Mikael Simons

Ceramide Triggers Budding of Exosome Vesicles into Multivesicular Endosomes

Intraluminal vesicles of multivesicular endosomes are either sorted for cargo degradation into lysosomes or secreted as exosomes into the extracellular milieu. The mechanisms underlying the sorting of membrane into the different populations of intraluminal vesicles are unknown. Here, we find that cargo is segregated into distinct subdomains on the endosomal membrane and that the transfer of exosome-associated domains into the lumen of the endosome did not depend on the function of the ESCRT (endosomal sorting complex required for transport) machinery, but required the sphingolipid ceramide. Purified exosomes were enriched in ceramide, and the release of exosomes was reduced after the inhibition of neutral sphingomyelinases. These results establish a pathway in intraendosomal membrane transport and exosome formation.

Exosomes--vesicular carriers for intercellular communication.

Cells release different types of vesicular carriers of membrane and cytosolic components into the extracellular space. These vesicles are generated within the endosomal system or at the plasma membrane. Among the various kinds of secreted membrane vesicles, exosomes are vesicles with a diameter of 40-100 nm that are secreted upon fusion of multivesicular endosomes with the cell surface. Exosomes transfer not only membrane components but also nucleic acid between different cells, emphasizing their role in intercellular communication. This ability is likely to underlie the different physiological and pathological events, in which exosomes from different cell origins have been implicated. Only recently light have been shed on the subcellular compartments and mechanisms involved in their biogenesis and secretion opening new avenues to understand their functions.

Selective transfer of exosomes from oligodendrocytes to microglia by macropinocytosis

The transfer of antigens from oligodendrocytes to immune cells has been implicated in the pathogenesis of autoimmune diseases. Here, we show that oligodendrocytes secrete small membrane vesicles called exosomes, which are specifically and efficiently taken up by microglia both in vitro and in vivo. Internalisation of exosomes occurs by a macropinocytotic mechanism without inducing a concomitant inflammatory response. After stimulation of microglia with interferon-γ, we observe an upregulation of MHC class II in a subpopulation of microglia. However, exosomes are preferentially internalised in microglia that do not seem to have antigen-presenting capacity. We propose that the constitutive macropinocytotic clearance of exosomes by a subset of microglia represents an important mechanism through which microglia participate in the degradation of oligodendroglial membrane in an immunologically ‘silent’ manner. By designating the capacity for macropinocytosis and antigen presentation to distinct cells, degradation and immune function might be assigned to different subtypes of microglia.

Neurotransmitter-triggered transfer of exosomes mediates oligodendrocyte-neuron communication.

Neuronal activity provokes myelinating oligodendrocytes to release exosomes by stimulation of ionotropic glutamate receptors, and that once released, these vesicles are internalized by neurons conveying neuroprotection.

Cholesterol and Alzheimer’s disease Is there a link?

Article abstract— The Aβ-amyloid peptide (Aβ), the main component of amyloid plaques, is derived by proteolytic cleavage from the amyloid precursor protein (APP). Epidemiologic and biochemical data suggest a link between cholesterol, APP processing, Aβ, and Alzheimer’s disease. Two recent epidemiologic studies indicate that there is a decreased prevalence of AD associated with the use of cholesterol-lowering drugs that inhibit 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase inhibitors or statins). Experiments in cell culture and in vivo demonstrate that treatment with statins reduces production of Aβ. The authors discuss how cholesterol might modulate Aβ deposit formation. As neurons receive only small amounts of exogenous cholesterol, statins that efficiently cross the blood–brain barrier may reduce the amount of neuronal cholesterol below a critical level. Decreased neuronal cholesterol levels inhibit the Aβ-forming amyloidogenic pathway possibly by removing APP from cholesterol- and sphingolipid-enriched membrane microdomains. In addition, depletion of cellular cholesterol levels reduces the ability of Aβ to act as a seed for further fibril formation. These intriguing relationships raise the hopes that cholesterol-lowering strategies may influence the progression of AD.

Efficient Inhibition of the Alzheimer's Disease β-Secretase by Membrane Targeting

β-Secretase plays a critical role in β-amyloid formation and thus provides a therapeutic target for Alzheimers disease. Inhibitor design has usually focused on active-site binding, neglecting the subcellular localization of active enzyme. We have addressed this issue by synthesizing a membrane-anchored version of a β-secretase transition-state inhibitor by linking it to a sterol moiety. Thus, we targeted the inhibitor to active β-secretase found in endosomes and also reduced the dimensionality of the inhibitor, increasing its local membrane concentration. This inhibitor reduced enzyme activity much more efficiently than did the free inhibitor in cultured cells and in vivo. In addition to effectively targeting β-secretase, this strategy could also be used in designing potent drugs against other membrane protein targets.

