• acid sphingomyelinase;
  • glia;
  • neurons;
  • Niemann Pick disease type A;
  • sphingolipids;
  • storage diseases


  1. Top of page
  2. Abstract
  3. Lipid alterations in ASMko mouse brains
  4. Consequences of ASM deficiency in neurons
  5. Consequences of ASM deficiency in glial cells
  6. Towards a therapy
  7. Concluding remarks
  8. Acknowledgements
  9. References

J. Neurochem. (2011) 116, 779–788.


Severe neurological involvement characterizes Niemann Pick disease (NPD) type A, an inherited disorder caused by loss of function mutations in the gene encoding acid sphingomyelinase (ASM). Mice lacking ASM, which mimic NPD type A, have provided important insights into the aberrant brain phenotypes induced by ASM deficiency. For example, lipid alterations, including the accumulation of sphingolipids, affect the membranes of different subcellular compartments of neurons and glial cells, leading to anomalies in signalling pathways, neuronal polarization, calcium homeostasis, synaptic plasticity, myelin production or immune response. These findings contribute to our understanding of the overall role of sphingolipids and their metabolic enzymes in brain physiology, and pave the way to design and test new therapeutic strategies for type A NPD and other neurodegenerative disorders. Some of these have already been tested in mice lacking ASM with promising results.

Abbreviations used:

acid sphingomyelinase


mice lacking ASM activity


detergent resistant membrane




neural progenitor cell


Niemann Pick disease



Mutations in the SMPD1 gene encoding acid sphingomyelinase (ASM) cause Niemann Pick diseases (NPD) types A and B (Brady et al. 1966). Both forms of the disorder are characterized by progressive visceral organ abnormalities, including hepatosplenomegaly, pulmonary insufficiency and cardiovascular disease (Schuchman and Desnick 2001). However, while NPD type B is a later-onset form in which patients exhibit little or no neurological involvement, NPD type A is the infantile form of ASM deficiency characterized by a rapidly progressive neurodegenerative course that leads to death in early childhood. The different clinical presentations of types A and B NPD are likely because of small differences in the amount of residual ASM activity. For example, while an effective in situ residual ASM activity of ∼ 5% results in NPD type B, a further reduction to ∼ 1–2% or less induces the severe type A phenotype (Graber et al. 1994). These observations highlight the fact that although low levels of ASM activity are sufficient to maintain intact neurological function, the absence of this activity has devastating consequences in the brain.

Acid sphingomyelinase is a lysosomal enzyme that converts sphingomyelin (SM) into ceramide and phosphorylcholine (Gatt 1963; Fowler 1969). Therefore, SM accumulation in lysosomes characterizes NPD patient cells, and both type A and B are classified as lysosomal storage disorders. It is assumed that lysosomal storage develops only when the residual activity of the lysosomal enzyme falls below a critical threshold and the substrate degradation rate is lower than the rate of influx (Conzelmann and Sandhoff 1983). SM influx in neural cells is lower than in liver, spleen or lymph nodes, and therefore it has been proposed that the low levels of residual activity in NPD type B would be sufficient to avoid lysosomal accumulation in neurons (Graber et al. 1994), thus precluding neurological involvement.

Unfortunately, despite these correlations of residual enzymatic activity with phenotype, in vitro ASM activity assays are not suitable to reliably predict the onset and extent of brain involvement in NPD patients. Moreover, even if the intralysosomal accumulation of unmetabolized substrates has been considered the primary cause of NPD, the molecular mechanisms leading from this event to the pathology are still obscure. Very likely the primary enzymatic defect results in multiple secondary biochemical and cellular abnormalities that could indeed be major contributing factors, or even the main cause, of tissue damage and death. Sphingolipids, including SM, exert many of their complex biological functions at the plasma membrane by modulating the lateral organization and biophysical properties of the membrane and by affecting the function of membrane-associated proteins or signaling complexes (Lingwood and Simons 2010). Thus, the lysosomal deficiency of ASM and resultant defects in lysosomal catabolism might directly lead to altered plasma membrane composition and function. On the other hand, the presence of an extralysosomal pool of ASM at the cell surface (Grassméet al. 2001; Gulbins 2003) suggests that irrespective of the lysosomal defect, plasma membrane alterations might arise when the enzyme is deficient. Data showing the ability of ASM to degrade SM within low density lipoprotein particles at physiologic pH (Schissel et al. 1998) and the possibility that acidified microenvironments may exist at the cell surface (Bourguignon et al. 2004; Steinert et al. 2008), support the notion that ASM deficiency at the plasma membrane may contribute directly to NPD pathology.

