A selection and optimization strategy for single-domain antibodies targeting the PHF6 linear peptide within the tau intrinsically disordered protein.

The use of variable domain of the heavy-chain of the heavy-chain-only antibodies (VHHs) as disease-modifying biomolecules in neurodegenerative disorders holds promises, including targeting of aggregation-sensitive proteins. Exploitation of their clinical values depends however on the capacity to deliver VHHs with optimal physico-chemical properties for their specific context of use. We described previously a VHH with high therapeutic potential in a family of neurodegenerative diseases called tauopathies. The activity of this promising parent VHH named Z70 relies on its binding within the central region of the tau protein. Accordingly, we carried out random mutagenesis followed by yeast two-hybrid screening to obtain optimized variants. The VHHs selected from this initial screen targeted the same epitope as VHH Z70 as shown using NMR spectroscopy and had indeed improved binding affinities according to dissociation constant values obtained by surface plasmon resonance spectroscopy. The improved affinities can be partially rationalized based on three-dimensional structures and NMR data of three complexes consisting of an optimized VHH and a peptide containing the tau epitope. Interestingly, the ability of the VHH variants to inhibit tau aggregation and seeding could not be predicted from their affinity alone. We indeed showed that the in vitro and in cellulo VHH stabilities are other limiting key factors to their efficacy. Our results demonstrate that only a complete pipeline of experiments, here described, permits a rational selection of optimized VHH variants, resulting in the selection of VHH variants with higher affinities and/or acting against tau seeding in cell models.

Reversal of RNA toxicity in myotonic dystrophy via a decoy RNA-binding protein with high affinity for expanded CUG repeats.

Myotonic dystrophy type 1 (DM1) is an RNA-dominant disease whose pathogenesis stems from the functional loss of muscleblind-like RNA-binding proteins (RBPs), which causes the formation of alternative-splicing defects. The loss of functional muscleblind-like protein 1 (MBNL1) results from its nuclear sequestration by mutant transcripts containing pathogenic expanded CUG repeats (CUGexp). Here we show that an RBP engineered to act as a decoy for CUGexp reverses the toxicity of the mutant transcripts. In vitro, the binding of the RBP decoy to CUGexp in immortalized muscle cells derived from a patient with DM1 released sequestered endogenous MBNL1 from nuclear RNA foci, restored MBNL1 activity, and corrected the transcriptomic signature of DM1. In mice with DM1, the local or systemic delivery of the RBP decoy via an adeno-associated virus into the animals' skeletal muscle led to the long-lasting correction of the splicing defects and to ameliorated disease pathology. Our findings support the development of decoy RBPs with high binding affinities for expanded RNA repeats as a therapeutic strategy for myotonic dystrophies.

[Extracellular vesicles in the central nervous system]. / Les vésicules extracellulaires - Actrices de la communication entre les cellules du système nerveux.

Extracellular Vesicles (EVs) are released by a wide diversity of cells. They contain proteins, RNAs and lipids that will be exchanged between these cells. They represent therefore a major form of intercellular communication in both physiological and pathological conditions. This is particularly relevant in the nervous system where neurons and glial cells form a very dense network where billions of connections are made. In this review, the different roles played by the EVs in a healthy brain to maintain cerebral homeostasis during development, synaptic transmission or axonal myelination will be discussed. In addition, the pathological aspects of EVs presence will also be addressed. In recent years, the EVs have emerged as major players in the spread of neurodegenerative diseases, in neuroinflammation and in tumor development, although they may also be beneficial in some conditions.

TITLE: Les vésicules extracellulaires - Actrices de la communication entre les cellules du système nerveux. ABSTRACT: Les vésicules extracellulaires (VE) sont libérées par une grande variété de cellules et contiennent des protéines, des ARN et des lipides, qui sont ainsi échangés entre ces cellules. Elles représentent donc un mode de communication intercellulaire majeur aussi bien en conditions physiologiques que pathologiques. C'est notamment le cas dans le système nerveux (SN) où les neurones et les cellules gliales forment un réseau très dense et où des milliards de connexions s'établissent. Cette revue fournit un aperçu des différents rôles joués par les VE dans un cerveau sain lors du renforcement des réseaux par exemple, mais également dans un cerveau malade où les VE participent, entre autres, à la progression des maladies neurodégénératives et tumorales.

