Mitochondria-associated membranes as hubs for neurodegeneration

Abstract

There is a growing appreciation that membrane-bound organelles in eukaryotic cells communicate directly with one another through direct membrane contact sites. Mitochondria-associated membranes are specialized subdomains of the endoplasmic reticulum that function as membrane contact sites between the endoplasmic reticulum and mitochondria. These sites have emerged as major players in lipid metabolism and calcium signaling. More recently also autophagy and mitochondrial dynamics have been found to be regulated at ER-mitochondria contact sites. Neurons critically depend on mitochondria-associated membranes as a means to exchange metabolites and signaling molecules between these organelles. This is underscored by the fact that genes affecting mitochondrial and endoplasmic reticulum homeostasis are clearly overrepresented in several hereditary neurodegenerative disorders. Conversely, the processes affected by the contact sites between the endoplasmic reticulum and mitochondria are widely implicated in neurodegeneration. This review will focus on the most recent data addressing the structural composition and function of the mitochondria-associated membranes. In addition, the 3D morphology of the contact sites as observed using volume electron microscopy is discussed. Finally, it will highlight the role of several key proteins associated with these contact sites that are involved not only in dementias, amyotrophic lateral sclerosis and Parkinson's disease, but also in axonopathies such as hereditary spastic paraplegia and Charcot-Marie-Tooth disease.

Fig. 1

3D morphology of ER-mitochondria contact sites. Mouse embryonic fibroblasts were imaged using a Zeiss Auriga Crossbeam focused ion beam scanning electron microscope (FIB-SEM). By automated serial block face imaging, large image stacks are generated at high resolution, allowing precise 3D reconstruction of a large cellular volume. In this dataset, over 600 10 nm z-slices were obtained resulting in a 3D dataset at 5 × 5 × 10 nm³ voxels. Mitochondria and their contact sites with the endoplasmic reticulum (ER) were manually segmented and 3D reconstructed in IMOD (http://bio3d.colorado.edu/imod/). A video of this dataset and the reconstruction can be seen in Supplementary movie 1. a The complete reconstruction of two mitochondria (transparent green) and their ER-mitochondria contact sites (magenta) is shown. A contact site is defined as a region where the ER and the mitochondrial membranes are in closer proximity than 30 nm. It is clear that a single mitochondrion makes multiple contacts with the ER and that these contacts are diverse in size, ranging from punctate sites to large patches of the outer mitochondrial membrane being juxtaposed to the ER. b–e Represent different examples of scanning electron micrographs extracted from the volume illustrating a section of the mitochondria and their contacts with the ER. The reconstructed mitochondria depicted in a are shown in transparent green. Magenta arrowheads mark the borders of the ER-mitochondria contact sites. The position of these slices is depicted in blue in a. Scale bar 200 nm

Fig. 2

Structural components of ER-mitochondria contact sites. Upper part in mammalian cells, dimers between endoplasmic reticulum (ER)-localized Mitofusin (MFN) 2 and mitochondrial MFN1/2 were the first proposed protein tethers. Charcot–Marie–Tooth (CMT)-causing mutations in MFN2 are believed to decrease ER-mitochondrial contact, contributing to the disease. The interaction of VAMP-associated protein B and C (VAPB) in the ER membrane with protein tyrosine phosphatase-interacting protein 51 (PTPIP51) in the outer mitochondrial membrane (OMM) also contributes to anchoring the mitochondria-associated membrane (MAM) to the mitochondrial membrane. This interaction is inhibited by TAR DNA-binding protein 43 (TDP43) in a glycogen synthase kinase 3β (GSK3β) dependent manner. Both these proteins are implied in neurodegeneration (see Table 1; Boxes 1, 2). The amyotrophic lateral sclerosis (ALS)-causing P56S mutation in VAPB on the other hand increases the physical interaction between the ER and mitochondria (Table 1; Box 2). Phosphofurin acidic cluster protein 2 (PACS2) is established as an essential component of these contacts. The levels of PACS2 are found to be altered in the brains of Alzheimer’s dementia (AD) patients (see Table 1; and Box 1). A functional rather than a structural component of the MAMs is formed by a complex between ER-resident inositol 1,4,5-triphosphate (IP3) channels and the mitochondria resident voltage-dependent anion channel (VDAC), which are bridged by Grp75 and important for calcium shuttling between ER and mitochondria (see Fig. 3). Lower part in Saccharomyces cerevisiae, two tethering complexes are known: the endoplasmic reticulum (ER) mitochondria encounter structure (ERMES), composed of the mitochondrial Mdm10 and Mdm34 proteins, the ER-based Mmm1 protein and the cytosolic Mdm12 protein was the first tether to be described. A second tether important for yeast phospholipid metabolism is achieved through the interaction between the ER membrane complex (EMC) and the outer mitochondrial membrane translocate complex 5 (TOM5). The Miro GTPase Gem1 is a regulatory subunit of ERMES and also Lam6 plays a modulating role determining the extent of membrane contact. It is currently not known whether mammalian orthologs of these components play similar roles in mammalian MAMs

