Gelman, Benjamin B MD, PhD*†; Soukup, Vicki M PhD‡; Holzer, Charles E III PhD§; Fabian, Roderic H MD‡; Schuenke, Kimberly W PhD*; Keherly, Michael J PhD*; Richey, Frances J RN*; Lahart, Christopher J MD∥
Brain white matter injury in HIV-1 infection may be clinically important because infected persons develop a subcortical type of neurocognitive impairment termed HIV-associated dementia (HAD).1-3 The nature of subcortical white matter changes in HIV-1-infected persons is not well characterized. A blood-brain barrier disturbance occurs in cerebral white matter that can produce abnormal histologic staining of myelinated nerve fibers in HIV-infected persons (“white matter pallor”).4-6 Histochemical data show structural damage of white matter axons,7 and magnetic resonance imaging shows that white matter volume is decreased, perhaps as a result of destruction of myelinated nerve fibers.8,9 Brain spectroscopy (MRS) shows that choline-containing compounds and myoinositol are increased in the white matter of HIV-infected persons, which is believed to reflect increased membrane turnover in white matter glia.10,11 These MRS anomalies may be driven by HIV infection, because they were reversed when HIV replication was suppressed.12 Diffusion tensor imaging in HIV-infected persons has revealed highly subtle structural changes in nerve fibers of white matter.13-15 Brain HIV-1 RNA loads are high in subcortical white matter relative to brain cortex,16 and there is preferential white matter brain pathology in monkeys with lentivirus encephalitis.17,18 There is an accumulation of lysosomes in white matter astrocytes, macrophages, and microglial and glial cells that is linked with the presence of HIV proviral DNA in brain tissue.19 Because membrane turnover is routed through the lysosomal compartment primarily,20 this expansion of the lysosomal apparatus is consonant with data suggesting that membrane turnover is increased in white matter.11,21 The relationship of white matter anomalies overall to brain dysfunction still is not clear. Here we show that an index of lysosomal expansion in white matter measured biochemically is indeed correlated with HIV-associated neurocognitive impairment.
Subjects and Neuropsychological Testing
Fifty-seven HIV-infected persons who had end-stage AIDS were studied. The subjects were treated with highly active antiretroviral therapy and underwent autopsy at 1 of 4 National NeuroAIDS Tissue Consortium (NNTC) brain repository sites, located in Galveston, Texas, Los Angeles, California, New York, New York, and San Diego, California (all in the United States). NNTC sites employed identical protocols for neuropsychological testing and brain dissection.22,23 Subjects underwent neuropsychological testing within 6 months prior to death using the NNTC neuropsychological test battery. Documentation of comorbid conditions, interrater reliability, general reliability, and sensitivity are reported elsewhere.2,23-25Table 1 details the cognitive domains that were tested. Raw scores were transformed into age-, education-, or ethnicity-corrected standardized scores; clinical impairment ratings were computed for each domain; a global clinical rating (GCR) and neurocognitive diagnosis was provided according to Heaton et al.2
Autopsy, Enzymology, and Virologic Assay
Subjects received a complete autopsy within 24 hours postmortem.22 Frozen samples of brain cortex and underlying gyral white matter from Brodmann area 8 were assayed enzymatically for β-glucuronidase activity.19 Brain specimens and correlative clinical data used in this study are available to the worldwide research community (available at: http://www.hivbrainbanks.org). Total RNA was isolated from frozen brain samples with UltraSpec RNAzol (Biotecx Laboratories, Houston, TX), and HIV RNA was assayed at the Johns Hopkins Medical Laboratories in Baltimore, MD, using the Amplicor HIV-1 Monitor UltraSensitive assay system (Roche Diagnostics, Indianapolis, IN). Blood and cerebrospinal fluid (CSF) were sent directly to the testing laboratory for HIV RNA assay. Pearson coefficients were used to compare neurocognitive impairment ratings with lysosomal enzyme activity.26
The GCR is a composite index of neurocognitive impairment as detected using the NNTC battery of tests. Fifty-one of 57 subjects (89%) had GCR scores >4, which is the cutoff for a minimal amount of neuropsychological impairment. Five subjects (9%) were not impaired. Four (7%) had subtle impairment that was not clinically apparent (“subsyndromic”). Sixteen (28%) had minor cognitive and motor disturbance. In 15 (26%), HAD was diagnosed. Comorbid conditions occur frequently in the NNTC population, so we studied 17 subjects who had comorbid conditions (diagnosed NPI-O for “neurocognitively impaired, other”). HIV-1 infection is still a potential cause of impairment in persons who were NPI-O; comorbid factors could have contributed in whole or part. We included these 17 subjects a priori in the analysis to determine whether they differed from those without comorbidity. Statistical validity of the hypotheses tested was not influenced by including them, and conclusions made were replicated with, and without, their inclusion.
