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Caveolae are plasma membrane invaginations that are enriched in cholesterol-binding proteins called caveolins. The presence of caveolae and caveolins in mixed cultures of human neurons and glia has not been investigated. Here, we sought to determine the presence of caveolae and caveolins in human NTera-2 (NT2/D1) cells, differentiated with retinoic acid into neuron-like (NT2/N) and astrocyte-like (NT2/A) cells. We found that while caveolin-3 mRNA levels remained relatively constant, caveolin-1 and -2 levels were upregulated in NT2/A and downregulated in NT2/N. No caveolin-1 immunoreactivity was detected in NT2/N. Electron microscopy revealed numerous flask-shaped invaginations (~86–102 nm in diameter) in the plasma membrane of NT2/A and NT2/N cells, while only few were detected in NT2/D1 cells. Immunoelectron microscopy localized caveolin-1 gold particles in the flask-shaped structures on plasmalemma and cytoplasmic vesicles of NT2/A cells. Furthermore, NT2/A endocytosed Alexa 488 conjugated-cholera toxin B subunit (CTX-B) through a caveolae- and clathrin-dependent pathway, whereas NT2/N endocytosed CTX-B through a caveolae-independent pathway. We have established that while NT2/A expressed functional caveolae, the molecular identity of the plasma membrane invaginations in NT2/N is unknown. The expression of caveolin proteins was differentially regulated in these cells. Taken together, our findings support the usefulness of the human NT2 model system to study the role of caveolins in neuron–glia communication, and their involvement in brain health and disease.

The identification of molecular biomarkers in CSF from individuals affected by Huntington disease may help improve predictions of disease onset, better define disease progression and could facilitate the evaluation of potential therapies. The primary objective of our study was to investigate novel CSF protein candidates and replicate previously reported protein biomarker changes in CSF from Huntington disease mutation carriers and healthy controls. Our secondary objective was to compare the discriminatory potential of individual protein analytes and combinations of CSF protein markers for stratifying individuals based on the severity of Huntington disease. We conducted a hypothesis-driven analysis of 26 pre-specified protein analytes in CSF from 16 manifest Huntington disease subjects, eight premanifest Huntington disease mutation carriers and eight healthy control individuals using parallel-reaction monitoring mass spectrometry. In addition to reproducing reported changes in previously investigated CSF biomarkers (NEFL, PDYN, and PENK), we also identified novel exploratory CSF proteins (C1QB, CNR1, GNAL, IDO1, IGF2, and PPP1R1B) whose levels were altered in Huntington disease mutation carriers and/or across stages of disease. Moreover, we report strong associations of select CSF proteins with clinical measures of disease severity in manifest Huntington disease subjects (C1QB, CNR1, NEFL, PDYN, PPP1R1B, and TTR) and with years to predicted disease onset in premanifest Huntington disease mutation carriers (ALB, C4B, CTSD, IGHG1, and TTR). Using receiver operating characteristic curve analysis, we identified PENK as being the most discriminant CSF protein for stratifying Huntington disease mutation carriers from controls. We also identified exploratory multi-marker CSF protein panels that improved discrimination of premanifest Huntington disease mutation carriers from controls (PENK, ALB and NEFL), early/mid-stage Huntington disease from premanifest mutation carriers (PPP1R1B, TTR, CHI3L1, and CTSD), and late-stage from early/mid-stage Huntington disease (CNR1, PPP1R1B, BDNF, APOE, and IGHG1) compared with individual CSF proteins. In this study, we demonstrate that combinations of CSF proteins can outperform individual markers for stratifying individuals based on Huntington disease mutation status and disease severity. Moreover, we define exploratory multi-marker CSF protein panels that, if validated, may be used to improve the accuracy of disease-onset predictions, complement existing clinical and imaging biomarkers for monitoring the severity of Huntington disease, and potentially for assessing therapeutic response in clinical trials. Additional studies with CSF collected from larger cohorts of Huntington disease mutation carriers are needed to replicate these exploratory findings.

