Previous studies of beta amyloid deposition in the AIDS brains have focused on the human β-amyloid precursor protein (APP) because this protein is considered to be a marker of neuronal degeneration. To date, several reports have described a significant increase in brain APP in AIDS, specifically in the axons present in the subcortical white matter tracts. While most investigators agree that increased levels of APP suggest axonal pathology, the relationship with HIV associated pathology in the brain is still controversial.
It has been postulated that inflammatory responses to HIV infection of the brain parenchyma can promote over-production and accumulation of APP [1,2]. This inflammatory response may be mediated by activated microglia, the resident macrophages of the brain, or astrocytes, the predominant glial cells of the brain. These cells secrete the cytokines IL-1α and S100 respectively, both of which have been shown to cause the overproduction of neuronal APP . It is theorized that the observed increase in APP may be due to cytokine production by microglia and astrocytes secondary to their activation by a generalized inflammatory response against virus in the brain.
Although a causative relationship with brain pathology has not been clearly identified, a strong association between HIV encephalitis (HIVE) and the presence of APP has been confirmed in several studies. The distribution of viral proteins, such as p24, correlates well with the distribution of APP aggregates that are often observed as intra-axonal globules. Results from human studies have been additionally confirmed in non-human primate models of disease. Using a simian immunodeficiency virus (SIV) model, Mankowski et al. have also found APP accumulation in degenerating axons .
In one of the first studies of APP in the HIV brain, An and colleagues  did not find a good correlation between APP and microgliosis, proposing that the axonal degeneration in HIV infection may not be due to direct microglial interactions. Instead, systemic factors may underlie the observed APP accumulation, reflecting mild pathological changes. In a series of in-vivo studies using double transgenic mice overexpressing human APP and the HIV envelope protein gp120, Mucke and Masliah have demonstrated that the hAPP may actually be protective against gliosis and synaptic loss . Finally, hAPP has been shown to be clearly protective against excitotoxicity, refuting the hypothesis that S100-mediated increases in neuronal calcium may induce toxic APP accumulation .
We analyzed the prevalence and distribution of β-amyloid plaques in the HIV+ brain, and determine whether they correlate with in-vivo neuro-behavioral assessments. In order to ascertain whether amyloid deposition was correlated to secondary pathology, we compared the brains of randomly selected HIV+ patients treated with highly active anti-retroviral therapy (HAART) to age and gender matched historical controls, who did not have access to modern combinatorial retroviral therapies. We hypothesized that the incidence of amyloid deposition will increase in the era of HAART despite reductions in viral burden, incidence of HIVE, decreased inflammatory cytokine production, and overall decreased morbidity related to HIV infection.
Materials and Methods
We analyzed archival brain tissues collected from 162 AIDS autopsies at UCSD and UCLA between 1983 and 2001. The brain regions examined included frontal cortex (gray and white matter), hippocampus, caudate, putamen and globus pallidus. The tissue blocks from UCSD were typical paraffin embedded histology cassettes while the UCLA cases were re-assembled into paraffin embedded “tissue microarrays” (TMA) as previously described . The majority of the patients died of respiratory failure due to bronchopneumonia and the general autopsy findings were consistent with AIDS. The associated pathology was most frequently due to systemic CMV, Kaposi sarcoma and liver cirrhosis.
From the 112 UCLA cases, 106 were males with an average age of 41.5 years (range 24–72). In this group, the prevalent brain pathologies were: microglial nodules (MGN) (n = 25), cytomegalovirus encephalitis (CMVE) (n = 17), multinucleated giant cells (MNGC) (n = 12), Cryptococcus (n = 9), CNS lymphoma (CNSL) (n = 7), progressive multifocal leukoencephalopathy (PML) (n = 4), toxoplasma (n = 4), while 45 patients had no significant neuropathology. In the 50 AIDS autopsy cases from UCSD, 46 were males, with an average age of 46.5 years (range 25–75). The main neuropathologic diagnoses were: MGN (n = 27), MNGC (n = 16), CMVE (n = 3), CNSL (n = 2), and PML (n = 2), while 15 cases were “normal”.
Briefly, the primary mouse monoclonal antibodies, 4G8 and 6E10 (Signet Labs, Dedham, MA, cat#9220 and 9330), diluted to 1:200, were incubated for 2 hours at room temperature (RT). Following another 30′ incubation with secondary antibodies (biotinylated goat-anti mouse) and 30′ ABC, color reactions were developed with NovaRed (Vector Labs).
The immunostaining results were scored on a 0 – 2 scale by at least two independent observers. Since the staining with the 4G8 antibody was significantly stronger than 6E10, it was used for all the statistical analyses. Each tissue section was reviewed by light microscopy (10–60X magnifications) and assessed for presence of beta-amyloid staining: 0 = no signal, 1 = intra-neuronal and vascular β-amyloid present (always in neurons and occasionally in vessel walls), 2 = β-amyloid extra-cellular plaques in addition to intra-neuronal staining.
