Many neurodegenerative diseases are linked to amyloid aggregation. In Huntington's disease (HD), neurotoxicity correlates with an increased aggregation propensity of a polyglutamine (polyQ) expansion in exon 1 of mutant huntingtin protein (mHtt). Here we establish how the domains flanking the polyQ tract shape the mHtt conformational landscape in vitro and in neurons. In vitro, the flanking domains have opposing effects on the conformation and stabilities of oligomers and amyloid fibrils. The N-terminal N17 promotes amyloid fibril formation, while the C-terminal Proline Rich Domain destabilizes fibrils and enhances oligomer formation. However, in neurons both domains act synergistically to engage protective chaperone and degradation pathways promoting mHtt proteostasis. Surprisingly, when proteotoxicity was assessed in rat corticostriatal brain slices, either flanking region alone sufficed to generate a neurotoxic conformation, while the polyQ tract alone exhibited minimal toxicity. Linking mHtt structural properties to its neuronal proteostasis should inform new strategies for neuroprotection in polyQ-expansion diseases.

Huntington disease (HD) is a neurodegenerative trinucleotide repeat disorder caused by an expanded poly-glutamine (polyQ) tract in the mutant huntingtin (mHTT) protein. The formation and topology of filamentous mHTT inclusions in the brain (hallmarks of HD implicated in neurotoxicity) remain elusive. Using cryo-electron tomography and subtomogram averaging, here we show that mHTT exon 1 and polyQ-only aggregates in vitro are structurally heterogenous and filamentous, similar to prior observations with other methods. Yet, we find filaments in both types of aggregates under ~2 nm in width, thinner than previously reported, and regions forming large sheets. In addition, our data show a prevalent subpopulation of filaments exhibiting a lumpy slab morphology in both aggregates, supportive of the polyQ core model. This provides a basis for future cryoET studies of various aggregated mHTT and polyQ constructs to improve their structure-based modeling as well as their identification in cells without fusion tags.

Corpora amylacea are cell-derived structures that appear physiologically in the aged human brain. While their histological identification is straightforward, their ultrastructural composition and microenvironment at the nanoscale have remained unclear so far, as has their relevance to aging and certain disease states that involve the sequestration of toxic cellular metabolites. Here, we apply correlative serial block-face scanning electron microscopy and transmission electron tomography to gain three-dimensional insight into the ultrastructure and surrounding microenvironment of cerebral Corpora amylacea in the human brainstem and hippocampal region. We find that cerebral Corpora amylacea are composed of dense labyrinth-like sheets of lipid membranes, contain vesicles as well as morphologically preserved mitochondria, and are in close proximity to blood vessels and the glymphatic system, primarily within the cytoplasm of perivascular glial cells. Our results clarify the nature of cerebral Corpora amylacea and provide first hints on how they may arise and develop in the aging brain.

In Huntington's disease, a mutated version of the huntingtin protein leads to cell death. Mutant huntingtin is known to aggregate, a process that can be inhibited by the eukaryotic chaperonin TRiC (TCP1-ring complex) in vitro and in vivo. A structural understanding of the genesis of aggregates and their modulation by cellular chaperones could facilitate the development of therapies but has been hindered by the heterogeneity of amyloid aggregates. Using cryo-electron microscopy (cryoEM) and single particle cryo-electron tomography (SPT) we characterize the growth of fibrillar aggregates of mutant huntingtin exon 1 containing an expanded polyglutamine tract with 51 residues (mhttQ51), and resolve 3-D structures of the chaperonin TRiC interacting with mhttQ51. We find that TRiC caps mhttQ51 fibril tips via the apical domains of its subunits, and also encapsulates smaller mhtt oligomers within its chamber. These two complementary mechanisms provide a structural description for TRiC's inhibition of mhttQ51 aggregation in vitro. DOI:http://dx.doi.org/10.7554/eLife.00710.001.

