Transfer of mesenchymal stem cell mitochondria to CD4+ T cells contributes to repress Th1 differentiation by downregulating T-bet expression.

BACKGROUND: Mesenchymal stem/stromal cells (MSCs) are multipotent cells with strong tissue repair and immunomodulatory properties. Due to their ability to repress pathogenic immune responses, and in particular T cell responses, they show therapeutic potential for the treatment of autoimmune diseases, organ rejection and graft versus host disease. MSCs have the remarkable ability to export their own mitochondria to neighboring cells in response to injury and inflammation. However, whether mitochondrial transfer occurs and has any role in the repression of CD4+ Th1 responses is unknown. METHODS AND RESULTS: In this report we have utilized CD4+ T cells from HNT TCR transgenic mice that develop Th1-like responses upon antigenic stimulation in vitro and in vivo. Allogeneic bone marrow-derived MSCs reduced the diabetogenic potential of HNT CD4+ T cells in vivo in a transgenic mouse model of disease. In co-culture experiments, we have shown that MSCs were able to reduce HNT CD4+ T cell expansion, expression of key effector markers and production of the effector cytokine IFNγ after activation. This was associated with the ability of CD4+ T cells to acquire mitochondria from MSCs as evidenced by FACS and confocal microscopy. Remarkably, transfer of isolated MSC mitochondria to CD4+ T cells resulted in decreased T cell proliferation and IFNγ production. These effects were additive with those of prostaglandin E2 secreted by MSCs. Finally, we demonstrated that both co-culture with MSCs and transfer of isolated MSC mitochondria prevent the upregulation of T-bet, the master Th1 transcription factor, on activated CD4+ T cells. CONCLUSION: The present study demonstrates that transfer of MSC mitochondria to activated CD4+ T cells results in the suppression of Th1 responses in part by downregulating T-bet expression. Furthermore, our studies suggest that MSC mitochondrial transfer might represent a general mechanism of MSC-dependent immunosuppression.

Loss of prion protein control of glucose metabolism promotes neurodegeneration in model of prion diseases.

Corruption of cellular prion protein (PrPC) function(s) at the plasma membrane of neurons is at the root of prion diseases, such as Creutzfeldt-Jakob disease and its variant in humans, and Bovine Spongiform Encephalopathies, better known as mad cow disease, in cattle. The roles exerted by PrPC, however, remain poorly elucidated. With the perspective to grasp the molecular pathways of neurodegeneration occurring in prion diseases, and to identify therapeutic targets, achieving a better understanding of PrPC roles is a priority. Based on global approaches that compare the proteome and metabolome of the PrPC expressing 1C11 neuronal stem cell line to those of PrPnull-1C11 cells stably repressed for PrPC expression, we here unravel that PrPC contributes to the regulation of the energetic metabolism by orienting cells towards mitochondrial oxidative degradation of glucose. Through its coupling to cAMP/protein kinase A signaling, PrPC tones down the expression of the pyruvate dehydrogenase kinase 4 (PDK4). Such an event favors the transfer of pyruvate into mitochondria and its conversion into acetyl-CoA by the pyruvate dehydrogenase complex and, thereby, limits fatty acids ß-oxidation and subsequent onset of oxidative stress conditions. The corruption of PrPC metabolic role by pathogenic prions PrPSc causes in the mouse hippocampus an imbalance between glucose oxidative degradation and fatty acids ß-oxidation in a PDK4-dependent manner. The inhibition of PDK4 extends the survival of prion-infected mice, supporting that PrPSc-induced deregulation of PDK4 activity and subsequent metabolic derangements contribute to prion diseases. Our study posits PDK4 as a potential therapeutic target to fight against prion diseases.

Prenatal exposure to low doses of fungicides corrupts neurogenesis in neonates.

