Morphological Adaptations of Mitochondria in Spatially Restricted Axons Revealed by Cryo-Electron Tomography

Neurons project axons to local and distal sites that can display heterogeneous morphologies with restricted physical dimensions that may influence the transport of large organelles, such as mitochondria. Although transport of mitochondria has been widely studied, whether spatial constraints within axons determines the distribution or morphological characteristics of mitochondria has not been systematically examined. The current study provides insight into the spatial environments of mitochondria within axons and presynaptic varicosities of cryopreserved hippocampal neurons imaged in their native state using 3D cryo-electron tomography (cryo-ET). Segmented tomographic reconstructions revealed major morphological adaptations of mitochondria due to restricted physical dimensions at the boundary between presynaptic varicosities and axons. Furthermore, segmentation of individual mitochondrial membranes revealed distinct structural changes between the inner and outer membranes during this modification, suggesting independent regulation of each membrane to adapt to the spatial constraints within axons. These findings offer a new perspective on the remarkable structural plasticity of mitochondrial membranes and the adaptability of mitochondrial morphology to navigate limited available space necessary for axonal transport. Significance Statement Restrictive physical dimensions within axons pose a significant problem as to how rather large organelles, such as mitochondria, navigate the constrained space for efficient axonal transport. Using cryo-electron tomography, we characterized native spatial environments within axons and presynaptic varicosities of hippocampal neurons to determine whether the magnitude of spatial restrictions within these compartments may influence the subcellular distribution and morphology of mitochondria. Major morphological adaptations of mitochondria and their membranes were revealed due to restricted available space within axons. These findings offer new perspectives on the influence of physical and spatial characteristics of cellular environments on mitochondrial morphology and highlights the remarkable structural plasticity of mitochondria to navigate the limited available space during axonal transport. Introduction Neurons are architecturally complex cells that can extend axonal projections with elaborate arborization for several hundreds of millimeters (and in some cases, meters) to form synaptic connections with local and distal targets (1-3). Depending on the target, presynaptic compartments can either be found at the end of axons (terminal boutons) or tracking along axons as intermediate swellings (en passant boutons or varicosities), such as in the unmyelinated CA3->CA1 axons of the hippocampus (3-7). The bead-like presynaptic varicosities are morphologically heterogeneous, displaying diameters that can range from 1-2 μm connected by thin axon segments that can have diameters less than 100 nm (4, 5, 8, 9). Tracking through the axons is a well-developed system of microtubules that mediate motor-driven anterograde and retrograde transport of signaling cargoes, protein complexes and organelles critical for function and homeostasis at distant synapses (10, 11). Transport of these intracellular components creates a spatially and temporally dynamic axonal environment that can contain a variety of organelles and cargoes of different shapes, sizes, and number (12, 13). As the axon segments interconnecting varicosities can be remarkably thin and the available space within axons can be occupied by various organelles and cargo, their overall diameter and changes in internal volume present an interesting problem for the efficient transport of large organelles, such as mitochondria, that can display an average diameter between 100-500 nm (14, 15). Axonal transport of mitochondria is particularly critical for synaptic development, function, and plasticity and movement is known to make frequent stops, or saltatory movements, at presynaptic compartments to provide local ATP synthesis and calcium regulation required for proper neurotransmission (16-20). Given the morphological complexity of axons, restricted physical dimensions and available space could potentially influence the subcellular localization, distribution, and transport of mitochondria required to meet local energy needs and for supporting synaptic transmission. Although mitochondria are morphologically dynamic organelles that can exist in a variety of shapes and sizes, how mitochondria adapt to the physical constraints presented in axons to navigate the spatially restricted environment has not been previously examined. Advances in microscopy and imaging techniques have played a pivotal role in revealing the three-dimensional architecture of neurons and their intracellular environments at resolutions reaching the nanometer scale (21, 22). In the current study, we employed the unique