P62 accumulates through neuroanatomical circuits in response to tauopathy propagation

Abstract

In Alzheimer's disease and related tauopathies, trans-synaptic transfer and accumulation of pathological tau from donor to recipient neurons is thought to contribute to disease progression, but the underlying mechanisms are poorly understood. Using complementary in vivo and in vitro models, we examined the relationship between these two processes and neuronal clearance. Accumulation of p62 (a marker of defective protein clearance) correlated with pathological tau accumulation in two mouse models of tauopathy spread; Entorhinal Cortex-tau (EC-Tau) mice where tau pathology progresses in time from EC to other brain regions, and PS19 mice injected with tau seeds. In both models and in several brain regions, p62 colocalized with human tau in a pathological conformation (MC1 antibody). In EC-Tau mice, p62 accumulated before overt tau pathology had developed and was associated with the presence of aggregation-competent tau seeds identified using a FRET-based assay. Furthermore, p62 accumulated in the cytoplasm of neurons in the dentate gyrus of EC-Tau mice prior to the appearance of MC1 positive tauopathy. However, MC1 positive tau was shown to be present at the synapse and to colocalize with p62 as shown by immuno electron microscopy. In vitro, p62 colocalized with tau inclusions in two primary cortical neuron models of tau pathology. In a three-chamber microfluidic device containing neurons overexpressing fluorescent tau, seeding of tau in the donor chamber led to tau pathology spread and p62 accumulation in both the donor and the recipient chamber. Overall, these data are in accordance with the hypothesis that the accumulation and trans-synaptic spread of pathological tau disrupts clearance mechanisms, preceding the appearance of obvious tau aggregation. A vicious cycle of tau accumulation and clearance deficit would be expected to feed-forward and exacerbate disease progression across neuronal circuits in human tauopathies.

Keywords: Clearance; Electron microscopy; Microfluidics; Spread; Tauopathy; p62.

Fig. 1

Tau transfer and clearance dysfunction in the seeded PS19 mouse. a DS9 (tau competent seeds) and DS1 (control) injections on each side of the dentate gyrus in the same PS19 mouse. Image source: Allen brain atlas. b–c Immunofluorescence images: MC1 (tau with a pathological conformation—green), p62 (marker of ubiquitinated cargos destined to degradation—white) and DAPI (nuclei—blue) staining on PS19 mice on the DS1 injected side (upper panel) and DS9 (lower panel) for the dentate gyrus. (b) and the entorhinal cortex (c). Scale bar represents 30 μm. d–e DAB staining of the hippocampus, entorhinal and perirhinal cortices of PS19 mice on the DS1 injected side (upper panel) and DS9 (lower panel) for MC1 (d) and p62 (e). Scale bar represents 200 μm. f–g Quantification of the number of positive neurons is shown for MC1 (f) and p62 (g) for the dentate gyrus, CA1, CA3, subiculum and the entorhinal, perirhinal and frontal cortices. Data is normalized to the DS1 side, paired t-test for each region. h Correlation between the number of MC1 and p62 positive neurons in each injected PS19 mouse and for each measured region, on both the DS1 and the DS9 side

Fig. 2

Tau transfer and clearance dysfunction in the EC-Tau mouse. a Tau pathology spread in the EC-Tau Mouse from the lateral EC to the frontal cortex. Image source: Allen brain atlas. b–c Immunofluorescence images: MC1 (green), p62 (white) and DAPI (blue) staining in EC-Tau mice with mild (upper panel), moderate (middle panel) and severe (bottom panel) pathology for the EC (b) and the CA3 (c). Scale bar represents 30 μm. d–e DAB staining of the hippocampus, entorhinal and frontal cortices on EC-Tau mice with mild (upper panel), moderate (middle panel) and severe (bottom panel) pathology for MC1 (d) and p62 (e). Scale bar represents 200 μm. f–g Quantification of the number of positive neurons is shown for MC1 (f) and p62 (g) for the dentate gyrus, CA1, CA3, subiculum and the entorhinal, perirhinal and frontal cortices. Absolute numbers, one-way ANOVA with post-hoc test for each region. h Correlation between the number of MC1 and p62 positive neurons in the EC-Tau mice and for each measured region

