LDLR-Mediated Targeting and Productive Uptake of siRNA-Peptide Ligand Conjugates In Vitro and In Vivo.

Small RNA molecules such as microRNA and small interfering RNA (siRNA) have become promising therapeutic agents because of their specificity and their potential to modulate gene expression. Any gene of interest can be potentially up- or down-regulated, making RNA-based technology the healthcare breakthrough of our era. However, the functional and specific delivery of siRNAs into tissues of interest and into the cytosol of target cells remains highly challenging, mainly due to the lack of efficient and selective delivery systems. Among the variety of carriers for siRNA delivery, peptides have become essential candidates because of their high selectivity, stability, and conjugation versatility. Here, we describe the development of molecules encompassing siRNAs against SOD1, conjugated to peptides that target the low-density lipoprotein receptor (LDLR), and their biological evaluation both in vitro and in vivo.

In the Rat Hippocampus, Pilocarpine-Induced Status Epilepticus Is Associated with Reactive Glia and Concomitant Increased Expression of CD31, PDGFRß, and Collagen IV in Endothelial Cells and Pericytes of the Blood-Brain Barrier.

In humans and animal models, temporal lobe epilepsy (TLE) is associated with reorganization of hippocampal neuronal networks, gliosis, neuroinflammation, and loss of integrity of the blood-brain barrier (BBB). More than 30% of epilepsies remain intractable, and characterization of the molecular mechanisms involved in BBB dysfunction is essential to the identification of new therapeutic strategies. In this work, we induced status epilepticus in rats through injection of the proconvulsant drug pilocarpine, which leads to TLE. Using RT-qPCR, double immunohistochemistry, and confocal imaging, we studied the regulation of reactive glia and vascular markers at different time points of epileptogenesis (latent phase-3, 7, and 14 days; chronic phase-1 and 3 months). In the hippocampus, increased expression of mRNA encoding the glial proteins GFAP and Iba1 confirmed neuroinflammatory status. We report for the first time the concomitant induction of the specific proteins CD31, PDGFRß, and ColIV-which peak at the same time points as inflammation-in the endothelial cells, pericytes, and basement membrane of the BBB. The altered expression of these proteins occurs early in TLE, during the latent phase, suggesting that they could be associated with the early rupture and pathogenicity of the BBB that will contribute to the chronic phase of epilepsy.

Neurotensin and Neurotensin Receptors in Stress-related Disorders: Pathophysiology & Novel Drug Targets.

Neurotensin (NT) is a 13-amino acid neuropeptide widely distributed in the CNS that has been involved in the pathophysiology of many neural and psychiatric disorders. There are three known neurotensin receptors (NTSRs), which mediate multiple actions, and form the neurotensinergic system in conjunction with NT. NTSR1 is the main mediator of NT, displaying effects in both the CNS and the periphery, while NTSR2 is mainly expressed in the brain and NTSR3 has a broader expression pattern. In this review, we bring together up-to-date studies showing an involvement of the neurotensinergic system in different aspects of the stress response and the main stress-related disorders, such as depression and anxiety, post-traumatic stress disorder (PTSD) and its associated symptoms, such as fear memory and maternal separation, ethanol addiction, and substance abuse. Emphasis is put on gene, mRNA, and protein alterations of NT and NTSRs, as well as behavioral and pharmacological studies, leading to evidence-based suggestions on the implicated regulating mechanisms as well as their therapeutic exploitation. Stress responses and anxiety involve mainly NTSR1, but also NTSR2 and NTSR3. NTSR1 and NTSR3 are primarily implicated in depression, while NTSR2 and secondarily NTSR1 in PTSD. NTSR1 is interrelated with substance and drug abuse and NTSR2 with fear memory, while all NTSRs seem to be implicated in ethanol consumption. Some of the actions of NT and NTSRs in these pathological settings may be driven through interactions between NT and corticotrophin releasing factor (CRF) in their regulatory contribution, as well as by NT's pro-inflammatory mediating actions.

Preparation and In Vitro Validation of a Cucurbit[7]uril-Peptide Conjugate Targeting the LDL Receptor.

