The Neuroprotective Role of Quinoa (Chenopodium quinoa, Wild) Supplementation in Hippocampal Morphology and Memory of Adolescent Stressed Rats.

Brain physiology and morphology are vulnerable to chronic stress, impacting cognitive performance and behavior. However, functional compounds found in food may alleviate these alterations. White quinoa (Chenopodium quinoa, Wild) seeds contain a high content of n-3 fatty acids, including alpha-linolenic acid. This study aimed to evaluate the potential neuroprotective role of a quinoa-based functional food (QFF) in rats. Prepubertal male Sprague-Dawley rats were fed with rat chow or QFF (50% rat chow + 50% dehydrated quinoa seeds) and exposed or not to restraint stress protocol (2 h/day; 15 days). Four experimental groups were used: Non-stressed (rat chow), Non-stressed + QFF, Stressed (rat chow) and Stressed + QFF. Weight gain, locomotor activity (open field), anxiety (elevated plus maze, light-dark box), spatial memory (Y-maze), and dendritic length in the hippocampus were measured in all animals. QFF intake did not influence anxiety-like behaviors, while the memory of stressed rats fed with QFF improved compared to those fed with rat chow. Additionally, QFF intake mitigated the stress-induced dendritic atrophy in pyramidal neurons located in the CA3 area of the hippocampus. The results suggest that a quinoa-supplemented diet could play a protective role in the memory of chronically stressed rats.

A kinesin-1 adaptor complex controls bimodal slow axonal transport of spectrin in Caenorhabditis elegans.

An actin-spectrin lattice, the membrane periodic skeleton (MPS), protects axons from breakage. MPS integrity relies on spectrin delivery via slow axonal transport, a process that remains poorly understood. We designed a probe to visualize endogenous spectrin dynamics at single-axon resolution in vivo. Surprisingly, spectrin transport is bimodal, comprising fast runs and movements that are 100-fold slower than previously reported. Modeling and genetic analysis suggest that the two rates are independent, yet both require kinesin-1 and the coiled-coil proteins UNC-76/FEZ1 and UNC-69/SCOC, which we identify as spectrin-kinesin adaptors. Knockdown of either protein led to disrupted spectrin motility and reduced distal MPS, and UNC-76 overexpression instructed excessive transport of spectrin. Artificially linking spectrin to kinesin-1 drove robust motility but inefficient MPS assembly, whereas impairing MPS assembly led to excessive spectrin transport, suggesting a balance between transport and assembly. These results provide insight into slow axonal transport and MPS integrity.

Capillary Electrophoresis Mass Spectrometry for Scalable Single-Cell Proteomics.

Understanding the biochemistry of the cell requires measurement of all the molecules it produces. Single-cell proteomics recently became possible through advances in microanalytical sample preparation, separation by nano-flow liquid chromatography (nanoLC) and capillary electrophoresis (CE), and detection using electrospray ionization (ESI) high-resolution mass spectrometry (HRMS). Here, we demonstrate capillary microsampling CE-ESI-HRMS to be scalable to proteomics across broad cellular dimensions. This study established proof-of-principle using giant, ∼250-µm-diameter cells from embryos of the frog Xenopus laevis and small, ∼35-µm-diameter neurons in culture from the mouse hippocampus. From ∼18 ng, or ∼0.2% of the total cellular proteome, subcellular analysis of the ventral-animal midline (V11) and equatorial (V12) cells identified 1,133 different proteins in a 16-cell embryo. CE-HRMS achieved ∼20-times higher sensitivity and doubled the speed of instrumental measurements compared to nanoLC, the closest neighboring single-cell technology of choice. Microanalysis was scalable to 722 proteins groups from ∼5 ng of cellular protein digest from identified left dorsal-animal midline cell (D11), supporting sensitivity for smaller cells. Capillary microsampling enabled the isolation and transfer of individual neurons from the culture, identifying 37 proteins between three different cells. A total of 224 proteins were detected from 500 pg of neuronal protein digest, which estimates to a single neuron. Serial dilution returned 157 proteins from sample amounts estimating to about half a cell (250 pg protein) and 70 proteins from ca. a quarter of a neuron (125 pg protein), suggesting sufficient sensitivity for subcellular proteomics. CE-ESI-HRMS complements nanoLC proteomics with scalability, sensitivity, and speed across broad cellular dimensions.

Data-Dependent Acquisition Ladder for Capillary Electrophoresis Mass Spectrometry-Based Ultrasensitive (Neuro)Proteomics.

