Targeting Inflammation, PHA-767491 Shows a Broad Spectrum in Protein Aggregation Diseases
Yu-Han Chung 1 • Chia-Wei Lin1 • Hsin-Yu Huang 1 • Shu-Ling Chen1 • Hei-Jen Huang2 • Ying-Chieh Sun 3 •
Guan-Chiun Lee 1 • Guey-Jen Lee-Chen 1 • Ya-Ching Chang 4 • Hsiu Mei Hsieh-Li1
Received: 27 August 2019 / Accepted: 28 February 2020
Ⓒ Springer Science+Business Media, LLC, part of Springer Nature 2020
Many protein aggregation diseases (PAD) affect the nervous system. Deposits of aggregated disease-specific proteins are found within or around the neuronal cells of neurodegenerative diseases. Although the main protein component is disease- specific, oligomeric aggregates are presumed to be the key agents causing the neurotoxicity. Evidence has shown that protein aggregates cause a chronic inflammatory reaction in the brain, resulting in neurodegeneration. Therefore, strategies targeting anti-inflammation could be beneficial to the therapeutics of PAD. PHA-767491 was originally identified as an inhibitor of CDC7/CDK9 and was found to reduce TDP-43 phosphorylation and prevent neurodegeneration in TDP-43 transgenic animals. We recently identified PHA-767491 as a GSK-3β inhibitor. In this study, we established mouse hippocampal primary culture with tau-hyperphosphorylation through the activation of GSK-3β using Wortmannin and GF109203X. We found that PHA-767491 significantly improved the neurite outgrowth of hippocampal primary neurons against the neurotoxicity induced by GSK-3β. We further showed that PHA-767491 had neuroprotective ability in hippocampal primary culture under oligomeric Aβ treatment. In addition, PHA-767491 attenuated the neuroinflammation in mouse cerebellar slice culture with human TBP-109Q agitation. Further study of SCA17 transgenic mice carrying human TBP-109Q showed that PHA-767491 ameliorated the gait ataxia and the inflammatory response both centrally and peripherally. Our findings suggest that PHA-767491 has a broad spectrum of activity in the treatment of different PAD and that this activity could be based on the anti-inflammation mechanism.Keywords Protein aggregation diseases . PHA-767491 . Inflammation . GSK-3β inhibitor
Yu-Han Chung and Chia-Wei Lin contributed equally to this work.Electronic supplementary material The online version of this article
Lines of evidence show pathological protein misfolding and aggregates occurring in many neurodegenerative diseases (Aguzzi and O’Connor 2010). These protein aggregation diseases (PAD) include polyglutamine (polyQ) expansion
diseases, such as Huntington’s disease ( HD) and
1 Department of Life Science, National Taiwan Normal University, Taipei, Taiwan
2 Department of Nursing, Mackay Junior College of Medicine, Nursing and Management, Taipei, Taiwan
3 Department of Chemistry, National Taiwan Normal University, Taipei, Taiwan
4 Department of Pharmacy, Taiwan Adventist Hospital, Taipei, Taiwan
spinocerebellar ataxias (SCAs), in which the pathological polyQ proteins accumulate within the nuclei and/or cyto- plasm; Alzheimer’s disease (AD), in which the aggregate pathological proteins tau and amyloid-β (Aβ) are present both intracellularly and extracellularly; and Parkinson’s dis- ease (PD), in which pathological α-synuclein is deposited in the cytoplasm (Aguzzi and O’Connor 2010). Although the amino-acid sequences of the aggregated proteins are diverse in PAD, all of the pathological proteins seem to adopt a similar, insoluble, highly ordered structure known as the cross-β spine (Sawaya et al. 2007). Around the aggregated proteins, gliosis of astrocytes and microglial are frequently observed (Muhleisen et al. 1995; Sapp et al. 2001). Pro- inflammatory cytokines such as IL-1β and IL-6 also accu- mulate in protein aggregates and increase in the brain (McGeer and McGeer 1995; Rubio-Perez and Morillas- Ruiz 2012). This leads to the brain-inflammation hypothesis that the accumulated protein aggregates cause a chronic in- flammatory reaction in the brain, further resulting in neuro- degeneration (Wyss-Coray and Mucke 2002). Since inflam- mation is a common feature of PAD, targeting the inflam- matory response is an attractive therapeutic strategy for dealing with the neurodegenerative diseases classified with- in the category of PAD, including AD and polyQ diseases.
PHA-767491 , a dual inhibitor of two cell cycle checkpoint kinases, cell division cycle kinase 7 (CDC7) and cyclin-dependent kinase 9 (CDK9), has been shown to have antitumor activity (Erbayraktar et al. 2016; Huggett et al. 2016; Montagnoli et al. 2008). TDP-43 was identified as a target of CDC7; therefore, PHA-767491 has been shown to reduce the phosphorylation of TDP-43 and to ameliorate the neurodegeneration of TDP-43 transgenic
C. elegans (Liachko et al. 2013). PHA-767491 was also identified by molecular dynamic simulations and was bio- chemically proved as a GSK-3β inhibitor in our recent study (Hsu et al. 2017). GSK-3β inhibitor has been pro- posed to have therapeutic potential in attenuating the ag- gregation of phosphorylated-tau (Llorens-Martin et al. 2014). In the current study, we showed that PHA-767491 can promote neuronal survival and neurite outgrowth in mouse hippocampal primary culture under the neurotoxic- ity of tau-hyperphosphorylation. We further found that PHA-767491 showed a protective effect against neuronal damage induced by oligomeric Aβ in mouse hippocampal primary culture. To further explore the neuroprotective ef- fect of PHA-767491, another PAD platform with SCA17 polyQ aggregation was also tested. SCA17 is a subtype of SCAs caused by polyQ overexpansion in the transcription factor called TATA box-binding protein (TBP) (Koide et al. 1999). The length of polyQ in a normal individual is be- tween 25 and 42 repeats (Gostout et al. 1993), and an individual suffering from SCA17 has a repeat number higher than 45 (Nakamura et al. 2001). The aggregation caused by polyQ expanded mutant TBP is usually local- ized in the nuclei. This is different from most other neuro- degenerative diseases that have aggregates mainly in the compartments of the cytoplasm (AD, PD, and HD) or ex- tracellularly (AD) (Kazantsev et al. 1999). The major clin- ical pathology of SCA17 is characterized by cerebellar at- rophy and Purkinje cell degeneration (Friedman et al. 2007). Gliosis is also a remarkable feature in the brains of SCA17 patients (Toyoshima et al. 2004). Our study showed that PHA-767491 alleviated the neuroinflamma- tion in SCA17 mouse cerebellar slice culture and in trans- genic mice with human TBP-109Q aggregation.
