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This work highlights the importance of CNS transduction for treatment of neurological diseases, a finding with significant clinical implications considering the long-lasting effects of gene therapy., Niemann–Pick C1 disease (NPC1) is a rare, fatal neurodegenerative disease caused by mutations in NPC1, which encodes the lysosomal cholesterol transport protein NPC1. Disease pathology involves lysosomal accumulation of cholesterol and lipids, leading to neurological and visceral complications. Targeting the central nervous system (CNS) from systemic circulation complicates treatment of neurological diseases with gene transfer techniques. Selected and engineered capsids, for example, adeno-associated virus (AAV)-PHP.B facilitate peripheral-to-CNS transfer and hence greater CNS transduction than parental predecessors. We report that systemic delivery to Npc1m1N/m1N mice using an AAV-PHP.B vector ubiquitously expressing NPC1 led to greater disease amelioration than an otherwise identical AAV9 vector. In addition, viral copy number and biodistribution of GFP-expressing reporters showed that AAV-PHP.B achieved more efficient, albeit variable, CNS transduction than AAV9 in Npc1m1N/m1N mice. This variability was associated with segregation of two alleles of the putative AAV-PHP.B receptor Ly6a in Npc1m1N/m1N mice. Our data suggest that robust improvements in NPC1 disease phenotypes occur even with modest CNS transduction and that improved neurotrophic capsids have the potential for superior NPC1 AAV gene therapy vectors.

The engineering of the AAV-PHP capsids was an important development for CNS research and the modulation of gene expression in the brain. They cross the blood brain barrier and transduce brain cells after intravenous systemic delivery, a property dependent on the genotype of Ly6a, the AAV-PHP capsid receptor. It is important to determine the transduction efficiency of a given viral preparation, as well as the comparative tropism for different brain cells; however, manual estimation of adeno-associated viral transduction efficiencies can be biased and time consuming. Therefore, we have used the Opera Phenix high-content screening system, equipped with the Harmony processing and analysis software, to reduce bias and develop an automated approach to determining transduction efficiency in the mouse brain. We used R Studio and ‘gatepoints’ to segment the data captured from coronal brain sections into brain regions of interest. C57BL/6J and CBA/Ca mice were injected with an AAV-PHP.B virus containing a green fluorescent protein reporter with a nuclear localization signal. Coronal sections at 600 μm intervals throughout the entire brain were stained with Hoechst dye, combined with immunofluorescence to NeuN and green fluorescent protein to identify all cell nuclei, neurons and transduced cells, respectively. Automated data analysis was applied to give an estimate of neuronal percentages and transduction efficiencies throughout the entire brain as well as for the cortex, striatum and hippocampus. The data from each coronal section from a given mouse were highly comparable. The percentage of neurons in the C57BL/6J and CBA/Ca brains was approximately 40% and this was higher in the cortex than striatum and hippocampus. The systemic injection of AAV-PHP.B resulted in similar transduction rates across the entire brain for C57BL/6J mice. Approximately 10–15% of all cells were transduced, with neuronal transduction efficiencies ranging from 5% to 15%, estimates that were similar across brain regions, and were in contrast to the much more localized transduction efficiencies achieved through intracerebral injection. We confirmed that the delivery of the AAV-PHP.B viruses to the brain from the vasculature resulted in widespread transduction. Our methodology allows the rapid comparison of transduction rates between brain regions producing comparable data to more time-consuming approaches. The methodology developed here can be applied to the automated quantification of any parameter of interest that can be captured as a fluorescent signal., Smith et al. used the Opera Phenix high-content screening system and R Studio to automate the quantification of viral transduction efficiency in the mouse brain. They show that the systemic delivery of AAV-PHP.B viruses results in widespread and even transduction throughout the brain and brain regions., Graphical Abstract Graphical Abstract

