Felipe Opazo

Pre-fibrillar α-synuclein variants with impaired β-structure increase neurotoxicity in Parkinson's disease models

The relation of α‐synuclein (αS) aggregation to Parkinsons disease (PD) has long been recognized, but the mechanism of toxicity, the pathogenic species and its molecular properties are yet to be identified. To obtain insight into the function different aggregated αS species have in neurotoxicity in vivo, we generated αS variants by a structure‐based rational design. Biophysical analysis revealed that the αS mutants have a reduced fibrillization propensity, but form increased amounts of soluble oligomers. To assess their biological response in vivo, we studied the effects of the biophysically defined pre‐fibrillar αS mutants after expression in tissue culture cells, in mammalian neurons and in PD model organisms, such as Caenorhabditis elegans and Drosophila melanogaster. The results show a striking correlation between αS aggregates with impaired β‐structure, neuronal toxicity and behavioural defects, and they establish a tight link between the biophysical properties of multimeric αS species and their in vivo function.

Endosomal sorting of readily releasable synaptic vesicles

Neurotransmitter release is achieved through the fusion of synaptic vesicles with the neuronal plasma membrane (exocytosis). Vesicles are then retrieved from the plasma membrane (endocytosis). It was hypothesized more than 3 decades ago that endosomes participate in vesicle recycling, constituting a slow endocytosis pathway required especially after prolonged stimulation. This recycling model predicts that newly endocytosed vesicles fuse with an endosome, which sorts (organizes) the molecules and buds exocytosis-competent vesicles. We analyzed here the endosome function using hippocampal neurons, isolated nerve terminals (synaptosomes), and PC12 cells by stimulated emission depletion microscopy, photooxidation EM, and several conventional microscopy assays. Surprisingly, we found that endosomal sorting is a rapid pathway, which appeared to be involved in the recycling of the initial vesicles to be released on stimulation, the readily releasable pool. In agreement with the endosomal model, the vesicle composition changed after endocytosis, with the newly formed vesicles being enriched in plasma membrane proteins. Vesicle proteins were organized in clusters both in the plasma membrane (on exocytosis) and in the endosome. In the latter compartment, they segregated from plasma membrane components in a process that is likely important for sorting/budding of newly developed vesicles from the endosome.

Limited intermixing of synaptic vesicle components upon vesicle recycling.

Synaptic vesicles recycle repeatedly in order to maintain synaptic transmission. We have previously proposed that upon exocytosis the vesicle components persist as clusters, which would be endocytosed as whole units. It has also been proposed that the vesicle components diffuse into the plasma membrane and are then randomly gathered into new vesicles. We found here that while strong stimulation (releasing the entire recycling pool) causes the diffusion of the vesicle marker synaptotagmin out of synaptic boutons, moderate stimulation (releasing ∼19% of all vesicles) is followed by no measurable diffusion. In agreement with this observation, synaptotagmin molecules labeled with different fluorescently tagged antibodies did not appear to mix upon vesicle recycling, when investigated by subdiffraction resolution stimulated emission depletion (STED) microscopy. Finally, as protein diffusion from vesicles has been mainly observed using molecules tagged with pH‐sensitive green fluorescent protein (pHluorin), we have also investigated the membrane patterning of several native and pHluorin‐tagged proteins. While the native proteins had a clustered distribution, the GFP‐tagged ones were diffused in the plasma membrane. We conclude that synaptic vesicle components intermix little, at least under moderate stimulation, possibly because of the formation of clusters in the plasma membrane. We suggest that several pHluorin‐tagged vesicle proteins are less well integrated in clusters.

Aptamers as potential tools for super-resolution microscopy

(a) The TfnR aptamer c2, the EGFR aptamer E07 and their respective control aptamers (random sequences) were incubated at 37°C with human A431 cells as described in Supplementary Methods. Similarly, HeLa cells stably transfected with a human PSMA construct were incubated with the PSMA A9 aptamer or its random control. The pairs of images (control and aptamer) are equally scaled to allow a direct visual comparison. The insets in the control images correspond to the same images, scaled to a level where autofluorescence can be visualized. Scale bar, 10 μm. (b) Colocalization of the different aptamers with endosomal labels. We co-incubated the cells (same as above) with aptamers against TfnR (c2) or PSMA (A9) and Alexa488-transferrin (Invitrogen), since the latter constitutes an ideal marker for early endosomes.

An RNA Alternative to Human Transferrin: A New Tool for Targeting Human Cells

The transferrin receptor, CD71, is an attractive target for drug development because of its high expression on a number of cancer cell lines and the blood brain barrier. To generate serum-stabilized aptamers that recognize the human transferrin receptor, we have modified the traditional aptamer selection protocol by employing a functional selection step that enriches for RNA molecules which bind the target receptor and are internalized by cells. Selected aptamers were specific for the human receptor, rapidly endocytosed by cells and shared a common core structure. A minimized variant was found to compete with the natural ligand, transferrin, for receptor binding and cell uptake, but performed ~twofold better than it in competition experiments. Using this molecule, we generated aptamer-targeted siRNA-laden liposomes. Aptamer targeting enhanced both uptake and target gene knockdown in cells grown in culture when compared to nonmodified or nontargeted liposomes. The aptamer should prove useful as a surrogate for transferrin in many applications including cell imaging and targeted drug delivery.

