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

The Cellular Prion Protein—ROCK Connection: Contribution to Neuronal Homeostasis and Neurodegenerative Diseases

International audienceAmyloid-based neurodegenerative diseases such as prion, Alzheimer's, and Parkinson's diseases have distinct etiologies and clinical manifestations, but they share common pathological events. These diseases are caused by abnormally folded proteins (pathogenic prions PrP Sc in prion diseases, β-amyloids/Aβ and Tau in Alzheimer's disease, α-synuclein in Parkinson's disease) that display β-sheet-enriched structures, propagate and accumulate in the nervous central system, and trigger neuronal death. In prion diseases, PrP Sc -induced corruption of the physiological functions exerted by normal cellular prion proteins (PrP C ) present at the cell surface of neurons is at the root of neuronal death. For a decade, PrP C emerges as a common cell surface receptor for other amyloids such as Aβ and α-synuclein, which relays, at least in part, their toxicity. In lipid-rafts of the plasma membrane, PrP C exerts a signaling function and controls a set of effectors involved in neuronal homeostasis, among which are the RhoA-associated coiled-coil containing kinases (ROCKs). Here we review (i) how PrP C controls ROCKs, (ii) how PrP C -ROCK coupling contributes to neuronal homeostasis, and (iii) how the deregulation of the PrP C -ROCK connection in amyloid-based neurodegenerative diseases triggers a loss of neuronal polarity, affects neurotransmitter-associated functions, contributes to the endoplasmic reticulum stress cascade, renders diseased neurons highly sensitive to neuroinflammation, and amplifies the production of neurotoxic amyloids

A PrPC-caveolin-Lyn complex negatively controls neuronal GSK3β and serotonin 1B receptor

International audienceThe cellular prion protein, PrPC, is a glycosylphosphatidylinositol-anchored protein, abundant in lipid rafts and highly expressed in the brain. While PrPC is much studied for its involvement under its abnormal PrPSc isoform in Transmissible Spongiform Encephalopathies, its physiological role remains unclear. Here, we report that GSK3β, a multifunctional kinase whose inhibition is neuroprotective, is a downstream target of PrPC signalling in serotonergic neuronal cells. We show that the PrPC-dependent inactivation of GSK3β is relayed by a caveolin-Lyn platform located on neuronal cell bodies. Furthermore, the coupling of PrPC to GSK3β potentiates serotonergic signalling by altering the distribution and activity of the serotonin 1B receptor (5-HT1BR), a receptor that limits neurotransmitter release. In vivo, our data reveal an increased GSK3β kinase activity in PrP-deficient mouse brain, as well as sustained 5-HT1BR activity, whose inhibition promotes an anxiogenic behavioural response. Collectively, our data unveil a new facet of PrPC signalling that strengthens neurotransmission

PDK1 decreases TACE-mediated alpha-secretase activity and promotes disease progression in prion and Alzheimer's diseases

α-secretase–mediated cleavage of amyloid precursor protein (APP) precludes formation of neurotoxic amyloid-β (Aβ) peptides, and α-cleavage of cellular prion protein (PrPC) prevents its conversion into misfolded, pathogenic prions (PrPSc). The mechanisms leading to decreased α-secretase activity in Alzheimer's and prion disease remain unclear. Here, we find that tumor necrosis factor-α–converting enzyme (TACE)-mediated α-secretase activity is impaired at the surface of neurons infected with PrPSc or isolated from APP-transgenic mice with amyloid pathology. 3-phosphoinositide–dependent kinase-1 (PDK1) activity is increased in neurons infected with prions or affected by Aβ deposition and in the brains of individuals with Alzheimer's disease. PDK1 induces phosphorylation and caveolin-1–mediated internalization of TACE. This dysregulation of TACE increases PrPSc and Aβ accumulation and reduces shedding of TNF-α receptor type 1 (TNFR1). Inhibition of PDK1 promotes localization of TACE to the plasma membrane, restores TACE-dependent α-secretase activity and cleavage of APP, PrPC and TNFR1, and attenuates PrPSc- and Aβ-induced neurotoxicity. In mice, inhibition or siRNA-mediated silencing of PDK1 extends survival and reduces motor impairment following PrPSc infection and in APP-transgenic mice reduces Alzheimer's disease-like pathology and memory impairment.8 page(s

Double-Edge Sword of Sustained ROCK Activation in Prion Diseases through Neuritogenesis Defects and Prion Accumulation

International audienceIn prion diseases, synapse dysfunction, axon retraction and loss of neuronal polarity precede neuronal death. The mechanisms driving such polarization defects, however, remain unclear. Here, we examined the contribution of RhoA-associated coiled-coil containing kinases (ROCK), key players in neuritogenesis, to prion diseases. We found that overactivation of ROCK signaling occurred in neuronal stem cells infected by pathogenic prions (PrPSc) and impaired the sprouting of neurites. In reconstructed networks of mature neurons, PrPSc-induced ROCK overactivation provoked synapse disconnection and dendrite/axon degeneration. This overactivation of ROCK also disturbed overall neurotransmitter-associated functions. Importantly, we demonstrated that beyond its impact on neuronal polarity ROCK overactivity favored the production of PrPSc through a ROCK-dependent control of 3-phosphoinositide-dependent kinase 1 (PDK1) activity. In non-infectious conditions, ROCK and PDK1 associated within a complex and ROCK phosphorylated PDK1, conferring basal activity to PDK1. In prion-infected neurons, exacerbated ROCK activity increased the pool of PDK1 molecules physically interacting with and phosphorylated by ROCK. ROCK-induced PDK1 overstimulation then canceled the neuroprotective α-cleavage of normal cellular prion protein PrPC by TACE α-secretase, which physiologically precludes PrPSc production. In prion-infected cells, inhibition of ROCK rescued neurite sprouting, preserved neuronal architecture, restored neuronal functions and reduced the amount of PrPSc. In mice challenged with prions, inhibition of ROCK also lowered brain PrPSc accumulation, reduced motor impairment and extended survival. We conclude that ROCK overactivation exerts a double detrimental effect in prion diseases by altering neuronal polarity and triggering PrPSc accumulation. Eventually ROCK emerges as therapeutic target to combat prion diseases

Early cortico-striatal synaptic disconnection after cortical fiber axotomy.

