3rd Prize: Nensi Alivodej

Mitochondria-ER contact sites in astrocytes: a 3D ultrastructural analysis and quantification using Focus Ion Beam-Scanning Electron Microscopy

By: Nensi Alivodej

Supervisor: Dr. Keith Murai

Astrocytes are essential non-neural cells of the central nervous system (CNS) that help maintain a healthy environment in the brain. Their main role in the cerebral cortex includes (1) providing metabolic support to neurons for efficient synaptic transmission and (2) regulating the interaction between the brain and the cerebrovasculature1. Focusing on the latter, astrocytes preserve the integrity of the blood-brain-barrier (BBB) through specialized processes called “endfeet”, which wrap around neighbouring cerebral blood vessels2,3. Through the regulation of extracellular ionic concentrations and the oversee of water, oxygen and glucose exchange, astrocytic endfeet are vital in ensuring that the brain is well oxygenated and receives enough nutrient supply to match the high energetic needs of active neurons4.

Unlike neurons, astrocytes are not electrically excitable; rather, they respond to neural and vascular activity with transient intracellular calcium (Ca2+) elevations5. In contrast to the rest of the cell, Ca2+ rises in the endfeet appear to have different dynamics as observed by members of Dr. Keith Murai and Dr. Hélène Girouard laboratories. Namely, they occur with a greater magnitude and at a frequency that is out of tune with spontaneous Ca2+ events in other microdomains of the astrocyte. Various ex vivo and in vivo studies have demonstrated that varying levels of endfoot Ca2+ are correlated with dilation and constriction of cerebral blood vessels, thereby influencing blood flow6,7. Currently, the role of endfoot Ca2+ in relation to vascular activity is an active area of research given that cerebrovascular tone and flow are potent indicators of health and activity in the brain. In fact, the cerebrovasculature is severely compromised following inflammatory changes associated with BBB breakdown in neurodegenerative disease, such as Alzheimer’s disease8.

From preliminary electron microscopy images acquired by the Murai laboratory, we observe tight physical associations between mitochondria and endoplasmic reticulum (ER) in the endfeet that contrasts those seen in the rest of the astrocyte. These mitochondria-ER contact sites (MERCS) are hosts of a variety of signaling functions, which include lipid synthesis, protein trafficking, and, most importantly, Ca2+ signaling9. However, very little is known about their functions in brain cells, and practically no studies have investigated them in astrocytes and their perivascular endfeet in tissue. Therefore, MERCS in astrocytes deserve further investigation as they represent an ideal biological platform to support the versatile functions of the endfeet.

Building on a previous project that involved the three dimensional (3D) reconstruction of astrocytes (Fig. 1A), we sought to resolve the 3D architecture of MERCS from serial scanning electron microscopy (SEM) images along with the quantification of their surface area. We hypothesized that the average surface area of MERCS would be significantly larger in the endfeet than in non-endfeet processes of the astrocyte. Given that the surface area of MERCS is correlated to the extent of their signaling functions as described in recent studies, a larger surface could indicate a specialization of their biological activities in the endfeet.

To verify our observations, we manually segmented and reconstructed 25 astrocytic mitochondria and 227 MERCS from three stacks of SEM images taken from the somatosensory barrel cortex of two adult mice and acquired using focus ion beam-scanning electron microscopy (FIB-SEM). MERCS were defined as ER membrane less than 30 nanometers away from the outer mitochondrial membrane. The surface area of individual MERCS was compared between endfeet and non-endfeet compartments of the astrocyte as defined by our anatomical description of the endfeet. Additionally, the total surface coverage of MERCS per mitochondrial surface area was measured and compared accordingly.

From our 3D ultrastructural reconstruction, MERCS appeared large and flat in the endfeet (Fig. 1B-C), but smaller and more tubular in non-endfeet processes. In both categories of processes, we observe multiples instances of MERCS wrapping around mitochondrial branching sites, which is consistent with previous studies implicating MERCS in mitochondrial fission10. Furthermore, we denote a stereotyped arrangement of organelles in the endfeet with the following configuration: basal lamina, ER, mitochondria. Such a distribution reflects a coordinated activity between organelles at discrete microdomains to support the synthesis of proteins, ensure their proper folding and subsequent trafficking to the basement membrane of the endfoot in addition to Ca2+ signaling. In agreement with our hypothesis, quantitative statistical results yielded an average individual surface area of MERCS significantly larger in the endfeet compared to non-endfeet (Fig. 1D) (P<0.001). In addition, the total surface coverage of MERCS per mitochondrial surface area was also found to be significantly larger in the endfeet (Fig. 1E) (P<0.05). An increased surface area of individual contact sites suggests an enhanced molecular tethering activity between mitochondria and ER membranes. The larger total surface coverage of MERCS per mitochondria in the endfeet also suggests an upregulation of the biological activities that are specific to MERCS. Large surfaces of MERCS could potentiate mitochondrial uptake of Ca2+ that is released from ER stores and impinge on the regulatory mechanisms associated to endfeet functions, such as energy turnover, biological stress sensing, and Ca2+ signaling tailored to vascular activity.