The tested PAD systems in this study covered tau- hyperphosphorylation, oligomeric Aβ and polyQ aggrega- tion. Our findings suggest that the broad neuroprotective ac- tivity of PHA-767491 for different PAD could be through the mechanism of anti-inflammation. This study helps in deciphering a new target for drug design and development related to the aggregation-mediated cell toxicity of PAD.
Methods and Materials
Animals and Ethics Statement
All of the animal experiments were conducted according to the guidelines and were approved by the Research Committee of the National Taiwan Normal University (No. 103022). C57BL/6J and FVB/N mice were purchased from the National Breeding Centre for Laboratory Animals (Taipei, Taiwan). The mice were housed in individual ventilated cages (IVC) with free access to food and water in a 12-h light/dark cycle (7 a.m. to 7 p.m.). SCA17 transgenic mice with human TBP (hTBP)-109Q were maintained by breeding heterozy- gous male mice with FVB/N wild-type female mice (Chang et al. 2011, 2016; Chen et al. 2019). SCA17 mice showed ataxia around 6 weeks of age, whereas Purkinje cell degener- ation occurred at 4 weeks of age (Chang et al. 2011, 2016; Huang et al. 2018b).
Hippocampal and Cerebellar Primary Cultures and Immunostaining
The primary culture and drug application were performed as described in our previous reports. Hippocampi were isolated from embryonic day 18–19 C57B/6 mice. Cytosine arabino- side (2 μM, Sigma) was added to the cultures to reduce the glial cell populations on days in vitro (DIV) 4. On DIV 9, oligomeric Aβ42 (1 μM) or Wortmannin and GF109203X (WM/GFX, 10 nM each, Sigma) were applied to the cells to mimic the conditions of AD with Aβ aggregation or tau- hyperphosphorylation (Huang et al. 2018b, c, 2019a, b). PHA-767491 (Sigma) was applied to the cells 30 min after the Aβ or WM/GFX insult. Cells were harvested 1 h later for immunostaining to examine the effect of PHA-767491.
For cerebellar primary culture, cerebella were isolated from 2-day-old SCA17 mice and their wild-type littermates and cultured as described (Chen et al. 2018, 2015; Kung et al. 2014). Cytosine arabinoside (2 μM, Sigma) was added to the cultures on DIV 4. PHA-767491 (0.5–1.5 μM) was ap- plied to the cells on DIV 5 and the cells were harvested on DIV 18 for immunostaining.
The harvested primary cells were fixed with 4% parafor- maldehyde and immunostained with primary antibodies [NeuN (Millipore, 1:1000), MAP2 (Millipore
1 PHA-767491 treatment alleviates the reduction of cell viability, neurite length, and neurite branches in the oligomeric Aβ-treated primary hippocampal cells. (A) The structure of PHA-767491. (B) Experimental timeline of PHA-767491 application to oligomeric Aβ-treated primary hippocampal cultures. The hippocampi of C57BL/6J mice were isolated at embryonic day 18–19. PHA-767491 (0.25 or 0.5 μM) was applied to the primary hippocampal cells 30 min after oligomeric Aβ42 treatment at DIV 9. Primary cells were harvested 1 h after PHA-767491 treatment. (C)
The representative images of immunofluorescent cytochemical (ICC) staining of primary hippocampal cultures with antibodies NeuN (red) and MAP2 (green); scale bar = 100 μM. (D) Quantification of the relative total cells, NeuN positive cells, neurite lengths, and neurite branches in primary cultures. Values represent the mean ± SEM. *p < 0.05;
***p < 0.001 vs. the vehicle group. #p < 0.05; ##p < 0.01; ###p < 0.001 vs. the oligomeric Aβ-treated group
GFAP (Millipore, 1:1000), Iba1 (Wako, 1:500), and calbindin (Santa Cruz, 1:1000)] and fluorescence-conjugated secondary antibodies (Invitrogen, 1:500) and DAPI (Sigma, 1:10,000). The staining results were observed using a high-content screening system (Molecular Devices). In each experiment, 1000 cells/mouse were analyzed (n = 3/group).
Organotypic Cerebellar Slice Culture and Immunostaining
The cerebellar slice culture was performed as described (Chen et al. 2015). Whole brains were isolated from postnatal day 7 SCA17 mice. The cerebellum was then cut into 350-μm
2 PHA-767491 treatment ameliorates the decreases of cell viability, neurite length, and branches in the primary hippocampal cells under tau- hyperphosphorylation induced by WM and GFX. (A) Experimental time- line of PHA-767491 application to the primary hippocampal cells under neurotoxicity of tau-hyperphosphorylation. The hippocampi of C57BL/ 6J mice were isolated at embryonic day 18–19. PHA-767491 (0.5, 1.0, or
1.5 μM) was applied to the primary hippocampal cells 30 min after WM/
GFX (10 nM) treatment at DIV 9. Primary cells were harvested1h after PHA-767491 treatment. (B) The representative ICC images of primary hippocampal cultures that were stained with antibodies NeuN (red) and MAP2 (green). (C) Quantification of the relative NeuN positive cells, neurite lengths, and branches in primary cultures. Values represent the mean ± SEM. **p < 0.01; ***p < 0.001 vs. the vehicle group. #p < 0.05 vs. the WM/GFX-induced group parasagittal sections with Vibratome (VT1200S, Leica). The slices were then cultured on 0.4-μm pore size culture plate inserts (Millipore) in six-well plates. PHA-767491 (0.5 μM) was ap- plied to the slices 24 h later. Once cultured for 7 days, the slices were immunostained with primary antibody [calbindin (Santa Cruz, 1:1000) and Iba1 (Wako, 1:500)], following fluorescence-conjugated secondary antibody (Invitrogen, 1:500) and DAPI (Sigma, 1:10,000). The staining results were observed using a confocal microscope (LSM 880, Zeiss). In each experi- ment, 4–6 slices per mouse were analyzed (n = 4/group).
