Impact of Neurons on Patient-Derived Cardiomyocytes Using Organ-On-A-Chip and iPSC Biotechnologies.

In the heart, cardiac function is regulated by the autonomic nervous system (ANS) that extends through the myocardium and establishes junctions at the sinus node and ventricular levels. Thus, an increase or decrease in neuronal activity acutely affects myocardial function and chronically affects its structure through remodeling processes. The neuro-cardiac junction (NCJ), which is the major structure of this system, is poorly understood and only a few cell models allow us to study it.

Genuine selective caspase-2 inhibition with new irreversible small peptidomimetics.

Caspase-2 (Casp2) is a promising therapeutic target in several human diseases, including nonalcoholic steatohepatitis (NASH) and Alzheimer's disease (AD). However, the design of an active-site-directed inhibitor selective to individual caspase family members is challenging because caspases have extremely similar active sites. Here we present new peptidomimetics derived from the VDVAD pentapeptide structure, harboring non-natural modifications at the P2 position and an irreversible warhead.

Dysregulated Neurotransmission induces Trans-synaptic degeneration in reconstructed Neuronal Networks.

Increasing evidence suggests that pathological hallmarks of chronic degenerative syndromes progressively spread among interconnected brain areas in a disease-specific stereotyped pattern. Functional brain imaging from patients affected by various neurological syndromes such as traumatic brain injury and stroke indicates that the progression of such diseases follows functional connections, rather than simply spreading to structurally adjacent areas. Indeed, initial damage to a given brain area was shown to disrupt the communication in related brain networks.

Nicotinamide riboside, a form of vitamin B, protects against excitotoxicity-induced axonal degeneration.

NAD depletion is a common phenomenon in neurodegenerative pathologies. Excitotoxicity occurs in multiple neurologic disorders and NAD was shown to prevent neuronal degeneration in this process through mechanisms that remained to be determined. The activity of nicotinamide riboside (NR) in neuroprotective models and the recent description of extracellular conversion of NAD to NR prompted us to probe the effects of NAD and NR in protection against excitotoxicity.

Acute amnestic encephalopathy in amyloid-β oligomer-injected mice is due to their widespread diffusion in vivo.

Amyloid-β (Aβ) oligomers are the suspected culprit as initiators of Alzheimer's disease (AD). However, their diffusion in the brain remains unknown. Here, we studied Aβ oligomers' dissemination and evaluated their in vivo toxicity.

Memantine plus vitamin D prevents axonal degeneration caused by lysed blood.

Intracranial hemorrhage, whether due to traumatic brain injury or ruptured cerebral aneurysm, is characterized by major neurological damage and a high mortality rate. Apart from cerebral vasospasm and mass effect, brain injury results from the release of unclotted blood that contacts neurons causing calcic stress. The combination of memantine with vitamin D, a neurosteroid hormone, may prevent blood neurotoxicity.

β-amyloid induces a dying-back process and remote trans-synaptic alterations in a microfluidic-based reconstructed neuronal network.

Introduction: Recent histopathological studies have shown that neurodegenerative processes in Alzheimer's and Parkinson's Disease develop along neuronal networks and that hallmarks could propagate trans-synaptically through neuronal pathways. The underlying molecular mechanisms are still unknown, and investigations have been impeded by the complexity of brain connectivity and the need for experimental models allowing a fine manipulation of the local microenvironment at the subcellular level.

Results: In this study, we have grown primary cortical mouse neurons in microfluidic (μFD) devices to separate soma from axonal projections in fluidically isolated microenvironments, and applied β-amyloid (Aβ) peptides locally to the different cellular compartments.

Combination of memantine and vitamin D prevents axon degeneration induced by amyloid-beta and glutamate.

The currently available drugs for treatment of Alzheimer's disease are symptomatic and only temporarily slow down the natural history of the disease process. Recently, its has been proposed that the combination of memantine with vitamin D, a neurosteroid hormone, may prevent amyloid-beta and glutamate neurotoxicity. Here, our purpose was to examine the potential protective effects of memantine and vitamin D against amyloid-beta peptide and glutamate toxicity in cortical neuronal cultures.

Synapto-protective drugs evaluation in reconstructed neuronal network.

Chronic neurodegenerative syndromes such as Alzheimer's and Parkinson's diseases, or acute syndromes such as ischemic stroke or traumatic brain injuries are characterized by early synaptic collapse which precedes axonal and neuronal cell body degeneration and promotes early cognitive impairment in patients. Until now, neuroprotective strategies have failed to impede the progression of neurodegenerative syndromes. Drugs preventing the loss of cell body do not prevent the cognitive decline, probably because they lack synapto-protective effects.

