An optimized dosing regimen of cimaglermin (neuregulin 1β3, glial growth factor 2) enhances molecular markers of neuroplasticity and functional recovery after permanent ischemic stroke in rats

Cimaglermin (neuregulin 1β3, glial growth factor 2) is a neuregulin growth factor family member in clinical development for chronic heart failure. Previously, in a permanent middle cerebral artery occlusion (pMCAO) rat stroke model, systemic cimaglermin treatment initiated up to 7 days after ischemia onset promoted recovery without reduced lesion volume. Presented here to extend the evidence are two studies that use a rat stroke model to evaluate the effects of cimaglermin dose level and dose frequency initiated 24 hr after pMCAO. Forelimb‐ and hindlimb‐placing scores (proprioceptive behavioral tests), body‐swing symmetry, and infarct volume were compared between treatment groups (n = 12/group). Possible mechanisms underlying cimaglermin‐mediated neurologic recovery were examined through axonal growth and synapse formation histological markers. Cimaglermin was evaluated over a wider dose range (0.02, 0.1, or 1.0 mg/kg) than doses previously shown to be effective but used the same dosing regimen (2 weeks of daily intravenous administration, then 1 week without treatment). The dose‐frequency study used the dose‐ranging study's most effective dose (1.0 mg/kg) to compare daily, once per week, and twice per week dosing for 3 weeks (then 1 week without treatment). Dose‐ and frequency‐dependent functional improvements were observed with cimaglermin without reduced lesion volume. Cimaglermin treatment significantly increased growth‐associated protein 43 expression in both hemispheres (particularly somatosensory and motor cortices) and also increased synaptophysin expression. These data indicate that cimaglermin enhances recovery after stroke. Immunohistochemical changes were consistent with axonal sprouting and synapse formation but not acute neuroprotection. Cimaglermin represents a potential clinical development candidate for ischemic stroke treatment. © 2015 The Authors. Journal of Neuroscience Research Published by Wiley Periodicals, Inc.     

Basal brain oxidative and nitrative stress levels are finely regulated by the interplay between superoxide dismutase 2 and p53

Superoxide dismutases (SODs) are the primary reactive oxygen species (ROS)‐scavenging enzymes of the cell and catalyze the dismutation of superoxide radicals O₂^– to H₂O₂ and molecular oxygen (O₂). Among the three forms of SOD identified, manganese‐containing SOD (MnSOD, SOD2) is a homotetramer located wholly in the mitochondrial matrix. Because of the SOD2 strategic location, it represents the first mechanism of defense against the augmentation of ROS/reactive nitrogen species levels in the mitochondria for preventing further damage. This study seeks to understand the effects that the partial lack (SOD2^?/+) or the overexpression (TgSOD2) of MnSOD produces on oxidative/nitrative stress basal levels in different brain isolated cellular fractions (i.e., mitochondrial, nuclear, cytosolic) as well as in the whole‐brain homogenate. Furthermore, because of the known interaction between SOD2 and p53 protein, this study seeks to clarify the impact that the double mutation has on oxidative/nitrative stress levels in the brain of mice carrying the double mutation (p53^?/? × SOD2^?/+ and p53^?/? × TgSOD2). We show that each mutation affects mitochondrial, nuclear, and cytosolic oxidative/nitrative stress basal levels differently, but, overall, no change or reduction of oxidative/nitrative stress levels was found in the whole‐brain homogenate. The analysis of well‐known antioxidant systems such as thioredoxin‐1 and Nrf2/HO‐1/BVR‐A suggests their potential role in the maintenance of the cellular redox homeostasis in the presence of changes of SOD2 and/or p53 protein levels. © 2015 Wiley Periodicals, Inc.     

