"Endothelial Antibody Factory" at the Blood Brain Barrier: Novel Approach to Therapy of Neurodegenerative Diseases.

The failures of anti-ß-amyloid immunotherapies suggested that the very low fraction of injected antibodies reaching the brain parenchyma due to the filtering effect of the BBB may be a reason for the lack of therapeutic effect. However, there is no treatment, as yet, for the amyotrophic lateral sclerosis (ALS) despite substantial evidence existing of the involvement of TDP-43 protein in the evolution of ALS. To circumvent this filtering effect, we have developed a novel approach to facilitate the penetration of antibody fragments (Fabs) into the brain parenchyma. Leveraging the homing properties of endothelial progenitor cells (EPCs), we transfected, ex vivo, such cells with vectors encoding anti-ß-amyloid and anti-TDP43 Fabs turning them into an "antibody fragment factory". When injected these cells integrate into the BBB, where they secrete anti-TDP43 Fabs. The results showed the formation of tight junctions between the injected engineered EPCs and the unlabeled resident endothelial cells. When the EPCs were further modified to express the anti-TDP43 Fab, we could observe integration of these cells into the vasculature and the secretion of Fabs. Results confirm that production and secretion of Fabs at the BBB level leads to their migration to the brain parenchyma where they might exert a therapeutic effect.

Secretion of proteins and antibody fragments from transiently transfected endothelial progenitor cells.

In neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, multiple sclerosis and amyotrophic lateral sclerosis, neuroinflammation can lead to blood-brain barrier (BBB) breakdown. After intravenous or intra-arterial injection into mice, endothelial progenitor cells (EPCs) home to the damaged BBB to promote neurovascular repair. Autologous EPCs transfected to express specific therapeutic proteins offer an innovative therapeutic option. Here, we demonstrate that EPC transfection by electroporation with plasmids encoding the reporter protein GFP or an anti-ß-amyloid antibody fragment (Fab) leads to secretion of each protein. We also demonstrate the secreted anti-ß-amyloid Fab protein functions in ß-amyloid aggregate solubilization.

Improved Genome Editing Efficiency and Flexibility Using Modified Oligonucleotides with TALEN and CRISPR-Cas9 Nucleases.

Genome editing has now been reported in many systems using TALEN and CRISPR-Cas9 nucleases. Precise mutations can be introduced during homology-directed repair with donor DNA carrying the wanted sequence edit, but efficiency is usually lower than for gene knockout and optimal strategies have not been extensively investigated. Here, we show that using phosphorothioate-modified oligonucleotides strongly enhances genome editing efficiency of single-stranded oligonucleotide donors in cultured cells. In addition, it provides better design flexibility, allowing insertions more than 100 bp long. Despite previous reports of phosphorothioate-modified oligonucleotide toxicity, clones of edited cells are readily isolated and targeted sequence insertions are achieved in rats and mice with very high frequency, allowing for homozygous loxP site insertion at the mouse ROSA locus in particular. Finally, when detected, imprecise knockin events exhibit indels that are asymmetrically positioned, consistent with genome editing taking place by two steps of single-strand annealing.

Genome Editing in Rats Using TALE Nucleases.

The rat is an important animal model to understand gene function and model human diseases. Since recent years, the development of gene-specific nucleases has become important for generating new rat models of human diseases, to analyze the role of genes and to generate human antibodies. Transcription activator-like (TALE) nucleases efficiently create gene-specific knockout rats and lead to the possibility of gene targeting by homology-directed recombination (HDR) and generating knock-in rats. We describe a detailed protocol for generating knockout and knock-in rats via microinjection of TALE nucleases into fertilized eggs. This technology is an efficient, cost- and time-effective method for creating new rat models.

Homology-directed repair in rodent zygotes using Cas9 and TALEN engineered proteins.