PHA-767491 Treatment in SCA17 Mice
SCA17 transgenic mice (TG) and their wild-type littermates (WT) were divided into 4 groups (WT vehicle, WT-PHA, TG-
vehicle, and TG-PHA, n = 12–15/group). Based on the effective dose (0.5 μM) on the slice culture, the fraction of drug absor- bance trough intraperitoneal injection (IP) (60%), and the releas- ing fraction from the protein association in the plasma (10%), PHA-767491 (71 μg/kg; in 0.5 μM NMP, IP) was applied daily to the mice from 4 to 18 weeks of age. The mouse body weight was measured every week. Behavioral analyses were performed during this period to evaluate the treatment effect.
Locomotor Activity Monitoring
The locomotor activity of the mice was evaluated when they were 12 weeks old. Each mouse was placed in a box (30 × 30 × 30 cm) and monitored under 2 Lux for 10 min. The total horizontal moving distance, velocity, and the path of
PHA-767491 (0.5, 1, 1.5 µM)DIV 0 DIV5 DIV18b PHA-767491 (µM)0 0.5 1.0 1.5PHA-767491 – 0.5 µM 1.0µM 1.5µM
3 PHA-767491 treatment attenuates the gliosis in SCA17 mouse cerebellar primary culture. (A) Experimental timeline of PHA-767491 treatment in SCA17 mouse cerebellar primary culture. The cerebella of the SCA17 mice were isolated at postnatal day 2. PHA-767491 (0.5, 1.0, or 1.5 μM) was applied to the cerebellar primary culture as of DIV 5 and cells were harvested on DIV 18. (B) The ICC images of Purkinje cells0PHA-767491 – 0.5 µM 1.0µM 1.5µMwere stained with calbindin antibody (green). (C) Quantification of the relative neurite length of Purkinje cells after PHA-767491 treatment. (D) The ICC images of astrocytes were stained with GFAP antibody (red). (E) Quantification of the relative GFAP positive cells of primary cultures after PHA-767491 treatment. Values represent the mean ± SEM. *p < 0.05 vs. the vehicle group movement of each mouse were recorded by using the tracking system (EthoVision, Noldus, Netherlands) (n = 12–15/group). The time spent in the central zone (15 × 15 cm) of the box was regarded as a sign of anxiolytic activity (Huang et al. 2018b, 2019a, b).
Mouse footprints were analyzed by the CatWalk XT system (Noldus) when the mice were 12 weeks old as previously described (Chen et al. 2015). Each mouse was allowed to walk on the glass plate three times, and the paw prints were record- ed and analyzed using the software (CatWalk XT 9.1, Noldus) (n = 12–15/group).
Immunofluorescent Staining of Mouse Cerebellar Cryosections After the behavioral analyses, the mice were perfused with 0.9% NaCl followed by 4% paraformaldehyde after being anesthe- tized with avertin (0.4 g/kg body weight). Immunofluorescent staining was performed as previously described (Chang et al. 2016; Chen et al. 2015). The cerebellar sections were incubated with primary antibodies [GFAP (Millipore, 1:1000) and Iba1(Wako, 1:500)], followed by secondary antibodies (1:500; Alexa Fluor dye-conjugated donkey anti-mouse, anti-rabbit or anti-goat IgG, Invitrogen). The cerebellar sections were mounted on gelatin-coated slides for observation under a confo- cal microscope (LSM 880, Zeiss). In each experiment, 4–6 sec- tions per mouse were analyzed (n = 3/group).
Data were shown as mean ± SEM. An independent t test and one-way analysis of variance (ANOVA) were performed with SPSS software to evaluate the significance. A p value cutoff of0.05 was considered statistically significant.
PHA-767491 Shows Neuroprotective Effects Against the Toxicity Induced by Oligomeric Aβ42 and Tau-Hyperphosphorylation in the Hippocampal Primary Neuronal Culture
First, we used oligomeric Aβ42 to treat the hippocampal primary neuronal culture to mimic the most well-known
. 4 PHA-767491 treatment reduced the microgliosis in the SCA17 mouse cerebellar slice culture. (A) The immunofluores- cence images of the SCA17 cere- bellar slice culture were stained with calbindin (green) and Iba1 (red) after PHA-767491 (0.5 μM) treatment for 6 days. (B) Quantification of the Iba1 levels in the slice after PHA-767491 treatment. Values represent the mean ± SEM. ***p < 0.001 vs. the vehicle group
calbindin Iba1 merge
pathological condition of Alzheimer’s disease as shown in the timeline (. 1(B)). The total cell viability, neuronal cell survival, neurite length, and branch numbers were significantly reduced in the primary hippocampal neuro- nal culture under the toxicity of oligomeric Aβ42 as com- pared to the control group (p < 0.05; 1(B–D)). The treatment of PHA-767491 at 0.25 μM (p < 0.01; 1(B– D)) and 0.5 μM (p < 0.001; 1(B–D)) improved the neuronal cell survival, neurite length, and branch numbers under oligomeric Aβ42. The results indicated that PHA- 767491 had a neuroprotective effect against the damage induced by oligomeric Aβ42.
We then evaluated the effect of PHA-767491 in the hippo- campal primary neuronal culture under another pathological co ndition of Alz h eime r ’ s d ise a se, t he ta u- hyperphosphorylation induced by WM/GFX treatment (Huang et al. 2019a; Li et al. 2006) as shown in 2(A). The WM/GFX treatment significantly decreased the neuronal cell viability, neurite length, and branch numbers (p < 0.01; 2(B, C)). PHA-767491 (1.5 μM) increased the average neurite length and branch numbers (p < 0.05; 2(B, C)) of the hippocampal primary neuronal cultures. These data further validated the neuroprotective effect of PHA-767491 in neuro- nal culture PHA-767491 Reduced the Neuroinflammation in Both Cerebellar Primary and Organotypic Slice Cultures
To test the effect of PHA-767491 on other neurodegener- ative diseases, we analyzed PHA-767491 in Purkinje neu- rons using SCA17 primary and organotypic slice cultures. PHA-767491 (0.5–1.5 μM) was applied to the SCA17 cerebellar primary culture on DIV 5 ( 3(A)). Cells were harvested on DIV 18 for immunostaining. We found no significant difference in the neurite outgrowth identi- fied in Purkinje neurons after treatment with PHA-767491 for 13 days (3(B, C)). However, the neuroinflamma- tion stained by GFAP was reduced in the primary culture after treatment with PHA-767491 (1.5 μM) (p < 0.05; 3(D, E)).
