Caspase-2 is upregulated after sciatic nerve transection and its inhibition protects dorsal root ganglion neurons from Apoptosis after serum withdrawal

Sciatic nerve (SN) transection-induced apoptosis of dorsal root ganglion neurons (DRGN) is one factor determining the efficacy of peripheral axonal regeneration and the return of sensation. Here, we tested the hypothesis that caspase-2(CASP2) orchestrates apoptosis of axotomised DRGN both in vivo and in vitro by disrupting the local neurotrophic supply to DRGN. We observed significantly elevated levels of cleaved CASP2 (C-CASP2), compared to cleaved caspase-3 (C-CASP3), within TUNEL+DRGN and DRG glia (satellite and Schwann cells) after SN transection. A serum withdrawal cell culture model, which induced 40% apoptotic death in DRGN and 60% in glia, was used to model DRGN loss after neurotrophic factor withdrawal. Elevated C-CASP2 and TUNEL were observed in both DRGN and DRG glia, with C-CASP2 localisation shifting from the cytosol to the nucleus, a required step for induction of direct CASP2-mediated apoptosis. Furthermore, siRNAmediated downregulation of CASP2 protected 50% of DRGN from apoptosis after serum withdrawal, while downregulation of CASP3 had no effect on DRGN or DRG glia survival. We conclude that CASP2 orchestrates the death of SN-axotomised DRGN directly and also indirectly through loss of DRG glia and their local neurotrophic factor support. Accordingly, inhibiting CASP2 expression is a potential therapy for improving both the SN regeneration response and peripheral sensory recovery

Single-Cell RNA-seq Reveals Profound Alterations in Mechanosensitive Dorsal Root Ganglion Neurons with Vitamin E Deficiency.

Ninety percent of Americans consume less than the estimated average requirements of dietary vitamin E (vitE). Severe vitE deficiency due to genetic mutations in the tocopherol transfer protein (TTPA) in humans results in ataxia with vitE deficiency (AVED), with proprioceptive deficits and somatosensory degeneration arising from dorsal root ganglia neurons (DRGNs). Single-cell RNA-sequencing of DRGNs was performed in Ttpa-/- mice, an established model of AVED. In stark contrast to expected changes in proprioceptive neurons, Ttpa-/- DRGNs showed marked upregulation of voltage-gated Ca2+ and K+ channels in mechanosensitive, tyrosine-hydroxylase positive (TH+) DRGNs. The ensuing significant conductance changes resulted in reduced excitability in mechanosensitive Ttpa-/- DRGNs. A highly supplemented vitE diet (600 mg dl-α-tocopheryl acetate/kg diet) prevented the cellular and molecular alterations and improved mechanosensation. VitE deficiency profoundly alters the molecular signature and functional properties of mechanosensitive TH+ DRGN, representing an intriguing shift of the prevailing paradigm from proprioception to mechanical sensation

Deep Recurrent Generative Decoder for Abstractive Text Summarization

We propose a new framework for abstractive text summarization based on a sequence-to-sequence oriented encoder-decoder model equipped with a deep recurrent generative decoder (DRGN). Latent structure information implied in the target summaries is learned based on a recurrent latent random model for improving the summarization quality. Neural variational inference is employed to address the intractable posterior inference for the recurrent latent variables. Abstractive summaries are generated based on both the generative latent variables and the discriminative deterministic states. Extensive experiments on some benchmark datasets in different languages show that DRGN achieves improvements over the state-of-the-art methods.Comment: 10 pages, EMNLP 201

Fast Blue and Diamidino Yellow as retrograde tracers in peripheral nerves: efficacy of combined nerve injection and capsule application to transected nerves in the adult rat

Capsule application of Diamidino Yellow (DY) to the cut end of the sciatic nerve immediately followed by capsule application of Fast Blue (FB) resulted in approximate to 95% double-labelled dorsal root ganglion neurones (DRGn) and motoneurones (Mn). Nerve injection of DY followed either immediately or 2 months later by capsule application of FB resulted in approximate to 90% double-labelled DRGn and Mn, indicating that DY and FB label similar populations of DRGn and Mn, and that insignificant DY fading occurred during this period. Inversing the order of application, however, i.e. nerve injection of FB followed immediately by capsule application of DY, resulted in double labelling in only approximate to 10% of the DRGn and Mn. These percentages increased to 70% of the DRGn and 60% of the Mn when the FB injection was followed 1 or 2 months after by the DY application, indicating that DY uptake is blocked by recent administration of FB. The results indicate that DY and FB might be useful for sequential labelling before and after nerve injury as a tool to investigate the accuracy of sensory and motor regeneration

Microglia become hypofunctional and release metalloproteases and tau seeds when phagocytosing live neurons with P301S tau aggregates.

