Microtubules are composed mainly of two polymer proteins and tubulin

Microtubules are composed mainly of two polymer proteins, α- and β-tubulin [10]. These proteins are regulated by posttranslational modifications (PTMs) including acetylation and phosphorylation. These PTMs can play crucial roles in the stability, guidance, and transport that take place along these axons. Because of this, the physiological process of microtubule-based axonal transport is of great interest as a functional indicator of neuronal health.
We and others have shown that microtubule-based axonal transport can be measured directly in vivo in models of AD using manganese-enhanced magnetic resonance imaging (MEMRI) [11–15]. Over the past 20 years, this technique has been used in vivo in rodent models to confirm in vitro and ex vivo impairments in axonal transport rate [12–14,16–18]. Thus, the MEMRI technique offers an in vivo methodology for microtubule-based axonal transport. Furthermore, microtubule-destabilizing agents (i.e., colchicine) can be used in vivo to confirm blockage of axonal transport using MEMRI. Recent studies have confirmed in vivo deficits in microtubule-based axonal transport are present before the onset of tau tangles in tauopathy models [13,16]. These deficits are highly correlated with PTMs of tau.
In addition to tau, tubulin is another key building block within microtubules that can be acetylated or deacetylated by histone deacetylase 6 (HDAC6) [19]. Evidence in patient samples indicates that this regulation is disrupted because of the level of HDAC6 being elevated in the brains of patients with AD, specifically by 52% in the cortex and 91% in the hippocampus, which is the center for learning and memory [20,21]. In addition, levels of acetylated α-tubulin protein are decreased in AD patient brains. HDAC6 has also been shown to interact with tau independent of its deacetylase activity and also helps to recruit chaperone proteins within the autophagic process to help clear protein aggregates. Studies also indicate that HDAC6 can directly modulate the phosphorylation and acetylation of tau as a protein [19,21,22]. These functions have been demonstrated in recent studies in multiple models of AD [21,23–26].
Because of the various functions of HDAC6 in neurons and its potential as a therapeutic target, a number of inhibitors have been developed. The first of these includes tubacin, which is specific to the α-tubulin regulation and to HDAC6; however, it has high lipophilicity and is difficult to synthesize [10,27]. The second is tubastatin A and its respective group of analogs. This family of drugs is less lipophilic and more selective for HDAC6 and its deacetylase activity, but dosing mice with this compound did not result in significant Anti-cancer Compound Library exposure [28]. However, a recent study compared these two inhibitors and did find that tubastatin A was most effective in the peripheral nervous system at rescuing distal axonal loss and muscle innervation in mouse models of Charcot-Marie-Tooth disease [29]. Most recently, Acetylon Pharmaceuticals has developed a series of selective and potent HDAC6 inhibitors that efficiently cross the blood-brain barrier. In a recent study, one of the compounds in this series, ACY-738, resulted in an antidepressant-like phenotype in a social defeat model of depression in mice. Thus, we sought to evaluate the effectiveness of ACY-738 in an amyloid mouse model of AD on axonal transport, behavior, and amyloid pathology.
Specifically, we chose the amyloid precursor protein/presenilin 1 (APP/PS1) mouse model because of the relevance of increased amyloid as an early marker of cognitive impairment in patients and in mice. In addition, amyloid has been highly associated with impairments in axonal transport and microtubule instability. Reduced axonal transport has been correlated with poor behavioral outcomes in mouse models of AD [8,12,30]. These correlations have linked Aß with tau proteins within dystrophic axons, leading to deficits in axonal transport. In addition, axonal transport deficits have been implicated as triggers for increased oxidative stress, and can lead to higher Aß deposition over time. Many of the proteins involved with this processing, including APP and PS1 have been observed to be accumulated in axons at presynaptic terminals, indicating that this transport process is necessary in the delivery of these cargo [31,32]. Specifically, the process of fast anterograde axonal transport is responsible for the delivery of these proteins. In addition, evidence suggests that APP directly interacts with kinesin in the development of microtubules and promotes axonal growth. Additional in vitro evidence of APP and PS1 manipulation in cell culture leads to altered axonal growth, morphologic changes, and swelling within neurons [33,34]. Finally, the APP/PS1 mouse model has been characterized using the MEMRI methodology for measurements of noninvasive, in vivo axonal transport rates, with rates decreasing beginning at age 3 months before overt biochemical changes in pathology as well as learning and memory deficits. Thus, we chose to evaluate the effects of ACY-738 at this time point, referred as 21 day or early treatment throughout the article. We selected a longer, 90-day treatment beginning at 3 months and evaluated at 6 months, referred to as the late or 90-day treatment throughout the article. Finally, axonal transport deficits have been linked to oxidative stress abnormalities before the onset of plaque depositions, all of which affect the microtubule network which ACY-738 targets through HDAC6 inhibition.