Here we report the coupling of a cyclic peptide (VH4127) targeting the low density lipoprotein (LDL) receptor (LDLR) noncompetitively to cucurbit[7]uril (CB[7]) to develop a new kind of drug delivery system (DDS), namely, CB[7]-VH4127, with maintained binding affinity to the LDLR. To evaluate the uptake potential of this bismacrocyclic compound, another conjugate was prepared comprising a high-affinity group for CB[7] (adamantyl(Ada)-amine) coupled to the fluorescent tracker Alexa680 (A680). The resulting A680-Ada·CB[7]-VH4127 supramolecular complex demonstrated conserved LDLR-binding potential and improved LDLR-mediated endocytosis and intracellular accumulation potential in LDLR-expressing cells. The combination of two technologies, namely, monofunctionalized CB[7] and the VH4127 LDLR-targeting peptide, opens new avenues in terms of targeting and intracellular delivery to LDLR-expressing tissues or tumors. The versatile transport capacity of CB[7], known to bind a large spectrum of bioactive or functional compounds, makes this new DDS suitable for a wide range of therapeutic or imaging applications.

High and low permeability of human pluripotent stem cell-derived blood-brain barrier models depend on epithelial or endothelial features.

The search for reliable human blood-brain barrier (BBB) models represents a challenge for the development/testing of strategies aiming to enhance brain delivery of drugs. Human-induced pluripotent stem cells (hiPSCs) have raised hopes in the development of predictive BBB models. Differentiating strategies are thus required to generate endothelial cells (ECs), a major component of the BBB. Several hiPSC-based protocols have reported the generation of in vitro models with significant differences in barrier properties. We studied in depth the properties of iPSCs byproducts from two protocols that have been established to yield these in vitro barrier models. Our analysis/study reveals that iPSCs derivatives endowed with EC features yield high permeability models while the cells that exhibit outstanding barrier properties show principally epithelial cell-like (EpC) features. We found that models containing EpC-like cells express tight junction proteins, transporters/efflux pumps and display a high functional tightness with very low permeability, which are features commonly shared between BBB and epithelial barriers. Our study demonstrates that hiPSC-based BBB models need extensive characterization beforehand and that a reliable human BBB model containing EC-like cells and displaying low permeability is still needed.

The Helico Maze Detects Early Impairment of Reference Memory at Three Months of Age in the 5XFAD Mouse Model of Alzheimer's Disease.

BACKGROUND: The 5XFAD model of Alzheimer's disease (AD) bearing five familial mutations of Alzheimer's disease on human APP and PSEN1 transgenes shows deposits of amyloid-ß peptide (Aß) as early as 2 months, while deficits in long-term memory can be detected at 4 months using the highly sensitive olfactory-dependent tests that we previously reported. OBJECTIVE: Given that detecting early dysfunctions in AD prior to overt pathology is of major interest in the field, we sought to detect memory deficits at earlier stages of the disease in 3-month-old male 5XFAD mice. METHODS: To this end, we used the Helico Maze, a behavioral task that was recently developed and patented. This device allows deeper analysis of learning and subcategories of hippocampal-dependent long-term memory using olfactory cues. RESULTS: Eight male 5XFAD and 6 male wild-type (WT: C57Bl6 background) mice of 3 months of age were tested in the Helico Maze. The results demonstrated, for the first time, a starting deficit of pure reference long-term memory. Interestingly, memory impairment was clearly correlated with Aß deposits in the hippocampus. While we also found significant differences in astrogliosis between 5XFAD and WT mice, this was not correlated with memory abilities. CONCLUSION: Our results underline the efficiency of this new olfactory-dependent behavioral task, which is easy to use, with a small cohort of mice. Using the Helico Maze may open new avenues to validate the efficacy of treatments that target early events related to the amyloid-dependent pathway of the disease and AD progression.

MT5-MMP promotes neuroinflammation, neuronal excitability and Aß production in primary neuron/astrocyte cultures from the 5xFAD mouse model of Alzheimer's disease.

