In vivo bioluminescence imaging of the intracerebral fibroin-controlled AAV-α-synuclein diffusion for monitoring the central nervous system and peripheral expression.

Among the several animal models of α-synucleinopathies, the well-known viral vector-mediated delivery of wild-type or mutated (A53T) α-synuclein requires new tools to increase the lesion in mice and follow up in vivo expression. To this end, we developed a bioluminescent expression reporter of the human A53T-α-synuclein gene using the NanoLuc system into an AAV2/9, embedded or not in a fibroin solution to stabilise its expression in space and time. We first verified the expression of the fused protein in vitro on transfected cells by bioluminescence and Western blotting. Next, two groups of C57Bl6Jr mice were unilaterally injected with the AAV-NanoLuc-human-A53T-α-synuclein above the substantia nigra combined (or not) with fibroin. We first show that the in vivo cerebral bioluminescence signal was more intense in the presence of fibroin. Using immunohistochemistry, we find that the human-A53T-α-synuclein protein is more restricted to the ipsilateral side with an overall greater magnitude of the lesion when fibroin was added. However, we also detected a bioluminescence signal in peripheral organs in both conditions, confirmed by the presence of viral DNA corresponding to the injected AAV in the liver using qPCR.

ApoE-fragment/Aß heteromers in the brain of patients with Alzheimer's disease.

Identification of endogenous pathological amyloid ß peptides (Aß) forms in the brains of patients with Alzheimer's disease (AD) is still unclear. In healthy brain, Aß can associate with Apolipoprotein E (ApoE) which is involved in its metabolism and clearance. In the brain of patients with AD, ApoE is cleaved and produces ApoE fragments. We studied the forms of Aß and their interaction with the ApoE fragments in post-mortem brains from control and AD patients by western blots and co-immunoprecipitation. Three Aß-containing peptides and three ApoE fragments were specifically found in the brain of AD patients. Co-immunoprecipitations showed that ApoE fragments and Aß1-42 peptides are co-partners in heteromers of 18 and 16 kDa while ApoE-fragments and Aß peptides of 12 kDa did not interact with each other. Formation of the 18 kDa ApoE-fragment/Aß heteromers is specifically increased in ApoE4 carriers and is a strong brain marker of AD while 16 kDa ApoE-fragment/Aß and Aß 12 kDa correlate to memory deficit. These data show that in patients with AD, ApoE fragmentation generates peptides that trap Aß in the brain. Inhibiting the fragmentation or targeting ApoE fragments could be exploited to define strategies to detect or reverse AD.

Detection of the Cyanotoxins L-BMAA Uptake and Accumulation in Primary Neurons and Astrocytes.

We show for the first time that a newly developed polyclonal antibody (pAb) can specifically target the cyanotoxin ß-methylamino-L-alanine (BMAA) and can be used to enable direct visualization of BMAA entry and accumulation in primary brain cells. We used this pAb to investigate the effect of acute and chronic accumulation, and toxicity of both BMAA and its natural isomer 2,4-diaminobutyric acid (DAB), separately or in combination, on primary cultures of rat neurons. We further present evidence that co-treatment with BMAA and DAB increased neuronal death, as measured by MAP2 fluorescence level, and appeared to reduce BMAA accumulation. DAB is likely to be acting synergistically with BMAA resulting in higher level of cellular toxicity. We also found that glial cells such as microglia and astrocytes are also able to directly uptake BMAA indicating that additional brain cell types are affected by BMAA-induced toxicity. Therefore, BMAA clearly acts at multiple cellular levels to possibly increase the risk of developing neurodegenerative diseases, including neuro- and gliotoxicity and synergetic exacerbation with other cyanotoxins.

In Vivo Imaging of Prostate Cancer Tumors and Metastasis Using Non-Specific Fluorescent Nanoparticles in Mice.

