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Deep sequencing identifies somatic activating mutations of MTOR in affected brain regions of FCDII patients that are sufficient to cause neuronal migration defects and epileptic seizures in mice. Focal cortical dysplasia type II (FCDII) is a sporadic developmental malformation of the cerebral cortex characterized by dysmorphic neurons, dyslamination and medically refractory epilepsy 1 , 2 . It has been hypothesized that FCD is caused by somatic mutations in affected regions 3 , 4 . Here, we used deep whole-exome sequencing (read depth, 412–668×) validated by site-specific amplicon sequencing (100–347,499×) in paired brain-blood DNA from four subjects with FCDII and uncovered a de novo brain somatic mutation, mechanistic target of rapamycin ( MTOR) c.7280T>C (p.Leu2427Pro) in two subjects. Deep sequencing of the MTOR gene in an additional 73 subjects with FCDII using hybrid capture and PCR amplicon sequencing identified eight different somatic missense mutations found in multiple brain tissue samples of ten subjects. The identified mutations accounted for 15.6% of all subjects with FCDII studied (12 of 77). The identified mutations induced the hyperactivation of mTOR kinase. Focal cortical expression of mutant MTOR by in utero electroporation in mice was sufficient to disrupt neuronal migration and cause spontaneous seizures and cytomegalic neurons. Inhibition of mTOR with rapamycin suppressed cytomegalic neurons and epileptic seizures. This study provides, to our knowledge, the first evidence that brain somatic activating mutations in MTOR cause FCD and identifies mTOR as a treatment target for intractable epilepsy in FCD.

Nanoparticle-mediated delivery of functional macromolecules is a promising method for treating a variety of human diseases. Among nanoparticles, cell-derived exosomes have recently been highlighted as a new therapeutic strategy for the in vivo delivery of nucleotides and chemical drugs. Here we describe a new tool for intracellular delivery of target proteins, named ‘exosomes for protein loading via optically reversible protein–protein interactions’ (EXPLORs). By integrating a reversible protein–protein interaction module controlled by blue light with the endogenous process of exosome biogenesis, we are able to successfully load cargo proteins into newly generated exosomes. Treatment with protein-loaded EXPLORs is shown to significantly increase intracellular levels of cargo proteins and their function in recipient cells in vitro and in vivo . These results clearly indicate the potential of EXPLORs as a mechanism for the efficient intracellular transfer of protein-based therapeutics into recipient cells and tissues. Exosomes have been identified as promising vehicles for the in vivo delivery of therapeutic molecules. Here the authors design a system to load protein cargos into exosomes during their biogenesis using optogenetic control of protein-protein interactions between the cargo and an exosome-localized partner.

Although the pathological contributions of reactive astrocytes have been implicated in Alzheimer’s disease (AD), their in vivo functions remain elusive due to the lack of appropriate experimental models and precise molecular mechanisms. Here, we show the importance of astrocytic reactivity on the pathogenesis of AD using GiD, a newly developed animal model of reactive astrocytes, where the reactivity of astrocytes can be manipulated as mild (GiDm) or severe (GiDs). Mechanistically, excessive hydrogen peroxide (H2O2) originated from monoamine oxidase B in severe reactive astrocytes causes glial activation, tauopathy, neuronal death, brain atrophy, cognitive impairment and eventual death, which are significantly prevented by AAD-2004, a potent H2O2 scavenger. These H2O2−-induced pathological features of AD in GiDs are consistently recapitulated in a three-dimensional culture AD model, virus-infected APP/PS1 mice and the brains of patients with AD. Our study identifies H2O2 from severe but not mild reactive astrocytes as a key determinant of neurodegeneration in AD.Chun et al. find that a severe model of reactive astrocytes overproduces hydrogen peroxide, leading to the development of Alzheimer’s disease-like pathologies, including neurodegeneration, tauopathy and memory impairment.

In Alzheimer's disease, increased MAOB activity in reactive astrocytes leads to enhanced GABA release, and blockage of this pathway ameliorates memory dysfunction in mouse models. In Alzheimer's disease (AD), memory impairment is the most prominent feature that afflicts patients and their families. Although reactive astrocytes have been observed around amyloid plaques since the disease was first described, their role in memory impairment has been poorly understood. Here, we show that reactive astrocytes aberrantly and abundantly produce the inhibitory gliotransmitter GABA by monoamine oxidase-B (Maob) and abnormally release GABA through the bestrophin 1 channel. In the dentate gyrus of mouse models of AD, the released GABA reduces spike probability of granule cells by acting on presynaptic GABA receptors. Suppressing GABA production or release from reactive astrocytes fully restores the impaired spike probability, synaptic plasticity, and learning and memory in the mice. In the postmortem brain of individuals with AD, astrocytic GABA and MAOB are significantly upregulated. We propose that selective inhibition of astrocytic GABA synthesis or release may serve as an effective therapeutic strategy for treating memory impairment in AD.

Corticotropin-releasing factor (CRF) that is released from the paraventricular nucleus (PVN) of the hypothalamus is essential for mediating stress response by activating the hypothalamic–pituitary–adrenal axis. CRF-releasing PVN neurons receive inputs from multiple brain regions that convey stressful events, but their neuronal dynamics on the timescale of behavior remain unknown. Here, our recordings of PVN CRF neuronal activity in freely behaving mice revealed that CRF neurons are activated immediately by a range of aversive stimuli. By contrast, CRF neuronal activity starts to drop within a second of exposure to appetitive stimuli. Optogenetic activation or inhibition of PVN CRF neurons was sufficient to induce a conditioned place aversion or preference, respectively. Furthermore, conditioned place aversion or preference induced by natural stimuli was significantly decreased by manipulating PVN CRF neuronal activity. Together, these findings suggest that the rapid, biphasic responses of PVN CRF neurons encode the positive and negative valences of stimuli.Animals must determine quickly whether any given environmental stimuli are beneficial or detrimental. This work reveals a novel strategy to encode opposing valences by a single population of CRF neurons in the hypothalamus.