BACKGROUND: Membrane-type matrix metalloproteinase 5 (MT5-MMP) deficiency in the 5xFAD mouse model of Alzheimer's disease (AD) reduces brain neuroinflammation and amyloidosis, and prevents deficits in synaptic activity and cognition in prodromal stages of the disease. In addition, MT5-MMP deficiency prevents interleukin-1 beta (IL-1ß)-mediated inflammation in the peripheral nervous system. In this context, we hypothesized that the MT5-MMP/IL-1ß tandem could regulate nascent AD pathogenic events in developing neural cells shortly after the onset of transgene activation. METHODS: To test this hypothesis, we used 11-14 day in vitro primary cortical cultures from wild type, MT5-MMP-/-, 5xFAD and 5xFAD/MT5-MMP-/- mice, and evaluated the impact of MT5-MMP deficiency and IL-1ß treatment for 24 h, by performing whole cell patch-clamp recordings, RT-qPCR, western blot, gel zymography, ELISA, immunocytochemistry and adeno-associated virus (AAV)-mediated transduction. RESULTS: 5xFAD cells showed higher levels of MT5-MMP than wild type, concomitant with higher basal levels of inflammatory mediators. Moreover, MT5-MMP-deficient cultures had strong decrease of the inflammatory response to IL-1ß, as well as decreased stability of recombinant IL-1ß. The levels of amyloid beta peptide (Aß) were similar in 5xFAD and wild-type cultures, and IL-1ß treatment did not affect Aß levels. Instead, the absence of MT5-MMP significantly reduced Aß by more than 40% while sparing APP metabolism, suggesting altogether no functional crosstalk between IL-1ß and APP/Aß, as well as independent control of their levels by MT5-MMP. The lack of MT5-MMP strongly downregulated the AAV-induced neuronal accumulation of the C-terminal APP fragment, C99, and subsequently that of Aß. Finally, MT5-MMP deficiency prevented basal hyperexcitability observed in 5xFAD neurons, but not hyperexcitability induced by IL-1ß treatment. CONCLUSIONS: Neuroinflammation and hyperexcitability precede Aß accumulation in developing neural cells with nascent expression of AD transgenes. MT5-MMP deletion is able to tune down basal neuronal inflammation and hyperexcitability, as well as APP/Aß metabolism. In addition, MT5-MMP deficiency prevents IL-1ß-mediated effects in brain cells, except hyperexcitability. Overall, this work reinforces the idea that MT5-MMP is at the crossroads of pathogenic AD pathways that are already incipiently activated in developing neural cells, and that targeting MT5-MMP opens interesting therapeutic prospects.

LDL receptor-peptide conjugate as in vivo tool for specific targeting of pancreatic ductal adenocarcinoma.

Despite clinical advances in diagnosis and treatment, pancreatic ductal adenocarcinoma (PDAC) remains the third leading cause of cancer death, and is still associated with poor prognosis and dismal survival rates. Identifying novel PDAC-targeted tools to tackle these unmet clinical needs is thus an urgent requirement. Here we use a peptide conjugate that specifically targets PDAC through low-density lipoprotein receptor (LDLR). We demonstrate by using near-infrared fluorescence imaging the potential of this conjugate to specifically detect and discriminate primary PDAC from healthy organs including pancreas and from benign mass-forming chronic pancreatitis, as well as detect metastatic pancreatic cancer cells in healthy liver. This work paves the way towards clinical applications in which safe LDLR-targeting peptide conjugate promotes tumor-specific delivery of imaging and/or therapeutic agents, thereby leading to substantial improvements of the PDAC patient's outcome.

Neurotensin receptor 2 is induced in astrocytes and brain endothelial cells in relation to neuroinflammation following pilocarpine-induced seizures in rats.