Single Domain Antibody Fragments as New Tools for the Detection of Neuronal Tau Protein in Cells and in Mice Studies.

Tau is a neuronal protein linked to pathologies called tauopathies, including Alzheimer's disease. In Alzheimer's disease, tau aggregates into filaments, leading to the observation of intraneuronal fibrillary tangles. Molecular mechanisms resulting in tau aggregation and in tau pathology spreading through the brain regions are still not fully understood. New tools are thus needed to decipher tau pathways involved in the diseases. In this context, a family of novel single domain antibody fragments, or VHHs, directed against tau were generated and characterized. Among the selected VHHs obtained from screening of a synthetic library, a family of six VHHs shared the same CDR3 recognition loop and recognized the same epitope, located in the C-terminal domain of tau. Affinity parameters characterizing the tau/VHHs interaction were next evaluated using surface plasmon resonance spectroscopy. The equilibrium constants KD were in the micromolar range, but despite conservation of the CDR3 loop sequence, a range of affinities was observed for this VHH family. One of these VHHs, named F8-2, was additionally shown to bind tau upon expression in a neuronal cell line model. Optimization of VHH F8-2 by yeast two-hybrid allowed the generation of an optimized VHH family characterized by lower KD than that of the F8-2 wild-type counterpart, and recognizing the same epitope. The optimized VHHs can also be used as antibodies for detecting tau in transgenic mice brain tissues. These results validate the use of these VHHs for in vitro studies, but also their potential for in-cell expression and assays in mouse models, to explore the mechanisms underlying tau physiopathology.

Potentiating tangle formation reduces acute toxicity of soluble tau species in the rat.

Tauopathies are neurodegenerative diseases characterized by the aggregation of tau protein. These pathologies exhibit a wide variety of clinical and anatomo-pathological presentations, which may result from different pathological mechanisms. Although tau inclusions are a common feature in all these diseases, recent evidence instead implicates small oligomeric aggregates as drivers of tau-induced toxicity. Hence in vivo model systems displaying either soluble or fibrillary forms of wild-type or mutant tau are needed to better identify their respective pathological pathways. Here we used adeno-associated viruses to mediate gene transfer of human tau to the rat brain to develop models of pure tauopathies. Two different constructs were used, each giving rise to a specific phenotype developing in less than 3 months. First, hTAUWT overexpression led to a strong hyperphosphorylation of the protein, which was associated with neurotoxicity in the absence of any significant aggregation. In sharp contrast, its co-expression with the pro-aggregation peptide TauRD-ΔK280 in the hTAUProAggr group strongly promoted its aggregation into Gallyas-positive neurofibrillary tangles, while preserving neuronal survival. Our results support the hypothesis that soluble tau species are key players of tau-induced neurodegeneration.

ß-Amyloid Precursor Protein Intracellular Domain Controls Mitochondrial Function by Modulating Phosphatase and Tensin Homolog-Induced Kinase 1 Transcription in Cells and in Alzheimer Mice Models.

