Blood neuron-derived exosomes as biomarkers of cognitive impairment in HIV

Abstract

Objective: To investigate proteins associated with neuronal damage in plasma neuron-derived exosomes (NDE) of HIV-infected study participants as a liquid biomarker for cognitive impairment.

Methods: Plasma NDE were isolated using precipitation and immunoadsorption with antibody to a cell surface-specific neuronal marker. Total exosomes and NDE were enumerated, characterized, and proteins extracted and targets quantified by ELISA.

Results: Plasma NDE from 23 HIV seropositive individuals of which 11 had mild cognitive impairment, and 12 HIV seronegative controls of which three had cognitive impairment were isolated. NDE were enriched for the neuronal markers neurofilament light (NF-L) and synaptophysin (SYP). Neuropsychologically impaired individuals had fewer NDE compared with neuropsychologically normal study participants. NDE from neuropsychologically impaired study participants had significantly higher levels of high-mobility group box 1 (HMGB1), NF-L, and amyloid β proteins compared with neuropsychologically normal individuals. NDE HMGB1 protein significantly decreased with age in HIV-infected individuals.

Conclusion: Plasma NDE were altered in several ways in HIV infection. Elevated HMGB1, NF-L, and amyloid β proteins could distinguish cognitive impairment. NDE contents reflect neuronal health in 'real time' and may be useful for following cognitive impairment and response to therapy in HIV infection.

Fig. 1. Exosome characterization and normalization

(a) Exosomes were counted with a nanoparticle tracking system NanoSight LM10. The range of total exosome counts was 9.4 – 3900 x10⁹/mL. The range of neuron-derived exosome (NDE) counts was 0.13 – 270 x10⁹/mL, about 5% of the total exosomes. Individuals with HIV (N=23) had fewer total exosomes than controls (C, N=12); neuropsychologically impaired (NPI) individuals (N=14) had fewer NDE than neuropsychologically normal individuals (NPN, N=21). The units are log10 transformed exosome counts x10⁹/mL plasma. HIV (△); Controls ( ). Horizontal bars indicate group means. (b) Box-whisker plots show tetraspanins CD63 and CD81 concentrations measured by ELISA. Plasma contained high levels of CD63 (ranged 317–896 pg/mL, N=5). Total exosomes were enriched with CD63. Although NDE showed low levels of CD63, the number of NDE was only 5.2% of total exosomes. CD63 was also enriched in NDE but different in HIV compared to C. Total exosomes and NDE from all subjects (N=35) were enriched with CD81. NDE from HIV infection showed higher levels of CD63 and lower levels of CD81. Therefore, exosome counts were used for normalization. Plasma data included 2 controls and 3 HIV-infected subjects for CD63 and 2 controls, 5 HIV-infected subjects for CD81 analyses. Because no difference was found between plasma control and HIV levels for either CD63 or CD81, the two groups were combined. Concentrations of CD63 and CD81 were log10 transformed. The box-whisker plot indicates the 25^th, 50^th and 75^th percentile within the box, whiskers extending from the boxes indicate extreme values within 1.5 inter-quartile range from the 25^th and 75^th percentile respectively and the circles indicate outliers. (c) The source of NDE was further confirmed with neuronal markers. Neurofilament-light (NF-L) and synaptophysin (SYP) were both enriched in NDE compared to total exosomes. Concentrations of NF-L and SYP were normalized to exosome counts and log10 transformed. Horizontal bars indicate group means. Two-tailed Student’s t test with Benjamini and Hochberg multiple test correction was used. *P<0.05, **P <0.01, ***P< 0.001.

Fig. 2. NDE markers for neuropsychological impairment (NPI)

Levels of markers in exosome lysates were quantified with ELISA. (a) NPI individuals (N=14) had significantly elevated levels of high mobility group box 1 (HMGB1), neurofilament-light (NF-L) and amyloid beta (Aβ) compared with neuropsychologically normal individuals (NPN, N=21). Synaptophysin (SYP) was not significant. (b) CD63 was increased in NPI and CD81 was trending elevated in NPI subjects. Total exosomes showed similar levels from NPN and NPI subjects in all analytes tested. n.s., not significant; Total, total exosomes; NDE, neuron-derived exosomes. Data are shown as box-whisker plots as explained in Fig. 1b. Concentrations of analytes were normalized to exosome counts of each sample. Two-tailed Student’s t test was used, *P< 0.05.

Figure 3. Aging and neuron-derived exosome (NDE) proteins

(a) Aging correlated with higher exosome counts in NDE from individuals with HIV. The units were log10 transformed exosome counts x10⁹ per milliliter plasma. (b) High mobility group box 1 (HMGB1) decreased significantly with age in NDE from HIV infection; neurofilament-light (NF-L), amyloid beta (Aβ) and tetraspanins CD63 and CD81 were trending decrease with age in HIV infection. No effect was seen in NDE isolated from controls. The range of ages in controls (51–65 years) was narrower than HIV (35–67 years). Exosome protein levels were log10 transformed. Non-parametric Spearman correlation was used.