Neurotensin (NT) acts as a primary neurotransmitter and neuromodulator in the CNS and has been involved in a number of CNS pathologies including epilepsy. NT mediates its central and peripheral effects by interacting with the NTSR1, NTSR2, and Sort1/NTSR3 receptor subtypes. To date, little is known about the precise expression of the NT receptors in brain neural cells and their regulation in pathology. In the present work, we studied the cellular distribution of the NTSR2 protein in the rat hippocampus and questioned whether its expression was modulated in conditions of neuroinflammation using a model of temporal lobe epilepsy induced by pilocarpine. This model is characterized by a rapid and intense inflammatory reaction with reactive gliosis in the hippocampus. We show that NTSR2 protein is expressed in hippocampal astrocytes and its expression increases together with astrocyte reactivity following induction of status epilepticus. NTSR2 immunoreactivity is also increased in astrocytes proximal to blood vessels and their end-feet, and in endothelial cells. Proinflammatory factors such as IL1ß and LPS induced NTSR2 mRNA and protein in cultured astroglial cells. Antagonizing NTSR2 with SR142948A decreased NTSR2 expression as well as astroglial reactivity. Together, our results suggest that NTSR2 is implicated in astroglial and gliovascular inflammation and that targeting the NTSR2 receptor may open new avenues in the regulation of neuroinflammation in CNS diseases.

MT5-MMP controls APP and ß-CTF/C99 metabolism through proteolytic-dependent and -independent mechanisms relevant for Alzheimer's disease.

We previously discovered the implication of membrane-type 5-matrix metalloproteinase (MT5-MMP) in Alzheimer's disease (AD) pathogenesis. Here, we shed new light on pathogenic mechanisms by which MT5-MMP controls the processing of amyloid precursor protein (APP) and the fate of amyloid beta peptide (Aß) as well as its precursor C99, and C83. We found in human embryonic kidney cells (HEK) carrying the APP Swedish familial mutation (HEKswe) that deleting the C-terminal non-catalytic domains of MT5-MMP hampered its ability to process APP and release the soluble 95 kDa form (sAPP95). Catalytically inactive MT5-MMP variants increased the levels of Aß and promoted APP/C99 sorting in the endolysosomal system, likely through interactions of the proteinase C-terminal portion with C99. Most interestingly, the deletion of the C-terminal domain of MT5-MMP caused a strong degradation of C99 by the proteasome and prevented Aß accumulation. These discoveries reveal new control of MT5-MMP over APP by proteolytic and non-proteolytic mechanisms driven by the C-terminal domains of the proteinase. The targeting of these non-catalytic domains of MT5-MMP could, therefore, provide new insights into the therapeutic regulation of APP-related pathology in AD.

The Helico Maze allows testing of early learning and subcategories of long-term memory in mice.

Different memory systems operate in parallel to support behaviour. To evaluate procedural and reference subcategories of long-term memory as early as possible in the mouse, the Helico Maze (HM) was developed. BALB/c AnNCrl (BALB), C57BL/6JRj (C57) and DBA/2 JRj (DBA) mice were trained on this new maze. The three strains learned how to use the HM (procedural memory), and they then learned and remembered four odour-reward associations (reference memory). The three strains differed in the number of correct responses. BALB mice showed better performance than C57 and DBA mice. The results of the first block of each session revealed that only the BALB and C57 mice remembered the odour-reward associations. DBA mice needed to relearn the associations each day. With this new apparatus, the number of olfactory cue-reward associations was increased from 2 to 4 in comparison to a previous olfactory tubing maze. Consequently, a supplementary effort of memory was required, and the chance level was decreased from 50 % to 25 %. Thus, in several important respects, the HM can be considered to measure the hippocampus-dependent behaviour of the mouse, allowing to study, as early as possible in young mice, the different subcategories of long-term memory, such as those observed in humans.

The actin binding protein α-actinin-2 expression is associated with dendritic spine plasticity and migrating granule cells in the rat dentate gyrus following pilocarpine-induced seizures.