ObjectivesThe objective of this study was to compare paediatric emergency airway management strategies in the simulation lab and operating room environments.MethodsThis was a two-part cluster cross-over randomised trial including simulation lab and operating room environments conducted between January 2017 and June 2018 in Portland, Oregon, USA. In simulated infant cardiac arrests, paramedic students placed an endotracheal tube, an i-gel or a laryngeal mask airway in random order. In the operating room, paramedic students placed a laryngeal mask airway or i-gel device in random order in sequential patients. The primary outcome for both portions of the study was time to ventilation. In the operating room portion, we also evaluated leak pressures and average initial tidal volumes.ResultsThere were 58 paramedic students who participated in the simulation lab and 22 who participated in the operating room study. The mean time to airway placement in the simulation lab was 48.5 s for the i-gel, 68.9 s for the laryngeal mask and 129.5 s for the endotracheal tube. In the operating room, mean time to i-gel placement was 34.3 s with 45.2 s for the laryngeal mask. In multivariable analysis of the simulation study, the laryngeal mask and i-gel were significantly faster than the endotracheal tube, and the i-gel was faster than the laryngeal mask. In the operating room, there was no significant difference in time to placement, leak pressure and average volume of the first five breaths between the i-gel and laryngeal mask.ConclusionsWe found that paramedic students were able to place supraglottic devices rapidly with high success rates in simulation lab and operating room environments. Supraglottic devices, particularly the i-gel, were rated as easy to use. The i-gel may be easiest to use since it lacks an inflable cuff and requires fewer steps to place.

We recently developed adeno-associated virus (AAV) capsids to facilitate efficient and noninvasive gene transfer to the central and peripheral nervous systems. However, a detailed protocol for generating and systemically delivering novel AAV variants was not previously available. In this protocol, we describe how to produce and intravenously administer AAVs to adult mice to specifically label and/or genetically manipulate cells in the nervous system and organs, including the heart. The procedure comprises three separate stages: AAV production, intravenous delivery, and evaluation of transgene expression. The protocol spans 8 d, excluding the time required to assess gene expression, and can be readily adopted by researchers with basic molecular biology, cell culture, and animal work experience. We provide guidelines for experimental design and choice of the capsid, cargo, and viral dose appropriate for the experimental aims. The procedures outlined here are adaptable to diverse biomedical applications, from anatomical and functional mapping to gene expression, silencing, and editing.

Rett syndrome is an incurable neurodevelopmental disorder caused by mutations in the gene encoding for methyl-CpG binding-protein 2 (MeCP2). Gene therapy for this disease presents inherent hurdles since MECP2 is expressed throughout the brain and its duplication leads to severe neurological conditions as well. Herein, we use the AAV-PHP.eB to deliver an instability-prone Mecp2 (iMecp2) transgene cassette which, increasing RNA destabilization and inefficient protein translation of the viral Mecp2 transgene, limits supraphysiological Mecp2 protein levels. Intravenous injections of the PHP.eB-iMecp2 virus in symptomatic Mecp2 mutant mice significantly improved locomotor activity, lifespan and gene expression normalization. Remarkably, PHP.eB-iMecp2 administration was well tolerated in female Mecp2 mutant or in wild-type animals. In contrast, we observed a strong immune response to the transgene in treated male Mecp2 mutant mice that was overcome by immunosuppression. Overall, PHP.eB-mediated delivery of iMecp2 provided widespread and efficient gene transfer maintaining physiological Mecp2 protein levels in the brain.

BACKGROUND Postoperative nausea and vomiting (PONV) is common after tonsillectomy in children. There is evidence that perioperative acupuncture at the pericardium 6 (P6) point is effective for preventing PONV in adults. Our goal was to determine if intraoperative acupuncture at the P6 point, in addition to usual antiemetics, is more effective than antiemetics alone in preventing PONV in children. METHODS In a randomized double-blind trial, 161 children age 3 through 9 years undergoing tonsillectomy with or without adenoidectomy were randomized to either bilateral acupuncture at P6 plus antiemetics (n = 86) or antiemetics only (n = 75). All participants received ondansetron 0.15 mg/kg and dexamethasone 0.25 mg/kg, up to 10 mg. The presence of nausea, retching, emesis and administration of additional antiemetics were recorded during phases I and II of PACU recovery. Follow-up calls occurred on postoperative day 1 (POD 1). RESULT During phase I and II recovery, the incidence of PONV was significantly less with acupuncture than without (7.0% vs 34.7%, RR: 0.2, 95% CI: 0.09-0.46; P