Production of intracellular amyloid-containing fragments in hippocampal neurons expressing human amyloid precursor protein and protection against amyloidogenesis by subtle amino acid substitutions in the rodent sequence.

A distinguishing feature of Alzheimers disease (AD) is the deposition of amyloid plaques in brain parenchyma. These plaques arise by the abnormal accumulation of beta A4, a proteolytic fragment of amyloid precursor protein (APP). Despite the fact that neurons are dramatically affected in the course of the disease, little is known about the neuronal processing of APP. To address this question we have expressed in fully mature, synaptically active rat hippocampal neurons, the neuronal form of human APP (APP695), two mutant forms of human APP associated with AD, and the mouse form of APP (a species known not to develop amyloid plaques). Protein expression was achieved via the Semliki Forest Virus system. Expression of wild type human APP695 resulted in the secretion of beta A4‐amyloid peptide and the intracellular accumulation of potential amyloidogenic and non‐amyloidogenic fragments. The relative amount of amyloid‐containing fragments increased dramatically during expression of the clinical mutants, while it decreased strongly when the mouse form of APP was expressed. ‘Humanizing’ the rodent APP sequence by introducing three mutations in the beta A4‐region also led to increased production of amyloid peptide to levels similar to those obtained with human APP. The single Gly601 to Arg substitution alone was sufficient to triple the ratio of beta A4‐peptide to non‐amyloidogenic p3‐peptide. Due to the capacity of these cells to secrete and accumulate intracellular amyloid fragments, we hypothesize that in the pathogenesis of AD there is a positive feed‐back loop where neurons are both producers and victims of amyloid, leading to neuronal degeneration and dementia.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell type- and brain region-resolved mouse brain proteome

Brain transcriptome and connectome maps are being generated, but an equivalent effort on the proteome is currently lacking. We performed high-resolution mass spectrometry–based proteomics for in-depth analysis of the mouse brain and its major brain regions and cell types. Comparisons of the 12,934 identified proteins in oligodendrocytes, astrocytes, microglia and cortical neurons with deep sequencing data of the transcriptome indicated deep coverage of the proteome. Cell type–specific proteins defined as tenfold more abundant than average expression represented about a tenth of the proteome, with an overrepresentation of cell surface proteins. To demonstrate the utility of our resource, we focused on this class of proteins and identified Lsamp, an adhesion molecule of the IgLON family, as a negative regulator of myelination. Our findings provide a framework for a system-level understanding of cell-type diversity in the CNS and serves as a rich resource for analyses of brain development and function.

Myelin Membrane Wrapping of CNS Axons by PI(3,4,5)P3-Dependent Polarized Growth at the Inner Tongue

Central nervous system myelin is a multilayered membrane sheath generated by oligodendrocytes for rapid impulse propagation. However, the underlying mechanisms of myelin wrapping have remained unclear. Using an integrative approach of live imaging, electron microscopy, and genetics, we show that new myelin membranes are incorporated adjacent to the axon at the innermost tongue. Simultaneously, newly formed layers extend laterally, ultimately leading to the formation of a set of closely apposed paranodal loops. An elaborated system of cytoplasmic channels within the growing myelin sheath enables membrane trafficking to the leading edge. Most of these channels close with ongoing development but can be reopened in adults by experimentally raising phosphatidylinositol-(3,4,5)-triphosphate levels, which reinitiates myelin growth. Our model can explain assembly of myelin as a multilayered structure, abnormal myelin outfoldings in neurological disease, and plasticity of myelin biogenesis observed in adult life.

Wrapping it up: the cell biology of myelination

During nervous system development, oligodendroglia in the central nervous system (CNS) and Schwann cells in the peripheral nervous system (PNS) synthesise large amounts of specific proteins and lipids to generate myelin, a specialised membrane that spirally ensheathes axons and facilitates fast conduction of the action potential. Myelination is initiated after glial processes have attached to the axon and polarisation of the plasma membrane has been triggered. Myelin assembly is a multi-step process that occurs in spatially distinct regions of the cell. We propose that assembly of myelin proteins and lipids starts during their transport through the biosynthetic pathway and continues at the plasma membrane aided by myelin-basic protein (MBP). These sequential processes create the special lipid and protein composition necessary for myelin to perform its insulating function during nerve conduction.