Measurement of broad types of proteins from a small number of cells to single cells would help to better understand the nervous system but requires significant leaps in sensitivity in high-resolution mass spectrometry (HRMS). Microanalytical capillary electrophoresis electrospray ionization (CE-ESI) offers a path to ultrasensitive proteomics by integrating scalability with sensitivity. Here, we systematically evaluate performance limitations in this technology to develop a data acquisition strategy with deeper coverage of the neuroproteome from trace amounts of starting materials than traditional dynamic exclusion. During standard data-dependent acquisition (DDA), compact migration challenged the duty cycle of second-stage transitions and redundant targeting of abundant peptide signals lowered their identification success rate. DDA was programmed to progressively exclude a static set of high-intensity peptide signals throughout replicate measurements, essentially forming rungs of a "DDA ladder." The method was tested for ∼500 pg portions of a protein digest from cultured hippocampal (primary) neurons (mouse), which estimated the total amount of protein from a single neuron. The analysis of ∼5 ng of protein digest over all replicates, approximating ∼10 neurons, identified 428 nonredundant proteins (415 quantified), an ∼35% increase over traditional DDA. The identified proteins were enriched in neuronal marker genes and molecular pathways of neurobiological importance. The DDA ladder enhances CE-HRMS sensitivity to single-neuron equivalent amounts of proteins, thus expanding the analytical toolbox of neuroscience.

Male-Specific cAMP Signaling in the Hippocampus Controls Spatial Memory Deficits in a Mouse Model of Autism and Intellectual Disability.

BACKGROUND: The prevalence of neurodevelopmental disorders is biased toward male individuals, with male-to-female ratios of 2:1 in intellectual disability and 4:1 in autism spectrum disorder. However, the molecular mechanisms of such bias remain unknown. While characterizing a mouse model for loss of the signaling scaffold coiled-coil and C2 domain-containing protein 1A (CC2D1A), which is mutated in intellectual disability and autism spectrum disorder, we identified biochemical and behavioral differences between male and female mice, and explored whether CC2D1A controls male-specific intracellular signaling. METHODS: CC2D1A is known to regulate phosphodiesterase 4D (PDE4D), which regulates cyclic adenosine monophosphate (cAMP) signaling. We tested for activation of PDE4D and downstream signaling molecules in the hippocampus of Cc2d1a-deficient mice. We then performed behavioral studies in female mice to analyze learning and memory, and then targeted PDE4D activation with a PDE4D inhibitor to define how changes in cAMP levels affect behavior in male and female mice. RESULTS: We found that in Cc2d1a-deficient male mice PDE4D is hyperactive, leading to a reduction in cAMP response element binding protein signaling, but this molecular deficit is not present in female mice. Cc2d1a-deficient male mice show a deficit in spatial memory, which is not present in Cc2d1a-deficient female mice. Restoring PDE4D activity using an inhibitor rescues cognitive deficits in male mice but has no effect on female mice. CONCLUSIONS: Our findings show that CC2D1A regulates cAMP intracellular signaling in a male-specific manner in the hippocampus, leading to male-specific cognitive deficits. We propose that male-specific signaling mechanisms are involved in establishing sex bias in neurodevelopmental disorders.

Enhanced Peptide Detection Toward Single-Neuron Proteomics by Reversed-Phase Fractionation Capillary Electrophoresis Mass Spectrometry.

The ability to detect peptides and proteins in single cells is vital for understanding cell heterogeneity in the nervous system. Capillary electrophoresis (CE) nanoelectrospray ionization (nanoESI) provides high-resolution mass spectrometry (HRMS) with trace-level sensitivity, but compressed separation during CE challenges protein identification by tandem HRMS with limited MS/MS duty cycle. Here, we supplemented ultrasensitive CE-nanoESI-HRMS with reversed-phase (RP) fractionation to enhance identifications from protein digest amounts that approximate to a few mammalian neurons. An ~1 to 20 µg neuronal protein digest was fractionated on a RP column (ZipTip), and 1 ng to 500 pg of peptides were analyzed by a custom-built CE-HRMS system. Compared with the control (no fractionation), RP fractionation improved CE separation (theoretical plates ~274,000 versus 412,000 maximum, resp.), which enhanced detection sensitivity (2.5-fold higher signal-to-noise ratio), minimized co-isolation spectral interferences during MS/MS, and increased the temporal rate of peptide identification by up to ~57%. From 1 ng of protein digest (<5 neurons), CE with RP fractionation identified 737 protein groups (1,753 peptides), or ~480 protein groups (~1,650 peptides) on average per analysis. The approach was scalable to 500 pg of protein digest (~a single neuron), identifying 225 protein groups (623 peptides) in technical triplicates, or 141 protein groups on average per analysis. Among identified proteins, 101 proteins were products of genes that are known to be transcriptionally active in single neurons during early development of the brain, including those involved in synaptic transmission and plasticity and cytoskeletal organization. Graphical abstract ᅟ.