Irrespective of these hypotheses, use of mice lacking ASM activity (ASMko), which mimic NPD type A disease (Horinouchi et al. 1995; Otterbach and Stoffel 1995), has led to the discovery of a number of anomalies in brain tissue and cells that could explain the severe mental retardation and neurodegeneration of NPD type A patients. It is the aim of this review to present and discuss these findings.

Lipid alterations in ASMko mouse brains

  1. Top of page
  2. Abstract
  3. Lipid alterations in ASMko mouse brains
  4. Consequences of ASM deficiency in neurons
  5. Consequences of ASM deficiency in glial cells
  6. Towards a therapy
  7. Concluding remarks
  8. Acknowledgements
  9. References

Sphingolipid metabolism is a complex network of interdependent events, and is tightly connected with the intracellular traffic of these lipids. Moreover, recycling of catabolic fragments originated in the lysosome for biosynthetic purposes is quantitatively relevant, and lysosomal membranes under certain conditions can directly contribute to the repair of plasma membrane. Thus, it can be expected that the blockade of proper sphingolipid catabolism at the lysosomal level would lead to the jamming of the overall flow of metabolites, with consequences on the sphingolipid composition in all cellular compartments, including the plasma membrane. It is also becoming clear that the in vivo mechanisms regulating sphingolipid levels in cells and tissue are multiple and complex. As a consequence, the loss of a single enzyme activity of sphingolipid metabolism can lead to highly unexpected effects. Several reports have analyzed the consequences of the absence of ASM on the lipid composition of brain cells. These are summarized in the next section.

Lipid changes in total brain and myelin from ASMko mice

The analysis of total brain extracts of ASMko mouse brains showed a 6-fold SM increase compared to wild type (wt) mice (Galvan et al. 2008; Scandroglio et al. 2008). Similar increase (6.9-fold) was observed in purified myelin from ASMko brains (Buccinnàet al. 2009). Interestingly, such increase was predominantly because of the accumulation of a single SM molecular species (d18:1/18:0) as determined by electrospray ionization mass spectrometry in purified myelin (Buccinnàet al. 2009) (Fig. 1) and by Thin Layer Chromatography in brain (unpublished results). The differential clustering of SM molecular species resulting in a differential accessibility to the activity of ASM and neutral sphingomyelinase could explain why SM containing stearic acid is preferentially accumulated.


Figure 1.  Specific increase of a single SM molecular species in ASMko mice brains. Analysis of SM in purified myelin from wt and ASMko mice was carried out by HPLC/electrospray ionization mass spectrometry as described in Valsecchi et al. (2007).

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The total brain ganglioside content also was slightly but significantly higher (1.2-fold) in ASMko compared to wt mice. However, the combined content of two minor monosialogangliosides, GM3 and GM2, was greatly increased (12-fold higher) (Scandroglio et al. 2008). Remarkably, a striking accumulation of GM3 and GM2 gangliosides was previously reported in the brain of a NPD type A patient (Rodriguez-Lafrasse and Vanier 1999). In addition, secondary accumulation of GM3 and/or GM2 has been observed in several other lysosomal storage disorders, including sphingolipidosis with or without primary defects in ganglioside catabolism, as well as lysosomal storage disorders not affecting sphingolipid degradation (α-mannosidosis and different types of mucopolysaccharidosis) (reviewed in Walkley 2004) (Table 1). A remarkable example is Niemann Pick C disease, where GM3 and GM2 accumulate in neurons from human patients and animal models (Zervas et al. 2001). Moreover, brain accumulation of GM3 and GM2 has been also detected in neurodegenerative pathologies without a clear lysosomal involvement including Alzheimer’s disease (Kracun et al. 1992; Barrier et al. 2007) and severe malignant autosomal recessive osteopetrosis (Prinetti et al. 2009). The literature is strangely silent on the mechanisms underlying GM3 and GM2 accumulation in these pathologies, however, fragmentary evidence points out, again, that alterations in intracellular lipid trafficking, especially at the level of the endosomal system, with a consequent lack of feedback regulation of sphingolipid synthesis within the Golgi/trans-Golgi network, might be responsible for these changes (Walkley 2004).