We further examined the activity of PHA-767491 in the SCA17 cerebellar organotypic slice culture ( 4), a system closer to the cerebellar environment than the primary culture. We applied PHA-767491 (0.5 μM) to the culture media at DIV 1 and analyzed the slice at DIV 7 by immunostaining. We found that the TBP aggregation in the Purkinje neurons was not significantly reduced after treatment (data not shown); however, the level of microglia was significantly reduced after treatment with PHA-767491 (p < 0.001; 4).a b
5 Evaluation of the effect of PHA-767491 treatment on the body weight and behaviors of SCA17 mice. (A) Experimental timeline of PHA-767491 treatment. PHA-767491 (71 μg/Kg) or vehicle (NMP,
0.5 μM) were injected intraperitoneally once a day into the SCA17 mice at 4–12 weeks old. The locomotor test was conducted before PHA- 767491 treatment, while both locomotor and footprint tests were evalu- ated at the end of treatment. (B) The body weight of the mice measured weekly showed no significant difference among the 4 groups of mice. (C)Results of moving distance in the locomotor test showed no significant difference among the 4 groups of mice. (D) Results of duration in the central zone during the locomotor test were shown and no significant difference was observed. (E–F) Results of footprint analysis. PHA- 767491 treatment significantly reduced the run duration and increased the swing speed of each pace in SCA17 mice. Values represent the mean
± SEM. *p < 0.05; **p < 0.01 vs. the WT vehicle group. #p < 0.05; ###p < 0.001 vs. the SCA17 vehicle group PHA-767491 Treatment Had No Notable Effect on Body Weight or Motor Activity in SCA17 MiceTo evaluate the in vivo effect of PHA-767491, we applied PHA-767491 (71 μg/kg, once every day) through IP injec- tion in SCA17 transgenic mice and their wild-type litter- mates for 8 weeks ( 5(A)). We monitored the mouse body weight during the treatment and no significant weight difference was identified among the 4 groups. Furthermore, the locomotor activity of the mice was analyzed before and after the administration of PHA-767491 in a 30 cm × 30 cm open field. There was no significant difference among the 4 groups of mice regard- less of the moving distance ( 5(C)) or the duration in the central zone (. 5(D)). These data indicated that the PHA-76749 treatment was safe for the mice; no side ef- fects were observed with respect to body weight, motor activity, or anxiety.a CTRL PHA-767491WT TG WT TGvehicle PHA-767491 vehicle PHA-767491
6 Evaluation of the effect of PHA-767491 treatment by Western blot analyses. (A) Western blot analysis of calbindin, GFAP, and Iba1 with proteins isolated from the mouse cerebellum. (B and B’) The calbindin levels were significantly reduced in the SCA17 mice but showed a higher tendency after the treatment of PHA-76749. (C and D) The expression ofGFAP and Iba1 was highly elevated in the SCA17 mice but significantly decreased after PHA-767491 treatment. Values represent the mean ± SEM. **p < 0.01; ***p < 0.001 vs. the WT vehicle group. ###p < 0.001 vs. the SCA17 vehicle groupPHA-767491 Treatment Ameliorated the Unsteady Gait of SCA17 MiceWe further conducted the footprint analysis to understand whether PHA-767491 treatment improved the gait ataxia of the SCA17 transgenic mice. We found the footprints of the PHA-767491-treated mice resumed a more-regular pattern. The lengthened “run duration” of the SCA17 mice was re- duced (p < 0.001; 5(E)). The significantly reduced “swing speed” of the 4 paws of the SCA17 mice was also recovered (p < 0.001; 5(F)). These data indicated that the PHA- 767491 treatment improved the steadiness of the gait of the SCA17 transgenic mice.
PHA-767491 Had an Anti-Inflammatory Effect on SCA17 MiceWith the improvement in the gait coordination, we deter- mined whether the PHA-767491 treatment altered the neu- roinflammation of the SCA17 mouse cerebellum in the same way as in the primary and slice cultures. Western blot analysis of the mouse cerebellum showed that the expres- sion level of calbindin, the marker of Purkinje neurons, was extensively decreased in the SCA17 cerebellum com- pared to the wild-type mouse cerebellum (6(A, B)). This indicated the severe degeneration of the Purkinje neu- rons of the SCA17 mice. While PHA-767491 had only amild effect in promoting the expression of calbindin ( 6(B–B’)), both markers of neuroinflammation, GFAP and Iba1, were highly reduced after the PHA-767491 treatment (6(A, C, and D)). Further, immunofluorescent staining of the mouse cerebellar sections also confirmed this data of Western analyses. PHA-767491 treatment ameliorated the highly elevated astrogliosis (stained by GFAP antibody) and microgliosis (stained by Iba1) of the SCA17 mice ( 7). These results indicated that PHA-767491 has an anti-inflammatory effect.
To further elucidate the anti-inflammatory effect of PHA-767491, we also examined the peripheral inflamma- tory factors of the mice. The TNF-α, IL-1β, and IL-6 were elevated in the SCA17 transgenic mice compared to the wild-type mice (. 8). The IL-1β and IL-6 levels in the SCA17 mouse plasma were both significantly higher than those of the wild-type mice (p < 0.05; 8(B, C)), which were all reduced by the treatment of PHA-767491, and significance was shown for TNF-α and IL-1β (p < 0.01; 8(A, B)). In addition, the IL-1β level was also reduced by the PHA-767491 effect in the wild-type mice. (p < 0.05; 8(B)). These data suggest that PHA-767491 has anti- inflammatory activity in both the central and peripheral systems of SCA17 mice.
In the present study, we demonstrated that the PHA-767491 has anti-inflammatory activity to ameliorate the SCA17 mouse gait impairment. In addition, PHA-767491 shows sig- nificant neuroprotective activity for hippocampal primary neurons under the neurotoxicity of Alzheimer’s conditions. Both SCA17 and Alzheimer’s disease are PAD; therefore, we suggest that PHA-767491 has potential in the treatment of PAD.
