Reprogramming of adult human dermal fibroblasts to induced dorsal forebrain precursor cells maintains aging signatures.

Introduction: With the increase in aging populations around the world, the development of in vitro human cell models to study neurodegenerative disease is crucial. A major limitation in using induced pluripotent stem cell (hiPSC) technology to model diseases of aging is that reprogramming fibroblasts to a pluripotent stem cell state erases age-associated features. The resulting cells show behaviors of an embryonic stage exhibiting longer telomeres, reduced oxidative stress, and mitochondrial rejuvenation, as well as epigenetic modifications, loss of abnormal nuclear morphologies, and age-associated features. Methods: We have developed a protocol utilizing stable, non-immunogenic chemically modified mRNA (cmRNA) to convert adult human dermal fibroblasts (HDFs) to human induced dorsal forebrain precursor (hiDFP) cells, which can subsequently be differentiated into cortical neurons. Analyzing an array of aging biomarkers, we demonstrate for the first time the effect of direct-to-hiDFP reprogramming on cellular age. Results: We confirm direct-to-hiDFP reprogramming does not affect telomere length or the expression of key aging markers. However, while direct-to-hiDFP reprogramming does not affect senescence-associated ß-galactosidase activity, it enhances the level of mitochondrial reactive oxygen species and the amount of DNA methylation compared to HDFs. Interestingly, following neuronal differentiation of hiDFPs we observed an increase in cell soma size as well as neurite number, length, and branching with increasing donor age suggesting that neuronal morphology is altered with age. Discussion: We propose direct-to-hiDFP reprogramming provides a strategy for modeling age-associated neurodegenerative diseases allowing the persistence of age-associated signatures not seen in hiPSC-derived cultures, thereby facilitating our understanding of neurodegenerative disease and identification of therapeutic targets.

Genomic regions underlying the species-specific mating songs of green lacewings.

Rapid species radiations provide insight into the process of speciation and diversification. The radiation of Chrysoperla carnea-group lacewings seems to be driven, at least in part, by their species-specific pre-mating vibrational duets. We associated genetic markers from across the genome with courtship song period in the offspring of a laboratory cross between Chrysoperla plorabunda and Chrysoperla adamsi, two species primarily differentiated by their mating songs. Two genomic regions were strongly associated with the song period phenotype. Large regions of chromosomes one and two were associated with song phenotype, as fewer recombination events occurred on these chromosomes relative to the other autosomes. Candidate genes were identified by functional annotation of proteins from the C. carnea reference genome. The majority of genes that are associated with vibrational courtship signals in other insects were found within QTL for lacewing song phenotype. Together these findings suggest that decreased recombination may be acting to keep together loci important to reproductive isolation between these species. Using wild-caught individuals from both species, we identified signals of genomic divergence across the genome. We identified several candidate genes both in song-associated regions and near divergence outliers including nonA, fruitless, paralytic, period, and doublesex. Together these findings bring us one step closer to identifying the genomic basis of a mating song trait critical to the maintenance of species boundaries in green lacewings.

Structures of highly flexible intracellular domain of human α7 nicotinic acetylcholine receptor.

The intracellular domain (ICD) of Cys-loop receptors mediates diverse functions. To date, no structure of a full-length ICD is available due to challenges stemming from its dynamic nature. Here, combining nuclear magnetic resonance (NMR) and electron spin resonance experiments with Rosetta computations, we determine full-length ICD structures of the human α7 nicotinic acetylcholine receptor in a resting state. We show that ~57% of the ICD residues are in highly flexible regions, primarily in a large loop (loop L) with the most mobile segment spanning ~50 Å from the central channel axis. Loop L is anchored onto the MA helix and virtually forms two smaller loops, thereby increasing its stability. Previously known motifs for cytoplasmic binding, regulation, and signaling are found in both the helices and disordered flexible regions, supporting the essential role of the ICD conformational plasticity in orchestrating a broad range of biological processes.

Regulation and drug modulation of a voltage-gated sodium channel: Pivotal role of the S4-S5 linker in activation and slow inactivation.