Recombinant viruses allow for targeted transgene expression in specific cell populations throughout the nervous system. The adeno-associated virus (AAV) is among the most commonly used viruses for neuroscience research. Recombinant AAVs (rAAVs) are highly versatile and can package most cargo composed of desired genes within the capsid's ∼5-kb carrying capacity. Numerous regulatory elements and intersectional strategies have been validated in rAAVs to enable cell type–specific expression. rAAVs can be delivered to specific neuronal populations or globally throughout the animal. The AAV capsids have natural cell type or tissue tropism and trafficking that can be modified for increased specificity. Here, we describe recently engineered AAV capsids and associated cargo that have extended the utility of AAVs in targeting molecularly defined neurons throughout the nervous system, which will further facilitate neuronal circuit interrogation and discovery.

Rett syndrome (RTT) is an incurable neurodevelopmental disorder caused by mutations in the gene encoding for methyl-CpG binding-protein 2 (MeCP2). Gene therapy for this disease presents inherent hurdles sinceMECP2is expressed throughout the brain and its duplication leads to severe neurological conditions as well. However, the recent introduction of AAV-PHP.eB, an engineered capsid with an unprecedented efficiency in crossing the blood-brain barrier upon intravenous injection, has provided an invaluable vehicle for gene transfer in the mouse nervous system. Herein, we use AAV-PHP.eB to deliver an instability-proneMecp2(iMecp2) transgene cassette which, increasing RNA destabilization and inefficient protein translation of the viralMecp2transgene, limits supraphysiological Mecp2 protein levels in transduced neural tissues. Intravenous injections of the PHP.eB-iMecp2virus in symptomatic male and femaleMecp2mutant mice significantly ameliorated the disease progression with improved locomotor activity, coordination, lifespan and normalization of altered gene expression and mTOR signaling. Remarkably, PHP.eB-iMecp2administration did not result in severe toxicity effects either in femaleMecp2mutant or in wild-type animals. In contrast, we observed a strong immune response to the transgene in treated maleMecp2mutant mice that was overcome by immunosuppression. Overall, PHP.eB-mediated delivery of theiMecp2cassette provided widespread and efficient gene transfer maintaining physiological Mecp2 protein levels in the brain. This combination defines a novel viral system with significant therapeutic efficacy and increased safety which can contribute to overcome the hurdles that are delaying clinical applications of gene therapy for RTT.One Sentence SummaryGlobal brain transduction of the instability-proneMecp2transgene by systemic AAV-PHP.eB administration is both safe and effective in protecting male and femaleMecp2mutant mice from the RTT disease phenotype.

Cell-type-specific expression of molecular tools and sensors is critical to construct circuit diagrams and to investigate the activity and function of neurons within the nervous system. Strategies for targeted manipulation include combinations of classical genetic tools such as Cre/loxP and Flp/FRT, use of cis-regulatory elements, targeted knock-in transgenic mice, and gene delivery by AAV and other viral vectors. The combination of these complex technologies with the goal of precise neuronal targeting is a challenge in the lab. This report will discuss the theoretical and practical aspects of combining current technologies and establish best practices for achieving targeted manipulation of specific cell types. Novel applications and tools, as well as areas for development, will be envisioned and discussed.

OBJECTIVE: To describe severity of pain during labor at previable estimated gestational age and to determine the effectiveness of various methods of analgesia in this population. METHODS: In this retrospective review of the records of women undergoing induction or augmentation of labor between 16 0/7 and 22 6/7 weeks of gestation, we examined pain scores (numeric rating scale 0–10) documented throughout labor and analgesia method (none, systemic, or neuraxial). We compared relevant patient and labor characteristics such as gestational age, fetal weight, time interval from induction to delivery as well as pain data including analgesia type and pain scores before and after analgesia. We compared analgesia response in patients who received neuraxial analgesia and those who did not. RESULTS: A total of 80 patients met inclusion criteria: four requested no analgesia, 56 used systemic analgesia only, 11 used systemic analgesia initially and then requested neuraxial analgesia, and nine used neuraxial without using systemic analgesia first. Median peak pain score was 7 among all patients. Patients who chose neuraxial analgesia had higher estimated gestational age (21 compared with 20 weeks of gestation, P=.03), fetal weight (362 compared with 268 g, P=.047), and admission-to-delivery time intervals (19.25 compared with 10.3 hours, P=.003) than those who chose no or systemic analgesia. Immediate preanalgesia peak pain scores were not different between groups, but patients who received neuraxial analgesia had lower immediate postanalgesia nadir scores than those who chose systemic pain relief. CONCLUSION: Pain during induction of labor at previable gestational ages may be greater than is commonly appreciated. Neuraxial analgesia may provide more optimal pain relief for patients with higher estimated gestational age and fetal weight, particularly after 22 weeks of estimated gestational age.