Table 1.   Secondary accumulation of gangliosides in diseases with neurological impairment
DiseaseLysosomal involvementEffect on gangliosidesReferences
  1. This table summarizes the alterations in glycosphingolipid metabolism leading to secondary ganglioside accumulation in several diseases characterized by neurological impairment, with or without impairment of lysosomal function.

Niemann Pick type AYesGM3 and GM2 accumulationRodriguez-Lafrasse and Vanier (1999); Scandroglio et al. (2008); Buccinnàet al. (2009)
Niemann Pick type CYesGM3 and GM2 accumulationSiegel and Walkley (1994); Sleat et al. (2004)
MucopolysaccharidosisYesGM3 and/or GM2 accumulationSiegel and Walkley (1994); Constantopoulos and Dekaban (1978); Constantopoulos et al. (1978, 1980); Liour et al. (2001)
α-MannosidosisYesGM3 and GM2 accumulationSiegel and Walkley (1994); Goodman et al. (1991)
Alzheimer’s diseaseNoReduced ganglioside concentration in several brain areas and altered ratios of a-series to b-series gangliosidesBrooksbank and McGovern (1989); Crino et al. (1989); Kalanj et al. (1991); Kracun et al. (1991, 1992); Svennerholm and Gottfries (1994)
Elevated levels of simpler gangliosides (GM3 and GM2)Kracun et al. (1992); Barrier et al. (2007)
Huntington’s diseaseNoReduced ganglioside concentration in erythrocytes, striatum and caudateWherrett and Brown (1969); Higatsberger et al. (1981); Desplats et al. (2007)
Abnormal expression of glycosyltransferase genesDesplats et al. (2007)
Increased GD3 levelsDesplats et al. (2007)
Prion diseasesNoReduced ganglioside content with a shift from complex to simpler species (GM3, GD3, GD2)Yu et al. (1974); Tamai et al. (1979); Ando et al. (1984); Di Martino et al. (1993); Ohtani et al. (1996)
Alterations in the long-chain base composition of gangliosidesDi Martino et al. (1993)
Severe malignant autosomal recessive osteopetrosisUncertainAccumulation of GM3 and GM2, no changes in lysosomal glycohydrolasesPrinetti et al. (2009)

Although ganglioside storage is, therefore, not exclusive of lysosomal storage disorders, and may represent a general feature of many neurological conditions, the findings in ASMko mice and other lysosomal diseases suggested that ganglioside reduction could be a unifying approach to treating their neurological symptoms. Indeed, this has been attempted by reducing the overall synthesis of glycosphingolipids via the use of synthetic enzyme inhibitors (i.e., substrate reduction therapy) (Andersson et al. 2004; Platt and Lachmann 2009). While some clinical efficacy of this approach has been demonstrated in lysosomal storage disease animal models, leading to regulatory approval for certain diseases (e.g., NPD type C) (Lachmann et al. 2004), the effects are limited, suggesting that other molecular changes strongly contribute to the disease pathology.

In contrast to gangliosides, the myelin-enriched sphingolipids, galactocerebroside and galactosulfocerebroside, decreased in the brain of ASMko mice compared to wt, in parallel with the mRNA levels of the two transferases responsible for their synthesis (Buccinnàet al. 2009). This is consistent with the defective myelin architecture and function reported in NPD type A patients (Landrieu and Saïd 1984; Folkerth 1999; Di Rocco et al. 2005).

As mentioned, the lack of ASM by itself is not sufficient to explain the unexpected changes in lipid composition observed in ASMko mice, that is, the preferential accumulation of a single SM molecular species and the accumulation of GM3 and GM2 gangliosides. The diversity in the structure of the ceramide moiety of cellular sphingolipids is likely because of both specificity of ceramide synthesis [at least six different ceramide synthases contribute to the fatty acid heterogeneity of ceramide in sphingolipids (Pewzner-Jung et al. 2006)] and selectivity in the traffic of ceramides with different molecular structure and metabolic fate [at least two different transport mechanisms, a vesicular one and a non-vesicular, ceramide transfer protein mediated one, are responsible for the structure-selective delivery of ceramide from the sites of synthesis to the sites of its metabolic utilization (Bartke and Hannun 2009)]. On the other hand, the de novo synthetic flow, the extent of recycling of catabolic fragments and the enzyme activities of both the biosynthetic and catabolic pathways of sphingolipids are likely subjected to a very complex regulation in vivo. The mechanisms underlying these differences still remain to be elucidated, but it is reasonable to predict that metabolic events apparently not related to the loss of ASM activity, and not necessarily restricted to the lysosome or to the catabolic pathway of sphingolipids, might be involved in the unexpected modifications of lipid levels and topology observed in ASMko mice brains.

