Huntington’s disease (HD) is an autosomal dominant disorder caused by polyglutamine expansion mutation containing CAG repeats in the huntingtin gene located on the short arm of chromosome 4 (1). Chorea, a dance-like movement disorder, is the major clinical hallmark of HD. Additionally, psychiatric disturbances, such as anxiety and depression, and cognitive impairment, are exhibited in the early stages (2). Despite the devastating effects HD has on patients and their families and despite extensive study, the underlying pathogenesis of the disease is still unknown, and there are no effective treatments for HD at this time. Various kinds of pharmaceutical compounds for potential HD therapy have been reported so far (3). Among them, histone deacetylase (HDAC) inhibitors, such as trichostatin A (TSA) and suberoylanilide hydroxamic acid, have neuroprotective effects (4). However, these pan-HDAC inhibitors commonly exhibit gastrointestinal, constitutional, hematologic and cardiac side effects (5, 6). Unlike other HDACs, HDAC6 is a unique cytoplasmic HDAC that deacetylates non-histone proteins, such as α-tubulin in microtubule and tau (7, 8). It is known that tubulin acetylation influences the binding and motility of motor protein kinesin-1 and enhances the recruitment of kinesin-1 to microtubule and increases axonal trafficking of kinesin-1 cargo protein JNK-interacting protein 1 (JIP1) in vitro (9). mHTT has been reported to impair axonal transport both in vivo and in vitro models of HD (10). Additionally, tubulin acetylation in HD brain tissue is lower than in wild-type (WT) mice, and TSA therapy improves HD phenotypes associated with transport- and release-related defects (11), suggesting that HDAC6 activity or expression increased only in the disease state. The phenotype of HDAC6 knockout mice was not demonstrated to be significant (12); therefore, if the selectivity for HDAC6 is guaranteed, the side effects of the HDAC6 inhibitor are anticipated to be negligible or insignificant. Based on this evidence, HDAC6 inhibition by increasing α-tubulin acetylation has been proposed as a therapeutic strategy for HD (11, 13).

In addition to HD, inhibition of HDAC6 has been shown to improve cognitive functions in various Alzheimer’s disease (AD) models (14) and a tauopathy model (rTg4510) (15) and to improve motor activities in axonal Charcot-Marie-Tooth disease (CMT) model (16). Tubastatin A (TBA) and ACY-1215, less toxic HDAC6 inhibitors, were developed as potential AD therapeutics (17), but TBA has limited brain penetration (18).

CKD-504 has been reported to selectively inhibit HDAC6 among HDAC families and to uniquely increase tubulin acetylation, but not histone acetylation (19). With its ability to cross the blood-brain barrier (BBB) (19), and low cytotoxicity, CKD-504 is an effective therapy option for neurodegenerative diseases in clinical settings. In fact, CKD-504 has been reported to degrade tau in an AD model (19) and to improve myelination of Schwann cells in a model of CMT disease type 1A (20). Therefore, in the present study the effect of CKD-504 on HD pathological phenotypes was tested.

For the in vivo study, YAC128 TG mice were used, a widely used HD animal model. Rather than HTT fragments, the YAC128 mouse expresses full-length human mHTT protein with 128 CAG repeats (21), which are predominant in the human HD brain (22). This model exhibits a motor deficit highly correlated with striatal neuronal loss (23) as well as corticostriatal (CS) dysfunction (24), similar to phenotypes reported in human HD. Motor symptom onset begins to appear after six months of age (21). Because YAC128 recapitulates the pathology of HD patients better than other HD animal models, it is currently considered to be one of the best models to verify the efficacy of drug treatments. Therefore, YAC128 TG mice were used for in vivo study and their neural stem cells (NSC) to validate the efficacy of CKD-504 for the in vitro study.

It was observed that CKD-504 improved the pathology of HD: it alleviated behavioral deficits, increased axonal transport and number of neurons, reduced mHTT accumulation and protein ubiquitination, inflammation, and tau hyperphosphorylation, and restored synaptic function in the CS circuit.

CKD-504 is effective in treating HD-related pathological phenotypes in YAC128 TG NSCs