Heart rate is under the precise control of the autonomic nervous system. However, the wiring of peripheral neural circuits that regulate heart rate is poorly understood. Here, we develop a clearing-imaging-analysis pipeline to visualize innervation of intact hearts in 3D and employed a multi-technique approach to map parasympathetic and sympathetic neural circuits that control heart rate in mice. We identify cholinergic neurons and noradrenergic neurons in an intrinsic cardiac ganglion and the stellate ganglia, respectively, that project to the sinoatrial node. We also report that the heart rate response to optogenetic versus electrical stimulation of the vagus nerve displays different temporal characteristics and that vagal afferents enhance parasympathetic and reduce sympathetic tone to the heart via central mechanisms. Our findings provide new insights into neural regulation of heart rate, and our methodology to study cardiac circuits can be readily used to interrogate neural control of other visceral organs., The wiring of peripheral neural circuits that regulate heart rate is poorly understood. In this study, authors used tissue clearing for high-resolution characterization of nerves in the heart in 3D and transgenic and novel viral vector approaches to identify peripheral parasympathetic and sympathetic neuronal populations involved in heart rate control in mice.

Chronic social isolation causes severe psychological effects in humans, but their neural bases remain poorly understood. 2 weeks (but not 24 hr) of social isolation stress (SIS) caused multiple behavioral changes in mice and induced brain-wide upregulation of the neuropeptide tachykinin 2 (Tac2)/neurokinin B (NkB). Systemic administration of an Nk3R antagonist prevented virtually all of the behavioral effects of chronic SIS. Conversely, enhancing NkB expression and release phenocopied SIS in group-housed mice, promoting aggression and converting stimulus-locked defensive behaviors to persistent responses. Multiplexed analysis of Tac2/NkB function in multiple brain areas revealed dissociable, region-specific requirements for both the peptide and its receptor in different SIS-induced behavioral changes. Thus, Tac2 coordinates a pleiotropic brain state caused by SIS via a distributed mode of action. These data reveal the profound effects of prolonged social isolation on brain chemistry and function and suggest potential new therapeutic applications for Nk3R antagonists.

We recently developed novel AAV capsids for efficient and noninvasive gene transfer across the central and peripheral nervous systems. In this protocol, we describe how to produce and systemically administer AAV-PHP viruses to label and/or genetically manipulate cells in the mouse nervous system and organs including the heart. The procedure comprises three separate stages: AAV production, intravenous delivery, and evaluation of transgene expression. The protocol spans eight days, excluding the time required to assess gene expression, and can be readily adopted by laboratories with standard molecular and cell culture capabilities. We provide guidelines for experimental design and choosing the capsid, cargo, and viral dose appropriate for the experimental aims. The procedures outlined here are adaptable to diverse biomedical applications, from anatomical and functional mapping to gene expression, silencing, and editing.

Adeno-associated viruses (AAVs) are commonly used for in vivo gene transfer. Nevertheless, AAVs that provide efficient transduction across specific organs or cell populations are needed. Here, we describe AAV-PHP.eB and AAV-PHP.S, capsids that efficiently transduce the central and peripheral nervous systems, respectively. In the adult mouse, intravenous administration of 1 × 1011 vector genomes (vg) of AAV-PHP.eB transduced 69% of cortical and 55% of striatal neurons, while 1 × 1012 vg of AAV-PHP.S transduced 82% of dorsal root ganglion neurons, as well as cardiac and enteric neurons. The efficiency of these vectors facilitates robust cotransduction and stochastic, multicolor labeling for individual cell morphology studies. To support such efforts, we provide methods for labeling a tunable fraction of cells without compromising color diversity. Furthermore, when used with cell-type-specific promoters and enhancers, these AAVs enable efficient and targetable genetic modification of cells throughout the nervous system of transgenic and non-transgenic animals.