Neurogenesis plays a crucial role during neurodevelopment and its dysfunction can lead to neurodevelopmental disorders. A recent hypothesis stipulates that exogenous factors could corrupt this process and predispose to neurodegenerative disorders later in life. The presence of pesticide residues in the diet represents a threat of which we have recently become aware of. Indeed, they could corrupt neurogenesis, especially during gestation, potentially leading to impaired neuronal and synaptic functions. Since the effects of this low-noise contamination have not yet been evaluated on the neurodevelopment, we investigated the impact of fungicide residues on WT mice exposed throughout gestation. Thus, mice were exposed to fungicides, cyprodinil, mepanipyrim and pyrimethanil, alone at 0.1 µg/L during gestation until P3. Besides, another group was exposed to a cocktail of these three fungicides (0.1 µg/L each) for the same time. Exposure was performed through drinking water at the regulatory limit dose of the European countries (0.1 µg/L). No general toxicity was observed in neonates on body and brain weight upon fungicide exposure. However, results showed that gestational exposure to fungicide residues substantially promoted an increase of neural precursor cells at P3. This corrupted neurogenesis was linked to increased levels of ß-catenin, likely through the crosstalk of the PI3K/Akt and Wnt/ß-catenin pathways, both involved in cell proliferation. Fungicide exposure also altered protein expression of PSD95 and NMDA receptors in P3 neonates, two targets of the ß-catenin signaling pathway. Adult neural stem cell extractions from mice treated with the fungicide cocktail, showed an increase proliferation and differentiation combined with a reduction of their migration properties. In addition, in vitro studies on hippocampal primary cell cultures treated with various concentrations of fungicides showed neurotoxic effects. To conclude, corruption of neurogenesis by this chemical assault could be a fertile ground for the development of neurological diseases later in life.

Tubulin modifying enzymes as target for the treatment of tau-related diseases

In the brain of patients with Alzheimer's disease (AD), the number and length of microtubules (MTs) are significantly and selectively reduced. MTs are involved in a wide range of cellular functions, and defects of the microtubular system have emerged as a unifying hypothesis for the heterogeneous and variable clinical presentations of AD. MTs orchestrate their numerous functions through the spatiotemporal regulation of the binding of specialised microtubule-associated proteins (MAPs) and molecular motors. Covalent posttranslational modifications (PTMs) on the tubulin C-termini that protrude at the surface of MTs regulate the binding of these effectors. In neurons, MAP tau is highly abundant and its abnormal dissociation from MTs in the axon, cellular mislocalization and hyperphosphorylation, are primary events leading to neuronal death. Consequently, compounds targeting tau phosphorylation or aggregation are currently evaluated but their clinical significance has not been demonstrated yet. In this review, we discuss the emerging link between tubulin PTMs and tau dysfunction. In neurons, high levels of glutamylation and detyrosination profoundly impact the physicochemical properties at the surface of MTs. Moreover, in patients with early-onset progressive neurodegeneration, deleterious mutations in enzymes involved in modifying MTs at the surface have recently been identified, underscoring the importance of this enzymatic machinery in neurology. We postulate that pharmacologically targeting the tubulin-modifying enzymes holds promise as therapeutic approach for the treatment of neurodegenerative diseases.

The cellular prion protein controls the mesenchymal-like molecular subtype and predicts disease outcome in colorectal cancer.

BACKGROUND: Comprehensive transcriptomic analyses have shown that colorectal cancer (CRC) is heterogeneous and have led to the definition of molecular subtypes among which the stem-cell, mesenchymal-like group is associated with poor prognosis. The molecular pathways orchestrating the emergence of this subtype are incompletely understood. In line with the contribution of the cellular prion protein PrPC to stemness, we hypothesize that deregulation of this protein could lead to a stem-cell, mesenchymal-like phenotype in CRC. METHODS: We assessed the distribution of the PrPC-encoding PRNP mRNA in two large CRC cohorts according to molecular classification and its association with patient survival. We developed cell-based assays to explore the impact of gain and loss of PrPC function on markers of the mesenchymal subtype and to delineate the signalling pathways recruited by PrPC. We measured soluble PrPC in the plasmas of 325 patients with metastatic CRC and probed associations with disease outcome. FINDINGS: We found that PRNP gene expression is enriched in tumours of the mesenchymal subtype and is associated with poor survival. Our in vitro analyses revealed that PrPC controls the expression of genes that specify the mesenchymal subtype through the recruitment of the Hippo pathway effectors YAP and TAZ and the TGFß pathway. We showed that plasma levels of PrPC are elevated in metastatic CRC and are associated with poor disease control. INTERPRETATION: Our findings define PrPC as a candidate driver of the poor-prognosis mesenchymal subtype of CRC. They suggest that PrPC may serve as a potential biomarker for patient stratification in CRC. FUNDING: Grant support was provided by the following: Cancéropôle Ile de France (grant number 2016-1-EMERG-36-UP 5-1), Association pour la Recherche sur le Cancer (grant number PJA 20171206220), SATT Ile de France Innov (grant number 415) as well as INSERM.

