Microtubule stabilising peptides rescue tau phenotypes in-vivo

The microtubule cytoskeleton is a highly dynamic, filamentous network underpinning cellular structure and function. In Alzheimer’s disease, the microtubule cytoskeleton is compromised, leading to neuronal dysfunction and eventually cell death. There are currently no disease-modifying therapies to slow down or halt disease progression. However, microtubule stabilisation is a promising therapeutic strategy that is being explored. We previously investigated the disease-modifying potential of a microtubule-stabilising peptide NAP (NAPVSIPQ) in a well-established Drosophila model of tauopathy characterised by microtubule breakdown and axonal transport deficits. NAP prevented as well as reversed these phenotypes even after they had become established. In this study, we investigate the neuroprotective capabilities of an analogous peptide SAL (SALLRSIPA). We found that SAL mimicked NAP’s protective effects, by preventing axonal transport disruption and improving behavioural deficits, suggesting both NAP and SAL may act via a common mechanism. Both peptides contain a putative ‘SIP’ (Ser-Ile-Pro) domain that is important for interactions with microtubule end-binding proteins. Our data suggests this domain may be central to the microtubule stabilising function of both peptides and the mechanism by which they rescue phenotypes in this model of tauopathy. Our observations support microtubule stabilisation as a promising disease-modifying therapeutic strategy for tauopathies like Alzheimer’s disease

The extracellular environment of the CNS : influence on plasticity, sprouting, and axonal regeneration after spinal cord injury

Melissa R Andrews and Shmma Quraishe are supported by a research grant from the Biotechnology and Biological Sciences Research Council (BBSRC).The extracellular environment of the central nervous system (CNS) becomes highly structured and organized as the nervous system matures. The extracellular space of the CNS along with its subdomains plays a crucial role in the function and stability of the CNS. In this review, we have focused on two components of the neuronal extracellular environment, which are important in regulating CNS plasticity including the extracellular matrix (ECM) and myelin. The ECM consists of chondroitin sulfate proteoglycans (CSPGs) and tenascins, which are organized into unique structures called perineuronal nets (PNNs). PNNs associate with the neuronal cell body and proximal dendrites of predominantly parvalbumin-positive interneurons, forming a robust lattice-like structure. These developmentally regulated structures are maintained in the adult CNS and enhance synaptic stability. After injury, however, CSPGs and tenascins contribute to the structure of the inhibitory glial scar, which actively prevents axonal regeneration. Myelin sheaths and mature adult oligodendrocytes, despite their important role in signal conduction in mature CNS axons, contribute to the inhibitory environment existing after injury. As such, unlike the peripheral nervous system, the CNS is unable to revert to a “developmental state” to aid neuronal repair. Modulation of these external factors, however, has been shown to promote growth, regeneration, and functional plasticity after injury. This review will highlight some of the factors that contribute to or prevent plasticity, sprouting, and axonal regeneration after spinal cord injury.Publisher PDFPeer reviewe

Soluble axoplasm enriched from injured CNS axons reveals the early modulation of the actin cytoskeleton

Axon injury and degeneration is a common consequence of diverse neurological conditions including multiple sclerosis, traumatic brain injury and spinal cord injury. The molecular events underlying axon degeneration are poorly understood. We have developed a novel method to enrich for axoplasm from rodent optic nerve and characterised the early events in Wallerian degeneration using an unbiased proteomics screen. Our detergent-free method draws axoplasm into a dehydrated hydrogel of the polymer poly(2-hydroxyethyl methacrylate), which is then recovered using centrifugation. This technique is able to recover axonal proteins and significantly deplete glial contamination as confirmed by immunoblotting. We have used iTRAQ to compare axoplasm-enriched samples from naïve vs injured optic nerves, which has revealed a pronounced modulation of proteins associated with the actin cytoskeleton. To confirm the modulation of the actin cytoskeleton in injured axons we focused on the RhoA pathway. Western blotting revealed an augmentation of RhoA and phosphorylated cofilin in axoplasm-enriched samples from injured optic nerve. To investigate the localisation of these components of the RhoA pathway in injured axons we transected axons of primary hippocampal neurons in vitro. We observed an early modulation of filamentous actin with a concomitant redistribution of phosphorylated cofilin in injured axons. At later time-points, RhoA is found to accumulate in axonal swellings and also colocalises with filamentous actin. The actin cytoskeleton is a known sensor of cell viability across multiple eukaryotes, and our results suggest a similar role for the actin cytoskeleton following axon injury. In agreement with other reports, our data also highlights the role of the RhoA pathway in axon degeneration. These findings highlight a previously unexplored area of axon biology, which may open novel avenues to prevent axon degeneration. Our method for isolating CNS axoplasm also represents a new tool to study axon biology

