Agonists of prostaglandin E2 receptors as potential first in class treatment for nephronophthisis and related ciliopathies.

Nephronophthisis (NPH) is an autosomal recessive tubulointerstitial nephropathy belonging to the ciliopathy disorders and known as the most common cause of hereditary end-stage renal disease in children. Yet, no curative treatment is available. The major gene, NPHP1, encodes a protein playing key functions at the primary cilium and cellular junctions. Using a medium-throughput drug-screen in NPHP1 knockdown cells, we identified 51 Food and Drug Administration-approved compounds by their ability to alleviate the cellular phenotypes associated with the loss of NPHP1; 11 compounds were further selected for their physicochemical properties. Among those compounds, prostaglandin E1 (PGE1) rescued ciliogenesis defects in immortalized patient NPHP1 urine-derived renal tubular cells, and improved ciliary and kidney phenotypes in our NPH zebrafish and Nphp1 knockout mouse models. Furthermore, Taprenepag, a nonprostanoid prostaglandin E2 receptor agonist, alleviated the severe retinopathy observed in Nphp1−/− mice. Finally, comparative transcriptomics allowed identification of key signaling pathways downstream PGE1, including cell cycle progression, extracellular matrix, adhesion, or actin cytoskeleton organization. In conclusion, using in vitro and in vivo models, we showed that prostaglandin E2 receptor agonists can ameliorate several of the pleotropic phenotypes caused by the absence of NPHP1; this opens their potential as a first therapeutic option for juvenile NPH-associated ciliopathies.

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. Using cortico-striatal neuronal networks reconstructed in a microfluidic environment, we investigated the role of glutamate signaling in activity-dependent neuronal survival and trans-synaptic degeneration processes. Using a variety of neuronal insults applied on cortical neurons, we demonstrate that acute injuries such as axonal trauma, focal ischemia, or alteration of neuronal rhythms, lead to glutamate-dependent striatal neuron dysfunction. Interestingly, focal pro-oxidant insults or chronic alteration of spontaneous cortical rhythms provoked dysfunction of distant striatal neurons through abnormal glutamate GluN2B-NMDAR-mediated signaling at cortico-striatal synapses. These results indicate that focal alteration of cortical functions can initiate spreading of dysfunction along neuronal pathways in the brain, reminiscent of diaschisis-like processes.

ß-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 ß-amyloid (Aß) peptides locally to the different cellular compartments. We observed that Aß application to the somato-dendritic compartment triggers a "dying-back" process, involving caspase and NAD(+) signalling pathways, whereas exposure of the axonal/distal compartment to Aß deposits did not induce axonal degeneration. In contrast, co-treatment with somatic sub-toxic glutamate and axonal Aß peptide triggered axonal degeneration. To study the consequences of such subcellular/local Aß stress at the network level we developed new µFD multi-chamber devices containing funnel-shaped micro-channels which force unidirectional axon growth and used them to recreate in vitro an oriented cortico-hippocampal pathway. Aß application to the cortical somato-dendritic chamber leads to a rapid cortical pre-synaptic loss. This happens concomitantly with a post-synaptic hippocampal tau-phosphorylation which could be prevented by the NMDA-receptor antagonist, MK-801, before any sign of axonal and somato-dendritic cortical alteration. CONCLUSION: Thanks to µFD-based reconstructed neuronal networks we evaluated the distant effects of local Aß stress on neuronal subcompartments and networks. Our data indicates that distant neurotransmission modifications actively take part in the early steps of the abnormal mechanisms leading to pathology progression independently of local Aß production. This offers new tools to decipher mechanisms underlying Braak's staging. Our data suggests that local Aß can play a role in remote tauopathy by distant disturbance of neurotransmission, providing a putative mechanism underlying the spatiotemporal appearance of pretangles.

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. Such locally initiated deleterious signal transduction pathways could theoretically spread through the cytoplasm. However, where axon-degenerative signals initiate, what these early signals are, and how they lead to axon degeneration are unanswered questions that limit our understanding of neurodegenerative diseases and our ability to identify novel therapeutic targets. Using a microfluidic culture device adapted to CNS primary neurons, allowing specific access to the axonal and somatodendritic compartments, we analyzed the molecular pathways involved in axonal degeneration of differentiated neurons. We show here that local application of proapoptotic stimuli on the somatodentritic compartment triggers a dying-back pattern involving caspase-dependent axonal degeneration. Using complementary pharmacological and genetic approaches, we further demonstrate that NAD(+) and grape wine polyphenols prevent axonal apoptosis and act via mitochondrial SirT3 activation in axons.

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. The absence of physiologically realistic neuronal network models which can be easily handled has hindered the development of synapto-protective drugs suitable for therapies. Here we describe a new microfluidic platform which makes it possible to study the consequences of axonal trauma of reconstructed oriented mouse neuronal networks. Each neuronal population and sub-compartment can be chemically addressed individually. The somatic, mid axon, presynaptic and postsynaptic effects of local pathological stresses or putative protective molecules can thus be evaluated with the help of this versatile "brain on chip" platform. We show that presynaptic loss is the earliest event observed following axotomy of cortical fibers, before any sign of axonal fragmentation or post-synaptic spine alteration. This platform can be used to screen and evaluate the synapto-protective potential of several drugs. For instance, NAD⁺ and the Rho-kinase inhibitor Y27632 can efficiently prevent synaptic disconnection, whereas the broad-spectrum caspase inhibitor zVAD-fmk and the stilbenoid resveratrol do not prevent presynaptic degeneration. Hence, this platform is a promising tool for fundamental research in the field of developmental and neurodegenerative neurosciences, and also offers the opportunity to set up pharmacological screening of axon-protective and synapto-protective drugs.

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". This array involves asymmetric micro-channels, imposing unidirectional axon connectivity with 97% selectivity. It allows the construction of complex, oriented neuronal networks not feasible with earlier platforms. Different neuronal subtypes could be co-cultivated for weeks, and sequential seeding of different cell populations reproduced physiological network development. To illustrate possible applications, we created and characterized a cortico-striatal oriented network. Functional synaptic connections were established. The activation of striatal differentiation by cortical axons, and the synchronization of neural activity were demonstrated. Each neuronal population and subcompartment could be chemically addressed individually. The directionality of neural pathways being a key feature of the nervous system organization, the axon diode concept brings in a paradigmatic change in neuronal culture platforms, with potential applications for studying neuronal development, synaptic transmission and neurodegenerative disorder such as Alzheimer and Parkinson diseases at the sub-cellular, cellular and network levels.

VEGF modulates NMDA receptors activity in cerebellar granule cells through Src-family kinases before synapse formation.

NMDA type glutamate receptors (NMDARs) are best known for their role in synaptogenesis and synaptic plasticity. Much less is known about their developmental role before neurons form synapses. We report here that VEGF, which promotes migration of granule cells (GCs) during postnatal cerebellar development, enhances NMDAR-mediated currents and Ca(2+) influx in immature GCs before synapse formation. The VEGF receptor Flk1 forms a complex with the NMDAR subunits NR1 and NR2B. In response to VEGF, the number of Flk1/NR2B coclusters on the cell surface increases. Stimulation of Flk1 by VEGF activates Src-family kinases, which increases tyrosine phosphorylation of NR2B. Inhibition of Src-family kinases abolishes the VEGF-dependent NR2B phosphorylation and amplification of NMDAR-mediated currents and Ca(2+) influx in GCs. These findings identify VEGF as a modulator of NMDARs before synapse formation and highlight a link between an activity-independent neurovascular guidance cue (VEGF) and an activity-regulated neurotransmitter receptor (NMDAR).