Many neurodegenerative diseases are caused by patho- logical protein aggregates that result from protein misfolding. The accumulation of abnormal proteins is the crucial pathogenic feature causing synaptic dysfunction, progressive loss of structure and function of neurons, and eventually neuronal degeneration (Takeuchi and Nagai 2017). Of the pathogenic proteins, mutations and post- translational modification are determinative factors in al- tering the stability, solubility, and the aggregation tendency of proteins. For example, mutations cluster around the β- and γ-secretase cleavage sites, increasing the production of insoluble Aβ42 (Shen and Kelleher 2007). Dynamic mu- tations involve the expansion of the number of CAG trinu- cleotides in the coding region of several genes that causea WT TG
bvehiclePHA-767491calbindin GFAP merge
calbindin GFAPmerge0vehicle PHA-767491
. 7 Evaluation of the effect of PHA-767491 treatment in gliosis by immunofluorescence staining. (A and B) The immunofluorescence im- ages of calbindin, GFAP, Iba1, and DAPI in the mouse cerebellum. The levels of GFAP and Ibal expression were significantly higher in the
SCA17 mice than in their WT littermates, but this expression was reduced after the PHA-767491 treatment. Values represent the mean ± SEM.
**p < 0.01; ***p < 0.001 vs. the WT vehicle group 200vehiclec60PHA-7674910vehiclePHA-76749140200vehiclePHA-767491 8 Evaluation of the anti-inflammatory effect of PHA-767491 treat- ment on SCA17 mice by ELISA. (A) The elevated TNF-α level in the SCA17 mouse plasma significantly decreased after PHA-767491 treat- ment. (B) The IL-1β level in the mouse plasma significantly decreased in both the WT and SCA17 groups after PHA-767491 treatment. (C) TheIL-6 level was highly elevated in the SCA17 mouse plasma and was reduced after the PHA-767491 treatment. Values represent the mean ± SEM. *p < 0.05; **p < 0.01 vs. the WT vehicle group. ##p < 0.01; ###p < 0.001 vs. the SCA17 vehicle groupthe polyQ diseases, including Huntington’s and many types of SCAs (Silva et al. 2018). The abnormally expand- ed polyQ stretch derived from the CAG repeats undergoes conformational transition to the β-sheet rich structure, as- sembling into insoluble aggregates and accumulating as inclusion bodies in neuronal cells (Takeuchi and Nagai 2017). Posttranslational modifications include phosphory- lation and glycosylation, which play a significant role in the solubility and aggregation of tau and prion proteins (Mietelska-Porowska et al. 2014; Ryan et al. 2019).
It is believed that substances released from damaged neu- rons trigger microglial activation. The activated microglia fur- ther affect neurons and glia macroglia, astrocytes, and oligo- dendrocytes through the secretion of cytotoxic substances such as oxygen radicals, nitric oxide, glutamate, proteases, and neurotoxic cytokines (TNF-α, IL-6), as well as cytoprotective agents (van Rossum and Hanisch 2004). The effects of microglia are themselves modulated by astrocytes and neurons through cytokines and neurotransmitters, thus giving rise to complex interactions among microglia, neurons,
and astrocytes (Moller 2010). It is believed that neuroinflam- mation will make a bad thing even worse (Moller 2010); therefore, neuroinflammation is an attractive target for phar- macological intervention in CNS disorders.
It was reported that the main characteristic of polyQ- mediated diseases is the accumulation of abnormal polyQ protein aggregation that could lead to inflammation in the CNS. Morphologically activated microglial cells were local- ized in the neostriatum, cortex, and globus palidus as well as in the adjoining white matter of HD patients. The accumula- tion of reactive microglia was increased with the degree of neuronal loss and grades of pathology (Sapp et al. 2001; Tai et al. 2007a; Tai et al. 2007b). Reactive astrocytes and activat- ed microglia were also found in SCA3 pons (Evert et al. 2001). The expression of the anti-inflammatory IL-1 receptor antagonist, the pro-inflammatory cytokine IL-1β, and the pro- inflammatory chemokine SDF1 was increased in the pontine neurons of SCA3 brains (Evert et al. 2001). In SCA17 pa- tients, histological examination identified neuronal loss and gliosis in several sites of the CNS, including the cerebella(Bruni et al. 2004; Lasek et al. 2006; Toyoshima et al. 2004). These results suggest that inflammatory processes are in- volved in the pathogenesis of SCAs.
Our previous reports have shown that the levels of both astrocytes and microglia were elevated in various AD models and SCA17 transgenic mice (Chang et al. 2011; Huang et al. 2015, 2018d). These findings suggest that neuroinflammation, present in SCA17 mice, is a potential target for SCA17 treat- ment. In this study, we found that PHA-767491 has a broad spectrum of neuroprotective activity in different PAD, includ- ing the hippocampal primary neuron under tau- hyperphosphorylation and oligomeric Aβ and both the polyQ-mediated SCA17 cerebellar slice culture and transgenic mice. Our results indicate that PHA-767491 attenuates the neu- roinflammation of the SCA17 slice culture and transgenic mice, suggesting that PHA-767491 could ameliorate the symptoms of different PAD through the mechanism of anti-inflammation. PHA-767491, a potent and selective ATP-competitive dual inhibitor of CDC7/CDK9, was shown to induce apoptosis of cancer cells in a p53-independent pathway (Montagnoli et al. 2008). It was proposed that CDK inhibitors induce immune cell death and thereby increase the resolution of inflammation. CDK9 inhibition is considered to play a potential role in com- batting inflammation (Schmerwitz et al. 2011). It was also hypothesized that the transcriptional inhibition of monocytes through CDK9 inhibition could influence the secretion of in- flammatory cytokines (Han et al. 2014). In addition, interfer- ing with the pathogenicity of atherosclerosis by targeting CDKs, CDK9 has been shown to exhibit marked characteris- tics in controlling inflammation (Schmerwitz et al. 2011). According to these studies, the anti-inflammatory effect of PHA-767491 could be achieved through the inhibition of
CDK9, which in turn alleviates the features of PAD.
PHA-767491 was previously identified as a GSK-3β in- hibitor with IC50 of 61 nM (Hsu et al. 2017). The GSK-3β inhibitor has been suggested to have therapeutic potential for attenuating the aggregation of phosphorylated-tau (Cai et al. 2012); however, our study showed that higher (1.5 μM), but not lower (0.5–1 μM), concentrations of PHA-767491 could benefit the neurite outgrowth and processing of the hippocam- pal primary culture presenting the tau-hyperphosphorylation. This result indicates that the action in the GSK-3β inhibitor might not be the main mechanism in the neuroprotective effect of PHA-767491 in the AD model with hyperphospho-tau. On the other hand, an extremely high concentration (70 μM) of PHA-767491 was reported to reduce TDP-43 phosphorylation through the inhibition of CDC7 and to prevent the neurode- generation in TDP-43 transgenic C. elegans (Liachko et al. 2013). In our study, the concentration of PHA-767491 bene- fits to all the models of PAD was under 1.5 μM, revealing that PHA-767491’s reduction of the phosphorylation of tau protein in this study might not have been through the inhibition of CDC7. Therefore, the protective effect of PHA-767491
probably was mediated by the pathway involved in anti- inflammation.
