Increased accumulation of intraneuronal amyloid beta in HIV-infected patients

Abstract

In recent years, human immunodeficiency virus (HIV)-infected patients under highly active anti-retroviral therapy (HAART) regimens have shown a markedly improved general clinical status; however, the prevalence of mild cognitive disorders has increased. We propose that increased longevity with HIV-mediated chronic inflammation combined with the secondary effects of HAART may increase the risk of early brain aging as shown by intraneuronal accumulation of abnormal protein aggregates like amyloid beta (Abeta), which might participate in worsening the neurodegenerative process and cognitive impairment in older patients with HIV. For this purpose, levels and distribution of Abeta immunoreactivity were analyzed in the frontal cortex of 43 patients with HIV (ages 38-60) and HIV- age-matched controls. Subcellular localization of the Abeta-immunoreactive material was analyzed by double labeling and confocal microscopy and by immunono-electron microscopy (EM). Compared to HIV- cases, in HIV+ cases, there was abundant intracellular Abeta immunostaining in pyramidal neurons and along axonal tracts. Cases with HIV encephalitis (HIVE) had higher levels of intraneuronal Abeta immunoreactivity compared to HIV+ cases with no HIVE. Moreover, levels of intracellular Abeta correlated with age in the group with HIVE. Double-labeling analysis showed that the Abeta-immunoreactive granules in the neurons co-localized with lysosomal markers such as cathepsin-D and LC3. Ultrastructural analysis by immuno-EM has confirmed that in these cases, intracellular Abeta was often found in structures displaying morphology similar to autophagosomes. These findings suggest that long-term survival with HIV might interfere with clearance of proteins such as Abeta and worsen neuronal damage and cognitive impairment in this population.

Fig. 1

Patterns of Aβ immunoreactivity in control and HIV+ cases. Panels are from the frontal cortex immunostained with the monoclonal antibody 4G8. a–c In an age-matched control HIV− case (42 year old) the neuronal cell bodies (a), axons in the white matter (b), and neuropil (c) are devoid of amyloid deposits. d–f Examples of intraneuronal (d) granular Aβ immunoreactivity in an older HIV+ case (47 year old). Aβ deposits can be found in axons (e) and in the neuropil (f) as diffuse plaques. g–i Examples of intraneuronal (g) and axonal (h) Aβ immunoreactivity (arrows) in an older HIV+ case (50 year old) with HIVE. The inset shows in greater detail the punctate appearance of the intraneuronal Aβ immunostaining. Diffuse amyloid plaques (i) were also detected in a few cases. Bar=10 μm

Fig. 2

Levels of intraneuronal Aβ immunoreactivity in older HIV+ cases. Images are from the frontal cortex immunostained with the monoclonal antibody 4G8. a–d Examples of the various levels (0–4) of intraneuronal Aβ immunoreactivity in HIV+ cases; the arrow indicates the enlarged image to the left displaying punctate appearance. e Compared to HIV+ with no HIVE, in cases with HIVE, there was an increase in the levels of intraneuronal Aβ immunoreactivity. Bar=5 μm

Fig. 3

Laser confocal microscopy imaging of the amyloid deposits in HIV+ cases. Examples are from the frontal cortex. a No evidence of amyloid deposits in HIV− age-matched control; b, c double-labeling with antibodies against the neuronal markers NeuN (red) and Aβ (green) showing neuronal amyloid deposits (b, arrows) and diffuse plaques (c, amyl) in HIV+ cases. d–f Comparative images in HIV− (d) and HIV+ (e, f) cases stained with thioflavine S. Bar=10 μm

Fig. 4

Linear regression analysis between intracellular Aβ and age. a In cases with HIVE, there was a significant correlation. b In cases with no HIVE, there was no significant correlation

Fig. 5

Laser confocal microscopy imaging of the intraneuronal Aβ and lysosomal markers in older HIV+ cases. Sections are from the frontal cortex double labeled with the Aβ (4G8) antibody (green) and lysosomal proteins cathepsin-D, LAMP2, and LC3 (red). a–c HIV− control case. d–l Co-localization (arrows) of intraneuronal Aβ and cathepsin-D (d–f), LAMP2 (g–i), or LC3 (j–l) in an HIV+ case. Bar=5 μm

Fig. 6

Ultrastructural analysis of the Aβ deposits in HIV+ cases. Sections from the frontal cortex were immunostained pre-embedding with 4G8 and analyzed with the electron microscope. a In a control, HIV−case, evidence of preservation of neuronal organelles such as lysosomes (lys) and mitochondria (m). b In an HIV+ case, abnormal electrodense membrane-bounded organelles (arrows) displaying Aβ immunoreactivity accumulate in the neuronal cell body and axons. c Additional view of abnormal electrodense membrane-bounded organelles displaying intraneuronal Aβ immunoreactivity in an HIV+ case. Bar=2 μm