Temporary inhibition of AMPA receptors induces a prolonged improvement of motor performance in a mouse model of juvenile Batten disease

Abstract

Mutations in the CLN3 gene cause juvenile Batten disease, a fatal pediatric neurodegenerative disorder. The Cln3-knockout (Cln3(Δex1-6)) mouse model of the disease displays many pathological characteristics of the human disorder including a deficit in motor coordination. We have previously found that attenuation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-type glutamate receptor activity in one-month-old Cln3(Δex1-6) mice resulted in an immediate improvement of their motor skills. Here we show that at a later stage of the disease, in 6-7-month-old Cln3(Δex1-6) mice, acute inhibition of AMPA receptors by a single intraperitoneal injection (1mg/kg) of the non-competitive AMPA antagonist, EGIS-8332, does not have an immediate effect. Instead, it induces a delayed but prolonged improvement of motor skills. Four days after the injection of the AMPA antagonist, Cln3(Δex1-6) mice reached the same motor skill level as their wild type (WT) counterparts, an improvement that persisted for an additional four days. EGIS-8332 was rapidly eliminated from the brain as measured by HPLC-MS/MS. Histological analysis performed 8 days after the drug administration revealed that EGIS-8332 did not have any impact upon glial activation or the survival of vulnerable neuron populations in 7-month-old Cln3(Δex1-6) mice. We propose that temporary inhibition of AMPA receptors can induce a prolonged correction of the pre-existing abnormal glutamatergic neurotransmission in vivo for juvenile Batten disease.

Fig. 1. A single intraperitoneal injection of the non-competitive AMPA receptor antagonist, EGIS-8332, induces a delayed but prolonged improvement of motor skills in 6-7-month-old Cln3^Δex1-6 mice

An accelerating rotarod (from 0 to 24 rpm in 240 s) was used to measure the motor skills of 6-7-month-old Cln3^Δex1-6 and wild type (WT) mice. Mice were intraperitoneally injected with either the non-competitive AMPA antagonist, EGIS-8332 (1 mg/kg) or the vehicle of the drug (Control). Data points represent mean ± S.E.M. of the time (s) mice were able to stay on the rotating rod. Repeated measures two-way ANOVA was applied to compare Control and EGIS-8332-treated Cln3^Δex1-6 mice (* p < 0.05, ** p < 0.01 and *** p < 0.001) as well as Control WT and Cln3^Δex1-6 mice. Repeated measures one-way ANOVA was used to compare the motor performances between two consecutive time points (# p < 0.05, ## p < 0.01). (A) EGIS-8332 induces a delayed but prolonged improvement of motor skills in 6-7-month-old Cln3^Δex1-6 mice (n=15). *: Control vs. EGIS-8332; #: comparison between two consecutive time points. (B) EGIS-8332 does not affect the motor skills of 6-7-month-old WT mice (n=12–13). #: comparison between two consecutive time points. (C) Control, vehicle-injected Cln3^Δex1-6 mice (n=15), differently from WT mice (n=13), had only a limited ability to improve their motor skills during the repeated rotarod test trials. *: WT vs. Cln3^Δex1-6; #: comparison between two consecutive time points. (D) Four days after the injection of EGIS-8332, Cln3^Δex1-6 mice reached the the same motor skill level as their WT counterparts and stayed at the WT skill level even four days later (*: WT vs. Cln3^Δex1-6).

Fig. 2. A single intraperitoneal injection of the non-competitive AMPA receptor antagonist, EGIS-8332, has no affect on the previously described neuropathological changes in 7-month-old Cln3^Δex1-6 mice

Four 7-month-old wild type (WT) mice were perfusion-fixed and their brains were histologically analyzed. Four vehicle-injected and four EGIS-8332-treated 7-month-old Cln3^Δex1-6 mice were perfusion-fixed eight days after the treatment, and their brains were also histologically analyzed. Columns and bars represent mean ± S.E.M. (A) EGIS-8332 has no effect on the selective neuron loss in the thalamus and cerebellum. Unbiased stereological estimates of the number of large projection neurons in the dorsal lateral geniculate nucleus of the thalamus and in the medial deep cerebellar nucleus. (B) EGIS-8332 does not affect astrocytic activation in the cortex. Quantitative determination of astrocytosis in the somatosensory barrelfield cortex and the primary visual cortex was performed by thresholding image analysis following immunohistochemical staining for the astrocytic marker, GFAP. (C) EGIS-8332 does not affect microgliosis in the cortex. Quantitative determination of microgliosis in the somatosensory barrelfield cortex was performed by thresholding image analysis following immunohistochemical staining for the microglial marker F4/80. Statistical analysis (one-way ANOVA with Bonferroni’s post-test) revealed that EGIS-8332 had no effect on the selective neuron loss, astrocytosis and microglial activation. Statistical significances between WT and Cln3^Δex1-6 mice are indicated in the graphs.