Several enzymes of the lysosomal class were assayed; all enzymes tested in this class were increased in AIDS brain specimens. 19 To avoid redundancy we focus on β-glucuronidase activity as illustrative of the results overall. β-Glucuronidase in white matter was positively correlated with 5 of 7 neurocognitive domains that were tested, and also with the GCR. Activity in gray matter showed hardly any correlation with neurocognitive test results (Table 1). Selectivity of white matter enzyme activity for significant correlation is shown in Figure 1. β-Glucuronidase was compared with the concentration of HIV-1 RNA in 3 body compartments in subjects for whom we could obtain those data (Fig. 2). HIV-1 RNA in brain frontal lobe was positively correlated with white matter β-glucuronidase (r = 0.4688, P < 0.021). A possible association between HIV-1 RNA in CSF and white matter β-glucuronidase was also found (r = 0.2657; P < 0.0677). Plasma HIV-1 RNA was not correlated. About 25% of the cohort had mildly increased β-glucuronidase in CSF, but it was not correlated with impairment (not illustrated).
The index of lysosome expansion in white matter was indeed correlated with HIV-associated neurocognitive impairment and with brain HIV RNA loading. These data add new biochemical evidence that white matter changes contribute to HIV-associated impairment. The scenario of metabolic activation of white matter glia in response to HIV-1 infection agrees with brain MRS data showing abnormal cellular metabolism of white matter27-29 that are believed to reflect increased membrane turnover.10,11,21 Because membrane turnover is routed almost exclusively through the endosome-lysosome system,20 expansion of the lysosome compartment could reflect increased membrane trafficking and organelle synthesis as suggested by MRS data. The anomaly is highly prevalent in autopsy specimens and it shows some relationship to assay of HIV-1 DNA and mRNA in brain tissue.19 MRS anomalies also are prevalent with HIV-1 infection and, because they improve after highly active antiretroviral therapy,12 also are related to HIV-1 replication.
One scenario that explains lysosome expansion is that white matter macrophages and microglia undergo increased lysosomal enzyme production and secretion when infected by HIV-1-a rudimentary sign of immunologic activation.30-33 In vitro data support that suggestion, as HIV-1 infection has a strong influence on the process of endosome trafficking, phagolysosome fusion, and virus budding.30,31,34,35 White matter astrocytes also exhibit lysosomal expansion19 and undergo limited infection by HIV-136; these cells also could participate in increased traffic through endosomal/lysosome pathways. The fact that white matter lysosome expansion is multicellular19 suggests that metabolic activation in white matter glial cells is broad based. That scenario is consistent with the theory that multicellular inflammatory cascades drive brain dysfunction and fits with the fact that inflammation seems to exert more influence on brain dysfunction than does the concentration of HIV-1 in the brain.37-39 Metabolic anomalies in white matter such as lysosomal expansion could drive neurocognitive impairment and may be visible in patients using MRS. The biochemistry of white matter and correlation with spectroscopic anomalies in HIV infection deserve further study.
