Physician/Scientist

The Biochemical Defect in
Types A and B Niemann-Pick Disease

The Biochemical Abnormalities in Types A and B NPD

The Accumulating Lipids

Sphingomyelin
Sphingomyelin is the major lipid that accumulates in the cells and tissues of patients with Types A and B NPD. In most normal tissues, sphingomyelin constitutes from 5 to 20 percent of the total cellular phospholipid content, but in tissues of Types A and B NPD patients, the sphingomyelin levels may be elevated up to fifty-fold, constituting about 70 percent of the total phospholipid fraction. The relative increases in sphingomyelin and cholesterol in various NPD tissues, including the brains of Type A patients, have been summarized by Spence and Callahan. Presumably, the accumulation of sphingomyelin in NPD is the result of abnormal turnover of cell membranes (the major component of the intracellular sphingomyelin pool) resulting from the ASM deficiency. Cells of the monocyte-macrophage system, particularly in the spleen and lymph nodes, accumulate the most sphingomyelin because they actively phagocytose sphingomyelin-rich membranous material. Storage in liver, brain, kidneys, and lungs also has been documented. Organs from Types A and B NPD patients contain about the same amounts of sphingomyelin, with the notable exception that Type B NPD patients have little or no lipid storage in their central nervous systems. Since Type B NPD patients die at a much later age than those with Type A NPD, the rate of sphingomyelin accumulation in Type A NPD individuals is much greater than that in Type B NPD patients.

Cholesterol
Tissue cholesterol levels are almost always increased in Types A and B NPD. The degree of storage varies, but it may be as much as 3 to 10 times the normal levels. The distribution of cholesterol storage is similar to that of sphingomyelin storage, with cells of the monocyte-macrophage system accumulating the most. In contrast to what is observed in sphingomyelin storage, cholesterol concentrations at the time of autopsy are significantly greater in Type A NPD patients than in those with Type B disease. Why cholesterol accumulates in the tissues of Type A and B NPD patients is not clear, since the metabolic trafficking of this sterol is clearly different from that of sphingomyelin. However, increased levels of cholesterol in various phospholipid storage disease patients had been recognized as far back as 1930, leading to an erroneous hypothesis that the biochemical defect in some of these diseases was in a "phospholipid/cholesterol" binding protein. More likely, the accumulation of cholesterol involves lipid-lipid interactions in biomembranes. Supporting this hypothesis, it has been demonstrated that sphingomyelin and cholesterol can form a complex with maximal van der Waals interactions between the sphingosine moiety and cholesterol carbons. Notably, the calculated distance between the phosphate groups in this complex compares well with the periodicity of the lamellar bodies seen in NPD cells. Cholesterol is a particularly good "lipid organizer," permitting strong interactions with other lipids. Thus, it is reasonable to assume that the primary storage of sphingomyelin in NPD cells leads to a secondary storage of cholesterol. Conversely, the accumulation of sphingomyelin in Types C and D NPD, which results from a cholesterol transport defect, may be a result of the same or a similar mechanism. It also has been found that patients with Type B NPD may have mild hypercholesterolnemia (McGovern et al, unpublished observations), although this phenomenon is rarely of clinical significance.

Bis(monoacylglycero)phosphate
Other than sphingomyelin and cholesterol, the major lipid that accumulates in the visceral tissues of most Type A and B NPD patients is bis(monoacylglycero)phosphate. In fact, this lipid may accumulate to higher levels than sphingomyelin, perhaps as much as 85 times greater than normal levels in the livers of patients with Type A NPD. Normal tissues have trace amounts of bis(monoacylglycero)phosphate localized in the lysosomes. Although this lipid's role in the pathogenesis of NPD is not understood, its accumulation has been attributed to the increased number of lysosomes in NPD cells. Notably, the storage of bis(monoacylglycero)phosphate is not seen in other lipid storage diseases, but has been found in drug-induced lipidoses.

Other Sphingolipids
There have been various reports of the accumulation of glycosphingolipids in tissues of patients with NPD. These substances include glucocerebroside and the gangliosides GM2 and GM3. In addition, lesser accumulations of lactosylceramide, globotriaosylceramide, and globotetraosylceramide also have been reported in liver and spleen from NPD patients.

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The Enzymatic Defect

Residual Acid Sphingomyelinase (ASM) Levels
Despite the fact that the enzymatic defect causing Types A and B NPD has been known for almost three decades, reports on the levels of residual ASM activity have been variable because of differences in the assay procedures and enzyme sources used. In general, patients with Type A NPD have ASM activity levels ranging from non-detectable to less than 5 percent of normal when determined in vitro using cultured fibroblasts and/or lymphoblasts as the enzyme source. Similar findings have been reported in extracts from tissues including liver, kidneys, and brain. The ASM activities in cells and tissues obtained from Type B NPD patients are more variable than they are in those of Type A. In general, the in vitro residual activities in Type B NPD may range from 2 to about 10 percent of normal when determined in cultured cells.

