Computational advancements in Cryo-Electron tomography : a quantitative characterization of parkinson’s disease hallmarks in the human brain

From physical motion to cognition, every physiological process of the human body is precisely controlled by our brain. To maintain its vital activity, our brain cells demand elevated levels of energy, which are tightly regulated to satisfy a critical homeostatic cellular balance. Thus, anomalies in brain homeostasis provoke devastating neurodegenerative diseases (NDs), which are among the most common cause of death in the Western world. To date, a cure for NDs does not exist. This is due to the current lack of knowledge on the understanding of the molecular mechanisms involved in normal brain ageing and the key players that trigger neuropathology. Human genetics have confirmed risk alleles associated with protein aggregation, mitochondrial biology and protein-quality control. A common root of NDs and senescence is protein misfolding and brain deposits of ordered protein structures referred to as amyloid.
Parkinson’s Disease (PD) is the second most common neurodegenerative disease affecting the central and peripheral nervous system. PD pathology is defined by the abnormal accumulation of the protein alpha-synuclein in neurons, nerve fibers and glial cells, characteristic of alpha-synucleinopathies. Key neuropathological hallmarks of PD are Lewy bodies (LBs) and Lewy neurites (LNs), which are alpha-synuclein-positive brain inclusions. Interestingly, the biological processes by which LBs and LNs are formed as well as their role in neurodegeneration await to be defined.
Overall, our knowledge on the ultrastructure of the human brain is extremely limited. Understanding human brain ultrastructure is central to describe its function, and therefore, identify physical alterations of senescence vs disease. This is essential to design novel therapies and translational animal and cellular models for NDs and ageing.
Electron microscopy (EM) visualizes cellular and tissue ultrastructure at high-resolution by two- (2D) and three-dimensional (3D) imaging. These studies have helped to unravel the nature of the structural components that compose human brain bodies, gaining insights into their possible origins and biological roles. However, a modern description of the ultrastructure of human brain aggregates by applying modern 3D EM technologies is largely lacking.
The first part of my thesis is focused on the ultrastructural characterization of human brain aggregates in PD and senile non-demented donors by means of 3D correlative light and electron microscopy (CLEM) approaches. In Chapter 2, we describe the detailed EM characterization of Lewy pathology alongside a multi-scale CLEM pipeline specifically developed for this study. Furthermore, in Chapter 3, we analysed the ultrastructural composition and cellular origins of the age-related brain body defined as Corpora amylacea (CA). Overall, these studies revealed an astonishing heterogeneity in Lewy pathology: previously described filament-containing LBs are identified, however, the vast majority of Lewy pathology, including LBs and LNs, possessed a predominant organellar nature composed of membrane stacks, disrupted cell organelles and dysmorphic cytoskeletal elements. In the case of CA, they are visualized as electron-dense granular aggregates composed of packed membranes and morphologically preserved cell organelles. Further, we provide strong indications that CA originate intracellularly in astrocytic feet forming the glymphatic system of the brain, pointing to a physiological role on sequestering toxic metabolites to prevent tissue inflammation via clearance through the cerebral spinal fluid.
The second part of this thesis is focused on cryo-electron tomography (cryo-ET) and the software developments implemented for 3D image processing and subtomogram averaging (STA). Cryo-ET has the enormous potential of in situ visualization of hydrated-frozen biological samples within their native context at high-resolution and in 3D. However, several bottlenecks are identified in the cryo-ET pipeline, such as sample preparation, data acquisition, tilt series alignment and STA, thus far preventing high-throughput cryo-ET. Usually specimen preparation and data acquisition are sample-dependent; however, tilt series alignment is a deterministic step that should be straightforward, but it is currently an important drawback in cryo-ET since alignment is not automated and errors require several rounds of manual intervention. Importantly, alignment errors result in a substantial decrease of the final resolution in the 3D reconstructed tomograms. Thus, in Part II, Chapter 2, we present an automatic tilt series alignment approach for high-resolution cryo-ET data sets. We described our algorithm and the factors to be considered when aiming for high-resolution STA. In Part II, Chapter 3, we implement computational tools and strategies for STA of membrane-bound protein macro-complexes. Furthermore, we present several user-friendly and flexible protocols for STA that can be applied to a wide spectrum of cryo-ET data sets.
The results comprised in this thesis highly improve our understanding of the structural basis and origins of human brain bodies in senescence and neurodegeneration. Alongside the biological results, a multi-scale pipeline that combines microscopy and advanced image processing approaches are established for nanoscale visualization of the human brain.