-quality crystals of the P1BD of Trypanosoma brucei PEX5 (TbPEX5) required surface entropy reduction mutagenesis. Each of the seven tetratricopeptide repeats appears to have a residue in the α_L conformation in the loop connecting helices A and B. Five crystal structures of the P1BD of TbPEX5 were determined, each in complex with a hepta- or decapeptide corresponding to a natural or nonnatural PTS1 sequence. The PTS1 peptides are bound between the two subdomains of the P1BD. These structures indicate precise recognition of the C-terminal Leu of the PTS1 motif and important interactions between the PTS1 peptide main chain and up to five invariant Asn side chains of PEX5. The TbPEX5 structures reported here reveal a unique hydrophobic pocket in the subdomain interface that might be explored to obtain compounds that prevent relative motions of the subdomains and interfere selectively with PTS1 motif binding or release in trypanosomatids, and would therefore disrupt glycosome biogenesis and prevent parasite growth.     

Interaction with 7SL RNA but not with HIV-1 genomic RNA or P bodies is required for APOBEC3F virion packaging

Human cytidine deaminase apolipoprotein B mRNA-editing catalytic polypeptide-like 3F (APOBEC3F, or A3F), like APOBEC3G, has broad antiviral activity against diverse retroelements, including Vif-deficient human immunodeficiency virus (HIV)-1. Its antiviral functions are known to rely on its virion encapsidation and be suppressed by HIV-1 Vif, which recruits Cullin5-based E3 ubiquitin ligases. However, the factors that mediate A3F virion packaging have not yet been identified. In this study, we demonstrate that A3F specifically interacts with cellular signal recognition particle (SRP) RNA (7SL RNA), which is selectively packaged into HIV-1 virions. Efficient packaging of 7SL RNA as well as A3F was mediated by the RNA-binding nucleocapsid domain of HIV-1 Gag. Reducing 7SL RNA packaging by overexpression of SRP19 protein inhibited A3F virion packaging. Although A3F has been shown to interact with P bodies and viral genomic RNA, our data indicated that P bodies and HIV-1 genomic RNA were not required for A3F packaging. Thus, in addition to its well-known function in SRPs, 7SL RNA, which is encapsidated into diverse retroviruses, also participates in the innate antiviral function of host cytidine deaminases.     

Molecular titration and ultrasensitivity in regulatory networks

Protein sequestration occurs when an active protein is sequestered by a repressor into an inactive complex. Using mathematical and computational modeling, we show how this regulatory mechanism (called “molecular titration”) can generate ultrasensitive or “all-or-none” responses that are equivalent to highly cooperative processes. The ultrasensitive nature of the input-output response is mainly determined by two parameters: the dimer dissociation constant and the repressor concentration. Because in vivo concentrations are tunable through a variety of mechanisms, molecular titration represents a flexible mechanism for generating ultrasensitivity. Using physiological parameters, we report how details of in vivo protein degradation affect the strength of the ultrasensitivity at steady state. Given that developmental systems often transduce signals into cell-fate decisions on timescales incompatible with steady state, we further examine whether molecular titration can produce ultrasensitive responses within physiologically relevant time intervals. Using Drosophila somatic sex determination as a developmental paradigm, we demonstrate that molecular titration can generate ultrasensitivity on timescales compatible with most cell-fate decisions. Gene duplication followed by loss-of-function mutations can create dominant negatives that titrate and compete with the original protein. Dominant negatives are abundant in gene regulatory circuits, and our results suggest that molecular titration might be generating an ultrasensitive response in these networks.     

Depolarization-induced differentiation of PC12 cells is mediated by phospholipase D2 through the transcription factor CREB pathway

The present study examined the role of phospholipase D2 (PLD2) in the regulation of depolarization-induced neurite outgrowth and the expression of growth-associated protein-43 (GAP-43) and synapsin I in rat pheochromocytoma (PC12) cells. Depolarization of PC12 cells with 50 mmol/L KCl increased neurite outgrowth and elevated mRNA and protein expression of GAP-43 and synapsin I. These increases were suppressed by inhibition of Ca²⁺-calmodulin-dependent protein kinase II (CaMKII), PLD, or mitogen-activated protein kinase kinase (MEK). Knockdown of PLD2 by small interfering RNA (siRNA) suppressed the depolarization-induced neurite outgrowth, and the increase in GAP-43 and synapsin I expression. Depolarization evoked a Ca²⁺ rise that activated various signaling enzymes and the cAMP response element-binding protein (CREB). Silencing CaMKIIδ by siRNA blocked KCl-induced phosphorylation of proline-rich protein tyrosine kinase 2 (Pyk2), Src kinase, and extracellular signal-regulated kinase (ERK). Inhibition of Src or MEK abolished phosphorylation of ERK and CREB. Furthermore, phosphorylation of Pyk2, ERK, and CREB was suppressed by the PLD inhibitor, 1-butanol and transfection of PLD2 siRNA, whereas it was enhanced by over-expression of wild-type PLD2. Depolarization-induced PLD2 activation was suppressed by CaMKII and Src inhibitors, but not by MEK or protein kinase A inhibitors. These results suggest that the signaling pathway of depolarization-induced PLD2 activation was downstream of CaMKIIδ and Src, and upstream of Pyk2(Y881) and ERK/CREB, but independent of the protein kinase A. This is the first demonstration that PLD2 activation is involved in GAP-43 and synapsin I expression during depolarization-induced neuronal differentiation in PC12 cells.     

