Alzheimer  >   8. The Roles of Microtubules and Ankyrin-G in Excitability

Chapter 7 has presented compelling evidence that elevated total and/or four-repeat (4R) Tau protein increase neuronal excitability. The Tau-induced hyperexcitability plays a key role in tauopathy, which includes Alzheimer's disease, Huntington's disease, progressive supranuclear palsy and corticobasal degeneration. Therefore, a better understanding of its mechanism should provide greater insights into the pathogenesis of Alzheimer's disease.

Tau is a microtubule-associated protein. Its influence on excitability could be mediated by microtubules. A microtubule is made up of tubulin dimers which are enriched with acidic residues (aspartate and glutamate). In a solution at the physiological pH value (~ 7), these amino acids become negatively charged. From its amino acid sequence, the net charge on a tubulin dimer can be calculated to be 50.9 e at pH = 6.7. Thus, a microtubule is highly negatively charged along the entire length (Article 1). This physical property is well-suited for the regulation of neuronal excitability.

Microtubule Model for Excitability (MTME)

The generation of nerve impulses requires opening of voltage-gated sodium channels. It has been well documented that action potentials initiate at the axon initial segment (AIS) (Article 2). Therefore, AIS is an ideal region for microtubules to modulate excitability. Fundamentally, the open probability of voltage-gated sodium channels is determined by electric fields acting on their voltage sensor (the S4 segment). In a nerve membrane, the electric fields may come from various sources, such as applied voltages or ions in the intracellular and extracellular solutions. Article 3 shows that the electric fields from microtubules can also influence excitability by acting on the voltage sensor of sodium channels, especially in pyramidal neurons that participate in long-range communication.

It is generally accepted that the threshold for generating action potentials is the lowest at AIS than at other regions. This explains why action potentials are initiated at AIS. However, there is a major disagreement on the mechanism underlying the lowest threshold at AIS. A few researchers believed that the AIS must contain high density of sodium channels to facilitate the generation of action potentials (Kole et al., 2008; Hu et al., 2009; Huang and Rasband, 2018), despite several studies indicating that the density of sodium channels at the AIS of pyramidal neurons is comparable to the soma (Colbert and Johnston, 1996; Colbert and Pan, 2002; Fleidervish et al., 2010). On the other hand, all studies revealed that in pyramidal neurons the AIS sodium channels were activated by less depolarization (~ 7 mV ) than somatic sodium channels, that is, the voltage dependence of sodium channels at AIS is shifted toward hyperpolarization. Colbert and Pan (2002) propose that this property, rather than a high density of channels at AIS determine the lowest threshold for action potential initiation. Katz et al. (2018) further suggests that the hyperpolarization shift probably reflects interactions of sodium channels with the proteins (e.g., Ankyrin-G) that characterize AIS.

Ankyrin-G is a master organizer of AIS. It may anchor various proteins to the AIS membrane, including ion channels and microtubules. In AIS, a number of microtubules usually forms a bundle, called "fascicle". Article 3 posits that in pyramidal neurons the open probability of AIS sodium channels is directly governed by the electric field from microtubule fascicle. Most AIS sodium channels would be closed when a fascicle associates with the membrane via Ankyrin-G. The open probability of AIS sodium channels will increase sharply as the microtubule fascicle moves away from the membrane due to disruption of the binding between fascicle and Ankyrin-G by electric fields. This hypothesis will be referred to as Microtubule Model for Excitability (MTME), which is illustrated in Figure 8-1.


Figure 8-1. The Microtubule Model for Excitability.
(A) Most AIS sodium channels would be closed when a negatively charged microtubule fascicle associates with the membrane via Ankyrin-G and EB1/3.
(B) The open probability of AIS sodium channels will increase sharply as the microtubule fascicle moves away from the membrane due to disruption of the binding between fascicle and Ankyrin-G by electric fields. EB1/3 may attach to either Ankyrin-G or fascicle.

According to MTME, the subthreshold depolarization does not activate AIS sodium channels by direct interaction with their voltage sensors. Rather, the depolarizing electric field may disrupt the Ankyrin-G/fascicle binding, allowing the fascicle to move away from the membrane. The electric field decreases with the square of distance. The distance between a fascicle and the membrane is about 40 nm when they are linked by Ankyrin-G. At this minimal distance, the fascicle produces a strong hyperpolarizing field to inactivate AIS sodium channels. The strength of the hyperpolarizing fields will reduce by an order of magnitude as the distance increases to 120 nm.

Ankyrin-G contains a flexible C-terminal tail that can extend into the intracellular AIS shaft at a maximum depth of ~140 nm, with an average of only 26 nm below the submembrane coat (Jones and Svitkina, 2016). The diameter of the AIS is about 1500 nm. Therefore, a large portion of the AIS shaft is beyond the reach of Ankyrin-G. The fascicle has ample space to move randomly and exert little influence on channel gating. As the fascicle moves toward the membrane and within the reach of Ankyrin-G, it will be anchored to the membrane, thereby reducing excitability.

Evidence for a Role of Ankyrin-G in Excitability

Based on MTME, decreased Ankyrin-G level should enhance excitability (Figure 8-2).


Figure 8-2. The effects of Ankyrin-G level on excitability.
(A) Ankyrin-G links the negatively charged microtubule fascicle to the membrane, thereby reducing excitability.
(B) Down-regulation of Ankyrin-G may increase the probability for the microtubule fascicle to detach from the membrane, consequently increasing excitability. Loss of all Ankyrin-G proteins would disrupt the AIS structure.

This model is supported by two studies that have demonstrated the effects of Ankyrin-G on excitability. The first study employed TsA201 cells to express Nav1.6 channels and/or Ankyrin-G. If only Nav1.6 channels are expressed, a significant persistent sodium current INaP (conducted by Nav1.6 channels) was observed. However, co-expression with Ankyrin-G reduced INaP (Shirahata et al., 2006). The second study investigated the effects of amyloid precursor protein (APP) over-expression in a transgenic mouse model. It was found that the APP over-expression up-regulates a microRNA, miR-342-5p, which in turn down-regulates the expression of Ankyrin-G (Sun et al., 2014). In agreement with MTME, the APP transgenic mice exhibited hyperexcitability (Wesson et al., 2011; Bezzina et al., 2015).

The Role of End-Binding Proteins

The microtubule end-binding protein EB1 or EB3 (denoted by EB1/3) is required for the binding between the microtubule and Ankyrin-G (Leterrier et al., 2011; Fréal et al., 2016). Normally, EB1/3 binds to the microtubule plus end, regulating its dynamic growth. However, at the AIS, the microtubule fascicle is decorated by EB1/3 over the entire length. This is because the microtubule also possesses other binding sites for EB1/3, albeit weaker. At low concentration, EB1/3 binds preferentially at the plus end which has stronger binding affinity. The weaker binding sites may be occupied only when the EB1/3 concentration is high (Bu and Su, 2001).

The Tau protein interacts with not only microtubules, but also EB1/3 (Sayas et al., 2015). Therefore, Tau could be recruited to the biding sites between microtubules and EB1/3. These sites are the anchor points that link microtubules to the membrane. As Tau proteins are recruited to anchor points, they could prevent the binding between Ankyrin-G and microtubule fascicles, thereby increasing excitability. Further details are discussed in the next chapter.

Related Articles

1. Introduction to Microtubules

2. Microtubules at the Axon Initial Segment

3. Why are Action Potentials initiated at AIS?

4. A Novel Function of α-Synuclein at the Axon Initial Segment


Author: Frank Lee
First published: May 23, 2015
Last updated: July 10, 2019