Lipid changes in particular neuronal populations and subcellular compartments in the absence of ASM activity

In addition to the analysis of lipid alterations in total brain and myelin, efforts have also been directed to elucidate whether and how such alterations affect particular neuronal populations. High levels of SM were confirmed in both cultured primary hippocampal and cerebellar granule neurons from ASMko mice compared to wt. However, ganglioside increase was not evident in these neurons indicating that such accumulation is not a direct consequence of the enzyme defect (Galvan et al. 2008; Scandroglio et al. 2008).

Further efforts have been aimed to characterize the lipid alterations in subcellular compartments of neuronal cells and tissues. Isolation of Golgi-enriched, lysosomal-enriched and lysosomal-free fractions from ASMko and wt mice brains revealed two interesting facts: (i) Golgi membranes are not significantly affected by SM accumulation in ASMko tissue, and (ii) SM accumulation is not restricted to lysosomes when ASM is lacking; that is, non-lysosomal membranes are also affected (6.1- and 4.8-fold higher SM content in ASMko versus wt lysosomal and non-lysosomal membranes, respectively) (Galvan et al. 2008). ASMko non-lysosomal membranes were also enriched in SM derivatives such as sphingosylphosphorylcholine and sphingosine compared to wt membranes. However, no significant differences were found in the content of other lipids (i.e., cholesterol, triglycerides, phospholipids or ceramide) (Galvan et al. 2008). In contrast, increased cholesterol content as indicated by filipin staining, was found in lysosomes of neurons (Sarna et al. 2001; Passini et al. 2005). The above reported findings suggested the presence of high SM, but not cholesterol levels, at the plasma membrane. This was confirmed in cultured hippocampal neurons by staining of non-permeabilized cells from ASMko and wt mice with lysenin or filipin, which respectively bind SM or cholesterol. A 3.8-fold increase in SM signal at the cell surface, but no significant changes in that of cholesterol, were evidenced in ASMko hippocampal neurons (Galvan et al. 2008).

Isolation of synaptosomes from ASMko and wt mice brains also led to the discovery that SM and sphingosine were increased (3- and 5-fold, respectively) in ASMko synaptic membranes (Camoletto et al. 2009). In recent years, evidence has accumulated indicating that, because of their chemical affinity, sphingolipids and cholesterol form microdomains in cellular membranes, which play a key role in cellular signaling (Lingwood and Simons 2010). Therefore, the lipid composition of detergent resistant membranes (DRMs), which are considered a biochemical correlate of such microdomains, has also been analyzed in ASMko total brain and cultured neurons. Increased sphingolipid content was found in DRMs from ASMko mice brains (Galvan et al. 2008; Scandroglio et al. 2008), and a higher detergent-to-protein ratio was needed to isolate them with respect to wt (Scandroglio et al. 2008), likely reflecting a reduced fluidity in restricted membrane areas.

Altogether, the above findings highlight the complexity of lipid abnormalities that ASM deficiency causes in the brain. They involve different kinds of neurons and glial cells and affect distinct cellular compartments. Next we discuss current information regarding the functional consequences that such alterations have in ASMko brain cells (Table 2).

Table 2.   Aberrant phenotypes in ASMko brain cells
Brain cell typeAberrant phenotypeReferences
  1. This table summarizes the aberrant phenotypes found until now in neurons, oligodendrocytes and astrocytes of ASMko mice brains. Corresponding references are also indicated.

NeuronsDeficient Ca2+ homeostasisGinzburg and Futerman (2005)
Altered molecular polarizationGalvan et al. (2008)
Impaired endocytosisGalvan et al. (2008)
Smaller synapsesCamoletto et al. (2009)
Altered pre-synaptic plasticityCamoletto et al. (2009)
Complete loss of Purkinje neuronsHorinouchi et al. (1995); Otterbach and Stoffel (1995); Sarna et al. (2001); Macauley et al. (2008)
Altered lipid compositionScandroglio et al. (2008); Galvan et al. (2008)
OligodendrocytesDecreased expression of myelin proteinsBuccinnàet al. (2009)
Altered lipid compositionBuccinnàet al. (2009)
AstrocytesComplete blockade of microparticle shedding and IL-1b releaseBianco et al. (2009)

