Inna Radzishevsky

Improved antimicrobial peptides based on acyl-lysine oligomers

We describe peptidomimetic oligomers that show rapid, nonhemolytic, broad-spectrum bactericidal properties in mice and do not induce the emergence of resistance. The oligomers contain acyl chains, which prevent the formation of stable secondary structure. This design appears advantageous over conventional antimicrobial peptides with respect to in vivo efficacy and safety, and may provide a convenient platform for the development of peptide antibiotics.

Neuronal d-Serine and Glycine Release Via the Asc-1 Transporter Regulates NMDA Receptor-Dependent Synaptic Activity

d-Serine and glycine are coagonists of NMDA receptors (NMDARs), but their relative contributions for several NMDAR-dependent processes are unclear. We now report that the alanine–serine–cysteine transporter-1 (Asc-1) mediates release of both d-serine and glycine from neurons, and, in turn, this modulates NMDAR synaptic activity. Asc-1 antiporter activity is enhanced by d-isoleucine (d-Ile), which releases d-serine and glycine from Asc-1-transfected cells, primary neuronal cultures, and hippocampal slices. d-Ile has no effect on astrocytes, which do not express Asc-1. We show that d-Ile enhances the long-term potentiation (LTP) in rat and mouse hippocampal CA1 by stimulating Asc-1-mediated endogenous d-serine release. d-Ile effects on synaptic plasticity are abolished by enzymatically depleting d-serine or by using serine racemase knock-out (SR-KO) mice, confirming its specificity and supporting the notion that LTP depends mostly on d-serine release. Conversely, our data also disclose a role of glycine in activating synaptic NMDARs. Although acute enzymatic depletion of d-serine also drastically decreases the isolated NMDAR synaptic potentials, these responses are still enhanced by d-Ile. Furthermore, NMDAR synaptic potentials are preserved in SR-KO mice and are also enhanced by d-Ile, indicating that glycine overlaps with d-serine binding at synaptic NMDARs. Altogether, our results disclose a novel role of Asc-1 in regulating NMDAR-dependent synaptic activity by mediating concurrent non-vesicular release of d-serine and glycine. Our data also highlight an important role of neuron-derived d-serine and glycine, indicating that astrocytic d-serine is not solely responsible for activating synaptic NMDARs.

Effects of Acyl versus Aminoacyl Conjugation on the Properties of Antimicrobial Peptides

ABSTRACT To investigate the importance of increased hydrophobicity at the amino end of antimicrobial peptides, a dermaseptin derivative was used as a template for a systematic acylation study. Through a gradual increase of the acyl moiety chain length, hydrophobicity was monitored and further modulated by acyl conversion to aminoacyl. The chain lengths of the acyl derivatives correlated with a gradual increase in the peptides global hydrophobicity and stabilization of its helical structure. The effect on cytolytic properties, however, fluctuated for different cells. Whereas acylation gradually enhanced hemolysis of human red blood cells and antiprotozoan activity against Leishmania major, bacteria displayed a more complex behavior. The gram-positive organism Staphylococcus aureus was most sensitive to intermediate acyl chains, while longer acyls gradually led to a total loss of activity. All acyl derivatives were detrimental to activity against Escherichia coli, namely, but not solely, because of peptide aggregation. Although aminoacyl derivatives behaved essentially similarly to the nonaminated acyls, they displayed reduced hydrophobicity, and consequently, the long-chain acyls enhanced activity against all microorganisms (e.g., by up to 12-fold for the aminolauryl derivative) but were significantly less hemolytic than their acyl counterparts. Acylation also enhanced bactericidal kinetics and peptide resistance to plasma proteases. The similarities and differences upon acylation of MSI-78 and LL37 are presented and discussed. Overall, the data suggest an approach that can be used to enhance the potencies of acylated short antimicrobial peptides by preventing hydrophobic interactions that lead to self-assembly in solution and, thus, to inefficacy against cell wall-containing target cells.