1. Filley CM. The Behavioral Neurology of White Matter. New York: Oxford University Press; 2001:247-267.
2. Heaton RK, Grant I, Butters N, et al. The HNRC 500: Neuropsychology of HIV infection at different disease stages. HIV Neurobehavioral Research Center. J Int Neuropsychol Soc. 1995;1:231-251.
3. Sacktor N, Tarwater PM, Skolasky RL, et al. CSF antiretroviral drug penetrance and the treatment of HIV-associated psychomotor slowing. Neurology. 2001;57:542-544.
4. Petito CK, Cash KS. Blood-brain barrier abnormalities in the acquired immunodeficiency syndrome: immunohistochemical localization of serum proteins in postmortem brain. Ann Neurol. 1992;32:658-666.
5. Power C, Kong PA, Crawford TO, et al. Cerebral white matter changes in acquired immunodeficiency syndrome dementia: alterations of the blood-brain barrier. Ann Neurol. 1993;34:339-350.
6. Olsen WL, Longo FM, Mills CM, et al. White matter disease in AIDS: findings at MR imaging. Radiology. 1988;169:445-448.
7. An SF, Giometto B, Groves M, et al. Axonal damage revealed by accumulation of beta-APP in HIV-positive individuals without AIDS. J Neuropathol Exp Neurol. 1997;56:1262-1268.
8. Aylward EH, Brettschneider PD, McArthur JC, et al. Magnetic resonance imaging measurement of gray matter volume reductions in HIV dementia. Am J Psychiatry. 1995;152:987-994.
9. Jernigan TL, Archibald S, Hesselink JR, et al. Magnetic resonance imaging morphometric analysis of cerebral volume loss in human immunodeficiency virus infection. The HNRC Group. Arch Neurol. 1993;50:250-255.
10. Baleja JD. Structure determination of membrane-associated proteins from nuclear magnetic resonance data. Anal Biochem. 2001;288:1-15.
11. Yiannoutsos CT, Ernst T, Chang L, et al. Regional patterns of brain metabolites in AIDS dementia complex. Neuroimage. 2004;23:928-935.
12. Chang L, Ernst T, Leonido-Yee M, et al. Highly active antiretroviral therapy reverses brain metabolite abnormalities in mild HIV dementia. Neurology. 1999;53:782-789.
13. Pomara N, Crandall DT, Choi SJ, et al. White matter abnormalities in HIV-1 infection: a diffusion tensor imaging study. Psychiatry Res. 2001;106:15-24.
14. Filippi CG, Ulug AM, Ryan E, et al. Diffusion tensor imaging of patients with HIV and normal-appearing white matter on MR images of the brain. AJNR Am J Neuroradiol. 2001;22:277-283.
15. Ragin AB, Storey P, Cohen BA, et al. Whole brain diffusion tensor imaging in HIV-associated cognitive impairment. AJNR Am J Neuroradiol. 2004;25:195-200.
16. Wiley CA, Soontornniyomkij V, Radhakrishnan L, et al. Distribution of brain HIV load in AIDS. Brain Pathol. 1998;8:277-284.
17. Xing HQ, Moritoyo T, Mori K, et al. Simian immunodeficiency virus encephalitis in the white matter and degeneration of the cerebral cortex occur independently in simian immunodeficiency virus-infected monkey. J Neurovirol. 2003;9:508-518.
18. Raghavan R, Cheney PD, Raymond LA, et al. Morphological correlates of neurological dysfunction in macaques infected with neurovirulent simian immunodeficiency virus. Neuropathol Appl Neurobiol. 1999;25:285-294.
19. Gelman BB, Wolf DA, Rodriguez-Wolf M, et al. Mononuclear phagocyte hydrolytic enzyme activity associated with cerebral HIV-1 infection. Am J Pathol. 1997;151:1437-1446.
20. Luzio JP, Poupon V, Lindsay MR, et al. Membrane dynamics and the biogenesis of lysosomes. Mol Membr Biol. 2003;20:141-154.