α-actinin-2 (α-actn-2) is an F-actin-crosslinking protein, localized in dendritic spines. In vitro studies suggested that it is involved in spinogenesis, morphogenesis, actin organization, cell migration and anchoring of the NR1 subunit of the N-methyl-D-aspartate (NMDA) receptors in dendritic spines. However, little is known regarding its function in vivo. We examined the levels of α-actn-2 expression within the dentate gyrus (DG) during the development of chronic limbic seizures (epileptogenesis) induced by pilocarpine in rats. In this model, plasticity of the DG glutamatergic granule cells including spine loss, spinogenesis, morphogenesis, neo-synaptogenesis, aberrant migration, and alterations of NMDA receptors have been well characterized. We showed that α-actn-2 immunolabeling was reduced in the inner molecular layer at 1-2 weeks post-status epilepticus (SE), when granule cell spinogenesis and morphogenesis occur. This low level persisted at the chronic stage when new functional synapses are established. This decreased of α-actn-2 protein is concomitant with the recovery of drebrin A (DA), another actin-binding protein, at the chronic stage. Indeed, we demonstrated in cultured cells that in contrast to DA, α-actn-2 did not protect F-actin destabilization and DA inhibited α-actn-2 binding to F-actin. Such alteration could affect the anchoring of NR1 in dendritic spines. Furthermore, we showed that the expression of α-actn-2 and NR1 are co-down-regulated in membrane fractions of pilocarpine animals at chronic stage. Last, we showed that α-actn-2 is expressed in migrating newly born granule cells observed within the hilus of pilocarpine-treated rats. Altogether, our results suggest that α-actn-2 is not critical for the structural integrity and stabilization of granule cell dendritic spines. Instead, its expression is regulated when spinogenesis and morphogenesis occur and within migrating granule cells. Our data also suggest that the balance between α-actn-2 and DA expression levels may modulate NR1 anchoring within dendritic spines.

Long-Term Pantethine Treatment Counteracts Pathologic Gene Dysregulation and Decreases Alzheimer's Disease Pathogenesis in a Transgenic Mouse Model.

The low-molecular weight thiol pantethine, known as a hypolipidemic and hypocholesterolemic agent, is the major precursor of co-enzyme A. We have previously shown that pantethine treatment reduces amyloid-ß (Aß)-induced IL-1ß release and alleviates pathological metabolic changes in primary astrocyte cultures. These properties of pantethine prompted us to investigate its potential benefits in vivo in the 5XFAD (Tg) mouse model of Alzheimer's disease (AD).1.5-month-old Tg and wild-type (WT) male mice were submitted to intraperitoneal administration of pantethine or saline control solution for 5.5 months. The effects of such treatments were investigated by performing behavioral tests and evaluating astrogliosis, microgliosis, Αß deposition, and whole genome expression arrays, using RNAs extracted from the mice hippocampi. We observed that long-term pantethine treatment significantly reduced glial reactivity and Αß deposition, and abrogated behavioral alteration in Tg mice. Moreover, the transcriptomic profiles revealed that after pantethine treatment, the expression of genes differentially expressed in Tg mice, and in particular those known to be related to AD, were significantly alleviated. Most of the genes overexpressed in Tg compared to WT were involved in inflammation, complement activation, and phagocytosis and were found repressed upon pantethine treatment. In contrast, pantethine restored the expression of a significant number of genes involved in the regulation of Αß processing and synaptic activities, which were downregulated in Tg mice. Altogether, our data support a beneficial role for long-term pantethine treatment in preserving CNS crucial functions altered by Aß pathogenesis in Tg mice and highlight the potential efficiency of pantethine to alleviate AD pathology.

Metalloproteinases and their tissue inhibitors in Alzheimer's disease and other neurodegenerative disorders.

As life expectancy increases worldwide, age-related neurodegenerative diseases will increase in parallel. The lack of effective treatment strategies may soon lead to an unprecedented health, social and economic crisis. Any attempt to halt the progression of these diseases requires a thorough knowledge of the pathophysiological mechanisms involved to facilitate the identification of new targets and the application of innovative therapeutic strategies. The metzincin superfamily of metalloproteinases includes matrix metalloproteinases (MMP), a disintegrin and metalloproteinase (ADAM) and ADAM with thrombospondin motifs (ADAMTS). These multigenic and multifunctional proteinase families regulate the functions of an increasing number of signalling and scaffolding molecules involved in neuroinflammation, blood-brain barrier disruption, protein misfolding, synaptic dysfunction or neuronal death. Metalloproteinases and their physiological inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), are therefore, at the crossroads of molecular and cellular mechanisms that support neurodegenerative processes, and emerge as potential new therapeutic targets. We provide an overview of current knowledge on the role and regulation of metalloproteinases and TIMPs in four major neurodegenerative diseases: Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis and Huntington's disease.

gH625-liposomes as tool for pituitary adenylate cyclase-activating polypeptide brain delivery.

