|The Role of Tubulin in Seizure Termination||MT|
Seizures are characterized by sudden surge of neural activities. They are also terminated abruptly. The mechanism of seizure progression is discussed in another article. This article highlights only the mechanism of seizure termination.
It has been well established that action potentials are initiated at the axon initial segment (AIS) which contains high density of voltage gated sodium channels (Buffington and Rasband, 2011), and several types of voltage-gated calcium channels (Bender et al., 2012; Yu et al., 2010). While neuronal firing also depends on synaptic activation at dendritic spines, the AIS has the veto power. Therefore, the best approach to terminate seizures is to act on AIS. Indeed, prolonged (3 hr) elevation of neural activity has been demonstrated to induce AIS shortening and attenuate excitability (Evans et al., 2015).
Beneath the plasma membrane of a neuron, there is a submembrane cytoskeleton composed of actin and spectrin. The actin/spectrin layer exists not only in axons (Xu et al., 2013), but also in the somatodendritic compartments of various neuronal types, across different animal species (He et al., 2016; Han et al., 2017). At AIS, the submembrane cytoskeleton also contains Ankyrin-G which plays a key role in anchoring microtubules to the AIS membrane (see Microtubules at the Axon Initial Segment). However, due to the presence of the submembrane cytoskeleton, microtubules cannot reach the membrane surface.
Tubulin is the building block of microtubules. It has two isoforms, α and β, that usually form a heterodimer. In a tubulin heterodimer, the number of negatively charged amino acids exceeds that of positively charged amino acids by about 50 (Minoura and Muto, 2006). As a result, a microtubule is highly negatively charged along the entire length (Baker et al., 2001). Although both actin and spectrin are also highly negatively charged (Elzinga et al., 1973; Speicher et al., 1983), their distance with the membrane surface is fixed. These charges do not contribute to the dynamic change of the membrane potential field that governs the opening of voltage-gated ion channels. By contrast, the AIS contains sparse microtubules (Palay et al., 1968), allowing a microtubule to bend or translocate significantly within the AIS. As discussed in a previous article, translocation of microtubules within the AIS is sufficient to influence channel gating. Closer to the membrane should have the same inhibitory effect as hyperpolarization.
The free tubulin dimer is a small molecular complex capable of penetrating the actin/spectrin layer. Therefore, free tubulin dimers should be able to exert a stronger inhibitory effect than microtubules. This may account for the reduced excitability by microtubule depolymerization (Sakai et al., 1985; Carletti et al., 2016). However, as mentioned above, both actin and spectrin are also highly negatively charged, which may repel tubulin out of the submembrane cytoskeleton. To overcome the repulsive force, the inhibition by tubulin should also require multivalent counterions (e.g., Ca2+) to mediate the attraction between two negatively charged molecules (Ha and Liu, 1999), as illustrated in Figure 1.
During seizures, the intensive neural activities often lead to Ca2+ overload. Ca2+ has been shown to bind with tubulin, inhibiting Tau-promoted microtubule polymerization (Lefèvre et al., 2011; Li and Rhoades, 2017). Therefore, not only can the elevated level of Ca2+ ions cause microtubule depolymerization (O'Brien et al., 1997) to produce free tubulin dimers, they also serve as the counterions mediating the attraction between the actin/spectrin layer and tubulin. The entry of tubulin into the submembrane cytoskeleton can then inhibit neuronal firing with their negative electric fields (Figure 2). We see that, through tubulin inhibition, the microtubule depolymerization resulting from Ca2+ overload provides a negative feedback to mitigate Ca2+ toxicity.
The above mechanism for seizure termination is supported by the finding that biallelic mutations in TBCD (encoding the tubulin folding cofactor D) reduces free tubulin level (Flex et al., 2016), and causes intractable seizures (Pode-Shakked et al., 2017). Furthermore, after seizure termination, the threshold to induce another seizure increases significantly at 2 and 4 hours inter-stimulation intervals (Minabe et al., 1989). That means, following seizure termination, the neurons become less excitable for a few hours. This inhibitory period could reflect the presence of free tubulin within the submembrane cytoskeleton. The neuronal excitability will gradually recover because the Ca2+ overload may fade away, allowing tubulin to exit the actin/spectrin layer.
Author: Frank Lee