Transmission electron microscopy characterization of fluorescently labelled amyloid β 1-40 and α-synuclein aggregates

<p>Abstract</p> <p>Background</p> <p>Fluorescent tags, including small organic molecules and fluorescent proteins, enable the localization of protein molecules in biomedical research experiments. However, the use of these labels may interfere with the formation of larger-scale protein structures such as amyloid aggregates. Therefore, we investigate the effects of some commonly used fluorescent tags on the morphologies of fibrils grown from the Alzheimer's disease-associated peptide Amyloid β 1-40 (Aβ40) and the Parkinson's disease-associated protein α-synuclein (αS).</p> <p>Results</p> <p>Using transmission electron microscopy (TEM), we verify that N-terminal labeling of Aβ40 with AMCA, TAMRA, and Hilyte-Fluor 488 tags does not prevent the formation of protofibrils and amyloid fibrils of various widths. We also measure the two-photon action cross-section of Aβ40 labelled with Hilyte Fluor 488 and demonstrate that this tag is suitable for use with two-photon fluorescence techniques. Similarly, we find that Alexa Fluor 488 labelling of αS variant proteins near either the N or C terminus (position 9 or 130) does not interfere with the formation of amyloid and other types of αS fibrils. We also present TEM images of fibrils grown from αS C-terminally labelled with enhanced green fluorescent protein (EGFP). Near neutral pH, two types of αS-EGFP fibrils are observed via TEM, while denaturation of the EGFP tag leads to the formation of additional species.</p> <p>Conclusions</p> <p>We demonstrate that several small extrinsic fluorescent tags are compatible with studies of amyloid protein aggregation. However, although fibrils can be grown from αS labelled with EGFP, the conformation of the fluorescent protein tag affects the observed aggregate morphologies. Thus, our results should assist researchers with label selection and optimization of solution conditions for aggregation studies involving fluorescence techniques.</p

India, Brazil, and public health: Rule‐making

This article analyzes the domestic drivers of regulatory state formation in India and Brazil and its consequences for the global rules governing pharmaceutical patents. We first analyze Indian and Brazilian politics of regulatory state formation; then, in light of the extent to which the two countries have built regulatory capacity and capability in the field of patent regulation, we explore whether and how they have been able to influence the existing intellectual property regime in health. We look into India's Section 3(d) and Brazil's prior consent requirement. Whereas India's Section 3(d) regulation has gained international regulatory influence by diffusing to other developing countries, the same cannot be said for Brazil's prior consent regulation, which has been caught by policy-reversals. The transition toward regulatory states in emerging countries is a bulky road and does not progress in linear ways. However, once regulatory capacity and capability have been solidified, domestic policy innovations can become internationally influential

The small molecule alpha-synuclein misfolding inhibitor, NPT200-11, produces multiple benefits in an animal model of Parkinson’s disease

Abstract Accumulation of alpha-synuclein (ASYN) in neurons and other CNS cell types may contribute to the underlying pathology of synucleinopathies including Parkinson’s disease (PD), dementia with Lewy bodies (DLB) and Multiple Systems Atrophy (MSA). In support of this hypothesis for PD, ASYN immunopositive aggregates are a prominent pathological feature of PD, and mutations and gene multiplications of human wild type (WT) ASYN cause rare familial autosomal-dominant forms of PD. Targeted therapeutics that reduce the accumulation of ASYN could prevent or slow the neurodegenerative processes in PD and other synucleinopathies. NPT200-11 is a novel small molecule inhibitor of ASYN misfolding and aggregation. The effects of NPT200-11 on ASYN neuropathology were evaluated in animal models over expressing human alpha synuclein. Longitudinal studies using retinal imaging in mice expressing a hASYN::GFP fusion protein revealed that 2 months of once daily administration of NPT200-11 (5 mg/kg IP) resulted in a time-dependent and progressive reduction in retinal ASYN pathology. The effects of NPT200-11 on ASYN pathology in cerebral cortex and on other disease-relevant endpoints was evaluated in the Line 61 transgenic mouse model overexpressing human wild type ASYN. Results from these studies demonstrated that NPT200-11 reduced alpha-synuclein pathology in cortex, reduced associated neuroinflammation (astrogliosis), normalized striatal levels of the dopamine transporter (DAT) and improved motor function. To gain insight into the relationship between dose, exposure, and therapeutic benefit pharmacokinetic studies were also conducted in mice. These studies demonstrated that NPT200-11 is orally bioavailable and brain penetrating and established target plasma and brain exposures for future studies of potential therapeutic benefit

Assessing the Subcellular Dynamics of Alpha-synuclein Using Photoactivation Microscopy

Alpha-synuclein (aSyn) is implicated in Parkinson’s disease and several other neurodegenerative disorders. To date, the function and intracellular dynamics of aSyn are still unclear. Here, we tracked the dynamics of aSyn using photoactivatable green fluorescent protein as a reporter. We found that the availability of the aSyn N terminus modulates its shuttling into the nucleus. Interestingly, familial aSyn mutations altered the dynamics at which the protein distributes throughout the cell. Both the A30P and A53T aSyn mutations increase the speed at which the protein moves between the nucleus and cytoplasm, respectively. We also found that specific kinases potentiate the shuttling of aSyn between nucleus and cytoplasm. A mutant aSyn form that blocks S129 phosphorylation, S129A, results in the formation of cytoplasmic inclusions, suggesting phosphorylation modulates aggregation in addition to modulating aSyn intracellular dynamics. Finally, we found that the molecular chaperone HSP70 accelerates the entry of aSyn into the nuclear compartment.peerReviewe

Visual stimuli-induced LTD of GABAergic synapses mediated by presynaptic NMDA receptors

Local GABA (gamma-aminobutyric acid) circuits contribute to sensory experience-dependent refinement of neuronal connections in the developing nervous system, but whether GABAergic synapses themselves can be rapidly modified by sensory stimuli is largely unknown. Here we report that repetitive light stimuli or theta burst stimulation (TBS) of the optic nerve in the developing Xenopus retinotectal system induces long-term potentiation (LTP) of glutamatergic inputs but long-term depression (LTD) of GABAergic inputs to the same tectal neuron. The LTD is due to a reduction in presynaptic GABA release and requires activation of presynaptic NMDA (N-methyl-D-aspartate) receptors (NMDARs) and coincident high-level GABAergic activity. Thus, the presynaptic NMDAR may function as a coincidence detector for adjacent glutamatergic and GABAergic activities, leading to coordinated synaptic modification by sensory experience

Extrasynaptic vesicle recycling in mature hippocampal neurons

Fast neuronal signalling relies on highly-regulated vesicle fusion and recycling at specialized presynaptic terminals. Recently, examples of non-classical neurotransmission have also been reported, where fusion of vesicles can occur at sites remote from conventional synapses. This has potentially broad biological implications, but the underlying mechanisms are not well established. Here, in mature hippocampal neurons, we demonstrate that a complete vesicle recycling pathway can occur at discrete axonal sites and that extrasynaptic fusion is a robust feature of native tissue. We show that laterally mobile vesicle clusters trafficking between synaptic terminals become transiently stabilized by evoked action potentials and undergo complete but delayed Ca2+-dependent fusion along axons. This fusion is associated with dynamic actin accumulation and subsequently, vesicles can be locally recycled, reacidified and re-used. Immunofluorescence and ultrastructural work demonstrates that extrasynaptic fusion sites can have apposed postsynaptic specializations, suggesting that mobile vesicle recycling may underlie highly dynamic neuron-neuron communication