Hyperphosphorylation of Tau Associates With Changes in Its Function Beyond Microtubule Stability

Abstract

Tau is a neuronal microtubule associated protein whose main biological functions are to promote microtubule self-assembly by tubulin and to stabilize those already formed. Tau also plays an important role as an axonal microtubule protein. Tau is an amazing protein that plays a key role in cognitive processes, however, deposits of abnormal forms of tau are associated with several neurodegenerative diseases, including Alzheimer disease (AD), the most prevalent, and Chronic Traumatic Encephalopathy (CTE) and Traumatic Brain Injury (TBI), the most recently associated to abnormal tau. Tau post-translational modifications (PTMs) are responsible for its gain of toxic function. Alonso et al. (1996) were the first to show that the pathological tau isolated from AD brains has prion-like properties and can transfer its toxic function to the normal molecule. Furthermore, we reported that the pathological changes are associated with tau phosphorylation at Ser199 and 262 and Thr212 and 231. This pathological version of tau induces subcellular mislocalization in cultured cells and neurons, and translocates into the nucleus or accumulated in the perinuclear region of cells. We have generated a transgenic mouse model that expresses pathological human tau (PH-Tau) in neurons at two different concentrations (4% and 14% of the total endogenous tau). In this model, PH-Tau causes cognitive decline by at least two different mechanisms: one that involves the cytoskeleton with axonal disruption (at high concentration), and another in which the apparent neuronal morphology is not grossly affected, but the synaptic terminals are altered (at lower concentration). We will discuss the putative involvement of tau in proteostasis under these conditions. Understanding tau's biological activity on and off the microtubules will help shed light to the mechanism of neurodegeneration and of normal neuronal function.

Keywords: PH-tau; hyperphosphorylation; microtubules; neurodegeneration; propagation; tau.

Figure 1

Microtubule disruption can be caused by hyperphosphorylation of tau. (A) Electron micrographs showing the products of microtubule assembly. The microtubules were negatively stained with phosphotungstic acid. Rat brain tubulin was placed in an in vitro reaction in the presence of (a) control acid-soluble tau, (b) Alzheimer disease (AD) acid-soluble tau, (c) AD Phosphorylated tau (AD P-tau) and (d) AD P-tau after dephosphorylation. Very few microtubules were observed in the presence of AD P-tau and dephosphorylation appears to slightly rescue microtubule disruption (Alonso et al., 1994). (B) Cartoon model of microtubule disruption due to P-tau sequestration of normal tau away from the tubulin resulting in instability and loss of cytoskeletal structure. Image reproduced as authorized by the editors of Alonso et al. (2016).

Figure 2

Pathological human tau (PH-Tau) mimics AD P-tau. (A) A hypothetical scheme of the phosphorylation-induced self-assembly of wild-type (left) and fronto-temporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17) mutated tau proteins (right). Tau self-assembles mainly through the microtubule binding domain (MTBD)/repeat R2 and R3. The β-structure of R2 and R3 is depicted at the cartoon on the bottom. Regions of tau molecule both N-terminal and C-terminal to the repeats are inhibitory of self-assembly. Hyperphosphorylation of tau neutralizes these basic inhibitory domains, enabling tau-tau interaction. The C-terminal region beyond Pro397 (398–441) is a highly acidic segment masks the repeats. Phosphorylation of tau at Ser396 and/or Ser404 opens this segment, allowing tau-tau interaction through the repeats. FTDP-17 mutations make tau a more favorable substrate for phosphorylation than the wild-type tau. The mutated tau proteins achieve the conformation required to self-assemble at a lower level of incorporated phosphate. Although the FTDP-17 mutant tau proteins have conformations that are more prone to polymerize, in the absence of hyperphosphorylation, the highly basic segments and the C-terminus interfere with polymerization. Phosphorylation sites are indicated by red Ps at Ser/Thr positions in tau (left panel): 199, 202, 205, 212, 231, 235, 262, 396, 404 and 422; and in FTDP-17 mutant tau (right panel): 199, 212, 231, 262 and 396, respectively. Image taken from Alonso et al. (2004). The Tau cartoon at the bottom indicates the pseudophosphorylated version of tau that is used in our studies, PH-Tau (Di et al., 2016). (B) AD P-tau inhibits regeneration of microtubules. To induce microtubule assembly 3T3 cells expressing AD P-tau were incubated with 15% fresh rat brain cytosol in buffer containing 1 mM GTP for 1 h at 37°C. Cells were processed for immunofluorescence staining by using DM1-A Ab against tubulin (green) and 134d rabbit polyclonal Ab against tau (red). AD P-tau, but not normal tau, inhibited microtubule assembly (Alonso et al., 2006). (C) PH-Tau, as AD P-tau, inhibits microtubule assembly when expressed in cells. CHO cells were transfected with either tau, PH-Tau on the normal tau background (PH-Tau(n)), or PH-Tau on the R406W background. After 48 h, the cells were permeabilized with 0.1% Nonidet P-40 before fixing and then processed for immunocytochemistry as described in (B). Merge is shown in yellow. Bar, 25 μm (Alonso et al., 2010). This research was originally published by Alonso et al. (2010).

Figure 3

Characterization of PH-Tau mouse model. (A) Bigenic mice (12-month old) were tested for behavior deficits in the Novel Object Recognition task. Significant decreases in spatial memory and memory storage were observed. (B) PH-Tau_low mice shows significant loss of synapses, decreased post-synaptic density and enlarged pre-synaptic portion. Decrease in the length of the post-synaptic density was observed in both PH-Tau_low and PH-Tau_high mice. Synapses in CA1 stratum radiatum area are shown in the magnified images of square boxes. Quantitation of the number of synapses in the CA1 stratum radiatum area is shown in the bottom of the figure. (C) Representative Western blot of mouse hippocampus homogenate. The levels of synaptophysin, PSD95 and β-III-tubulin were measured. Loss of synaptic proteins was observed in PH-Tau_low mice. (D) Coronal slices of hippocampus stained with antibody NeuN recognizing nucleus of neurons (Left). Scale bar = 50 μm. Counts of the NeuN positive cells in both the CA1 and CA3 regions. *p < 0.05, **p < 0.01, ***p < 0.001; right; Di et al. .

Figure 4

Proposed mechanisms of neurodegeneration. (A) Hyperphosphorylation of tau affects multiple cellular processes. PH-tau aggregates are formed when the balance between kinases and phosphorylases is disrupted. The aggregates can begin to disrupt the stability of the microtubules. This toxic molecule has also been shown to translocate into the nucleus, cause intracellular degeneration, protein aggregation and vacuole formation. The presence of tau in the nucleus might be involved in alterations of protein expression. Disruption of the actin cytoskeleton can lead to membrane zeiosis and tau can be released from the cells, potentially propagating the disease to neighboring cells. Image reproduced from Alonso et al. (2016). Permissions were obtained through RightsLink License Number: 4358781157689. (B) Left panel: low level of PH-tau expression results in translocation to the nucleus, synaptic dysfunction and mitochondrial disruption. The presence of tau in the nucleus might be involved in alterations of protein expression. Right panel: high levels of PH-Tau expression results in protein aggregation, microtubule disruption and loss of synapses. Loss of cytoskeletal stability leads to neurodegeneration (Alonso et al., 2017). Permission granted to reproduce figure from Alonso et al. (2017).