With the growing interest in the use of nanoparticles (NPs) in nanomedicine, there is a crucial need for imaging and targeted therapies to determine NP distribution in the body after systemic administration, and to achieve strong accumulation in tumors with low background in other tissues. Accumulation of NPs in tumors results from different mechanisms, and appears extremely heterogeneous in mice models and rather limited in humans. Developing new tumor models in mice, with their low spontaneous NP accumulation, is thus necessary for screening imaging probes and for testing new targeting strategies. In the present work, accumulation of LipImageTM 815, a non-specific nanosized fluorescent imaging agent, was compared in subcutaneous, orthotopic and metastatic tumors of RM1 cells (murine prostate cancer cell line) by in vivo and ex vivo fluorescence imaging techniques. LipImageTM 815 mainly accumulated in liver at 24 h but also in orthotopic tumors. Limited accumulation occurred in subcutaneous tumors, and very low fluorescence was detected in metastasis. Altogether, these different tumor models in mice offered a wide range of NP accumulation levels, and a panel of in vivo models that may be useful to further challenge NP targeting properties.

In vivo imaging of prostate cancer using an anti-PSMA scFv fragment as a probe.

We aimed to evaluate a fluorescent-labeled single chain variable fragment (scFv) of the anti-PSMA antibody as a specific probe for the detection of prostate cancer by in vivo fluorescence imaging. An orthotopic model of prostate cancer was generated by injecting LNCaP cells into the prostate lobe. ScFvD2B, a high affinity anti-PSMA antibody fragment, was labeled using a near-infrared fluorophore to generate a specific imaging probe (X770-scFvD2B). PSMA-unrelated scFv-X770 was used as a control. Probes were injected intravenously into mice with prostate tumors and fluorescence was monitored in vivo by fluorescence molecular tomography (FMT). In vitro assays showed that X770-scFvD2B specifically bound to PSMA and was internalized in PSMA-expressing LNCaP cells. After intravenous injection, X770-scFvD2B was detected in vivo by FMT in the prostate region. On excised prostates the scFv probe co-localized with the cancer cells and was found in PSMA-expressing cells. The PSMA-unrelated scFv used as a control did not label the prostate cancer cells. Our data demonstrate that scFvD2B is a high affinity contrast agent for in vivo detection of PSMA-expressing cells in the prostate. NIR-labeled scFvD2B could thus be further developed as a clinical probe for imaging-guided targeted biopsies.

TRPP2 modulates ryanodine- and inositol-1,4,5-trisphosphate receptors-dependent Ca2+ signals in opposite ways in cerebral arteries.

TRPP2 is a cationic channel expressed in plasma membrane and in sarcoplasmic reticulum. In several cell lines, TRPP2 is described as a reticulum Ca(2+) leak channel but it also interacts with ryanodine and inositol 1,4,5-trisphosphate (InsP3) receptors to inhibit and increase the release of Ca(2+) stores, respectively. TRPP2 is known to be expressed in vascular smooth muscle cells, however its function in Ca(2+) signals remains poorly described in native cells, principally because the pharmacology is not developed. TRPP2 was expressed in cerebral arteries. Triptolide evoked Ca(2+) responses in a Ca(2+)-free solution as well as permeabilized arteries. This Ca(2+) signal was inhibited in presence of antisense oligonucleotide and siRNA directed against TRPP2 and antibody directed against the first loop of TRPP2. The partial inhibition of TRPP2 expression increased both the caffeine-evoked Ca(2+) responses and in vivo contraction. It also decreased the InsP3-evoked Ca(2+) responses. Finally, aging affected the regulations in which TRPP2 is engaged, whereas the triptolide-evoked Ca(2+) response was not modified. Taken together, our results have shown that TRPP2 is implicated in triptolide-induced Ca(2+) release from intracellular Ca(2+) stores. TRPP2 functionally interacts with both ryanodine and InsP3 receptors. These interactions were not similar in adult and old mice.

Gipc1 has a dual role in Vangl2 trafficking and hair bundle integrity in the inner ear.