Upregulation of Epac-1 in Hepatic Stellate Cells by Prostaglandin E2 in Liver Fibrosis Is Associated with Reduced Fibrogenesis.

Exchange protein activated by cAMP (Epac-1) is an important signaling mechanism for cAMP-mediated effects, yet factors that change Epac-1 levels are unknown. Such factors are relevant because it has been postulated that Epac-1 directly affects fibrogenesis. Prostaglandin E2 (PGE2) is a well-known cAMP activator, and we therefore studied the effects of this cyclo-oxygenase product on Epac-1 expression and on fibrogenesis within the liver. Liver fibrosis was induced by 8 weeks carbon tetrachloride (CCL4) administration to mice. In the last 2 weeks, mice received vehicle, PGE2, the cyclo-oxygenase-2 inhibitor niflumic acid (NFA), or PGE2 coupled to cell-specific carriers to hepatocytes, Kupffer cells, or hepatic stellate cells (HSC). Results showed antifibrotic effects of PGE2 and profibrotic effects of NFA in CCL4 mice. Western blot analysis revealed reduced Epac-1 protein expression in fibrotic livers of mice and humans compared with healthy livers. PGE2 administration to fibrotic mice completely restored intrahepatic Epac-1 levels and also led to reduced Rho kinase activity, a downstream target of Epac-1. Cell-specific delivery of PGE2 to either hepatocytes, Kupffer cells, or HSC identified the latter cell as the key player in the observed effects on Epac-1 and Rho kinase. No significant alterations in protein kinase A expressions were found. In primary isolated HSC, PGE2 elicited Rap1 translocation reflecting Epac-1 activation, and Epac-1 agonists attenuated platelet-derived growth factor-induced proliferation and migration of these cells. These studies demonstrate that PGE2 enhances Epac-1 activity in HSC, which is associated with significant changes in (myo)fibroblast activities in vitro and in vivo. Therefore, Epac-1 is a potential target for antifibrotic drugs.

Microtubule-regulating proteins and cAMP-dependent signaling in neuroblastoma differentiation.

Neurons are highly differentiated cells responsible for the conduction and transmission of information in the nervous system. The proper function of a neuron relies on the compartmentalization of their intracellular domains. Differentiated neuroblastoma cells have been extensively used to study and understand the physiology and cell biology of neuronal cells. Here, we show that differentiation of N1E-115 neuroblastoma cells is more pronounced upon exposure of a chemical analog of cyclic AMP (cAMP), db-cAMP. We next analysed the expression of key microtubule-regulating proteins in differentiated cells and the expression and activation of key cAMP players such as EPAC, PKA and AKAP79/150. Most of the microtubule-promoting factors were up regulated during differentiation of N1E-115 cells, while microtubule-destabilizing proteins were down regulated. We observed an increase in tubulin post-translational modifications related to microtubule stability. As expected, db-cAMP increased PKA- and EPAC-dependent signalling. Consistently, pharmacological modulation of EPAC activity instructed cell differentiation, number of neurites, and neurite length in N1E-115 cells. Moreover, disruption of the PKA-AKAP interaction reduced these morphometric parameters. Interestingly, PKA and EPAC act synergistically to induce neuronal differentiation in N1E-115. Altogether these results show that the changes observed in the differentiation of N1E-115 cells proceed by regulating several microtubule-stabilizing factors, and the acquisition of a neuronal phenotype is a process involving concerted although independent functions of EPAC and PKA.

Exchange Protein Directly Activated by cAMP (EPAC) Regulates Neuronal Polarization through Rap1B.

