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1.8.1 Microtubule stabilisation as a therapeutic

Microtubule-stabilising drugs have been proposed as a therapy for AD and other neurodegenerative diseases with axonal transport impairments (Ballatore et al., 2012; Trojanowski et al., 2005). Reduced microtubule stability is an early pathological change in response to Aβ toxicity, likely a result of tau hyperphosphorylation. In addition, the destabilisation of microtubules is implicated in axon degeneration (Tang-Schomer et al., 2010). There are a number of ways that microtubule stabilisation can be targeted. Firstly, there are microtubule-stabilising agents that bind directly to microtubules and can promote stability of microtubules and prevent them from depolymerising (Yu et al., 2013). Research into drugs that bind to the same site as tau, such as taxanes and epothilones, has demonstrated their effects in reducing pathology in tauopathy mouse models (Bakota and Brandt, 2016; Brunden et al., 2010; Erez et al., 2014; Zhang et al., 2012a). However, it is unknown whether binding by these agents cause a conformational change to microtubules which promotes stability. Alternatively, drugs that can target PTMs of tubulin such as acetylation and glutamylation, as well as MAPs such as tau, may promote stability and are also attractive targets for intervention.

An important feature of any drug for neurodegenerative disease is that it must be able to cross the blood brain barrier. Many microtubule-stabilising drugs were first used to treat cancers by causing cell cycle arrest, so while they may have been developed for human use, their ability to access the CNS needs to be determined. Taxanes and epothilones are two of the two main classes of microtubule-stabilising agents, however their properties differ. Taxol has

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poor permeability to the blood brain barrier, whereas epothilone B and D have been shown to cross the blood brain barrier and are a potential therapeutic for AD.

1.8.2 Microtubule modifying drugs

Taxol

Taxol was first isolated from Taxus brevifolia in 1971 and was identified as an anti-leukemic and anti-tumour drug (Wani et al., 1971). It is now known that it enhances microtubule assembly and length, as well as stabilising microtubules (Parness and Horwitz, 1981; Schiff and Horwitz, 1981). Taxol has been found to assemble tubulin subunits of microtubules in the absence of GTP or MAPs, such as tau (Schiff and Horwitz, 1981). Without taxol, in in vitro preparations, tubulin is predominantly in the depolymerised state, however, with the addition of taxol, polymerisation increases, along with GTP hydrolysis (Hamel et al., 1981). When GTP is absent, tubulin still forms microtubule structures when taxol and MAPs are present (Hamel et al., 1981). Taxol binds to tubulin polymers between the ‘M-loop’ and the ‘central helix’ of beta-tubulin (Amos and Lowe, 1999; Nogales et al., 1999). A number of studies have investigated the therapeutic potential of taxol in injury models to the brain, spinal cord and peripheral nerves. In a needle stick model of acute damage to the somatosensory cortex, taxol was an effective therapeutic agent to protect against neurodegeneration by reducing neurite loss via microtubule stabilisation of axons (Adlard et al., 2000). As taxol improves axonal elongation, the potential of taxol to improve axon regeneration of neurons following injury has also been investigated. In adult rats with a dorsal hemisection spinal cord injury, taxol treatment increased the number of axons regenerating, and increased axon outgrowth (Hellal et al., 2011). Moreover, when taxol was administered prior to axon stretch injury in vitro, it delayed axon degeneration was delayed compared to untreated injured axons (Tang-Schomer et al., 2010). Taxol restored facial nerve function after nerve crush injury in

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Wistar rats, through the stabilisation of microtubules, which was evaluated through motor function and the degree of axonal branching (Grosheva et al., 2008). Furthermore, taxol administration to an optic nerve crush injury site, allowed axon regeneration past the lesion area because of microtubule stabilisation (Sengottuvel et al., 2011). At spinal cord lesions sites in rodents, microtubule stabilisation promoted axonal growth, and increased levels of detyrosinated and acetylated tubulin, at the lesion site of the spinal cord in rodents (Ruschel et al., 2015), suggesting that axon growth may have been due to the reduction of scar tissue, which contains axon growth-inhibitory factors (Ruschel et al., 2015), although astrogliosis was shown to be unaffected by microtubule stabilisation treatment. The effect of microtubule- stabilising agents on glial cells has not been extensively investigated in disease models. The effects of taxol have also been studied in a T44 tau mouse model where fast axonal transport was improved, and microtubule density and detyrosinated tubulin levels increased (Zhang et al., 2005).

