Nanoscopic Characterisation of Individual Endogenous Protein Aggregates in Human Neuronal Cells.

The aberrant misfolding and subsequent conversion of monomeric protein into amyloid aggregates characterises many neurodegenerative disorders, including Parkinson's and Alzheimer's diseases. These aggregates are highly heterogeneous in structure, generally of low abundance and typically smaller than the diffraction limit of light (≈250 nm). To overcome the challenges these characteristics pose to the study of endogenous aggregates formed in cells, we have developed a method to characterise them at the nanometre scale without the need for a conjugated fluorophore. Using a combination of DNA PAINT and an amyloid-specific aptamer, we demonstrate that this technique is able to detect and super-resolve a range of aggregated species, including those formed by α-synuclein and amyloid-ß. Additionally, this method enables endogenous protein aggregates within cells to be characterised. We found that neuronal cells derived from patients with Parkinson's disease contain a larger number of protein aggregates than those from healthy controls.

Impaired intracellular trafficking defines early Parkinson's disease.

Parkinson's disease (PD) is an insidious and incurable neurodegenerative disease, and represents a significant cost to individuals, carers, and ageing societies. It is defined at post-mortem by the loss of dopamine neurons in the substantia nigra together with the presence of Lewy bodies and Lewy neurites. We examine here the role of α-synuclein and other cellular transport proteins implicated in PD and how their aberrant activity may be compounded by the unique anatomy of the dopaminergic neuron. This review uses multiple lines of evidence from genetic studies, human tissue, induced pluripotent stem cells, and refined animal models to argue that prodromal PD can be defined as a disease of impaired intracellular trafficking. Dysfunction of the dopaminergic synapse heralds trafficking impairment.

Induction of the unfolded protein response by α-synuclein in experimental models of Parkinson's disease.

Accumulation of misfolded proteins in the endoplasmic reticulum (ER) is the main event leading to the induction of the ER stress-related unfolded protein response (UPR). Recent postmortem evaluation, showing that the UPR pathway is activated in nigral dopaminergic neurons bearing α-synuclein inclusions in the brain of Parkinson's disease (PD) patients, suggests that the activation of the UPR may be induced by the accumulation of α-synuclein. In this study, we show that the misfolded protein-sensor/UPR activator glucose-regulated protein 78/immunoglobulin heavy chain-binding protein was bound to α-synuclein and was increased in 'in vitro' and 'in vivo' models showing aggregated α-synuclein accumulation. Moreover, α-synuclein accumulation induced the expression of the UPR-related activating transcription factor 4/cAMP-responsive element-2. These findings indicate that activation of the UPR pathway in the PD brain is associated with α-synuclein accumulation occurring in part within the ER.

Late onset distal axonal swelling in YFP-H transgenic mice.

Axonal swellings, or spheroids, are a feature of central nervous system (CNS) axon degeneration during normal aging and in many disorders. The direct cause and mechanism are unknown. The use of transgenic mouse line YFP-H, which expresses yellow-fluorescent protein (YFP) in a subset of neurons, greatly facilitates longitudinal imaging and live imaging of axonal swellings, but it has not been established whether long-term expression of YFP itself contributes to axonal swelling. Using conventional methods to compare YFP-H mice with their YFP negative littermates, we found an age-related increase in swellings in discrete CNS regions in both genotypes, but the presence of YFP caused significantly more swellings in mice aged 8 months or over. Increased swelling was found in gracile tract, gracile nucleus and dorsal roots but not in lateral columns, olfactory bulb, motor cortex, ventral roots or peripheral nerve. Thus, long-term expression of YFP accelerates age-related axonal swelling in some axons and data reliant on the presence of YFP in these CNS regions in older animals needs to be interpreted carefully. The ability of a foreign protein to exacerbate age-related axon pathology is an important clue to the mechanisms by which such pathology can arise.

Nurr1 co-localizes with EphB1 receptors in the developing ventral midbrain, and its expression is enhanced by the EphB1 ligand, ephrinB2.

Both ephrins and the transcription factor, Nurr1, are critically involved in CNS development and, particularly, in the ontogenesis of the nigro-striatal system. Here we examined whether the ephrin receptor, EphB1, and Nurr1 share a similar expression pattern in the embryonic brain and whether expression of Nurr1 is under the control of EphB1 activation. The transcripts of EphB1 receptor and Nurr1 showed a similar pattern of expression in the ventral midbrain of mice at early stages of embryonic development (E11.5 and E12.5). At later stages (E15.5), only Nurr1 mRNA could still be detected in significant amounts in the A9-A10 regions of the ventral midbrain, whereas the two transcripts still showed a similar pattern of expression in discrete regions of the hindbrain. To examine whether activation of EphB1 receptor could induce the expression of Nurr1 in the ventral midbrain, we applied the EphB1 ligand, ephrinB2, to explants of embryonic mouse ventral midbrain. Low concentrations of clustered ephrinB2 (0.25 microg/mL) enhanced Nurr1 mRNA and protein levels, whereas higher concentrations were inactive. We conclude that activation of EphB1 receptors by appropriate concentrations of its ligand ephrinB2 might contribute to the acquisition of a dopaminergic fate in developing midbrain ventral neurones.