Proteomic Studies of a Single CNS Synapse Type: The Parallel Fiber/Purkinje Cell Synapse

Precise neuronal networks underlie normal brain function and require distinct classes of synaptic connections. Although it has been shown that certain individual proteins can localize to different classes of synapses, the biochemical composition of specific synapse types is not known. Here, we have used a combination of genetically engineered mice, affinity purification, and mass spectrometry to profile proteins at parallel fiber/Purkinje cell synapses. We identify approximately 60 candidate postsynaptic proteins that can be classified into 11 functional categories. Proteins involved in phospholipid metabolism and signaling, such as the protein kinase MRCKγ, are major unrecognized components of this synapse type. We demonstrate that MRCKγ can modulate maturation of dendritic spines in cultured cortical neurons, and that it is localized specifically to parallel fiber/Purkinje cell synapses in vivo. Our data identify a novel synapse-specific signaling pathway, and provide an approach for detailed investigations of the biochemical complexity of central nervous system synapse types.     

Biochemical and structural basis for inhibition of Enterococcus faecalis hydroxymethylglutaryl-CoA synthase, mvaS, by hymeglusin

Hymeglusin (1233A, F244, L-659-699) is established as a specific β-lactone inhibitor of eukaryotic hydroxymethylglutaryl-CoA synthase (HMGCS). Inhibition results from formation of a thioester adduct to the active site cysteine. In contrast, the effects of hymeglusin on bacterial HMG-CoA synthase, mvaS, have been minimally characterized. Hymeglusin blocks growth of Enterococcus faecalis. After removal of the inhibitor from culture media, a growth curve inflection point at 3.1 h is observed (vs 0.7 h for the uninhibited control). Upon hymeglusin inactivation of purified E. faecalis mvaS, the thioester adduct is more stable than that measured for human HMGCS. Hydroxylamine cleaves the thioester adduct; substantial enzyme activity is restored at a rate that is 8-fold faster for human HMGCS than for mvaS. Structural results explain these differences in enzyme-inhibitor thioester adduct stability and solvent accessibility. The E. faecalis mvaS-hymeglusin cocrystal structure (1.95 ?) reveals virtually complete occlusion of the bound inhibitor in a narrow tunnel that is largely sequestered from bulk solvent. In contrast, eukaryotic (Brassica juncea) HMGCS binds hymeglusin in a more solvent-exposed cavity.     

Secreted meningeal chemokines,but not VEGFA,modulate the migratory properties of medulloblastoma cells

Leptomeningeal metastasis is a cause of morbidity and mortality in medulloblastoma, but the understanding of molecular mechanisms driving this process is nascent. In this study, we examined the secretory chemokine profile of medulloblastoma cells (DAOY) and a meningothelial cell line (BMEN1). Conditioned media (CM) of meningothelial cells increased adhesion, spreading and migration of medulloblastoma. VEGFA was identified at elevated levels in the CM from BMEN1 cells (as compared to DAOY CM); however, recombinant VEGFA alone was insufficient to enhance medulloblastoma cell migration. In addition, bevacizumab, the VEGFA scavenging monoclonal antibody, did not block the migratory phenotype induced by the CM. These results reveal that paracrine factors secreted by meningothelial cells can influence migration and adherence of medulloblastoma tumor cells, but VEGFA may not be a specific target for therapeutic intervention in this context.     

Association of three highly mutable genes involving pigmentation in Anthirrhinum majus

Unstable alleles at three unlinked loci (delilah, nivea and pallida) can mutate somatically to allow the production of clones of pigmented cells (“flakes”) in particular parts of the corolla. The size of the flakes depends on the timing of the mutations during the development of the flower.     

Structural consequences of the inhibitor-resistant Ser130Gly substitution in TEM beta-lactamase

Beta-lactamase confers resistance to penicillin-like antibiotics by hydrolyzing their beta-lactam bond. To combat these enzymes, inhibitors covalently cross-linking the hydrolytic Ser70 to Ser130 were introduced. In turn, mutant beta-lactamases have emerged with decreased susceptibility to these mechanism-based inhibitors. Substituting Ser130 with glycine in the inhibitor-resistant TEM (IRT) mutant TEM-76 (S130G) prevents the irreversible cross-linking step. Since the completely conserved Ser130 is thought to transfer a proton important for catalysis, its substitution might be hypothesized to result in a nonfunctional enzyme; this is clearly not the case. To investigate how TEM-76 remains active, its structure was determined by X-ray crystallography to 1.40 A resolution. A new water molecule (Wat1023) is observed in the active site, with two configurations located 1.1 and 1.3 A from the missing Ser130 Ogamma; this water molecule likely replaces the Ser130 side-chain hydroxyl in substrate hydrolysis. Intriguingly, this same water molecule is seen in the IRT TEM-32 (M69I/M182T), where Ser130 has moved significantly. TEM-76 shares other structural similarities with various IRTs; like TEM-30 (R244S) and TEM-84 (N276D), the water molecule activating clavulanate for cross-linking (Wat1614) is disordered (in TEM-30 it is actually absent). As expected, TEM-76 has decreased kinetic activity, likely due to the replacement of the Ser130 side-chain hydroxyl with a water molecule. In contrast to the recently determined structure of the S130G mutant in the related SHV-1 beta-lactamase, in TEM-76 the key hydrolytic water (Wat1561) is still present. The conservation of similar accommodations among IRT mutants suggests that resistance arises from common mechanisms, despite the disparate locations of the various substitutions.     