Consequences of ASM deficiency in neurons

  1. Top of page
  2. Abstract
  3. Lipid alterations in ASMko mouse brains
  4. Consequences of ASM deficiency in neurons
  5. Consequences of ASM deficiency in glial cells
  6. Towards a therapy
  7. Concluding remarks
  8. Acknowledgements
  9. References

High susceptibility of Purkinje neurons in the cerebellum

The first observations of aberrant phenotypes in the brains of ASMko mice were those of the accumulation of distended lysosomes within the cytoplasm of neurons and the complete loss of the ganglionic cell layer of Purkinje cells, leading to severe impairment of neuromotor coordination (Horinouchi et al. 1995; Otterbach and Stoffel 1995). In addition, it was shown that even as the ASMko mice age and the degree of storage pathology affected neurons in all brain regions (Macauley et al. 2008), a subset of Purkinje cells (those zebrin II-negative) was particularly and very early compromised (Sarna et al. 2001). Although the reason for the differential susceptibility, even between morphological identical cells (Purkinje neurons) but with different molecular phenotypes (zebrin positive or negative), is not completely elucidated, several possibilities have been postulated. For example, the topographical distribution of the small heat shock protein Hsp25 (Armstrong et al. 2001) and the p75 nerve growth factor receptor (Dusart et al. 1994) matched that of zebrin positive or negative Purkinje cells, respectively. Hence, it has been proposed that while the high expression of Hsp25 would protect zebrin II positive neurons, a possible disruption of the prosurvival p75 signalling in the zebrin negative neurons would trigger their preferential early death (Sarna et al. 2001). The ratio between ceramide and sphingomyelin has been suggested to be critical in regulating plasma membrane-dependent signaling events (Zhang et al. 2009), and in fact, ceramide and lipid microdomains have a prominent role in signalling via p75 (Brann et al. 1999). Therefore, their alterations because of ASM deficiency could underlie p75 impaired signalling in ASMko zebrin negative Purkinje neurons. Further research is needed to confirm this point.

Defective calcium homeostasis

Alterations of calcium (Ca2+) homeostasis are a feature in several sphingolipid storage diseases. Ginzburg and Futerman (2005) demonstrated that this is also the case in ASMko mice brains. These alterations seem to concern Ca2+ release, but not its uptake. Interestingly, Ca2+ abnormalities affected the cerebellum but not the cortex of ASMko mice. Such specificity correlated with the decreased expression of the sarco/endoplasmic reticulum Ca2+-ATPase and of the major Ca2+ release channel in the cerebellum Inositol 1,4,5-triphosphate receptor (Ginzburg and Futerman 2005). Importantly, the reduction in these Ca2+ related molecules was dramatic in the Purkinje cell layer, where they are particularly abundant, and preceded the loss of zebrin II expression. Therefore, alterations in Ca2+ homeostasis could be another reason for the preferential susceptibility of the cerebellum in NPD type A. How changes in this pathway affect neuronal viability is not clear. Altered intracellular Ca2+ concentrations may affect signal transduction pathways and the opening of ion channels at the plasma membrane (Berridge et al. 2003). On the other hand, high cytosolic Ca2+ can also lead to cell death because of induction of oxidative stress (Mattson and Chan 2003). The observations that addition of exogenous SM did not affect Ca2+ modulation by sarco/endoplasmic reticulum Ca2+-ATPase or Inositol 1,4,5-triphosphate receptor and that the levels of its lyso-derivative sphingosylphosphorylcholine needed to induce a small effect were significantly higher than those observed in NPD type A brains (Ginzburg and Futerman 2005), made unlikely a direct effect of storage lipids on the proteins involved in regulating Ca2+ homeostasis. However, this needs to be more carefully evaluated.

