ATRIVIEW®, the CNS regenerative drug screening platform, and properties of its hit trametinib (SNR1611)
Because drugs against AD aiming for Aβ clearance or interference of its production have not been successful in restoring already damaged cognitive functions, we hypothesized that enhancing adult neurogenesis from endogenous NSCs will restore neuronal network integrity in affected brain regions of AD patients. Thus, we developed a phenotypic drug discovery platform, ATRIVIEW®, aiming to identify small molecular compounds inducing neuronal differentiation of adult NSCs derived from AD model mice (AD-NSCs) (Fig. 1a). Using NSCs derived from Tg2576 AD mouse model, we screened 994 small molecules from the FDA-approved drug library to identify small molecules inducing neuronal differentiation. By comparing the level of Tuj1 (class III beta-tubulin, neuronal marker) expression using fluorescent immunocytochemical analysis, 48 compounds were found to increase neuronal differentiation (at least 1.7-fold increase to the DMSO-treated control) (Supplementary Fig. 1a). Among them, the most effective drug inducing the neuronal differentiation of NSCs was SNR1611, which turned out to be trametinib (Mekinist®), a specific MEK1/2 inhibitor and FDA-approved anti-cancer drug. Upon immunocytochemistry, it was observed that trametinib treatment induced morphological changes of NSCs into neuron-like cells (Fig. 1a). Another MEK1 inhibitor, Cobimetinib 28, and a CDK inhibitor, Dinaciclib 29, were among the selected drugs. Semagacestat 30, a g-secretase inhibitor, and Duloxetine 31, a serotonin-norepinephrine reuptake inhibitor, which have been known for their therapeutic potential for AD were also screened (Supplementary Fig. 1a). In the presence of growth factors in the medium (20 ng/ml EGF and 20 ng/ml bFGF), trametinib inhibited proliferation (assessed by PCNA level) and induced neuronal differentiation (by Tuj1 level) but not astrocytic differentiation (by GFAP level) of adult NSCs from C57B/L6 mice (Supplementary Fig. 1b). Regardless of Aβ1− 42 oligomer (10 mM) presence, both MEK1/2 inhibition by trametinib (Supplementary Fig. 1c) and MEK1/2 knock-down (Supplementary Fig. 1d) induced neuronal differentiation of NSCs. These findings indicate that inhibition of MEK/ERK signaling by trametinib is a potential approach to induce neuronal differentiation of adult NSCs.
Inhibition of MEK/ERK signaling by trametinib induces neuronal differentiation via induction of P15INK4b and Neurog2 expression and protects against cell death
It has been reported that MEK/ERK signaling is activated in the brain of AD patients compared to the normal brain 32,33 and Aβ plaques activate this signaling 34. To find out whether MEK/ERK signaling is also activated in NSCs of the AD model mice with Ab plaque phenotypes, we measured the pERK level in the brain of WT and AD model mice, 5XFAD. It was confirmed that the MEK/ERK signaling pathway was activated in the SGZ (Fig. 1b, c) and SVZ (Fig. 1d, e) of 7.5-month-old 5XFAD mice compared with WT. Administration of trametinib (for 2.5 months to 5-month-old 5XFAD mice) reduced the level of pERKs in both areas (Fig. 1b-e). Thus, we further tested whether trametinib induces the neuronal differentiation of adult NSCs isolated from the 5XFAD mouse. The size of neurospheres derived from 5XFAD were smaller than that from wild type (WT) (Fig. 1f, g). In addition, trametinib strongly increased the level of Tuj1 but not the level of GFAP in NSCs isolated from 5XFAD mouse, indicating its induction of neuronal but not astrocytic differentiation. (Fig. 1h, i). Meanwhile, we confirmed that other MEK1/2 inhibitors (AZD8330, PD184352, Refametinib, PD318088, AS703026) also induced neuronal differentiation (Supplementary Fig. 2) in embryonic NSCs, indicating that MEK1/2 inhibition activates neuronal differentiation. Notably, we observed that trametinib was the most effective MEK inhibitors to neuronal differentiation.
