In vivo imaging of injured cortical axons reveals a rapid onset form of Wallerian degeneration.

BACKGROUND: Despite the widespread occurrence of axon and synaptic loss in the injured and diseased nervous system, the cellular and molecular mechanisms of these key degenerative processes remain incompletely understood. Wallerian degeneration (WD) is a tightly regulated form of axon loss after injury, which has been intensively studied in large myelinated fibre tracts of the spinal cord, optic nerve and peripheral nervous system (PNS). Fewer studies, however, have focused on WD in the complex neuronal circuits of the mammalian brain, and these were mainly based on conventional endpoint histological methods. Post-mortem analysis, however, cannot capture the exact sequence of events nor can it evaluate the influence of elaborated arborisation and synaptic architecture on the degeneration process, due to the non-synchronous and variable nature of WD across individual axons. RESULTS: To gain a comprehensive picture of the spatiotemporal dynamics and synaptic mechanisms of WD in the nervous system, we identify the factors that regulate WD within the mouse cerebral cortex. We combined single-axon-resolution multiphoton imaging with laser microsurgery through a cranial window and a fluorescent membrane reporter. Longitudinal imaging of > 150 individually injured excitatory cortical axons revealed a threshold length below which injured axons consistently underwent a rapid-onset form of WD (roWD). roWD started on average 20 times earlier and was executed 3 times slower than WD described in other regions of the nervous system. Cortical axon WD and roWD were dependent on synaptic density, but independent of axon complexity. Finally, pharmacological and genetic manipulations showed that a nicotinamide adenine dinucleotide (NAD+)-dependent pathway could delay cortical roWD independent of transcription in the damaged neurons, demonstrating further conservation of the molecular mechanisms controlling WD in different areas of the mammalian nervous system. CONCLUSIONS: Our data illustrate how in vivo time-lapse imaging can provide new insights into the spatiotemporal dynamics and synaptic mechanisms of axon loss and assess therapeutic interventions in the injured mammalian brain.

In vivo whole brain, cellular and molecular imaging in nonhuman primate models of neuropathology.

Rodents have been the principal model to study brain anatomy and function due to their well-mapped brain architecture, rapid reproduction and amenability to genetic modification. However, there are clear limitations, for example their simpler neocortex, necessitating the need to adopt a model that is closer to humans in order to understand human cognition and brain conditions. Nonhuman primates (NHPs) are ideally suited as they are our closest relatives in the animal kingdom but in vivo imaging technologies to study brain structure and function in these species can be challenging. With the surge in NHP research in recent years, scientists have begun adapting imaging technologies, such as two-photon microscopy, for these species. Here we review the various NHP models that exist as well as their use in advanced microscopic and mesoscopic studies. We discuss the challenges in the field and investigate the opportunities that lie ahead.

Bacurd1/Kctd13 and Bacurd2/Tnfaip1 are interacting partners to Rnd proteins which influence the long-term positioning and dendritic maturation of cerebral cortical neurons.

BACKGROUND: The development of neural circuits within the embryonic cerebral cortex relies on the timely production of neurons, their positioning within the embryonic cerebral cortex as well as their terminal differentiation and dendritic spine connectivity. The RhoA GTPases Rnd2 and Rnd3 are important for neurogenesis and cell migration within the embryonic cortex (Nat Commun 4:1635, 2013), and we recently identified the BTB/POZ domain-containing Adaptor for Cul3-mediated RhoA Degradation family member Bacurd2 (also known as Tnfaip1) as an interacting partner to Rnd2 for the migration of embryonic mouse cortical neurons (Neural Dev 10:9, 2015). FINDINGS: We have extended this work and report that Bacurd1/Kctd13 and Bacurd2/Tnfaip1 are interacting partners to Rnd2 and Rnd3 in vitro. Given that these genes are expressed during cortical development, we performed a series of in utero electroporation studies in mice and found that disruptions to Bacurd1/Kctd13 or Bacurd2/Tnfaip1 expression impair the long-term positioning of E14.5-born cortical neurons within the postnatal (P17) mouse cerebral cortex. We also find that forced expression of Bacurd1/Kctd13 and Bacurd2/Tnfaip1 alters the branching and dendritic spine properties of layer II/III projection neurons. CONCLUSIONS: We identify Bacurd1/Kctd13 and Bacurd2/Tnfaip1 as interacting partners to Rnd proteins which influence the development of cortical neurons. Their neurodevelopmental functions are likely to be relevant to human brain development and disease.

