Neuron-to-neuron wild-type Tau protein transfer through a trans-synaptic mechanism: relevance to sporadic tauopathies

Abstract

Background: In sporadic Tauopathies, neurofibrillary degeneration (NFD) is characterised by the intraneuronal aggregation of wild-type Tau proteins. In the human brain, the hierarchical pathways of this neurodegeneration have been well established in Alzheimer's disease (AD) and other sporadic tauopathies such as argyrophilic grain disorder and progressive supranuclear palsy but the molecular and cellular mechanisms supporting this progression are yet not known. These pathways appear to be associated with the intercellular transmission of pathology, as recently suggested in Tau transgenic mice. However, these conclusions remain ill-defined due to a lack of toxicity data and difficulties associated with the use of mutant Tau.

Results: Using a lentiviral-mediated rat model of hippocampal NFD, we demonstrated that wild-type human Tau protein is axonally transferred from ventral hippocampus neurons to connected secondary neurons even at distant brain areas such as olfactory and limbic systems indicating a trans-synaptic protein transfer. Using different immunological tools to follow phospho-Tau species, it was clear that Tau pathology generated using mutated Tau remains near the IS whereas it spreads much further using the wild-type one.

Conclusion: Taken together, these results support a novel mechanism for Tau protein transfer compared to previous reports based on transgenic models with mutant cDNA. It also demonstrates that mutant Tau proteins are not suitable for the development of experimental models helpful to validate therapeutic intervention interfering with Tau spreading.

Figure 1

Neuron-to-cell spread of WT Tau in a microfluidic device. The microfluidic device used in our study comprised two compartments separated by a physical barrier containing microgrooves that facilitate the passage of axons, but not neuronal cell bodies, during neuronal differentiation. (a) Primary culture of embryonic rat cortical neurons seeded in the first compartment (somatodendritic) was infected at DIV 7 with LVs encoding V5-hTau46^WT. The flow was then reversed and a second rat primary embryonic neuronal culture cells was seeded in the axonal compartment. (b) Forty-eight hours post-infection, the cells were processed for immunofluorescence analysis using anti-V5 antibodies and an Alexa Fluor 488-labeled secondary antibody (green). The nuclei were counterstained with DAPI (blue). The scale bar is indicated on the figure. These data showed that V5 is found in axons in primary neurons and in cell bodies of secondary neurons in the axonal compartment.

Figure 2

The transfer of V5-hTau46^WT protein is correlated to brain area connected to the IS (caudal part of the CA1 layer). (a) Dextran amines (10 kD) are anterograde tracers: they were injected into the rat brain (n = 3), and one week later, the animals were sacrificed and their brains were processed to reveal fluorescent axons efferent from the IS. Brains were virtually divided into five sections: bregma +5.20 to +1.40, bregma +1.20 to -1.40, bregma -1.80 to -4.30, bregma -4.52 to -6.04 and bregma -6.30 to -7.80. (b) LVs encoding V5-hTau46^WT were bilaterally injected into the CA1 layer (IS; bregma -5.3) of rat brains (n = 3). Five months later, the animals were sacrificed, and the whole brain was processed for immunohistochemical analysis using a rabbit polyclonal anti-V5 antibody. The brains were virtually divided into five sections as in (a). The bar scale is indicated on the figure. The drawing showing the extension of WT V5-Tau protein transfer from the IS to extreme rostral and caudal positions is illustrated in the lower part of the figure. These data showed that V5-hTau46^WT Tau is transported throughout the brain using neural networks. (c) The V5-immunoreactivity is summarized in a cartoon drawing of several coronal sections at different bregma coordinates. The different blue intensities (level 1 to 3) indicate the density of fibres and the red stars indicate the presence of V5-immunopositive cellular bodies indicating a trans-cellular transfer of V5-hTau46^WT Tau.

Figure 3

In vivo trans-synaptic transfer of WT Tau protein. LVs encoding V5-hTau46^WT were bilaterally injected into the CA1 layer (IS; bregma -5.3) of rat brains (n = 3 rats per group). One, three and five months later, the animals were sacrificed, and the whole brains were processed for immunohistochemical analysis using an antibody to total V5-Tau. Sections from the prelimbic or orbital cortex (bregma +4.7), the olfactory bulb (bregma +5.2) and the CA1 (bregma -5.3, IS) are shown. The scale bars are indicated on the figure. These data showed that V5-hTau46^WT is transported from primary to secondary neurons in a time-dependent manner.