Alterations in axonal polarity and impaired endocytosis

As mentioned above, the sphingolipid content of DRMs is elevated in ASMko brains (Galvan et al. 2008; Scandroglio et al. 2008), and given the crucial role of these lipid domains in cell signalling, such alterations may underlie the aberrant neurological phenotypes in NPD type A. Some examples (i.e., p75 signalling) have been already discussed and further research is necessary to fully characterize these changes. In addition, DRMs and SM are involved in the establishment of neuronal polarity by contributing to the sorting of DRM enriched molecules to the axons (Ledesma et al. 1998, 1999). Among such molecules are the ganglioside GM1 and glycosyl phosphatidyl inositol-anchored proteins like the Prion protein (Galvan et al. 2005). Analysis of the distribution of these molecules in ASMko cultured hippocampal neurons revealed their presence both in axons and dendrites, different from their almost exclusive axonal distribution in wt neurons (Galvan et al. 2008). The aberrant distribution of Prion protein was also observed in situ in the hippocampus of ASMko mice, strongly suggesting that similar alterations might also exist in the neurons of NPD type A patients. The molecular mechanism underlying these alterations implied the impaired endocytosis from dendrites because of deficient membrane attachment and activation of the small GTPase RhoA (Galvan et al. 2008). In this case, a direct effect of SM accumulation on the aberrant phenotype was demonstrated. Hence, addition of SM to cultured wt hippocampal neurons diminished RhoA membrane attachment and impaired DRM molecule endocytosis and polarized axonal distribution. Consistent with this observation, addition of exogenous sphingomyelinase to ASMko neuronal cultures rescued the aberrant phenotypes (Galvan et al. 2008). It is likely that altered polarization of DRM molecules will affect their function, and more work is needed to fully clarify this issue.

Altered pre-synaptic plasticity

It has been shown that sphingolipid accumulation also affects synaptic membranes from ASMko mice (Camoletto et al. 2009). This observation opened the possibility that synaptic alterations might occur in NPD type A. Supporting this view, a careful analysis on the time course of the disease progression in ASMko mice brains revealed that axonal synaptic terminals are the first parts of the neuronal cells to show signs of degeneration (Macauley et al. 2008). Electrophysiological recordings in ASMko and wt hippocampal slices showed enhanced paired-pulse facilitation and post-tetanic potentiation in the former, whereas basal synaptic transmission and synaptic depression were not significantly changed (Camoletto et al. 2009). This was consistent with a decreased probability of neurotransmitter release. Electron microscopy analysis of ASMko and wt CA1 hippocampal areas indeed revealed a reduced number of docked vesicles in ASMko pre-synaptic terminals, which is an anatomical correlate of neurotransmitter release in the hippocampus (Camoletto et al. 2009). The molecular mechanism underlying this aberrant phenotype involved the altered interaction of the cytosolic factor Munc 18 and the soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor syntaxin1, which form a platform for vesicular docking (Toonen and Verhage 2007). This alteration was due, in turn, to the conformational change promoted in syntaxin1 by sphingosine accumulation. Exogenous addition of this SM derivative to wt cultured hippocampal neurons or slices was indeed capable of altering Munc18-syntaxin1 interaction, of impairing synaptic vesicle release, and of enhancing paired-pulse facilitation (Camoletto et al. 2009). These evidences in ASMko mice strongly indicate that synaptic alterations occur in NPD type A. In addition, the fact that not only the pre-synaptic, but also the post-synaptic, termini are significantly smaller in ASMko mice compared to wt (Camoletto et al. 2009) opens the perspective that dendritic spines, structures that are key in learning and memory processes, could also be affected.

Consequences of ASM deficiency in glial cells

  1. Top of page
  2. Abstract
  3. Lipid alterations in ASMko mouse brains
  4. Consequences of ASM deficiency in neurons
  5. Consequences of ASM deficiency in glial cells
  6. Towards a therapy
  7. Concluding remarks
  8. Acknowledgements
  9. References

Deficient myelination of the CNS has been observed in NPD type A patients (Landrieu and Saïd 1984; Folkerth 1999; Di Rocco et al. 2005). This, together with the neuronal anomalies described above, could contribute to explain the severe mental retardation in the disease. Hence, the analysis of myelin producing and other glial cells appears to be key to fully understand brain pathology in the absence of ASM activity.

Reduced expression of myelin specific proteins

Oligodendrocytes are the myelin producing cells in the CNS. Myelination is a two-step process in which first oligodendrocyte precursors proliferate and differentiate until they are mature enough to then acquire the capacity of producing the myelin sheath (Folkerth 1999). A delay or failure in the myelination procedure may thus result from deficiency in the differentiation process, leading to reduced numbers of mature oligodendrocytes, or from changes in myelin sheath assembly and maintenance arising from metabolic alterations in mature oligodendrocytes. Buccinnàet al. (2009) offered evidence to discriminate between these two alternatives by analyzing time dependent protein and mRNA expression of oligodendrocyte specific proteins involved in myelin architecture and function (myelin basic protein, myelin associated glycoprotein, 2′-3′-cyclic nucleotide 3′-phosphodiesterase and proteolipid protein). They observed that such expression did not change between ASMko and wt mice at birth, but became significantly reduced at later postnatal times in ASMko conditions. These findings suggested that oligodendrocyte differentiation during embryogenesis would be insensitive to ASM deficiency. This is consistent with the fact that neurological function of the ASMko mice appears normal at birth, as does that of the type A NPD children, but rapidly progresses postnatally. Although further evaluation of the number of oligodendrocyte precursors in ASMko brains would be necessary to confirm this hypothesis, the fact that ASMko brains express normal levels of the transcription factors involved in oligodendrocyte differentiation (Buccinnàet al. 2009), makes unlikely that there is a loss of oligodendrocyte during myelination. Instead, the findings described above pave the way to investigate metabolic alterations of mature ASMko oligodendrocytes that could explain hypomyelination in NPD type A patients.