Acquisition of neuronal polarity is a complex process involving cellular and molecular events. The second messenger cAMP is involved in axonal specification through activation of protein kinase A. However, an alternative cAMP-dependent mechanism involves the exchange protein directly activated by cAMP (EPAC), which also responds to physiological changes in cAMP concentration, promoting activation of the small Rap GTPases. Here, we present evidence that EPAC signaling contributes to axon specification and elongation. In primary rat hippocampal neurons, EPAC isoforms were expressed differentially during axon specification. Furthermore, 8-pCPT, an EPAC pharmacological activator, and genetic manipulations of EPAC in neurons induced supernumerary axons indicative of Rap1b activation. Moreover, 8-pCPT-treated neurons expressed ankyrin G and other markers of mature axons such as synaptophysin and axonal accumulation of vGLUT1. In contrast, pharmacological inhibition of EPAC delayed neuronal polarity. Genetic manipulations to inactivate EPAC1 using either shRNA or neurons derived from EPAC1 knock-out (KO) mice led to axon elongation and polarization defects. Interestingly, multiaxonic neurons generated by 8-pCPT treatments in wild-type neurons were not found in EPAC1 KO mice neurons. Altogether, these results propose that EPAC signaling is an alternative and complementary mechanism for cAMP-dependent axon determination. SIGNIFICANCE STATEMENT: This study identifies the guanine exchange factor responsible for Rap1b activation during neuronal polarization and provides an alternate explanation for cAMP-dependent acquisition of neuronal polarity.

The role of small GTPases in neuronal morphogenesis and polarity.

The highly dynamic remodeling and cross talk of the microtubule and actin cytoskeleton support neuronal morphogenesis. Small RhoGTPases family members have emerged as crucial regulators of cytoskeletal dynamics. In this review we will comprehensively analyze findings that support the participation of RhoA, Rac, Cdc42, and TC10 in different neuronal morphogenetic events ranging from migration to synaptic plasticity. We will specifically address the contribution of these GTPases to support neuronal polarity and axonal elongation.

Effect of the environment on the dendritic morphology of the rat auditory cortex.

The present study aimed to identify morphological correlates of environment-induced changes at excitatory synapses of the primary auditory cortex (A1). We used the Golgi-Cox stain technique to compare pyramidal cells dendritic properties of Sprague-Dawley rats exposed to different environmental manipulations. Sholl analysis, dendritic length measures, and spine density counts were used to monitor the effects of sensory deafness and an auditory version of environmental enrichment (EE). We found that deafness decreased apical dendritic length leaving basal dendritic length unchanged, whereas EE selectively increased basal dendritic length without changing apical dendritic length. On the contrary, deafness decreased while EE increased spine density in both basal and apical dendrites of A1 Layer 2/3 (LII/III) neurons. To determine whether stress contributed to the observed morphological changes in A1, we studied neural morphology in a restraint-induced model that lacked behaviorally relevant acoustic cues. We found that stress selectively decreased apical dendritic length in the auditory but not in the visual primary cortex. Similar to the acoustic manipulation, stress-induced changes in dendritic length possessed a layer-specific pattern displaying LII/III neurons from stressed animals with normal apical dendrites but shorter basal dendrites, while infragranular neurons (Layers V and VI) displayed shorter apical dendrites but normal basal dendrites. The same treatment did not induce similar changes in the visual cortex, demonstrating that the auditory cortex is an exquisitely sensitive target of neocortical plasticity, and that prolonged exposure to different acoustic as well as emotional environmental manipulation may produce specific changes in dendritic shape and spine density.

Chronic stress induces dendritic atrophy in the rat medial geniculate nucleus: effects on auditory conditioning.

Chronic stress induces dendritic atrophy in the inferior colliculus (IC, auditory mesencephalon) and impairs auditory avoidance conditioning. The aim of this study was to determine in Golgi preparations and in cued fear conditioning whether stress affects other auditory components, like the thalamic medial geniculate nucleus (MG) or the posterior thalamic nucleus (PO), in Sprague-Dawley rats. Chronic restraint stress produced a significant dendritic atrophy in the MG (stress: 407+/-55 microm; control: 808+/-120 microm; p<0.01) but did not affect auditory fear conditioning. The last result was in apparent contrast with the fact that stress impairs both the acquisition of auditory avoidance conditioned responses and the dendritic structure in two major nuclei of the auditory system. In order to analyze this disagreement, we investigated whether the stress-related freezing to tone occurring in the fear conditioning protocol corresponded to a conditioned or an unconditioned fear response, using changes in tone instead of light throughout conditioning trials. Chronic stress significantly enhanced visual fear conditioning in stressed animals compared to controls (stress: 58.9+/-8.42%, control: 23.31+/-8.01%; p<0.05), but this fear enhancement was related to unconditioned fear. Conversely, chronic stress did not affect the morphology of the PO (subserving both auditory and somatosensory information) or the corresponding auditory and somatosensory unconditioned responses (acoustic startle response and escape behavior). Our results suggest that the auditory conditioned stimulus can be processed in part independently of the IC and MG in the stressed animals, and sent to the amygdala via the PO inducing unconditioned fear. Comparable alterations could be produced in major depression.