Efficacy of epothilones in central nervous system trauma treatment: what has age got to do with it?

Jayden Clark, Zhendan Zhu, Jyoti Chuckowree, Tracey Dickson,Catherine Blizzard

Abstract Central nervous system injury, specifically traumatic brain and spinal cord injury, can have significant long lasting effects. There are no comprehensive treatments to combat the injury and sequalae of events that occurring following a central nervous system trauma.Herein we discuss the potential for the epothilone family of microtubule stabilizing agents to improve outcomes following experimentally induced trauma. These drugs, which are able to cross the blood-brain barrier, may hold great promise for the treatment of central nervous system trauma and the current literature presents the extensive range of beneficial effects these drugs may have following trauma in animal models. Importantly,the effect of the epothilones can vary and our most recent contributions to this field indicate that the efficacy of epothilones following traumatic brain injury is dependent upon the age of the animals. Therefore, we present a case for a greater emphasis to be placed upon age when using an intervention aimed at neural regeneration and highlight the importance of tailoring the therapeutic regime in the clinic to the age of the patient to promote improved patient outcomes.

Key Words: aging; epothilones; glial; microtubule stablization; neuron; neuronal regeneration; spinal cord injury; traumatic brain injury

Traumatic brain and spinal cord injuries continue to be a leading cause of death and disability in developed nations(Stocchetti and Zanier, 2016). This trauma can result in primary damage and complex secondary pathologies, which can cause prolonged or life-long motor and/or cognitive impairments. There are currently no available therapeutics that are able to prevent, minimize or reverse the deficits that develop following central nervous system (CNS) trauma.Effective therapeutic strategies are not being discovered fast enough and many promising drugs in the preclinical setting are not proving effective in clinical trial. A recurrent theme over the last decade has been to focus on why so many promising new therapeutics are failing to jump the gap from bench to bedside. One possible limitation to preclinical trials,that may not be accounted for appropriately, is the effect of age on the heterogeneity of symptoms and functional deficits following a CNS insult (Sun et al., 2020). Here we examine the evidence for a promising therapy for treating CNS trauma, the epothilones, which have potent neuronal and non-neuronal cell actions principally through microtubule stabilization, and propose that the efficacy of these drugs and likelihood of success in translating to the clinic may depend upon stratifying for age at insult.

A consequence of CNS trauma is neuronal cell loss and axonal injury, and the activation and reaction of glial populations(Spain et al., 2010; Wang and Ma, 2010; Greer et al., 2011;Hassannejad et al., 2019). Following injury microglia,oligodendroglial precursors, meningeal cells and astrocytes concentrate within the injury site. These cells can have both favorable and detrimental effects on the neuronal regenerative response (Ng and Lee, 2019). The formation of the glial scar presents a chemical and physical barrier to stop the spread of the injury, while conversely inhibiting regeneration of disconnected axons (Fawcett and Asher, 1999). Axons are particularly vulnerable to structural injury due to their relative long length and size. Injury-induced axonal degeneration can have devastating and far-reaching effects. In severe trauma axons are severed, and this disconnection can lead to a starvation of the post synaptic connection and disruption and retraction of the proximal neuron (Blennow et al., 2016).In mild to moderate traumatic brain injury, axons are often subtly disrupted at the time of injury, with immediate changes including mechanically induced alterations in permeability,deregulation of ionic homeostasis and compression of the cytoskeleton that can finally lead to disconnection (Smith et al., 2013). Hence, regardless of the initial injury severity cytoskeletal misalignment or loss and the accumulation or compaction of cytoskeletal components is a common event. The microtubule cytoskeleton has been shown to be particularly disrupted following structural injury: Microtubule alterations after injury include microtubule disruption and detachment with associated defects in axonal transport(Smith et al., 1999; Tang-Schomer et al., 2010) and the loss of microtubule-associated proteins such as Tau and microtubuleassociated protein 2 (Farkas and Povlishock, 2007; Bradke et al., 2012; Smith et al., 2013).