Laser-Mediated Microlesions in Mouse Neocortex to Investigate Neuronal Degeneration and Regeneration.

In vivo two-photon (2P) imaging enables neural circuitry to be repeatedly visualized in both normal conditions and following trauma. This protocol describes how laser-mediated neuronal microlesions can be created in the cerebral cortex using an ultrafast laser without causing a significant inflammatory reaction or compromising the blood-brain barrier. Furthermore, directives are provided for the acute and chronic in vivo imaging of the lesion site, as well as for post-hoc analysis of the lesion site in fixed tissue, which can be correlated with the live imaging phase.

In vivo single branch axotomy induces GAP-43-dependent sprouting and synaptic remodeling in cerebellar cortex.

Plasticity in the central nervous system in response to injury is a complex process involving axonal remodeling regulated by specific molecular pathways. Here, we dissected the role of growth-associated protein 43 (GAP-43; also known as neuromodulin and B-50) in axonal structural plasticity by using, as a model, climbing fibers. Single axonal branches were dissected by laser axotomy, avoiding collateral damage to the adjacent dendrite and the formation of a persistent glial scar. Despite the very small denervated area, the injured axons consistently reshape the connectivity with surrounding neurons. At the same time, adult climbing fibers react by sprouting new branches through the intact surroundings. Newly formed branches presented varicosities, suggesting that new axons were more than just exploratory sprouts. Correlative light and electron microscopy reveals that the sprouted branch contains large numbers of vesicles, with varicosities in the close vicinity of Purkinje dendrites. By using an RNA interference approach, we found that downregulating GAP-43 causes a significant increase in the turnover of presynaptic boutons. In addition, silencing hampers the generation of reactive sprouts. Our findings show the requirement of GAP-43 in sustaining synaptic stability and promoting the initiation of axonal regrowth.

Increased axonal bouton dynamics in the aging mouse cortex.

Aging is a major risk factor for many neurological diseases and is associated with mild cognitive decline. Previous studies suggest that aging is accompanied by reduced synapse number and synaptic plasticity in specific brain regions. However, most studies, to date, used either postmortem or ex vivo preparations and lacked key in vivo evidence. Thus, whether neuronal arbors and synaptic structures remain dynamic in the intact aged brain and whether specific synaptic deficits arise during aging remains unknown. Here we used in vivo two-photon imaging and a unique analysis method to rigorously measure and track the size and location of axonal boutons in aged mice. Unexpectedly, the aged cortex shows circuit-specific increased rates of axonal bouton formation, elimination, and destabilization. Compared with the young adult brain, large (i.e., strong) boutons show 10-fold higher rates of destabilization and 20-fold higher turnover in the aged cortex. Size fluctuations of persistent boutons, believed to encode long-term memories, also are larger in the aged brain, whereas bouton size and density are not affected. Our data uncover a striking and unexpected increase in axonal bouton dynamics in the aged cortex. The increased turnover and destabilization rates of large boutons indicate that learning and memory deficits in the aged brain arise not through an inability to form new synapses but rather through decreased synaptic tenacity. Overall our study suggests that increased synaptic structural dynamics in specific cortical circuits may be a mechanism for age-related cognitive decline.

Nanobodies®: proficient tools in diagnostics.

