AMPK is abnormally activated in tangle- and pre-tangle-bearing neurons in Alzheimer's disease and other tauopathies

Abstract

Tauopathies represent a class of neurodegenerative disorders characterized by abnormal tau phosphorylation and aggregation into neuronal paired helical filaments (PHFs) and neurofibrillary tangles. AMP-activated protein kinase (AMPK) is a metabolic sensor expressed in most mammalian cell types. In the brain, AMPK controls neuronal maintenance and is overactivated during metabolic stress. Here, we show that activated AMPK (p-AMPK) is abnormally accumulated in cerebral neurons in 3R+4R and 3R tauopathies, such as Alzheimer's disease (AD), tangle-predominant dementia, Guam Parkinson dementia complex, Pick's disease, and frontotemporal dementia with parkinsonism linked to chromosome 17, and to a lesser extent in some neuronal and glial populations in the 4R tauopathies, progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), and argyrophilic grain disease. In AD brains, p-AMPK accumulation decorated neuropil threads and dystrophic neurites surrounding amyloid plaques, and appeared in more than 90% of neurons bearing pre-tangles and tangles. Granular p-AMPK immunoreactivity was also observed in several tauopathies in apparently unaffected neurons devoid of tau inclusion, suggesting that AMPK activation preceded tau accumulation. Less p-AMPK pathology was observed in PSP and CBD, where minimal p-AMPK accumulation was also found in tangle-positive glial cells. p-AMPK was not found in purified PHFs, indicating that p-AMPK did not co-aggregate with tau in tangles. Finally, in vitro assays showed that AMPK can directly phosphorylate tau at Thr-231 and Ser-396/404. Thus, activated AMPK abnormally accumulated in tangle- and pre-tangle-bearing neurons in all major tauopathies. By controlling tau phosphorylation, AMPK might regulate neurodegeneration and therefore could represent a novel common determinant in tauopathies.

Fig. 1

p-AMPK antibody characterization. a Wild type (WT) and α1/2AMPK knockout (AMPK KO) fibroblasts were treated for 24 h with the indicated concentrations of AMPK activator AICAR [41]. Cell extracts were then analyzed by WB for p-AMPK [using Abcam and Cell Signaling (c.s.) antibodies, Table 1], the AMPK target phospho-acetyl-CoA carboxylase (p-ACC), and actin. b WB analysis of temporal cortex sample extracts from three controls (CTRL) and three AD patients using antibodies directed against p-AMPK (Abcam, upper panel) and p-tau (PHF1, lower panel). c WB analysis of purified PHFs using antibodies directed against p-tau (PHF1, left panel) and p-AMPK [Abcam and Cell Signaling (c.s.), middle and right panels, respectively]

Fig. 2

Immunostaining of p-AMPK in AD brains. p-AMPK immunostaining in the CA1 (a, e–i), dentate gyrus (b), entorhinal region (c), and temporal isocortex (d) in floating brain sections of an AD case (Braak stage VI). Arrowheads indicate p-AMPK staining in the neuritic corona of amyloid plaques (a, b). p-AMPK staining was found in intracytoplasmic vesicles (e), flame-shaped tangles (f, g), extracellular ghost tangles (h), and neuropil threads (i). Scale bar 50 μm (a–d), 10 μm (e–i)

Fig. 3

Immunostaining of p-AMPK in normal, AD, and primary tauopathy brains. p-AMPK immunoreactivity in a control individual (a–c), or in AD (d, e), FTDP-17 (f–h), Guam PDC (i, j), tangle-predominant dementia (k, l), CBD (m), PSP (n), AGD (o), and PiD (p–r) patients in different brain regions: Dentate gyrus (a, f, p), CA1 (b, d, g, i, k, o, q), temporal isocortex (c, e, h, j, l, r), basal ganglia (m, n). Arrowheads indicate p-AMPK immunoreactivity in dystrophic neurites surrounding amyloid plaques in AD (d, e). Pick bodies in PiD are strongly immunostained with p-AMPK (p–r). Paraffin sections were counterstained with toluidine blue. Scale bar 50 μm

Fig. 4

p-AMPK and p-tau co-localization in AD. Immunohistochemical co-localization of p-AMPK with p-tau, labeled with CP13 (a, c, d, f) or PHF1 (b, e, g) in AD patients (CA1 region, Braak stage V/VI). p-AMPK (green) partially or fully co-localized with p-tau (red) in tangle-bearing neurons (c, e), and in dystrophic neurites surrounding amyloid plaques (d). p-AMPK was also present in intracytoplasmic vesicles (f, g, arrowheads) where no (or very weak) staining for p-tau was present. Scale bar 50 μm (a, b), 10 μm (c–g)

Fig. 5

p-AMPK and p-tau co-localization in primary tauopathies. Immunohistochemical co-localization of p-AMPK (green) with p-tau, labeled with CP13 (red) in tangle-predominant dementia (a–c), FTDP-17 (d), Guam PDC (e, f), AGD (g–i), PiD (j, k), CBD (l, m), and PSP (n–p) patients. a–j CA1, k dentate gyrus, l–p basal ganglia. Scale bar 10 μm

Fig. 6

AMPK phosphorylates tau in vitro. WB analysis of recombinant tau incubated in vitro with AMPK or GSK3β using antibodies directed against total tau (CP27), or tau phosphorylated on Ser-396/404 (PHF1), Thr-231 (2E12), Ser-202 (CP13), Ser-409 (PG5), Ser-214 (CP3), or Ser-235 (MC6)