NAD+ acts on mitochondrial SirT3 to prevent axonal caspase activation and axonal degeneration.

In chronic degenerative syndromes, neuronal death occurs over long periods, during which cells progressively lose their axons and, ultimately, their cell bodies. Although apoptosis is recognized as a key event in neuronal death, the molecular mechanisms involved in CNS axons degeneration are poorly understood. Due to the highly polarized phenotypes of CNS neurons, the different neuronal subcompartments are likely to be targeted by light repetitive and localized aggression.

Effectiveness of the combination of memantine plus vitamin D on cognition in patients with Alzheimer disease: a pre-post pilot study.

Objective: To determine whether treatment with memantine plus vitamin D is more effective than memantine or vitamin D alone in improving cognition among patients with Alzheimer disease (AD).

Methods: We studied 43 white outpatients (mean 84.7 ± 6.

Axon diodes for the reconstruction of oriented neuronal networks in microfluidic chambers.

Various experimental models are used to study brain development and degeneration. They range from whole animal models, which preserve anatomical structures but strongly limit investigations at the cellular level, to dissociated cell culture systems that allow detailed observation of cell phenotypes but lack the highly ordered physiological neuron connection architecture. We describe here a platform comprising independent cell culture chambers separated by an array of "axonal diodes".

A "dry and wet hybrid" lithography technique for multilevel replication templates: Applications to microfluidic neuron culture and two-phase global mixing.

A broad range of microfluidic applications, ranging from cell culture to protein crystallization, requires multilevel devices with different heights and feature sizes (from micrometers to millimeters). While state-of-the-art direct-writing techniques have been developed for creating complex three-dimensional shapes, replication molding from a multilevel template is still the preferred method for fast prototyping of microfluidic devices in the laboratory. Here, we report on a "dry and wet hybrid" technique to fabricate multilevel replication molds by combining SU-8 lithography with a dry film resist (Ordyl).

Induction of early Purkinje cell dendritic differentiation by thyroid hormone requires RORα.

Background: The active form (T3) of thyroid hormone (TH) controls critical aspects of cerebellar development, such as migration of postmitotic neurons and terminal dendritic differentiation of Purkinje cells. The effects of T3 on early dendritic differentiation are poorly understood.

Results: In this study, we have analyzed the influence of T3 on the progression of the early steps of Purkinje cell dendritic differentiation in postnatal day 0 organotypic cerebellar cultures.

Wallerian-like degeneration of central neurons after synchronized and geometrically registered mass axotomy in a three-compartmental microfluidic chip.

Degeneration of central axons may occur following injury or due to various diseases and it involves complex molecular mechanisms that need to be elucidated. Existing in vitro axotomy models are difficult to perform, and they provide limited information on the localization of events along the axon. We present here a novel experimental model system, based on microfluidic isolation, which consists of three distinct compartments, interconnected by parallel microchannels allowing axon outgrowth.

Phosphorylation and transcriptional activity regulation of retinoid-related orphan receptor alpha 1 by protein kinases C.

Retinoid-related orphan receptor alpha1 (RORalpha1) is a member of the nuclear receptor superfamily. It is highly expressed in CNS particularly in the cerebellum. Absence of this transcription factor in mice leads to several abnormalities, such as cerebellar atrophy linked to Purkinje cell death and impaired differentiation.

Beta-amyloid(1-42) induces a reduction in the parallel fiber responses of Purkinje cells: possible involvement of pro-inflammatory processes.

In Alzheimer's disease there is an increased production of the toxic beta-amyloid peptides (Abeta), especially the longer forms such as Abeta(1-42). Using the patch-clamp technique we have studied the contribution of early pro-inflammatory processes to the acute effects of 1 microM Abeta(1-42) on the parallel fiber EPSC (PF-EPSC) of Purkinje cells in cerebellar slices. Abeta(1-42) induces a decrease in the PF-EPSC amplitude.

Silencing of the Charcot-Marie-Tooth associated MTMR2 gene decreases proliferation and enhances cell death in primary cultures of Schwann cells.