With the advent of new antibody engineering technologies, conventional antibodies have been minimized into smaller antibody formats. Small size is an important advantage for current and future diagnostic development. Nanobodies® (Ablynx) are among the smallest known antigen-binding antibody fragments, and are derived from the heavy-chain only antibodies that occur naturally in the serum of Camelidae. Endowed by natural evolution, these Nanobodies inherently exhibit unique biophysical, biochemical and pharmacological characteristics. In addition to their excellent potential as molecules in drug development, Nanobodies possess very attractive functional properties that aid in their development for diagnostic tools. Here we present several examples of currently available applications of Nanobodies to the field of immunosensor for cancer, immunoaffinity chromatography, in vivo and intracellular imaging.

Targeting the EGF receptor ectodomain in the context of cancer.

With the discovery of the involvement of the ErbB family of transmembrane growth factor receptors in tumour malignancy, major efforts have been undertaken to develop agents able to specifically target these receptors. With varying success, these agents have been applied to either detect the presence of ErbB receptors on cancer cells or to neutralize receptor function in order to put a hold on the unbridled tumour growth. The two most exploited classes of ErbB-targeting agents are monoclonal antibodies binding the extracellular portion of the receptor and small molecules able to interfere with the intracellular tyrosine kinase activity. Here we focus on the various kinds of agents that have recently been developed to target the extracellular region of the EGFR, the best characterised member of the ErbB family. Because clinical successes are relatively poor, alternative but less developed approaches for receptor targeting are being evaluated. Much effort has been put into the development of smaller antibody fragments. In this context, EGFR-binding nanobodies and affibodies may prove to be a more efficient novel approach in targeting EGFR-positive tumours for therapeutic and diagnostic use.

Comparison of the biodistribution and tumor targeting of two 99mTc-labeled anti-EGFR nanobodies in mice, using pinhole SPECT/micro-CT.

UNLABELLED: Camelidae possess an unusual class of antibodies devoid of light chains. Nanobodies are intact antigen-binding fragments that are stable, easily generated against different targets, and fully functional. Their rapid clearance from the blood circulation favors their use as imaging agents. We compared the in vivo tumor uptake and biodistribution of 2 anti-epidermal growth factor receptor (anti-EGFR) Nanobodies, (99m)Tc-7C12 and (99m)Tc-7D12. METHODS: Nanobodies were labeled via their hexahistidine tail with (99m)Tc-tricarbonyl ((99m)Tc(CO)(3)) generated from a kit. Mice bearing subcutaneous A431 (EGFR-positive) and R1M (EGFR-negative) xenografts were intravenously injected with (99m)Tc-7C12 and (99m)Tc-7D12 on separate days. Pinhole SPECT/micro-CT images were acquired at 1 h after administration to assess noninvasively the biodistribution and tumor targeting of the labeled compounds. Pinhole SPECT and micro-CT images from the same mouse were automatically fused on the basis of a mathematic rigid-body-transformation algorithm using six (57)Co sources. Images were quantified, and tracer uptake was expressed as percentage injected activity per gram per cubic centimeter (%IA/cm(3)) of tissue. Ex vivo biodistribution of mice bearing A431 injected with either (99m)Tc-7C12 or (99m)Tc-7D12 was also assessed; activity in the tumor and organs was recorded and expressed as percentage injected activity per gram (%IA/g). RESULTS: Binding of both tracers was receptor-specific. Image analysis showed high and similar tumor uptake values for both (99m)Tc-7C12 and (99m)Tc-7D12 (4.55+/-0.24 %IA/cm(3) and 4.62+/-0.36 %IA/cm(3), respectively) in A431 xenografts, whereas the uptake in the negative tumor (R1M) was low (1.16+/-0.14 for (99m)Tc-7C12 and 1.49+/-0.60 for (99m)Tc-7D12). (99m)Tc-7C12 showed significantly higher kidney uptake (63.48+/-2.36 vs. 56.25+/-2.46 %IA/cm(3)) and lower liver uptake (2.55+/-0.26 vs. 4.88+/-0.86 %IA/cm(3)) than did (99m)Tc-7D12. The ex vivo analysis confirmed the image quantification with high tumor-to-background ratio; however, (99m)Tc-7C12 showed higher tumor uptake (9.11+/-1.12 %IA/g) than did (99m)Tc-7D12 (6.09+/-0.77 %IA/g). (99m)Tc-7D12 demonstrated significantly higher blood activity than did (99m)Tc-7C12, but both showed short plasma half-lives (<10 min). CONCLUSION: The Nanobody fragments used here show high tumor uptake, low liver uptake, and rapid blood clearance. Nanobodies are promising probes for noninvasive radioimmunodetection of specific targets early after administration. On the basis of its favorable biodistribution, (99m)Tc-7C12 was selected for further studies.

