Chronic clemastine treatment rescues cognitive deficits of APP/PS1 Transgenic Mice
We first examined whether clemastine might attenuate the cognitive deficits in APP/PS1 transgenic mice. APP/PS1 transgenic mice exhibit appearance of Aβ plaques from 3-month old. They develop AD-like pathology such as extensive Aβ plaques, neuroinflammation and cognitive deficits at 7-month old (Zhang et al., 2014). Thus, we have taken a prophylactic strategy as an early invention in which four-month-old APP/PS1 transgenic mice were administered clemastine mixed in the food for 3 months. Clemastine exhibits beneficial effect in social isolation-caused depression model mice and in SOD1-G93A mice, a transgenic mouse model of amyotrophic lateral sclerosis (ALS) at 10 mg/kg/day(Apolloni et al., 2016). Thus, to prove the concept that whether clemastine might attenuate AD pathology, APP/PS1 mice were treated orally with clemastine at 10 mg/kg/day. APP/PS1 mice and age-matched WT mice fed with normal food were used as the control. In Morris water maze test, APP/PS1 mice showed impaired learning, as indicated by the increased escape latencies (Fig. 1A) and swimming distances (Fig. 1B, C) in the consecutive trials compared with WT mice. In contrast, clemastine-treated APP/PS1 mice showed shorter escape latencies (Fig. 1A), and decreased swimming distances (Fig. 1B, C) compared with control APP/PS1 mice, even to a level comparable to WT mice. In the probe trials, clemastine-treated APP/PS1 mice exhibited improved memory retention as indicated that they spent longer time in target quadrant (Fig. 1D) and swam to cross over the target site more times than control APP/PS1 mice (Fig. 1E), which are comparable to WT mice. The differences among these groups of mice were not due to the distinct swimming capability, since the swimming speed of these groups of mice was similar (Fig. 1F). In novel object recognition tests, no significant difference was observed in location preference during the training phase, indicating that the location of the objects does not affect the exploratory behavior of mice (Fig. 1H). In the testing phase, as demonstrated previously, control APP/PS1 mice displayed a reduced recognition index (RI) than WT mice, which was rescued by treatment with clemastine (Fig. 1H). It is noteworthy that clemastine treatment showed no effects on WT mice in the above-mentioned tests (Fig. 1A-H). These results indicate that the chronic treatment with clemastine rescues cognitive deficits in APP/PS1 mice.
Chronic clemastine treatment attenuates Aβ accumulation in APP/PS1 mice
Accumulation of Aβ is the central initiator of AD pathogenesis (Musiek and Holtzman, 2015). We thus examined whether clemastine treatment would decrease Aβ accumulation. The coronal section of the hippocampus and cortex of clemastine-treated APP/PS1 transgenic mice and the control mice were stained with an antibody against Aβ (6E10) (Fig. 2A, D). Results showed that the numbers (Fig. 2C, F) and size (Fig. 2B, E) of Aβ plaques were decreased in the hippocampus (Fig. 2A-C) and cortex (Fig. 2D-F) of clemastine-treated APP/PS1 transgenic mice, compared to that in control APP/PS1 transgenic mice. These results indicate that chronic treatment with clemastine decreases the densities of Aβ plaques. We further examined whether the reduced densities of Aβ plaques were due to decreased Aβ concentrations in the brain. ELISA analysis showed that the levels of both soluble and insoluble Aβ42 and Aβ40 in the cortex and hippocampus of clemastine-treated APP/PS1 transgenic mice decreased, compared with those in control transgenic mice. These results indicate that chronic treatment with clemastine decreases accumulation of Aβ.
Chronic clemastine treatment attenuates neuroinflammation in APP/PS1 mice
Neuroinflammation is an essential contributor to the pathogenesis of AD. Microglia and astrocytes, the main types of cells in the inflammatory response in the central nervous system, are activated in the brains of AD patients and AD model mice (Calsolaro and Edison, 2016;Shadfar et al., 2015). Activated microglia and astrocytes accumulate around Aβ plaques and produce pro-inflammatory cytokines and chemokines, which cause synaptic dysfunction and neurodegeneration (Calsolaro and Edison, 2016). Anti-inflammatory therapy has therefore been credited as a strategy for reducing the risk or slowing the progression of AD (Shadfar et al., 2015). Clemastine has been reported to decrease microgliosis and expression of microglia-related inflammatory genes in the model mice of ALS (Apolloni et al., 2016). We thus examined whether clemastine treatment might also decrease neuroinflammation in APP/PS1 transgenic mice. The densities of astrocytes and microglia as indicated by the volume of GFAP+ (a marker for astrocytes, Fig. 3A, C) and Iba-1+ (a marker for microglia, Fig. 3B, D) cells decreased in both the hippocampus and the cortex of clemastine-treated APP/PS1 transgenic mice. These results indicate that chronic treatment with clemastine attenuates neuroinflammation in the brains of APP/PS1 transgenic mice.