Epothilones

Another family of microtubule-stabilising agents, epothilones, have similar characteristics and mechanisms to taxol, although they are able to cross the blood brain barrier (Bollag et al., 1995). Epothilone, originally an anti-tumour drug, is produced by the gram-negative bacteria Sorangium cellulosum, and has undergone both phases I and II clinical trials (Beer et al., 2007; Bollag et al., 1995; Konner et al., 2012). Taxol and epothilones have a similar structure and bind to beta-tubulin at a common site (Cheng et al., 2008). Like taxol, epothilone, has been shown to prevent microtubule disassembly when calcium is present. Taxol and epothilone also reduce the amount of tau bound to microtubules, suggesting that these drugs also compete with tau (Mukhtar et al., 2014; Ross et al., 2004). Due to their success in treating cancer, epothilones are being investigated as a treatment for

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neurodegenerative diseases. As Parkinson’s disease shares some pathological features with AD, epothilone D was shown to reduce microtubule defects and increase detyrosinated microtubules in dopaminergic neurons in a mouse model of Parkinson’s disease (Cartelli et al., 2013). In a mouse model of schizophrenia, epothilone D was also shown to improve short- term memory, suggesting that epothilone D may affect synapses in the hippocampus (Andrieux et al., 2006). Some research has suggested that the effectiveness of treatment with epothilone B depends on the type and age of the neuron (Jang et al., 2016). Many mouse models of varying neurodegenerative diseases have shown successful microtubule stabilisation, resulting in reduced pathology. For example, amyloid plaque pathology may be hindered by microtubule stabilisation, as APP is transported along microtubules in the same compartment as its cleaving enzymes (Kamal et al., 2001), and that loss of microtubules may cause altered processing of APP, leading to possible increased Ab production (Busciglio et al., 1995). Therefore, stabilisation of microtubules with taxanes or epothilones could potentially alter APP processing by preventing microtubule depolymerisation. Epothilone D has increased microtubule density in axons, decreased axonal dystrophy and reduced cognitive defects in a PS19 mouse model of frontotemporal lobar degeneration (Zhang et al., 2012a). Tau acetylation leads to destabilisation of the axon initial segment cytoskeleton and leads to mis-localisation of tau to the somatodendritic compartment. The axon initial segment provides a barrier from the axon and the somatodendritic compartment and allows retention of axonal proteins. Cytoskeletal proteins Ankyrin G and βIV-spectrin, which are part of the axon initial segment, are reduced in AD and are associated with increased tau acetylation in tau transgenic mouse models. However, in primary neuronal cultures, epothilone D treatment restored the axon initial segment barrier function, preventing tau from being mis-localised (Sohn et al., 2016). Another type of microtubule-stabilising agent, NAPVSIPQ (Bassan et al., 1999) has been shown to reduce tau pathology and enhance cognitive function (Matsuoka et

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al., 2008). Epothilones may also have an effect on mobility of glial cells, such as microglia, due to over-stabilisation of the cytoskeleton. One study demonstrated that epothilone B attenuated microglia activation through redistributing the cytoskeleton, causing morphological transition and suppressed expression of pro-inflammatory factors such as interleukin-1b and tumour necrosis factor-a (Yu et al., 2018). This suggests that epothilones may not alter mobility of microglia, but can have an effect on morphology. Epothilone D also had a similar effect in an in vitro model of multiple system atrophy. Epothilone D was shown to reduce microglial-mediated transport of alpha synuclein by inhibiting cytoskeletal dynamics (Valdinocci et al., 2018).

