|The Role of Tubulin in Parkinson's Disease||Alzheimer|
Parkinson's disease (PD) is a neurodegenerative disorder that affects dopaminergic (DA) neurons in the substantia nigra pars compacta (SNc). Its symptoms include tremor, rigidity and other forms of motor dysfunction. Pathologically, PD is characterized by the presence of Lewy bodies comprising mainly α-synuclein, which is a protein located primarily at the presynaptic terminal (Iwai et al., 1995). Their aggregation into oligomers is the major cause of neurodegeneration. A plethora of possible mechanisms were proposed to explain the toxicity of oligomeric α-synuclein, but no consensus has been reached yet (Roberts and Brown, 2015). Several lines of evidence suggest that tubulin plays a pivotal role.
The wide spectrum of symptoms in Huntington's disease and Guam-type ALS demonstrate the common origin of neurodegenerative disorders. They may share the same pathogenic cascade from BDNF deficiency to hyperexcitability described in Chapter 12. In line with this notion, PD is also associated with BDNF deficiency (Howells et al., 2000), miR-132 down-regulation Gillardon et al., 2008; Wanet et al., 2012) and hyperexcitability (Blandini et al., 1996). Various neurodegenerative disorders could differentiate at the neurons that exhibit hyperexcitability.
Hyperexcitability in Subthalamic Nucleus
There is substantial evidence that the loss of DA neurons in SNc arises from glutamate-mediated excitotoxicity (Blandini et al., 1996; Ambrosi et al., 2014). The subthalamic nucleus (STN) provides major glutamatergic inputs to SNc (Lee and Tepper, 2009). Moreover, STN plays a critical role in long range synchronization at the beta band (Ahn et al., 2016) - the brain rhythm responsible for motor timing. Computer simulation suggests that the beta rhythm is generated by bidirectional interaction between STN and external Globus Pallidus (GPe), together with excitatory input from the motor cortex to STN (Holgado et al., 2010; Pavlides et al., 2015). The idea that STN could be the initial site of Parkinson's disease is further supported by the observation that excessive beta activity is associated with Parkinson's disease, resulting from hyperactive STN (Kühn et al., 2006).
The Over-stimulated Dopaminergic Neurons
The hyperactive STN can over-stimulate the DA neurons in SNc. When a neuron fires, Ca2+ may enter the neuron through various voltage-gated calcium channels located at different neuronal compartments: dendrites, axon initial segment (AIS) and axon terminals. Obviously, the Ca2+ ions that enter a compartment may diffuse to another, but its concentration will be reduced. Furthermore, in the course of Ca2+ diffusion, they may encounter Ca2+ buffering proteins that prevent activation of Ca2+-dependent enzymes. Calpain plays a crucial role in Tau hyperphosphorylation. Its activation requires high Ca2+ concentration. Normal Tau proteins are present only in the axon. In the distal part of the axon, most Tau proteins are tightly bound with microtubules, making them hard to be phosphorylated by protein kinases. In contrast, the unique structure of AIS contains high proportion of free Tau proteins, which are easily accessible by GSK-3 and Cdk5 - the major targets of calpain (Chapter 12). Therefore, generally speaking, Tau pathology (neurofibrillary tangles) is induced by Ca2+ influx at AIS.
TDP-43 localizes predominately to the nucleus and under some conditions it may translocate to the cytoplasm. Its pathology also depends on Ca2+ (Appendix B). In Figure C-1, the contribution from Ca2+ overload at the axon (either AIS or terminals) is omitted because axonal contribution is expected to be small compared to the Ca2+ overload at dendrites. The DA neurons in SNc do not participate in long range synchronization. Their AIS may contain only low level of free Tau or voltage-gated calcium channels, consistent with the finding that neurofibrillary tangles in SNc are far less than STN (Ishino and Otsuki, 1975). TDP-43 pathology in SNc DA neurons is possible (McKee et al., 2010), but not as common as in motor neurons, probably due to higher level of Ca2+ buffering proteins and/or lower level of TDP-43. Degeneration of SNc DA neurons is mainly caused by α-synuclein pathology.
From Ca2+ overload to α-synuclein aggregation
At the nerve terminal, Ca2+ entry is required to trigger the release of neurotransmitters, including dopamine (Okita et al., 2000). In most cases, Ca2+ enters the nerve terminal via L-type channels, whose antagonists are protective for Parkinson's disease (Ilijic et al., 2011; Swart and Hurley, 2016). This supports the view that Ca2+ overload at the nerve terminal leads to α-synuclein pathology. Mechanistically, the terminal Ca2+ overload, together with induced oxidative stress, promote α-synuclein aggregation (Nath et al., 2011; Goodwin et al., 2013; Rcom-H'cheo-Gauthier et al., 2014; Rcom-H'cheo-Gauthier et al., 2016).