The generation of genetically-modified organisms has been revolutionized by the development of new genome editing technologies based on the use of gene-specific nucleases, such as meganucleases, ZFNs, TALENs and CRISPRs-Cas9 systems. The most rapid and cost-effective way to generate genetically-modified animals is by microinjection of the nucleic acids encoding gene-specific nucleases into zygotes. However, the efficiency of the procedure can still be improved. In this work we aim to increase the efficiency of CRISPRs-Cas9 and TALENs homology-directed repair by using TALENs and Cas9 proteins, instead of mRNA, microinjected into rat and mouse zygotes along with long or short donor DNAs. We observed that Cas9 protein was more efficient at homology-directed repair than mRNA, while TALEN protein was less efficient than mRNA at inducing homology-directed repair. Our results indicate that the use of Cas9 protein could represent a simple and practical methodological alternative to Cas9 mRNA in the generation of genetically-modified rats and mice as well as probably some other mammals.

Local control of the host immune response performed with mesenchymal stem cells: perspectives for functional intracerebral xenotransplantation.

Foetal pig neuroblasts are interesting candidates as a cell source for transplantation, but xenotransplantation in the brain requires the development of adapted immunosuppressive treatments. As systemic administration of high doses of cyclosporine A has side effects and does not protect xenotransplants forever, we focused our work on local control of the host immune responses. We studied the advantage of cotransplanting syngenic mesenchymal stem cells (MSC) with porcine neuroblasts (pNb) in immunocompetent rat striata. Two groups of animals were transplanted, either with pNb alone or with both MSC and pNb. At day 63, no porcine neurons were detected in the striata that received only pNb, while four of six rats transplanted with both pNb and MSC exhibited healthy porcine neurons. Interestingly, 50% of the cotransplanted rats displayed healthy grafts with pNF70+ and TH+ neurons at 120 days post-transplantation. qPCR analyses revealed a general dwindling of pro- and anti-inflammatory cytokines in the striata that received the cotransplants. Motor recovery was also observed following the transplantation of pNb and MSC in a rat model of Parkinson's disease. Taken together, the present data indicate that the immunosuppressive properties of MSC are of great interest for the long-term survival of xenogeneic neurons in the brain.

Efficient gene targeting by homology-directed repair in rat zygotes using TALE nucleases.

The generation of genetically modified animals is important for both research and commercial purposes. The rat is an important model organism that until recently lacked efficient genetic engineering tools. Sequence-specific nucleases, such as ZFNs, TALE nucleases, and CRISPR/Cas9 have allowed the creation of rat knockout models. Genetic engineering by homology-directed repair (HDR) is utilized to create animals expressing transgenes in a controlled way and to introduce precise genetic modifications. We applied TALE nucleases and donor DNA microinjection into zygotes to generate HDR-modified rats with large new sequences introduced into three different loci with high efficiency (0.62%-5.13% of microinjected zygotes). Two of these loci (Rosa26 and Hprt1) are known to allow robust and reproducible transgene expression and were targeted for integration of a GFP expression cassette driven by the CAG promoter. GFP-expressing embryos and four Rosa26 GFP rat lines analyzed showed strong and widespread GFP expression in most cells of all analyzed tissues. The third targeted locus was Ighm, where we performed successful exon exchange of rat exon 2 for the human one. At all three loci we observed HDR only when using linear and not circular donor DNA. Mild hypothermic (30°C) culture of zygotes after microinjection increased HDR efficiency for some loci. Our study demonstrates that TALE nuclease and donor DNA microinjection into rat zygotes results in efficient and reproducible targeted donor integration by HDR. This allowed creation of genetically modified rats in a work-, cost-, and time-effective manner.

Gene targeting in rats using transcription activator-like effector nucleases.

The rat is a model of choice to understanding gene function and modeling human diseases. Since recent years, successful engineering technologies using gene-specific nucleases have been developed to gene edit the genome of different species, including the rat. This development has become important for the creation of new rat animals models of human diseases, analyze the role of genes and express recombinant proteins. Transcription activator-like (TALE) nucleases are designed nucleases consist of a DNA binding domain fused to a nuclease domain capable of cleaving the targeted DNA. We describe a detailed protocol for generating knockout rats via microinjection of TALE nucleases into fertilized eggs. This technology is an efficient, cost- and time-effective method for creating new rat models.

