Pregnenolone-methyl-ether enhances CLIP170 and microtubule functions improving spine maturation and hippocampal deficits related to CDKL5 deficiency

Abstract

Mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5) cause CDKL5 deficiency disorder (CDD), a neurodevelopmental disease characterized by severe infantile seizures and intellectual disability. The absence of CDKL5 in mice causes defective spine maturation that can at least partially explain the cognitive impairment in CDKL5 patients and CDD mouse models. The molecular basis for such defect may depend on the capacity of CDKL5 to regulate microtubule (MT) dynamics through its association with the MT-plus end tracking protein CLIP170. Indeed, we here demonstrate that the absence of CDKL5 causes CLIP170 to be mainly in a closed inactive conformation that impedes its binding to MTs. Previously, the synthetic pregnenolone analogue, pregnenolone-methyl-ether (PME), was found to have a positive effect on CDKL5-related cellular and neuronal defects in vitro. Here we show that PME induces the open active conformation of CLIP170 and promotes the entry of MTs into dendritic spines in vitro. Furthermore, the administration of PME to symptomatic Cdkl5-knock-out mice improved hippocampal-dependent behavior and restored spine maturation and the localization of MT-related proteins in the synaptic compartment. The positive effect on cognitive deficits persisted for one week after treatment withdrawal. Altogether, our results suggest that CDKL5 regulates spine maturation and cognitive processes through its control of CLIP170 and MT dynamics, which may represent a novel target for the development of disease modifying therapies.

Loss of CDKL5 Causes Synaptic GABAergic Defects That Can Be Restored with the Neuroactive Steroid Pregnenolone-Methyl-Ether

“…The presence of GABA A Rs on the neuronal membrane is determined by the dynamic balance of delivery, recycling and degradation, which altogether depends on complex regulatory mechanisms involving the interaction of various proteins, including gephyrin, with MTs [28,29]. We previously showed that various neuronal defects linked to CDKL5 deficiency in vitro and in vivo can be restored through treatment with PME, which promotes MT dynamics [13][14][15]. We therefore found it intriguing to analyse whether the treatment of Cdkl5-KO neurons with PME could restore the observed defects at the inhibitory synapse.…”

“…By treating symptomatic Cdkl5-KO mice with PME, we previously found that CDKL5related hippocampal-dependent behavioural and excitatory synaptic defects benefit from increased MT dynamics in vivo [15]. Intrigued by the positive effect of PME on inhibitory synapses in vitro, we proceeded to evaluate its effect in vivo, also.…”

“…As shown in Figure 5 we observed a dramatic decrease in the density of GABA A R γ 2 clusters in the dentate gyrus of the vehicle-treated Cdkl5-KO mice with respect to the WT mice; interestingly, upon treatment with PME, the number of GABA A R γ 2 clusters was similar between the two genotypes. By treating symptomatic Cdkl5-KO mice with PME, we previously found that CDKL5-related hippocampal-dependent behavioural and excitatory synaptic defects benefit from increased MT dynamics in vivo [15]. Intrigued by the positive effect of PME on inhibitory synapses in vitro, we proceeded to evaluate its effect in vivo, also.…”

“…Various recent data have shown that CDKL5 is involved in regulating MT dynamics [13,[39][40][41]. Interestingly, the possibility of targeting MT interacting proteins, the function of which is impaired in the absence of CDKL5, seems to represent an interesting disease modifying therapeutic strategy for CDD [14,15,42].…”

“…These defects depend in part on altered microtubule (MT) dynamics leading to the impaired invasion of MTs into dendritic spines and reduced spine maturation. Indeed, we previously demonstrated that CDKL5 regulates the MT-binding of the plus-end tracking protein CLIP170, thus impacting MT dynamics [13][14][15]. Importantly, the modulation of MT dynamics in vitro and in vivo, mediated by treatment with the pregnenolone analogue pregnenolone-methyl-ether (PME), which promotes CLIP170 functioning, can restore several CDKL5-related defects, including spine maturation, expression of synaptic proteins and hippocampal-dependent learning and memory [13][14][15].…”