Voltage-gated sodium (NaV) channels control excitable cell functions. While structural investigations have revealed conformation details of different functional states, the mechanisms of both activation and slow inactivation remain unclear. Here, we identify residue T140 in the S4-S5 linker of the bacterial voltage-gated sodium channel NaChBac as critical for channel activation and drug effects on inactivation. Mutations at T140 either attenuate activation or render the channel nonfunctional. Propofol, a clinical anesthetic known to inhibit NaChBac by promoting slow inactivation, binds to a pocket between the S4-S5 linker and S6 helix in a conformation-dependent manner. Using 19F-NMR to quantify site-specific binding by saturation transfer differences (STDs), we found strong STDs in inactivated, but not activated, NaChBac. Molecular dynamics simulations show a highly dynamic pocket in the activated conformation, limiting STD buildup. In contrast, drug binding to this pocket promotes and stabilizes the inactivated states. Our results provide direct experimental evidence showing distinctly different associations between the S4-S5 linker and S6 helix in activated and inactivated states. Specifically, an exchange occurs between interaction partners T140 and N234 of the same subunit in activation, and T140 and N225 of the domain-swapped subunit in slow inactivation. The drug action on slow inactivation of prokaryotic NaV channels seems to have a mechanism similar to the recently proposed "door-wedge" action of the isoleucine-phenylalanine-methionine (IFM) motif on the fast inactivation of eukaryotic NaV channels. Elucidating this gating mechanism points to a possible direction for conformation-dependent drug development.

Central nervous system pathology in preclinical MPS IIIB dogs reveals progressive changes in clinically relevant brain regions.

Mucopolysaccharidosis type IIIB (MPS IIIB; Sanfilippo syndrome B) is an autosomal recessive lysosomal storage disorder caused by the deficiency of alpha-N-acetylglucosaminidase activity, leading to increased levels of nondegraded heparan sulfate (HS). A mouse model has been useful to evaluate novel treatments for MPS IIIB, but has limitations. In this study, we evaluated the naturally occurring canine model of MPS IIIB for the onset and progression of biochemical and neuropathological changes during the preclinical stages (onset approximately 24-30 months of age) of canine MPS IIIB disease. Even by 1 month of age, MPS IIIB dogs had elevated HS levels in brain and cerebrospinal fluid. Analysis of histopathology of several disease-relevant regions of the forebrain demonstrated progressive lysosomal storage and microglial activation despite a lack of cerebrocortical atrophy in the oldest animals studied. More pronounced histopathology changes were detected in the cerebellum, where progressive lysosomal storage, astrocytosis and microglial activation were observed. Microglial activation was particularly prominent in cerebellar white matter and within the deep cerebellar nuclei, where neuron loss also occurred. The findings in this study will form the basis of future assessments of therapeutic efficacy in this large animal disease model.

Membrane cholesterol dependence of cannabinoid modulation of glycine receptor.

Cannabinoids exert therapeutic effects on several diseases such as chronic pain and startle disease by targeting glycine receptors (GlyRs). Our previous studies have shown that cannabinoids target a serine residue at position 296 in the third transmembrane helix of the α1/α3 GlyR. This site is located on the outside of the ion channel protein at the lipid interface where the cholesterol concentrates. However, whether membrane cholesterol regulates cannabinoid-GlyR interaction remains unknown. Here, we show that GlyRs are closely associated with cholesterol/caveolin-rich domains at subcellular levels. Membrane cholesterol reduction significantly inhibits cannabinoid potentiation of glycine-activated currents in cultured spinal neurons and in HEK 293T cells expressing α1/α3 GlyRs. Such inhibition is fully rescued by cholesterol replenishment in a concentration-dependent manner. Molecular docking calculations further reveal that cholesterol regulates cannabinoid enhancement of GlyR function through both direct and indirect mechanisms. Taken together, these findings suggest that cholesterol is critical for the cannabinoid-GlyR interaction in the cell membrane.

Pathomechanisms in the neuronal ceroid lipofuscinoses.