To test whether CKD-504 is effective in treating HD neurons, we used neural stem cells (NSCs) prepared from YAC128 mice. CKD-504 was treated in the NSCs at 0.1, 0.3, 1 and 3 μM and TBA at 0.3 μM. TBA, a well-known HDAC6 inhibitor, was used as a positive control (25). Immunocytochemistry (Fig. 1A), together with Western blot analysis (Fig. 1B, C), revealed that CKD-504 significantly increased the level of α-tubulin acetylation in a dose dependent manner in both WT and TG NSCs. TBA also increased the level of α-tubulin acetylation in the TG NSCs (Fig. 1B, C). To investigate the effect of CKD-504 on mitochondrial transport, live cell imaging was performed using Mito Tracker dye both in WT and TG NSCs. Mitochondrial movement in the YAC128 TG NSCs was decreased compared to that of WT, and both CKD-504 and TBA rescued the mitochondrial movement in TG. Treatment of TG NSCs with CKD-504 at 0.1, 0.3, and 1 M and TBA at 0.3 M significantly recovered the reduced mitochondrial movement in TG as shown by kymographs (Fig. 1D) and decreased mitochondrial velocity (Fig. 1E), indicating that -tubulin acetylation regulates mitochondrial transport in YAC128 TG NSCs. Next, the expression of the mHTT protein level was examined using the EM48 antibody to detect mHTT aggregates in HD models. Immunocytochemical (ICC) analysis showed that mHTT was highly expressed in YAC128 TG, compared to WT. In addition, EM48-positive mHTT level was reduced by treatment with CKD-504 at 3 μM and TBA at 0.3 μM compared to TG (Fig. 1F). Consistent with the EM48 ICC data, Western blot analysis exhibited that EM48 expression was significantly increased in YAC128 TG compared to WT, which was significantly reduced to the level of WT by treatment with CKD-504 or TBA at 3 μM and 0.3 μM, respectively (Fig. 1G). In this study, it was demonstrated that the relative magnitude of acetylated tubulin in SH-SY5Y cells was increased by treatment with CKD-504 in a dose-dependent manner from 0.1 to 10 μM, but CKD-504 did not affect histone H4 acetylation up to 10 μM (Supplementary Fig. 1A). We also confirmed that CKD-504 treatment increased the level of acetylated -tubulin in a dose-dependent manner using a pair of WT STHdhQ7/HdhQ7 and mHTT-expressing STHdhQ111/HdhQ111 striatal cell lines, as shown by ICC analysis (Supplementary Fig. 1B) and Western blot analysis (Supplementary Fig. 1C). This showed that CKD-504 increased cellular α -tubulin acetylation in both WT and mutant striatal cell lines, though at a lower concentration than TBA. To evaluate whether CKD-504 can penetrate the brain-blood barrier, we analyzed the in vivo brain distribution using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) methods. Exposure of CKD-504 in the brain following intraperitoneal administration of 1, 2.5, and 10 mg/kg generally increased its brain/plasma ratio in a dose-dependent manner, 1.88, 1.83, and 2.78 at each concentration, respectively (Supplementary Fig. 1D), similar to the value previously reported (19). The results showed that CKD-504 efficiently penetrates the brain.

CKD-504 is effective in treating motor and cognitive deficits in YAC128 mouse model

To test whether CKD-504 treatment improves behavior and synaptic functions in HD animal models, we used 6-month-old YAC128 TG mice. YAC128 mice were treated with CKD-504 at 1, 2.5, or 10 mg/kg for three months and subjected to various behavior tests at the indicated times. First, we tested motor functions using rotarod and grip strength tests. CKD-504 treatments led to a significant increase in motor function, compared to WT and sham (Veh) groups (Fig. 2A, B). Significant muscle strength recovery was noted in both tests, beginning as early as two months after the 2.5 mg and 10 mg doses were administered (Fig. 2A, B). To examine cognitive functions, simple swim test and novel object recognition test were performed. Impairment of cognitive function in both tests was observed in TG mice compared to WT mice (Fig. 2C, D). Treatments of CKD-504 significantly recovered memory impairment at 2.5 mg or 10 mg/kg in the simple swim test and at 10 mg/kg in the novel object recognition test (Fig. 2C, D). To assess the effect of CKD-504 on emotional function, we employed the forced swim test (FST) and the elevated plus maze (EPM) tests. In FST, TG mice remained inactive for a longer period of time than sham (Veh) mice did. However, CKD-504 treated group decreased immobility time in a dose-dependent manner (Fig. 2E). In the EPM test, TG mice exhibited anxiety-like phenotypes with reduced activity in open arms. The activity in the open arms increased in a dose-dependent manner in the CKD-504 treated group (Fig. 2F). In both tests, the significance was observed at the highest dose (Fig. 2E, F).

CKD-504 is effective in improving axonal transport in vivo

We also tested whether the improved axonal transport observed in the cells (Fig. 1D, E) could also be observed by CKD-504 treatment in vivo. Labeling with Fluoro-Gold (FG) is widely used to measure damage to axonal transport, trafficking efficiency, and neuronal integrity (26) since accumulation of the tracer in cell bodies depends on the retrograde transport of axons. As a result, FG was administered to the substantia nigra of TG mice that had been given CKD-504 as well as to the WT and sham-treated groups. In the striatum near −0.3 mm to the bregma, no difference was observed in the number of FG-labeled cells between the saline-treated group and drug-treated groups in the TG mice (data not shown). However, in the anterior area of the striatum (AP: +1.0-+0.8), the number of FG-labeled cells was significantly different between TG and WT mice. At dosages of 10 and 2.5 mg/kg, respectively, the number of FG-labeled cells was 26.0% and 23.6% greater than that of the untreated mice (Fig. 2G).