Topographic organization and possible function of the posterior optic tubercles in the brain of the desert locust Schistocerca gregaria

Migrating desert locusts, Schistocerca gregaria, are able to use the skylight polarization pattern for navigation. They detect polarized light with a specialized dorsal rim area in their compound eye. After multistage processing, polarization signals are transferred to the central complex, a midline‐spanning brain area involved in locomotor control. Polarization‐sensitive tangential neurons (TB‐neurons) of the protocerebral bridge, a part of the central complex, give rise to a topographic arrangement of preferred polarization angles in the bridge, suggesting that the central complex acts as an internal sky compass. TB‐neurons connect the protocerebral bridge with two adjacent brain areas, the posterior optic tubercles. To analyze the polarotopic organization of the central complex further, we investigated the number and morphologies of TB‐neurons and the presence and colocalization of three neuroactive substances in these neurons. Triple immunostaining with antisera against Diploptera punctata allatostatin (Dip‐AST), Manduca sexta allatotropin (Mas‐AT), and serotonin (5HT) raised in the same host species revealed three spatially distinct TB‐neuron clusters, each consisting of 10 neurons per hemisphere: cluster 1 and 3 showed Dip‐AST/5HT immunostaining, whereas cluster 2 showed Dip‐AST/Mas‐AT immunostaining. Five subtypes of TB‐neuron could be distinguished based on ramification patterns. Corresponding to ramification domains in the protocerebral bridge, the neurons invaded distinct but overlapping layers within the posterior optic tubercle. Similarly, neurons interconnecting the tubercles of the two hemispheres also targeted distinct layers of these neuropils. From these data we propose a neuronal circuit that may be suited to stabilize the internal sky compass in the central complex of the locust. J. Comp. Neurol. 523:1589–1607, 2015. © 2015 Wiley Periodicals, Inc.     

Dopamine D1 receptor expression is bipolar cell type‐specific in the mouse retina

In the retina, dopamine is a key molecule for daytime vision. Dopamine is released by retinal dopaminergic amacrine cells and transmits signaling either by conventional synaptic or by volume transmission. By means of volume transmission, dopamine modulates all layers of retinal neurons; however, it is not well understood how dopamine modulates visual signaling pathways in bipolar cells. Here we analyzed Drd1a‐tdTomato BAC transgenic mice and found that the dopamine D1 receptor (D1R) is expressed in retinal bipolar cells in a type‐dependent manner. Strong tdTomato fluorescence was detected in the inner nuclear layer and localized to type 1, 3b, and 4 OFF bipolar cells and type 5‐2, XBC, 6, and 7 ON bipolar cells. In contrast, type 2, 3a, 5‐1, 9, and rod bipolar cells did not express Drd1a‐tdTomato. Other interneurons were also found to express tdTomato including horizontal cells and a subset (25%) of AII amacrine cells. Diverse visual processing pathways, such as color or motion‐coded pathways, are thought to be initiated in retinal bipolar cells. Our results indicate that dopamine sculpts bipolar cell performance in a type‐dependent manner to facilitate daytime vision. J. Comp. Neurol. 524:2059–2079, 2016. © 2015 Wiley Periodicals, Inc.     

Distribution of the SynDIG4/proline‐rich transmembrane protein 1 in rat brain

The modulation of AMPA receptor (AMPAR) content at synapses is thought to be an underlying molecular mechanism of memory and learning. AMPAR content at synapses is highly plastic and is regulated by numerous AMPAR accessory transmembrane proteins such as TARPs, cornichons, and CKAMPs. SynDIG (synapse differentiation‐induced gene) defines a family of four genes (SynDIG1–4) expressed in distinct and overlapping patterns in the brain. SynDIG1 was previously identified as a novel transmembrane AMPAR‐associated protein that regulates synaptic strength. The related protein SynDIG4 [also known as Prrt1 (proline‐rich transmembrane protein 1)] has recently been identified as a component of AMPAR complexes. In this study, we show that SynDIG1 and SynDIG4 have distinct yet overlapping patterns of expression in the central nervous system, with SynDIG4 having especially prominent expression in the hippocampus and particularly within CA1. In contrast to SynDIG1 and other traditional AMPAR auxiliary subunits, SynDIG4 is de‐enriched at the postsynaptic density and colocalizes with extrasynaptic GluA1 puncta in primary dissociated neuron culture. These results indicate that, although SynDIG4 shares sequence similarity with SynDIG1, it might act through a unique mechanism as an auxiliary factor for extrasynaptic GluA1‐containing AMPARs. J. Comp. Neurol. 524:2266–2280, 2016. © 2015 Wiley Periodicals, Inc.     