Systematic database searches of PubMed and Web of Science were performed to identify valid peer reviewed studies with no limitations on year of publication. Keyword terms used focused on CNS aging, CNS trauma, traumatic brain injury,microtubule stabilisation, epothilones and taxanes, axonal regeneration and neuronal regeneration. We used a narrow selection criteria for this review, and other variables should be considered to gain a greater understanding on age dependant CNS trauma and therapeutic targeting.

Critical to the treatment of CNS trauma is the restoration of neuronal and glial function. Microtubule stabilizing drugs, including Food and Drug Administration approved taxanes and the epothilones, hold the potential to effect both neuronal and glial populations. Taxanes and the epothilones are already used at relatively high doses in the treatment of various cancers where, due to their hyperstabilization of microtubules, they act to arrest cell division and slow or prevent cancer cell growth (Goodin et al., 2004;Kolman, 2004). However, it is their action at low doses that holds particular promise for treating CNS injury. At low concentrations these same drugs can protect against microtubule depolarization and dissolution, and encourage polarization and structural stability (Brunden et al., 2010;Hellal et al., 2011; Sengottuvel and Fischer, 2011; Baas and Ahmad, 2013). This quality could be particularly beneficial for protecting axons following structural injury. Indeed,paclitaxel, one of the taxanes, has been shown to improve outcomes following models of nerve injury by promoting axonal elongation and regeneration and also reducing glial scar formation (Hellal et al., 2011; Sengottuvel et al., 2011).However, paclitaxel has poor blood-brain barrier permeability,making it a sub-optimal candidate for CNS delivery. The epothilones, a group of microtubule stabilizing compounds found naturally in the myxobacterium Sorangium cellulosum(Bollag et al., 1995), offer an attractive alternative solution.Numerous epothilones have now been identified, including Epothilone B (EpoB) and Epothlione D (EpoD), which share a mode of action similar to paclitaxel for the binding site on β-tubulin (Brunden et al., 2011). Due to their increased water solubility, the epothilones readily crosses the blood-brain barrier and can be retained within the CNS for several days(Andrieux et al., 2006; Cortes and Baselga, 2007; Brunden et al., 2012).

An exciting publication in 2015 cemented the epothilones as a promising therapeutic intervention following CNS injury. Using EpoB, administered following spinal cord injury, the authors convincingly demonstrated EpoB therapy led to increased axon outgrowth, reduced scarring and improved functional recovery in female rats (Ruschel et al., 2015). The axonal outgrowth was improved through increased microtubule polymerization, and consequently microtubule protrusion into regenerating axons. This work has been supported with evidence for similar efficacy of EpoD (Ruschel and Bradke,2018; Sandner et al., 2018), its effect on other cell types (Zhao et al., 2017) and otherin vivoexperimental models of axonal injury such as spinal and corneal nerve injury (Li and Wu, 2017;Wang et al., 2018). A positive effect has also been reported in EpoB administered following intracerebral hemorrhage,in which it restored the integrity of the nigrostriatal pathway neuronal circuit and improve fine motor functional recovery after injury in mice (Yang et al., 2018). These findings follow similar promising results seen in neurodegenerative disease;EpoD treatment resulted in improved outcomes inin vivomodels of Parkinson’s disease (Cartelli et al., 2013), tauopathy and Alzheimer’s disease (Brunden et al., 2010; Zhang et al.,2012), and schizophrenia (Andrieux et al., 2006; Fournet et al., 2012). However, a collection of studies also reports subtle or adverse effects of epothilones in some neurodegenerative models (Mao et al., 2017; Clark et al., 2018). This discrepancy in findings raises the question of why does the effect of epothilone administration vary across different experimental investigations?