An NO Donor Approach to Neuroprotective and Procognitive Estrogen Therapy Overcomes Loss of NO Synthase Function and Potentially Thrombotic Risk

Selective estrogen receptor modulators (SERMs) are effective therapeutics that preserve favorable actions of estrogens on bone and act as antiestrogens in breast tissue, decreasing the risk of vertebral fractures and breast cancer, but their potential in neuroprotective and procognitive therapy is limited by: 1) an increased lifetime risk of thrombotic events; and 2) an attenuated response to estrogens with age, sometimes linked to endothelial nitric oxide synthase (eNOS) dysfunction. Herein, three 3^rd generation SERMs with similar high affinity for estrogen receptors (ERα, ERβ) were studied: desmethylarzoxifene (DMA), FDMA, and a novel NO-donating SERM (NO-DMA). Neuroprotection was studied in primary rat neurons exposed to oxygen glucose deprivation; reversal of cholinergic cognitive deficit was studied in mice in a behavioral model of memory; long term potentiation (LTP), underlying cognition, was measured in hippocampal slices from older 3×Tg Alzheimer''s transgenic mice; vasodilation was measured in rat aortic strips; and anticoagulant activity was compared. Pharmacologic blockade of GPR30 and NOS; denudation of endothelium; measurement of NO; and genetic knockout of eNOS were used to probe mechanism. Comparison of the three chemical probes indicates key roles for GPR30 and eNOS in mediating therapeutic activity. Procognitive, vasodilator and anticoagulant activities of DMA were found to be eNOS dependent, while neuroprotection and restoration of LTP were both shown to be dependent upon GPR30, a G-protein coupled receptor mediating estrogenic function. Finally, the observation that an NO-SERM shows enhanced vasodilation and anticoagulant activity, while retaining the positive attributes of SERMs even in the presence of NOS dysfunction, indicates a potential therapeutic approach without the increased risk of thrombotic events.     

A systematic protocol for the characterization of Hsp90 modulators

Several Hsp90 modulators have been identified including the N-terminal ligand geldanamycin (GDA), the C-terminal ligand novobiocin (NB), and the co-chaperone disruptor celastrol. Other Hsp90 modulators elicit a mechanism of action that remains unknown. For example, the natural product gedunin and the synthetic anti-spermatogenic agent H2-gamendazole, recently identified Hsp90 modulators, manifest biological activity through undefined mechanisms. Herein, we report a series of biochemical techniques used to classify such modulators into identifiable categories. Such studies provided evidence that gedunin and H2-gamendazole both modulate Hsp90 via a mechanism similar to celastrol, and unlike NB or GDA.     

Generating Recombinant Antibodies against Putative Biomarkers of Retinal Injury

Candidate biomarkers, indicative of disease or injury, are beginning to overwhelm the process of validation through immunological means. Recombinant antibodies developed through phage-display offer an alternative means of generating monoclonal antibodies faster than traditional immunization of animals. Peptide segments of putative biomarkers of laser induced injury in the rabbit, discovered through mass spectrometry, were used as targets for a selection against a library of phage-displayed human single-chain variable fragment (scFv) antibodies. Highly specific antibodies were isolated to four of these unique peptide sequences. One antibody against the retinal protein, Guanine Nucleotide-Binding Protein Beta 5 (GBB5), had a dissociation constant ~300 nM and recognized the full-length endogenous protein in retinal homogenates of three different animal species by western blot. Alanine scanning of the peptide target identified three charged and one hydrophobic amino acid as the critical binding residues for two different scFvs. To enhance the utility of the reagent, one scFv was dimerized through a Fragment-crystallizable hinge region (i.e., Fc) and expressed in HEK-293 cells. This dimeric reagent yielded a 25-fold lower detection limit in western blots.     

Rapid microstructural plasticity in the cortical semantic network following a short language learning session

Despite the clear importance of language in our life, our vital ability to quickly and effectively learn new words and meanings is neurobiologically poorly understood. Conventional knowledge maintains that language learning—especially in adulthood—is slow and laborious. Furthermore, its structural basis remains unclear. Even though behavioural manifestations of learning are evident near instantly, previous neuroimaging work across a range of semantic categories has largely studied neural changes associated with months or years of practice. Here, we address rapid neuroanatomical plasticity accompanying new lexicon acquisition, specifically focussing on the learning of action-related language, which has been linked to the brain’s motor systems. Our results show that it is possible to measure and to externally modulate (using transcranial magnetic stimulation (TMS) of motor cortex) cortical microanatomic reorganisation after mere minutes of new word learning. Learning-induced microstructural changes, as measured by diffusion kurtosis imaging (DKI) and machine learning-based analysis, were evident in prefrontal, temporal, and parietal neocortical sites, likely reflecting integrative lexico-semantic processing and formation of new memory circuits immediately during the learning tasks. These results suggest a structural basis for the rapid neocortical word encoding mechanism and reveal the causally interactive relationship of modal and associative brain regions in supporting learning and word acquisition.

This combined neuroimaging and brain stimulation study reveals rapid and distributed microstructural plasticity after a single immersive language learning session, demonstrating the causal relevance of the motor cortex in encoding the meaning of novel action words.     

Sex-biased dispersal and the speed of two-sex invasions

Population models that combine demography and dispersal are important tools for forecasting the spatial spread of biological invasions. Current models describe the dynamics of only one sex (typically females). Such models cannot account for the sex-related biases in dispersal and mating behavior that are typical of many animal species. In this article, we construct a two-sex integrodifference equation model that overcomes these limitations. We derive an explicit formula for the invasion speed from the model and use it to show that sex-biased dispersal may significantly increase or decrease the invasion speed by skewing the operational sex ratio at the invasion's low-density leading edge. Which of these possible outcomes occurs depends sensitively on complex interactions among the direction of dispersal bias, the magnitude of bias, and the relative contributions of females and males to local population growth.