Whether PHA-767491 can penetrate the blood-brain- barrier to act directly on the brain cells is not clear. It was reported PHA-767491 could inhibit cell proliferation and in- duce apoptosis of glioblastoma which was intracranial im- planted in the mouse brain (Li et al. 2018), but no description in that report whether PHA-767491 can pass the blood-brain barrier (BBB). It was described that hydrophobic compounds have good chance to be transmitted through BBB with a mo- lecular weight less than 400 (Bellettato and Scarpa 2018; Pardridge 2012). The molecular weight of PHA-767491 is
213.24. In addition, we used the BBB predictor (http://www. cbligand.org/BBB/; Prof. Xiang-Qun (Sean) Xie’s laboratory) (Liu et al. 2014) and predicts the PHA-767491 could pass the BBB as shown in supplementary S1. Furthermore, it has been known the BBB is destroyed in certain neurodegenera- tive diseases including AD and HD (Dong 2018; Sweeney et al. 2018). Another study pointed out the importance of BBB permeability of CNS drugs is decreased during a long- term chronic administration of drugs, and even poorly perme- able drugs can have significant pharmacological effects (Reichel 2009). In our study, PHA-767491 does improve the symptoms of SCA17 mice. To understand the BBB penetra- tion of PHA-767491, further studies need to be conducted as described (Kiss et al. 2017; Stanimirovic et al. 2018). In addi- tion, studies suggest that peripheral inflammation and central inflammation are closely related (Kamer 2010). In addition, evidence has shown that anti-inflammatory drugs peripherally can rapidly attenuate inflammatory responses in the brain and improve cognition (Huang et al. 2018a). Therefore, the reduc- tion in neuroinflammation by PHA-767491 could be attribut- ed to the systematic effect of anti-inflammation. In sum, our study suggests that PHA-767491 exerts a neuroprotective ef- fect in PAD mediated by the pathway involved in anti-inflam- mation, which might be achieved through the inhibition of CDK9.
Acknowledgments Our gratitude is extended to the Molecular Imaging Core Facility of the National Taiwan Normal University under the aus- pices of the Ministry of Science and Technology.Funding Information This work was supported by research grants MOST 107-2320-B-003-007, 107-2320-B-003-009, and 104-2320-B-
003-009-MY3 from the Ministry of Science and Technology.
Compliance with Ethical Standards
All of the animal experiments were conducted according to the guidelines and were approved by the Research Committee of the National Taiwan Normal University (No. 103022).Conflict of Interest The authors declare that they have no conflicts of interest.Abbreviations AD, Alzheimer’s disease; ANOVA, analysis of variance; DIV, days in vitro; DRPLA, dentatorubral-pallidoluysian atrophy; FDA, Food and Drug Administration; GFAP, glial fibrillary acidic protein; GRAS, generally recognized as safe; HD, Huntington’s disease; LSD, least significant difference; PD, Parkinson’s disease; polyQ, polyglutamine; SBMA, spinal and bulbar muscular atrophy; SCA, spinocerebellar ataxias; SCA17, spinocerebellar ataxia type 17; TBP, TATA box-binding protein; TBS, tris-buffered saline; TG, transgenic; WT, wild-type
Aguzzi A, O’Connor T (2010) Protein aggregation diseases: pathogenic- ity and therapeutic perspectives. Nat Rev Drug Discov 9:237–248
Bellettato CM, Scarpa M (2018) Possible strategies to cross the blood- brain barrier. Ital J Pediatr 44:131
Bruni AC, Takahashi-Fujigasaki J, Maltecca F, Foncin JF, Servadio A, Casari G, D’Adamo P, Maletta R, Curcio SA, De Michele G, Filla A, El Hachimi KH, Duyckaerts C (2004) Behavioral disorder, demen- tia, ataxia, and rigidity in a large family with TATA box-binding protein mutation. Arch Neurol 61:1314–1320
Cai Z, Zhao Y, Zhao B (2012) Roles of glycogen synthase kinase 3 in Alzheimer’s disease. Curr Alzheimer Res 9:864–879
Chang YC, Lin CY, Hsu CM, Lin HC, Chen YH, Lee-Chen GJ, Su MT, Ro LS, Chen CM, Hsieh-Li HM (2011) Neuroprotective effects of granulocyte-colony stimulating factor in a novel transgenic mouse model of SCA17. J Neurochem 118:288–303
Chang YC, Lin CW, Hsu CM, Lee-Chen GJ, Su MT, Ro LS, Chen CM, Huang HJ, Hsieh-Li HM (2016) Targeting the prodromal stage of spinocerebellar ataxia type 17 mice: G-CSF in the prevention of motor deficits via upregulating chaperone and autophagy levels. Brain Res 1639:132–148
Chen ZZ, Wang CM, Lee GC, Hsu HC, Wu TL, Lin CW, Ma CK, Lee- Chen GJ, Huang HJ, Hsieh-Li HM (2015) Trehalose attenuates the gait ataxia and gliosis of spinocerebellar ataxia type 17 mice. Neurochem Res 40:800–810
Chen CM, Chen WL, Hung CT, Lin TH, Chao CY, Lin CH, Wu YR, Chang KH, Yao CF, Lee-Chen GJ, Su MT, Hsieh-Li HM (2018) The indole compound NC009-1 inhibits aggregation and promotes neurite outgrowth through enhancement of HSPB1 in SCA17 cells and ameliorates the behavioral deficits in SCA17 mice. Neurotoxicology 67:259–269
Chen CM, Chen WL, Hung CT, Lin TH, Lee MC, Chen IC, Lin CH, Chao CY, Wu YR, Chang KH, Hsieh-Li HM, Lee-Chen GJ (2019) Shaoyao Gancao Tang (SG-Tang), a formulated Chinese medicine, reduces aggregation and exerts neuroprotection in spinocerebellar ataxia type 17 (SCA17) cell and mouse models. Aging (Albany NY) 11:986–1007