The neuronal ceroid lipofuscinoses (NCLs) are a group of inherited neurodegenerative lysosomal storage disorders (LSDs), traditionally grouped together based on shared clinical symptoms. The recent emergence of new forms of NCL along with an improved understanding of endo-lysosomal system function have necessitated the reassessment of their classification and pathogenesis. Novel clinical findings, as well as observations in various animal models of NCL, have revealed significant pathological changes in regions outside the brain, as well as progression of disease along connected anatomical pathways. The characterization of animal models of NCLs has not only highlighted the vulnerability of certain neuron populations but has also revealed glial cells to be adversely affected and actively contribute to disease progression. While the lysosome has been thought of as being the 'waste-disposal' unit of the cell, recent evidence of the endo-lysosomal system playing a crucial role in nutrient sensing and cellular homeostasis have shown that NCL mutations have far-ranging effects on cellular functions including autophagy and synaptic dysfunction. The discovery of the machinery controlling endo-lysosomal function via transcription factor EB (TFEB) and mTORC1, have also shed light on potential mechanisms by which NCL mutations may exert their effect. While the NCLs share many common down-stream pathologies, there is a growing body of evidence for unique pathogenic pathways in each form. In light of the rapid advances in therapeutic strategies for the NCLs and LSDs, these new lessons learnt about unique NCL pathomechanisms will be key for informing the targeting, timing and strategies for future treatments.

Combined Anti-inflammatory and Neuroprotective Treatments Have the Potential to Impact Disease Phenotypes in Cln3 -/- Mice.

Batten disease, or juvenile NCL, is a fatal neurodegenerative disorder that occurs due to mutations in the CLN3 gene. Because the function of CLN3 remains unclear, experimental therapies for JNCL have largely concentrated upon the targeting of downstream pathomechanisms. Neuron loss is preceded by localized glial activation, and in this proof-of-concept study we have investigated whether targeting this innate immune response with ibuprofen in combination with the neuroprotective agent lamotrigine improves the previously documented beneficial effects of immunosuppressants alone. Drugs were administered daily to symptomatic Cln3 -/- mice over a 3 month period, starting at 6 months of age, and their impact was assessed using both behavioral and neuropathological outcome measures. During the treatment period, the combination of ibuprofen and lamotrigine significantly improved the performance of Cln3 -/- mice on the vertical pole test, slowing the disease-associated decline, but had less of an impact upon their rotarod performance. There were also moderate and regionally dependent effects upon astrocyte activation that were most pronounced for ibuprofen alone, but there was no overt effect upon microglial activation. Administering such treatments for longer periods will enable testing for any impact upon the neuron loss that occurs later in disease progression. Given the partial efficacy of these treatments, it will be important to test further drugs of this type in order to find more effective combinations.

19F Paramagnetic Relaxation-Based NMR for Quaternary Structural Restraints of Ion Channels.

Quaternary distance restraints are essential to define the three-dimensional structures of protein assemblies. These distances often fall within a range of 10-18 Å, which challenges the high and low measurement limits of conventional nuclear magnetic resonance (NMR) and double electron-electron resonance electron spin resonance spectroscopies. Here, we report the use of 19F paramagnetic relaxation enhancement (PRE) NMR in combination with 19F/paramagnetic labeling to equivalent sites in different subunits of a protein complex in micelles to determine intersubunit distances. The feasibility of this strategy was evaluated on a pentameric ligand-gated ion channel, for which we found excellent agreement of the 19F PRE NMR results with previous structural information. The study suggests that 19F PRE NMR is a viable tool in extracting distance restraints to define quaternary structures.

Structural basis of neurosteroid anesthetic action on GABAA receptors.

Type A γ-aminobutyric acid receptors (GABAARs) are inhibitory pentameric ligand-gated ion channels in the brain. Many anesthetics and neurosteroids act through binding to the GABAAR transmembrane domain (TMD), but the structural basis of their actions is not well understood and no resting-state GABAAR structure has been determined. Here, we report crystal structures of apo and the neurosteroid anesthetic alphaxalone-bound desensitized chimeric α1GABAAR (ELIC-α1GABAAR). The chimera retains the functional and pharmacological properties of GABAARs, including potentiation, activation and desensitization by alphaxalone. The apo-state structure reveals an unconventional activation gate at the intracellular end of the pore. The desensitized structure illustrates molecular determinants for alphaxalone binding to an inter-subunit TMD site. These structures suggest a plausible signaling pathway from alphaxalone binding at the bottom of the TMD to the channel gate in the pore-lining TM2 through the TM1-TM2 linker. The study provides a framework to discover new GABAAR modulators with therapeutic potential.