Feedback inactivation of D-serine synthesis by NMDA receptor-elicited translocation of serine racemase to the membrane

D-serine is a physiological coagonist of N-methyl D-aspartate receptors (NMDARs) that plays a major role in several NMDAR-dependent events. In this study we investigate mechanisms regulating D-serine production by the enzyme serine racemase (SR). We now report that NMDAR activation promotes translocation of SR to the plasma membrane, which dramatically reduces the enzyme activity. Membrane-bound SR isolated from rat brain is not extracted from the membrane by high detergent and salt concentration, indicating a strong association. Colocalization studies indicate that most membrane-bound SR is located at the plasma membrane and dendrites, with much less SR observed in other types of membrane. NMDAR activation promotes translocation of the cytosolic SR to the membrane, resulting in reduced D-serine synthesis, and this effect is averted by blockade of NMDARs. In primary neuronal cultures, SR translocation to the membrane is blocked by a palmitoylation inhibitor, indicating that membrane binding is mediated by fatty acid acylation of SR. In agreement, we found that SR is acylated in transfected neuroblastoma cells using [3H]palmitate or [3H]octanoic acid as precursors. In contrast to classical S-palmitoylation of cysteines, acylation of SR occurs through the formation of an oxyester bond with serine or threonine residues. In addition, we show that phosphorylation of Thr-227 is also required for steady-state binding of SR to the membrane under basal, nonstimulated condition. We propose that the inhibition of D-serine synthesis caused by translocation of SR to the membrane provides a fail-safe mechanism to prevent NMDAR overactivation in vicinal cells or synapses.

Analogous oligo-acyl-lysines with distinct antibacterial mechanisms

Bactericidal properties were recently shown to emerge from hydrophobicity and charge buildup in oligo‐acyl‐lysine (OAK) peptide mimetics. Toward understanding the attributes that govern the activity of this novel antimicrobial system, we compared the functional and mechanistic properties of a known octamer and a newly generated hexamer analog. The data provide strong evidence for multiple similarities that included high tissue stability, low hemolysis, large‐spectrum antibacterial activity in vitro, and the ability to prevent Escherichia coli‐induced mortality in vivo. Despite these similarities, however, the octamer mode of action involved membrane disruption, unlike the hexamer, which acted predominantly through inhibition of DNA functions with characteristically slower bactericidal kinetics. Collectively, the data support the view that the analogous OAKs induced bacterial death by distinct mechanisms and further suggest that relatively minor differences in the sequence of host defense peptides are responsible for selecting one mechanism over another, possibly in conjunction with differential binding affinities to the external and/or cytoplasmic membrane.—Rotem, S., Radzishevsky, I. S., Bourdetsky, D., Navon‐Venezia, S., Carmeli, Y., Mor, A. Analogous oligo‐acyl‐lysines with distinct antibacterial mechanisms. FASEB J. 22, 2652–2661 (2008)

Structure-Activity Relationships of Antibacterial Acyl-Lysine Oligomers

We describe structure-activity relationships that emerged from biophysical data obtained with a library of antimicrobial peptide mimetics composed of 103 oligoacyllysines (OAKs) designed to pin down the importance of hydrophobicity (H) and charge (Q). Based on results obtained with OAKs displaying minimal inhibitory concentration < or = 3 microM, the data indicate that potent inhibitory activity of the gram-negative Escherichia coli and the gram-positive Staphylococcus aureus required a relatively narrow yet distinct window of HQ values where the acyl length played multiple and critical roles, both in molecular organization and in selective activity. Thus, incorporation of long-but not short-acyl chains within a peptide backbone is shown to lead to rigid supramolecular organization responsible for poor antibacterial activity and enhanced hemolytic activity. However, sequence manipulations, including introduction of a tandem lysine motif into the oligomer backbone, enabled disassembly of aggregated OAKs and subsequently revealed tiny, nonhemolytic, yet potent antibacterial derivatives.

Identity of the NMDA receptor coagonist is synapse specific and developmentally regulated in the hippocampus.

Significance NMDA receptors (NMDARs) support patterning and activity of synapses throughout life and are central to many brain disorders. The NMDAR activation requires the concomitant binding of glutamate and a coagonist glycine or d-serine. To date, whether a preference for one coagonist at specific connections occurs remains unsolved. Here, we sought to determine when and where d-serine and glycine enter into play at hippocampal synapses. We demonstrate that the identity of the NMDAR coagonist is synapse specific and developmentally regulated. Remarkably, this segregation coincides with the subunit composition of postsynaptic NMDARs and the maturation of the tripartite synapse. These results point out the importance of the spatial and temporal switch in coagonist identity for therapeutic interventions aimed at treating deficits in NMDAR activity. NMDA receptors (NMDARs) require the coagonists d-serine or glycine for their activation, but whether the identity of the coagonist could be synapse specific and developmentally regulated remains elusive. We therefore investigated the contribution of d-serine and glycine by recording NMDAR-mediated responses at hippocampal Schaffer collaterals (SC)–CA1 and medial perforant path–dentate gyrus (mPP–DG) synapses in juvenile and adult rats. Selective depletion of endogenous coagonists with enzymatic scavengers as well as pharmacological inhibition of endogenous d-amino acid oxidase activity revealed that d-serine is the preferred coagonist at SC–CA1 mature synapses, whereas, unexpectedly, glycine is mainly involved at mPP–DG synapses. Nevertheless, both coagonist functions are driven by the levels of synaptic activity as inferred by recording long-term potentiation generated at both connections. This regional compartmentalization in the coagonist identity is associated to different GluN1/GluN2A to GluN1/GluN2B subunit composition of synaptic NMDARs. During postnatal development, the replacement of GluN2B- by GluN2A-containing NMDARs at SC–CA1 synapses parallels a change in the identity of the coagonist from glycine to d-serine. In contrast, NMDARs subunit composition at mPP–DG synapses is not altered and glycine remains the main coagonist throughout postnatal development. Altogether, our observations disclose an unprecedented relationship in the identity of the coagonist not only with the GluN2 subunit composition at synaptic NMDARs but also with astrocyte activity in the developing and mature hippocampus that reconciles the complementary functions of d-serine and glycine in modulating NMDARs during the maturation of tripartite glutamatergic synapses.

