Direct angiotensin AT2 receptor stimulation using a novel AT2 receptor agonist, compound 21, evokes neuroprotection in conscious hypertensive rats

Background: In this study, the neuroprotective effect of a novel nonpeptide AT2R agonist, C21, was examined in a conscious model of stroke to verify a class effect of AT2R agonists as neuroprotective agents. Methods and Results: Spontaneously hypertensive rats (SHR) were pre-treated for 5 days prior to stroke with C21 alone or in combination with the AT2R antagonist PD123319. In a separate series of experiments C21 was administered in a series of 4 doses commencing 6 hours after stroke. A focal reperfusion model of ischemia was induced in conscious SHR by administering endothelin-1 to the middle cerebral artery (MCA). Motor coordination was assessed at 1 and 3 days after stroke and post mortem analyses of infarct volumes, microglia activation and neuronal survival were performed at 72 hours post MCA occlusion. When given prior to stroke, C21 dose dependently decreased infarct volume, which is consistent with the behavioural findings illustrating an improvement in motor deficit. During the pre-treatment protocol C21 was shown to enhance microglia activation, which are likely to be evoking protection by releasing brain derived neurotrophic factor. When drug administration was delayed until 6 hours after stroke, C21 still reduced brain injury. Conclusion: These results indicate that centrally administered C21 confers neuroprotection against stroke damage. This benefit is likely to involve various mechanisms, including microglial activation of endogenous repair and enhanced cerebroperfusion. Thus, we have confirmed the neuroprotective effect of AT2R stimulation using a nonpeptide compound which highlights the clinical potential of the AT2R agonists for future development

Spontaneous Development of Full Weight-Supported Stepping after Complete Spinal Cord Transection in the Neonatal Opossum, Monodelphis domestica

Spinal cord trauma in the adult nervous system usually results in permanent loss of function below the injury level. The immature spinal cord has greater capacity for repair and can develop considerable functionality by adulthood. This study used the marsupial laboratory opossum Monodelphis domestica, which is born at a very early stage of neural development. Complete spinal cord transection was made in the lower-thoracic region of pups at postnatal-day 7 (P7) or P28, and the animals grew to adulthood. Injury at P7 resulted in a dense neuronal tissue bridge that connected the two ends of the cord; retrograde neuronal labelling indicated that supraspinal and propriospinal innervation spanned the injury site. This repair was associated with pronounced behavioural recovery, coordinated gait and an ability to use hindlimbs when swimming. Injury at P28 resulted in a cyst-like cavity encased in scar tissue forming at the injury site. Using retrograde labelling, no labelled brainstem or propriospinal neurons were found above the lesion, indicating that detectable neuronal connectivity had not spanned the injury site. However, these animals could use their hindlimbs to take weight-supporting steps but could not use their hindlimbs when swimming. White matter, demonstrated by Luxol Fast Blue staining, was present in the injury site of P7- but not P28-injured animals. Overall, these studies demonstrated that provided spinal injury occurs early in development, regrowth of supraspinal innervation is possible. This repair appears to lead to improved functional outcomes. At older ages, even without detectable axonal growth spanning the injury site, substantial development of locomotion was still possible. This outcome is discussed in conjunction with preliminary findings of differences in the local propriospinal circuits following spinal cord injury (demonstrated with fluororuby labelling), which may underlie the weight bearing locomotion observed in the apparent absence of axons bridging the lesion site in P28-injured Monodelphis

First Reported Prairie Dog–to-Human Tularemia Transmission, Texas, 2002

A tularemia outbreak, caused by Francisella tularensis type B, occurred among wild-caught, commercially traded prairie dogs. F. tularensis microagglutination titers in one exposed person indicated recent infection. These findings represent the first evidence for prairie-dog-to-human tularemia transmission and demonstrate potential human health risks of the exotic pet trade

Spatio-Temporal Progression of Grey and White Matter Damage Following Contusion Injury in Rat Spinal Cord

Cellular mechanisms of secondary damage progression following spinal cord injury remain unclear. We have studied the extent of tissue damage from 15 min to 10 weeks after injury using morphological and biochemical estimates of lesion volume and surviving grey and white matter. This has been achieved by semi-quantitative immunocytochemical methods for a range of cellular markers, quantitative counts of white matter axonal profiles in semi-thin sections and semi-quantitative Western blot analysis, together with behavioural tests (BBB scores, ledged beam, random rung horizontal ladder and DigiGait™ analysis). We have developed a new computer-controlled electronic impactor based on a linear motor that allows specification of the precise nature, extent and timing of the impact. Initial (15 min) lesion volumes showed very low variance (1.92±0.23 mm3, mean±SD, n = 5). Although substantial tissue clearance continued for weeks after injury, loss of grey matter was rapid and complete by 24 hours, whereas loss of white matter extended up to one week. No change was found between one and 10 weeks after injury for almost all morphological and biochemical estimates of lesion size or behavioural methods. These results suggest that previously reported apparent ongoing injury progression is likely to be due, to a large extent, to clearance of tissue damaged by the primary impact rather than continuing cell death. The low variance of the impactor and the comprehensive assessment methods described in this paper provide an improved basis on which the effects of potential treatment regimes for spinal cord injury can be assessed

