Altered sensitivity of cerebellar granule cells to glutamate receptor overactivation in the Cln3(Δex7/8)-knock-in mouse model of juvenile neuronal ceroid lipofuscinosis

Abstract

The juvenile onset form of neuronal ceroid lipofuscinoses (JNCL) is a recessively inherited lysosomal storage disorder characterized by progressive neurodegeneration. JNCL results from mutations in the CLN3 gene that encodes a lysosomal membrane protein with unknown function. Utilizing a Cln3-knock-out mouse model of JNCL that was created on the 129S6/SvEv genetic background, we have previously demonstrated that CLN3-deficient cerebellar granule cells (CGCs) have a selectively increased sensitivity to AMPA-type glutamate receptor-mediated toxicity. Our recent findings that CGCs from 129S6/SvEv and C57BL/6J wild type (WT) mice have significant differences in glutamate receptor expression and in excitotoxic vulnerability indicated that the genetic background possibly have a strong influence on how glutamate receptor function is dysregulated in CLN3-deficient neurons. Indeed, here we show that in the Cln3(Δex7/8)-knock-in mouse model, that is on the C57BL/6J genetic background, mimics the most frequent mutation observed in JNCL patients and considered a null mutant, the sensitivity of CGCs to both AMPA- and NMDA-type glutamate receptor overactivations is altered. Cultured wild type and Cln3(Δex7/8) CGCs were equally sensitive to AMPA toxicity after 2 or 3 weeks in vitro, whereas the subunit-selective AMPA receptor agonist, CPW-399, induced significantly more cell death in mature, 3-week-old Cln3(Δex7/8) cultures. NMDA receptor-mediated toxicity changed during in vitro development: Cln3(Δex7/8) CGCs were less sensitive to high concentration of NMDA after 2 weeks in culture but became more vulnerable than their WT counterparts after 3 weeks in vitro. Abnormally altered glutamate receptor function in the cerebellum may result in motor deficits, and we confirmed that 7-week-old Cln3(Δex7/8) mice, similarly to Cln3-knock-out mice, have a motor coordination deficit as measured by an accelerating rotarod. Our results demonstrate altered glutamate receptor function in Cln3(Δex7/8) neurons and suggest that both AMPA and NMDA receptors are potential therapeutic targets in JNCL.

Fig. 1. Cln3 ^Δex7-8 and WT cerebellar granule cells have similar sensitivity to AMPA-induced cell death

AMPA-induced cell death was investigated in primary cultures of CGCs prepared from seven-day-old WT (solid bars) and Cln3 ^Δex7-8 (open bars) mice. Cultures were maintained in Neurobasal medium with B27 serum replacement. On the 14^th (A) or 21^st (B) day in vitro (DIV14 or DIV21), cultures were exposed to the indicated concentrations of AMPA for two hours in Neurobasal medium without B27. Cyclothiazide (CTZ) was added at a concentration of 100 μM to prevent desensitization of AMPA receptors. Treatments were done in the presence of 50 μM MK-801 to prevent transactivation of NMDA receptors. Upon completion of the two-hour treatment, medium was replaced with complete, B27-containing Neurobasal medium. Cell viability was determined using the MTT viability assay twenty-four hours later. Results presented here represent mean ± S.E.M. of four separate culture preparations (n = 9–22). No statistically significant differences between WT and Cln3 ^Δex7-8 cultures were found by two-way ANOVA with Bonferroni’s post-test for multiple comparisons.

Fig. 2. Cln3 ^Δex7-8 cerebellar granule cells are significantly more sensitive to the subtype-selective AMPA receptor agonist, CPW-399, in mature, three-week-old cultures

CPW-399-induced cell death was investigated in primary cultures of CGC prepared from seven-day-old WT (solid bars) and Cln3 ^Δex7-8 (open bars) mice. Cultures were maintained in Neurobasal medium with B27 serum replacement. On the 14^th (A) or 21^st (B) day in vitro (DIV14 or DIV21), cultures were exposed to the indicated concentrations of CPW-399 for two hours in Neurobasal medium without B27. CPW-399 is a non-desensitizing, subtype-selective AMPA receptor agonist. Treatments were done in the presence of 50 μM MK-801 to prevent transactivation of NMDA receptors. Upon completion of the two-hour treatment, medium was replaced with complete, B27-containing Neurobasal medium. Cell viability was determined using the MTT viability assay twenty-four hours later. Results presented here represent mean ± S.E.M. of four separate culture preparations (n = 8–22). Statistical significance was determined by two-way ANOVA with Bonferroni’s post-test for multiple comparisons: * p<0.05, *** p<0.001.