<p><b>a–f</b>: Cortical presynaptic structures (v-GLUT1, red) affixed to striatal dendrites (MAP-2, blue) in control conditions (<b>a</b>), three hours after axotomy (<b>b</b>)<i>scale bar: 20. </i><i>µm.</i> Note the disappearance of v-GLUT1 labeling suggesting cortical presynaptic degeneration. <b>c–f</b>: Representative high magnification images in control conditions (<b>c</b>) and two hours (<b>d</b>), four hours (<b>e</b>) and six hours (<b>f</b>) after axotomy. Note the fast cortico-striatal disconnection as soon as two hours after axotomy. <i>scale bar: 5. </i><i>µm. </i><b>g–n</b>: Live imaging of GFP-expressing striatal neurons at time 0h (<b>g,k</b>), 2h (<b>h,l</b>), 4h (<b>i,m</b>) and 6h (<b>j,n</b>) in control (<b>g–j</b>) or axotomy (<b>k–n</b>) conditions. Note the stability of striatal dendritic spines (arrow) in both conditions. s<i>cale bar: 15. </i><i>μm.</i></p

Kinetics of axonal degeneration following cortical axotomy in a network.

<p><b>a,d</b>: Cortical, <b>b,e</b>: central and <b>c,f</b>: striatal, chambers of normal (<b>a–c</b>) or axotomized (<b>d–f</b>) reconstructed cortico-striatal networks (div14). Neurons were stained for MAP-2 (blue), α-tubulin (green) and v-GLUT1 (red). <b>a–c</b>: Cortical neurons send axons efficiently through the micro-channels and the central chamber to reach, and connect with, striatal neurons. <b>d–f</b>: Two hours after axotomy, no axons are present in the central chamber but axons inside the micro-channels and in the striatal chamber remain intact. <i>scale bar = 75 </i><i>µm </i><b>g–j</b>: Kinetics of degeneration of the distal part of the cortical axons following axotomy of cortical fibers in the receiving chamber. No axonal fragmentation (assessed by α-tubulin, green) is observed either in non-axotomized condition (<b>g</b>), or two hours after axotomy (<b>h</b>). The first tubulin beads (blebs) on cortical axons appear four hours after axotomy (<b>i</b>), and after six hours, all cortical axons disconnected from their soma are fragmented whereas striatal morphology remains intact (MAP-2, bleu) (<b>j</b>). <i>scale bar = 20 </i><i>µm.</i></p

Pharmacological screen of synapto-protective drugs in 3C-microfluidic chip.

<p><b>a–j</b>: Representative high magnification images of cortical presynaptic structures (v-GLUT1, red) affixed to striatal dendrites (MAP-2, blue) in non-axotomized (<b>a–e</b>) and 3 hours post-axotomy (<b>f–j</b>) conditions, without treatment (<b>a,f</b>), or with resveratrol (20 µ<i>M </i><b>b,g</b>), 50 µ<i>M</i> z-VAD (<b>c,h</b>), 10 µ<i>M</i> Y27632 (<b>d,i</b>) or 5 m<i>M</i> NAD⁺ (<b>e,j</b>) pre-treatment. <i>scale bar: 5 </i><i>µm</i>. <b>k</b>: Quantification of cortical presynaptic structures affixed to MAP-2-positive striatal dendrites after cortical axotomy. The graph represents the relative number of synapses remaining compared to non-axotomized condition. Y27632 and NAD+ delay cortical synaptic loss whereas no significant protection is observed with resveratrol or z-VAD pre-treatment.(ANOVA 2, *p-value<0.05, **p-value<0.01). Synaptic degeneration was quantified using SynD software (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071103#pone.0071103.s001" target="_blank">Figure S1</a>) which allows automatic segmentation of striatal dendritic trees and co-localization of VGLUT1 positive synapses docked to the dendrites <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071103#pone.0071103-Schmitz1" target="_blank">[23]</a>.</p

Reconstruction of oriented neuronal networks in 3C-microfluidic chip.

<p><b>a</b>: Microfluidic neuronal culture devices are made up of two separate cell culture chambers (blue) interconnected by a series of asymmetrical micro-channels interrupted by a central narrow (50 µm-wide) channel that gives access to the central part of the axons. Each chamber is perfused individually by two reservoirs (indicated by R). <b>b</b>: Phase contrast image of a reconstructed oriented neuronal network in 3C-chip. Cortical neurons are seeded in the left chamber and connect striatal neurons seeded in the right chamber (red stars). The size of asymmetrical micro-channels allows the outgrowth of axons but prevents the entry of cell bodies; micro-channels allow the passage of axons only in a left-to-right direction (axonal diodes) <i>scale bar = 50 </i><i>µm</i>. <b>c–c’</b>: Immunofluorescent images of the receiving (right) chamber. Cortical axons exit micro-channels (green: α-tubulin) and cluster presynaptic markers (red: v-GLUT1) on striatal dendrites (blue: MAP-2). <b>c’</b>: Enlargement of the clustering of cortical presynaptic labeling on striatal dendrites, suggesting cortico-striatal synapse establishment. <i>scale bar = 20 μm.</i></p