SUMMARY Excessive and unresolved neuroinflammation is a key component of the pathological cascade in brain injuries such as ischemic stroke. Here, we report that TRIM9, a brain-specific tripartite motif (TRIM) protein, was highly expressed in the peri-infarct areas shortly after ischemic insults in mice, but expression was decreased in aged mice, which are known to have increased neuroinflammation after stroke. Mechanistically, TRIM9 sequestered β-transducin repeat-containing protein (β-TrCP) from the Skp-Cullin-F-box ubiquitin ligase complex, blocking IκBα degradation and thereby dampening nuclear factor κB (NF-κB)-dependent proinflammatory mediator production and immune cell infiltration to limit neuroinflammation. Consequently, Trim9-deficient mice were highly vulnerable to ischemia, manifesting uncontrolled neuroinflammation and exacerbated neuropathological outcomes. Systemic administration of a recombinant TRIM9 adeno-associated virus that drove brain-wide TRIM9 expression effectively resolved neuroinflammation and alleviated neuronal death, especially in aged mice. These findings reveal that TRIM9 is essential for resolving NF-κB-dependent neuroinflammation to promote recovery and repair after brain injury and may represent an attractive therapeutic target., Graphical Abstract, In Brief Neuroinflammation drives pathology during brain injury. Zeng et al. show that TRIM9 is induced after ischemic insults in young mice, but not old mice, and promotes resolution of neuroinflammation. AAV-mediated TRIM9 therapy in aged mice restricts neuroinflammation and alleviates stroke damage, representing a potential therapeutic target for brain injury.

Widespread genetic modification of cells in the central nervous system (CNS) with a viral vector has become possible and increasingly more efficient. We previously applied an AAV9 vector with the cytomegalovirus/chicken beta-actin (CBA) hybrid promoter and achieved wide-scale CNS transduction in neonatal and adult rats. However, this method transduces a variety of tissues in addition to the CNS. Thus we studied intravenous AAV9 gene transfer with a synapsin promoter to better target the neurons. We noted in systematic comparisons that the synapsin promoter drives lower level expression than does the CBA promoter. The engineered adeno-associated virus (AAV)-PHP.B serotype was compared with AAV9, and AAV-PHP.B did enhance the efficiency of expression. Combining the synapsin promoter with AAV-PHP.B could therefore be advantageous in terms of combining two refinements of targeting and efficiency. Wide-scale expression was used to model a disease with widespread pathology. Vectors encoding the amyotrophic lateral sclerosis (ALS)-related protein transactive response DNA-binding protein, 43 kDa (TDP-43) with the synapsin promoter and AAV-PHP.B were used for efficient CNS-targeted TDP-43 expression. Intracerebroventricular injections were also explored to limit TDP-43 expression to the CNS. The neuron-selective promoter and the AAV-PHP.B enhanced gene transfer and ALS disease modeling in adult rats.

SUMMARY The successful planning and execution of adaptive behaviors in mammals may require long-range coordination of neural networks throughout cerebral cortex. The neuronal implementation of signals that could orchestrate cortex-wide activity remains unclear. Here, we develop and apply methods for cortex-wide Ca2+ imaging in mice performing decision-making behavior and identify a global cortical representation of task engagement encoded in the activity dynamics of both single cells and superficial neuropil distributed across the majority of dorsal cortex. The activity of multiple molecularly defined cell types was found to reflect this representation with type-specific dynamics. Focal optogenetic inhibition tiled across cortex revealed a crucial role for frontal cortex in triggering this cortex-wide phenomenon; local inhibition of this region blocked both the cortex-wide response to task-initiating cues and the voluntary behavior. These findings reveal cell-type-specific processes in cortex for globally representing goal-directed behavior and identify a major cortical node that gates the global broadcast of task-related information.