Fig. 3. Relative sensitivity of Cln3 ^Δex7-8 cerebellar granule cells to NMDA receptor-mediated toxicity changes during in vitro development

NMDA receptor-mediated cell death was investigated in primary cultures of CGC prepared from seven-day-old WT (solid bars) and Cln3 ^Δex7-8 (open bars) mice. Cultures were maintained in Neurobasal medium with B27 serum replacement. On the 14^th (A) or 21^st (B) day in vitro (DIV14 or DIV21), cultures were exposed to the indicated concentrations of NMDA for two hours in Neurobasal medium without B27. Upon completion of the two-hour treatment, medium was replaced with complete, B27-containing Neurobasal medium. Cell viability was determined using the MTT viability assay twenty-four hours later. Results presented here represent mean ± S.E.M. of four separate culture preparations (n = 11–19). Statistical significance was determined by two-way ANOVA with Bonferroni’s post-test for multiple comparisons: ** p<0.01, *** p<0.001.

Fig. 4. Surface/Total ratio of the GluR1 AMPA receptor subunit is significantly higher in the cerebellum of one-month-old Cln3 ^Δex7-8 mice

Surface and intracellular expression levels of the GluR1, GluR2, and GluR4 AMPA receptor subunits were investigated using acutely isolated slices from the cerebella of one-month-old WT (solid bars) and Cln3 ^Δex7-8 (open bars) mice. Slices were subjected to surface cross-linking and then analyzed by Western blot for GluR1, GluR2, and GluR4. A: Western blots showing separation of surface (cross-linked) and intracellular receptor subunits. B-D: Quantification of surface, intracellular, and total (surface + intracellular) subunit expression levels. E-G: Comparison of relative surface/total subunit expression ratios. For graphs B-G, Subunit expression levels were normalized to total protein as determined by Ponceau S staining. All measurements for Cln3 ^Δex7-8 samples are expressed as a percentage of WT. Four mice were analyzed for each genotype; histogram bars represent mean ± S.E.M. Statistical significance was determined by two-way ANOVA with Bonferroni’s post-test for multiple comparisons (B-D) or unpaired t-test (E-G), * p<0.05.

Fig. 5. Expression levels of the NR1, NR2A and NR2B NMDA receptor subunits in the cerebellum of one-month-old Cln3 ^Δex7-8 and WT mice

Surface and intracellular expression levels of the NR1, NR2A, and NR2B NMDA receptor subunits were investigated using acutely isolated slices from the cerebella of one-month-old WT (solid bars) and Cln3 ^Δex7-8 (open bars) mice. Slices were subjected to surface cross-linking and then analyzed by Western blot for NR1, NR2A, and NR2B. A: Western blots showing separation of surface (cross-linked) and intracellular receptor subunits. B-D: Quantification of surface, intracellular, and total (surface + intracellular) subunit expression levels. E-G: Comparison of relative surface/total subunit expression ratios. For graphs B-G, Subunit expression levels were normalized to total protein as determined by Ponceau S staining. All measurements for Cln3 ^Δex7-8 samples are expressed as a percentage of WT. Four mice were analyzed for each genotype; histogram bars represent mean ± S.E.M. Statistical significance was determined by two-way ANOVA with Bonferroni’s post-test for multiple comparisons (B-D) or unpaired t-test (E-G).

Fig. 6. Cln3 ^Δex7/8 mice have a motor coordination deficit

An accelerating rotarod (from 0 to 37 rpm in 120 s) was used to measure the motor skills of 7-week-old WT and Cln3 ^Δex7-8 male mice. Data are plotted as the time at which animals fell from the rotarod. Cln3 ^Δex7/8 mice had a reduced ability to remain on the rod as it accelerated. Columns and bars represent mean ± S.E.M. (n = 12). The data sets passed the normality test (α level 0.05), and therefore, statistical significance was determined by unpaired t-test.