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

Abstract

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. We observed that Aβ 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 Aβ deposits did not induce axonal degeneration. In contrast, co-treatment with somatic sub-toxic glutamate and axonal Aβ peptide triggered axonal degeneration. To study the consequences of such subcellular/local Aβ 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. Aβ 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 Aβ 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 Aβ production. This offers new tools to decipher mechanisms underlying Braak's staging. Our data suggests that local Aβ can play a role in remote tauopathy by distant disturbance of neurotransmission, providing a putative mechanism underlying the spatiotemporal appearance of pretangles.

Figure 1

Somatic applications of Aβ peptides induce a dying-back pattern. a. Schematic representation of a two-chamber microfluidic device (2-C μFD) with two cell culture chambers interconnected by funnel-shaped micro-channels [8] (a’: cross-section of the microfluidic device. A neuron is represented. Note that only axons can reach the right chamber). b-f. Fluorescence microscopy analysis of axonal degeneration vs somatic status after addition of Aβ peptide in the left (soma) vs right (distal part of axons) chambers. Cortical neurons were grown for 12 days in two-compartment chips and each chamber was treated for 48 h with Aβ peptides as indicated. The two left micrographs show the somato-dendritic chamber after staining with anti-MAP2 (red), DAPI (blue) and Thioflavine S (green). The right rectangular micrographs show the distal chamber after immunostaining with anti-α-tubulin and labeling with Thioflavine S. Each central scheme represents the device with indicated localization of treatment (left chamber vs right chamber): b) control cultures (Ø/Ø), c) somato-dendritic treatment with Aβ 25–35 peptide (Aβ/Ø, 30 μM), d) axonal treatment with Aβ 25–35 peptide (Ø/Aβ, 30 μM), e) somato-dendritic treatment with glutamate (Glut/Ø, 10 μM), f) somato-dendritic treatment with glutamate combined with axonal treatment Aβ 25–35 (Glut/Aβ, 10 μM/30 μM). Scale bar: 20 μm. g) Quantification of axonal fragmentation. Aβ25-35, glutamate (Glut), and control Aβ35-25 were added, or not (Ø), to the chamber as indicated. Axonal fragmention index was calculated as described in Methods section (n = 3; **P < 0.01; ***P < 0.001, ANOVA).

Figure 2

Axonal administration of NAD+, broad-spectrum caspase inhibitor, and JNK inhibitor reduces Aβ-induced axonal degeneration. Cortical (Cx) neurons were cultured for 12 days in μFD chambers as in Figure 1. Somatic Aβ peptide treatment (48 h) induced a strong axonal fragmentation, which could be reversed by pre-treatment of the axonal compartment with 5 mM NAD⁺, 50 μM z-VAD-fmk or 50 μM SP600125 (n = 5; *P < 0.01; **P < 0.001, ANOVA).

Figure 3

Cortical Aβ-peptide exposures induce early synaptic loss in a reconstructed cortical-hippocampal network. a. Phase contrast picture of the microfluidic device comprising funnel-shaped micro-channels allowing unidirectional axonal growth. Cortical neurons (Cx) were seeded in the left chamber, hippocampal neurons (Hi) in the right chamber, and were cultured for 14 days to reconstruct a cortical-hippocampal network. A cartoon representing one cortical neuron connected to one hippocampal neuron is inserted for clarity. b-e. Effect of Aβ42 oligomers and Aβ25-35 peptides on synaptic connections. Cortical and hippocampal neurons were cultured in μFD chambers as shown in a. b,c) Representative fluorescence micrographs from somato-dendritic compartment of Cx neurons (left panels) and from Hi neurons in the distal chamber receiving cortical fibers (right panels). Cx chambers were treated with sham (b, Ø/ Ø) or Aβ25-35 (c, Aβ /Ø). Neurons, axons and synapses were immunodetected using anti-MAP2 (blue), anti-α-tubulin (green), and anti-α-synuclein (red) respectively. Scale bar: 20 μm. Similar presynaptic patterns were observed with anti-synaptophysin labeling (not shown). d) Representative higher magnification showing presynaptic clusters (anti-α-synuclein, red) affixed to hippocampal dendrites (anti-MAP2, blue) in hippocampal neurons cultured alone (Hi) connected as in b (Hi-Cx) and connected plus treated with Aβ as in c (Hi-Cx + Aβ). e) Quantification of presynaptic structures affixed to hippocampal dendrites after cortical exposure to oligomeric Aβ1-42 (10 nM), fibrillar Aβ1-42 (10 nM), Aβ25-35 peptides (10 μM), and Aβ35-25 peptides (10 μM). (n = 3; **P < 0.01; ***P < 0.001, ANOVA).

Figure 4

Exposure of cortical somata to Aβ-peptide induces an axonal loss in a reconstructed cortical-hippocampal network. Cortical (Cx) and hippocampal (Hi) neurons were cultured for 14 days in μFD chambers as in Figure 2. Hippocampal neurons and projecting cortical axons were immunodetected using anti-MAP2 (red), anti-α-tubulin (green). Cx chambers were treated with sham (a, Ø/ Ø) or 10 μM Aβ42 oligomers (b, Aβ /Ø) for 48 hours. Representative fluorescence micrographs from the distal hippocampal chamber receiving cortical fibers. Scale bar: 20 μm.

Figure 5

Cortical Aβ peptide deposition induces glutamate-dependent hippocampal Tau phosphorylation. Cortical (Cx) and hippocampal (Hi) neurons were cultured in μFD chambers as in Figure 2a. a,c. Representative fluorescence micrograph ofcortical neurons (left μFD chamber) after immunostaining of phosphorylated Tau (pTau Thr231, red; β-tubulin, green). Effect of Aβ42 oligomers and Aβ25-35 peptides on synaptic connections. Cortical and hippocampal neurons were cultured in μFD chambers as shown in a. b,c) Representative fluorescence micrographs from somato-dendritic compartment of Cx neurons (left panels) and from Hi neurons in the distal chamber receiving cortical fibers (right panels). b,d. Representative fluorescence micrograph of hippocampal neurons receiving cortical axons after immunostaining of pTau (Thr231; pseudo-colors). b) Control conditions (Ø/Ø), d) cortical treatment with Aβ25-35 (10 nM) for 24 h. Scale bar: 20 μm. e. Quantification of pTau-positive hippocampal neurons after 24 h cortical exposure to fibrillar Aβ25-35, oligomeric Aβ42 or fibrillar Aβ42 (10 nM, each) in the presence or absence of the NMDAR antagonist MK801 (10 μM). The results were compared to pTau-positive neurons induced by exposure of Hi neurons to okadaic acid (1 nM, 24 h) (n = 3 **P < 0.01; ***P < 0.001, ANOVA).

Figure 6

Hypothetical model of synaptic, post-synaptic and axonal pathological events induced by Aβ in a cortico-hippocampal neuronal network. β-amyloid application to the somato-dendritic compartment triggers a so-called “dying-back” leading to a rapid cortical presynaptic loss concomitant with a postsynaptic hippocampal Tau-phosphorylation which could be prevented by the NMDA-receptor antagonist, MK-801, before any sign of axonal and somato-dendritic cortical alteration.