The blood-brain barrier (BBB) regulates the traffic of molecules into the central nervous system (CNS) and also limits the drug delivery. Due to their flexible properties, liposomes are an attractive tool to deliver drugs across the BBB. We previously characterized gH625, a peptide derived from Herpes simplex virus 1. The present study investigates the efficiency of liposomes functionalized on their surface with gH625 to promote the brain uptake of neuroprotective peptide PACAP (pituitary adenylate cyclase-activating polypeptide). Using a rat in vitro BBB model, we showed that the liposomes preparations were non-toxic for the endothelial cells, as assessed by analysis of tight junction protein ZO1 organization and barrier integrity. Next, we found that gH625 improves the transfer of liposomes across endothelial cell monolayers, resulting in both low cellular uptake and increased transport of PACAP. Finally, in vivo results demonstrated that gH625 ameliorates the efficiency of liposomes to deliver PACAP to the mouse brain after intravenous administration. gH625-liposomes improve both PACAP reaching and crossing the BBB, as showed by the higher number of brain cells labelled with PACAP. gH625-liposomes represent a promising strategy to deliver therapeutic agents to CNS and to provide an effective imaging and diagnostic tool for the brain.

High levels of serum soluble TWEAK are associated with neuroinflammation during multiple sclerosis.

BACKGROUND: Inflammation and demyelination are the main processes in multiple sclerosis. Nevertheless, to date, blood biomarkers of inflammation are lacking. TWEAK, a transmembrane protein that belongs to the TNF ligand family, has been previously identified as a potential candidate. METHODS: Twenty-eight patients (9 males, 19 females) were prospectively included after a first clinical episode suggestive of multiple sclerosis and clinically followed during 3 years. Fifty-seven healthy controls were also included. TWEAK serum levels and MRI exams including magnetization transfer imaging were performed at baseline, 6- and 12-month follow-up. RESULTS: TWEAK serum levels were significantly increased in the patient group (mean baseline = 1086 ± 493 pg/mL, mean M6 = 624 ± 302 pg/mL and mean M12 = 578 ± 245 pg/mL) compared to healthy controls (mean = 467 ± 177 pg/mL; respectively p < 0.0001, 0.01 and 0.06). Serum levels of soluble TWEAK were significantly increased during relapses, compared to time periods without any relapse (respectively 935 ± 489 pg/mL and 611 ± 292 pg/mL, p = 0.0005). Moreover, patients presenting at least one gadolinium-enhanced CNS lesion at baseline (n = 7) displayed significantly increased serum TWEAK levels in comparison with patients without any gadolinium-enhanced lesion at baseline (n = 21) (respectively 1421 ± 657 pg/mL vs 975 ± 382 pg/mL; p = 0.02). Finally, no correlation was evidenced between TWEAK serum levels and the extent of brain tissue damage assessed by magnetization transfer ratio. CONCLUSIONS: The present study showed that TWEAK serum levels are increased in MS patients, in relation to the disease activity. This simple and reproducible serum test could be used as a marker of ongoing inflammation, contributing in the follow-up and the care of MS patients. Thus, TWEAK is a promising serum marker of the best window to perform brain MRI, optimizing the disease control in patients.

Proamyloidogenic effects of membrane type 1 matrix metalloproteinase involve MMP-2 and BACE-1 activities, and the modulation of APP trafficking.