Vangl2 is one of the central proteins controlling the establishment of planar cell polarity in multiple tissues of different species. Previous studies suggest that the localization of the Vangl2 protein to specific intracellular microdomains is crucial for its function. However, the molecular mechanisms that control Vangl2 trafficking within a cell are largely unknown. Here, we identify Gipc1 (GAIP C-terminus interacting protein 1) as a new interactor for Vangl2, and we show that a myosin VI-Gipc1 protein complex can regulate Vangl2 traffic in heterologous cells. Furthermore, we show that in the cochlea of MyoVI mutant mice, Vangl2 presence at the membrane is increased, and that a disruption of Gipc1 function in hair cells leads to maturation defects, including defects in hair bundle orientation and integrity. Finally, stimulated emission depletion microscopy and overexpression of GFP-Vangl2 show an enrichment of Vangl2 on the supporting cell side, adjacent to the proximal membrane of hair cells. Altogether, these results indicate a broad role for Gipc1 in the development of both stereociliary bundles and cell polarization, and suggest that the strong asymmetry of Vangl2 observed in early postnatal cochlear epithelium is mostly a 'tissue' polarity readout.

Combined macro-/mesoporous microelectrode arrays for low-noise extracellular recording of neural networks.

Microelectrode arrays (MEAs) are appealing tools to probe large neural ensembles and build neural prostheses. Microelectronics microfabrication technologies now allow building high-density MEAs containing several hundreds of microelectrodes. However, several major problems become limiting factors when the size of the microelectrodes decreases. In particular, regarding recording of neural activity, the intrinsic noise level of a microelectrode dramatically increases when the size becomes small (typically below 20-µm diameter). Here, we propose to overcome this limitation using a template-based, single-scale meso- or two-scale macro-/mesoporous modification of the microelectrodes, combining the advantages of an overall small geometric surface and an active surface increased by several orders of magnitude. For this purpose, standard platinum MEAs were covered with a highly porous platinum overlayer obtained by lyotropic liquid crystal templating possibly in combination with a microsphere templating approach. These porous coatings were mechanically more robust than Pt-black coating and avoid potential toxicity issues. They had a highly increased active surface, resulting in a noise level ∼3 times smaller than that of conventional flat electrodes. This approach can thus be used to build highly dense arrays of small-size microelectrodes for sensitive neural signal detection.

Artificial CSF motion ensures rhythmic activity in the developing CNS ex vivo: a mechanical source of rhythmogenesis?

Spontaneous rhythmic activity is a ubiquitous feature of developing neural structures that has been shown to be essential for the establishment of functional CNS connectivity. However, the primordial origin of these rhythms remains unknown. Here, we describe two types of rhythmic activity in distinct parts of the developing CNS isolated ex vivo on microelectrode arrays, the expression of which was found to be strictly dependent upon the movement of the artificial CSF (aCSF) flowing over the inner wall of the ventricles or over the outer surface of the CNS. First, whole embryonic mouse hindbrain-spinal cord preparations (stages E12.5-E15.5) rhythmically expressed waves of activity originating in the hindbrain and propagating in the spinal cord. Interestingly enough, the frequency of this rhythm was completely determined by the speed of the aCSF flow. In particular, at all stages considered, hindbrain activity was abolished when the perfusion was stopped. Immature rhythmic activity was also recorded in the isolated newborn (P0-P8) mouse cortex under normal aCSF perfusion. Again, this rhythm was abolished when the perfusion flow was stopped. In both structures, this phenomenon was not due to changes in temperature, oxygen level, or pH of the bath, but to the movement itself of the aCSF. These observations challenge the so-called "spontaneous" nature of rhythmic activity in immature neural networks and suggest that the movement of CSF in the ventricles and around the brain in vivo may mechanically drive rhythmogenesis in the developing CNS.

NeuroMap: A Spline-Based Interactive Open-Source Software for Spatiotemporal Mapping of 2D and 3D MEA Data.

