Altered glutamate receptor function in the cerebellum of the Ppt1^−/− mouse, a murine model of infantile neuronal ceroid lipofuscinosis

Abstract

The neuronal ceroid lipofuscinoses (NCLs) are a family of devastating pediatric neurodegenerative disorders and currently represent the most common form of pediatric-onset neurodegeneration. Infantile NCL (INCL), the most aggressive of these disorders, is caused by mutations in the CLN1 gene that encodes the enzyme palmitoyl protein thioesterase 1 (PPT1). Previous studies have suggested that glutamatergic neurotransmission may be disrupted in INCL, and therefore, the present study investigates glutamate receptor function in the Ppt1−/− mouse model of INCL by comparing the sensitivity of cultured WT and Ppt1−/− cerebellar granule cells to glutamate receptor-mediated toxicity. Ppt1−/− neurons were significantly less sensitive to AMPA receptor-mediated toxicity but markedly more vulnerable to NMDA receptor-mediated cell death. Since glutamate receptor function is primarily regulated by the surface expression level of the receptor, the surface level of AMPA and NMDA receptor subunits in the cerebella of WT and Ppt1−/− mice was also examined. Western blotting of surface cross-linked cerebellar samples showed a significantly lower surface level of the GluR4 AMPA receptor subunit in Ppt1−/− mice, providing a plausible explanation for the decreased vulnerability of Ppt1−/− cerebellar neurons to AMPA receptor-mediated cell death. The surface expression of the NR1, NR2A and NR2B NMDA receptor subunits was similar in the cerebella of WT and Ppt1−/− mice, indicating that there is another mechanism behind the increased sensitivity of Ppt1−/− cerebellar granule cells to NMDA toxicity. Our results indicate an AMPA receptor hypo- and NMDA receptor hyperfunction phenotype in Ppt1−/− neurons and provide new therapeutic targets for INCL.

Decreased sensitivity of palmitoyl protein thioesterase 1-deficient neurons to chemical anoxia

“…The MTT assay reliably measures neuronal viability in primary neuronal cultures as we have shown in several previous studies (12, 15-19). The main advantage of the MTT assay is that cell death is quantified independently of the mode of cell death (apoptosis, necrosis, atypical modes of cell death).…”

“…Primary cultures of cerebellar granule neurons were prepared from seven-day-old WT and Ppt1 −/− mouse pups as previously described (12, 14, 15). Briefly, meninges were removed from cerebella and the tissue was minced with a tissue chopper (McIlwain Tissue Chopper, Brinkmann).…”

“…The results of the MTT viability assay correlate well with what was seen in the preliminary visual inspection. The assay was performed as previously described (12, 14, 15). Briefly, at the end of the 24-h treatments, culture medium was removed and replaced with a 0.3 mg/ml solution of MTT dissolved in Neurobasal medium containing 25 mM KCl, 0.5 mM glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin.…”

“…Although, PPT1 is ubiquitously expressed (5) and active in diverse cell types (6, 7), PPT1 deficiency causes selective neurodegeneration. Despite clues on the neuron-specific functions of PPT1 (8-12) it is still unclear why neurons are selectively vulnerable to the loss of PPT1 enzyme activity.…”

Treatment of the Ppt1–/– Mouse Model of Infantile Neuronal Ceroid Lipofuscinosis With the N-methyl-d-aspartate (NMDA) Receptor Antagonist Memantine

“…Our previous results demonstrating an increase in NMDA receptor activity 25 coupled with the extensive upregulation of microglial activation in Ppt1 −/− brains reported by others 8–9 suggest that memantine, a compound originally described as a NMDA receptor antagonist 27 and more recently shown to decrease pathological microglial activation, 28 has great potential as a treatment for infantile neuronal ceroid lipofuscinosis. As Ppt1 −/− mice exhibit significant neural pathology before the onset of an observable motor coordination deficit 9 and changes on the molecular level occur even earlier, 10 we first treated mice at time points significantly before the age at which we observed a measureable rotarod phenotype.…”