Adeno-associated viral (AAV) vectors are a rapidly emerging gene therapy platform for the treatment of neurological diseases. In preclinical studies, transgenes encoding therapeutic proteins, microRNAs, antibodies or gene-editing machinery have been successfully delivered to the central nervous system with natural or engineered viral capsids via various routes of administration. Importantly, initial clinical studies have demonstrated encouraging safety and efficacy in diseases such as Parkinson disease and spinal muscular atrophy, as well as durability of transgene expression. Here, we discuss key considerations and challenges in the future design and development of therapeutic AAV vectors, highlighting the most promising targets and recent clinical advances.

Background Therapeutic gene delivery to the central nervous system (CNS) remains a challenge in Huntington’s disease research. The blood brain barrier restricts the delivery of therapeutic agents to various cell types in the CNS known to be involved in disease progression. Recombinant adeno-associated viral vectors (rAAVs) are frequently used for in vivo gene delivery in neurodegenerative research but can have poor or limited tropism in the CNS when delivered systemically. When injected directly into the CNS, AAVs are efficient at transducing a variety of cell types though with limited spread. Conversely, when naturally occurring AAVs are injected systematically they lead to widespread transgene expression but with low transduction efficiency. Aims The aim of this experiment was to utilise a novel AAV variant, ssAAV-PHP.B:CAG-GFP, to investigate and compare its transduction efficiency in brains of wild type (WT) and R6/2 mice. Methods Four week old WT and R6/2 mice were intravenously injected through the tail vein with 5 × 10 9 vg/g with ssAAV-CAG-NLS-GFP packaged into AAV-PHP.B. Three weeks later, the mice were perfused and the brains processed for immunohistochemistry. GFP expression was assessed using images captured by a laser scanning confocal microscope. Results We show that systemic delivery of this AAV variant results in widespread and efficient GFP expression in different cell types throughout the CNS in both WT and R6/2 mice. Currently, we are assessing the impact of age and disease stage upon transduction efficiency. Conclusion Our results demonstrate the potential for use of this AAV vector as an approach to knock-down or deliver genes of interest to the CNS. Funding CHDI Foundation

Recombinant adeno-associated viruses (rAAVs) are commonly used vehicles for in vivo gene transfer. However, the tropism repertoire of naturally occurring AAVs is limited, prompting the development of novel AAV capsids with more desirable transduction characteristics. We have developed a capsid selection method, called Cre-recombination-based AAV targeted evolution (CREATE), that enables the identification of AAV capsids that more efficiently transduce defined cell populations in vivo (Deverman et al. in press, Nature Biotechnology). We generated AAV capsid libraries and used CREATE to identify variants that cross the blood brain barrier and efficiently and widely transduce astrocytes in the mouse central nervous system (CNS) after intravenous injection. One variant, AAVPHP. B, transfers genes throughout the adult CNS with an efficiency that is 40- to 92-fold greater (depending on the CNS region) than that of the current standard, AAV9. It transduces the majority of astrocytes and neurons across multiple CNS regions, and in vitro, it transduces human neurons and astrocytes more efficiently than does AAV9. We are now evolving AAV-PHP.B for even greater transduction of specific CNS cell types as a means to both develop more effective vectors and to gain insight into the mechanism of enhanced transduction. Our identification of AAV-PHP.B and several other enhanced vectors after only two rounds of selection establishes CREATE as a powerful method to customize AAV vectors for biomedical applications.

