cAMP-PKA phosphorylation of tau confers risk for degeneration in aging association cortex

Abstract

The pattern of neurodegeneration in Alzheimer's disease (AD) is very distinctive: neurofibrillary tangles (NFTs) composed of hyperphosphorylated tau selectively affect pyramidal neurons of the aging association cortex that interconnect extensively through glutamate synapses on dendritic spines. In contrast, primary sensory cortices have few NFTs, even in late-stage disease. Understanding this selective vulnerability, and why advancing age is such a high risk factor for the degenerative process, may help to reveal disease etiology and provide targets for intervention. Our study has revealed age-related increase in cAMP-dependent protein kinase (PKA) phosphorylation of tau at serine 214 (pS214-tau) in monkey dorsolateral prefrontal association cortex (dlPFC), which specifically targets spine synapses and the Ca(2+)-storing spine apparatus. This increase is mirrored by loss of phosphodiesterase 4A from the spine apparatus, consistent with increase in cAMP-Ca(2+) signaling in aging spines. Phosphorylated tau was not detected in primary visual cortex, similar to the pattern observed in AD. We also report electron microscopic evidence of previously unidentified vesicular trafficking of phosphorylated tau in normal association cortex--in axons in young dlPFC vs. in spines in aged dlPFC--consistent with the transneuronal lesion spread reported in genetic rodent models. pS214-Tau was not observed in normal aged mice, suggesting that it arises with the evolutionary expansion of corticocortical connections in primates, crossing the threshold into NFTs and degeneration in humans. Thus, the cAMP-Ca(2+) signaling mechanisms, needed for flexibly modulating network strength in young association cortex, confer vulnerability to degeneration when dysregulated with advancing age.

Fig. 1.

Tau phosphorylation increases with age in monkey dlPFC. (A) The aged dlPFC (31 y) presents intense pS214-tau immunoreactivity along apical and basal pyramidal dendrites (arrowheads) and diffuse reactivity in the neuropil. (B) In contrast, pS214-tau is not detected in aged V1 (same hemisphere shown; arrowheads point to an apical dendrite for comparison). (Scale bar: 20 μm.) (C) Immunoblots of heat-stable tau preparations from dlPFC show an increase with age of tau phosphorylation at S214 (Left) and T231 (Right). (D) ImmunoEM demonstrates age-related aggregation of pS214-tau (red oval in Inset) along dendritic microtubules (arrows). Ax, axon; Den, dendrite. (Scale bars: 0.5 μm.) The bottom graphs summarize the distribution of pS214-tau in cellular profiles as the percentage of immunoreactive profiles in the neuropil of young vs. aged dlPFC (SI Materials and Methods).

Fig. 2.

Ultrastructural localization of pS214-tau in dendritic spines in monkey dlPFC. (A and B) In aged spines, p-tau (arrowheads) aggregates directly over the postsynaptic membrane of asymmetric, presumed glutamatergic synapses (black arrows and Insets); bracket in A, Inset denotes an asynaptic adherens junction for comparison. Symmetric synapses onto spines, as in synaptic triads, were not labeled (B, white arrows). (C–E) In addition to the selective accumulation at asymmetric spine synapses, p-tau in aged dlPFC aggregates over the SA (asterisk). Note that the limiting membrane of the apparatus reticular cisterns is labeled, as is the spine postsynaptic membrane in E (double arrowheads). Ax, axon; Sp, spine. (Scale bars: 200 nm.)

Fig. 3.

Vesicular trafficking of pS214-tau in monkey dlPFC. (A and B) Axons in young dlPFC contained 60 nm p-tau–reactive vesicles (red arrowheads) that fused with the axolemma to form exocytotic omega-shaped profiles (B, white arrowheads). (C and D) In aged dlPFC, p-tau vesicular profiles (red arrowheads) were no longer present in axons but found in spines, fusing with the perisynaptic membrane flanking presumed glutamatergic synapses (C, white arrowhead), and within the synapse per se (D, Inset). Arrows point to asymmetric axospinous synapses. Ax, axon; Sp, spine. (Scale bars: A–C, 100 nm; D, 200 nm; and D, Inset, 50 nm.)

Fig. 4.

PDE4A is lost from dendritic spines in aged monkey dlPFC. (A and B) In young dlPFC, PDE4A (green arrowheads) is localized next to the SA (asterisk); shown are immunogold and immunoperoxidase, respectively. (Scale bars: 200 nm.) (C) PDE4A is widely expressed in layer III spines in young, but not in aged monkey dlPFC (green circles, PDE4A spines; white circles, unlabeled spines; green rectangle, PDE4A dendrite). (Scale bar: 0.5 μm.) The graphs illustrate percentage of PDE4A spines in 10-y vs. 25-y monkey dlPFC (Upper), averaged from 46-μm² electron microscopic fields (20 fields from five tissue blocks per animal; SI Materials and Methods) of layer III. Young (Left): PDE4A spines/46 μm² = 5.95 (± 2.13 SD). Aged (Right): PDE4A spines/46 μm² = 2.05 (± 1.24 SD).

Fig. 5.

PDE4A expression and physiological effect decrease with age in PFC. (A) Immunoblot and quantification of decreased PDE4A5 protein expression with age in monkey dlPFC, normalized to GAPDH, 7–11 y vs. 20–28 y, *P < 0.05. Individual data points in arbitrary units: 7 y = 0.93; 11 y = 1.06; 20 y = 0.61 and 0.38; and 28 y = 0.42. (B) Low doses of the PDE4 inhibitor etazolate (5–10 nA) decreased task-related firing of dlPFC neurons in young but not aged monkeys, whereas a high dose (25 nA) reduced firing in all neurons (n = 39). Shown are significant effects of dose (F = 6.95, P = 0.002) and age (F = 12.27, P = 0.001); significant changes from 0 nA, *P < 0.05, ***P < 0.009; and firing rates percentage of young control. (C) Quantification of postsynaptic density (PSD) preparations from mouse frontal cortex shows significant decrease of PDE4A5 in the PSD fraction with age, **P < 0.05, which remains significant (P < 0.05) when normalized by PSD95 levels to account for presumed spine loss. (D) PDE4 inhibitor, rolipram, increased pS214-tau in mouse primary cortical neurons. Activation of PKA by forskolin (Fsk) produced a dose-related increase in pS214-tau [F(4,60) = 8.920, P < 0.0001] that was significantly increased by rolipram [10 μM; F(1,60) = 8.882, P = 0.0042].

Fig. 6.

Synopsis of age-related alterations in layer III corticocortical network synapses in primate dlPFC. (A) Schematic illustration of young dlPFC, with PDE4A positioned to regulate feedforward cAMP-Ca²⁺-K⁺ signaling near NMDAR glutamate synapses in dendritic spines (K⁺ channels near synapse not shown). pS214-Tau is distributed sparsely along axonal and dendritic microtubules and in trafficking vesicles in axons (red circle). (B) Representative immunoEM of pS214-tau (red arrowheads) in young dlPFC. (C) Schematic illustration of changes in aged dlPFC, with loss of PDE4A, dysregulation of cAMP-Ca²⁺-K⁺ signaling, and aggregation of pS214-tau in the synapse and the SA (asterisk) and along microtubules in distal dendrites. pS214-Tau endoplasmic vesicles are no longer present in axons but appear in spines perisynaptically and within the synapse. (D) Representative immunoEM of pS214-tau in aged dlPFC. Ax, axon; Den, dendrite; Sp, spine; arrows point to asymmetric axospinous synapses. (Scale bars: 200 nm.)