Loss of function of the myotubularin (MTM)-related protein 2 (MTMR2) in Schwann cells causes Charcot-Marie-Tooth disease type 4B1, a severe demyelinating neuropathy, but the consequences of MTMR2 disruption in Schwann cells are unknown. We established the expression profile of MTMR2 by real-time RT-PCR during rat myelination and showed it to be preferentially expressed at the onset of the myelination period. We developed a model in which MTMR2 loss of function was reproduced in primary cultures of Schwann cells by RNA interference.

RORalpha, a pivotal nuclear receptor for Purkinje neuron survival and differentiation: from development to ageing.

RORalpha (Retinoid-related Orphan Receptor) is a transcription factor belonging to the superfamily of nuclear receptors. The spontaneous staggerer (sg) mutation, which consists of a deletion in the Rora gene, has been shown to cause the loss of function of the RORalpha protein. The total loss of RORalpha expression leads to cerebellar developmental defects, particularly to a dramatic decreased survival of Purkinje cells and an early block in the differentiation process.

Human retinoic acid receptor-related orphan receptor alpha1 overexpression protects neurones against oxidative stress-induced apoptosis.

Retinoic acid receptor-related orphan receptor alpha (RORalpha) is a transcription factor belonging to the superfamily of nuclear receptors. Disruption of the Rora gene in the mouse results in a defect in the development of Purkinje cells leading to a cerebellar atrophy, which suggests a neuroprotective role for RORalpha. To test this hypothesis, the survival rate of lentiviral-mediated human RORalpha1-overexpressing neurones has been evaluated in response to different stressors disturbing the redox homeostasis, such as beta-amyloid peptide, c(2)-ceramide and H(2)O(2).

IGF-I protects cortical neurons against ceramide-induced apoptosis via activation of the PI-3K/Akt and ERK pathways; is this protection independent of CREB and Bcl-2?

Current understanding of IGF-I-mediated neuroprotection implies the activation of phosphatidylinositol-3-kinase (PI-3K), which leads to the activation of Akt/Protein Kinase B. In non-neuronal cells, Akt phosphorylates and activates the transcription factor CREB, implicated in the transcription of the anti-apoptotic bcl-2 gene. This paper further analyses the anti-apoptotic IGF-I action in neurons.

Activation of the JNK-c-Jun pathway during the early phase of neuronal apoptosis induced by PrP106-126 and prion infection.

Prion diseases are neurodegenerative pathologies characterized by apoptotic neuronal death. Although the late execution phase of neuronal apoptosis is beginning to be characterized, the sequence of events occurring during the early decision phase is not yet well known. In murine cortical neurons in primary culture, apoptosis was first induced by exposure to a synthetic peptide homologous to residues 106-126 of the human prion protein (PrP), PrP106-126.

Dynamic analysis of apoptosis in primary cortical neurons by fixed- and real-time cytofluorometry.

We describe here a cytofluorometric technology for the characterization of decision, execution, and degradation steps of neuronal apoptosis. Multiparametric flow cytometry was developed and combined to detailed fluorescence microscopy observations to establish the chronology and hierarchy of death-related events: neuron morphological changes, mitochondrial transmembrane potential (DeltaPsi(m)) collapse, caspase-3 and -9 activation, phosphatidyl-serine exposure, nuclear dismantling and final plasma membrane permeabilization. Moreover, we developed a reliable real-time flow cytometric monitoring of DeltaPsi(m) and plasma membrane integrity in response to neurotoxic insults including MPTP treatment.

C-Jun N-terminal kinases/c-Jun and p38 pathways cooperate in ceramide-induced neuronal apoptosis.

Understanding the regulation of the apoptotic program in neurons by intracellular pathways is currently a subject of great interest. Recent results suggest that c-Jun N-terminal kinases (JNK), mitogen-activated protein kinases and the transcription factor c-Jun are important regulators of this cell death program in post-mitotic neurons following survival-factor withdrawal. Our study demonstrates that ceramide levels increase upon survival-factor withdrawal in primary cultured cortical neurons.

Activation of mitogen-activated protein kinase pathways during the death of PC12 cells is dependent on the state of differentiation.

PC12 cells that are differentiated with NGF and cAMP become totally dependent on these factors for their survival, unlike those that are differentiated with NGF alone. We have asked whether the MAP Kinases, ERKs, JNKs and p38s play a role in the cell death induced by withdrawal of trophic factors on NGF- and NGF/cAMP-differentiated PC12 cells. By Western-blot analyses with antibodies directed against the activated forms of these kinases, we show that when the trophic factors were withdrawn, ERK phosphorylation was reduced to very low levels within 1 h in both cases.