(J-399) Establishing an okadaic acid model of progressive neurodegeneration using hTau phosphorylation dynamics for neuroengineering applications

The protein Tau is known to play an essential role in stabilization of microtubules in neuronal axons. Tau undergoes regular post translational modifications within homeostatic neurons, such as phosphorylation and dephosphorylation. However, a defining characteristic of Alzheimer’s and other Tauopathies is the phosphorylation of Tau which is known to lead to microtubule dissociation [1]. The addition of phosphate groups causes Tau to bind to itself which creates neurofibrillary tangles [2]. To model Tauopathies in brain cell cultures, okadaic acid (OA) has been used in previous studies to inhibit protein phosphatase 2A (PP2A), which results in the accumulation of phosphate groups on the protein Tau [1]. In the current study, we examine the phosphorylation of human Tau in primary rat neurons following OA treatment in order to establish a live-cell, time-dependent dementia model that mirrors the progression of the disease state as it advances. We utilized immunostaining to examine the phosphorylation levels of exogenous human Tau within a primary rat neuron model. Threonine 231 (T231) and Serine 404 (S404) are highly conserved phosphorylation sites in Tauopathic forms of neurodegeneration [3-5] and thus are the central focus of imaging in this study. We strive to correlate increased Tau hyperphosphorylation and apparent stages of degeneration of neurons within cell culture in order to establish a model for the future exploration of engineering solutions to Tauopathies.

We first dissociated and seed E18 primary rat hippocampal neurons in nine, 3-mm PDMS wells bonded to glass bottom 35-mm Petri dishes treated with 0.005% poly-L-lysine. Neurons were transduced with 5004 VG/cell of hTau40-Tdtomato BacMam virus (Montana Molecular, USA) for 24 hours beginning on DIV 2. On DIV 3, the virus is removed and the samples are incubated with fresh media. On DIV 9, neurons are treated with 100 nM Okadaic Acid (OA) for 1 hour and washed 3x with fresh media. Cells are then fixed with 4% paraformaldehyde at 0, 12, and 24 hours after OA removal. Cells are then permeabilized and a blocking solution applied before incubating with primary antibodies (rabbit anti-rat hTau, T231 phosphoTau, and S404 phosphoTau, AbCam), followed  by secondary antibody incubation (goat anti-rabbit Alexa488, AbCam) and DAPI wash prior to mounting. The phospho-Tau antibodies are specific to two highly conserved phosphorylation sites within Tauopathic neurodegeneration [3]. Expression of hTau40-tdTomato (filter: TXR) and antibody binding (filter: GFP) were visualized using a Leica DMi8 fluorescent microscope. Images are processed in ImageJ to assess fluorescent intensity levels both from the tdTomato reporter protein and antibodies. We created a region of interest based on hTau40-expressing cells in ImageJ and applied it to both the image from the TXR channel and the GFP channel. Mean channel intensity was then measured within these ROIs (IhTau and IAB, respectively) in order to assess the ratio between the Tau antibody presence and total hTau40 expression over time.

Results & Conclusions 

By treating hippocampal neurons with 100nM OA for 1 hour, we found higher rates of phosphorylation and general neurite degeneration compared to controls. We calculated the ratio between IAB/IhTau between these two mean values to assess the phosphoTau presence at each phosphorylation site relative to the total hTau40-Tdtomato intensity. The results of this analysis can be seen in Figure 1a. While both the T231 and S404 sites appear to experience similar levels of phosphorylation at 24 hours, T231 trends toward rapid and consistent phosphorylation whereas the S404 binding site shows a more gradual increase in phosphorylation after OA removal (Fig 1a). Furthermore, we can see that while control and 12 hour post-OA cells still possess healthy and fluorescing neurites while 24 hour samples have markedly decreased neurite fluorescence, show large tau aggregates, and signs of degeneration (Fig 1b). 

Discussion 

OA is a common tool for modeling neurodegeneration, but traditionally only at one time point and immediately after exposure [3-5]. The effect of OA on neurons over time, to our knowledge, has not been fully examined. Studies have examined phosphorylation of the same binding sites used in this study, but only at a single time point after OA exposure (12 hours with 10 nM OA [4], 3 hours with 100 nM OA [5]).  This study examines how phosphorylation levels of human Tau change following exposure to OA. Both T231 and S404 phosphorylation show an increase in relative abundance 24 hours post-OA. However, phosphorylation of S404 shows a gradual increase over time, where phosphorylation of T231 increases immediately, though not significantly, following OA exposure. This may imply two different mechanisms for hyperphosphorylation at these sites. Further, qualitative observation of neurons 24 hours post-OA reveals deterioration of neuronal networks consistent with progressive neurodegeneration. This study establishes a time-dependent model of Tau hyperphosphorylation which can be utilized for the development of biomedical engineering interventions at progressive stages of neurodegeneration.

1. 
   

   Kunze, A.; Meissner, R.; Brando, S.; Renaud, P. Co-pathological connected primary neurons in a microfluidic device for Alzheimer studies. Biotechnol. Bioeng. 2011, 108, 2241–2245.

   
   


2. 
   

   Kunze, A.; Peter, T.; Chanya, G.; Coleman, M.; Anna, C.Engineering Cortical Neuron Polarity with Nanomagnets on a Chip. ACS Nano 2015, 9, 3664– 3676,  DOI: 10.1021/nn505330w

   
   


3. 
   

   Trushina, N. I., Bakota, L., Mulkidjanian, A. Y., & Brandt, R. (2019). The evolution of tau phosphorylation and interactions. Frontiers in aging Neuroscience, 11, 256.

   
   


4. 
   

   Yu, U. Y., Yoo, B. C., & Ahn, J. H. (2014). Regulatory B subunits of protein phosphatase 2A are involved in site-specific regulation of tau protein phosphorylation. The Korean journal of physiology & pharmacology: official journal of the Korean Physiological Society and the Korean Society of Pharmacology, 18(2), 155.

   
   


5. 
   

   Qian, W., Shi, J., Yin, X., Iqbal, K., Grundke-Iqbal, I., Gong, C. X., & Liu, F. (2010). PP2A regulates tau phosphorylation directly and also indirectly via activating GSK-3β. Journal of Alzheimer's Disease, 19(4), 1221-1229.