Here, we characterize a conditional null mutant of the mouse Npc1 gene, and demonstrate that gene deletion restricted to Purkinje cells is sufficient to cause cell autonomous neuronal loss. Purkinje cell specific null mutants display impaired motor function, but not weight loss or early death, indicating that cerebellar degeneration accounts for limited aspects of the NPC phenotype in mice. Our data also establish that Purkinje cells in the anterior cerebellar lobules exhibit vulnerability to the toxicity of Npc1 deficiency, whereas those in the posterior lobules unexpectedly show remarkable resistance. Finally, we demonstrate that Npc1-deficient Purkinje cells in both susceptible and resistant lobules display normal electrophysiological activity prior to their degeneration, indicating that defects in ion homeostasis and energy metabolism do not underlie their loss. Our findings demonstrate that Npc1 deficiency leads to cell autonomous, selective neuronal vulnerability and suggest that the ataxic symptoms of NPC disease arise from Purkinje cell death rather than cellular dysfunction.
Studies of a diverse array of neurodegenerative disorders have yielded increasing evidence that neuronal dysfunction and death can arise from defects extrinsic to the neurons that are lost. Among the most compelling evidence in support of this conclusion comes from studies in animal models. For example, expression of polyglutamine-expanded ataxin-7, the cause of spinocerebellar ataxia type 7, only in Bergmann glia is sufficient to trigger Purkinje cell degeneration in mice (34
). Similarly, the deletion of mutant SOD1 from microglia (35
) or astrocytes (36
) slows disease progression in a mouse model of familial amyotrophic lateral sclerosis. Additionally, studies in a transgenic mouse model of Huntington disease indicate that pathological interactions between neurons are important for cortical pathology (37
). These observations and others have lead to a model in which neurodegeneration can be caused by cell autonomous mechanisms, defects in supporting glia, aberrant interactions between neurons or a combination of these (38
The data reported here establish that Purkinje cell degeneration in NPC mice is cell autonomous. Our findings support conclusions from the analysis of a chimeric mouse model of NPC disease (15
), and extend this work by showing the extent of selective vulnerability of Purkinje cell subpopulations. As Npc1
deletion mediated by the Pcp2-Cre
transgene occurs in post-developmental Purkinje cells (26
), we also conclude that this neuronal loss is independent of events during embryonic or early postnatal development. Prior studies raised the possibility that degeneration of Purkinje cells in NPC mice may arise from developmental defects, perhaps mediated by decreased production of neurosteroids to guide neuronal maturation (20
). Our findings do not support this conclusion. Whereas Npc1
is deleted weeks later in Npc1flox/–;Pcp2-Cre+
mice than in Npc1Δ/−
mice, the rate of Purkinje cell degeneration is similar in both sets of animals. Cell loss is therefore independent of the cumulative time following Npc1
deletion, and instead likely reflects a requirement for Npc1 only after Purkinje cells reach maturity.
It is notable that Npc1flox/–;Pcp2-Cre+
mice do not exhibit weight loss or early death. Although there has been some speculation that cerebellar ataxia impairs feeding ability of NPC mice, in turn causing weight loss and death, our data are inconsistent with this notion. Prior work established that weight loss and death are due to Npc1 deficiency in the nervous system (10
), yet the identity of the specific cellular population(s) responsible for these aspects of the phenotype remains enigmatic. It is possible that weight loss stems from dysfunction of feeding centers in the hypothalamus, and that early death results from degeneration of distinct brain regions required for support life, such as the brainstem (39
). Our data suggest that therapies targeted to Purkinje cells would be expected to rescue limited aspects of the neurological phenotype, particularly those reflecting ataxia. Weight loss and early death are likely mediated by impairment of other cell types, and further studies are needed to clarify their identity.
Our observation that Purkinje cells in posterior cerebellar lobules exhibit resistance to the toxicity of Npc1 deficiency prompted us to consider the possibility that electrophysiological dysfunction contributed to the differential survival of Purkinje cell subpopulations in NPC. To test this notion, we examined the spontaneous firing of Purkinje cells in acute cerebellar slices from 10-week Npc1flox/–;Pcp2-Cre+
mice. Surprisingly, Purkinje cells from both anterior and posterior cerebellar lobules exhibited normal spontaneous firing activity. These data indicate that electrophysiological defects do not underlie neuronal vulnerability, and that Purkinje cells can function despite the presence of filipin-positive lipid storage material. On the basis of these findings, we suggest that the cerebellar ataxia that develops in these mice is largely dependent upon Purkinje cell death rather than cellular dysfunction. Consistent with this interpretation, Npc1flox/–;Pcp2-Cre+
mice develop symptoms only after the loss of a substantial proportion of their Purkinje cells. This is in marked contrast with other cerebellar disorders, including many of the spinocerebellar ataxias, episodic ataxias and paraneoplastic ataxia, wherein symptoms become evident prior to, or even in the absence of, frank Purkinje cell loss (40
). This observation raises the possibility that therapies directed at preventing neuron death (41
) may be as valuable as those aimed at relieving the primary lipid trafficking defect (21
) for treating aspects of the neurological symptoms in NPC disease.