Protein Synthesis in the Developing Neocortex at Near-Atomic Resolution Reveals Ebp1-Mediated Neuronal Proteostasis at the 60S Tunnel Exit.

Protein synthesis must be finely tuned in the developing nervous system as the final essential step of gene expression. This study investigates the architecture of ribosomes from the neocortex during neurogenesis, revealing Ebp1 as a high-occupancy 60S peptide tunnel exit (TE) factor during protein synthesis at near-atomic resolution by cryoelectron microscopy (cryo-EM). Ribosome profiling demonstrated Ebp1-60S binding is highest during start codon initiation and N-terminal peptide elongation, regulating ribosome occupancy of these codons. Membrane-targeting domains emerging from the 60S tunnel, which recruit SRP/Sec61 to the shared binding site, displace Ebp1. Ebp1 is particularly abundant in the early-born neural stem cell (NSC) lineage and regulates neuronal morphology. Ebp1 especially impacts the synthesis of membrane-targeted cell adhesion molecules (CAMs), measured by pulsed stable isotope labeling by amino acids in cell culture (pSILAC)/bioorthogonal noncanonical amino acid tagging (BONCAT) mass spectrometry (MS). Therefore, Ebp1 is a central component of protein synthesis, and the ribosome TE is a focal point of gene expression control in the molecular specification of neuronal morphology during development.

Srsf1 and Elavl1 act antagonistically on neuronal fate choice in the developing neocortex by controlling TrkC receptor isoform expression.

The seat of higher-order cognitive abilities in mammals, the neocortex, is a complex structure, organized in several layers. The different subtypes of principal neurons are distributed in precise ratios and at specific positions in these layers and are generated by the same neural progenitor cells (NPCs), steered by a spatially and temporally specified combination of molecular cues that are incompletely understood. Recently, we discovered that an alternatively spliced isoform of the TrkC receptor lacking the kinase domain, TrkC-T1, is a determinant of the corticofugal projection neuron (CFuPN) fate. Here, we show that the finely tuned balance between TrkC-T1 and the better known, kinase domain-containing isoform, TrkC-TK+, is cell type-specific in the developing cortex and established through the antagonistic actions of two RNA-binding proteins, Srsf1 and Elavl1. Moreover, our data show that Srsf1 promotes the CFuPN fate and Elavl1 promotes the callosal projection neuron (CPN) fate in vivo via regulating the distinct ratios of TrkC-T1 to TrkC-TK+. Taken together, we connect spatio-temporal expression of Srsf1 and Elavl1 in the developing neocortex with the regulation of TrkC alternative splicing and transcript stability and neuronal fate choice, thus adding to the mechanistic and functional understanding of alternative splicing in vivo.

Socrates: A Novel N-Ethyl-N-nitrosourea-Induced Mouse Mutant with Audiogenic Epilepsy.

Epilepsy is one of the common neurological diseases that affects not only adults but also infants and children. Because epilepsy has been studied for a long time, there are several pharmacologically effective anticonvulsants, which, however, are not suitable as therapy for all patients. The genesis of epilepsy has been extensively investigated in terms of its occurrence after injury and as a concomitant disease with various brain diseases, such as tumors, ischemic events, etc. However, in the last decades, there are multiple reports that both genetic and epigenetic factors play an important role in epileptogenesis. Therefore, there is a need for further identification of genes and loci that can be associated with higher susceptibility to epileptic seizures. Use of mouse knockout models of epileptogenesis is very informative, but it has its limitations. One of them is due to the fact that complete deletion of a gene is not, in many cases, similar to human epilepsy-associated syndromes. Another approach to generating mouse models of epilepsy is N-Ethyl-N-nitrosourea (ENU)-directed mutagenesis. Recently, using this approach, we generated a novel mouse strain, soc (socrates, formerly s8-3), with epileptiform activity. Using molecular biology methods, calcium neuroimaging, and immunocytochemistry, we were able to characterize the strain. Neurons isolated from soc mutant brains retain the ability to differentiate in vitro and form a network. However, soc mutant neurons are characterized by increased spontaneous excitation activity. They also demonstrate a high degree of Ca2+ activity compared to WT neurons. Additionally, they show increased expression of NMDA receptors, decreased expression of the Ca2+-conducting GluA2 subunit of AMPA receptors, suppressed expression of phosphoinositol 3-kinase, and BK channels of the cytoplasmic membrane involved in protection against epileptogenesis. During embryonic and postnatal development, the expression of several genes encoding ion channels is downregulated in vivo, as well. Our data indicate that soc mutation causes a disruption of the excitation-inhibition balance in the brain, and it can serve as a mouse model of epilepsy.