Codon swapping of zinc finger nucleases confers expression in primary cells and in vivo from a single lentiviral vector.

BACKGROUND: Zinc finger nucleases (ZFNs) are promising tools for genome editing for biotechnological as well as therapeutic purposes. Delivery remains a major issue impeding targeted genome modification. Lentiviral vectors are highly efficient for delivering transgenes into cell lines, primary cells and into organs, such as the liver. However, the reverse transcription of lentiviral vectors leads to recombination of homologous sequences, as found between and within ZFN monomers. METHODS: We used a codon swapping strategy to both drastically disrupt sequence identity between ZFN monomers and to reduce sequence repeats within a monomer sequence. We constructed lentiviral vectors encoding codon-swapped ZFNs or unmodified ZFNs from a single mRNA transcript. Cell lines, primary hepatocytes and newborn rats were used to evaluate the efficacy of integrative-competent (ICLV) and integrative-deficient (IDLV) lentiviral vectors to deliver ZFNs into target cells. RESULTS: We reduced total identity between ZFN monomers from 90.9% to 61.4% and showed that a single ICLV allowed efficient expression of functional ZFNs targeting the rat UGT1A1 gene after codon-swapping, leading to much higher ZFN activity in cell lines (up to 7-fold increase compared to unmodified ZFNs and 60% activity in C6 cells), as compared to plasmid transfection or a single ICLV encoding unmodified ZFN monomers. Off-target analysis located several active sites for the 5-finger UGT1A1-ZFNs. Furthermore, we reported for the first time successful ZFN-induced targeted DNA double-strand breaks in primary cells (hepatocytes) and in vivo (liver) after delivery of a single IDLV encoding two ZFNs. CONCLUSION: These results demonstrate that a codon-swapping approach allowed a single lentiviral vector to efficiently express ZFNs and should stimulate the use of this viral platform for ZFN-mediated genome editing of primary cells, for both ex vivo or in vivo applications.

Human dental pulp stem cells cultured in serum-free supplemented medium.

UNLABELLED: Growing evidence show that human dental pulp stem cells (DPSCs) could provide a source of adult stem cells for the treatment of neurodegenerative pathologies. In this study, DPSCs were expanded and cultured with a protocol generally used for the culture of neural stem/progenitor cells. METHODOLOGY: DPSC cultures were established from third molars. The pulp tissue was enzymatically digested and cultured in serum-supplemented basal medium for 12 h. Adherent (ADH) and non-adherent (non-ADH) cell populations were separated according to their differential adhesion to plastic and then cultured in serum-free defined N2 medium with epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). Both ADH and non-ADH populations were analyzed by FACS and/or PCR. RESULTS: FACS analysis of ADH-DPSCs revealed the expression of the mesenchymal cell marker CD90, the neuronal marker CD56, the transferrin receptor CD71, and the chemokine receptor CXCR3, whereas hematopoietic stem cells markers CD45, CD133, and CD34 were not expressed. ADH-DPSCs expressed transcripts coding for the Nestin gene, whereas expression levels of genes coding for the neuronal markers ß-III tubulin and NF-M, and the oligodendrocyte marker PLP-1 were donor dependent. ADH-DPSCs did not express the transcripts for GFAP, an astrocyte marker. Cells of the non-ADH population that grew as spheroids expressed Nestin, ß-III tubulin, NF-M and PLP-1 transcripts. DPSCs that migrated out of the spheroids exhibited an odontoblast-like morphology and expressed a higher level of DSPP and osteocalcin transcripts than ADH-DPSCs. CONCLUSION: Collectively, these data indicate that human DPSCs can be expanded and cultured in serum-free supplemented medium with EGF and bFGF. ADH-DPSCs and non-ADH populations contained neuronal and/or oligodendrocyte progenitors at different stages of commitment and, interestingly, cells from spheroid structures seem to be more engaged into the odontoblastic lineage than the ADH-DPSCs.

Impaired spatial memory in mice lacking CD3ζ is associated with altered NMDA and AMPA receptors signaling independent of T-cell deficiency.