SPECT imaging with 99mTc-labeled EGFR-specific nanobody for in vivo monitoring of EGFR expression.

PURPOSE: Overexpression of the epidermal growth factor receptor (EGFR) occurs with high incidence in various carcinomas. The oncogenic expression of the receptor has been exploited for immunoglobulin-based diagnostics and therapeutics. We describe the use of a llama single-domain antibody fragment, termed Nanobody, for the in vivo radioimmunodetection of EGFR overexpressing tumors using single photon emission computed tomography (SPECT) in mice. METHODS: Fluorescence-activated cell sorting (FACS) analysis was performed to evaluate the specificity and selectivity of 8B6 Nanobody to bind EGFR on EGFR overexpressing cells. The Nanobody was then labeled with (99m)Tc via its C-terminal histidine tail. Uptake in normal organs and tissues was assessed by ex vivo analysis. In vivo tumor targeting of (99m)Tc-8B6 Nanobody was evaluated via pinhole SPECT in mice bearing xenografts of tumor cells with either high (A431) or moderate (DU145) overexpression of EGFR. RESULTS: FACS analysis indicated that the 8B6 Nanobody only recognizes cells overexpressing EGFR. In vivo blood clearance of (99m)Tc-8B6 Nanobody is relatively fast (half-life, 1.5 h) and mainly via the kidneys. At 3 h postinjection, total kidney accumulation is high (46.6+/-0.9%IA) compared to total liver uptake (18.9+/-0.6%IA). Pinhole SPECT imaging of mice bearing A431 xenografts showed higher average tumor uptake (5.2+/-0.5%IA/cm(3)) of (99m)Tc-8B6 Nanobody compared to DU145 xenografts (1.8+/-0.3%IA/cm(3), p<0.001). CONCLUSION: The EGFR-binding Nanobody investigated in this study shows high specificity and selectivity towards EGFR overexpressing cells. Pinhole SPECT analysis with (99m)Tc-8B6 Nanobody enabled in vivo discrimination between tumors with high and moderate EGFR overexpression. The favorable biodistribution further corroborates the suitability of Nanobodies for in vivo tumor imaging.

Antibody Fragments as Probe in Biosensor Development.

Today's proteomic analyses are generating increasing numbers of biomarkers, making it essential to possess highly specific probes able to recognize those targets. Antibodies are considered to be the first choice as molecular recognition units due to their target specificity and affinity, which make them excellent probes in biosensor development. However several problems such as difficult directional immobilization, unstable behavior, loss of specificity and steric hindrance, may arise from using these large molecules. Luckily, protein engineering techniques offer designed antibody formats suitable for biomarker analysis. Minimization strategies of antibodies into Fab fragments, scFv or even single-domain antibody fragments like VH, VL or VHHs are reviewed. Not only the size of the probe but also other issues like choice of immobilization tag, type of solid support and probe stability are of critical importance in assay development for biosensing. In this respect, multiple approaches to specifically orient and couple antibody fragments in a generic one-step procedure directly on a biosensor substrate are discussed.

Efficient inhibition of EGFR signaling and of tumour growth by antagonistic anti-EFGR Nanobodies.