21. Jansen SM, Groener JE, Bax W, et al. Biosynthesis of phosphatidylcholine from a phosphocholine precursor pool derived from the late endosomal/lysosomal degradation of sphingomyelin. J Biol Chem. 2001;276:18722-18727.
22. Morgello S, Gelman BB, Kozlowski PB, et al. The National NeuroAIDS Tissue Consortium: a new paradigm in brain banking with an emphasis on infectious disease. Neuropathol Appl Neurobiol. 2001;27:326-335.
23. Woods SP, Rippeth JD, Frol AB, et al. Interrater reliability of clinical ratings and neurocognitive diagnoses in HIV. J Clin Exp Neuropsychol. 2004;26:759-778.
24. Heaton RK, Grant I, Anthony WZ, et al. A comparison of clinical and automated interpretation of the Halstead-Reitan Battery. J Clin Neuropsychol. 1981;3:121-141.
25. Heaton RK, Velin RA, McCutchan JA, et al. Neuropsychological impairment in human immunodeficiency virus-infection: implications for employment. HNRC Group. HIV Neurobehavioral Research Center. Psychosom Med. 1994;56:8-17.
26. Remington R, Schork M. Statistics With Applications to the Biological Health Sciences. Englewood Cliffs, NJ: Prentice-Hall; 1970:253-281.
27. Chang L, Ernst T, Leonido-Yee M, et al. Cerebral metabolite abnormalities correlate with clinical severity of HIV-1 cognitive motor complex. Neurology. 1999;52:100-108.
28. Ernst T, Chang L, Arnold S. Increased glial metabolites predict increased working memory network activation in HIV brain injury. Neuroimage. 2003;19:1686-1693.
29. Lee PL, Yiannoutsos CT, Ernst T, et al. A multi-center 1H MRS study of the AIDS dementia complex: validation and preliminary analysis. J Magn Reson Imaging. 2003;17:625-633.
30. Moorjani H, Craddock BP, Morrison SA, et al. Impairment of phagosome-lysosome fusion in HIV-1-infected macrophages. J Acquir Immune Defic Syndr Hum Retrovirol. 1996;13:18-22.
31. Pittis MG, Sternik G, Sen L, et al. Impaired phagolysosomal fusion of peripheral blood monocytes from HIV-infected subjects. Scand J Immunol. 1993;38:423-427.
32. Banati RB, Rothe G, Valet G, et al. Detection of lysosomal cysteine proteinases in microglia: flow cytometric measurement and histochemical localization of cathepsin B and L. Glia. 1993;7:183-191.
33. Machaiah JP. Activation of lysosomal enzymes in chemotactically elicited rat peritoneal macrophages. Indian J Biochem Biophys. 1989;26:343-347.
34. Morita E, Sundquist WI. Retrovirus budding. Annu Rev Cell Dev Biol. 2004;20:395-425.
35. Pelchen-Matthews A, Kramer B, Marsh M. Infectious HIV-1 assembles in late endosomes in primary macrophages. J Cell Biol. 2003;162:443-455.
36. Sabri F, Tresoldi E, Di Stefano M, et al. Nonproductive human immunodeficiency virus type 1 infection of human fetal astrocytes: independence from CD4 and major chemokine receptors. Virology. 1999;264:370-384.
37. Glass JD, Fedor H, Wesselingh SL, et al. Immunocytochemical quantitation of human immunodeficiency virus in the brain: correlations with dementia. Ann Neurol. 1995;38:755-762.
38. Williams KC, Hickey WF. Central nervous system damage, monocytes and macrophages, and neurological disorders in AIDS. Annu Rev Neurosci. 2002;25:537-562.
39. Gorry PR, Ong C, Thorpe J, et al. Astrocyte infection by HIV-1: mechanisms of restricted virus replication, and role in the pathogenesis of HIV-1-associated dementia. Curr HIV Res. 2003;1:463-473.
© 2005 Lippincott Williams & Wilkins, Inc.