Gaining insight to pathologically relevant processes in continuous volumes of unstained brain tissue is important for a better understanding of neurological diseases. Many pathological processes in neurodegenerative disorders affect myelinated axons, which are a critical part of the neuronal circuitry. Cryo ptychographic X-ray computed tomography in the multi-keV energy range is an emerging technology providing phase contrast at high sensitivity, allowing label-free and non-destructive three dimensional imaging of large continuous volumes of tissue, currently spanning up to 400,000 μm3. This aspect makes the technique especially attractive for imaging complex biological material, especially neuronal tissues, in combination with downstream optical or electron microscopy techniques. A further advantage is that dehydration, additional contrast staining, and destructive sectioning/milling are not required for imaging. We have developed a pipeline for cryo ptychographic X-ray tomography of relatively large, hydrated and unstained biological tissue volumes beyond what is typical for the X-ray imaging, using human brain tissue and combining the technique with complementary methods. We present four imaged volumes of a Parkinson's diseased human brain and five volumes from a non-diseased control human brain using cryo ptychographic X-ray tomography. In both cases, we distinguish neuromelanin-containing neurons, lipid and melanic pigment, blood vessels and red blood cells, and nuclei of other brain cells. In the diseased sample, we observed several swellings containing dense granular material resembling clustered vesicles between the myelin sheaths arising from the cytoplasm of the parent oligodendrocyte, rather than the axoplasm. We further investigated the pathological relevance of such swollen axons in adjacent tissue sections by immunofluorescence microscopy for phosphorylated alpha-synuclein combined with multispectral imaging. Since cryo ptychographic X-ray tomography is non-destructive, the large dataset volumes were used to guide further investigation of such swollen axons by correlative electron microscopy and immunogold labeling post X-ray imaging, a possibility demonstrated for the first time. Interestingly, we find that protein antigenicity and ultrastructure of the tissue are preserved after the X-ray measurement. As many pathological processes in neurodegeneration affect myelinated axons, our work sets an unprecedented foundation for studies addressing axonal integrity and disease-related changes in unstained brain tissues.

Morphologically distinct TDP-43 aggregates occur in clinically different FTLD-TDP subtypes, yet the mechanism of their emergence and contribution to clinical heterogeneity are poorly understood. Several lines of evidence suggest that pathological TDP-43 follows a prion-like cascade, but the molecular determinants of this process remain unknown. We use advanced microscopy techniques to compare the seeding properties of pathological FTLD-TDP-A and FTLD-TDP-C aggregates. Upon inoculation of patient-derived aggregates in cells, FTLD-TDP-A seeds amplify in a template-dependent fashion, triggering neoaggregation more efficiently than those extracted from FTLD-TDP-C patients, correlating with the respective disease progression rates. Neoaggregates are sequentially phosphorylated with N-to-C directionality and with subtype-specific timelines. The resulting FTLD-TDP-A neoaggregates are large and contain densely packed fibrils, reminiscent of the pure compacted fibrils present within cytoplasmic inclusions in postmortem brains. In contrast, FTLD-TDP-C dystrophic neurites show less dense fibrils mixed with cellular components, and their respective neoaggregates are small, amorphous protein accumulations. These cellular seeding models replicate aspects of the patient pathological diversity and will be a useful tool in the quest for subtype-specific therapeutics.

Positron Emission Tomography (PET) ligands have advanced Alzheimer's disease (AD) diagnosis and treatment. Using autoradiography and cryo-EM, we identified AD brain tissue with elevated tau burden, purified filaments, and determined the structure of second-generation high avidity PET ligand MK-6240 at 2.31 Å resolution, which bound at a 1:1 ratio within the cleft of tau paired-helical filament (PHF), engaging with glutamine 351, lysine K353, and isoleucine 360. This information elucidates the basis of MK-6240 PET in quantifying PHF deposits in AD and may facilitate the structure-based design of superior ligands against tau amyloids.

The aggregation of α-synuclein is associated with progression of Parkinson's disease. We have identified submicrometer supramolecular structures that mediate the early stages of the overall mechanism. The sequence of structural transformations between metastable intermediates were captured and characterized by atomic force microscopy guided by a fluorescent probe sensitive to preamyloid species. A novel ~0.3-0.6 μm molecular assembly, denoted the acuna, nucleates, expands, and liberates fibers with distinctive segmentation and a filamentous fuzzy fringe. These fuzzy fibers serve as precursors of mature amyloid fibrils. Cryo-electron tomography resolved the acuna inner structure as a scaffold of highly condensed colloidal masses interlinked by thin beaded threads, which were perceived as fuzziness by atomic force microscopy. On the basis of the combined data, we propose a sequential mechanism comprising molecular, colloidal, and fibrillar stages linked by reactions with disparate temperature dependencies and distinct supramolecular forms. We anticipate novel diagnostic and therapeutic approaches to Parkinson's and related neurodegenerative diseases based on these new insights into the aggregation mechanism of α-synuclein and intermediates, some of which may act to cause and/or reinforce neurotoxicity.

Discovering ligands for amyloid fibrils, such as those formed by the tau protein, is an area of much current interest. In recent structures, ligands bind in stacks in the tau fibrils to reflect the rotational and translational symmetry of the fibril itself; in these structures the ligands make few interactions with the protein but interact extensively with each other. To exploit this symmetry and stacking, we developed SymDOCK, a method to dock molecules that follow the protein's symmetry. For each prospective ligand pose, we apply the symmetry operation of the fibril to generate a self-interacting and fibril-interacting stack, checking that doing so will not cause a clash between the original molecule and its image. Absent a clash, we retain that pose and add the ligand-ligand van der Waals energy to the ligand's docking score (here using DOCK3.8). We can check these geometries and energies using an implementation of ANI, a neural network-based quantum-mechanical evaluation of the ligand stacking energies. In retrospective calculations, symmetry docking can reproduce the poses of three tau PET tracers whose structures have been determined. More convincingly, in a prospective study SymDOCK predicted the structure of the PET tracer MK-6240 bound in a symmetrical stack to AD PHF tau before that structure was determined; the docked pose was used to determine how MK-6240 fit the cryo-EM density. In proof-of-concept studies, SymDOCK enriched known ligands over property-matched decoys in retrospective screens without sacrificing docking speed, and can address large library screens that seek new symmetrical stackers. Future applications of this approach will be considered.