Transcranial Photosensitizer-Free Laser Treatment of Glioblastoma in Rat Brain.

Over sixty years, laser technologies have undergone a technological revolution and become one of the main tools in biomedicine, particularly in neuroscience, neurodegenerative diseases and brain tumors. Glioblastoma is the most lethal form of brain cancer, with very limited treatment options and a poor prognosis. In this study on rats, we demonstrate that glioblastoma (GBM) growth can be suppressed by photosensitizer-free laser treatment (PS-free-LT) using a quantum-dot-based 1267 nm laser diode. This wavelength, highly absorbed by oxygen, is capable of turning triplet oxygen to singlet form. Applying 1267 nm laser irradiation for a 4 week course with a total dose of 12.7 kJ/cm2 firmly suppresses GBM growth and increases survival rate from 34% to 64%, presumably via LT-activated apoptosis, inhibition of the proliferation of tumor cells, a reduction in intracranial pressure and stimulation of the lymphatic drainage and clearing functions. PS-free-LT is a promising breakthrough technology in non- or minimally invasive therapy for superficial GBMs in infants as well as in adult patients with high photosensitivity or an allergic reaction to PSs.

The murine ortholog of Kaufman oculocerebrofacial syndrome protein Ube3b regulates synapse number by ubiquitinating Ppp3cc.

Kaufman oculocerebrofacial syndrome (KOS) is a severe autosomal recessive disorder characterized by intellectual disability, developmental delays, microcephaly, and characteristic dysmorphisms. Biallelic mutations of UBE3B, encoding for a ubiquitin ligase E3B are causative for KOS. In this report, we characterize neuronal functions of its murine ortholog Ube3b and show that Ube3b regulates dendritic branching in a cell-autonomous manner. Moreover, Ube3b knockout (KO) neurons exhibit increased density and aberrant morphology of dendritic spines, altered synaptic physiology, and changes in hippocampal circuit activity. Dorsal forebrain-specific Ube3b KO animals show impaired spatial learning, altered social interactions, and repetitive behaviors. We further demonstrate that Ube3b ubiquitinates the catalytic γ-subunit of calcineurin, Ppp3cc, the overexpression of which phenocopies Ube3b loss with regard to dendritic spine density. This work provides insights into the molecular pathologies underlying intellectual disability-like phenotypes in a genetically engineered mouse model.

TrkC-T1, the Non-Catalytic Isoform of TrkC, Governs Neocortical Progenitor Fate Specification by Inhibition of MAP Kinase Signaling.

Neocortical projection neurons are generated by neural progenitor cells (NPCs) within the ventricular and subventricular zone. While early NPCs can give rise to both deep and upper layer neurons, late progenitors are restricted to upper layer neurogenesis. The molecular mechanisms controlling the differentiation potential of early versus late NPCs are unknown. Here, we report a novel function for TrkC-T1, the non-catalytic isoform of the neurotrophin receptor TrkC, that is distinct from TrkC-TK+, the full-length isoform. We provide direct evidence that TrkC-T1 regulates the switch in NPC fate from deep to upper layer neuron production. Elevated levels of TrkC-T1 in early NPCs promote the generation of deep layer neurons. Conversely, downregulation of TrkC-T1 in these cells promotes upper layer neuron fate. Furthermore, we show that TrkC-T1 exerts this control by interaction with the signaling adaptor protein ShcA. TrkC-T1 prevents the phosphorylation of Shc and the downstream activation of the MAP kinase (Erk1/2) pathway. In vivo manipulation of the activity of ShcA or Erk1/2, directly affects cortical neuron cell fate. We thus show that the generation of upper layer neurons by late progenitors is dependent on the downregulation of TrkC-T1 in late progenitor cells and the resulting activation of the ShcA/Erk1/2 pathway.

