It has been demonstrated that the sphingomyelin-ceramide balance in brain becomes progressively disrupted with HAND in accordance with the severity of neurocognitive dysfunction (Haughey et al, 2004
). These studies found that multiple species of sphingomyelin were increased in the middle frontal and temporal gyrus of HIV-infected patients with mild cognitive dysfunction, and multiple forms of sphingomylein, ceramide, and the lipid peroxidation product 4-hydroxynoneal were increased in those with moderate to severe cognitive dysfunction (Haughey et al, 2004
). Measures of CSF sphingolipids (as surrogate markers for brain sphingolipid metabolism) in patients living with HIV demonstrated that sphingomyelin accumulated with mild and stable cognitive dysfunction, and increased ceramide levels were associated with ongoing cognitive decline (Bandaru et al, 2007
). Additionally, there were greater accumulations of sterols sphingomyleins and ceramides in brains of subjects with an apolipoprotein E4 (ApoE4) genotype, suggesting that a genetic component may contribute to perturbed sphingolipid metabolism in HAND (Cutler et al, 2004a
). Because each of these studies used a combinational score (Memorial Sloan Kettering Scale) that combines data from tasks that assess multiple cognitive domains and motor functions, it was not known if alterations in sphingomyelin and ceramide content associated with deficits in particular cognitive and/or motor functions. In this study we addressed this question and found that changes in the sphingomyelin:ceramide ratio for acyl chain lengths of C16, C18, C22, and C24 were associated with worse performance on one or more indices of memory, including the total, trial 5, and immediate and delayed recall scores, on the RAVLT. The strongest of these associations was for the sphingomylein: ceramide ratio of C18:0 that was associated with performance on each of the RAVLT memory tasks. These findings suggest that the sphingomyelin:ceramide ratio for C18 may be a sensitive marker for neurocognitive impairment in HIV-infected subjects, and that the levels of C18 may be a reasonable surrogate marker for memory function. However, we cannot rule out possible associations for sphingolipids with other cognitive functions that were not specifically tested in this group of subjects.
To our knowledge, this is the first report to use the ratio of sphingomyelin to ceramide as a diagnostic/prognostic marker. We reasoned that because sphingomyelin and ceramide are directly related metabolic pathways (ceramide is a precursor to sphingomyelin and sphingomyelin can be hydrolyzed to create ceramide), the ratio of sphingomyelin to ceramide would be a useful metric to measure the status of sphingomyelin metabolism. Because these reactions most often modify the head group and not the acyl chain length, we only compared identical tail compositions. Using this metric we found that the sphingomyelin:ceramide ratio for C16:0, C18:0, C22:0, and C24:0 were associated with worse performance on several indices of memory, with C18:0 showing the most consistent effects. A ration of sphingomyelin:ceramide for the particular chain length of C18:0 may be a useful marker for HAND, since other neurodegenerative disease with a known disruption in ceramide metabolism have reported that very long chain ceramide accumulate in brain and CSF. Perturbed sphingolipid balance has been identified in a number of neurodegenerative conditions that include multiple sclerosis, Alzheimer’s disease (AD), amyotrophic lateral sclerosis, and HAND (Cutler et al, 2002
; Bandaru et al, 2007
; Haughey et al, 2004
; Wheeler et al, 2008
). In each of these neurodegenerative conditions there appears to be disease-specific alterations in particular species and forms of sphigolipids. For example, C24:0 ceramide and sphingomylein are inversely and progressively disrupted in AD with increasing disease severity (Cutler et al, 2004b
; He et al, 2008
). In contrast, subjects with HAND who showed evidence of mild and stable cognitive impairment had increased C18:0 to C24:0 sphingomylein with no apparent changes in ceramide. Those with HAND and frank dementia showed increased C18:0 to C24:0 sphingomylein and ceramide (Bandaru et al, 2007
; Cutler et al, 2004b
; Haughey et al, 2004
). Our current findings suggest that a ratio of C18 sphingomylein: ceramide may be a sensitive biomarker for cognitive dysfunction in HAND that specifically relates to memory function. Further studies should be aimed to clarify the potential usefulness of this metric in larger and diverse populations of HIV-infected subjects.