BACKGROUND: Mitophagy and mitochondrial dynamics alterations are two major hallmarks of neurodegenerative diseases. Dysfunctional mitochondria accumulate in Alzheimer's disease-affected brains by yet unexplained mechanisms. METHODS: We combined cell biology, molecular biology, and pharmacological approaches to unravel a novel molecular pathway by which presenilins control phosphatase and tensin homolog-induced kinase 1 (Pink-1) expression and transcription. In vivo approaches were carried out on various transgenic and knockout animals as well as in adeno-associated virus-infected mice. Functional readout and mitochondrial physiology (mitochondrial potential) were assessed by combined procedures including flow cytometry, live imaging analysis, and immunohistochemistry. RESULTS: We show that presenilins 1 and 2 trigger opposite effects on promoter transactivation, messenger RNA, and protein expression of Pink-1. This control is linked to γ-secretase activity and ß-amyloid precursor protein but is independent of phosphatase and tensin homolog. We show that amyloid precursor protein intracellular domain (AICD) accounts for presenilin-dependent phenotype and upregulates Pink-1 transactivation in cells as well as in vivo in a Forkhead box O3a-dependent manner. Interestingly, the modulation of γ-secretase activity or AICD expression affects Pink-1-related control of mitophagy and mitochondrial dynamics. Finally, we show that parkin acts upstream of presenilins to control Pink-1 promoter transactivation and protein expression. CONCLUSIONS: Overall, we delineate a molecular cascade presenilins-AICD-Forkhead box O3a linking parkin to Pink-1. Our study demonstrates AICD-mediated Pink-1-dependent control of mitochondrial physiology by presenilins. Furthermore, it unravels a parkin-Pink-1 feedback loop controlling mitochondrial physiology that could be disrupted in neurodegenerative conditions.

Tunneling nanotube (TNT)-mediated neuron-to neuron transfer of pathological Tau protein assemblies.

A given cell makes exchanges with its neighbors through a variety of means ranging from diffusible factors to vesicles. Cells use also tunneling nanotubes (TNTs), filamentous-actin-containing membranous structures that bridge and connect cells. First described in immune cells, TNTs facilitate HIV-1 transfer and are found in various cell types, including neurons. We show that the microtubule-associated protein Tau, a key player in Alzheimer's disease, is a bona fide constituent of TNTs. This is important because Tau appears beside filamentous actin and myosin 10 as a specific marker of these fine protrusions of membranes and cytosol that are difficult to visualize. Furthermore, we observed that exogenous Tau species increase the number of TNTs established between primary neurons, thereby facilitating the intercellular transfer of Tau fibrils. In conclusion, Tau may contribute to the formation and function of the highly dynamic TNTs that may be involved in the prion-like propagation of Tau assemblies.

Tau monoclonal antibody generation based on humanized yeast models: impact on Tau oligomerization and diagnostics.

A link between Tau phosphorylation and aggregation has been shown in different models for Alzheimer disease, including yeast. We used human Tau purified from yeast models to generate new monoclonal antibodies, of which three were further characterized. The first antibody, ADx201, binds the Tau proline-rich region independently of the phosphorylation status, whereas the second, ADx215, detects an epitope formed by the Tau N terminus when Tau is not phosphorylated at Tyr(18). For the third antibody, ADx210, the binding site could not be determined because its epitope is probably conformational. All three antibodies stained tangle-like structures in different brain sections of THY-Tau22 transgenic mice and Alzheimer patients, and ADx201 and ADx210 also detected neuritic plaques in the cortex of the patient brains. In hippocampal homogenates from THY-Tau22 mice and cortex homogenates obtained from Alzheimer patients, ADx215 consistently stained specific low order Tau oligomers in diseased brain, which in size correspond to Tau dimers. ADx201 and ADx210 additionally reacted to higher order Tau oligomers and presumed prefibrillar structures in the patient samples. Our data further suggest that formation of the low order Tau oligomers marks an early disease stage that is initiated by Tau phosphorylation at N-terminal sites. Formation of higher order oligomers appears to require additional phosphorylation in the C terminus of Tau. When used to assess Tau levels in human cerebrospinal fluid, the antibodies permitted us to discriminate patients with Alzheimer disease or other dementia like vascular dementia, indicative that these antibodies hold promising diagnostic potential.

A fiber-modified adenoviral vector interacts with immunoevasion molecules of the B7 family at the surface of murine leukemia cells derived from dormant tumors.