INTRODUCTION Antisense oligonucleotides (ASOs) have emerged as a promising novel drug modality that aims to address unmet medical needs. A record of six ASO drugs have been approved since 2016, and more candidates are in clinical development. ASOs are the most advanced class within the RNA-based therapeutics field. AREAS COVERED This review highlights the two major backbones that are currently used to build the most advanced ASO platforms - the phosphorodiamidate morpholino oligomers (PMOs) and the phosphorothioates (PSs). The absorption, distribution, metabolism, and excretion (ADME) properties of the PMO and PS platforms are discussed in detail. EXPERT OPINION Understanding the ADME properties of existing ASOs can foster further improvement of this cutting-edge therapy, thereby enabling researchers to safely develop ASO drugs and enhancing their ability to innovate. ABBREVIATIONS 2'-MOE, 2'-O-methoxyethyl; 2'PS, 2 modified PS; ADME, absorption, distribution, metabolism, and excretion; ASO, antisense oligonucleotide; AUC, area under the curve; BNA, bridged nucleic acid; CPP, cell-penetrating peptide; CMV, cytomegalovirus; CNS, central nervous system; CYP, cytochrome P; DDI, drug-drug interaction; DMD, Duchenne muscular dystrophy; FDA, Food and Drug Administration; GalNAc3, triantennary N-acetyl galactosamine; IT, intrathecal; IV, intravenous; LNA, locked nucleic acid; mRNA, messenger RNA; NA, not applicable; PBPK, physiologically based pharmacokinetics; PD, pharmacodynamic; PK, pharmacokinetic; PMO, phosphorodiamidate morpholino oligomer; PMOplus, PMOs with positionally specific positive molecular charges; PPMO, peptide-conjugated PMO; PS, phosphorothioate; SC, subcutaneous; siRNA, small-interfering RNA; SMA, spinal muscular atrophy.

This study aimed to evaluate the feasibility and accuracy of a technique for atherosclerosis imaging using local delivery of relatively small quantities (0.04–0.4 mg/kg) of labeled-specific imaging tracers targeting ICAM-1 and unpolymerized type I collagen or negative controls in 13 rabbits with atheroma induced by balloon injury in the abdominal aorta and a 12-week high-cholesterol diet. Immediately after local infusion, in vivo intravascular ultrasonography (IVUS)-NIRF imaging was performed at different time-points over a 40-minute period. The in vivo peak NIRF signal was significantly higher in the molecular tracer-injected rabbits than in the control-injected animals (P Ex vivo peak NIRF signal was significantly higher in the ICAM-1 probe-injected rabbits than in controls (P = 0.04), but not in the collagen probe-injected group (P = 0.29). NIRF signal discrimination following dual-probe delivery was also shown to be feasible in a single animal and thus offers the possibility of combining several distinct biological imaging agents in future studies. This innovative imaging strategy using in vivo local delivery of low concentrations of labeled molecular tracers followed by IVUS-NIRF catheter-based imaging holds potential for detection of vulnerable human coronary artery plaques.

Optical imaging offers high sensitivity and portability at low cost. The design of 'smart' or 'activatable' probes can decrease the background noise and increase the specificity of the signal. By conjugating a fluorescent dye and a compatible quencher on each side of an enzyme's substrate, the signal remains in its 'off ' state until it reaches the area where a specific enzyme is expressed. However, the signal can leak from that area unless the dye is attached to a molecule able to bind to a specific target also presented in that area. The aim of this study was to (i) specifically conjugate the quencher on the α-amino group of the peptide's N-terminus, (ii) conjugate the dye on the e-amino group of a lysine in C-terminus, and (iii) conjugate the carboxyl group of the peptide's C-terminus to an amino group present on an antibody, using carbodiimide chemistry. The use of protecting groups, such as Boc or Fmoc, to allow site-specific conjugation, presents several drawbacks including 'on beads labeling', additional steps required for deprotection and removal from the resin, decreased yield, and dye degradation. A method of preferential labeling of α-amino N-terminal group in slightly acidic solution, proposed by Selo et al. (1996) has partially solved the problem. The present study reports improvements of the method allowing to (i) avoid the homo-bilabeling, (ii) increase the yield of the N-terminal labeling by two folds, and (iii) decrease the cost by 44-fold. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

This article was updated to correct the spelling of B. Gino Fallone's name; it is correct as displayed above. Correction to: Mol Imaging Biol (2017). DOI: https://doi.org/10.1007/s11307-017-1140-4.