CKD-504 improves synaptic function in the CS circuit of dorsomedial striatum in YAC-128 mice

We assessed synaptic strength in cortico-striatal (CS) slices from the same TG mice used in behavior tests to see whether CKD-504 affects synaptic function. Field excitatory postsynaptic potentials (fEPSPs) were evoked by electrical stimulation in fibers of the dorsomedial striatum from the cortex with a bipolar stimulating electrode located at the border of corpus callosum. The strength of CS input to the dorsomedial striatum in slices prepared from each experimental group was compared by measuring the fEPSP: fiber volley (FV) ratio at different stimulus intensities (Fig. 2Ha). In slices from the TG mice compared to the WT mice, the I/O relationship for the CS input to the dorsomedial striatum displayed a significantly gentler slope, indicating a depression in the synaptic strength of the CS input to the dorsomedial striatum in the TG mice. Furthermore, CKD-504 treatment of TG mice significantly restored the slope of the I/O relationship for CS input to the TG mice’s dorsomedial striatum, implying that CKD-504 may play a therapeutic role in the deterioration of CS synaptic function in HD (Fig. 2Ha). To determine the effect of CKD-504 on LTP in CS input to the dorsomedial striatum in TG mice, the potentiation of CS fEPSP evoked by TBS was investigated. LTP was measured as a ratio of the amplitude of fEPSP after TBS to that before the stimulation. As shown in Fig. 2Hb, LTP in the CS input was significantly attenuated in the TG mice compared to the vehicle-treated controls. This attenuation of CS LTP in the TG mice was significantly inhibited by administration of CKD-504. These findings suggested that CKD-504 effectively treats synaptic dysfunction in HD model mice by restoring the ability of LTP in CS inputs to the dorsomedial striatum.

CKD-504 improves pathological features of Huntington’s disease in YAC 128 TG mice

In the progression of HD, the degeneration of nerve cells is accelerated, which was also observed in the brains of YAC128 TG mice. We measured the number of neuronal and microglia cells in the YAC128 TG mice after CKD-504 treatment. In the anterior striatum (AP: +1.2-+0.5 mm), the number of NeuN-positive neurons of TG mice treated with 2.5 and 10 mg CKD-504 was significantly increased to a level similar to the that of the WT (Fig. 3A). An increase in the inflammatory response is one of the most typical pathological changes in HD. We also investigated the effect of CKD-504 on the inflammatory response based on the number of the Iba1⁺ microglia in the striatum. The number of Iba1+ cells in the TG mice increased by about 199.8% compared to that of the WT and significantly decreased compared to the sham group at all doses in a dose-dependent manner after three months of CKD-504 treatment (Fig. 3B). The mHTT aggregates that can be detected by the EM48 antibody are predictors of cell death and markers of regional HD neuropathology. There was a significant difference in the intensity of HTT aggregation between the WT group and the TG sham group (Fig. 3C). In comparison to the sham group, we observed significant drops in mHTT aggregates in the 2.5 and 10 mg treatment groups but not in the 1 mg treatment group (Fig. 3C). Tau protein is a microtubule-associated protein that is widely expressed in the central nervous system, particularly in neurons, where it regulates microtubule dynamics, neurite outgrowth, and axonal transport (27). Tau hyperphosphorylation and aggregation are strongly associated with neuronal dysfunction and progressive neuronal death (28), which are also known to be involved in HD (27). To find out, we used immunohistochemistry and the AT8 monoclonal antibody against phosphorylated tau to look at tau phosphorylation in the YAC128 TG mouse brain. The intensity of AT8-positive signals significantly increased in the TG brains compared to WT and significantly decreased in the 2.5- and 10-mg-treated TG mouse groups (Fig. 3D), suggesting that CKD-504 can modulate tau phosphorylation. Furthermore, the ubiquitinated protein level was significantly increased in the striatum of TG mice, compared to that of WT, which was again decreased by CKD-504 treatment in the 10 mg-treated TG mice (Fig. 3E). These results showed that CKD-504 treatment decreased ubiquitinated protein levels in YAC128 mice, suggesting proteasomal degradation via ubiquitination. Taken together, these results can strongly suggest that CKD-504 treatment restored neural cells in the brains of HD mice.

Further detailed information is provided in the Supplementary data information.

This work was supported by an internal funding from the CKD Research Institute and by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. 2021M3A9G2015885) and by the Technological Innovation R&D Program (S3305828) funded by the Ministry of SMEs and Startups (MSS, Korea). We thank Dongchul Shin (iPS Bio) for the interpretation of electrophysiological data analysis.

JS is the founder and CEO of iPS Bio, Inc., and SL and WS are the employees of iPS Bio, Inc. H-RS, JHJ, JK, NH, SK and KHR are the employees of Chong Kun Dang Pharmaceutical Corp. The other authors indicated no competing interests.