Since the determination of ASM activities in vitro has proven problematic, a number of laboratories have developed in situ cell-loading assays to determine ASM activity. In these systems, radioactive or fluorescent sphingomyelin is added to the culture media of fibroblasts or lymphoblasts for uptake and transport to the lysosomes. Following a chase period, the cells are harvested, the lipids are extracted, and the amount of sphingomyelin converted to ceramide is determined. Gatt and coworkers have developed a modification of this technique using fluorescently-labeled (pyrene) sphingomyelin and ApoE to target the substrate to lysosomes efficiently via the LDL receptor. The advantage of this technique is that it avoids hydrolysis of the exogenous substrate by contamination of the neutral sphingomyelinase activity present on the plasma membrane. Using this in situ assay, the residual activities in cultured cells obtained from nine Type A and six Type B NPD patients were determined. Cells from Type A NPD patients hydrolyzed only about 1 to 3 percent of the delivered sphingomyelin, whereas cells from Type B patients hydrolyzed from 10 to 60 percent of the substrate, providing substantial proof that cells from Type B NPD patients have higher levels of residual ASM activity than those from patients with Type A NPD. Presumably, the higher levels of residual ASM activity in Type B NPD patients prevent the development of neurologic symptoms. Notably, there also have been reports that the residual ASM activity in Type B NPD cells can be enhanced to about normal levels by dimethylsulfoxide (DMSO) and cannabidiol. Although these reports are intriguing, they have not been adequately confirmed.

Immunologic Studies of the Mutant ASM
A variety of antibody preparations have been made against ASM and used to study the CRM in cultured cells from Types A and B NPD patients. However, because there are difficulties in obtaining highly purified ASM, the quality of the antibodies used in these early studies remains suspect. Furthermore, since ASM is known to form aggregates and a wide range of molecular weight species has been reported, interpretation of the published immunologic studies has proven difficult. A number of NPD patients have been recently analyzed for the presence of CRM using a highly specific antibody preparation raised against recombinant human ASM purified from the overexpressing CHO cells. To date, all of the Type A or B patients analyzed (other than two Type A patients who were homoallelic for nonsense mutations leading to truncated ASM polypeptides) have had about normal levels of ASM CRM. The availability of these high-titer, monospecific anti-ASM antibodies should facilitate further immunologic studies of the enzyme defects underlying Types A and B NPD.

Sphingolipids and Signal Transduction:
Implications for Niemann-Pick Disease
About 10 years ago, sphingosine was discovered to be a potent inhibitor of protein kinase C activity and of phorbol ester binding in mixed micellar assays. Similar findings were soon reported in human platelets, neutrophils, and HL-60 cells, raising the possibility that sphingoid bases may be important regulators of protein kinase C-mediated signal transduction. Recent reports documenting the effects of sphingolipids on protein kinase C-mediated signal transduction have been published, and it also has been suggested that some of the cellular pathology in the various sphingolipidoses may be due to sphingolipid-induced alterations in signal transduction.

Much of the interest in the sphingolipid-mediated signal transduction field has focused on the role of ceramide (generated by sphingomyelin hydrolysis) as a second messenger in these pathways. This so-called "sphingomyelin pathway" is a ubiquitous, evolutionarily conserved signaling system, and the Mg2+-dependent and -independent neutral sphingomyelinases, as well as ASM, have been implicated in the activation of this pathway. For example, activation of ASM has now been associated with signaling via Fas, CD28, and the interleukin-1 (IL-1) receptor. The recent findings that ASM is actively secreted by many cell types, is stable at physiologic pH, and can be rapidly re-internalized and sequested in endosomal compartments are supportive of a role for this enzyme in signal transduction, since it is widely assumed that the pool of ceramide that functions as a second messenger is generated at or near the cell surface, not within lysosomes.

Of particular relevance to NPD, it was recently shown that lymphoblasts from Type B NPD patients failed to respond to ionizing radiation with ceramide generation and apoptosis (Santana et al. 1996). These abnormalities were reversible upon restoration of ASM activity by retroviral-mediated gene transfer of the human ASM cDNA. The ASM knockout mice also expressed defects in radiation-induced ceramide generation and apoptosis in vivo.

These results in the animal model system suggest that NPD patients may have subtle abnormalities in various signaling pathways and that these abnormalities could be exacerbated by stress (e.g., radiation, infection, etc.). While there is currently no clinical evidence to support this hypothesis, future research will undoubtedly focus on the role of apoptosis in the NPD pathophysiology.

Lysosphingolipids and the NPD Pathophysiology
Lysosphingolipids, which differ from their respective parental sphingolipids by not having the amide-linked fatty acid at the 2-amino position of the sphingoid base, also have been shown to be potent and reversible inhibitors of protein kinase C activity. They are degraded by the same hydrolytic enzymes as the respective parent sphingolipids, and thus the deficiency of a particular hydrolytic enzyme leads to the accumulation of the sphingolipid and the derivative lysosphingolipid. It has been hypothesized that the accumulation of lysosphingolipids may result in cell dysfunction and cell death. In support of this hypothesis, a number of lysosphingolipids have been shown to have a role in the pathophysiology of certain sphingolipidoses, including psychosine (galactosylsphingosine) in Krabbe's disease and glucosylsphingosine in Gaucher disease.

To date, there has only been one lysosphingolipid, sphingosylphosphocholine (SPC), which has been shown to accumulate in Type A NPD. Of note, SPC is a potent mitogen that, among other things, increases intracellular free Ca2+ uptake and induces neuite outgrowth. Moreover, it has been shown that SPC also increases the DNA-binding activity of the AP-1 transcription activator AP-1 (Berger et al. 1995), and that AP-1 binding sites can be found within the ASM promoter (see "The ASM Gene").