We have shown that neuron-targeted gene rescue in a mouse model of NPC disease is sufficient to correct CNS cholesterol accumulation, prevent neurodegeneration, reduce glial activity, and significantly improve the health of the animal. Thus, we conclude, loss of NPC1 function in neurons is predominantly responsible for the CNS pathogenesis in mice. Therapies, including enzyme replacement to correct secondary enzyme defects (Devlin et al., 2010
) and future genetic interventions that target neurons in a NPC1 disease patient may prove optimal for treatment.
The neuron-mediated progression of neurodegenerative disease is surprising considering that defects in glia-to-neuron interaction may be involved (Rossi and Volterra, 2009
). Previous studies on NPC disease have documented glial dysfunction such as defective steroidogenesis (Chen et al., 2007
) and abnormal lipid trafficking (Lloyd-Evans et al., 2008
) that can negatively affect neurons. Also, it has been reported that an astrocyte-targetted GFAP promoter driven Npc1
can triple the lifespan of Npc1−/−
mice (Zhang et al., 2008
). However, since transgenes are subject to variegating and non-target expression (Bao-Cutrona and Moral, 2009
; Martin and Whitelaw, 1996
), neurons in these mice could have produced some NPC1, especially since neuronal cholesterol was reported as reduced; moreover, GFAPNpc1
was not tagged to allow for more precise cellular detection of NPC1 within tissue. Thus, it may not have been astrocyte rescue alone that ameliorated the disease.
To test whether neuron rescue is sufficient to correct the disease, we generated various neuron-specific rescue lines using the Tet system. This system was chosen over other gene induction systems because of the wide commercial and academic availability of driver transgenes for the targeted expression of a single reporter transgene. We chose drivers whose cell type specificity and expression profiles have been previously reported (). Although we cannot be absolutely certain that NPC1-YFP was absent from all other cell types except neurons, the use of multiple mouse lines with different drivers to induce varied NPC1-YFP expression patterns in the brain allowed the demonstration of full neuron autonomy. In an Npc1−/− mouse, NPC1-YFP produced in a specific neuron population corrected the cholesterol accumulation phenotype only within those neurons () and the production of NPC1-YFP in neighboring astrocytes did not change neuronal cholesterol accumulation (). Along with the reduction of cholesterol in the specific brain areas (), the anatomical location of neuron rescue in Npc1−/− mice could be identified by local reduction of inflammation (). In addition, the lack of improvement in weight and lifespan of the R; N; Npc1−/− mice (), which produced NPC1-YFP in virtually all tissues except the brain (), allowed us to exclude potential confounding effects of non-nervous system rescue.
Our study does not exclude the possibility that glial NPC1 is required for the overall health of neurons, but it appears that the loss of NPC1 does not significantly affect glial function that is critical for neuron survival. Prior work has shown that the secretion of sterols was not inhibited in Npc1−/−
astrocytes. Lipoproteins generated by Npc1−/−
glia were capable of supporting axon elongation in vitro
(Karten et al., 2005
; Mutka et al., 2004
). Prior work has also demonstrated the cell-autonomous death of PNs by utilizing chimeric mice, mice comprised of a mixture of wild-type and Npc1−/−
cells (Ko et al., 2005
), and conditional knockout mice, wild-type mice with Npc1 gene deletion in PNs (Elrick et al., 2009
). However, these studies did not address whether PNs can survive alone despite the loss of NPC1 from all other neurons, glia cells, or other cells of the body. Here we show in vivo
that despite glial NPC1 deficiency, cerebellar PNs, which are sensitive to many genetic and acquired disorders as well as toxic environmental factors (Sarna and Hawkes, 2003
), survived as long as they produced NPC1-YFP ().
The targeted rescue of PNs in an Npc1−/−
mouse allowed us to observe the benefit cerebellar improvements alone could have on NPC disease. PNs are the sole neuronal output of the cerebellar cortex and loss of PNs in an otherwise normal brain has long been known to cause motor abnormalities. Current research has begun to suggest that the cerebellum can regulate non-motor brain functions as well (Strick et al., 2009
), including the early development of the whole brain. Thus, rescuing cerebellar function in a disease that also affects the cerebellum could have broad and significantly beneficial therapeutic outcomes. The benefit PN rescue alone had on weight gain, nest building activity, motor ability and lifespan in P; N; Npc1−/−
mice supports this view and points to an important cerebellar involvement in the severity of NPC disease progression.