[Figure: see text]

Artificial Ontogenies: A Computational Model of the Control and Evolution of Development

Understanding the behaviour of biological systems is a challenging task. Gene regulation, development and evolution are each a product of nonlinear interactions between many individual agents: genes, cells or organisms. Moreover, these three processes are not isolated, but interact with one another in an important fashion. The development of an organism involves complex patterns of dynamic behaviour at the genetic level. The gene networks that produce this behaviour are subject to mutations that can alter the course of development, resulting in the production of novel morphologies. Evolution occurs when these novel morphologies are favoured by natural selection and survive to pass on their genes to future generations. Computational models can assist us to understand biological systems by providing a framework within which their behaviour can be explored. Many natural processes, including gene regulation and development, have a computational element to their control. Constructing formal models of these systems enables their behaviour to be simulated, observed and quantified on a scale not otherwise feasible. This thesis uses a computational simulation methodology to explore the relationship between development and evolution. An important question in evolutionary biology is how to explain the direction of evolution. Conventional explanations of evolutionary history have focused on the role of natural selection in orienting evolution. More recently, it has been argued that the nature of development, and the way it changes in response to mutation, may also be a significant factor. A network-lineage model of artificial ontogenies is described that incorporates a developmental mapping between the dynamics of a gene network and a cell lineage representation of a phenotype. Three series of simulation studies are reported, exploring: (a) the relationship between the structure of a gene network and its dynamic behaviour; (b) the characteristic distributions of ontogenies and phenotypes generated by the dynamics of gene networks; (c) the effect of these characteristic distributions on the evolution of ontogeny. The results of these studies indicate that the model networks are capable of generating a diverse range of stable behaviours, and possess a small yet significant sensitivity to perturbation. In the context of developmental control, the intrinsic dynamics of the model networks predispose the production of ontogenies with a modular, quasi-systematic structure. This predisposition is reflected in the structure of variation available for selection in an adaptive search process, resulting in the evolution of ontogenies biased towards simplicity. These results suggest a possible explanation for the levels of ontogenetic complexity observed in biological organisms: that they may be a product of the network architecture of developmental control. By quantifying complexity, variation and bias, the network-lineage model described in this thesis provides a computational method for investigating the effects of development on the direction of evolution. In doing so, it establishes a viable framework for simulating computational aspects of complex biological systems

Implantable and transcutaneous photobiomodulation promote neuroregeneration and recovery of lost function after spinal cord injury

Spinal cord injury (SCI) is a cause of profound and irreversible damage, with no effective therapy to promote functional recovery. Photobiomodulation (PBM) may provide a viable therapeutic approach using red or near-infrared light to promote recovery after SCI by mitigating neuroinflammation and preventing neuronal apoptosis. Our current study aimed to optimize PBM dose regimens and develop and validate the efficacy of an invasive PBM delivery paradigm for SCI. Dose optimization studies were performed using a serum withdrawal model of injury in cultures of primary adult rat dorsal root ganglion neurons (DRGN). Implantable and transcutaneous PBM delivery protocols were developed and validated using cadaveric modeling. The efficacy of PBM in promoting recovery after SCI in vivo was studied in a dorsal column crush injury model of SCI in adult rats. Optimal neuroprotection in vitro was achieved between 4 and 22 mW/cm2. 11 mW/cm2 for 1 min per day (0.66 J/cm2) increased cell viability by 45% over 5 days (p <0.0001), increasing neurite outgrowth by 25% (p < 0.01). A method for invasive application of PBM was developed using a diffusion-tipped optogenetics fiber optic. Delivery methods for PBM were developed and validated for both invasive (iPBM) and noninvasive (transcutaneous) (tcPBM) application. iPBM and tcPBM (24 mW/cm2 at spinal cord, 1 min per day (1.44 J/cm2) up to 7 days) increased activation of regeneration-associated protein at 3 days after SCI, increasing GAP43+ axons in DRGN from 18.0% (control) to 41.4% ± 10.5 (iPBM) and 45.8% ± 3.4 (tcPBM) (p < 0.05). This corresponded to significant improvements at 6 weeks post-injury in functional locomotor and sensory function recovery (p < 0.01), axonal regeneration (p < 0.01), and reduced lesion size (p < 0.01). Our results demonstrated that PBM achieved a significant therapeutic benefit after SCI, either using iPBM or tcPBM application and can potentially be developed for clinical use in SCI patients

Cytoplasmic expression systems triggered by mRNA yield increased gene expression in post-mitotic neurons

Non-viral vectors are promising vehicles for gene therapy but delivery of plasmid DNA to post-mitotic cells is challenging as nuclear entry is particularly inefficient. We have developed and evaluated a hybrid mRNA/DNA system designed to bypass the nuclear barrier to transfection and facilitate cytoplasmic gene expression. This system, based on co-delivery of mRNA(A64) encoding for T7 RNA polymerase (T7 RNAP) with a T7-driven plasmid, produced between 10- and 2200-fold higher gene expression in primary dorsal root ganglion neuronal (DRGN) cultures isolated from Sprague–Dawley rats compared to a cytomegalovirus (CMV)-driven plasmid, and 30-fold greater expression than the enhanced T7-based autogene plasmid pR011. Cell-free assays and in vitro transfections highlighted the versatility of this system with small quantities of T7 RNAP mRNA required to mediate expression at levels that were significantly greater than with the T7-driven plasmid alone or supplemented with T7 RNAP protein. We have also characterized a number of parameters, such as mRNA structure, intracellular stability and persistence of each nucleic acid component that represent important factors in determining the transfection efficiency of this hybrid expression system. The results from this study demonstrate that co-delivery of mRNA is a promising strategy to yield increased expression with plasmid DNA, and represents an important step towards improving the capability of non-viral vectors to mediate efficient gene transfer in cell types, such as in DRGN, where the nuclear membrane is a significant barrier to transfection