Modelling Tauopathies in Drosophila: Insights from the Fruit Fly

Drosophila melanogaster is an experimentally tractable model organism that has been used successfully to model aspects of many human neurodegenerative diseases. Drosophila models of tauopathy have provided valuable insights into tau-mediated mechanisms of neuronal dysfunction and death. Here we review the findings from Drosophila models of tauopathy reported over the past ten years and discuss how they have furthered our understanding of the pathogenesis of tauopathies. We also discuss the multitude of technical advantages that Drosophila offers, which make it highly attractive as a model for such studies

Far-field Unlabelled Super-Resolution Imaging with Superoscillatory Illumination

Unlabelled super-resolution is the next grand challenge in imaging. Stimulated emission depletion and single-molecule microscopies have revolutionised the life sciences but are still limited by the need for reporters (labels) embedded within the sample. While the Veselago-Pendry “super-lens” using a negative-index metamaterial is a promising idea for imaging beyond the diffraction limit, there are substantial technological challenges to its realisation. Another route to far-field subwavelength focusing is using optical superoscillations: engineered interference of multiple coherent waves creating an, in principle, arbitrarily small hotspot. Here we demonstrate microscopy with superoscillatory illumination of the object and describe its underlying principles. We show that far-field images taken with superoscillatory illumination are themselves superoscillatory and hence can reveal fine structural details of the object that are lost in conventional far-field imaging. We show that the resolution of a superoscillatory microscope is determined by the size of the hotspot, rather than the bandwidth of the optical instrument. We demonstrate high-frame-rate polarisation-contrast imaging of unmodified living cells with resolution significantly exceeding that achievable with conventional instruments. This non-algorithmic, low-phototoxicity imaging technology is a powerful tool both for biological research and for super-resolution imaging of samples that do not allow labelling, such as the interior of silicon chips

Distinct phenotypes of three-repeat and four-repeat human tau in a transgenic model of tauopathy.

Tau exists as six closely related protein isoforms in the adult human brain. These are generated from alternative splicing of a single mRNA transcript and they differ in the absence or presence of two N-terminal and three or four microtubule binding domains. Typically all six isoforms have been considered functionally similar. However, their differential involvement in particular tauopathies raises the possibility that there may be isoform-specific differences in physiological function and pathological role. To explore this, we have compared the phenotypes induced by the 0N3R and 0N4R isoforms in Drosophila. Expression of the 3R isoform causes more profound axonal transport defects and locomotor impairments, culminating in a shorter lifespan than the 4R isoform. In contrast, the 4R isoform leads to greater neurodegeneration and impairments in learning and memory. Furthermore, the phosphorylation patterns of the two isoforms are distinct, as is their ability to induce oxidative stress. These differences are not consequent to different expression levels and are suggestive of bona fide physiological differences in isoform biology and pathological potential. They may therefore explain isoform-specific mechanisms of tau-toxicity and the differential susceptibility of brain regions to different tauopathies

The sHsp expression signature in the brain and modulation in models of chronic neurodegeneration