Despite significant benefits, cerebellar PN survival ultimately did not halt disease progression or prevent premature death of P; N; Npc1−/− mice. Improved motor coordination in these mice was temporary as ataxia seemed to eventually increase with age () and weight gain was unsustainable (). E; N; Npc1−/− mice also showed a similar worsening of condition with age but these mice exhibited an extraordinary increase in lifespan marked by a more delayed and gradual weight loss (). The broad neuronal rescue in E; N; Npc1−/−mice would suggest that P; N; Npc1−/− mice eventually succumb to the effect of neurological deterioration of other brain regions.
With the given data, we conclude that rescue of thalamic pathways is essential for the prolonged survival of Npc1−/− mice. This deduction is based, in part, on the persistent inflammation in the thalamus of P; N; Npc1−/− and C; N; Npc1−/− mice but not in E; N; Npc1−/− mice, which can survive several weeks longer than either P; N; Npc1−/− or C; N; Npc1−/− mice (, ). Therefore, rescue of neurons that are present or synapse in the thalamic region correlated with the most pronounced effect on lifespan.
Caution is necessary in attributing all the survival effect to thalamic rescue. Considering the complexity and interconnectivity of the nervous system, it is likely that other brain regions rescued in E; N; Npc1−/−
mice may have contributed to improved lifespan. Nevertheless, it is reasonable to conclude that thalamic rescue would be beneficial. Thalamic atrophy, which occurs in many common neurodegenerative diseases including Parkinson’s and multiple sclerosis (Halliday, 2009
; Rocca et al., 2010
), can affect motor functions, consciousness, arousal, and sleep. In NPC disease mice, it is known that the sensory thalamus is extremely vulnerable (Yamada et al., 2001
) with degeneration of thalamic neurons starting early in life and possibly preceding a dying-back type degeneration of afferent neurons in the brainstem or elsewhere (Ohara et al., 2004
). It would be interesting to determine if thalamus-specific neuron rescue would be sufficient to extend animal lifespan and to document the behavioral consequence of correcting only the thalamus.
With the use of the tetO-Npc1-YFP
transgenic mouse strain generated for this study, future studies can take advantage of gene delivery of a tTA transgene, or other existing mouse driver lines, to target the thalamus or other CNS areas affected in Npc1−/−
mice, such as the brainstem (Luan et al., 2008
). The targeting of increasingly discrete neuronal networks and the development of assays to measure specific behaviors will be useful in order to associate rescue of a particular neural circuit with improvements to health-related quality of life, prolonged lifespan, and inhibition of neurological signs. For example, dystonia, which can be caused by damage to multiple brain regions including the basal ganglia, thalamus, brainstem, and cerebellum (Breakefield et al., 2008
) was not greatly suppressed in P; N; Npc1−/−
mice, despite improvements in cerebellar pathology (), or in C; N; Npc1−/−
mice, even though NPC1-YFP was produced in major areas of the forebrain and other brain regions (). Thus, it remains unclear which neuronal networks are necessary to reduce dystonia. More precise region-specific control over neuron rescue and more quantitative criteria for measuring dystonia may begin to address this issue.
Our work may be relevant to other lysosomal storage disorders that cause neurological disease. Recent genetic studies on a mouse model of Gaucher disease determined that microglia are not the primary determinant of CNS pathology (Enquist et al., 2007
). For NPC disease, we have observed that the inflammatory process is selective and responds to local neuronal dysfunction and degeneration without inevitably harming healthy or functional neurons. This is exemplified in the cerebellum, where the presence of reactive glia in the molecular layer occurs almost exclusively in areas of PN loss, despite accumulation of reactive microglia and astrocytes in the granule layer (). Although, correction of NPC1 loss in astrocytes did curtail glial reactivity, increased inflammation and neuron loss was unavoidable unless neuronal NPC1 loss was corrected. In other lysosomal storage diseases and, possibly, more common neurological disorders with lysosomal system dysfunction such as Alzheimer’s and Parkinson’s (Dehay et al., 2010
; Nixon et al., 2001
), defects within neurons themselves may be the central cause of neurological decay. Restoring or enhancing the right cellular function in neurons may prove to be the best neuroprotective strategy.