Distinct subcellular localization of alternative splicing variants of EFA6D,a guanine nucleotide exchange factor for Arf6, in the mouse brain

EFA6D (guanine nucleotide exchange factor for ADP‐ribosylation factor 6 [Arf6]D) is also known as EFA6R, Psd3, and HCA67. It is the fourth member of the EFA6 family with guanine nucleotide exchange activity for Arf6, a small guanosine triphosphatase (GTPase) that regulates endosomal trafficking and actin cytoskeleton remodeling. We propose a classification and nomenclature of 10 EFA6D variants deposited in the GenBank database as EFA6D1a, 1b, 1c, 1d, 1s, 2a, 2b, 2c, 2d, and 2s based on the combination of N‐terminal and C‐terminal insertions. Polymerase chain reaction analysis showed the expression of all EFA6D variants except for variants a and d in the adult mouse brain. Immunoblotting analysis with novel variant‐specific antibodies showed the endogenous expression of EFA6D1b, EFA6D1c, and EFA6D1s at the protein level, with the highest expression being EFA6D1s, in the brain. Immunoblotting analysis of forebrain subcellular fractions showed the distinct subcellular distribution of EFA6D1b/c and EFA6D1s. The immunohistochemical analysis revealed distinct but overlapping immunoreactive patterns between EFA6D1b/c and EFA6D1s in the mouse brain. In immunoelectron microscopic analyses of the hippocampal CA3 region, EFA6D1b/c was present predominantly in the mossy fiber axons of dentate granule cells, whereas EFA6D1s was present abundantly in the cell bodies, dendritic shafts, and spines of hippocampal pyramidal cells. These results provide the first anatomical evidence suggesting the functional diversity of EFA6D variants, particularly EFA6D1b/c and EFA6D1s, in neurons. J. Comp. Neurol. 524:2531–2552, 2016. © 2016 Wiley Periodicals, Inc.     

Coexpression of auxiliary subunits KChIP and DPPL in potassium channel Kv4‐positive nociceptors and pain‐modulating spinal interneurons

Subthreshold A‐type K⁺ currents (I_SAs) have been recorded from the somata of nociceptors and spinal lamina II excitatory interneurons, which sense and modulate pain, respectively. Kv4 channels are responsible for the somatodendritic I_SAs. Accumulative evidence suggests that neuronal Kv4 channels are ternary complexes including pore‐forming Kv4 subunits and two types of auxiliary subunits: K⁺ channel‐interacting proteins (KChIPs) and dipeptidyl peptidase‐like proteins (DPPLs). Previous reports have shown Kv4.3 in a subset of nonpeptidergic nociceptors and Kv4.2/Kv4.3 in certain spinal lamina II excitatory interneurons. However, whether and which KChIP and DPPL are coexpressed with Kv4 in these I_SA‐expressing pain‐related neurons is unknown. In this study we mapped the protein distribution of KChIP1, KChIP2, KChIP3, DPP6, and DPP10 in adult rat dorsal root ganglion (DRG) and spinal cord by immunohistochemistry. In the DRG, we found colocalization of KChIP1, KChIP2, and DPP10 in the somatic surface and cytoplasm of Kv4.3(+) nociceptors. KChIP3 appears in most Aβ and Aδ sensory neurons as well as a small population of peptidergic nociceptors, whereas DPP6 is absent in sensory neurons. In the spinal cord, KChIP1 is coexpressed with Kv4.3 in the cell bodies of a subset of lamina II excitatory interneurons, while KChIP1, KChIP2, and DPP6 are colocalized with Kv4.2 and Kv4.3 in their dendrites. Within the dorsal horn, besides KChIP3 in the inner lamina II and lamina III, we detected DPP10 in most projection neurons, which transmit pain signal to brain. The results suggest the existence of Kv4/KChIP/DPPL ternary complexes in I_SA‐expressing nociceptors and pain‐modulating spinal interneurons. J. Comp. Neurol. 524:846–873, 2016. © 2015 Wiley Periodicals, Inc.     