The immunoreceptor-associated protein CD3ζ is known for its role in immunity and has also been implicated in neuronal development and synaptic plasticity. However, the mechanism by which CD3ζ regulates synaptic transmission remains unclear. In this study, we showed that mice lacking CD3ζ exhibited defects in spatial learning and memory as examined by the Barnes maze and object location memory tasks. Given that peripheral T cells have been shown to support cognitive functions and neural plasticity, we generated CD3ζ(-/-) mice in which the peripheral T cells were repopulated to a normal level by syngeneic bone marrow transplantation. Using this approach, we showed that T-cell replenishment in CD3ζ(-/-) mice did not restore spatial memory defects, suggesting that the cognitive deficits in CD3ζ(-/-) mice were most likely mediated through a T-cell-independent mechanism. In support of this idea, we showed that CD3ζ proteins were localized to glutamatergic postsynaptic sites, where they interacted with the NMDAR subunit GluN2A. Loss of CD3ζ in brain decreased GluN2A-PSD95 association and GluN2A synaptic localization. This effect was accompanied by a reduced interaction of GluN2A with the key NMDAR downstream signaling protein calcium/calmodulin-dependent protein kinase II (CaMKII). Using the glycine-induced, NMDA-dependent form of chemical long-term potentiation (LTP) in cultured cortical neurons, we showed that CD3ζ was required for activity-dependent CaMKII autophosphorylation and for the synaptic recruitment of the AMPAR subunit GluA1. Together, these results support the model that the procognitive function of CD3ζ may be mediated through its involvement in the NMDAR downstream signaling pathway leading to CaMKII-dependent LTP induction.

Generation of Rag1-knockout immunodeficient rats and mice using engineered meganucleases.

Despite the recent availability of gene-specific nucleases, such as zinc-finger nucleases (ZFNs) and transcription activator-like nucleases (TALENs), there is still a need for new tools to modify the genome of different species in an efficient, rapid, and less costly manner. One aim of this study was to apply, for the first time, engineered meganucleases to mutate an endogenous gene in animal zygotes. The second aim was to target the mouse and rat recombination activating gene 1 (Rag1) to describe, for the first time, Rag1 knockout immunodeficient rats. We microinjected a plasmid encoding a meganuclease for Rag1 into the pronucleus of mouse and rat zygotes. Mutant animals were detected by PCR sequencing of the targeted sequence. A homozygous RAG1-deficient rat line was generated and immunophenotyped. Meganucleases were efficient, because 3.4 and 0.6% of mouse and rat microinjected zygotes, respectively, generated mutated animals. RAG1-deficient rats showed significantly decreased proportions and numbers of immature and mature T and B lymphocytes and normal NK cells vs. littermate wild-type controls. In summary, we describe the use of engineered meganucleases to inactivate an endogenous gene with efficiencies comparable to those of ZFNs and TALENs. Moreover, we generated an immunodeficient rat line useful for studies in which there is a need for biological parameters to be analyzed in the absence of immune responses.

Expression of heme oxygenase-1 in neural stem/progenitor cells as a potential mechanism to evade host immune response.

Besides their therapeutic benefit as cell source, neural stem/progenitor cells (NSPCs) exhibit immunosuppressive properties of great interest for modulating immune response in the central nervous system. To decipher the mechanisms of NSPC-mediated immunosuppression, activated T cells were exposed to NSPCs isolated from fetal rat brains. Analyses revealed that NSPCs inhibited T-cell proliferation and interferon-gamma production in a dose-dependent manner. A higher proportion of helper T cells (CD4+ T cells) was found in the presence of NSPCs, but analyses of FoxP3 population indicated that T-cell suppression was not secondary to an induction of suppressive regulatory T cells (FoxP3+ CD4+ CD25+). Conversely, induction of the high affinity interleukin-2 (IL-2) receptor (CD25) and the inability of IL-2 to rescue T-cell proliferation suggest that NSPCs display immunosuppressive activity without affecting T-cell activation. Cultures in Transwell chambers or addition of NSPC-conditioned medium to activated T cells indicated that part of the suppressive activity was not contact dependent. We therefore searched for soluble factors that mediate NSPC immunosuppression. We found that NSPCs express several immunosuppressive molecules, but the ability of these cells to inhibit T-cell proliferation was only counteracted by heme oxygenase (HO) inhibitors in association or not with nitric oxide synthase inhibitors. Taken together, our findings highlight a dynamic crosstalk between NSPCs and T lymphocytes and provide the first evidence of an implication of HO-1 in mediating the immunosuppressive effects of the NSPCs.

