Polarized α-synuclein trafficking and transcytosis across Brain Endothelial Cells via Rab7-decorated carriers

Abstract

Parkinsons disease is mainly caused by aggregation of alpha-synuclein (α-syn) in the brain. Exchange of α-syn between the brain and peripheral tissues could have important pathophysiological and therapeutic implications, but the trafficking mechanism of α-syn across the blood brain barrier (BBB) remains unclear. In this study, we therefore investigated uptake and transport mechanisms of α-syn monomers and oligomers across an in vitro BBB model system. Both α-syn monomers and oligomers were internalized by primary brain endothelial cells, with increased restriction of oligomeric over monomeric transport. To enlighten the trafficking route of monomeric α-syn in brain endothelial cells, we investigated co-localization of α-syn and intracellular markers of vesicular transport. Here, we observed the highest colocalization with clathrin, Rab7 and VPS35, suggesting a clathrin-dependent internalization, preferentially followed by a late endosome retromer-connected trafficking pathway. Furthermore, STED microscopy revealed monomeric α-syn trafficking via Rab7-decorated carriers. Knockdown of Caveolin1, VPS35, and Rab7 using siRNA did not affect monomeric α-syn uptake into endothelial cells. However, it significantly reduced transcytosis of monomeric α-syn in the luminal-abluminal direction, suggesting a polarized regulation of monomeric α-syn vesicular transport. Our findings suggest a direct role for Rab7 in polarized trafficking of monomeric α-syn across BBB endothelium, and the potential of Rab7 directed trafficking to constitute a target pathway for new therapeutic strategies against Parkinsons disease and related synucleinopathies.

The impact of α-synuclein aggregates on blood-brain barrier integrity in the presence of neurovascular unit cells

“…Consistent with this, our data show that brain endothelial cells, with more anionic membrane to maintain the brain homeostasis, provide lower permeability [75] but also more interaction with labeled αSN compared to HUVEC cells. Recently, the mechanism and trafficking route of αSN across BBB have been demonstrated, indicating monomeric αSN internalization via clathrin-dependent endocytosis and subsequent retromer-mediated endosome, as well as its polarized trafficking [21]. Furthermore, internalization and bidirectional transportation of αSN in monomeric and oligomeric form have been shown [21].…”

“…Recently, the mechanism and trafficking route of αSN across BBB have been demonstrated, indicating monomeric αSN internalization via clathrin-dependent endocytosis and subsequent retromer-mediated endosome, as well as its polarized trafficking [21]. Furthermore, internalization and bidirectional transportation of αSN in monomeric and oligomeric form have been shown [21].…”

“…It is possible that early toxic aggregated species of αSN are produced in peripheral organs and cross BBB. αSN is known to cross the BBB in both directions [19, 20], although recent work shows that monomeric αSN, taken up in a clathrin-dependent pathway, is transported in a polarized fashion, largely in a luminal-abluminal direction [21]. On the other hand, oligomeric αSN is transported across the BBB to a much smaller extent [21], and transport of fibrillar species is likely to be even less efficient.…”

“…αSN is known to cross the BBB in both directions [19, 20], although recent work shows that monomeric αSN, taken up in a clathrin-dependent pathway, is transported in a polarized fashion, largely in a luminal-abluminal direction [21]. On the other hand, oligomeric αSN is transported across the BBB to a much smaller extent [21], and transport of fibrillar species is likely to be even less efficient. Thus, αSN aggregates could also be formed as a secondary response to neuronal deterioration and αSN clearance.…”

4-Oxo-2-nonenal-Induced α-Synuclein Oligomers Interact with Membranes in the Cell, Leading to Mitochondrial Fragmentation

“…40 It has also been shown that α-syn monomers colocalize with markers for late endosome/lysozyme Rab7 and the retromer protein VPS35 in brain endothelial cells after uptake from media. 41 Furthermore, mitochondrial fragmentation has been observed in a PD disease mouse model with a heterozygous (D620N) mutation of Vacuolar Protein Sorting 35 (VPS35). 42 PD with disease mutation D620N VPS35 resembles sporadic PD, indicating that common mechanisms may underlie both familial and sporadic PD.…”