Fig. 3

Lipid metabolism at MAMs. Historically, MAMs were identified as essential regions for phospholipid metabolism. For phosphatidylethanolamine (PE) synthesis, phosphatidylserine (PS) synthesized in the endoplasmic reticulum (ER) from phosphatidic acid (PA) by the PS synthetases 1 and 2 (Pss1/2) needs to be shuttled to the inner mitochondrial membrane (IMM), where it is decarboxylated to PE by the PS decarboxylase (Psd). This PE can then be shuttled back to the ER and used in further lipid metabolism, such as conversion to phosphatidylcholine (PC) by the PE-N-methyltransferase (PEMT). In addition, the synthesis of sterols requires the import of cholesterol (chol) from the ER into mitochondria

Fig. 4

Calcium signaling at ER-mitochondria membrane contact sites. Transfer of calcium to mitochondria requires calcium hotspots, which can be achieved at mitochondria-associated membranes (MAMs) owing to the quasi-synaptic structure of this membrane contact site (MCS). Shuttling of calcium is needed during a variety of responses, as it can both stimulate ATP synthesis and promote mitophagy and apoptosis. Efficient import of calcium at MAMs is mediated by Grp75, which brings the openings of the inositol 1,4,5-triphosphate receptor (IP3R) calcium channels in the endoplasmic reticulum (ER) in close vicinity to the voltage dependent anion channel (VDAC) in the outer mitochondrial membrane (OMM). Opening of the IP3Rs is regulated by a set of proteins present at or recruited to MAMs, including calnexin (CNX), the Sigma non-opioid intracellular receptor 1 (Sigma1R), presenilin 1 and 2 (PS1 and PS2). The presence of CNX at MAMs in turn is regulated by the sorting proteins Rab32 and phosphofurin acidic cluster protein 2 (PACS2), as well as by palmitoylation of CNX. The association of Sigma1R with MAMs on the other hand depends on cholesterol levels. Dysregulation of calcium signaling at MAMs is strongly implied in neurodegenerative disorders. PS1 and PS2 play a complex role regulating both the extent of ER-mitochondrial contact and calcium signaling. The Parkinson’s disease (PD)-associated Parkin, DJ1 and α-synuclein (α-Syn) were also found to affect calcium transfer at these contacts. Finally, defects in mitofusin 2 (MFN2) or VAMP-associated protein B and C (VAPB), which contribute to the structural integrity of the MCSs, affect the calcium crosstalk between the ER and mitochondria

Fig. 5

MAMs control mitochondrial dynamics. Endoplasmic reticulum (ER)-mitochondria contact sites are involved in mitochondrial fission and transport of mitochondria along cytoskeletal tracks. During fission, mitochondria-associated membranes (MAMs) determine the site of scission by contributing to the mitochondrial constriction required for dynamin-related protein 1 (Drp1) assembly. This constriction is accomplished through the actin-modulating activity of Spire1C and inverted forming 2 (INF2) at the membrane contact sites (MCSs). At least two other MAM proteins, syntaxin 17 (STX17) and Rab32, contribute to the regulation of mitochondrial fission by controlling Drp1 activity. Mitochondrial transport is primarily mediated by the mitochondrial Rho GTPases 1 and 2 (Miro1/2), which connect the mitochondria to motor proteins such as kinesins through the trafficking kinesin protein (TRAK) adaptor proteins. Miro depends on mitofusin1 and 2 (MFN1/2) to facilitate transport, and PTEN-induced putative kinase 1 (PINK1)-Parkin-dependent ubiquitination of Miro1/2 and MFN2 blocks mitochondrial movement. In zones of high calcium concentrations, such as those that occur at MAMs, calcium binding to the Miro1/2 EF-hand motifs releases the mitochondria from the cytoskeleton and halts their migration. The P56S mutation in VAMP-associated protein B and C (VAPB), which results in a higher degree of ER-mitochondria contact and calcium crosstalk, consequently results in axonal transport defects of mitochondria

Fig. 6

MAMs as sites for autophagosome biogenesis. Mitochondria-associated membranes (MAMs) were identified as a membrane source for autophagosome biogenesis. In a first instance, phosphatidylethanolamine (PE) synthesis at MAMs was believed to be a crucial lipid source for these emerging organelles. However, ER-mitochondria contact sites appear to play a more direct role. Syntaxin 17 (STX17) at MAMs recruits early autophagosome markers including Atg14L, Vps15, Vps34 and Beclin1. In addition, the omegasome marker double FYVE-containing protein 1 (DFCP1) translocates to these sites upon autophagy induction. Underscoring the relevance of ER-mitochondria membrane contact sites (MCSs), loss of phosphofurin acidic cluster sorting protein 2 (PACS2) or mitofusin 2 (MFN2) abrogates autophagosome biogenesis