Dong X (2018) Current strategies for brain drug delivery. Theranostics. 8: 1481–1493
Erbayraktar Z, Alural B, Erbayraktar RS, Erkan EP (2016) Cell division cycle 7-kinase inhibitor PHA-767491 hydrochloride suppresses glioblastoma growth and invasiveness. Cancer Cell Int 16:88
Evert BO, Vogt IR, Kindermann C, Ozimek L, de Vos RA, Brunt ER, Schmitt I, Klockgether T, Wullner U (2001) Inflammatory genes are upregulated in expanded ataxin-3-expressing cell lines and spinocerebellar ataxia type 3 brains. J Neurosci 21:5389–5396
Friedman MJ, Shah AG, Fang ZH, Ward EG, Warren ST, Li S, Li XJ (2007) Polyglutamine domain modulates the TBP-TFIIB interac- tion: implications for its normal function and neurodegeneration. Nat Neurosci 10:1519–1528
Gostout B, Liu Q, Sommer SS (1993) “Cryptic” repeating triplets of purines and pyrimidines (cRRY(i)) are frequent and polymorphic: analysis of coding cRRY(i) in the proopiomelanocortin (POMC)and TATA-binding protein (TBP) genes. Am J Hum Genet 52: 1182–1190
Han Y, Zhan Y, Hou G, Li L (2014) Cyclin-dependent kinase 9 may as a novel target in downregulating the atherosclerosis inflammation (re- view). Biomed Rep 2:775–779
Hsu CJ, Hsu WC, Lee DJ, Liu AL, Chang CM, Shih HJ, Huang WH, Lee-Chen GJ, Hsieh-Li HM, Lee GC, Sun YC (2017) Investigation of the bindings of a class of inhibitors with GSK3beta kinase using thermodynamic integration MD simulation and kinase assay. Chem Biol Drug Des 90:272–281
Huang HJ, Chen SL, Hsieh-Li HM (2015) Administration of NaHS at- tenuates footshock—induced pathologies and emotional and cogni- tive dysfunction in triple transgenic Alzheimer’s mice. Front Behav Neurosci 9:312
Huang C, Irwin MG, Wong GTC, Chang RCC (2018a) Evidence of the impact of systemic inflammation on neuroinflammation from a non- bacterial endotoxin animal model. J Neuroinflammation 15:147
Huang HJ, Chen SL, Chang YT, Chyuan JH, Hsieh-Li HM (2018b) Administration of Momordica charantia enhances the neuroprotec- tion and reduces the side effects of LiCl in the treatment of Alzheimer’s disease. Nutrients 10
Huang HJ, Chen SL, Huang HY, Sun YC, Lee GC, Lee-Chen GJ, Hsieh- Li HM, Su MT (2018c) Chronic low dose of AM404 ameliorates the cognitive impairment and pathological features in hyperglycemic 3xTg-AD mice. Psychopharmacology
Huang HJ, Huang HY, Hsieh-Li HM (2018d) MGCD0103, a selective histone deacetylase inhibitor, coameliorates oligomeric Abeta25-35
-induced anxiety and cognitive deficits in a mouse model. CNS Neurosci Ther
Huang HJ, Chen SL, Huang HY, Sun YC, Lee GC, Lee-Chen GJ, Hsieh- Li HM, Su MT (2019a) Chronic low dose of AM404 ameliorates the cognitive impairment and pathological features in hyperglycemic 3xTg-AD mice. Psychopharmacology 236:763–773
Huang HJ, Huang HY, Hsieh-Li HM (2019b) MGCD0103, a selective histone deacetylase inhibitor, coameliorates oligomeric Abeta25-35
-induced anxiety and cognitive deficits in a mouse model. CNS Neurosci Ther 25:175–186
Huggett MT, Tudzarova S, Proctor I, Loddo M, Keane MG, Stoeber K, Williams GH, Pereira SP (2016) Cdc7 is a potent anti-cancer target in pancreatic cancer due to abrogation of the DNA origin activation checkpoint. Oncotarget 7:18495–18507
Kamer AR (2010) Systemic inflammation and disease progression in Alzheimer disease. Neurology 74:1157 author reply 1157-8
Kazantsev A, Preisinger E, Dranovsky A, Goldgaber D, Housman D (1999) Insoluble detergent-resistant aggregates form between path- ological and nonpathological lengths of polyglutamine in mamma- lian cells. Proc Natl Acad Sci U S A 96:11404–11409
Kiss L, Bocsik A, Walter FR, Ross J, Brown D, Mendenhall BA, Crews SR, Lowry J, Coronado V, Thompson DE, Sipos P, Szabo-Revesz P, Deli MA, Petrikovics I (2017) From the cover: in vitro and in vivo blood-brain barrier penetration studies with the novel cyanide anti- dote candidate dimethyl trisulfide in mice. Toxicol Sci 160:398–407
Koide R, Kobayashi S, Shimohata T, Ikeuchi T, Maruyama M, Saito M, Yamada M, Takahashi H, Tsuji S (1999) A neurological disease caused by an expanded CAG trinucleotide repeat in the TATA- binding protein gene: a new polyglutamine disease? Hum Mol Genet 8:2047–2053
Kung PJ, Tao YC, Hsu HC, Chen WL, Lin TH, Janreddy D, Yao CF, Chang KH, Lin JY, Su MT, Wu CH, Lee-Chen GJ, Hsieh-Li HM (2014) Indole and synthetic derivative activate chaperone expres- sion to reduce polyQ aggregation in SCA17 neuronal cell and slice culture models. Drug Des Devel Ther 8:1929–1939
Lasek K, Lencer R, Gaser C, Hagenah J, Walter U, Wolters A, Kock N, Steinlechner S, Nagel M, Zuhlke C, Nitschke MF, Brockmann K, Klein C, Rolfs A, Binkofski F (2006) Morphological basis for the spectrum of clinical deficits in spinocerebellar ataxia 17 (SCA17). Brain 129:2341–2352
Li X, Lu F, Tian Q, Yang Y, Wang Q, Wang JZ (2006) Activation of glycogen synthase kinase-3 induces Alzheimer-like tau hyperphosphorylation in rat hippocampus slices in culture. J Neural Transm (Vienna) 113:93–102