Whole-body tissue stabilization and selective extractions via tissue-hydrogel hybrids for high-resolution intact circuit mapping and phenotyping

To facilitate fine-scale phenotyping of whole specimens, we describe here a set of tissue fixation-embedding, detergent-clearing and staining protocols that can be used to transform excised organs and whole organisms into optically transparent samples within 1–2 weeks without compromising their cellular architecture or endogenous fluorescence. PACT (passive CLARITY technique) and PARS (perfusion-assisted agent release in situ) use tissue-hydrogel hybrids to stabilize tissue biomolecules during selective lipid extraction, resulting in enhanced clearing efficiency and sample integrity. Furthermore, the macromolecule permeability of PACT- and PARS-processed tissue hybrids supports the diffusion of immunolabels throughout intact tissue, whereas RIMS (refractive index matching solution) grants high-resolution imaging at depth by further reducing light scattering in cleared and uncleared samples alike. These methods are adaptable to difficult-to-image tissues, such as bone (PACT-deCAL), and to magnified single-cell visualization (ePACT). Together, these protocols and solutions enable phenotyping of subcellular components and tracing cellular connectivity in intact biological networks

Targeting the Intrinsically Disordered Structural Ensemble of a-Synuclein by Small Molecules as a Potential Therapeutic Strategy for Parkinson's Disease

Abstract The misfolding of intrinsically disordered proteins such as a-synuclein, tau and the Ab peptide has been associated with many highly debilitating neurodegenerative syndromes including Parkinson's and Alzheimer's diseases. Therapeutic targeting of the monomeric state of such intrinsically disordered proteins by small molecules has, however, been a major challenge because of their heterogeneous conformational properties. We show here that a combination of computational and experimental techniques has led to the identification of a drug-like phenyl-sulfonamide compound (ELN484228), that targets a-synuclein, a key protein in Parkinson's disease. We found that this compound has substantial biological activity in cellular models of a-synuclein-mediated dysfunction, including rescue of a-synuclein-induced disruption of vesicle trafficking and dopaminergic neuronal loss and neurite retraction most likely by reducing the amount of a-synuclein targeted to sites of vesicle mobilization such as the synapse in neurons or the site of bead engulfment in microglial cells. These results indicate that targeting a-synuclein by small molecules represents a promising approach to the development of therapeutic treatments of Parkinson's disease and related conditions

Ndfip1 regulates nuclear Pten import in vivo to promote neuronal survival following cerebral ischemia

PTEN (phosphatase and tensin homologue deleted on chromosome TEN) is the major negative regulator of phosphatidylinositol 3-kinase signaling and has cell-specific functions including tumor suppression. Nuclear localization of PTEN is vital for tumor suppression; however, outside of cancer, the molecular and physiological events driving PTEN nuclear entry are unknown. In this paper, we demonstrate that cytoplasmic Pten was translocated into the nuclei of neurons after cerebral ischemia in mice. Critically, this transport event was dependent on a surge in the Nedd4 family–interacting protein 1 (Ndfip1), as neurons in Ndfip1-deficient mice failed to import Pten. Ndfip1 binds to Pten, resulting in enhanced ubiquitination by Nedd4 E3 ubiquitin ligases. In vitro, Ndfip1 overexpression increased the rate of Pten nuclear import detected by photobleaching experiments, whereas Ndfip1⁻/⁻ fibroblasts showed negligible transport rates. In vivo, Ndfip1 mutant mice suffered larger infarct sizes associated with suppressed phosphorylated Akt activation. Our findings provide the first physiological example of when and why transient shuttling of nuclear Pten occurs and how this process is critical for neuron survival.Jason Howitt, Jenny Lackovic, Ley-Hian Low, Adam Naguib, Alison Macintyre, Choo-Peng Goh, Jennifer K. Callaway, Vicki Hammond, Tim Thomas, Matthew Dixon, Ulrich Putz, John Silke, Perry Bartlett, Baoli Yang, Sharad Kumar, Lloyd C. Trotman, and Seong-Seng Ta

The contribution of X-linked coding variation to severe developmental disorders

Abstract: Over 130 X-linked genes have been robustly associated with developmental disorders, and X-linked causes have been hypothesised to underlie the higher developmental disorder rates in males. Here, we evaluate the burden of X-linked coding variation in 11,044 developmental disorder patients, and find a similar rate of X-linked causes in males and females (6.0% and 6.9%, respectively), indicating that such variants do not account for the 1.4-fold male bias. We develop an improved strategy to detect X-linked developmental disorders and identify 23 significant genes, all of which were previously known, consistent with our inference that the vast majority of the X-linked burden is in known developmental disorder-associated genes. Importantly, we estimate that, in male probands, only 13% of inherited rare missense variants in known developmental disorder-associated genes are likely to be pathogenic. Our results demonstrate that statistical analysis of large datasets can refine our understanding of modes of inheritance for individual X-linked disorders