Cerebrovascular inflammation is often involved in the pathogenesis of neurodegenerative disorders such as Alzheimer's disease (AD). Non-invasive and sensitive molecular imaging of cerebrovascular inflammation biomarkers therefore represents a potential AD diagnostic and therapeutic monitoring method. Here, we describe the development of a novel aptamer-based near infrared fluorescence imaging probe targeting Vascular Cell Adhesion Molecule-1 (VCAM-1), an adhesion molecule overexpressed by the activated cerebrovasculature during inflammation. A SELEX-type screening of a random ssDNA library against human VCAM-1 identified a high-affinity ssDNA aptamer with a dissociation constant of 49 nM. We demonstrated that the Cy5.5-labeled aptamer binds to activated endothelial cells, with no affinity to non-activated cells. A scrambled aptamer labeled with Cy5.5 did not image activated and non-activated endothelial cells, confirming the sequence specificity of the targeting. In vivo, the aptameric imaging agent targeting VCAM-1 successfully identified inflammation associated with amyloid-β plaques deposition in the vessels of the cerebellum of transgenic AD mice. It exhibited excellent retention by remaining bound to vessels 4 hours post-injection, indicating its effectiveness in in vivo imaging and its potential in early detection of cerebrovascular inflammation.

Diagnosis of glioblastoma multiform (GBM) with MRI lacks molecular information and requires a biopsy for pathologic confirmation. The EGFRvIII, is a constitutively active mutant of the EGF receptor, identified in a high percentage of brain cancers and associated with increased invasiveness and resistance, making it a good target to improve imaging and diagnosis. The present study shows that conjugation of near-infrared quantum dot (Qd800) to an anti-EGFRvIII single domain antibody, made of the variable region with an extra cysteine for site-specific conjugation (EG2-Cys), increased its internalization in U87MG-EGFRvIII cells in vitro compared to Qd800 conjugated with the Fc region of the antibody (EG2-hFc) or unconjugated. EG2-Cys also improved the contrast in Near-Infrared Imaging of mice bearing orthotopic glioblastoma. The increased accumulation was confirmed by fluorescence microscopy of brain sections. The specificity of EG2-Cys in brain tumor expressing the EGFRvIII mutant receptor may provide an accurate less invasive diagnosis and determine the level of tumor aggressiveness and resistance.

We report our newly developed low-temperature synthesis of colloidal photoluminescent (PL) CuInS2 nanocrystals (NCs) and their in vitro and in vivo imaging applications. With diphenylphosphine sulphide (SDPP) as a S precursor made from elemental S and diphenylphosphine, this is a noninjection based approach in 1-dodecanethiol (DDT) with excellent synthetic reproducibility and large-scale capability. For a typical synthesis with copper iodide (CuI) as a Cu source and indium acetate (In(OAc)3) as an In source, the growth temperature was as low as 160 °C and the feed molar ratios were 1Cu-to-1In-to-4S. Amazingly, the resulting CuInS2 NCs in toluene exhibit quantum yield (QY) of ~23% with photoemission peaking at ~760 nm and full width at half maximum (FWHM) of ~140 nm. With a mean size of ~3.4 nm (measured from the vertices to the bases of the pyramids), they are pyramidal in shape with a crystal structure of tetragonal chalcopyrite. In situ (31)P NMR (monitored from 30 °C to 100 °C) and in situ absorption at 80 °C suggested that the Cu precursor should be less reactive toward SDPP than the In precursor. For our in vitro and in vivo imaging applications, CuInS2/ZnS core-shell QDs were synthesized; afterwards, dihydrolipoic acid (DHLA) or 11-mercaptoundecanoic acid (MUA) were used for ligand exchange and then bio-conjugation was performed. Two single-domain antibodies (sdAbs) were used. One was 2A3 for in vitro imaging of BxPC3 pancreatic cancer cells. The other was EG2 for in vivo imaging of a Glioblastoma U87MG brain tumour model. The bioimaging data illustrate that the CuInS2 NCs from our SDPP-based low-temperature noninjection approach are good quality.

Atherosclerotic cardiovascular diseases are characterized by the formation of a plaque in the arterial wall. Intravascular ultrasound (IVUS) provides high-resolution images allowing delineation of atherosclerotic plaques. When combined with near infrared fluorescence (NIRF), the plaque can also be studied at a molecular level with a large variety of biomarkers. In this work, we present a system enabling automated volumetric histology imaging of excised aortas that can spatially correlate results with combined IVUS/NIRF imaging of lipid-rich atheroma in cholesterol-fed rabbits. Pullbacks in the rabbit aortas were performed with a dual modality IVUS/NIRF catheter developed by our group. Ex vivo three-dimensional (3D) histology was performed combining optical coherence tomography (OCT) and confocal fluorescence microscopy, providing high-resolution anatomical and molecular information, respectively, to validate in vivo findings. The microscope was combined with a serial slicer allowing for the imaging of the whole vessel automatically. Colocalization of in vivo and ex vivo results is demonstrated. Slices can then be recovered to be tested in conventional histology.