A single-component optogenetic tool controls calcium fluxes with high dynamic range. Calcium (Ca 2+ ) signals that are precisely modulated in space and time mediate a myriad of cellular processes, including contraction, excitation, growth, differentiation and apoptosis 1 . However, study of Ca 2+ responses has been hampered by technological limitations of existing Ca 2+ -modulating tools. Here we present OptoSTIM1, an optogenetic tool for manipulating intracellular Ca 2+ levels through activation of Ca 2+ -selective endogenous Ca 2+ release−activated Ca 2+ (CRAC) channels. Using OptoSTIM1, which combines a plant photoreceptor 2 , 3 and the CRAC channel regulator STIM1 (ref. 4 ), we quantitatively and qualitatively controlled intracellular Ca 2+ levels in various biological systems, including zebrafish embryos and human embryonic stem cells. We demonstrate that activating OptoSTIM1 in the CA1 hippocampal region of mice selectively reinforced contextual memory formation. The broad utility of OptoSTIM1 will expand our mechanistic understanding of numerous Ca 2+ -associated processes and facilitate screening for drug candidates that antagonize Ca 2+ signals.

The infralimbic cortex (IL) is known to facilitate the formation of extinction memory through reciprocal interactions with the amygdala, which produces fear responses such as freezing. Thus, whether presynaptic input from the amygdala and post-synaptic output of IL neurons are functionally dissociated in extinction memory formation remains unclear. Here, we demonstrated that photostimulation of IL inputs from BLA did not change freezing responses to conditioned stimuli (CS) during training, but did facilitate extinction memory, measured as a reduction in freezing responses to the CS 1 day later. On the other hand, photostimulation of somata of IL neurons induced an immediate reduction in freezing to CS, but this did not affect extinction memory tested the next day. These results provide in vivo evidence for IL-dependent facilitation of extinction memory without post-synaptic modulation of freezing circuits.

Sustainable swine manure management is critical to reducing adverse environmental impacts on surrounding ecosystems, particularly in regions of intensive production. Conventional swine manure management practices contribute to agricultural greenhouse gas (GHG) emissions and aquatic eutrophication. There is a lack of full-scale research of the thermochemical conversion of solid-separated swine manure. This study utilizes a consequential life cycle assessment (CLCA) to investigate the environmental impacts of the thermal gasification of swine manure solids as a manure management strategy. CLCA is a modeling tool for a comprehensive estimation of the environmental impacts attributable to a production system. The present study evaluates merely the gasification scenario as it includes manure drying, syngas production, and biochar field application. The assessment revealed that liquid storage of manure had the highest contribution of 57.5% to GHG emissions for the entire proposed manure management scenario. Solid-liquid separation decreased GHG emissions from the manure liquid fraction. Swine manure solids separation, drying, and gasification resulted in a net energy expenditure of 12.3 MJ for each functional unit (treatment of 1 metric ton of manure slurry). Land application of manure slurry mixed with biochar residue could potentially be credited with 5.9 kg CO2-eq in avoided GHG emissions, and 135 MJ of avoided fossil fuel energy. Manure drying had the highest share of fossil fuel energy use. Increasing thermochemical conversion efficiency was shown to decrease overall energy use significantly. Improvements in drying technology efficiency, or the use of solar or waste-heat streams as energy sources, can significantly improve the potential environmental impacts of manure solids gasification.

The motor cortex orchestrates simple to complex motor behaviors through its output projections to target areas. The primary (MOp) and secondary (MOs) motor cortices are known to produce specific output projections that are targeted to both similar and different target areas. These projections are further divided into layer 5 and 6 neuronal outputs, thereby producing four cortical outputs that may target other areas in a combinatorial manner. However, the precise network structure that integrates these four projections remains poorly understood. Here, we constructed a whole-brain, three-dimensional (3D) map showing the tract pathways and targeting locations of these four motor cortical outputs in mice. Remarkably, these motor cortical projections showed unique and separate tract pathways despite targeting similar areas. Within target areas, various combinations of these four projections were defined based on specific 3D spatial patterns, reflecting anterior-posterior, dorsal-ventral and core-capsular relationships. This 3D topographic map ultimately provides evidence for the relevance of comparative connectomics: motor cortical projections known to be convergent are actually segregated in many target areas with unique targeting patterns, a finding that has anatomical value for revealing functional subdomains that have not been classified by conventional methods.

Enhanced NMDA receptor function and social interaction deficits are observed in mice lacking the excitatory postsynaptic scaffolding protein IRSp53. Reducing NMDAR activity by pharmacological methods rescues the impaired social interaction observed in these mice. This suggests that enhanced NMDA receptor function may be associated with social deficits. Social deficits are observed in diverse psychiatric disorders, including autism spectrum disorders and schizophrenia. We found that mice lacking the excitatory synaptic signaling scaffold IRSp53 (also known as BAIAP2) showed impaired social interaction and communication. Treatment of IRSp53 −/− mice, which display enhanced NMDA receptor (NMDAR) function in the hippocampus, with memantine, an NMDAR antagonist, or MPEP, a metabotropic glutamate receptor 5 antagonist that indirectly inhibits NMDAR function, normalized social interaction. This social rescue was accompanied by normalization of NMDAR function and plasticity in the hippocampus and neuronal firing in the medial prefrontal cortex. These results, together with the reduced NMDAR function implicated in social impairments, suggest that deviation of NMDAR function in either direction leads to social deficits and that correcting the deviation has beneficial effects.