Li Q, Xie W, Wang N, Li C, Wang M (2018) CDC7-dependent transcrip- tional regulation of RAD54L is essential for tumorigenicity and radio-resistance of glioblastoma. Transl Oncol 11:300–306
Liachko NF, McMillan PJ, Guthrie CR, Bird TD, Leverenz JB, Kraemer BC (2013) CDC7 inhibition blocks pathological TDP-43 phosphor- ylation and neurodegeneration. Ann Neurol 74:39–52
Liu H, Wang L, Lv M, Pei R, Li P, Pei Z, Wang Y, Su W, Xie XQ (2014) Alz Platform: an Alzheimer ’s disease domain-specific chemogenomics knowledgebase for polypharmacology and target identification research. J Chem Inf Model 54:1050–1060
Llorens-Martin M, Jurado J, Hernandez F, Avila J (2014) GSK-3beta, a pivotal kinase in Alzheimer disease. Front Mol Neurosci 7:46
McGeer PL, McGeer EG (1995) The inflammatory response system of brain: implications for therapy of Alzheimer and other neurodegen- erative diseases. Brain Res Brain Res Rev 21:195–218
Mietelska-Porowska A, Wasik U, Goras M, Filipek A, Niewiadomska G (2014) Tau protein modifications and interactions: their role in func- tion and dysfunction. Int J Mol Sci 15:4671–4713
Moller T (2010) Neuroinflammation in Huntington’s disease. J Neural Transm 117:1001–1008Montagnoli A, Valsasina B, Croci V, Menichincheri M, Rainoldi S, Marchesi V, Tibolla M, Tenca P, Brotherton D, Albanese C, Patton V, Alzani R, Ciavolella A, Sola F, Molinari A, Volpi D, Avanzi N, Fiorentini F, Cattoni M, Healy S, Ballinari D, Pesenti E, Isacchi A, Moll J, Bensimon A, Vanotti E, Santocanale C (2008) A Cdc7 ki- nase inhibitor restricts initiation of DNA replication and has antitu- mor activity. Nat Chem Biol 4:357–365
Muhleisen H, Gehrmann J, Meyermann R (1995) Reactive microglia in Creutzfeldt-Jakob disease. Neuropathol Appl Neurobiol 21:505– 517
Nakamura K, Jeong SY, Uchihara T, Anno M, Nagashima K, Nagashima T, Ikeda S, Tsuji S, Kanazawa I (2001) SCA17, a novel autosomal dominant cerebellar ataxia caused by an expanded polyglutamine in TATA-binding protein. Hum Mol Genet 10:1441–1448
Pardridge WM (2012) Drug transport across the blood-brain barrier. J Cereb Blood Flow Metab 32:1959–1972
Reichel A (2009) Addressing central nervous system (CNS) penetration in drug discovery: basics and implications of the evolving new con- cept. Chem Biodivers 6:2030–2049
Rubio-Perez JM, Morillas-Ruiz JM (2012) A review: inflammatory pro- ces s i n A lz hei m er ’s d is eas e, role of cytokines . ScientificWorldJournal 2012:756357
Ryan P, Xu M, Davey AK, Danon JJ, Mellick GD, Kassiou M, Rudrawar S (2019) O-GlcNAc modification protects against protein misfolding and aggregation in neurodegenerative disease. ACS Chem Neurosci 10:2209–2221
Sapp E, Kegel KB, Aronin N, Hashikawa T, Uchiyama Y, Tohyama K, Bhide PG, Vonsattel JP, DiFiglia M (2001) Early and progressive accumulation of reactive microglia in the Huntington disease brain. J Neuropathol Exp Neurol 60:161–172
Sawaya MR, Sambashivan S, Nelson R, Ivanova MI, Sievers SA, Apostol MI, Thompson MJ, Balbirnie M, Wiltzius JJ, McFarlane HT, Madsen AO, Riekel C, Eisenberg D (2007) Atomic structures of amyloid cross-beta spines reveal varied steric zippers. Nature 447: 453–457
Schmerwitz UK, Sass G, Khandoga AG, Joore J, Mayer BA, Berberich N, Totzke F, Krombach F, Tiegs G, Zahler S, Vollmar AM, Furst R (2011) Flavopiridol protects against inflammation by attenuating leukocyte-endothelial interaction via inhibition of cyclin- dependent kinase 9. Arterioscler Thromb Vasc Biol 31:280–288
Shen J, Kelleher RJ 3rd (2007) The presenilin hypothesis of Alzheimer’s disease: evidence for a loss-of-function pathogenic mechanism. Proc Natl Acad Sci U S A 104:403–409
Silva A, de Almeida AV, Macedo-Ribeiro S (2018) Polyglutamine expan- sion diseases: more than simple repeats. J Struct Biol 201:139–154 Stanimirovic DB, Sandhu JK, Costain WJ (2018) Emerging technologies for delivery of biotherapeutics and gene therapy across the blood-
brain barrier. BioDrugs. 32:547–559
Sweeney MD, Sagare AP, Zlokovic BV (2018) Blood-brain barrier break- down in Alzheimer disease and other neurodegenerative disorders. Nat Rev Neurol 14:133–150
Tai YF, Pavese N, Gerhard A, Tabrizi SJ, Barker RA, Brooks DJ, Piccini P (2007a) Imaging microglial activation in Huntington’s disease. Brain Res Bull 72:148–151
Tai YF, Pavese N, Gerhard A, Tabrizi SJ, Barker RA, Brooks DJ, Piccini P (2007b) Microglial activation in presymptomatic Huntington’s disease gene carriers. Brain 130:1759–1766
Takeuchi T, Nagai Y (2017) Protein misfolding and aggregation as a therapeutic target for polyglutamine diseases. Brain Sci 7
Toyoshima Y, Yamada M, Onodera O, Shimohata M, Inenaga C, Fujita N, Morita M, Tsuji S, Takahashi H (2004) SCA17 homozygote showing Huntington’s disease-like phenotype. Ann Neurol 55: 281–286
van Rossum D, Hanisch UK (2004) Microglia. Metab Brain Dis 19:393– 411
Wyss-Coray T, Mucke L (2002) Inflammation in neurodegenerative disease—a double-edged sword. Neuron 35:419–432Publisher’s PHA-767491 Note Springer Nature remains neutral with regard to jurisdic- tional claims in published maps and institutional affiliations.