Deamidation of two asparagines activates a conditional PEST sequence to target Bcl-xL for degradation., The cellular concentration of Bcl-xL is among the most important determinants of treatment response and overall prognosis in a broad range of tumors as well as an important determinant of the cellular response to several forms of tissue injury. We and others have previously shown that human Bcl-xL undergoes deamidation at two asparaginyl residues and that DNA-damaging antineoplastic agents as well as other stimuli can increase the rate of deamidation. Deamidation results in the replacement of asparginyl residues with aspartyl or isoaspartyl residues. Thus deamidation, like phosphorylation, introduces a negative charge into proteins. Here we show that the level of human Bcl-xL is constantly modulated by deamidation because deamidation, like phosphorylation in other proteins, activates a conditional PEST sequence to target Bcl-xL for degradation. Additionally, we show that degradation of deamidated Bcl-xL is mediated at least in part by calpain. Notably, we present sequence and biochemical data that suggest that deamidation has been conserved from the simplest extant metazoans through the human form of Bcl-xL, underscoring its importance in Bcl-xL regulation. Our findings strongly suggest that deamidation-regulated Bcl-xL degradation is an important component of the cellular rheostat that determines susceptibility to DNA-damaging agents and other death stimuli., Author Summary Cellular levels of the pro-survival protein Bcl-xL are an important determinant of cellular susceptibility to many death stimuli, including most cancer therapies. We previously showed that human Bcl-xL undergoes deamidation – the conversion of two neutral asparaginyl side-chains into negatively charged aspartyl side-chains – a process that occurs spontaneously but is accelerated by the treatment of tumor cells with DNA-damaging agents. Here, we show that deamidation activates a hitherto undetected signal sequence within Bcl-xL that targets it for degradation by a pathway involving the proteolytic enzyme calpain. This increased degradation of Bcl-xL, and the consequent enhanced cellular susceptibility to programmed cell death, may contribute to the ability of DNA-damaging agents to kill tumors. We also demonstrate that deamidation of Bcl-xL has likely been conserved from the simplest metazoans to humans, underscoring the importance of deamidation in the regulation of Bcl-xL.

New central nervous system (CNS) neurons and glia are generated throughout adulthood from endogenous neural stem and progenitor cells. These progenitors can respond to injury, but their ability to proliferate, migrate, differentiate and survive is usually insufficient to replace lost cells and restore normal function. Potentiating the progenitor response with exogenous factors is an attractive strategy for the treatment of nervous system injuries and neurodegenerative and demyelinating disorders. Previously we reported that delivery of leukemia inhibitory factor (LIF) to the CNS stimulates the self-renewal of neural stem cells as well as the proliferation of parenchymal glial progenitors. Here we identify these parenchymal glia as oligodendrocyte (OL) progenitor cells (OPCs) and show that LIF delivery stimulates their proliferation through the activation of gp130 receptor signaling within these cells. Importantly, this effect of LIF on OPC proliferation can be harnessed to enhance the generation of OLs that express myelin proteins and reform nodes of Ranvier in the context of chronic demyelination in the adult mouse hippocampus. Our findings, considered together with the known beneficial effects of LIF on OL and neuron survival, suggest that LIF has both reparative and protective activities that make it a promising potential therapy for CNS demyelinating disorders and injuries.

The discovery two decades ago that the Philadelphia chromosome encodes an oncogenic fusion of Bcr and Abl remains among the most important contributions to our understanding of the process of malignant transformation. We now know that Bcr-Abl is one of more than 30 aberrantly activated tyrosine kinases that are expressed in a variety of tumors. Conventional treatment of the tumors in which these proteins are expressed is usually doomed to failure because the activated tyrosine kinases render the tumor cells stubbornly resistant to apoptosis. In this context, it is notable that Zhao and coworkers have uncovered a novel weapon in the resistance armamentarium of these rogue kinases, the suppression of the inactivating deamidation of Bcl-xL (this issue of Cancer Cell).

SummaryUnderstanding the structure-function relationships at cellular, circuit, and organ-wide scale requires 3D anatomical and phenotypical maps, currently unavailable for many organs across species. At the root of this knowledge gap is the absence of a method that enables whole-organ imaging. Herein, we present techniques for tissue clearing in which whole organs and bodies are rendered macromolecule-permeable and optically transparent, thereby exposing their cellular structure with intact connectivity. We describe PACT (passive clarity technique), a protocol for passive tissue clearing and immunostaining of intact organs; RIMS (refractive index matching solution), a mounting media for imaging thick tissue; and PARS (perfusion-assisted agent release in situ), a method for whole-body clearing and immunolabeling. We show that in rodents PACT, RIMS, and PARS are compatible with endogenous-fluorescence, immunohistochemistry, RNA single-molecule FISH, long-term storage, and microscopy with cellular and subcellular resolution. These methods are applicable for high-resolution, high-content mapping and phenotyping of normal and pathological elements within intact organs and bodies.