Tumor cells can escape the immune system by overexpressing molecules of the B7 family, e.g. B7-H1 (PD-L1 or CD86), which suppresses the anti-tumor T-cell responses through binding to the PD-1 receptor, and similarly for B7.1 (CD80), through binding to CTLA-4. Moreover, direct interactions between B7-H1 and B7.1 molecules are also likely to participate in the immunoevasion mechanism. In this study, we used a mouse model of tumor dormancy, DA1-3b leukemia cells. We previously showed that a minor population of DA1-3b cells persists in equilibrium with the immune system for long periods of time, and that the levels of surface expression of B7-H1 and B7.1 molecules correlates with the dormancy time. We found that leukemia cells DA1-3b/d365 cells, which derived from long-term dormant tumors and overexpressed B7-H1 and B7.1 molecules, were highly permissive to Ad5FB4, a human adenovirus serotype 5 (Ad5) vector pseudotyped with chimeric human-bovine fibers. Both B7-H1 and B7.1 were required for Ad5FB4-cell binding and entry, since (i) siRNA silencing of one or the other B7 gene transcript resulted in a net decrease in the cell binding and Ad5FB4-mediated transduction of DA1-3b/d365; and (ii) plasmid-directed expression of B7.1 and B7-H1 proteins conferred to Ad5FB4-refractory human cells a full permissiveness to this vector. Binding data and flow cytometry analysis suggested that B7.1 and B7-H1 molecules played different roles in Ad5FB4-mediated transduction of DA1-3b/d365, with B7.1 involved in cell attachment of Ad5FB4, and B7-H1 in Ad5FB4 internalization. BRET analysis showed that B7.1 and B7-H1 formed heterodimeric complexes at the cell surface, and that Ad5FB4 penton, the viral capsomere carrying the fiber projection, could negatively interfere with the formation of B7.1/B7-H1 heterodimers, or modify their conformation. As interactors of B7-H1/B7.1 molecules, Ad5FB4 particles and/or their penton capsomeres represent potential therapeutic agents targeting cancer cells that had developed immunoevasion mechanisms.

Liver X receptor activation induces the uptake of cholesteryl esters from high density lipoproteins in primary human macrophages.

OBJECTIVE: Liver X receptors (LXRs) are oxysterol-activated nuclear receptors regulating reverse cholesterol transport, in part by modulating cholesterol efflux from macrophages to apoAI and HDL via the ABCA1 and ABCG1/ABCG4 pathways. Moreover, LXR activation increases intracellular cholesterol trafficking via the induction of NPC1 and NPC2 expression. However, implication of LXRs in the selective uptake of cholesteryl esters from lipoproteins in human macrophages has never been reported. METHODS AND RESULTS: Our results show that (1) selective CE uptake from HDL(3) is highly efficient in human monocyte-derived macrophages; (2) surprisingly, HDL(3)-CE uptake is strongly increased by LXR activation despite antiatherogenic effects of LXRs; (3) HDL(3)-CE uptake increase is not linked to SR-BI expression modulation but it is dependent of proteoglycan interactions; (4) HDL(3)-CE uptake increase is associated with increased expression and secretion of apoE and LPL, two proteins interacting with proteoglycans; (5) HDL(3)-CE uptake increase depends on the integrity of raft domains and is associated with an increased caveolin-1 expression. CONCLUSIONS: Our study identifies a new role for LXRs in the control of cholesterol homeostasis in human macrophages. LXR activation results in enhanced dynamic intracellular cholesterol fluxes through an increased CE uptake from HDL and leads to an increased cholesterol availability to efflux to apoAI and HDL.

Intracellular trafficking of a fiber-modified adenovirus using lipid raft/caveolae endocytosis.