A major characteristic of neural networks is the complexity of their organization at various spatial scales, from microscopic local circuits to macroscopic brain-scale areas. Understanding how neural information is processed thus entails the ability to study them at multiple scales simultaneously. This is made possible using microelectrodes array (MEA) technology. Indeed, high-density MEAs provide large-scale coverage (several square millimeters) of whole neural structures combined with microscopic resolution (about 50 µm) of unit activity. Yet, current options for spatiotemporal representation of MEA-collected data remain limited. Here we present NeuroMap, a new interactive Matlab-based software for spatiotemporal mapping of MEA data. NeuroMap uses thin plate spline interpolation, which provides several assets with respect to conventional mapping methods used currently. First, any MEA design can be considered, including 2D or 3D, regular or irregular, arrangements of electrodes. Second, spline interpolation allows the estimation of activity across the tissue with local extrema not necessarily at recording sites. Finally, this interpolation approach provides a straightforward analytical estimation of the spatial Laplacian for better current sources localization. In this software, coregistration of 2D MEA data on the anatomy of the neural tissue is made possible by fine matching of anatomical data with electrode positions using rigid-deformation-based correction of anatomical pictures. Overall, NeuroMap provides substantial material for detailed spatiotemporal analysis of MEA data. The package is distributed under GNU General Public License and available at http://sites.google.com/site/neuromapsoftware.

The planar polarity protein Scribble1 is essential for neuronal plasticity and brain function.

Scribble (Scrib) is a key regulator of apicobasal polarity, presynaptic architecture, and short-term synaptic plasticity in Drosophila. In mammals, its homolog Scrib1 has been implicated in cancer, neural tube closure, and planar cell polarity (PCP), but its specific role in the developing and adult nervous system is unclear. Here, we used the circletail mutant, a mouse model for PCP defects, to show that Scrib1 is located in spines where it influences actin cytoskeleton and spine morphing. In the hippocampus of these mutants, we observed an increased synapse pruning associated with an increased number of enlarged spines and postsynaptic density, and a decreased number of perforated synapses. This phenotype was associated with a mislocalization of the signaling pathway downstream of Scrib1, leading to an overall activation of Rac1 and defects in actin dynamic reorganization. Finally, Scrib1-deficient mice exhibit enhanced learning and memory abilities and impaired social behavior, two features relevant to autistic spectrum disorders. Our data identify Scrib1 as a crucial regulator of brain development and spine morphology, and suggest that Scrib1(crc/+) mice might be a model for studying synaptic dysfunction and human psychiatric disorders.

Matrix metalloproteinase 3 is present in the cell nucleus and is involved in apoptosis.

Matrix metalloproteinase (MMP)-3 is a protease involved in cancer progression and tissue remodeling. Using immunofluorescence and immunoelectron microscopy, we identified nuclear localization of MMP-3 in several cultured cell types and in human liver tissue sections. Western blot analysis of nuclear extracts revealed two immunoreactive forms of MMP-3 at 35 and 45 kd, with the 35-kd form exhibiting caseinolytic activity. By transient transfection, we expressed active MMP-3 fused to the enhanced green fluorescent protein (EGFP/aMMP-3) in Chinese hamster ovary cells. We showed that EGFP/aMMP-3 translocates into the nucleus. A functional nuclear localization signal was demonstrated by the loss of nuclear translocation after site-directed mutagenesis of a putative nuclear localization signal and by the ability of the MMP-3 nuclear localization signal to drive a heterologous protein into the nucleus. Finally, expression by Chinese hamster ovary cells of EGFP/aMMP-3 induced a twofold increase of apoptosis rate, compared with EGFP/pro-MMP-3, which does not translocate to the nucleus. Increased apoptosis was abolished by site-directed mutagenesis of the catalytic site of MMP-3 or by using the MMP inhibitor GM6001. This study elucidates for the first time the mechanisms of nuclear localization of a MMP and shows that nuclear MMP-3 can induce apoptosis via its catalytic activity.

Identification and characterization of two dipeptidyl-peptidase III isoforms in Drosophila melanogaster.