Propofol inhibits the voltage-gated sodium channel NaChBac at multiple sites.

Voltage-gated sodium (NaV) channels are important targets of general anesthetics, including the intravenous anesthetic propofol. Electrophysiology studies on the prokaryotic NaV channel NaChBac have demonstrated that propofol promotes channel activation and accelerates activation-coupled inactivation, but the molecular mechanisms of these effects are unclear. Here, guided by computational docking and molecular dynamics simulations, we predict several propofol-binding sites in NaChBac. We then strategically place small fluorinated probes at these putative binding sites and experimentally quantify the interaction strengths with a fluorinated propofol analogue, 4-fluoropropofol. In vitro and in vivo measurements show that 4-fluoropropofol and propofol have similar effects on NaChBac function and nearly identical anesthetizing effects on tadpole mobility. Using quantitative analysis by 19F-NMR saturation transfer difference spectroscopy, we reveal strong intermolecular cross-relaxation rate constants between 4-fluoropropofol and four different regions of NaChBac, including the activation gate and selectivity filter in the pore, the voltage sensing domain, and the S4-S5 linker. Unlike volatile anesthetics, 4-fluoropropofol does not bind to the extracellular interface of the pore domain. Collectively, our results show that propofol inhibits NaChBac at multiple sites, likely with distinct modes of action. This study provides a molecular basis for understanding the net inhibitory action of propofol on NaV channels.

Solution NMR Studies of Anesthetic Interactions with Ion Channels.

NMR spectroscopy is one of the major tools to provide atomic resolution protein structural information. It has been used to elucidate the molecular details of interactions between anesthetics and ion channels, to identify anesthetic binding sites, and to characterize channel dynamics and changes introduced by anesthetics. In this chapter, we present solution NMR methods essential for investigating interactions between ion channels and general anesthetics, including both volatile and intravenous anesthetics. Case studies are provided with a focus on pentameric ligand-gated ion channels and the voltage-gated sodium channel NaChBac.

Ketamine Inhibition of the Pentameric Ligand-Gated Ion Channel GLIC.

Ketamine inhibits pentameric ligand-gated ion channels (pLGICs), including the bacterial pLGIC from Gloeobacter violaceus (GLIC). The crystal structure of GLIC shows R-ketamine bound to an extracellular intersubunit cavity. Here, we performed molecular dynamics simulations of GLIC in the absence and presence of R- or S-ketamine. No stable binding of S-ketamine in the original cavity was observed in the simulations, largely due to its unfavorable access to residue D154, which provides important electrostatic interactions to stabilize R-ketamine binding. Contrary to the symmetric binding shown in the crystal structure, R-ketamine moved away from some of the binding sites and was bound to GLIC asymmetrically at the end of simulations. The asymmetric binding is consistent with the experimentally measured negative cooperativity of ketamine binding to GLIC. In the presence of R-ketamine, all subunits showed changes in structure and dynamics, irrespective of binding stability; the extracellular intersubunit cavity expanded and intersubunit electrostatic interactions involved in channel activation were altered. R-ketamine binding promoted a conformational shift toward closed GLIC. Conformational changes near the ketamine-binding site were propagated to the interface between the extracellular and transmembrane domains, and further to the pore-lining TM2 through two pathways: pre-TM1 and the ß1-ß2 loop. Both signaling pathways have been predicted previously using the perturbation-based Markovian transmission model. The study provides a structural and dynamics basis for the inhibitory modulation of ketamine on pLGICs.

Structural Basis of Alcohol Inhibition of the Pentameric Ligand-Gated Ion Channel ELIC.