HDAC6 inhibitors

Another microtubule-stabilising agent which has been postulated as a therapeutic is trichostatin A (Simões-Pires et al., 2013). Trichostatin A inhibits the de-acetylating enzyme HDAC6, responsible for deacetylation, therefore promoting acetylation (Liu et al., 2015a). Research has shown that HDAC6 inhibition by use of trichostatin A or tubastatin A in neurodegenerative mouse models, have rescued axonal transport and locomotor deficits (Benoy et al., 2018; Godena et al., 2014) and neurite outgrowth (Hasan et al., 2013). Several HDAC6 inhibitors such as tubastatin A and tubacin have been used as a treatment in various diseases, including Japanese encephalitis virus (Lu et al., 2017), lymphoproliferative disease (Cosenza and Pozzi, 2018) and Charcot-Marie-Tooth disease (Benoy et al., 2018). Axonal transport has been shown to be restored after treatment with trichostatin A (Godena et al., 2014). In an APP/PS1 mouse model, with a knock-out of HDAC6, Aβ-induced deficits in mitochondrial trafficking were rescued (Govindarajan et al., 2013).

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Sodium selenate is an agonist to PP2A, enhancing phosphatase activity. Serine/threonine- specific PP2A is a major phosphatase implicated in tau dephosphorylation and is reduced in protein and mRNA levels and activity in the AD brain (van Eersel et al., 2010; Vogelsberg- Ragaglia et al., 2001). Altered expression of PP2A is also correlated with AD pathology (Sontag and Sontag, 2014). Sodium selenate acts upon the phosphatase, thereby stabilising the tau-PP2A complex, and reducing hyperphosphorylated tau (Shultz et al., 2015). In a tauopathy mouse model, mice treated with sodium selenate had reduced levels of both phosphorylated tau and total tau levels in both the hippocampus and amygdala compared with controls (Corcoran et al., 2010). The TAU441 mice also exhibited significantly improved spatial learning and memory. Sodium selenate is currently undergoing a Phase II clinical trial for mild-moderate AD (Malpas et al., 2016). More recently, sodium selenate has been used to treat hyperexcitability in mouse models of Lafora disease, where some motor and memory deficits were ameliorated and neurodegeneration and the glial response were reduced (Sánchez-Elexpuru et al., 2017). Other AD pathology has been reduced with sodium selenate treatment, where in a 3xTg-AD mouse model, sodium selenate repressed amyloid beta formation, via the Wnt/β-catenin signalling pathway (Jin et al., 2017).

Developing microtubule-modifying drugs as therapeutics for neurodegenerative disease

Microtubule stabilisation has been successful in treating many forms of cancer, as it causes cell cycle arrest at G2/M transition phase (Fanale et al., 2015; Rohrer Bley et al., 2013; Zhao et al., 2016). Microtubule stabilisation as a therapeutic has undergone many phase I and II clinical trials to treat solid tumours, lymphoma, malignancies, metastatic prostate cancer and advanced colorectal cancer. (Beer et al., 2007; Eng et al., 2004; Holen et al., 2004; Konner et al., 2012; Rubin et al., 2005; Spriggs et al., 2003).

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Although these studies suggest that microtubule stabilisation with taxol, epothilone D or trichostatin A may be protective in a number of conditions, the use of taxol and epothilones as anti-cancer drugs has shown adverse side effects such as neutropenia, fatigue, nausea, dizziness as well as abdominal pain, when high doses of epothilone D were administered (Bedard et al., 2010; Holen et al., 2004). In addition, administration of these drugs can cause peripheral neuropathy. Most doses required to cause cell cycle arrest in cancer are approximately 100 mg/m2 _{weekly,however these doses are considered to be relatively high} compared to only a low dose needed for neurodegenerative disease (Cartelli et al., 2013; Ruschel et al., 2015). A number of clinical studies using epothilone as a microtubule- stabilising drug have demonstrated that these toxic effects are dose-dependent and when used at a lower dosage, the side effects are minimised (Fumoleau et al., 2007). Importantly, the concentrations of taxol or epothilone required to stabilise microtubules in axons or to promote axon regeneration are much lower than those required to prevent division of cancer cells (Brunden et al., 2012).