Fig. 3

P62 accumulation precedes MC1 positive tau pathology in the EC-Tau mouse. a DAB staining of the entorhinal, perirhinal and frontal cortices on EC-Tau mice with moderate (upper panel) and severe pathology (bottom panel) for MC1 (left panel) and p62 (right panel). Scale bar represents 200 μm. b Quantification of the number of positive neurons for MC1 and p62 is shown in EC-Tau mice with mild, moderate and severe pathology for the entorhinal, perirhinal and frontal cortices. Absolute numbers, two-way ANOVA with post-hoc test. c FACS sorting of Tau biosensor HEK cells treated with brain lysate from the entorhinal (left panel) and frontal cortices (right panel) of EC-Tau mice with mild (top panel) and severe pathology (bottom panel). d Quantification of the seeding ability (% FRET in the biosensor cells) for the hippocampus and entorhinal, perirhinal and frontal cortices of EC-Tau mice with mild, moderate and severe pathology. One-way ANOVA with post-hoc test

Fig. 4

Tau and p62 subcellular localization in the EC-Tau mouse dentate gyrus. a Low magnification immunohistochemistry image of the middle molecular layer (MML) of an EC-Tau mouse with low pathology (restricted to the EC): conformational abnormal human tau was labelled with antibody MC1 (red). Image shows co-labelling with an antibody against p62 (green) and DAPI labelled nuclei (blue). Scale bar represents 15 μm. b Representative immuno electron microcopy (iEM) images of synapses in the middle molecular layer (MML) of an 18 month old EC-Tau mouse double labelled with p62 (6 nm gold beads) and MC1 (10 nm gold beads). Image shows tau pathology (MC1 antibody) only. Scale bar represents 200 nm. c Representative iEM images of the colocalization of p62 (red arrow) and MC1 (blue arrow) in the middle molecular layer (MML) of an 18 month old EC-Tau mouse double labelled with p62 (6 nm gold beads) and MC1 (10 nm gold beads). Scale bar represents 200 nm.

Fig. 5

Tau pathology and clearance deficits in two primary neuron models of tauopathy. a Immunofluorescence images of MC1 (pathological tau—red) and α-tubulin (microtubules—white) in DIV 15 (days of culture) cortical neurons from PS19 mice (overexpressing human tau P301S) exposed to DS1 seeds (upper panel) or to DS9 seeds (lower panel). Scale bar represents 30 μm. b Co-localization between MC1 (red) and p62 (green) in DIV 15 neuronal dendrites (labelled with Map2, white) from PS19 cortical neurons exposed to DS9 seeds. Scale bar represents 10 μm. c Rat neurons treated with a lentivirus overexpressing the tau repeat domain tagged with yellow fluorescent protein (TauRD-YFP) showing immunofluorescence images of tau tagged with YFP (red), p62 (green) and Map2 (white) in DIV 18 cortical neurons exposed to WT mouse brain lysate (upper panel) or rTg4510 (overexpressing human tau P301L) brain lysate (lower panel). Notice the aggregation of TauRD-YFP in the presence of pathological brain lysate. Scale bar represents 30 μm. d–f Images showing that increasing doses of pharmacological inhibitors of the proteasome—epoxomicin (1 to 20 nM) or MG132 (10 nM to 1 µM)—triggered an increase of the percentage of neurons with TauRD-YFP inclusions. Images are shown for epoxomicin. Scale bar represents 30 μm  (e); quantification is shown for both (e–f)

Fig. 6

Transsynaptic spread of pathological tau inhibits clearance mechanisms in recipient neurons grown in a microfluidic device. a Timecourse study of the spread of TauRD-YFP inclusions in cortico-cortical network reconstructed in microfluidic devices. Rat cortical neurons in the donor (left) chamber connect via their axons to cortical neuron dendrites in the recipient (right) chamber via synaptic connections in the middle chamber. The three panels show the same cortico-cortical network at DIV8, DIV14 and DIV16. White arrows highlight the progressive aggregation of TauRD-YFP in neurons in the recipient chamber. b Quantification of progressive TauRD-YFP aggregation in neurons in the donor chamber (top panel) and in the recipient chamber (bottom panel). c Confocal immunofluorescence images showing the colocalization between TauRD-YFP aggregates (red), p62 (green) and Map2 (white), in the donor (top panel) and recipient (bottom panel) neuron chambers at DIV 16 in a microfluidic device