The development of a number of different solid tumours is associated with over-expression of ErbB1, or the epidermal growth factor receptor (EGFR), and this over-expression is often correlated with poor prognosis of patients. Therefore, this receptor tyrosine kinase is considered to be an attractive target for antibody-based therapy. Indeed, antibodies to the EGFR have already proven their value for the treatment of several solid tumours, especially in combination with chemotherapeutic treatment regimens. Variable domains of camelid heavy chain-only antibodies (called Nanobodies) have superior properties compared with classical antibodies in that they are small, very stable, easy to produce in large quantities and easy to re-format into multi-valent or multi-specific proteins. Furthermore, they can specifically be selected for a desired function by phage antibody display. In this report, we describe the successful selection and the characterisation of antagonistic anti-EGFR Nanobodies. By using a functional selection strategy, Nanobodies that specifically competed for EGF binding to the EGFR were isolated from "immune" phage Nanobody repertoires. The selected antibody fragments were found to efficiently inhibit EGF binding to the EGFR without acting as receptor agonists themselves. In addition, they blocked EGF-mediated signalling and EGF-induced cell proliferation. In an in vivo murine xenograft model, the Nanobodies were effective in delaying the outgrowth of A431-derived solid tumours. This is the first report describing the successful use of untagged Nanobodies for the in vivo treatment of solid tumours. The results show that functional phage antibody selection, coupled to the rational design of Nanobodies, permits the rapid development of novel anti-cancer antibody-based therapeutics.

Engineering camel single-domain antibodies and immobilization chemistry for human prostate-specific antigen sensing.

The specificity and affinity characteristics of antibodies make them excellent probes in biosensor applications. Unfortunately, their large size, unstable behavior, and random immobilization properties create numerous problems. The single-domain antigen-binding fragment derived from heavy-chain antibodies of camelids (termed VHH) offers special advantages in terms of size, stability, and ease of generating different antibody constructs. In this study, we show the potential of those VHHs in sensing human prostate-specific antigen (hPSA) by SPR technology. Different VHH constructs were immobilized onto commercial and custom-built sensor surfaces by metal chelation, biotin-streptavidin interaction, or covalent coupling. The detection of subnanogram per milliliter hPSA concentrations could be attained on a covalently coupled three-dimensional dextran surface. Moreover, the ratio of different hPSA isoform concentrations could be assessed via a sandwich assay and resulted in the detection of clinically significant antigen concentrations within 15 min. In addition, for the first time, the intrinsic protein stability is presented as an important probe design factor, since our results reveal that higher intrinsic stability offers higher resistance to harsh regeneration conditions. In conclusion, we present VHHs as a novel class of biosensor probes rivaling conventional antibodies and their derived antibody fragments.

Prostate-specific antigen immunosensing based on mixed self-assembled monolayers, camel antibodies and colloidal gold enhanced sandwich assays.

Prostate-specific antigen (PSA) is a valuable biomarker for prostate cancer screening. We developed a PSA immunoassay on a commercially available surface plasmon resonance biosensor. Our PSA receptor molecule consists of a single domain antigen-binding fragment, cAbPSA-N7, derived from dromedary heavy-chain antibodies and identified after phage display. It binds PSA with a high k(on) value of 1.9x10(6) M-1 s-1, and was covalently immobilised on a gold substrate via a mixed self-assembled monolayer (SAM) of alkanethiols by using carbodiimide-coupling chemistry in 10mM acetate buffer pH 5.5 to obtain an optimal pre-concentration. The best performing and optimised mixed SAM consisted of (10%) 16-mercapto-1-hexadecanoic acid (16-MHA) for covalent cAbPSA-N7 immobilisation and (90%) 11-mercapto-1-undecanol (11-MUOH) to minimise non-specific adsorption of the analyte. In this way, two advantages are incorporated in a single coupling layer. Up to 28 fmol/mm2 of cAbPSA-N7 could be immobilised and 30% of its binding sites participate actively in PSA interaction. In addition, the optimised layer showed also optimal performance to assess physiological samples. Although PSA concentrations as low as 10 ng/ml could be detected directly, this detection limit could be enhanced to PSA levels in the sub ng/ml range by introducing a sandwich assay involving a biotinylated secondary antibody and streptavidin modified gold nanoparticles. This approach realizes the PSA detection at clinical relevant concentrations.