The Lurcher mutation transforms the GRID2 receptor into a constitutively opened channel. In Lurcher heterozygous mice, cerebellar Purkinje cells are permanently depolarized, a characteristic that has been thought to be the primary cause of their death, which occurs from the second postnatal week onward. The more dramatic phenotype of Lurcher homozygotes is thought to be due to a simple gene dosage effect of the mutant allele. We have analyzed the phenotype of Lurcher/hotfoot heteroallelic mutants bearing only one copy of the Lurcher allele and no wild-type Grid2. Our results show that the absence of wild-type GRID2 receptors in these heteroallelic mutants induces an early and massive Purkinje cell death that is correlated with early signs of autophagy. This neuronal death is independent of depolarization and can be explained by the direct activation of autophagy by Lurcher GRID2 receptors through the recently discovered signaling pathway formed by GRID2, n-PIST, and Beclin1.

Research efforts to deduce the function of the prion protein (PrPc) in knock-out mouse mutants have revealed that large deletions in the PrPc genome result in the ectopic neuronal expression of the prion-like protein Doppel (Dpl). In our analysis of one such line of mutant mice, Ngsk Prnp0/0 (NP0/0), we demonstrate that the ectopic expression of Dpl in brain neurons induces significant levels of cerebellar Purkinje cell (PC) death as early as six months after birth. To investigate the involvement of the mitochondrial proapoptotic factor BAX in the Dpl-induced apoptosis of PCs, we have analyzed the progression of PC death in aging NP0/0:Bax-/- double knockout mutants. Quantitative analysis of cell numbers showed that significantly more PCs survived in NP0/0:Bax-/- double mutants than in the NP0/0:Bax+/+ mutants. However, PC numbers were not restored to wildtype levels or to the increased number of PCs observed in Bax-/- mutants. The partial rescue of NP0/0 PCs suggests that the ectopic expression of Dpl induces both BAX-dependent and BAX-independent pathways of cell death. The activation of glial cells that is shown to be associated topographically with Dpl-induced PC death in the NP0/0:Bax+/+ mutants is abolished by the loss of Bax expression in the double mutant mice, suggesting that chronic inflammation is an indirect consequence of Dpl-induced PC death.

Abnormalities in the formation and function of cerebellar circuitry potentially contribute to cognitive deficits in humans. In the adult, the activity of the sole output neurons of the cerebellar cortex - the Purkinje cells (PCs) - is shaped by the balance of activity between local excitatory and inhibitory circuits. However, how this balance is established during development remains poorly understood. Here, we investigate the role of interleukin-1 receptor accessory protein-like 1 (IL1RAPL1), a protein linked to cognitive function which interacts with neuronal calcium sensor 1 (NCS-1) in the development of mouse cerebellum. Using Il1rapl1-deficient mice, we found that absence of IL1RAPL1 causes a transient disinhibition of deep cerebellar nuclei neurons between postnatal days 10 and 14 (P10/P14). Upstream, in the cerebellar cortex, we found developmental perturbations in the activity level of molecular layer interneurons (MLIs), resulting in the premature appearance of giant GABAA-mediated inhibitory post-synaptic currents capable of silencing PCs. Examination of feed-forward recruitment of MLIs by parallel fibres shows that during this P10/P14 time window, MLIs were more responsive to incoming excitatory drive. Thus, we conclude that IL1RAPL1 exerts a key function during cerebellar development in establishing local excitation/inhibition balance.

The pro-apoptotic factor BAX has recently been shown to contribute to Purkinje cell (PC) apoptosis induced by the neurotoxic prion-like protein Doppel (Dpl) in the prion-protein-deficient Ngsk Prnp(0/0) (NP(0/0)) mouse. In view of cellular prion protein (PrP(c)) ability to counteract Dpl neurotoxicity and favor neuronal survival like BCL-2, we investigated the effects of the anti-apoptotic factor BCL-2 on Dpl neurotoxicity by studying the progression of PC death in aging NP(0/0)-Hu-bcl-2 double mutant mice overexpressing human BCL-2 (Hu-bcl-2). Quantitative analysis showed that significantly more PCs survived in NP(0/0)-Hu-bcl-2 double mutants compared with the NP(0/0) mutants. However, number of PCs remained inferior to wild-type levels and to the increased number of PCs observed in Hu-bcl-2 mutants. In the NP(0/0) mutants, Dpl-induced PC death occurred preferentially in the aldolase C-negative parasagittal compartments of the cerebellar cortex. Activation of glial cells exclusively in these compartments, which was abolished by the expression of Hu-bcl-2 in the double mutants, suggested that chronic inflammation is an indirect consequence of Dpl-induced PC death. This partial rescue of NP(0/0) PCs by Hu-bcl-2 expression was similar to that observed in NP(0/0):Bax(-/-) double mutants with bax deletion. Taken together, these data strongly support the involvement of BCL-2 family-dependent apoptotic pathways in Dpl neurotoxicity. The capacity of BCL-2 to compensate PrP(c) deficiency by rescuing PCs from Dpl-induced death suggests that the BCL-2-like property of PrP(c) may impair Dpl-like neurotoxic pathways in wild-type neurons.

In Ngsk prion protein (PrP)-deficient mice (NP(0/0)), ectopic expression of PrP-like protein Doppel (Dpl) in central neurons induces significant Purkinje cell (PC) death resulting in late-onset ataxia. NP(0/0) PC death is partly prevented by either knocking-out the apoptotic factor BAX or overexpressing the anti-apoptotic factor BCL-2 suggesting that apoptosis is involved in Dpl-induced death. In this study, Western blotting and immunohistofluorescence show that both before and during significant PC loss, the scrapie-responsive gene 1 (Scrg1)--potentially associated with autophagy--and the autophagic markers LC3B and p62 increased in the NP(0/0) PCs whereas RT-PCR shows stable mRNA expression, suggesting that the degradation of autophagic products is impaired in NP(0/0) PCs. At the ultrastructural level, autophagic-like profiles accumulated in somatodendritic and axonal compartments of NP(0/0), but not wild-type PCs. The most robust autophagy was observed in NP(0/0) PC axon compartments in the deep cerebellar nuclei suggesting that it is initiated in these axons. Our previous and present data indicate that Dpl triggers autophagy and apoptosis in NP(0/0) PCs. As observed in amyloid neurodegenerative diseases, upregulation of autophagic markers as well as extensive accumulation of autophagosomes in NP(0/0) PCs are likely to reflect a progressive dysfunction of autophagy that could trigger apoptotic cascades.