The structural basis for alcohol modulation of neuronal pentameric ligand-gated ion channels (pLGICs) remains elusive. We determined an inhibitory mechanism of alcohol on the pLGIC Erwinia chrysanthemi (ELIC) through direct binding to the pore. X-ray structures of ELIC co-crystallized with 2-bromoethanol, in both the absence and presence of agonist, reveal 2-bromoethanol binding in the pore near T237(6') and the extracellular domain (ECD) of each subunit at three different locations. Binding to the ECD does not appear to contribute to the inhibitory action of 2-bromoethanol and ethanol as indicated by the same functional responses of wild-type ELIC and mutants. In contrast, the ELIC-α1ß3GABAAR chimera, replacing the ELIC transmembrane domain (TMD) with the TMD of α1ß3GABAAR, is potentiated by 2-bromoethanol and ethanol. The results suggest a dominant role of the TMD in modulating alcohol effects. The X-ray structures and functional measurements support a pore-blocking mechanism for inhibitory action of short-chain alcohols.

Direct Pore Binding as a Mechanism for Isoflurane Inhibition of the Pentameric Ligand-gated Ion Channel ELIC.

Pentameric ligand-gated ion channels (pLGICs) are targets of general anesthetics, but molecular mechanisms underlying anesthetic action remain debatable. We found that ELIC, a pLGIC from Erwinia chrysanthemi, can be functionally inhibited by isoflurane and other anesthetics. Structures of ELIC co-crystallized with isoflurane in the absence or presence of an agonist revealed double isoflurane occupancies inside the pore near T237(6') and A244(13'). A pore-radius contraction near the extracellular entrance was observed upon isoflurane binding. Electrophysiology measurements with a single-point mutation at position 6' or 13' support the notion that binding at these sites renders isoflurane inhibition. Molecular dynamics simulations suggested that isoflurane binding was more stable in the resting than in a desensitized pore conformation. This study presents compelling evidence for a direct pore-binding mechanism of isoflurane inhibition, which has a general implication for inhibitory action of general anesthetics on pLGICs.

Ensemble-based virtual screening for cannabinoid-like potentiators of the human glycine receptor α1 for the treatment of pain.

The human glycine receptors (hGlyRs) are chloride-selective ion channels that mediate inhibitory neurotransmission in the brain stem and spinal cord. They are also targets for compounds of potential use in analgesic therapies. Here, we develop a strategy to discover analgesic drugs via structure-based virtual screening based on the recently published NMR structure of the hGlyR-α1 transmembrane domain (PDB ID: 2M6I ) and the critical role of residue S296 in hGlyR-α1 potentiation by Δ(9)-tetrahydrocannabinol (THC). We screened 1549 FDA-approved drugs in the DrugBank database on an ensemble of 180 hGlyR-α1 structures generated from molecular dynamics simulations of the NMR structure of the hGlyR-α1 transmembrane domain in different lipid environments. Thirteen hit compounds from the screening were selected for functional validation in Xenopus laevis oocytes expressing hGlyR-α1. Only one compound showed no potentiation effects; seven potentiated hGlyR-α1 at a level greater than THC at 1 µM. Our virtual screening protocol is generally applicable to drug targets with lipid-facing binding sites.

Obligatory duetting behaviour in the Chrysoperla carnea-group of cryptic species (Neuroptera: Chrysopidae): its role in shaping evolutionary history.

An unusual system of communication has evolved in green lacewings of the Chrysoperla carnea-group, triggering rapid proliferation of numerous cryptic species across all of the Northern Hemisphere and large portions of Africa. The system is based on sexually monomorphic, substrate-borne vibrational signals, produced by abdominal oscillation. These low-frequency signals are exchanged between courting individuals in a precise duetting format. The song of each of the more than 20 described species exhibits a unique acoustical phenotype that reproductively isolates the taxon from all other species with which it might come into contact. Here, we review what is known about duetting behaviour in the carnea-group, emphasizing the dominant role that duetting has played in the evolution, ecology, and speciation of the complex. Included are descriptions and discussions of song diversity and its impact on reproductive isolation among species, the genetic basis of interspecific song differences, partitioning of acoustic space among sympatric species, parallel song evolution in allopatric species pairs, and modes of speciation within the complex. We also emphasize the importance of correctly identifying by song all species of the carnea-group that are to be used either in experimental studies or programs of biological control, while acknowledging the continuing relevance of morphology to carnea-group systematics.