The immune molecule CD3zeta and its downstream effectors ZAP-70/Syk mediate ephrin signaling in neurons to regulate early neuritogenesis.

Recent studies have highlighted the key role of the immune protein CD3ζ in the maturation of neuronal circuits in the CNS. Yet, the upstream signals that might recruit and activate CD3ζ in neurons are still unknown. In this study, we show that CD3ζ functions early in neuronal development and we identify ephrinA1-dependent EphA4 receptor activation as an upstream regulator of CD3ζ. When newly born neurons are still spherical, before neurite extension, we found a transient CD3ζ aggregation at the cell periphery matching the initiation site of the future neurite. This accumulation of CD3ζ correlated with a stimulatory effect on filopodia extension via a Rho-GEF Vav2 pathway and a repression of neurite outgrowth. Conversely, cultured neurons lacking CD3ζ isolated from CD3ζ(-/-) mice showed a decreased number of filopodia and an enhanced neurite number. Stimulation with ephrinA1 induces the translocation of both CD3ζ and its activated effector molecules, ZAP-70/Syk tyrosine kinases, to EphA4 receptor clusters. EphrinA1-induced growth cone collapse was abrogated in CD3ζ(-/-) neurons and was markedly reduced by ZAP-70/Syk inhibition. Moreover, ephrinA1-induced ZAP-70/Syk activation was inhibited in CD3ζ(-/-) neurons. Altogether, our data suggest that CD3ζ mediates the ZAP-70/Syk kinase activation triggered by ephrinA-activated pathway to regulate early neuronal morphogenesis.

Effects of Human Alpha-Synuclein A53T-A30P Mutations on SVZ and Local Olfactory Bulb Cell Proliferation in a Transgenic Rat Model of Parkinson Disease.

A transgenic Sprague Dawley rat bearing the A30P and A53T α-synuclein (α-syn) human mutations under the control of the tyrosine hydroxylase promoter was generated in order to get a better understanding of the role of the human α-syn mutations on the neuropathological events involved in the progression of the Parkinson's disease (PD). This rat displayed olfactory deficits in the absence of motor impairments as observed in most early PD cases. In order to investigate the role of the mutated α-syn on cell proliferation, we focused on the subventricular zone (SVZ) and the olfactory bulbs (OB) as a change of the proliferation could affect OB function. The effect on OB dopaminergic innervation was investigated. The human α-syn co-localized in TH-positive OB neurons. No human α-syn was visualized in the SVZ. A significant increase in resident cell proliferation in the glomerular but not in the granular layers of the OB and in the SVZ was observed. TH innervation was significantly increased within the glomerular layer without an increase in the size of the glomeruli. Our rat could be a good model to investigate the role of human mutated α-syn on the development of olfactory deficits.

Mesenchymal stem cell transplantation and DMEM administration in a 3NP rat model of Huntington's disease: morphological and behavioral outcomes.

Transplantation of mesenchymal stem cells (MSCs) may offer a viable treatment for Huntington's disease (HD). We tested the efficacy of MSC transplants to reduce deficits in a 3-nitropropionic acid (3NP) rat model of HD. Five groups of rats (Sham, 3NP, 3NP+vehicle, 3NP+TP(low), 3NP+TP(high)), were given PBS or 3NP intraperitoneally, twice daily for 42 days. On day 28, rats in all groups except Sham and 3NP, received intrastriatal injections of either 200,000 MSCs (TP(low)), 400,000 (TP(high)) MSCs or DMEM (VH, the vehicle for transplantation). MSCs survived 72 days without inducing a strong inflammatory response from the striatum. Behavioral sparing was observed on tests of supported-hindlimb-retraction, unsupported-hindlimb-retraction, visual paw placement and stepping ability for 3NP+TP(low) rats and on the unsupported-hindlimb-retraction and rotarod tasks for 3NP+VH rats. Relative to 3NP controls, all treated groups were protected from 3NP-induced enlargement of the lateral ventricles. In vitro, MSCs expressed transcripts for numerous neurotrophic factors. In vivo, increased striatal labeling in BDNF, collagen type-I and fibronectin (but not GDNF or CNTF) was observed in the brains of MSC-transplanted rats but not in DMEM-treated rats. In addition, none of the transplanted MSCs expressed neural phenotypes. These findings suggest that factors other than neuronal replacement underlie the behavioral sparing observed in 3NP rats after MSC transplantation.