A possible explanation for the specificity of C18:0 to associate with memory impairment in HAND has been provided by findings that suggest differential functions that depend on the biophysical properties of sphingolipids with particular carbon chain lengths. For instance, the length and saturation of acyl chains can influence the width of bilayers, fluidity of membranes, interactions with other lipids, and lipid-protein interactions (Aittoniemi et al, 2007
; Niemela et al, 2006
). Additionally, there is evidence of considerable tissue, cellular, and subcellular specificities for particular molecular species of sphingolipids, suggesting that the pattern of disruption in sphingolipid metabolism may identify specific organ and organelle dysfunctions (Kaiser et al, 2009
; Meer and Hoetzl, 2009
). Consistent with the regulated subcellular distribution of sphingolipids, there is an increasing amount of data that suggest that sphingolipids with particular carbon chain lengths can differentially regulate signal transduction, cell motility, neurite outgrowth, synpatogenesis, and cellular fusion events important for protein trafficking and synaptic transmitter release (Gallegos et al, 2008
; Galvan et al, 2005
; Nygren et al, 2005
; Wheeler et al, 2009
). The findings from these mechanistic studies together with our biochemical and clinical observations suggest that disrupted sphingomyelin:ceramide balance in HAND may reflect perturbed neural function that becomes manifest as deficits in verbal memory. Although we cannot make any definite conclusions why verbal memory was better correlated to surrogate markers of sphingoipid metabolism compared with visual memory, a review of 56 studies that used neuropsychological tests to investigate areas of function affected by central nervous system dysfunction in HIV-infected subjects found evidence for some dysfunction in verbal memory (27% of studies), executive function (43%), motor performance (20%), and information processing (44%) among subjects who were otherwise asymptomatic. Subjects with more advanced HIV infection showed consistent evidence of abnormal functioning in the areas of verbal (48% of studies) and visual (43%) memory, executive functioning (71%), complex attention (62%), motor performance (37%), and information processing (69%) (Dunbar and Brew, 1997
). Thus, in HIV-infected subjects there may be impairments of verbal memory early in disease progression that occur independent of visual memory.
The specific association of sphingomylein:ceramide ratios for C18 and impairment on memory in the RAVLT also provides clues to the specific enzymes responsible for perturbations of sphingomyelin:ceramide balance in HAND. There are a multitude of enzymes involved in sphingomyelin and ceramide metabolism, including the following: Sphingomyelinases that mediate the hydrolysis of sphingomyelin to ceramide; five different sphingomyelinase that differ in pH optimum, metal dependence, and subcellular localization are known (Marchesini and Hannun, 2004
). Ceramidases that cleave fatty acids from ceramide to produce sphingosine; seven different ceramidase have been identified (Mao and Obeid, 2008
). Sphingomyelin synthases (SMS) that catalyze the conversion of ceramide and phosphatidylcholine to sphingomyelin and diacylglycerol; two SMS have been identified, SMS1 that is localized to the Golgi, SMS2 that resides primarily at the plasma membrane (Huitema et al, 2004
; Takeuchi et al, 1995
). Dihydroceramide desaturases (ceramide synthase; CerS) that are involved in the de novo
production of ceramide; six CerS have been identified (Pewzner-Jung et al, 2006
). Of these enzymes, only CerS are known to have preferences for particular acyl chain lengths, with each of CerS family members utilizing a relatively restricted subset of fatty acyl–coenzyme As (CoAs) (see Mizutani et al, 2006
, for a review). Of particular interest to the present work are CerS1 and CerS3 that preferentially use C18 as a substrate. Our findings suggest that a defect in CerS1 and/or CerS3 may be prominently involved in the perturbation of the sphingomylein:ceramide C18:0 ratio in subjects with HAND. The identification of specific enzymes involved with disturbances of sphingomylein metabolism in HAND may identify new therapeutic targets for this neurodegenerative disease.