Given Traumatic brain and spinal cord injuries can be acquired at any stage of life, age may play a critical role in outcomes following CNS trauma (Sun et al., 2020). Based on our recent studies, we propose that age is not only an important factor in dictating outcomes following CNS trauma, but also plays a role in determining the efficacy of microtubule stabilizers, such as EpoD, as a post injury intervention. When studying the effect of EpoDin vitro, we found that the vulnerability of cortical neurons to EpoD increased as the age of the neuron increased(Clark et al., 2020). These findings are in line with those of Jang et al. (2016), who found an age-related contribution in response to EpoB treatment in both cortical and dorsal root ganglion cellsin vitro. Moreover, using anin vivomodel of trauma (lateral fluid percussion brain injury) we have shown that a single peripherally administered dose of EpoD targeted central neurons, specifically increasing the density of mushroom spines on layer 5 cortical pyramidal neurons, with an absence of astroglial effects (Chuckowree et al., 2018). To add further evidence to these findings, we have previously investigated the protective effect of EpoD in a mouse model of amyotrophic lateral sclerosis (Clark et al., 2018). This therapy led to worse survival outcomes, greater functional deficits and an increase in microglial and astroglial activation at end stage–approximately 6 months old. However, this treatment initially prevented motor neuron loss and axonal degeneration at 2 months of age, suggesting that there may be a differing effect as either the disease progressed or the mouse aged. Together,these studies suggest that age may differentially impact the efficacy of EpoD on neuronal and glial populations.

To directly address the effect of age on EpoD efficacy, we exposed young, adult and aged mice to anin vivobrain injury (lateral fluid percussion brain injury). We found that the degree of axonal degeneration as well as astrogliosis and microglial activation were age dependent (Zhu et al., 2020).Critically, we determined that EpoD administration had very different effects in young (1.5 months)versusadult (3 months)mice. In young mice, EpoD administration trended to confer protection from axonal degeneration. However, when the same dose was given to adult mice, EpoD had a detrimental effect – axonal degeneration in the internal capsule white matter tract was significantly increased. Collectively, these studies provide compelling evidence that age is an important contributor to outcomes following therapeutic intervention with epothilone derivatives.

How exactly age is affecting epothilone efficacy, is not clear.Within the neuron, microtubules contribute to a range of neuronal functions including neurite outgrowth, neuronal polarity, axonal transport and regulating gene expression and signaling pathways (Dubey et al., 2015). Interestingly microtubule loss has not only been seen in Alzheimer’s disease but can also be present in cases of normal aging (Cash et al., 2003). How microtubule function changes over time and how microtubule stabilizing agents bind remains to be defined. Furthermore, the mechanism of the effect of age is multifactorial; likely to involve glial cells (Ritzel et al., 2019; Sun et al., 2019; Webster et al., 2019) as well as neurons (Sun et al.,2019). EpoD has been shown to reduce the glial scar and affect both fibroblasts and immune cells such as microglia following spinal cord injury (Ruschel et al., 2015; Mao et al., 2017). The function of microglia changes throughout the lifespan, and these alterations can exacerbate the injury response in an aged system (Morganti-Kossmann et al., 2019; Sun et al., 2020). This altered microglial response can be correlated to a decrease in functional recovery following injury, demonstrating the impact of the aging system on outcomes (Ritzel et al., 2019; Sun et al.,2019). It is important to also note that the heterogeneity of the response to epothilone treatment may not be limited to ageing.The response of the CNS to trauma can be sex-dependent(Inampudi et al., 2020) – how this affects therapeutic intervention outcomes is largely unexplored.

In conclusion, mounting evidence in both trauma and neurodegenerative disease models highlights the need for future studies determining how the efficacy of therapeutic microtubule stabilization is altered across the lifespan.Understanding how age contributes to therapeutic intervention following trauma could pave the way to providing a more tailored therapeutic regime in the clinic that is specific to the age of the patient.

Author contributions:CB, JC and ZZ wrote the manuscript. JC and TD edited the manuscript. All authors approved the final manuscript.

Conflicts of interest:The authors declare no conflicts of interest.

Financial support:None.

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Peer review:Externally peer reviewed.

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