Types of spectroscopy and microscopy techniques for cancer diagnosis: a review.

Cancer is a life-threatening disease that has claimed the lives of many people worldwide. With the current diagnostic methods, it is hard to determine cancer at an early stage, due to its versatile nature and lack of genomic biomarkers. The rapid development of biophotonics has emerged as a potential tool in cancer detection and diagnosis. Using the fluorescence, scattering, and absorption characteristics of cells and tissues, it is possible to detect cancer at an early stage. The diagnostic techniques addressed in this review are highly sensitive to the chemical and morphological changes in the cell and tissue during disease progression. These changes alter the fluorescence signal of the cell/tissue and are detected using spectroscopy and microscopy techniques including confocal and two-photon fluorescence (TPF). Further, second harmonic generation (SHG) microscopy reveals the morphological changes that occurred in non-centrosymmetric structures in the tissue, such as collagen. Again, Raman spectroscopy is a non-destructive method that provides a fingerprinting technique to differentiate benign and malignant tissue based on Raman signal. Photoacoustic microscopy and spectroscopy of tissue allow molecule-specific detection with high spatial resolution and penetration depth. In addition, terahertz spectroscopic studies reveal the variation of tissue water content during disease progression. In this review, we address the applications of spectroscopic and microscopic techniques for cancer detection based on the optical properties of the tissue. The discussed state-of-the-art techniques successfully determines malignancy to its rapid diagnosis.

A Surface Plasmon Resonance Biosensor Based on Directly Immobilized Hemoglobin and Myoglobin.

Immobilization of proteins on a surface plasmon resonance (SPR) transducer is a delicate procedure since loss of protein bioactivity can occur upon contact with the untreated metal surface. Solution to the problem is the use of an immobilization matrix having a complex structure. However, this is at the expense of biosensor selectivity and sensitivity. It has been shown that the matrix-assisted pulsed laser evaporation (MAPLE) method has been successfully applied for direct immobilization (without a built-in matrix) of proteins, preserving their bioactivity. So far, MAPLE deposition has not been performed on a gold surface as required for SPR biosensors. In this paper we study the impact of direct immobilization of heme proteins (hemoglobin (Hb) and myoglobin (Mb)) on their bioactivity. For the purpose, Hb and Mb were directly immobilized by MAPLE technique on a SPR transducer. The bioactivity of the ligands immobilized in the above-mentioned way was assessed by SPR registration of the molecular reactions of various Hb/Mb functional groups. By SPR we studied the reaction between the beta chain of the Hb molecule and glucose, which shows the structural integrity of the immobilized Hb. A supplementary study of films deposited by FTIR and AFM was provided. The experimental facts showed that direct immobilization of an intact molecule was achieved.

Colonizing the Wild West: Low Diversity of Complete Mitochondrial Genomes in Western North Pacific Killer Whales Suggests a Founder Effect.

In the North Pacific, fish-eating R-type "resident" and mammal-eating T-type "transient" killer whales do not interbreed and differ in ecology and behavior. Full-length mitochondrial genomes (about 16.4 kbp) were sequenced and assembled for 12 R-type and 14 T-type killer whale samples from different areas of the western North Pacific. All R-type individuals had the same haplotype, previously described for R-type killer whales from both eastern and western North Pacific. However, haplotype diversity of R-type killer whales was much lower in the western North Pacific than in the Aleutian Islands and the eastern North Pacific. T-type whales had 3 different haplotypes, including one previously undescribed. Haplotype diversity of T-type killer whales in the Okhotsk Sea was also much lower than in the Aleutian Islands and the eastern North Pacific. The highest haplotype diversity for both R- and T-type killer whales was observed in the Aleutian Islands. We discuss how the environmental conditions during the last glacial period might have shaped the history of killer whale populations in the North Pacific. Our results suggest the recent colonization or re-colonization of the western North Pacific by small groups of killer whales originating from the central or eastern North Pacific, possibly due to favorable environmental changes after the Last Glacial Maximum.