The inheritance of mating songs in two cryptic, sibling lacewing species (Neuroptera: Chrysopidae: Chrysoperla).

Speciation often involves incremental responses to natural selection and results in large scale genomic changes, but it may also occur abruptly and with little genetic imprint, as seen in some complexes of cryptic species. Recent attention has focused on sexual selection in rapid speciation, because it can disrupt premating signals that mediate reproductive isolation. Some models require that environmental adaptation assist sexual selection during speciation, while others show that populations can diverge through mate choice alone. We propose that speciation involving environmental adaptation in premating signals is likely to have a polygenic basis, while speciation due to arbitrary changes in premating signals could be accompanied by changes at just a few loci. The sibling species Chrysoperla plorabunda and Chrysoperla johnsoni belong to a large complex of cryptic species of green lacewings, and meet all criteria for speciation via sexual selection. We perform a genetic analysis of line crosses between them, focusing on their substrate-borne premating songs. Measurements of seven song features and their principal components are compared among parentals, F1 and F2 hybrids, and backcrosses. The distributions of phenotypes are consistent with a model of more than one, but not many, genes. Sex linkage and/or maternal effects are negligible. C. plorabunda is dominant for most traits affecting mate choice. Bayesian analyses of segregation variance show significant additive and epistatic effects on line means. A Bayesian Castle-Wright estimate suggests that relatively few effective factors are responsible for variation in volley period (x = two factors), number of volleys per song (x = 1), and PC-1 (x = 4). Our results are consistent with simple genetic architecture of songs, supporting a role for major genes in premating isolation and strengthening the notion that mate choice alone, without significant environmental adaptation, has been responsible for generating new lacewing species.

CONVERGENT EVOLUTION OF COURTSHIP SONGS AMONG CRYPTIC SPECIES OF THE CARNEA GROUP OF GREEN LACEWINGS (NEUROPTERA: CHRYSOPIDAE: CHRYSOPERLA).

Although traits of related species are likely to be similar due to common ancestry, mating signals are an exception. In singing insects, for example, song similarity has been documented only for allopatric or allochronic species pairs, and even then, not often. Where song similarity does occur, it has been logically attributed to the inheritance of ancestral traits rather than convergence. It is quite common for related, sympatric insect species to differ dramatically in calling song, which is predicted by evolutionary theory to maximize intraspecific mating success. Given that there are a limited number of ways to make sounds on anatomically similar organs and given that there would be no selective pressure for songs to differ in widely separated geographic areas, convergence in songs among related species living on different continents might be expected. Here we present the first well-documented case of such convergence, in a group of sibling, cryptic species characterized by substrate-borne vibrational mating songs. In this example from green lacewings of the carnea group of the genus Chrysoperla, a variety of statistical tests shows that one species in North America and another in Asia possess songs that are strikingly similar to each other. DNA data demonstrate that the species involved belong to divergent speciose lineages, and behavioral data demonstrate that the convergent songs are readily accepted by members of both species.

THE ROLE OF COURTSHIP SONGS IN REPRODUCTIVE ISOLATION AMONG POPULATIONS OF GREEN LACEWINGS OF THE GENUS CHRYSOPERLA (NEUROPTERA: CHRYSOPIDAE).

Male and female lacewings tremulate during courtship, establishing duets that always precede copulation. Three distinct courtship songs are found in populations of the green lacewing Chrysoperla plorabunda (P1, P2 and P3 song morphs). Analysis of five features of the songs for individuals collected from Connecticut, Idaho, Oregon and California showed few differences within song morphs, but sympatric song morphs differed significantly in temporal features of the songs and their mode of presentation. Playback experiments using recorded songs were performed on females with all possible sympatric and allopatric combinations of females and recorded songs. The results showed that females strongly prefer to duet with recordings of males of their own song type and usually showed no responses to songs of other types. Thus, song differences are effective barriers to reproduction between the sympatric morphs. Our results support the hypothesis that the three song morphs are true biological species.