Since trametinib was discovered as a drug to induce the CDK inhibitor (such as P15INK4b)-mediated cell cycle arrest by inhibiting MEK1/2 35, we tested whether it would induce neuronal differentiation through cell cycle arrest in NSCs as well. We found that trametinib induced P15INK4b (cell cycle arrest) and Neurog2 (proneuronal factors) expression and increased the protein levels of P15INK4b and Neurod 1 (neuronal differentiation 1) in adult NSCs from 5XFAD mice (Fig. 1j-m). Whole brain RNA-Seq analysis was also supported that inhibition of MEK/ERK signaling is the mechanism responsible for the neuronal differentiation. In Go term analysis, the second week of trametinib administration appeared to be the critical period for neuron development, pyramidal neuron differentiation and regulation of neuron migration (Supplementary Fig. 3). Among the 107 genes whose expression was increased by 1.5 times or more, it was confirmed that the expression of Ebf1, Nhlh2, Irx5, Ebf3, Irx3, Sox14, Cdh1, Tead4 genes, known as the target gene of Ngn2, was also increased by trametinib administration (Supplementary Table 1).
To investigate whether apoptosis was induced by Aβ accumulation as in the previous results 22 in adult NSCs isolated from 5XFAD mice and whether trametinib protects against this, we examined the expression of the apoptosis markers. The Aβ accumulation was observed in the adult NSCs from 5XFAD mice, and active caspase-3 was also observed simultaneously. Trametinib treatment, however, reduced the level of Aβ accumulation and active caspase-3. In addition, the level of cleaved Poly (ADP-ribose) polymerase (PARP), another apoptosis marker) was also decreased by trametinib treatment (Supplementary Fig. 4a-e). MEK1/2 knock-down by shRNA also reduced the expression of active caspase 3 (Supplementary Fig. 4f, g).
Taken together, these results demonstrate that activation of MEK/ERK signaling is related to the apoptosis of adult NSCs of 5XFAD mice and that trametinib protects against cell death. Furthermore, trametinib enhanced neuronal differentiation through induction of CDK inhibitors and proneuronal factors.
Trametinib induces hippocampal and SVZ neurogenesis in 5XFAD mice
Since trametinib induced the neuronal differentiation of NSCs derived from AD model mice, we examined whether oral administration of trametinib also induces hippocampal neurogenesis in 5XFAD mice. Five-month-old 5XFAD mice were administered 0.1mg/kg trametinib for 2.5 months, after which all mice were sacrificed at the age of 7.5 months (Fig. 2a). In the DG of the vehicle-administered 5XFAD mice, Sox2+/GFAP + NSCs 36 increased, but the number of both Neurod1 + neuroblasts and Tuj1 + immature neurons decreased compared with WT control (Fig. 2b, c). In contrast, the number of neuroblasts and immature neurons in the same brain region of the trametinib-administered 5XFAD mice increased, while the number of radial glial cells decreased (Fig. 2b, c).
Next, we asked whether trametinib also induces neuronal differentiation of NSCs in the SVZ of 5XFAD mice 37. The number of NSCs expressingSox2 and GFAP (type B1 cells) in the SVZ of 5XFAD mice decreased compared with WT control mice (Fig. 2d, e). Also, the number of Dcx + neuroblasts (Type A cells) significantly decreased in 5XFAD mice. Trametinib increased the number of Sox2+/GFAP + NSCs, Ki67 + proliferating cells and Dcx + neuroblasts in the SVZ of 5XFAD mice to a level comparable to that observed in WT (Fig. 2d, e). These data indicate that the generation of type A neuroblasts from NSCs is impaired in 5XFAD, and trametinib restores the transition to facilitate adult neurogenesis in the SVZ.
Then we asked further whether trametinib induced the migration of newly formed neuroblasts in the SVZ to the olfactory bulb (OB). While the number of Dcx + neuroblasts was significantly reduced in the OB granular layer of 5XFAD mice, trametinib indeed recovered the neuroblast population (Fig. 2d). These data demonstrate that inducing adult neurogenesis in the SVZ by trametinib restores migration of neuroblasts to the OB, which is disrupted in AD conditions. Altogether, trametinib administration evidently restores adult hippocampal and SVZ neurogenesis, which is impaired in 5XFAD mice.