A simple two-spindle based lathe system for the preparation of cylindrical samples intended for X-ray tomography is presented. The setup can operate at room temperature as well as under cryogenic conditions, allowing the preparation of samples down to 20 and 50 µm in diameter, respectively, within minutes. Case studies are presented involving the preparation of a brittle biomineral brachiopod shell and cryogenically fixed soft brain tissue, and their examination by means of ptychographic X-ray computed tomography reveals the preparation method to be mainly free from causing artefacts. Since this lathe system easily yields near-cylindrical samples ideal for tomography, a usage for a wide variety of otherwise challenging specimens is anticipated, in addition to potential use as a time- and cost-saving tool prior to focused ion-beam milling. Fast sample preparation becomes especially important in relation to shorter measurement times expected in next-generation synchrotron sources.

The human brain contains a wide array of billions of neurons and interconnections, which are often simplified for analysis in vitro using compartmentalized microfluidic devices for neuronal cell culturing, to better understand neuronal development and disease. However, such devices are traditionally incompatible for high-pressure freezing and high-resolution nanoscale imaging and analysis of their sub-cellular processes by methods including electron microscopy. Here we develop a novel compartmentalized neuronal co-culture platform allowing reconstruction of neuronal networks with high variable spatial control, which is uniquely compatible for high-pressure freezing. This cryo-fixation method is well-established to enable high-fidelity preservation of the reconstructed neuronal networks and their sub-cellular processes in a near-native vitreous state without requiring chemical fixatives. To direct the outgrowth of neurites originating from two distinct groups of neurons growing in the two different compartments, polymer microstructures akin to microchannels are fabricated atop of sapphire disks. Two populations of neurons expressing either enhanced green fluorescent protein (EGFP) or mCherry were grown in either compartment, facilitating the analysis of the specific interactions between the two separate groups of cells. Neuronally differentiated PC12 cells, murine hippocampal and striatal neurons were successfully used in this context. The design of this device permits direct observation of entire neuritic processes within microchannels by optical microscopy with high spatial and temporal resolution, prior to processing for high-pressure freezing and electron microscopy. Following freeze substitution, we demonstrate that it is possible to process the neuronal networks for ultrastructural imaging by electron microscopy. Several key features of the embedded neuronal networks, including mitochondria, synaptic vesicles, axonal terminals, microtubules, with well-preserved ultrastructures were observed at high resolution using focused ion beam - scanning electron microscopy (FIB-SEM) and serial sectioning - transmission electron microscopy (TEM). These results demonstrate the compatibility of the platform with optical microscopy, high-pressure freezing and electron microscopy. The platform can be extended to neuronal models of brain disease or development in future studies, enabling the investigation of subcellular processes at the nanoscale within two distinct groups of neurons in a functional neuronal pathway, as well as pharmacological testing and drug screening.

Discovering ligands for amyloid fibrils, such as those formed by the tau protein, is an area of great current interest. In recent structures, ligands bind in stacks in the tau fibrils to reflect the rotational and translational symmetry of the fibril itself; in these structures, the ligands make few interactions with the protein but interact extensively with each other. To exploit this symmetry and stacking, we developed SymDOCK, a method to dock molecules that follow the protein's symmetry. For each prospective ligand pose, we apply the symmetry operation of the fibril to generate a self-interacting and fibril-interacting stack, checking that doing so will not cause a clash between the original molecule and its image. Absent a clash, we retain that pose and add the ligand-ligand van der Waals energy to the ligand's docking score (here using DOCK3.8). We can check these geometries and energies using an implementation of ANI, a neural-network-based quantum-mechanical evaluation of the ligand stacking energies. In retrospective calculations, symmetry docking can reproduce the poses of three tau PET tracers whose structures have been determined. More convincingly, in a prospective study, SymDOCK predicted the structure of the PET tracer MK-6240 bound in a symmetrical stack to AD PHF tau before that structure was determined; the docked pose was used to determine how MK-6240 fit the cryo-EM density. In proof-of-concept studies, SymDOCK enriched known ligands over property-matched decoys in retrospective screens without sacrificing docking speed and can address large library screens that seek new symmetrical stackers. Future applications of this approach will be considered.