Figure 4

Cell-to-cell protein transfer is specific to Tau protein WT. (a) eGFP is not transported in secondary connected neurons. LVs encoding eGFP were bilaterally injected into the CA1 layer of rat brains (IS bregma -5.3, n = 3). Eight months later, the animals were sacrificed, and brains sections processed for immunofluorescence assays to detect eGFP. Sections from the prelimbic (bregma +4.7), external capsule (bregma -1.8), CA1 (IS, bregma -5.3) and ectorhinal cortex (bregma -7.8) are shown. The scale bars are indicated on the figure. (b) Restricted lentiviral insertion in the vicinity of the IS. PBS (left panel, n = 3) or LVs encoding hTau46^WT (middle panel, n = 3) or hTau46^P301L (right panel, n = 3) were bilaterally injected into the CA1 layer of rat brains. One month later, the brains were dissected, and coronal sections of 1 mm thickness (indicated as the position relative to bregma) were prepared using an acrylic rat brain matrix. Total RNA was extracted from these sections to generate cDNA using RT-PCR. cDNAs were amplified using oligonucleotides specific to human Tau (hTau, 117 base pairs) or murine Tau (70 base pairs). The positive control was prepared by amplifying the plasmid containing hTau sequence. The lower parts represent the mean +/-SEM of the relative density (hTau/mTau) coming from the three rats. These data showed that Tau transport is a specific mechanism since neither eGFP nor viral genome is found associated to distant secondary brain areas.

Figure 5

Differential Tau spreading between WT and mutated Tau species. LVs encoding hTau46^WT (n = 5, sacrificed at 2 months; n = 5, sacrificed at 8 months) (a), V5-hTau46^WT (n = 3, sacrificed at 8 months) (b) or hTau46^P301L (n = 5, sacrificed at 8 months) (c) were bilaterally injected into the CA1 layer of rat brains. After sacrifice, the whole brain was processed for immunohistochemical analysis using AT8, MC1 or AT100 phosphorylated Tau antibodies. Among the positive rats, the rostralmost and caudalmost brain coordinates (from bregma) labeled by each antibody were determined for each brain. The figures represent the mean values +/-SEM of the rostralmost of caudalmot brain coordinates. These data showed that whereas hTau46^P301L diffusion is restricted to the vicinity of the IS, hTau46^WT has spread throughout the brain. Numbers of immunopositive rats per group are indicated on the right of each mapping.

Figure 6

Spatiotemporal progression of the Tau pathology through neural networks. LVs encoding hTau46^WT were bilaterally injected into the CA1 layer of rat brains (n = 5). Eight months later, the animals were sacrificed, and the whole brain was processed for immunohistochemical analysis to show AT8-related pTau. The brains were virtually separated into five sections: (a) bregma +5.20 to +1.40, (b) bregma +1.20 to -1.40, (c) bregma -1.80 to -4.30, (d) bregma -4.52 to -6.04 and (e) bregma -6.30 to -7.80. The bar scale is indicated on the figure. These data showed that phospho-hTau46^WT is found all over the brain eight months post-LVs delivery.

Figure 7

Transferred Tau species are mainly in a dephosphorylated state. LVs encoding V5-hTau46^WT were bilaterally injected into the CA1 layer (IS; bregma -5.3) of rat brains (n = 3). Five months later, the animals were sacrificed, and the rostral areas of the brain were processed for immunofluorescence analysis using a rabbit polyclonal anti-V5 antibody to detect total Tau and a mouse monoclonal antibody to pTau (AT8). The V5-Tau proteins were visualised in green using the corresponding secondary antibody Alexa Fluor-488 labelled goat anti-rabbit IgG (a and e) and the phospho-Tau in red using Alexa Fluor-568 labelled goat anti-mouse IgG (b and f). The scale bars are indicated on the figure. Adjacent brain slides from the rostral areas were assessed by immunohistochemistry using an antibody directed against the N-terminal portion of Tau containing a dephosphorylated tyrosine 18 residue (ADx215) (c) or an antibody to total V5-Tau (d). Most of hTau46WT Tau species found in secondary neurons are dephosphorylated.