Stress-Induced Stroke and Stomach Cancer: Sex Differences in Oxygen Saturation.

Sex differences in stress-related diseases such as stroke and stomach cancer are well established, but the mechanisms underlying this phenomenon remain unknown. Despite the fact that sexual hormones play an important role in the high resistance of females to harmful effects of stress compared with males, the regulation of oxygenation status can be a potential factor, which might explain sex differences in stress-induced cerebrovascular catastrophes in newborn rats and in mutagens activation in adult rats with stomach cancer.

Hypoxia and Neonatal Haemorrhagic Stroke: Experimental Study of Mechanisms.

We studied the level of blood oxygen saturation (SpO2) in the brain in newborn rats in the pre- and post-stroke periods, as well as the changes in cerebral blood flow and beta-arrestin-1 as a marker of hypoxic stress. Our results show that mild hypoxia precedes the stroke development and is associated with venous relaxation and decrease blood outflow from the brain resulting in the elevation of synthesis of beta-arrestin-1 in the brain. The incidence of stroke is characterized by severe hypoxia, which is accompanied by the progression of pathological changes in cerebral veins and the high level of beta-arrestin-1.

Photodynamic inactivation of pathogenic species Pseudomonas aeruginosa and Candida albicans with lutetium (III) acetate phthalocyanines and specific light irradiation.

Photodynamic inactivation (PDI) is a light-associated therapeutic approach suitable for treatment of local acute infections. The method is based on specific light-activated compound which by specific irradiation and in the presence of molecular oxygen produced molecular singlet oxygen and other reactive oxygen species, all toxic for pathogenic microbial cells. The study presents photodynamic impact of two recently synthesized water-soluble cationic lutetium (III) acetate phthalocyanines (LuPc-5 and LuPc-6) towards two pathogenic strains, namely, the Gram-negative bacterium Pseudomonas aeruginosa and a fungus Candida albicans. The photodynamic effect was evaluated for the cells in suspensions and organized in 48-h developed biofilms. The relatively high levels of uptakes of LuPc-5 and LuPc-6 were determined for fungal cells compared to bacterial cells. The penetration depths and distribution of both LuPcs into microbial biofilms were investigated by means of confocal fluorescence microscopy. The photoinactivation efficiency was studied for a wide concentration range (0.85-30 µM) of LuPc-5 and LuPc-6 at a light dose of 50 J cm-2 from red light-emitting diode (LED; 665 nm). The PDI study on microbial biofilms showed incomplete photoinactivation (<3 logs) for the used gentle drug-light protocol.

Protein translation rate determines neocortical neuron fate.

The mammalian neocortex comprises an enormous diversity regarding cell types, morphology, and connectivity. In this work, we discover a post-transcriptional mechanism of gene expression regulation, protein translation, as a determinant of cortical neuron identity. We find specific upregulation of protein synthesis in the progenitors of later-born neurons and show that translation rates and concomitantly protein half-lives are inherent features of cortical neuron subtypes. In a small molecule screening, we identify Ire1α as a regulator of Satb2 expression and neuronal polarity. In the developing brain, Ire1α regulates global translation rates, coordinates ribosome traffic, and the expression of eIF4A1. Furthermore, we demonstrate that the Satb2 mRNA translation requires eIF4A1 helicase activity towards its 5'-untranslated region. Altogether, we show that cortical neuron diversity is generated by mechanisms operating beyond gene transcription, with Ire1α-safeguarded proteostasis serving as an essential regulator of brain development.

From Empire-wide integration to regional localization: A synthetic and quantitative study of heterogeneous amphora data in Roman Germania reveals centuries-long change in regional patterns of production and consumption.

We present novel insights into trade in amphorae-borne products over a 550-year period in Germania along the frontier of the Roman Empire, derived through probabilistic aoristic methods to study temporal changes in archaeological materials. Our data analysis reveals highly detailed differential patterns of consumption and production within the German market. We show how connections to far-flung regions such as the Eastern Mediterranean or the Iberian Peninsula wax and wane through time, and how the local German producers start to compete with these imported products. These chronological patterns provide important insight into a regional market within the larger Roman economy and provide an important case study in changing economic connections over a long period, demonstrating in a transparent and reproducible way a geographical and chronological pulsation in market activity that was otherwise unknown and undemonstrated.