Statistical analysis was performed using the 145 male patients. Individuals were divided into two groups: those with access to combinatorial anti-retroviral therapy (n = 46) and those without (n = 99). Following independent analysis by at least two investigators, the two groups were further subdivided into 3 categories based on neuropahtological assessment scores of 0, 1 or 2. χ2 analysis for categorical data was performed between groups for age and deposition score. Yates’ correction for continuity was used with χ2 analysis for grouped, ordinal data. Multiple linear regression analysis and ANOVA was performed to determine the correlation between amyloid deposition scores and in-vivo neuro-behavioral test scores. P Values less than 0.05 (P < 0.05) were considered significant. Statistical analysis was performed using SPSS v.11 for Macintosh OS X, and the results depicted with SPSS and Excel v.X.
Immunostaining for β-amyloid showed significant deposition in the frontal cortex of almost half of the AIDS brains studied. Similar but less abundant findings were observed in the hippocampus and occasionally in the basal ganglia. For consistency, we chose to do statistical analysis on the frontal cortex of 150 males from our original study group of 162. The distribution of β-amyloid identified immunocytochemistry was predominantly in the soma and axonal processes (Fig. 1d).
Extracellular plaques were often perivascular but a clear distribution could not be determined. In a few cases, β-amyloid was detected in the walls of brain vessels in addition to intra-neuronal deposition. None of the AIDS brains analyzed were positive for neurofibrillary tangles. ApoE genotyping was available in a subset of the patients studied. Due to the limited number of cases studied in this category the correlation analysis could not reach statistical significance although a trend could be determined: all ApoE 3,4 cases had beta-amyloid in the brain.
In comparison to the abundant parenchymal, extra-cellular deposition typical of AD (Fig. 1a), in the AIDS brains we studied, β-amyloid was found both in plaques (Figs 1b and c) and inside neurons (1d). The intra-cellular deposition of β-amlyoid (score = 1) was the most frequent finding, followed by combined intra-neuronal amyloid and plaques. There was no clear association with HIV related pathology although a trend suggests that many of the 4G8 positive cases had also microglial nodules in the brain. While the number of the cases studied with confirmed, consistent, long-term HAART is relatively low, a clear trend suggests increased β-amyloid deposition in the HAART era (Fig. 2).
We found that there is a statistically significant increase in β-amyloid deposition in the HAART era (Fig. 2) and in older patients. Detailed neuro-psychiatric evaluations were available for 18 patients, of whom 3 had medical histories of HIV associated dementia (HAD). While we have identified significant, presumably amyloid associated neuropathology in these patients, it does not appear to be associated with HAD or the minor motor-cognitive impairment previously described. It is possible that individual variability in the ability to compensate for amyloid mediated toxicity obscured any relationship between the two findings in this small sample.
The first study to identify changes similar to mild AD was reported by Esiri et al.  who found β-amyloid plaques in HIV patients by using argyrophilic and thioflavine stainings. Another study using a more comprehensive methodology that included staining with the 4G8 antibody, Congo red and Thioflavine-S  showed that in 15 AIDS cases (5 with HIVE), 3 had perivascular plaques positive for 4G8 but not Congo Red or thioflavine-S. Based on these findings, the authors concluded that the neurodegeneration associated with HIV infection could be primarily of vascular origin. Neuronal degeneration is a major feature of HIV infection, and APP is considered to be a marker of axonal transport injury . Axonal spheroids positive for APP by ICC, are associated with microglial activation and are believed to represent early signs of neurodegeneration . Intracellular APP is moved by fast axonal transport and it may be the most accurate marker of neuronal cytoskeletal breakdown and dysfunctional processing [13,14].
Several studies showed that APP deposition may precede β-amyloid accumulation  in the vicinity of synapses suggesting that APP may be processed at these sites. Other investigators believe that beta-amyloid deposition is the primary event that, in combination with reactive surrounding glia, may lead to APP accumulation in degenerating neurites . In vitro, β-amyloid treatments induce degeneration of neurites similar to AD , possibly by inhibiting fast axonal anterograde transport .
In conclusion, we observed a statistically significant increase in parenchymal β-amyloid deposition in the HAART era. It has been proposed that HIV persists in the brain during HAART therapy, and our findings suggest that local inflammatory responses to HIV in the brain could lead to increased APP production and susceptibility to amyloid deposition. Alternatively, HAART therapy itself may contribute to an overall increase in amyloid deposition. We speculate that this may be mediated by inhibition of insulin degradation enzyme. We interpret our results as suggesting that β-amyloid may be a good indicator of early neuronal (axonal) degeneration but not necessarily a causative agent. Future longitudinal studies of long-term survivors on HAART will most likely resolve this issue.
We are grateful to Dr. David B. Seligson from the UCLA Tissue Micro-array Facility for assistance with TMA preparations. Drs. Igor Grant, Ron Ellis and the HNRC in San Diego offered key support in assembling the data for the study cohort. Jessica Schindelar (University of Pittsburgh), Anthony Adame and Salomon Maya (UCSD) provided excellent technical help.
Support: MH58528 to CLA, HNRC Developmental Grant to DAG, P50 AG 16570, and MACS Contract #U01 AI/CA 35040 to HVV.
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