Kappa opioids promote the proliferation of astrocytes via Gβγ and β‐arrestin 2‐dependent MAPK‐mediated pathways

GTP binding regulatory protein (G protein)‐coupled receptors can activate MAPK pathways via G protein‐dependent and ‐independent mechanisms. However, the physiological outcomes correlated with the cellular signaling events are not as well characterized. In this study, we examine the involvement of G protein and β‐arrestin 2 pathways in kappa opioid receptor‐induced, extracellular signal‐regulated kinase 1/2 (ERK1/2)‐mediated proliferation of both immortalized and primary astrocyte cultures. As different agonists induce different cellular signaling pathways, we tested the prototypic kappa agonist, U69593 as well as the structurally distinct, non‐nitrogenous agonist, C(2)‐methoxymethyl salvinorin B (MOM‐Sal‐B). In immortalized astrocytes, U69593, activated ERK1/2 by a rapid (min) initial stimulation that was sustained over 2 h and increased proliferation. Sequestration of activated Gβγ subunits attenuated U69593 stimulation of ERK1/2 and suppressed proliferation in these cells. Furthermore, small interfering RNA silencing of β‐arrestin 2 diminished sustained ERK activation induced by U69593. In contrast, MOM‐Sal‐B induced only the early phase of ERK1/2 phosphorylation and did not affect proliferation of immortalized astrocytes. In primary astrocytes, U69593 produced the same effects as seen in immortalized astrocytes. MOM‐Sal‐B elicited sustained ERK1/2 activation which was correlated with increased primary astrocyte proliferation. Proliferative actions of both agonists were abolished by either inhibition of ERK1/2, Gβγ subunits or β‐arrestin 2, suggesting that both G protein‐dependent and ‐independent ERK pathways are required for this outcome.     

TRAIL-related neurotoxicity implies interaction with the Wnt pathway in human neuronal cells in vitro

Tumor necrosis factor related apoptosis inducing ligand (TRAIL) is involved in amyloid beta dependent neurotoxicity via the extrinsic pathway. Recently, several genes modulating TRAIL cytotoxicity have been characterized, providing evidence for a role of wingless-type mouse mammary tumor virus integration site family (Wnt), Jun-N-terminal kinase and other pathways in increased cell susceptibility to the cytokine. We investigated whether neurotoxic effects of TRAIL could be due to modulation of the Wnt signaling pathway. Western blot analysis of Wnt in SH-SY5Y human neuroblastoma cells showed significantly decreased Wnt expression in cultures treated with TRAIL. Correspondingly, both phosphorylation of glycogen synthase kinase 3 beta and degradation of cytoplasmic β-catenin were increased, as well as phosphorylation of the τ protein, bringing about the picture of neuronal damage. As a counterproof of the interaction of TRAIL with the Wnt pathway, the addition of the specific glycogen synthase kinase 3 beta inhibitor SB216763 resulted in rescue of a significant percent of cells from TRAIL-induced apoptosis. The rescue was total when the caspase 8 inhibitor z-IETD-FMK was added in combination with SB216763. Results show that, probably, in addition to triggering caspase signaling, TRAIL also interferes with the Wnt pathway, additionally concurring to neuronal damage. These data suggest that the Wnt pathway substantially contributes to the TRAIL-related neurotoxicity and indicate the TRAIL system as a candidate target for pharmacological treatment of Alzheimer's disease and related disorders.     

Mitogen-activated protein kinase activity may not be necessary for the neuropathology of Niemann-Pick type C mice

Hyperphosphorylation of neurofilament and tau, and formation of cytoskeletal lesions, are notable features of several human neurodegenerative diseases, including Niemann-Pick Disease Type C (NPC). Previous studies suggested that the MAPKs, extracellular signal regulated kinase 1 and 2 (ERK1/2) may play a significant role in this aspect of NPC. To test this idea, we treated npc mice with PD98059, a specific and potent inhibitor of MAPK activation. Although activity of ERK1/2 was inhibited by 40%, a 2-week intracerebroventricular infusion of PD98059 just prior to onset of cytoskeletal pathology and symptoms in npc mice did not delay or inhibit prominent hallmarks of NPC. Unexpectedly, ERK1/2 inhibition led to aggravation of tau hyperphosphorylation, particularly in oligodendroctyes, in a manner similar to that of certain human tauopathies. Our results suggest that ERK1/2 does not play a major role in NPC neuropathology, and therefore, that MAPK inhibition is unlikely to be a useful strategy for managing the disease.