native state preservation afforded by cryopreservation and the resolution provided by cryo-electron tomography (cryo-ET) to visualize three-dimensional spatial relationships and organellar structure within cultured hippocampal axons. Our results show that axon morphology and physically restrictive intracellular dimensions present a previously unrecognized influence on mitochondria residing within axonal subcompartments, revealing dramatic adaptations mitochondria must undertake to navigate the available space during transport. Results Preparation of primary hippocampal neurons for cryo-ET Primary neuronal cultures from E18 rat hippocampi were grown on holey carbon grids prior to cryo-preservation. At 10 days post plating, primary hippocampal neurons have extended dendritic and axonal processes and have established pre-synaptic varicosities and initial excitatory synaptic connections (23). Figure 1A and B show representative bright field images of neurons cultured on a Quantifoil grid where widespread elaboration of processes is evident. Figure 1C and D demonstrate images of companion grids that were fixed and immunolabeled with antibodies to calcium/calmodulin dependent protein kinase II alpha (CaMKIIa) and synapsin 1 to visualize the excitatory neuron population and presynaptic varicosities, respectively. CaMKII largely labels soma and processes, while the synapsin 1 antibody shows distinct puncta representing enrichment of synaptic vesicles at presynaptic varicosities. For cryo-ET, fiducial gold markers were applied to prepared grids and cryopreserved by plunge freezing in liquid ethane. Cryopreservation conserves the near native state of the preparation permitting a fixation free assessment of spatial relationships of organelles present in different intracellular environments within hippocampal neurons. Low magnification images were first collected (Fig. 1E) and montaged to provide maps for targeting areas of interest for data collection. A higher magnification image reveals the distribution of presynaptic varicosities and axonal processes (Fig. 1F). Varicosities and axons can be seen residing on both the carbon and overlying the grid holes. To provide maximum contrast, tomographic data collection was targeted to cryopreserved structures within the grid holes. In these preparations, the increased thickness of the ice surrounding the soma and proximal dendrites prevented imaging of these structures. In contrast, the ice thickness surrounding the axonal processes and varicosities was ideal, permitting a detailed assessment of the spatial relationships of cytoskeletal structures and organelles within these subcompartments. Organelle populations of pre-synaptic varicosities in primary hippocampal neurons are heterogeneous Areas were randomly chosen, and tilt series were collected from varicosities and axon segments overlying grid holes to visualize cytoskeletal and organellar structures. Figure 2A shows a slice through a representative tomographic reconstruction with various resident organelles and structures visible, including two mitochondria, a multi-vesicular body (MVB), microtubules, endoplasmic reticulum (ER) and a collection of vesicles. To determine the three-dimensional (3D) relationship between the different structures, segmentation was accomplished of the tomographic reconstruction of both the presynaptic varicosity and the adjoining axon (Fig. 2B; Movie S1). The reconstruction demonstrates microtubules (light blue) forming a continuous set of tracks traveling from one end of the varicosity to the other. Microtubules appear less constrained within the varicosity where they exhibit greater curvature, while gathering together and straightening when entering the adjoining axon segment. The ER (yellow) also forms a reticulated and continuous structure spanning the entire length of the varicosity, consistent with other reports on the ubiquitous presence of ER in axons and synaptic terminals (21). Mitochondria, MVB, and Golgi, exhibit more random distributions within the varicosity, while vesicles appeared to be somewhat clustered. To determine if the presence of each of these identifiable organelles was consistent across varicosities, we analyzed their distribution in an additional 77 additional tomographic reconstructions. ER and microtubules were present in 100% of presynaptic varicosities, vesicles were present in 97%, mitochondria in 82%, Golgi bodies in 38%, multi-vesicular bodies in 21%, lamellar bodies in 9%, and autophagosomes in 4% (exemplars shown in Fig. S1). Variations in distinct sub-features of the observed organelle populations also emerged in 3D at high resolution. Specifically, the ubiquitous ER was observed in close apposition with almost every other organelle within the varicosities, consistent with ER-membrane contact sites described by others (21). The vesicular population was heterogeneous in number, ranging from 1 to >100 vesicles per varicosity. 75% of varicosities contained <50 vesicles, 10% had 50-100, and 6% contained greater t