New and effective therapeutics that cross the blood-brain barrier (BBB) are critically needed for treatment of many brain diseases. We characterize here a novel drug development platform that is broadly applicable for the development of new therapeutics with increased brain penetration. The platform is based on the Angiopep-2 peptide, a sequence derived from ligands that bind to low-density lipoprotein receptor-related protein-1 (LRP-1), a receptor expressed on the BBB. Fluorescent imaging studies of a Cy5.5Angiopep-2 conjugate and immunohistochemical studies of injected Angiopep-2 in mice demonstrated efficient transport across the BBB into brain parenchyma and subsequent co-localization with the neuronal nuclei-selective marker NeuN and the glial marker glial fibrillary acidic protein (GFAP). Uptake of [125I]-Angiopep-2 into brain endothelial cells occurred by a saturable mechanism involving LRP-1. The primary sequence and charge of Angiopep-2 were crucial for its passage across the BBB. Overall, the results demonstrate the significant potential of this platform for the development of novel neurotherapeutics.

Background and purpose: The overexpression of epidermal growth factor receptor (EGFR) and its mutated variant EGFRvIII occurs in 50% of glioblastoma multiforme. We developed antibody fragments against EGFR/EGFRvIII for molecular imaging and/or therapeutic targeting applications. Experimental approach: An anti–EGFR/EGFRvIII llama single-domain antibody (EG2) and two higher valency format constructs, bivalent EG2-hFc and pentavalent V2C-EG2 sdAbs, were analysed in vitro for their binding affinities using surface plasmon resonance and cell binding studies, and in vivo using pharmacokinetic, biodistribution, optical imaging and fluorescent microscopy studies. Key results: Kinetic binding analyses by surface plasmon resonance revealed intrinsic affinities of 55 nM and 97 nM for the monovalent EG2 to immobilized extracellular domains of EGFR and EGFRvIII, respectively, and a 10- to 600-fold increases in apparent affinities for the multivalent binders, V2C-EG2 and EG2-hFc, respectively. In vivo pharmacokinetic and biodistribution studies in mice revealed plasma half-lives for EG2, V2C-EG2 and EG2-hFc of 41 min, 80 min and 12.5 h, respectively, as well as a significantly higher retention of EG2-hFc compared to the other two constructs in EGFR/EGFRvIII-expressing orthotopic brain tumours, resulting in the highest signal in the tumour region in optical imaging studies. Time domain volumetric optical imaging fusion with high-resolution micro-computed tomography of microvascular brain network confirmed EG2-hFc selective accumulation/retention in anatomically defined tumour regions. Conclusions: Single domain antibodies can be optimized for molecular imaging applications by methods that improve their apparent affinity and prolong plasma half-life and, at the same time, preserve their ability to penetrate tumour parenchyma.

The development of some solid tumors is associated with overexpression of the epidermal growth factor receptor (EGFR) and often correlates with poor prognosis. Near field scanning optical microscopy, a technique with subdiffraction-limited optical resolution, was used to examine the influence of two inhibitors (the chimeric 225 antibody and tyrosine phosphorylation inhibitor AG1478) on the nanoscale clustering of EGFR in HeLa cells. The EGFR is organized in small clusters, average diameter of 150 nm, on the plasma membrane for both control and EGF-treated cells. The numbers of receptors in individual clusters vary from as few as one or two proteins to greater than 100. Both inhibitors yield an increased cluster density and an increase in the fraction of clusters with smaller diameters and fewer receptors. Exposure to AG1478 also decreases the fraction of EGFR that colocalizes with both rafts and caveolae. EGF stimulation results in a significant loss of the full-length EGFR from the plasma membrane with the concomitant appearance of low molecular mass proteolytic products. By contrast, AG1478 reduces the level of EGFR degradation. Changes in receptor clustering provide one mechanism for regulating EGFR signaling and are relevant to the design of strategies for therapeutic interventions based on modulating EGFR signaling.