Heart Dysfunction in Essential Hypertension Depends on Systemic Proinflammatory Influences: A Retrospective Clinical Pathophysiological Study.

Low-intensity systemic inflammation is an important element of heart failure pathogenesis. The aim of this study is to assess proinflammatory status serum indicators (C-reactive protein (CRP), tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6)) in middle-aged males (M) and females (F) with essential hypertension (HTN) depending on left ventricular (LV) diastolic dysfunction (LVDD). The main group comprised 55 M and 49 F with the first- to second-severity grade HTN with mild heart failure and a preserved LV ejection fraction ≥50%. Patients had sinus rhythm, first or second-severity degree LVDD, LV hypertrophy, left atrium dilatation, and NT-proBNP > 125 pg/mL. Comparison group: 30 hypertensives without cardiac dysfunction; control group: 31 normotensives. Quantitative features were compared using the Mann−Whitney test, median χ2, ANOVA module. Spearman's rank correlation coefficients were determined to identify the relationship between the proinflammatory pattern and exercise tolerance. Hypertensive M had markedly higher CRP, TNF-α, and IL-6 levels compared to F. All mean values corresponded to reference range. In patients with second-degree LVDD, CRP, TNF-α, and IL-6 levels were significantly greater than in subjects with first-degree LVDD (both within M and within F samples). Significant negative associations between CRP, IL-6, and TNF-α levels and the 6 min walk test existed in hypertensive M and F. The study demonstrated a close relationship between the proinflammatory pattern and LVDD and exercise tolerance indicators, regardless of the hypertensive patient's sex.

PCSK9 deficiency alters brain lipid composition without affecting brain development and function.

PCSK9 induces lysosomal degradation of the low-density lipoprotein (LDL) receptor (LDLR) in the liver, hereby preventing removal of LDL cholesterol from the circulation. Accordingly, PCSK9 inhibitory antibodies and siRNA potently reduce LDL cholesterol to unprecedented low levels and are approved for treatment of hypercholesterolemia. In addition, PCSK9 inactivation alters the levels of several other circulating lipid classes and species. Brain function is critically influenced by cholesterol and lipid composition. However, it remains unclear how the brain is affected long-term by the reduction in circulating lipids as achieved with potent lipid lowering therapeutics such as PCSK9 inhibitors. Furthermore, it is unknown if locally expressed PCSK9 affects neuronal circuits through regulation of receptor levels. We have studied the effect of lifelong low peripheral cholesterol levels on brain lipid composition and behavior in adult PCSK9 KO mice. In addition, we studied the effect of PCSK9 on neurons in culture and in vivo in the developing cerebral cortex. We found that PCSK9 reduced LDLR and neurite complexity in cultured neurons, but neither PCSK9 KO nor overexpression affected cortical development in vivo. Interestingly, PCSK9 deficiency resulted in changes of several lipid classes in the adult cortex and cerebellum. Despite the observed changes, PCSK9 KO mice had unchanged behavior compared to WT controls. In conclusion, our findings demonstrate that altered PCSK9 levels do not compromise brain development or function in mice, and are in line with clinical trials showing that PCSK9 inhibitors have no adverse effects on cognitive function.

A critical period of translational control during brain development at codon resolution.

Translation modulates the timing and amplification of gene expression after transcription. Brain development requires uniquely complex gene expression patterns, but large-scale measurements of translation directly in the prenatal brain are lacking. We measure the reactants, synthesis and products of mRNA translation spanning mouse neocortex neurogenesis, and discover a transient window of dynamic regulation at mid-gestation. Timed translation upregulation of chromatin-binding proteins like Satb2, which is essential for neuronal subtype differentiation, restricts protein expression in neuronal lineages despite broad transcriptional priming in progenitors. In contrast, translation downregulation of ribosomal proteins sharply decreases ribosome biogenesis, coinciding with a major shift in protein synthesis dynamics at mid-gestation. Changing activity of eIF4EBP1, a direct inhibitor of ribosome biogenesis, is concurrent with ribosome downregulation and affects neurogenesis of the Satb2 lineage. Thus, the molecular logic of brain development includes the refinement of transcriptional programs by translation. Modeling of the developmental neocortex translatome is provided as an open-source searchable resource at https://shiny.mdc-berlin.de/cortexomics .