Intrinsic protein folding pathways are modulated by molecular chaperones, such as the diversegroup of heat shock proteins (Hsps). Among these is the small heat shock protein (sHsp)family which in the mammalian genome consists of 10 low molecular weight (15-30kDa)members. The sHsps have classical chaperone functions but additionally contribute topathways that protect against cellular stresses, maintain the cytoskeleton, prevent proteinaggregation and regulate apoptosis. They contain a characteristic C-terminal ?-crystallindomain, which is exclusive to the sHsp family. In addition to their constitutive expressionunder physiological (non-disease) conditions, they are also induced under conditions ofstress/heat shock which is thought to play a role in response to protein misfolding thatunderpins disease. There are a wide range of diseases in which the sHsps function or aredysfunctional by mutations, such as neurodegenerative disorders, cataract, and desmin relatedmyopathy.Each of the 10 sHsps is believed to have a unique expression profile. Seven of the sHsps areexpressed in heart and muscle, but little is known about their precise expression and/orphysiological role in the CNS. In the present study the expression of the mammalian sHsps invarious mouse tissues including the brain was investigated. This provided evidence for theconstitutive expression of 4 sHsps in the brain. In situ hybridization using naïve adult micerevealed a distinct white matter (oligodendrocyte) specific expression pattern for HspB5 (?Bcrystallin).HspB1 (Hsp25) and HspB8 (Hsp22) demonstrated overlapping expression in thelateral and dorsal ventricles of the brain, as well as expression in a distinct set of motorneurons in the ventral horn of the spinal cord. Further, cellular immunostaining and subfractionationof brain tissue supports a distinct cellular and subcellular protein expression ofHspB1, HspB5, HspB6 (Hsp20) and HspB8 in the brain. Both HspB5 and HspB6 wereenriched in the myelin fraction. In view of the potential for induction of these sHsps by stressand modulation in chronic brain diseases we systematically investigated the sHsp signature intwo distinct models of intracellular (R6/2) and extracellular (ME7) proteinopathies. Thesemodels recapitulate key features of Huntington’s and prion disease, respectively.Analysis of the sHsps in the R6/2 Huntington’s disease (HD) mouse model showed a specificdown-regulation of HspB5 in the white matter at all time points analyzed. All other sHspsinvestigated did not change in this model of HD. Analysis of the sHsps in ME7 prion diseaseshowed up-regulation of HspB1, HspB5 and HspB8 in the hippocampus. For HspB1, this wasselective to an anatomically defined sub-population of astrocytes distributed in the stratumradiatum. In contrast, all GFAP positive astrocytes throughout the hippocampus exhibitedinduced expression of HspB5 and HspB8. Based on QT-PCR data, the changes in expressionof the sHsps in either model was not under transcriptional control, suggesting translation/posttranslationalregulation. The differing results in the two models suggest that the presence ofintracellular (R6/2) or extracellular (ME7) aggregates may dictate the sHsp responseassociated with non-neuronal cells. In view of the emerging significance of non-neuronal cellsin chronic diseases the data supports adaptive and differential responses that might contributeto and/or provide a route to therapy of distinct aspects of neurodegeneration

What is the pathological significance of tau oligomers?

Insoluble aggregates of the microtubule-associated protein tau characterize a number of neurodegenerative diseases collectively termed tauopathies. These aggregates comprise abnormally hyperphosphorylated and misfolded tau proteins. Research in this field has traditionally focused on understanding how hyperphosphorylated and aggregated tau mediates dysfunction and toxicity in tauopathies. Recent findings from both Drosophila and rodent models of tauopathy suggest that large insoluble aggregates such as tau filaments and tangles may not be the key toxic species in these diseases. Thus some investigators have shifted their focus to study pre-filament tau species such as tau oligomers and hyperphosphorylated tau monomers. Interestingly, tau oligomers can exist in a variety of states including hyperphosphorylated and unphosphorylated forms, which can be both soluble and insoluble. It remains to be determined which of these oligomeric states of tau are causally involved in neurodegeneration and which signal the beginning of the formation of inert/protective filaments. It will be important to better understand this so that tau-based therapeutic interventions can target the most toxic tau species