Cell-based therapy against prion diseases.

Despite multiple efforts to find treatments, prion diseases are still incurable. The currently available therapeutic strategies are mostly based on compounds to inhibit pathological PrP (PrPSc) accumulation, and cellular PrP (PrPC) conversion into PrPSc. However, they cannot reverse the pathological changes already present in the brain. Cell-based therapeutic strategies could promote the repair of the pre-existing brain damage. The few available data come mostly from preclinical studies using neural stem cells, bone marrow-derived microglia and mesenchymal stem cells, as cell sources. Moreover, the benefits of cell-based therapeutic strategies could be linked not only to the replacement of damaged cells, but also to the secretion of trophic factors by the grafted cells that might modulate inflammation, cell death, or endogenous neurogenesis.

Prion diseases and adult neurogenesis: how do prions counteract the brain's endogenous repair machinery?

Scientific advances in stem cell biology and adult neurogenesis have raised the hope that neurodegenerative disorders could benefit from stem cell-based therapy. Adult neurogenesis might be part of the physiological regenerative process, however it might become impaired by the disease's mechanism and therefore contribute to neurodegeneration. In prion disorders this endogenous repair system has rarely been studied. Whether adult neurogenesis plays a role or not in brain repair or in the propagation of prion pathology remains unclear. We have recently investigated the status of adult neural stem cells isolated from prion-infected mice. We were able to show that neural stem cells accumulate and replicate prions thus resulting in an alteration of their neuronal destiny. We also reproduced these results in adult neural stem cells, which were infected in vitro. The fact that endogenous adult neurogenesis could be altered by the accumulation of misfolded prion protein represents another great challenge. Inhibiting prion propagation in these cells would thus help the endogenous neurogenesis to compensate for the injured neuronal system. Moreover, understanding the endogenous modulation of the neurogenesis system would help develop effective neural stem cell-based therapies.

Systemic delivery of siRNA down regulates brain prion protein and ameliorates neuropathology in prion disorder.

One of the main challenges for neurodegenerative disorders that are principally incurable is the development of new therapeutic strategies, which raises important medical, scientific and societal issues. Creutzfeldt-Jakob diseases are rare neurodegenerative fatal disorders which today remain incurable. The objective of this study was to evaluate the efficacy of the down-regulation of the prion protein (PrP) expression using siRNA delivered by, a water-in-oil microemulsion, as a therapeutic candidate in a preclinical study. After 12 days rectal mucosa administration of Aonys/PrP-siRNA in mice, we observed a decrease of about 28% of the brain PrP(C) level. The effect of Aonys/PrP-siRNA was then evaluated on prion infected mice. Several mice presented a delay in the incubation and survival time compared to the control groups and a significant impact was observed on astrocyte reaction and neuronal survival in the PrP-siRNA treated groups. These results suggest that a new therapeutic scheme based an innovative delivery system of PrP-siRNA can be envisioned in prion disorders.

High Dub3 expression in mouse ESCs couples the G1/S checkpoint to pluripotency.

The molecular mechanism underlying G1/S checkpoint bypass in mouse embryonic stem cells (ESCs) remains unknown. DNA damage blocks S phase entry by inhibiting the CDK2 kinase through destruction of its activator, the Cdc25A phosphatase. We observed high Cdc25A levels in G1 that persist even after DNA damage in mouse ESCs. We also found higher expression of Dub3, a deubiquitylase that controls Cdc25A protein abundance. Moreover, we demonstrate that the Dub3 gene is a direct target of Esrrb, a key transcription factor of the self-renewal machinery. We show that Dub3 expression is strongly downregulated during neural conversion and precedes Cdc25A destabilization, while forced Dub3 expression in ESCs becomes lethal upon differentiation, concomitant to cell-cycle remodeling and lineage commitment. Finally, knockdown of either Dub3 or Cdc25A induced spontaneous differentiation of ESCs. Altogether, these findings couple the self-renewal machinery to cell-cycle control through a deubiquitylase in ESCs.