Chronic clemastine treatment decreases β-amyloidosis of APP processing in vivo
Chronic treatment with clemastine decreases Aβ accumulation, neuroinflammation and cognitive deficits of APP/PS1 transgenic mice. Among these pathological processes, Aβ accumulation is the upstream cause (Musiek and Holtzman, 2015). We thus examined the mechanisms underlying that clemastine reduces Aβ accumulation. To further confirm that clemastine affects Aβ generation in neurons, primary cortical neurons derived from embryonic 17 days (E17) APP/PS1 transgenic mice were treated with clemastine. The result showed that Aβ40 levels in culture medium were reduced by treatment with clemastine (Fig. 4A), confirming that clemastine can reduce Aβ levels in neurons, which is independent on glial cells. Moreover, the levels of neprilysin and insulin-degrading enzyme (IDE), two enzymes account for Aβ degradation, remained unchanged in the hippocampus and cortex of APP/PS1 transgenic mice upon treatment with clemastine (Fig. 4B-D), suggesting that clemastine may not being involved in Aβ clearance. Since the ratio of Aβ40/Aβ42, which can be altered by γ-secretase cleavage of APP (Borchelt et al., 1996), remained identical levels in between clemastine-treated and control APP/PS1 transgenic mice (Fig. 4K), indicating that clemastine treatment does not alter γ-secretase activity. We then examined the cleavage of APP by α-/β-secretase. APP is cleaved by α- or β-secretase at the extracellular domain, generating two fragments called α- or β-CTF, respectively (Musiek and Holtzman, 2015). The levels of both α- and β-CTF, but not full-length APP, were decreased in the hippocampus and cortex of clemastine-treated APP/PS1 transgenic mice, compared to that in control transgenic mice (Fig. 4E-H). β-CTF is produced from cleavage of APP by BACE1 (Yan, 2016;Yan et al., 2016). Consistently, treatment with clemastine decreased BACE1 levels in the hippocampus and cortex of APP/PS1 transgenic mice (Fig. 4I, 4J). These results indicate that chronic treatment with clemastine reduces Aβ generation through suppressing cleavage of APP, especially by BACE1.
Clemastine treatment induces ATG5-dependent autophagy
Autophagy is a highly conserved catabolic process in which proteins and organelles are engulfed in double-membraned vacuoles called autophagosomes and then transported to lysosomes for degradation. Autophagy plays broad functions in neurodegenerative disease (Nixon, 2013). Both α-/β-CTFs and BACE1 are degraded through autophagy (Tian et al., 2013;Wu et al., 2015). Moreover, co-treatment with H1R antagonist astemizole and histamine induces autophagy (Jakhar et al., 2016), suggesting that histamine signaling is involved in autophagy induction. Thus, to investigate the underlying molecular mechanisms of therapeutic effects of clemastine in AD pathogenesis, we examined the effect of clemastine on autophagy. Microtubule-associated protein light chain 3 (LC3) was used as a marker of autophagy induction, because cytosolic LC3-I is processed to its lipidated LC3-II form upon autophagy induction. LC3-II then locates to newly forming autophagophores and subsequently be present in mature autophagosomes (Wu et al., 2015). Therefore, processing of LC3 and a punctuate LC3 pattern represent the formation of autophagosomes and autophagic responses. HeLa cells transfected with LC3-GFP were treated with either 30 μM clemastine or DMSO. HeLa cells exhibited much more LC3-GFP+ puncta 12 h after clemastine treatment (Fig. 5A, 5B), indicating that clemastine induces formation of autophagosomes. In addition, treatment with clemastine increases LC3-II levels in a dose-dependent manner (Fig. 5C, 5D). The increasement of LC3-II by clemastine was further enhanced in presence of chloroquine, a lysosomal inhibitor which inhibits fusion of autophagosomes to lysosomes, indicating that the increasement of LC3-II by clemastine is due to an enhanced autophagic influx. Consistently, levels of P62, a substrate of autophagy, were decreased by clemastine dose-dependently (Fig. 5C, 5E). ATG5 is an initial factor in autophagy induction. We then examined whether clemastine induced autophagy in a way dependent on ATG5. ATG5+/+ and ATG5-/- MEF cells were treated with clemastine. The results showed that, like in HeLa cells, clemastine treatment increased LC3-II levels (Fig. 5H, 5J), whereas decreasing P62 levels (Fig. 5H, 5J) indicating an enhanced autophagy influx. In contrast, clemastine failed to do so in ATG5-/- MEF cells (Fig. 5H-5J), indicating that clemastine induces ATG5-dependent autophagy. Consistent with the fact that autophagy is impaired in AD (Nixon, 2013), APP/PS1 transgenic exhibited increased levels of LC3-II. In contrast, chronic treatment with clemastine increased LC3-II (Fig. 5K, 5M) while decreasing P62 levels (Fig. 5K, 5L), as it did in cultured cells. Therefore, these results indicate that clemastine enhances autophagy. However, clemastine failed to increase the LC3-II levels in WT mice, indicating clemastine enhances autophagy in a context-dependent manner.
Clemastine enhances autophagy via the mTOR pathway both in vivo and vitro
We further explored molecular mechanisms underlying that clemastine induces autophagy. Target of rapamycin (mTOR) signaling, when being suppressed, is one of central pathways in autophagy induction (Zhu et al., 2019). We thus examined first whether clemastine could affect mTOR signaling. HeLa cells treated with distinct concentrations of clemastine revealed that clemastine decreased levels of phosphorylated mTOR (p-mTOR, Ser2448) (Fig. 6A, B) and phosphorylated P70S6K (p-P70S6K) (Fig. 6A, 6C) in a dose-dependent manner. In contrast, the levels of total mTOR (Fig. 6A, 6D) and P70S6K (Fig. 6A, 6E) remained unchanged upon treatment with clemastine. Similar results were observed in clemastine-treated APP/PS1 transgenic mice. The levels of p-mTOR and p-P70S6K, but not total mTOR and P70S6K, were decreased in the hippocampus and cortex of clemastine-treated APP/PS1 transgenic mice, compared to control transgenic mice (Fig. 6F-6H). However, clemastine did not alter mTOR signaling in WT mice, suggesting that clemastine suppresses mTOR signaling in an environment-dependent manner. Thus, these results indicate that clemastine suppresses mTOR signaling, a central pathway inducing autophagy.