AUF1 and Hu proteins in the developing rat brain: implication in the proliferation and differentiation of neural progenitors.

Posttranscriptional events such as RNA stabilization are important for cell differentiation, but little is known about the impact of AU-rich binding proteins (AUBPs) on the fate of neural cells. Expression of destabilizing AUBPs such as AUF1 and neuronal-specific stabilizing proteins such as HuB, HuC and HuD was therefore analyzed in the developing central nervous system. Real-time RT-PCR indicated a specific developmental pattern in the postnatal cerebellum, with a progressive down-regulation of AUF1 from P1, whereas HuB was strongly up-regulated at about P7. These changes were accompanied by a progressive increase in AUF1p45 and the disappearance of one HuB isoform from P15, suggesting particular roles for these AUBPs in the developing cerebellum. AUF1 was detected in the three main cerebellar layers, whereas Hu proteins were found only in postmitotic neurons. A role for Hu proteins in the early stages of neuronal differentiation is further supported by arrest of cell proliferation following induction of HuB or HuD expression in a neural stem cell line. The decrease in nestin expression suggest that HuD, but not HuB, favors the transition of neural progenitors into early neuroblasts, but other factors are most probably required for their full differentiation into neurons, insofar as GAP-43 was not detected in HuD-transfected cells. These data suggest critical roles for HuB at the very earliest stages of neuronal differentiation, such as cell cycle exit, and HuD might also be involved in the transition of neural progenitors into early neuroblasts. Taken together, the present results strengthen the importance of AUBPs in brain ontogenesis.

Mesenchymal stem cells induce a weak immune response in the rat striatum after allo or xenotransplantation.

Mesenchymal stem cells (MSCs) have attracted attention for their potential use in regenerative medicine such as brain transplantation. As MSCs are considered to be hypoimmunogenic, transplanted MSCs should not trigger a strong host inflammatory response. To verify this hypothesis, we studied the brain immune response after transplantation of human or rat MSCs into the rat striatum and MSC fate at days 5, 14, 21 and 63 after transplantation. Flow cytometry analysis indicated that both MSCs express CD90 and human leucocyte antigen (MHC) class I, but no MHC class II molecules. They do not express CD45 or CD34 antigens. However, MSC phenotype varies with passage number. Human MSCs have mRNAs for interleukin (IL)-6, IL-8, IL-12, tumour necrosis factor (TNF)-alpha and TGF-beta(1), whereas rat MSCs express IL-6-, IL-10-, IL-12- and TGF-beta(1)-mRNAs. The quantification shows higher levels of mRNAs for the anti-inflammatory molecules IL-6 and TGF-beta(1) than for pro-inflammatory cytokines IL-8 and IL-12; ELISA analysis showed no IL-12 whereas TGF-beta(1) and IL-6 were detected. Transplant size did not significantly vary between 14 and 63 days after transplantation, indicating an absence of immune rejection of the grafts. Very few mast cells and moderate macrophage and microglial infiltrations, observed at day 5 remained stable until day 63 after transplantation in both rat and human MSC grafts. The observations of very few dendritic cells, T alphabeta-cells, and no T gammadelta-lymphocytes, all three being associated with Tp rejection in the brain, support the contention that MSCs are hypoimmunogenic. Our results suggest that MSCs are of great interest in regenerative medicine in a (xeno)transplantation setting.