Most adenoviral vectors (HAdvs) elaborated for gene therapy are derived from serotype 5 viruses that use clathrin-coated vesicle endocytosis for cell entry. However, it appears that adenoviral vectors are able to take advantage of lipid raft/caveolae endocytosis to infect cells. In vivo targeting of a therapeutic gene to specific cells by vector engineering has become a major focus of gene therapy research. Yet, modification of adenoviral tropism, especially fiber gene engineering, can induce deficient intracellular trafficking of the viral particle, with a shift in subcellular localization resulting in extensive exocytosis. In this study we demonstrate that uptake of a fiber-modified adenovirus using lipid raft/caveolae endocytosis leads to non-altered intracellular trafficking without endosomal retention. Moreover, activation of lipid raft structures by this vector leads to the formation of "mega-caveosomes". These results demonstrate that, by forcing adenoviruses to take advantage of a non-clathrin, non-classical endocytic pathway, it is possible to compensate for the deficiency in endosomolysis that is associated with the use of some of the fiber-modified adenoviral constructs. Moreover, it renders such vectors ideal candidates for infecting human coxsackie and adenoviruses receptor (hCAR) negative cells.

Improved gene delivery to B lymphocytes using a modified adenovirus vector targeting CD21.

Gene transfer by adenoviruses, which are widely used for gene therapy, may provide an alternative approach to treatment of several hematopoietic malignancies. However, a major limitation of adenovirus 5-based gene therapy lies in the natural tropism of the virus for the widely expressed hCAR receptor. The efficacy of adenoviral vectors could be improved if viral vectors that exhibit tissue-specific gene delivery were developed. For efficient gene transfer it is essential that every step from binding of virus to target cells to transgene expression is successfully accomplished. We developed a specific vector targeting the CD21 receptor, by inserting a CD21 binding sequence, derived from the EBV GP350/220 protein, into the HI loop of the HAdV5 fiber protein. This vector, HAdV5-CD21HIloop, binds specifically to CD21-positive cells and results in enhanced expression of the transgene in these cells and reduced expression in CD21-negative cells. Viral infection is highly correlated with the presence of CD21 receptors. Taken together, these results demonstrate that HAdV5-CD21HIloop is able to transduce CD21-positive cells specifically with reduced infection of nontarget cells. This is the result of the maintenance of the intracellular trafficking of the genetically modified adenovirus without vesicular retention, leading to enhanced nuclear transfer.

Efficient species C HAdV infectivity in plasmocytic cell lines using a clathrin-independent lipid raft/caveola endocytic route.

Hematopoietic cells are known to be refractory to species C human adenovirus (HAdV) infection; however, the reason for this has not been clearly established. We have previously demonstrated that this nonpermissivity is the consequence of inefficient HAdV particle uptake, notably in B lymphocytes. We noted that while the protein clathrin is observed in association with membranes in epithelial cells, it is found predominantly in the cytoplasm of hematopoietic cell lines. So it appears that altered clathrin-coated pit endocytosis could explain the weak HAdV uptake in B cells. In contrast, mature B cell plasmocytes are permissive to HAdV. However, this is not the result of clathrin-coated pit endocytosis since this process is also inefficient in these cells. Confocal microscopy showed colocalization between HAdV particles and caveolae/lipid rafts in plasmocytes. Moreover, inhibiting caveola endocytosis by depletion of cholesterol or expression of dominant negative caveolin-1 in these cells results in a 50-70% reduction in HAdV infectivity. It appears that caveola endocytosis and nonclathrin noncaveola endocytosis are used by HAdV to enter plasmocytes in response to a loss of the clathrin-dependent pathway. Thus targeting of caveolae by modifying the capsid of HAdV may represent an alternative approach to enhancing uptake in most hematopoietic cells for future gene therapy.

Abolition of hCAR-dependent cell tropism using fiber knobs of Atadenovirus serotypes.