Prion replication occurs in endogenous adult neural stem cells and alters their neuronal fate: involvement of endogenous neural stem cells in prion diseases.

Prion diseases are irreversible progressive neurodegenerative diseases, leading to severe incapacity and death. They are characterized in the brain by prion amyloid deposits, vacuolisation, astrocytosis, neuronal degeneration, and by cognitive, behavioural and physical impairments. There is no treatment for these disorders and stem cell therapy therefore represents an interesting new approach. Gains could not only result from the cell transplantation, but also from the stimulation of endogenous neural stem cells (NSC) or by the combination of both approaches. However, the development of such strategies requires a detailed knowledge of the pathology, particularly concerning the status of the adult neurogenesis and endogenous NSC during the development of the disease. During the past decade, several studies have consistently shown that NSC reside in the adult mammalian central nervous system (CNS) and that adult neurogenesis occurs throughout the adulthood in the subventricular zone of the lateral ventricle or the Dentate Gyrus of the hippocampus. Adult NSC are believed to constitute a reservoir for neuronal replacement during normal cell turnover or after brain injury. However, the activation of this system does not fully compensate the neuronal loss that occurs during neurodegenerative diseases and could even contribute to the disease progression. We investigated here the status of these cells during the development of prion disorders. We were able to show that NSC accumulate and replicate prions. Importantly, this resulted in the alteration of their neuronal fate which then represents a new pathologic event that might underlie the rapid progression of the disease.

Stem cell therapy extends incubation and survival time in prion-infected mice in a time window-dependant manner.

Prion diseases, which are mostly represented in humans by Creutzfeldt-Jakob disease, are transmissible neurodegenerative disorders characterized by vacuolization and neuronal loss, as well as by the accumulation of an abnormal form of the prion protein. These disorders have yet no effective treatment, and drugs that block prion replication in vitro do not significantly slow down the progression of the disease when used in vivo at late stages. Cell therapy that has been already tested in other neurodegenerative disorders therefore represents an interesting alternative approach. In this study, we showed for the first time in prion diseases that intracerebral transplantation of fetal neural stem cells significantly extended both incubation and survival time. This result was dependant on the time window chosen for the engraftment and was obtained with both genetically modified and wild-type stem cells, therefore forging a path toward efficient stem cell therapy for human prion diseases.

Comparative proteomic analysis of human mesenchymal and embryonic stem cells: towards the definition of a mesenchymal stem cell proteomic signature.

Mesenchymal stem cells (MSC) are adult multipotential progenitors which have a high potential in regenerative medicine. They can be isolated from different tissues throughout the body and their homogeneity in terms of phenotype and differentiation capacities is a real concern. To address this issue, we conducted a 2-DE gel analysis of mesenchymal stem cells isolated from bone marrow (BM), adipose tissue, synovial membrane and umbilical vein wall. We confirmed that BM and adipose tissue derived cells were very similar, which argue for their interchangeable use for cell therapy. We also compared human mesenchymal to embryonic stem cells and showed that umbilical vein wall stem cells, a neo-natal cell type, were closer to BM cells than to embryonic stem cells. Based on these proteomic data, we could propose a panel of proteins which were the basis for the definition of a mesenchymal stem cell proteomic signature.

Trehalose impairs aggregation of PrPSc molecules and protects prion-infected cells against oxidative damage.

Neurodegenerative disorders such as Alzheimer's, Huntington's, and prion diseases are characterized by abnormal protein deposits in the brain of affected patients. In prion diseases, a key event in the pathogenesis is the conversion of the normal prion protein (PrP(c)) into abnormal protease resistant PrP(Sc) deposits, a phenomenon associated with a higher sensitivity to oxidative stress in vitro. In cellular models of Alzheimer and Huntington diseases, the disaccharide trehalose has been shown to be effective in inhibiting huntingtin and Abeta peptide aggregates and reducing their associated toxicity. We show in this study that trehalose treatment of prion-infected cells decreases the size of de novo produced PrP(Sc) aggregates and modify their subcellular localization. Despite the fact that trehalose does not modify the protease resistance properties of PrP(Sc) molecules, it significantly protects prion-infected cells from induced oxidative damage, suggesting that this compound is of therapeutic interest.