Dipeptidyl-peptidase III (DPP III) hydrolyses small peptides with a broad substrate specificity. It is thought to be involved in a major degradation pathway of the insect neuropeptide proctolin. We report the purification and characterization of a soluble DPP III from 40 g Drosophila melanogaster. Western blot analysis with anti-(DPP III) serum revealed the purification of two proteins of molecular mass 89 and 82 kDa. MS/MS analysis of these proteins resulted in the sequencing of 45 and 41 peptide fragments, respectively, confirming approximately 60% of both annotated D. melanogaster DPP III isoforms (CG7415-PC and CG7415-PB) predicted at 89 and 82 kDa. Sequencing also revealed the specific catalytic domain HELLGH in both isoforms, indicating that they are both effective in degrading small peptides. In addition, with a probe specific for D. melanogaster DPP III, northern blot analysis of fruit fly total RNA showed two transcripts at approximately 2.6 and 2.3 kb, consistent with the translation of 89-kDa and 82-kDa DPP III proteins. Moreover, the purified enzyme hydrolyzed the insect neuropeptide proctolin (Km approximately 4 microm) at the second N-terminal peptide bound, and was inhibited by the specific DPP III inhibitor tynorphin. Finally, anti-(DPP III) immunoreactivity was observed in the central nervous system of D. melanogaster larva, supporting a functional role for DPP III in proctolin degradation. This study shows that DPP III is in actuality synthesized in D. melanogaster as 89-kDa and 82-kDa isoforms, representing two native proteins translated from two alternative mRNA transcripts.

Thrombin up-regulates tissue factor pathway inhibitor-2 synthesis through a cyclooxygenase-2-dependent, epidermal growth factor receptor-independent mechanism.

The serine proteinase inhibitor tissue factor pathway inhibitor-2 (TFPI-2) inhibits the tissue factor-factor VIIa complex and thereby impairs factor Xa and subsequently thrombin generation. Here we show that thrombin itself up-regulates TFPI-2 mRNA and protein expression in human liver myofibroblasts, a cell type shown to express high levels of TFPI-2 (Neaud, V., Hisaka, T., Monvoisin, A., Bedin, C., Balabaud, C., Foster, D. C., Desmoulière, A., Kisiel, W., and Rosenbaum, J. (2000) J. Biol. Chem. 275, 35565-35569). This effect required thrombin catalytic activity, as shown by its abolition with hirudin. Although the thrombin effect could be mimicked by agonists of both protease-activated receptor (PAR)-1 and PAR-4, it was largely blocked by a PAR-1 blocking antibody. Transactivation of the epidermal growth factor (EGF) receptor has been reported as a common event in thrombin signaling. However, thrombin did not detectably transactivate the EGF receptor in liver myofibroblasts, and blocking the EGF receptor did not affect TFPI-2 induction. On the other hand, thrombin increased the expression of cyclooxygenase-2 (COX-2) mRNA via a MAPK-dependent pathway, and a specific COX-2 inhibitor abolished the effect of thrombin on TFPI-2 expression. Thus, thrombin, through PAR-1 signaling, up-regulates the synthesis of TFPI-2 via a MAPK/COX-2-dependent pathway. The up-regulation of TFPI-2 expression by thrombin could in turn down-regulate thrombin generation and contribute to limit blood coagulation.

Characterization of a functionally expressed dipeptidyl aminopeptidase III from Drosophila melanogaster.

A Drosophila melanogaster cDNA clone (GH01916) encoding a putative 723-residue long (82 kDa) protein (CG 7415) and displaying 50% identity with mammalian cytosolic dipeptidyl aminopeptidase (DPP) III was functionally expressed in Schneider S2 cells. Immunocytochemical studies using anti-(rat liver DPP III) Ig indicated the expression of this putative DPP III at the outer cell membrane and into the cytosol of transfected cells. Two protein bands (82 and 86 kDa) were immunologically detected after PAGE and Western blot of cytosol or membrane prepared from transfected cells. Western blot analysis of partially purified D. melanogaster DPP III confirmed the overexpression of these two protein bands into the cytosol and on the membranes of transfected cells. Despite the identification of six potential glycosylation sites, PAGE showed that these protein bands were not shifted after deglycosylation experiments. The partially purified enzyme hydrolysed the insect myotropic neuropeptide proctolin (Arg-Tyr-Leu-Pro-Thr) at the Tyr-Leu bond (Km approximately 4 micro m). In addition, low concentration of the specific DPP III inhibitor tynorphin prevented proctolin degradation (IC50 = 0.62 +/- 0.15 micro m). These results constitute the first characterization of an evolutionarily conserved insect DPP III that is expressed as a cytosolic and a membrane peptidase involved in proctolin degradation.