Polarization and depolarization metrics as optical markers in support to histopathology of ex vivo colon tissue.

Tissue polarimetry holds great promise to improve the effectiveness of conventional cancer diagnostics and staging, being a fast, minimally invasive, and low-cost optical technique. We introduce an enhanced diagnostic method for ex vivo colon specimens assessment by utilizing Stokes and Mueller matrix polarimetry. The proposed method makes use of experimental Mueller matrices, measured from healthy and tumor zones of a colon specimen, as input data for post-processing algorithms that include physical realisability filtering, symmetric decomposition and estimation of various polarization and depolarization metrics for colon specimen diagnostics. We validated our results with the gold standard histological diagnostics provided by pathologists. It was found that the Stokes-Mueller matrix polarimetry, combined with the appropriate filtering, decomposition algorithms and polarization/depolarization metrics calculations provides relevant optical markers of the colon tissue pathological conditions (healthy versus cancer), as confirmed by histopathology analysis. This approach potentially provides physicians with valuable and complementary information that holds promises in helping with the diagnostics of colon tissue specimens.

Adhesion dynamics in the neocortex determine the start of migration and the post-migratory orientation of neurons.

The neocortex is stereotypically organized into layers of excitatory neurons arranged in a precise parallel orientation. Here we show that dynamic adhesion both preceding and following radial migration is essential for this organization. Neuronal adhesion is regulated by the Mowat-Wilson syndrome-associated transcription factor Zeb2 (Sip1/Zfhx1b) through direct repression of independent adhesion pathways controlled by Neuropilin-1 (Nrp1) and Cadherin-6 (Cdh6). We reveal that to initiate radial migration, neurons must first suppress adhesion to the extracellular matrix. Zeb2 regulates the multipolar stage by transcriptional repression of Nrp1 and thereby downstream inhibition of integrin signaling. Upon completion of migration, neurons undergo an orientation process that is independent of migration. The parallel organization of neurons within the neocortex is controlled by Cdh6 through atypical regulation of integrin signaling via its RGD motif. Our data shed light on the mechanisms that regulate initiation of radial migration and the postmigratory orientation of neurons during neocortical development.

Split Chloramphenicol Acetyl-Transferase Assay Reveals Self-Ubiquitylation-Dependent Regulation of UBE3B.

Split reporter protein-based genetic section systems are widely used to identify and characterize protein-protein interactions (PPI). The assembly of split markers that antagonize toxins, rather than required for synthesis of missing metabolites, facilitates the seeding of high density of cells and selective growth. Here we present a newly developed split chloramphenicol acetyltransferase (split-CAT) -based genetic selection system. The N terminus fragment of CAT is fused downstream of the protein of interest and the C terminus fragment is tethered upstream to its postulated partner. We demonstrate the system's advantages for the study of PPIs. Moreover, we show that co-expression of a functional ubiquitylation cascade where the target and ubiquitin are tethered to the split-CAT fragments results in ubiquitylation-dependent selective growth. Since proteins do not have to be purified from the bacteria and due to the high sensitivity of the split-CAT reporter, detection of challenging protein cascades and post-translation modifications is enabled. In addition, we demonstrate that the split-CAT system responds to small molecule inhibitors and molecular glues (GLUTACs). The absence of ubiquitylation-dependent degradation and deubiquitylation in E. coli significantly simplify the interpretation of the results. We harnessed the developed system to demonstrate that like NEDD4, UBE3B also undergoes self-ubiquitylation-dependent inactivation. We show that self-ubiquitylation of UBE3B on K665 induces oligomerization and inactivation in yeast and mammalian cells respectively. Finally, we showcase the advantages of split-CAT in the study of human diseases by demonstrating that mutations in UBE3B that cause Kaufman oculocerebrofacial syndrome exhibit clear E. coli growth phenotypes.