Impaired microparticle release from ASMko astrocytes

Microglia has been considered the immune cells of the CNS, which release microparticles from the plasma membrane containing crucial cytokines (IL-1β) in CNS inflammatory events. However, it has been recently shown that such microparticles are also released by astrocytes in an ASM dependent manner (Bianco et al. 2009). Accordingly, a complete blockade of microparticle shedding and IL-1β release was observed in ASMko astrocytes (Bianco et al. 2009). This is consistent with the reduced levels of IL-1β found in the brain of ASMko mice (Ng and Griffin 2006). These observations led to the suggestion that the increased frequency of infections observed in NPD type A patients (Minai et al. 2000) might be related with the capability of ASM to control cytokine release and therefore to regulate the immune responses.

Towards a therapy

  1. Top of page
  2. Abstract
  3. Lipid alterations in ASMko mouse brains
  4. Consequences of ASM deficiency in neurons
  5. Consequences of ASM deficiency in glial cells
  6. Towards a therapy
  7. Concluding remarks
  8. Acknowledgements
  9. References

Niemann Pick disease type A is a devastating disease that always results in early childhood death, usually by 2–3 years of age. At the present time there is no treatment. Early attempts at bone marrow transplantation, amniotic cell, and liver transplantation did not impact the neurological disease course in type A NPD patients, and new approaches are clearly needed. When considering these approaches several factors must be taken into account. First, this is a very early onset disease. While patients are generally born without evidence of neurological dysfunction, by 3–6 months this is clearly evident. Since it is unlikely that damage incurred by ASM deficiency will be reversible, any successful therapies must therefore be initiated at birth or shortly thereafter. Second, therapies must be carefully judged with regard to significant improvement of quality of life. This is particularly important when translating results obtained in the ASMko mouse model (below) to patients. For example, in the mouse even modest improvements in neurological function may be considered an experimental ‘success’; however, similar results in patients might ultimately lead to more pain and suffering for families by slowing (but not preventing) the rate of neurological decline, ultimately prolonging the disease. On the positive side, as discussed above, for ASM deficiency it is known that very low levels of residual enzymatic activity (∼ 5%) can recover neurological function. In fact, recent studies using mutation specific mouse models (Jones et al. 2008) have shown that as little as 8% ASM activity can completely prevent the occurrence of neurological disease. Thus, the therapeutic threshold is low, provided that the enzyme can be delivered globally throughout the CNS to the proper sites of pathology, and that the therapy can be initiated prior to the time when irreversible damage occurs.

Enzyme replacement therapy, administered by intravenous infusion of the recombinant enzyme into deficient patients, is a successful strategy for several lysosomal storage diseases. However, the presence of the brain blood barrier generally renders the brain irresponsive to this form of therapy. This was confirmed in the ASMko mice in which intravenous ASM replacement therapy (at doses up to 10 mg/kg) resulted in the improvement of visceral but not brain pathology (Miranda et al. 2000). Thus, while enzyme replacement therapy is currently being evaluated in NPD type B patients (non-neurological), and it may alter the visceral disease in type A NPD, it is likely not to affect the severe neurological course in these patients.

The fact that NPD type A is a single-gene disorder also opened the possibility for gene replacement by intracranial injection of viral vectors. Adeno-associated virus is particularly suited for brain applications since besides being non-toxic and neurotropic, it has the potential to sustain long-term expression in the CNS. Several adeno-associated virus serotypes and different brain injection sites have been tested in ASMko mice to induce the expression of human ASM and alter the course of the mouse disease (Dodge et al. 2005; Passini et al. 2005). Such studies showed that wide-spread enzyme distribution could be obtained throughout the mouse CNS, leading to alleviation of storage pathology, rescue of Purkinje cells and correction of behavioural deficits. Thus, enzyme replacement by gene therapy is envisioned as candidate for future clinical trials. One challenge, however, is to scale up this approach from the mouse to primate brain in order to achieve similar biodistribution of the virus and ASM expression.