We previously demonstrated that membrane type 1 (MT1) matrix metalloproteinase (MMP) was up-regulated in the hippocampus of the model of transgenic mice bearing 5 familial mutations on human amyloid precursor protein (APP) and presenilin 1 of Alzheimer disease (AD), and that the proteinase increased the levels of amyloid ß peptide (Aß) and its APP C-terminal fragment of 99 aa in a heterologous cell system. Here we provide further evidence that MT1-MMP interacts with APP and promotes amyloidogenesis in a proteolytic-dependent manner in Swedish APP-expressing human embryonic kidney 293 (HEKswe) cells. MT1-MMP-mediated processing of APP releases a soluble APP fragment, sAPP95. This process partly requires the activation of endogenous MMP-2 but is independent of ß-site APP cleaving enzyme 1 (BACE-1) or α-secretase activities. In contrast, MT1-MMP-mediated increase of Aß levels involved BACE-1 activity and was inhibited by tissue inhibitor of MMP-2, a natural inhibitor of both MT1-MMP and MMP-2. Interestingly, near abolishment of basal Aß production upon BACE-1 inhibition was rescued by MT1-MMP, indicating that the latter could mimic ß-secretase-like activity. Moreover, MT1-MMP promoted APP/Aß localization in endosomes, where Aß production mainly occurs. These data unveil new mechanistic insights to support the proamyloidogenic role of MT1-MMP based on APP processing and trafficking, and reinforce the idea that this proteinase may become a new potential therapeutic target in AD.-Paumier, J.-M., Py, N. A., González, L. G., Bernard, A., Stephan, D., Louis, L., Checler, F., Khrestchatisky, M., Baranger, K., Rivera, S. Proamyloidogenic effects of membrane type 1 matrix metalloproteinase involve MMP-2 and BACE-1 activities, and the modulation of APP trafficking.

Syngeneic Transplantation of Olfactory Ectomesenchymal Stem Cells Restores Learning and Memory Abilities in a Rat Model of Global Cerebral Ischemia.

Stem cells are considered as promising tools to repair diverse tissue injuries. Among the different stem cell types, the "olfactory ectomesenchymal stem cells" (OE-MSCs) located in the adult olfactory mucosa stand as one of the best candidates. Here, we evaluated if OE-MSC grafts could decrease memory impairments due to ischemic injury. OE-MSCs were collected from syngeneic F344 rats. After a two-step global cerebral ischemia, inducing hippocampal lesions, learning abilities were evaluated using an olfactory associative discrimination task. Cells were grafted into the hippocampus 5 weeks after injury and animal's learning abilities reassessed. Rats were then sacrificed and the brains collected for immunohistochemical analyses. We observed significant impairments in learning and memory abilities following ischemia. However, 4 weeks after OE-MSC grafts, animals displayed learning and memory performances similar to those of controls, while sham rats did not improve them. Immunohistochemical analyses revealed that grafts promoted neuroblast and glial cell proliferation, which could permit to restore cognitive functions. These results demonstrated, for the first time, that syngeneic transplantations of OE-MSCs in rats can restore cognitive abilities impaired after brain injuries and provide support for the development of clinical studies based on grafts of OE-MSCs in amnesic patients following brain injuries.

Correction: Metabolic changes and inflammation in cultured astrocytes from the 5xFAD mouse model of Alzheimer's disease: Alleviation by pantethine.

[This corrects the article DOI: 10.1371/journal.pone.0175369.].

Identification and characterization of highly versatile peptide-vectors that bind non-competitively to the low-density lipoprotein receptor for in vivo targeting and delivery of small molecules and protein cargos.

Insufficient membrane penetration of drugs, in particular biotherapeutics and/or low target specificity remain a major drawback in their efficacy. We propose here the rational characterization and optimization of peptides to be developed as vectors that target cells expressing specific receptors involved in endocytosis or transcytosis. Among receptors involved in receptor-mediated transport is the LDL receptor. Screening complex phage-displayed peptide libraries on the human LDLR (hLDLR) stably expressed in cell lines led to the characterization of a family of cyclic and linear peptides that specifically bind the hLDLR. The VH411 lead cyclic peptide allowed endocytosis of payloads such as the S-Tag peptide or antibodies into cells expressing the hLDLR. Size reduction and chemical optimization of this lead peptide-vector led to improved receptor affinity. The optimized peptide-vectors were successfully conjugated to cargos of different nature and size including small organic molecules, siRNAs, peptides or a protein moiety such as an Fc fragment. We show that in all cases, the peptide-vectors retain their binding affinity to the hLDLR and potential for endocytosis. Following i.v. administration in wild type or ldlr-/- mice, an Fc fragment chemically conjugated or fused in C-terminal to peptide-vectors showed significant biodistribution in LDLR-enriched organs. We have thus developed highly versatile peptide-vectors endowed with good affinity for the LDLR as a target receptor. These peptide-vectors have the potential to be further developed for efficient transport of therapeutic or imaging agents into cells -including pathological cells-or organs that express the LDLR.