Altogether, our 3D reconstructions allowed us to identify, characterize and map the distribution of novel subcellular structures, namely MERCS, in cortical astrocytes. To our knowledge, our study is the first to provide evidence for the specialization of MERCS in astrocytes that is supported by a quantitative analysis of their 3D ultrastructural characteristics. MERCS are novel structures that have not been extensively studied in the brain so far, and their signaling mechanisms remain to be discovered. We propose that these structures deserve further investigation for their implication in endfoot Ca2+ activity and local protein trafficking in astrocytes. In addition, we herein illustrate that FIB-SEM technology is an innovative approach to study and resolve the complexity of subcellular organelles. Such advancement in 3D imaging allows the precise morphological description of individual organelles and the quantification of biological factors, such as gap width between membranes, that are, otherwise, difficult to obtain from light microscopy. The identification and quantification of MERCS in the endfeet of astrocytes would not have been possible without the high resolution offered by FIB-SEM.



1. Mishra, A. (2017). Binaural blood flow control by astrocytes : listening to synapses and the vasculature, 6, 1885–1902. https://doi.org/10.1113/JP270979

2. Mathiisen, T. M., Lehre, K. P., Danbolt, N. C., & Ottersen, O. P. (2010). The perivascular astroglial sheath provides a complete covering of the brain microvessels: An electron microscopic 3D reconstruction. Glia, 58(9), 1094–1103. https://doi.org/10.1002/glia.20990

3. Wong, A. D., Ye, M., Levy, A. F., Rothstein, J. D., Bergles, D. E., & Searson, P. C. (2013). The blood-brain barrier: an engineering perspective. Frontiers in Neuroengineering, 6(August), 1–22. https://doi.org/10.3389/fneng.2013.00007

4. Boison, D., & Masino, S. (2015). Homeostatic Control of Brain Function, (1967), 656. https://doi.org/10.1093/med/9780199322299.001.0001

5. Bazargani, N., & Attwell, D. (2016). Astrocyte calcium signaling : the third wave. Nature Neuroscience, 19, 182–189. https://doi.org/10.1038/nn.4201

6. Mishra, A., Reynolds, J. P., Chen, Y., Gourine, A. V., Rusakov, D. A., & Attwell, D. (2016). Astrocytes mediate neurovascular signaling to capillary pericytes but not to arterioles. Nature Neuroscience, 19(12), 1619–1627. https://doi.org/10.1038/nn.4428

7. Girouard, H., Bonev, A. D., Hannah, R. M., Meredith, A., Aldrich, R. W., & Nelson, M. T. (2010). Astrocytic endfoot Ca2+ and BK channels determine both arteriolar dilation and constriction. Proceedings of the National Academy of Sciences, 107(8), 3811–3816. https://doi.org/10.1073/pnas.0914722107

8. Roher, A. E., Lowenson, J. D., Clarke, S., Woods, A. S., Cotter, R. J., Gowing, E., & Ball, M. J. (1993). beta-Amyloid-(1-42) is a major component of cerebrovascular amyloid deposits: implications for the pathology of Alzheimer disease. Proceedings of the National Academy of Sciences, 90(22), 10836–10840. https://doi.org/10.1073/pnas.90.22.10836

9. Giacomello, M., & Pellegrini, L. (2016). The coming of age of the mitochondria–ER contact: a matter of thickness. Cell Death and Differentiation, 23(9), 1417–1427. https://doi.org/10.1038/cdd.2016.52

10. Chakrabarti, R., Ji, W. K., Stan, R. V., Sanz, J. de J., Ryan, T. A., & Higgs, H. N. (2018). INF2-mediated actin polymerization at the ER stimulates mitochondrial calcium uptake, inner membrane constriction, and division. Journal of Cell Biology, 217(1), 251–268. https://doi.org/10.1083/jcb.201709111