Intracerebral transplantation of bone marrow-derived mesenchymal stem cells (Jin et al. 2002) or adult mouse neural progenitor cells (NPCs) (Shihabuddin et al. 2004) expressing the human ASM also has been assessed in the ASMko mice. These studies demonstrated that genetically engineered cells can serve as a vehicle for enzymatic cross-correction and reversal of storage pathology of host brain cells in the mouse model. Hence, human ASM-encoding mesenchymal stem cells s injected in the hippocampus and cerebellum migrated away from the injection sites and survived at least 6 months after transplant. Greater survival times and significantly delayed Purkinje cell loss were observed in the transplanted animals (Jin et al., 2002). Also promising were the results obtained upon NPC transplantation. Transplanted, ASM expressing NPCs survived, migrated and showed region-specific differentiation in the host brain. Although the levels of hASM were barely detectable by immunostaining they were sufficient for uptake and cross-correction of host cells leading to reversal of distended lysosomal pathology and regional clearance of SM and cholesterol storage (Shihabuddin et al. 2004).

Future approaches for type A NPD that could be evaluated in the mouse models also include small molecule approaches aimed at enzyme enhancement (e.g., chaperone) or substrate reduction, as well as enhanced methods to deliver recombinant ASM or gene therapy vectors across the blood brain barrier. Clearly, for all of these approaches research on the aberrant neurological phenotypes in ASMko mice will provide read out systems that can be used to test their efficacy, and will hopefully reveal new targets for genetic and/or pharmacological intervention.

Concluding remarks

  1. Top of page
  2. Abstract
  3. Lipid alterations in ASMko mouse brains
  4. Consequences of ASM deficiency in neurons
  5. Consequences of ASM deficiency in glial cells
  6. Towards a therapy
  7. Concluding remarks
  8. Acknowledgements
  9. References

Observations on type A NPD, dating back nearly 100 years, have highlighted the important and essential role of ASM in the brain, and studies in the ASMko mouse model are beginning to elucidate the molecular mechanisms leading to severe neurological dysfunction in this disorder. The combined efforts of different disciplines, ranging from protein and lipid biochemistry and biophysics to molecular biology, has begun to unravel the complex biology underlying the brain abnormalities in these patients. Abnormalities involve neurons and glial cells, as well as lysosomal and non-lysosomal membranes. In this regard, the results discussed in this review open the question on whether type A NPD should be still considered a lysosomal storage disorder. Lysosomal dysfunction because of SM accumulation will certainly contribute to the pathology and the lack of ASM at the lysosomal level is surely responsible, at least in part, for its onset. However, the recent demonstrations of dramatic changes in the lipid content of the plasma and synaptic membranes, which could explain many of the aberrant brain phenotypes reported and are likely independent of the lysosomes, point to non-lysosomal events as the main and/or initiating cause for this neurological disease. Such findings are valuable tools for the design and testing of new therapeutic strategies for type A NPD, and some of these have already shown promising results in the mouse model, opening new opportunities for the treatment of this devastating disease. Moreover, since patients with several common neurological disorders (e.g., Alzheimer’s disease) also exhibit abnormalities in the sphingolipid pathway, including alterations in ASM activity, it is likely that findings in the ASMko mice will influence these diseases as well.


  1. Top of page
  2. Abstract
  3. Lipid alterations in ASMko mouse brains
  4. Consequences of ASM deficiency in neurons
  5. Consequences of ASM deficiency in glial cells
  6. Towards a therapy
  7. Concluding remarks
  8. Acknowledgements
  9. References

We thank the support of Ministerio de Ciencia e Innovacion (grant SAF 2008-01473) and the Fundación Ramón Areces to M.D.L; CARIPLO and AIRC to S.S; NPD research in the E.H.S. laboratory is supported by the National Institutes of Health (5 R01 HD28607) and Genzyme Corporation. EHS is a consultant for Genzyme and an inventor on patents that have been licensed to Genzyme for the treatment of NPD.


  1. Top of page
  2. Abstract
  3. Lipid alterations in ASMko mouse brains
  4. Consequences of ASM deficiency in neurons
  5. Consequences of ASM deficiency in glial cells
  6. Towards a therapy
  7. Concluding remarks
  8. Acknowledgements
  9. References
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