Effective gene therapy in a mouse model of prion diseases.

Classical drug therapies against prion diseases have encountered serious difficulties. It has become urgent to develop radically different therapeutic strategies. Previously, we showed that VSV-G pseudotyped FIV derived vectors carrying dominant negative mutants of the PrP gene are efficient to inhibit prion replication in chronically prion-infected cells. Besides, they can transduce neurons and cells of the lymphoreticular system, highlighting their potential use in gene therapy approaches. Here, we used lentiviral gene transfer to deliver PrPQ167R virions possessing anti-prion properties to analyse their efficiency in vivo. Since treatment for prion diseases is initiated belatedly in human patients, we focused on the development of a curative therapeutic protocol targeting the late stage of the disease, either at 35 or 105 days post-infection (d.p.i.) with prions. We observed a prolongation in the lifespan of the treated mice that prompted us to develop a system of cannula implantation into the brain of prion-infected mice. Chronic injections of PrPQ167R virions were done at 80 and 95 d.p.i. After only two injections, survival of the treated mice was extended by 30 days (20%), accompanied by substantial improvement in behaviour. This delay was correlated with: (i) a strong reduction of spongiosis in the ipsilateral side of the brain by comparison with the contralateral side; and (ii) a remarkable decrease in astrocytic gliosis in the whole brain. These results suggest that chronic injections of dominant negative lentiviral vectors into the brain, may be a promising approach for a curative treatment of prion diseases.

Cellular pathogenesis in prion diseases.

Prion diseases are characterised by neuronal loss, vacuolation (spongiosis), reactive astrocytosis, microgliosis and in most cases by the accumulation in the central nervous system of the abnormal prion protein, named PrP(Sc). In this review on the "cellular pathogenesis in prion diseases", we have chosen to highlight the main mechanisms underlying the impact of PrP(C)/PrP(Sc) on neurons: the neuronal dysfunction, the neuronal cell death and its relation with PrP(Sc) accumulation, as well as the role of PrP(Sc) in the microglial and astrocytic reaction.

[Prions: where do we stand 20 years after the appearance of bovine spongiform encephalopathy?]. / Les prions: état des lieux 20 ans après l'apparition de l'encéphalopathie spongiforme bovine.

Bovine spongiform encephalopathy (BSE) is a transmissible spongiform encephalopathy (TSE) identified twenty years ago in the British cattle herds. Creutzfeldt-Jakob disease (CJD) is a TSE that occurs in humans. In 1996, scientists found a possible link between BSE and a new variant of CJD (vCJD). The fact that the non conventional infectious agent of TSE, named prions, could cross the species barrier from cattle to human through meat consumption, raised a tremendous concern for public safety in Europe. This led to the development in the following two decades of substantial and expensive measures to contain BSE and prevent its transmission to humans. In parallel, scientific programs have been funded to progress through the comprehension of the physiopathology of these fatal disorders. In Europe, the BSE epidemics is now ending and the number of cases is decreasing thanks to the strict control of animal foodstuff that was the main source of prion contamination. Only a small number of vCJD have been detected, however, additional concerns have been raised recently for public safety as secondary transmission of CJD through medical procedure and blood transfusion is possible. In addition, the possibility that the BSE was transmitted to other animals including small ruminants is also worrisome. Research efforts are now focussing on decontamination and ante mortem diagnosis of TSE to prevent animal and human transmission. However, needs for fundamental research are still important as many questions remain to be addressed to understand the mechanism of prion transmission, as well as its pathogenesis.

Peripheral circulation of the prion infectious agent in transgenic mice expressing the ovine prion protein gene in neurons only.