Trametinib induces cortical neurogenesis in the 5XFAD mouse brain
We asked whether trametinib can induce adult cortical neurogenesis in 5XFAD mice which is characterized by neuronal loss in the brain cortical layer V 38. Previous studies demonstrated that the production of new neurons occurs in the brain cortex after cortical injury, and those new neurons originate from cortical glial cells or NSCs migrating from the SVZ 39–41. Therefore, we investigated which cell population differentiated into cortical neurons. We counted the number of Sox2, GFAP, and Neurod1 triple-positive cells in the sagittal sections of the 5XFAD mouse somatosensory cortex. The Sox2 and GFAP double-positive cell is well known as a neural stem/progenitor cell in the DG and SVZ 42. Sox2, especially, is known as a reprogramming factor in the adult brain 43, and Neurod1 is expressed in dividing neuro-progenitor cells 43. Thus, these triple positive (Neurod1+/Sox2+/GFAP+) cells may act as neurogenic progenitors in the cortex. After 0.1 mg/kg trametinib was administered to five-month-old 5XFAD mice for 2.5 months, mice were sacrificed at the age of 7.5 months (Fig. 3a). Interestingly, triple-positive cells were significantly increased by trametinib administration in the 5XFAD mouse cortex (Fig. 3b, c). We also confirmed cortical neurogenesis using EdU incorporation analysis. We administered trametinib for 1.5 months to 7-month-old 5XFAD mice while giving them 200mg/kg of EdU injection 30 days before sacrifice at 8.5 months of age (Fig. 3d). The number of EdU-positive cells in the cortex increased with trametinib administration (Fig. 3e, f). Furthermore, the number of EdU/NeuN double-positive cells significantly increased (Fig. 3g, h), suggesting that trametinib distinctly induced neurogenesis and produced new neurons in the cortex. We also questioned whether trametinib can induce cortical neurogenesis in the late-symptomatic stage of AD and administered trametinib to 9-month-old 5XFAD mice for 1.5 months (Fig. 3i). The level of Dcx in the cortex was increased by trametinib administration (Fig. 3j, k), and EdU/NeuN double-positive cells were also increased (Fig. 3l, m). These results strongly suggest that NPCs exist in the AD brain cortex and that trametinib activates cortical neurogenesis in 5XFAD mice.
Trametinib induces functional rescue of AD pathogenesis by restoring neuron numbers and neuronal structure
To investigate whether adult neurogenesis, especially cortical neurogenesis, induced by trametinib supports the restoration of neurons in 5XFAD mice, we administered daily 0.1 mg/kg of trametinib to 5-month-old 5XFAD mice for 2.5 months (Fig. 4a) or 12-month-old 5XFAD mice for 1 month (Fig. 4d). The number and axonal length of neurons in cortical layer V of the somatosensory cortex was decreased in the 7.5-month-old trametinib-treated 5XFAD mice, whereas 0.1 mg/kg of trametinib administration for 2.5 months to 5-month-old 5XFAD mice improved neuronal numbers and axonal length (Fig. 4b, c). The number and axonal length of neurons in cortical layer V of the cortex and subiculum were also significantly increased in 13-month-old 5XFAD upon trametinib administration compared with vehicle-treated 5XFAD mice (Fig. 4e, f). This was an unexpected effect of trametinib because, at this late stage, neuronal loss in the cortex is believed to be too severe to allow restoration.
Consequently, we further examined whether trametinib administration recovers AD pathologies and improves cognitive functions in 5XFAD mice. In the fear conditioning test and novel object recognition test, vehicle-administered 5XFAD mice showed cognitive impairment, whereas administration of 0.1 mg/kg trametinib for 1.5 months to 9-month-old 5XFAD or for 1.5 months to 7-month-old 5XFAD mice improved cognitive functions as shown by an increase in the percentage of freezing or discrimination index measures (Fig. 4g, h) with no changes in locomotor functions (Supplementary Fig. 5a, b). These data indicate that trametinib recovers damaged neurons and cognitive function in 5XFAD AD model mice.
Trametinib induces neurogenic differentiation of AD patient iPSC derived-NSCs
Next, we asked if the neurogenic effect of trametinib observed in 5XFAD AD model mice has human relevance using NSCs derived from human iPSCs. When we treated AD patient iPSC-derived NSCs with trametinib, the level of pERKs was significantly decreased (Fig. 5a-c). The level of SOX2 did not change in the NSCs, but the level of an early post-mitotic neuronal marker Dcx was increased by trametinib (Figure. 5D and 5E). To assess the neurogenic effect of trametinib on those NSCs, cells were immunostained for Dcx 2 days after trametinib treatment. The number of Dcx + cells was increased by trametinib (Fig. 5f, g). The level of DCX mRNA also increased in trametinib-treated NSCs (Fig. 5h). These neurogenic effects of trametinib are also observed in healthy donor iPSC derived-NSCs (Supplementary Fig. 6). From these data, we suggest that trametinib has potential to induce neurogenic differentiation in AD patients.