From α-synuclein aggregation to microtubule deficiency
As mentioned at the beginning of this article, several lines of evidence suggest that tubulin may play a central role in the toxicity of α-synuclein aggregation. A tubulin dimer contains about 50 more negatively than positively charged residues (Minoura and Muto, 2006). In a solution, tubulin dimers are surrounded by counterions and polar water molecules which may reduce the electrostatic interaction with external fields. The effective charge on a tubulin dimer was estimated to be 12 - 20 e– (van den Heuvel et al., 2006; Minoura and Muto, 2006 ). Theoretical calculation shows that the hyperpolarizing field from the negative charges is comparable to the membrane potential field when tubulin is near the membrane (see Introduction to Microtubules). Therefore, at the axon terminal, depolymerized free tubulin dimers may move close to the membrane to inhibit neuronal firing via their hyperpolarizing fields. Tubulin polymerization into microtubules will reduce the amount of free tubulin dimers, thereby enhancing excitability. Oligomeric α-synuclein has been shown to inhibit tubulin polymerization (Chen et al., 2007). This explains why α-synuclein oligomers reduce neuronal excitability (Kaufmann et al., 2016) and why dopamine release is impaired in Parkinson's disease.
In Tau pathology, hyperexcitability due to excess Tau proteins eventually leads to hypoexcitability caused by Tau hyperphosphorylation (see Paper 2). Similarly, in α-synuclein pathology, over-stimulation of SNc DA neurons by hyperactive STN also leads to hypoexcitability, but in this case it is caused by high level of free tubulin dimers. Reduction in tubulin polymerization affects not only excitability, but also the amount of functional microtubules. Microtubule deficiency may impair axonal transport of essential cellular components (Pellegrini et al., 2017), eventually leading to neuronal death.
The Role of Parkin
Parkin is an E3 ubiquitin ligase which has been shown to enhance α/β-tubulin ubiquitination and degradation via direct interaction. The parkin mutants found in PD patients do not ubiquitinate or degrade either tubulin (Ren et al., 2003). Tubulin exists in the postsynaptic density (PSD), a structure just beneath the postsynaptic membrane (Sahyoun et al., 1986; Yun-Hong et al., 2011, Figure 9). As explained in the last section, the tubulin near a nerve membrane may reduce excitability through its negative electric field. Therefore, degradation of tubulin by parkin is expected to enhance excitability. This prediction agrees with the experimental findings that synaptic excitability decreases in the absence of parkin (Goldberg et al., 2003) and parkin gene inactivation inhibits glutamate neurotransmission (Itier et al., 2003).
The Role of LRRK2
LRRK2 is a large protein with over 2,500 amino acids, and exists as a dimer under native conditions (Greggio et al., 2008). The bound LRRK2/tubulin complex is unlikely to enter PSD. Therefore, at the dendritic spines, LRRK2 may enhance excitability by preventing tubulin from entering PSD (Figure C-2). In the axon, there is an actin layer under the plasma membrane (Xu et al., 2013). This actin layer could also exclude the LRRK2/tubulin complex, while allowing the free tubulin to penetrate. Hence, overexpression of wild-type LRRK2 is expected to enhance excitability by reducing the amount of free tubulin inside PSD and/or actin layer, consistent with observations (Li et al., 2010; Plowey et al., 2014). However, the effect of LRRK2 mutants on excitability is harder to predict. It depends not only on the binding of LRRK2 mutant with the free tubulin, but also with other competing components such as microtubules. Experimentally, both negative (Li et al., 2010; Chou et al., 2014; Yue et al., 2015) and positive effects (Plowey et al., 2014) have been reported.
LRRK2 PD-linked mutations have been demonstrated to enhance its microtubule association (Kett et al., 2012), particularly at the stable region (Blanca Ramírez et al., 2017). These microtubule-associated LRRK2 forms filamentous structures, which could impair microtubule functions. Furthermore, the aggregation of LRRK2 to microtubules would reduce the amount of LRRK2 for the association with free tubulin. This mechanism may account for the negative impact of LRRK2 mutations on excitability.
LRRK2 plays an important role in axon and dendrite growth (Sepulveda et al., 2013; Häbig et al., 2013). During neurite growth, massive tubulins are produced for the polymerization of microtubules. However, large amount of free tubulins inside PSD or actin layer could inhibit neuronal firing. LRRK2 may have a normal function to retain tubulin at the growth cone for microtubule polymerization and proper excitability.
Author: Frank Lee