BACKGROUND: For prion diseases, even if a large body of evidence indicates that both the lymphoreticular system (LRS) and peripheral nerves are involved in scrapie neuroinvasion, the processes by which prions invade the central nervous system are only partially understood. METHODS: Transgenic Tg(OvPrP4) mice, which express the ovine prion protein (PrP) gene under the rat neuron-specific enolase promoter on a knockout background, were used to study prion extracerebral circulation after scrapie prions were inoculated via the intracerebral (ic) and the intraperitoneal (ip) route. RESULTS: Surprisingly, PrP(Sc) was detected in the spleens of mice inoculated ic with prions. Moreover, the absence of the ovine PrP(C) in nonneural tissue at the periphery did not stop neuroinvasion after ip challenge. Additionally, pilot studies performed in Tg(OvPrP4) mice that had undergone splenectomy before ic prion inoculation showed that the time course of the disease is delayed. CONCLUSIONS: Given that these mice express the ovine PrP gene in neuronal cells but not in nonnervous tissue, our results suggest that PrP(C) expressed by cells of the LRS are not necessary for neuroinvasion or for their ability to accumulate PrP(Sc) and emphasize the importance of extracerebral circulation of PrP(C) or PrP(Sc) for the development of the disease.

Scrapie strain transmission studies in ovine PrP transgenic mice reveal dissimilar susceptibility.

The Tg(OvPrP4) mouse line, expressing the sheep prion protein, is a sensitive model crucial for the identification of the bovine spongiform encephalopathy agent possibly present in natural sheep spongiform encephalopathies. It was also previously demonstrated as susceptible to infection with natural scrapie isolates from sheep harbouring various genotypes. The performance of this new transgenic mouse line in scrapie strain characterization was further assessed by intracranial inoculation of five groups of Tg(OvPrP4) mice with brain homogenate of the wild type mouse-adapted scrapie strains, C506M3, 22A, 79A, 87V, or Chandler. The Tg(OvPrP4) mice were susceptible to the scrapie agent transmitted using mouse-adapted scrapie strains but not equivalently. Strains 87V and Chandler were most readily transmissible followed by 79A and C506M3. Strain 22A was the least transmissible. Clinical signs, survival data, spongiosis, and PrP(sc) distribution were also reported. These various data demonstrate the possibility of distinguishing between scrapie strains. Our findings are discussed with regard to agent strain and host factors and already demonstrate the dissimilar susceptibilities of Tg(OvPrP4) mice to the different murine strains studied, thus, reinforcing their potential use in strain typing studies.

A novel copper-hydrogen peroxide formulation for prion decontamination.

With the appearance of variant Creutzfeldt-Jakob disease (CJD) and the detection of infectious prions in the peripheral organs of persons with sporadic CJD, the development of decontamination methods that are compatible with medical equipment has become a major issue. Here, we show that a formulation of copper metal ions in combination with hydrogen peroxide dramatically reduces the level of prion protein (PrP)(Sc) (the scrapie isoform of PrP) present in homogenates of samples from prion-infected brains, including brain samples from humans with CJD. An animal bioassay confirmed the reduction in prion infectivity, indicating that this novel Cu(2+)-H(2)O(2) formulation has great potential for prion decontamination.

The truncated 23-230 form of the prion protein localizes to the nuclei of inducible cell lines independently of its nuclear localization signals and is not cytotoxic.

The mechanisms of prion-induced neurological dysfunction observed in prion diseases are poorly understood. Transgenic mice expressing a truncated form of the prion protein (23-230 PrP) acquire cerebellar degeneration (Ma and Lindquist, Science, 2002). To decipher the mechanisms of neurodegeneration induced by 23-230 PrP, we established inducible cell lines expressing this truncated form of PrP. We found that 23-230 PrP, expected to be cytosolic, accumulated mostly in the nucleus of the cells and was not cytotoxic. Nuclear localization of this mutant form of PrP is independent of its predicted nuclear localization signals. In contrast to what we previously described for PrPSc, nuclear accumulation of 23-230 PrP does not require a functional microtubule network. We observed that 23-230 PrP interacts with chromatin in vivo, as already described for recombinant PrP and for PrPSc. Our data demonstrate that the 23-230 PrP model does not reflect the situation of a cytosolic PrP but could represent a very useful tool to understand the consequences of the accumulation of the prion protein in the nucleus.