Association of Aβ with ceramide-enriched astrosomes mediates Aβ neurotoxicity
Ahmed Elsherbini, Haiyan Qin, Zhihui Zhu, Priyanka Tripathi, Simone M. Crivelli, and Erhard Bieberich
Department of Physiology, University of Kentucky, Lexington, KY
Amyloid beta (Aβ) is a pathologic hallmark of Alzheimer’s disease (AD), however, the mechanism of Aβ neurotoxicity is not fully understood. Exosomes associate with Aβ, but it is not clear how this association would affect Aβ neurotoxicity. We report that the sphingolipid ceramide mediates neurotoxicity of Aβ. We show that sera and brains from AD transgenic mouse model (5xFAD) and sera from AD patients, but not the WT or healthy controls, contain a subpopulation of astrocyte-derived exosomes that are enriched with ceramide and are prone to aggregation (termed astrosomes) as confirmed by nanoparticle tracking and cluster analyses. When taken up by Neuro2A cells and human iPS cell-derived neurons, these astrosomes are shuttled to mitochondria where they induce mitochondria clustering, evident by elevation of expression of the fission protein dynamin related protein1 (Drp1). Using proximity ligation assays (PLA), we show that Aβ associates with voltage dependent anion channel 1 (VDAC1), a key protein in mitochondria-mediated apoptosis. PLA signals colocalized with ceramide cotransported with Aβ by astrosomes. The interaction between Aβ and VDAC1 leads to caspase3 activation and subsequently apoptosis. This effect was mitigated by removal of the ceramide enriched exosomes from the exosomes pool. Interestingly, the novel ceramide analog N-oleoyl serinol (S18) prevented the aggregation of exosomes, and Aβ association with astrosomes, and reduced Aβ interaction with VDAC1. Our data suggests that association of Aβ with ceramide in astrosomes enhances Aβ interaction with VDAC1 and mediates Aβ neurotoxicity in AD, which can be prevented by novel ceramide analogs.
This work is supported by NIH R01AG034389 and R01NS095215, and VA 1 I01 BX003643.
Reciprocal regulation of cell cycle and sphingolipid metabolism
Gabriel Soares Matos, Mónica Montero Lomeli
Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro
Sphingolipids are central bioactive lipids that have important functions in cell division. However, the control sphingolipid metabolism occurs during cell cycle is not well understood. To answer this question, we used S. cerevisiae as a model. We first studied the gene expression profile of key enzymes involved in the ceramide synthesis pathway and found, by qRT-PCR experiments, that genes involved in the synthesis of long chain bases (LCBs) and ceramides are periodically expressed during the mitotic cell cycle, having a peak at G1/S. This transcription peak coincides the canonical targets of the SBF complex, which is formed by the transcription factor SWI4 and its regulator SWI6, and which play a crucial role in G1/S transition. The transcription of sphingolipid genes was decreased in SBF mutants furthermore, SBF mutants were sensitive to myriocin, which inhibits the first step of sphingolipid synthesis. In addition, HPLC- MS/MS data indicated that the swi4Δ mutant had decreased levels of dihydrosphingosine, phytosphingosine (PHS) and total ceramides. A decrease in the length of ceramide fatty acids and in the hydroxylation of its long chain bases was also observed. The reduced sphingolipid content in swi4Δ strain was mimicked in WT cells by myriocin treatment and resulted in cell cycle arrest at the G2/M phase, which was reversed by addition of PHS to the media. Our results, overall suggests that the SWI4 transcription factor is important to coordinate transcription and synthesis of sphingolipids, which may affect not just membrane architecture, but also the biological activity of ceramides which are reported to control a myriad of phenomena including cell cycle progression.
Sphingosine-1-phoshate signaling in astrocytes is a critical modulator
of stroke outcome
Matuskova H. 1,2,3,4, Matthes F.3,4, Petzold G. C.1,2 and Meissner A.3,4
1 Department of Neurology, University Hospital Bonn, Germany
2 German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany
3 Department of Experimental Medical Sciences, Lund University, Sweden
4 Wallenberg Centre for Molecular Medicine, Lund University, Sweden
Stroke remains a leading cause of long-term disability worldwide. Due to its complexity, treatment options are sparse. The bioactive phospholipid sphingosine-1-phosphate (S1P) is involved in variety of physiological processes particularly, in vascular and immune cell responses. Moreover, altered S1P levels have been reported in several cardiovascular and inflammation-associated diseases, including stroke. As astrocytes play a critical role in the regulation of both vascular and immune responses in the injured brain, we sought to investigate astrocytic S1P signaling and its contribution to stroke. In a mouse model of transient middle cerebral artery occlusion (MCAo), we discovered significantly increased expression of one of S1P’s receptors (S1PR3) 24 hrs post-ischemia in the ipsilateral hemisphere. Vessel-parenchyma fractionation of brain tissue revealed the majority of S1PR3 protein associated to cerebral vessels. Astrocyte-specific RiboTag analysis confirmed an augmentation of ipsilateral S1PR3 expression 24 and 72 hrs post-stroke. This was further supported by colocalization of Gfap, a marker of reactive astrocytes, and S1PR3 in the ischemic hemisphere by RNA scope technique. Moreover, single administration of an S1PR3 antagonist 4 hrs after permanent MCAo revealed significant improvements of regional cerebral blood flow in the ipsilateral hemisphere 24 hrs that persisted after 72 hrs. Consequently, infarct size was markedly reduced in mice treated with S1PR3 antagonist. In conclusion, our findings point to an important involvement of the S1P/S1PR3 signaling axis during stroke, and a potential contribution of astrocytes. Modulating S1PR3-mediated vascular and inflammatory responses may emerge as viable target to improving stroke outcome.
Sphingomyelin serves a critical role in the repair of damaged organelles
Patrick Niekamp, Joost Holthuis
Molecular Cell Biology Division, University of Osnabrück, 49076 Osnabrück,, Germany
Sphingomyelin (SM) displays a strict asymmetric distribution across cellular membranes, with
the bulk localized to the exoplasmic leaflet of late secretory and endocytic organelles. Using an engineered SM-binding probe, we and others (Ellison et al., 2020, Curr Biol 30, 1-10) found that SM is readily exposed to the cytosolic surface of these organelles upon membrane damage inflicted by pathogenic bacteria, lysosomotropic drugs or a two-photon laser. Remarkably, cytosolic exposure of SM at sites of membrane damage precedes the recruitment of galectins, ESCRT proteins and other components of the membrane repair machinery, suggesting that a break in SM asymmetry may serve a fundamental role in the mechanism by which cells detect and repair damaged organelles. By monitoring the recovery of LysoTracker fluorescence in lysosomes transiently exposed to lysosomotropic drugs, we found that cells lacking SM displayed a significant defect in the repair of damaged lysosomal membranes in comparison to control cells. Moreover, SM-deficient cells showed an enhanced sensitivity to lysosomotropic drugs and a prolonged retention of ESCRT-III components on their damaged lysosomes. These phenotypes could be suppressed by restoring SM biosynthesis. Analogous to the role of phosphatidylserine displayed on the surface of damaged cells, we postulate that cytosolic SM serves as a key indicator of damaged organelles and actively participates in their repair. In this talk, I will present our latest findings regarding the mechanism by which SM contributes to the mending of damaged organelles.