Resolving bundled microtubules using anti-tubulin nanobodies

Microtubules are hollow biopolymers of 25-nm diameter and are key constituents of the cytoskeleton. In neurons, microtubules are organized differently between axons and dendrites, but their precise organization in different compartments is not completely understood. Super-resolution microscopy techniques can detect specific structures at an increased resolution, but the narrow spacing between neuronal microtubules poses challenges because most existing labelling strategies increase the effective microtubule diameter by 20–40 nm and will thereby blend neighbouring microtubules into one structure. Here we develop single-chain antibody fragments (nanobodies) against tubulin to achieve super-resolution imaging of microtubules with a decreased apparent diameter. To test the resolving power of these novel probes, we generate microtubule bundles with a known spacing of 50–70 nm and successfully resolve individual microtubules. Individual bundled microtubules can also be resolved in different mammalian cells, including hippocampal neurons, allowing novel insights into fundamental mechanisms of microtubule organization in cell- and neurobiology.

Accumulation and clearance of α‐synuclein aggregates demonstrated by time‐lapse imaging

Aggregates of α‐synuclein are the pathological hallmark of sporadic Parkinson’s disease (PD), and mutations in the α‐synuclein gene underlie familial forms of the disease. To characterize the formation of α‐synuclein aggregates in living cells, we developed a new strategy to visualize α‐synuclein by fluorescence microscopy: α‐synuclein was tagged with a six amino acid PDZ binding motif and co‐expressed with the corresponding PDZ domain fused to enhanced green fluorescent protein (EGFP). In contrast to the traditional approach of α‐synuclein‐EGFP fusion proteins, this technique provided several‐fold higher sensitivity; this allowed us to compare α‐synuclein variants and perform time‐lapse imaging. A C‐terminally truncated α‐synuclein variant showed the highest prevalence of aggregates and toxicity, consistent with stabilization of the α‐synuclein monomer by its C‐terminus. Time‐lapse imaging illustrated how cells form and accumulate aggregates of α‐synuclein. A substantial number of cells also reduced their aggregate load, primarily through formation of an aggresome, which could itself be cleared from the cell. The molecular chaperone Hsp70 not only prevented the formation of aggregates, but also increased their reduction and clearance, underlining the therapeutic potential of similar strategies. In contrast to earlier assumptions build‐up, reduction and clearance of α‐synuclein aggregation thus appear a highly dynamic process.

Super-resolution imaging for cell biologists

The recent 2014 Nobel Prize in chemistry honored an era of discoveries and technical advancements in the field of super-resolution microscopy. However, the applications of diffraction-unlimited imaging in biology have a long road ahead and persistently engage scientists with new challenges. Some of the bottlenecks that restrain the dissemination of super-resolution techniques are tangible, and include the limited performance of affinity probes and the yet not capillary diffusion of imaging setups. Likewise, super-resolution microscopy has introduced new paradigms in the design of projects that require imaging with nanometer-resolution and in the interpretation of biological images. Besides structural or morphological characterization, super-resolution imaging is quickly expanding towards interaction mapping, multiple target detection and live imaging. Here we review the recent progress of biologists employing super-resolution imaging, some pitfalls, implications and new trends, with the purpose of animating the field and spurring future developments.

Rapid microtubule bundling and stabilization by the Streptococcus pneumoniae neurotoxin pneumolysin in a cholesterol-dependent, non-lytic and Src-kinase dependent manner inhibits intracellular trafficking.

Streptococcus pneumoniae is the most frequent cause of bacterial meningitis, leading to permanent neurological damage in 30% and lethal outcome in 25% of patients. The cholesterol‐dependent cytolysin pneumolysin is a major virulence factor of S. pneumoniae. It produces rapid cell lysis at higher concentrations or apoptosis at lower concentrations. Here, we show that sublytic amounts of pneumolysin produce rapid bundling and increased acetylation of microtubules (signs of excessive microtubule stabilization) in various types of cells – neuroblastoma cells, fibroblasts and primary astrocytes. The bundling started perinuclearly and extended peripherally towards the membrane. The effect was not connected to pneumolysins capacity to mediate calcium influx, macropore formation, apoptosis, or RhoA and Rac1 activation. Cellular cholesterol depletion and neutralization of the toxin by pre‐incubation with cholesterol completely inhibited the microtubule phenotype. Pharmacological inhibition of Src‐family kinases diminished microtubule bundling, suggesting their involvement in the process. The relevance of microtubule stabilization to meningitis was confirmed in an experimental pneumococcal meningitis animal model, where increased acetylation was observed. Live imaging experiments demonstrated a decrease in organelle motility after toxin challenge in a manner comparable to the microtubule‐stabilizing agent taxol, thus proposing a possible pathogenic mechanism that might contribute to the CNS damage in pneumococcal meningitis.

Membrane-permeable Bcl-xL prevents MPTP-induced dopaminergic neuronal loss in the substantia nigra

The anti‐apoptotic Bcl‐xL is a promising agent to prevent neurodegeneration in Parkinson’s disease, which is characterized by a demise of dopaminergic neurons. We linked Bcl‐xL to a peptide that allows its delivery across biological membranes and the blood–brain barrier. We tested the fusion protein in two models of Parkinson’s Disease. Cell‐permeable Bcl‐xL protected neuroblastoma cells from the selective neurotoxin 1‐methyl‐4‐phenylpyridinium. Furthermore, its systemic application in aged mice protected dopaminergic neurons following administration of MPTP as revealed by counting of tyrosine hydroxylase‐immunoreactive neurons in the substantia nigra pars compacta. Hence, we present that a cell‐permeable form of an anti‐apoptotic protein can be delivered to CNS neurons through its systemic application, and we provide the proof that the delivery of this protein to the CNS neurons effectively prevents neuronal cell death in models of chronic neurodegenerative diseases.