Detection and Characterization of Different Brain-Derived Subpopulations of Plasma Exosomes by Surface Plasmon Resonance Imaging

The use of exosomes for diagnostic and disease monitoring purposes is becoming particularly appealing in biomedical research because of the possibility to study directly in biological fluids some of the features related to the organs from which exosomes originate. A paradigmatic example are brain-derived exosomes that can be found in plasma and used as a direct read-out of the status of the central nervous system (CNS). Inspired by recent remarkable development of plasmonic biosensors, we have designed a surface plasmon resonance imaging (SPRi) assay that, taking advantage of the fact that exosome size perfectly fits within the surface plasmon wave depth, allows the detection of multiple exosome subpopulations of neural origin directly in blood. By use of an array of antibodies, exosomes derived from neurons and oligodendrocytes were isolated and detected with good sensitivity. Subsequently, by injecting a second antibody on the immobilized vesicles, we were able to quantify the amount of CD81 and GM1, membrane components of exosomes, on each subpopulation. In this way, we have been able to demonstrate that they are not homogeneously expressed but exhibit a variable abundance according to the exosome cellular origin. These results confirm the extreme variability of exosome composition and demonstrate how SPRi can provide an effective tool for their characterization. Besides, our work paves the road toward more precise clinical studies on the use of exosomes as potential biomarkers of neurodegenerative diseases

Neuromelanins of Human Brain Have Soluble and Insoluble Components with Dolichols Attached to the Melanic Structure

<div><p>Neuromelanins (NMs) are neuronal pigments of melanic-lipidic type which accumulate during aging. They are involved in protective and degenerative mechanisms depending on the cellular context, however their structures are still poorly understood. NMs from nine human brain areas were analyzed in detail. Elemental analysis led to identification of three types of NM, while infrared spectroscopy showed that NMs from neurons of substantia nigra and locus coeruleus, which selectively degenerate in Parkinson’s disease, have similar structure but different from NMs from brain regions not targeted by the disease. Synthetic melanins containing Fe and bovine serum albumin were prepared to model the natural product and help clarifying the structure of NMs. Extensive nuclear magnetic resonance spectroscopy studies showed the presence of dolichols both in the soluble and insoluble parts of NM. Diffusion measurements demonstrated that the dimethyl sulfoxide soluble components consist of oligomeric precursors with MWs in the range 1.4–52 kDa, while the insoluble part contains polymers of larger size but with a similar composition. These data suggest that the selective vulnerability of neurons of substantia nigra and locus coeruleus in Parkinson’s disease might depend on the structure of the pigment. Moreover, they allow to propose a pathway for NM biosynthesis in human brain.</p> </div

Solid state ¹³C NMR spectra of NM from PUT.

<p>(A) regions of interest in the <i>zg</i> spectrum; (B) high power decoupled spectrum. The intense signal at 110 ppm derives from the teflon insert, * indicates the spinning side band. The second (right hand) spinning side band (at ∼ 30 ppm) disappears beneath sample signals.</p

Transmission electron microscopy image of NM-containing organelles.

<p>Transmission electron microscopy image of a pigmented organelle from SN containing the NM pigment (white arrow) and lipid droplets (black arrow). The tissue was prepared as described in ref. 2 (scale bar 500 nm).</p

Summary of COSY, HSCQ and HMBC interactions ^a.

a<p>The asterisks (*) indicate the interaction was not observable in all NMs spectra, possibly due to low concentration of the pigment.</p

Elemental composition and C/N ratio of NM of SN, CAL and other brain areas ^a.

a<p>Average of 2–9 samples per brain area.</p

IR spectra of the three NM types.

<p>Comparison of IR spectra of SN-NM, other-NM (from PAL) and CAL-NM. Typical absorptions are indicated by asterisks (1230, 1194, 1163 cm⁻¹) and arrows (1774, 1015 cm⁻¹), for assignments see text.</p

Experimental ¹³C ppm values of NM pigments as determined by 2D ¹H-¹³C HSQC, compared to known values of bovine liver dolichol [27].

a<p>Quaternary carbon atom as determined by HMBC NMR.</p

1D proton HR-MAS and solution NMR spectra of NM from PUT.

<p>(A) HR-MAS water-presaturation pulse sequence with composite pulse; (B) solution spectrum with water suppression by gradient-tailored excitation. In the HR-MAS spectrum most major signals are shifted slightly upfield.</p

Theoretical percentage ^a of the various components in SN-NM, other-NM, and CAL-NM.

a<p>As calculated from elemental analysis data considering varying eu/pheo ratio for each NM type and assuming all nitrogen and sulphur derives from melanin.</p