Most adenoviral vectors use in gene therapy protocols derive from species C. However, expression of the primary receptor (human Coxsackievirus and Adenovirus receptor, hCAR) for these AdV is variable on cancer cells. In vivo targeting of a therapeutic gene to specific cells has then become a major issue in gene therapy. The Ad fiber protein largely determines viral tropism through interaction with specific receptors. Hereto, we constructed a set of HAdV5 vectors carrying chimeric fibers with knob domains from nonhuman AdV, namely from the FAdV-1 (Aviadenovirus), DAdV-1, and BAdV-4 (Atadenovirus). Correspondents viruses were produced using an established new HEK293 cell line, which express the HAdV2 fiber. Recombinant HAdV harboring chimeric fibers constituted of the N-terminal domain of HAdV2, and knob domain of bovine adenovirus type 4 (BAdV-4) demonstrated the greatest reduction in fiber-mediated gene transfer into human cells expressing the hCAR. Moreover, this vector infects with a better efficiency than vector with wild-type fiber, the Chinese Hamster Ovarian (CHO) and SKOV3 cell lines, both from ovarian origin, hamster and human, respectively. These studies support the concept that changing the fiber knob domain to ablate hCAR interaction should result in a de- or retargeted adenoviral vector. The adenoviral vector with the chimeric HAdV2/BAdV-4 fiber lacking hCAR interaction and with an ovarian cell tropism could be a nice candidate to elaborate vectors for ovarian tumor therapy.

Factors involved in the sensitivity of different hematopoietic cell lines to infection by subgroup C adenovirus: implication for gene therapy of human lymphocytic malignancies.

Gene transfer approaches using viruses such human adenovirus (HAdV) may provide an alternative treatment for diseases involving hematopoietic cells. Better understanding of the cellular mechanisms by which the HAdV introduces DNA into these cells should help in vector design. We examined HAdV intracellular delivery in several cell lines including B and T lymphocytes. We demonstrated that HAdV resistance in most B lymphocytes is the result of moderate HAdV uptake. In contrast, high levels of coxsackie and HAdV receptor (hCAR) are expressed on the surface of HSB2 (T cells), allowing efficient binding and uptake but no transgene expression, probably because of deficient endosomolysis and subsequent exocytose. This work demonstrates the existence of hCAR-dependent and -independent endocytic route in hematopoietic cells. Moreover, it precises the intracellular barriers to be overcome by HAdV in such cells to be infectious and gives previous information's to design new vectors for gene transfer.

Nuclear localisation sequence templated nonviral gene delivery vectors: investigation of intracellular trafficking events of LMD and LD vector systems.

The impact of a peptide that contains a nuclear localisation sequence (NLS) on intracellular DNA trafficking was studied. We used the adenoviral core peptide mu and an SV40 NLS peptide to condense plasmid DNA (pDNA) prior to formulation with 3beta-[N-(N', N'-dimethylaminoethane)carbamoyl]cholesterol/dioleoyl-L-alpha-phosphatidyl ethanolamine (DC-Chol/DOPE) liposomes to give LMD and LND vectors, respectively. Fluorescent-labelled lipid and peptides plus dye-labelled pDNA components were used to investigate gene delivery in dividing and S-phase growth-arrested cells. Confocal microscopic analyses reveal little difference in intracellular trafficking events. Strikingly, mu peptide associates with nuclei and nucleoli of cells within less than 15 mins incubation of LMD with cells, which suggests that mu peptide has an NLS function. These NLS properties were confirmed by cloning of a mu-beta-galactosidase fusion protein that localises in the nuclei of cells after cytosolic translation. In dividing cells both LMD and LND deliver pDNA(Cy3) to nuclei within 30-45 min incubation with cells. By contrast, pDNA is detected only in the cytoplasm in growth-arrested cells over the period of time investigated, and not in the nuclei. LD systems prepared from DC-Chol/DOPE cationic liposomes and pDNA(Cy3) behave similarly to LMD systems, which suggests that mu peptide is unable to influence trafficking events in this current LMD formulation, in spite of its strong NLS capacity. We further describe the effect of polyethyleneglycol (PEG) on cellular uptake. "Stealth" systems obtained by post-coating LMD particles with fluorescent-labelled PEG molecules (0.5, 5 and 10 mol % fluorescein-PEG(5000)-N-hydroxysuccinimide) were prepared and shown to be internalised rapidly (mins) by cells, without detectable transgene expression. This result indicates that PEG blocks intracellular trafficking of pDNA.