The serine shuttle between glia and neurons: implications for neurotransmission and neurodegeneration

D-Serine is a physiological co-agonist of NMDARs (N-methyl-D-aspartate receptors) required for neurotransmission, synaptic plasticity and neurotoxicity. There is no consensus, however, on the relative roles of neurons and astrocytes in D-serine signalling. The effects of D-serine had been attributed to its role as a gliotransmitter specifically produced and released by astrocytes. In contrast, recent studies indicate that neurons regulate their own NMDARs by releasing D-serine via plasma membrane transporters and depolarization-sensitive pathways. Only a minority of astrocytes contain authentic D-serine, whereas neuronal D-serine accounts for up to 90% of the total D-serine pool. Neuronal and glial D-serine production requires astrocytic L-serine generated by a 3-phosphoglycerate dehydrogenase-dependent pathway. These findings support a model whereby astrocyte-derived L-serine shuttles to neurons to fuel the synthesis of D-serine by serine racemase. We incorporate these new findings in a revised model of serine dynamics, called the glia-neuron serine shuttle, which highlights the role of glia-neuron cross-talk for optimal NMDAR activity and brain development.

In vivo fate and therapeutic efficacy of PF-4/CTF microspheres in an orthotopic human glioblastoma model

The correlation between glioma grade and angiogenesis suggests that antiangiogenic therapies are potentially therapeutically effective for these tumors. However, to achieve tumor suppression, antiangiogenic therapies need to be administered daily using high systemic quantities. We designed a biodegradable polymeric device that overcomes those barriers by providing sustained local delivery of a C‐terminal fragment of platelet factor 4 (PF‐4/CTF), an antiangiogenic agent. Fluorescent‐labeled microspheres composed of poly lactic‐coglycolic acid (PLGA) were loaded with rhodamine‐labeled PF‐4/CTF and formulated to release their contents over time. Fluorescent labeling enabled the correlation between the in vitro to the in vivo kinetic and release studies. PF‐4/CTF microspheres were injected into established intracranial human glioma tumors in nude mice. Noninvasive magnetic resonance imaging (MRI) was used to assess the therapeutic response. Tumor size, microvessel density, proliferation, and apoptosis rate were measured by histological analysis. Intracranially, the microspheres were located throughout the tumor bed and continuously released PF‐4/CTF during the entire experimental period. MRI and histological studies showed that a single injection of microspheres containing PF‐4/CTF caused a 65.2% and 72% reduction in tumor volume, respectively, with a significant decrease in angiogenesis and an increase in apoptosis. Our data demonstrate that polymeric microspheres are an effective therapeutic approach for delivering antiangiogenic agents that result in the inhibition of glioma tumor growth.—Benny, O., Kim, S. K., Gvili, K., Radzishevsky, I. S., Mor, A., Verduzco, L., Menon, L. G., Black, P. M., Machluf, M., Carroll, R.S. In vivo fate and therapeutic efficacy of PF‐4/CTF microspheres in an orthotopic human glioblastoma model. FASEB J. 22, 488–499 (2008)

Effect of peptides bearing nuclear localization signals on therapeutic ultrasound mediated gene delivery

One of the major limitations of nonviral gene delivery methods is nuclear transport of plasmid DNA (pDNA). Peptides bearing nuclear localization signal (NLS) were shown to mediate nuclear import of macromolecules. We have explored the use of cell‐permeable peptides (CPP) bearing NLS sequences to enhance transfection mediated by a nonviral approach: therapeutic ultrasound (TUS).