Micro‐RNA‐30a regulates ischemia‐induced cell death by targeting heat shock protein HSPA5 in primary cultured cortical neurons and mouse brain after stroke

Micro‐RNAs (miRs) have emerged as key gene regulators in many diseases, including stroke. We recently reported that miR‐30a protects N2A cells against ischemic injury, in part through enhancing beclin 1‐mediated autophagy. The present study explores further the involvement of miR‐30a in ischemia‐induced apoptosis and its possible mechanisms in primary cortical neurons and stroked mouse brain. We demonstrate that miR‐30a level is significantly decreased in cortical neurons after 1‐hr oxygen–glucose deprivation (OGD)/24‐hr reoxygenation. Overexpression of miR‐30a aggravated the OGD‐induced neuronal cell death, whereas inhibition of miR‐30a attenuated necrosis and apoptosis as determined by 3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐di‐phenyl‐2H‐tetrazolium bromide, lactate dehydrogenase, TUNEL, and cleaved caspase‐3. The amount of HSPA5 protein, which is predicted to be a putative target of miR‐30a by TargetScan, could be reduced by pre‐miR‐30a, whereas it was increased by anti‐miR‐30a. Furthermore, the luciferase reporter assay confirmed that miR‐30a directly binds to the predicted 3′‐UTR target sites of the hspa5 gene. The cell injury regulated by miR‐30a in OGD‐treated cells could be aggravated by HSPA5 siRNA. We also observed an interaction of HSPA5 and caspase‐12 by coimmunoprecipitation and speculate that HSPA5 might be involved in endoplasmic reticulum stress‐induced apoptosis. In vivo, reduced miR‐30a increased the HSPA5 level and attenuated ischemic brain infarction in focal ischemia‐stroked mice. Downregulation of miR‐30a could prevent neural ischemic injury through upregulating HSPA5 protein expression, and decreased ER stress‐induced apoptosis might be one of the mechanisms underlying HSPA5‐mediated neuroprotection. © 2015 Wiley Periodicals, Inc.     

Neurochemistry of neurons in the ventrolateral medulla activated by hypotension: Are the same neurons activated by glucoprivation?

Previous studies have demonstrated that a range of stimuli activate neurons, including catecholaminergic neurons, in the ventrolateral medulla. Not all catecholaminergic neurons are activated and other neurochemical content is largely unknown hence whether stimulus specific populations exist is unclear. Here we determine the neurochemistry (using in situ hybridization) of catecholaminergic and noncatecholaminergic neurons which express c‐Fos immunoreactivity throughout the rostrocaudal extent of the ventrolateral medulla, in Sprague Dawley rats treated with hydralazine or saline. Distinct neuronal populations containing PPCART, PPPACAP, and PPNPY mRNAs, which were largely catecholaminergic, were activated by hydralazine but not saline. Both catecholaminergic and noncatecholaminergic neurons containing preprotachykinin and prepro‐enkephalin (PPE) mRNAs were also activated, with the noncatecholaminergic population located in the rostral C1 region. Few GlyT2 neurons were activated. A subset of these data was then used to compare the neuronal populations activated by 2‐deoxyglucose evoked glucoprivation (Brain Structure and Function (2015) 220:117). Hydralazine activated more neurons than 2‐deoxyglucose but similar numbers of catecholaminergic neurons. Commonly activated populations expressing PPNPY and PPE mRNAs were defined. These likely include PPNPY expressing catecholaminergic neurons projecting to vasopressinergic and corticotrophin releasing factor neurons in the paraventricular nucleus, which when activated result in elevated plasma vasopressin and corticosterone. Stimulus specific neurons included noncatecholaminergic neurons and a few PPE positive catecholaminergic neuron but neurochemical codes were largely unidentified. Reasons for the lack of identification of stimulus specific neurons, readily detectable using electrophysiology in anaesthetized preparations and for which neural circuits can be defined, are discussed.     

The gyri of the octopus vertical lobe have distinct neurochemical identities

The cephalopod vertical lobe is the largest learning and memory structure known in invertebrate nervous systems. It is part of the visual learning circuit of the central brain, which also includes the superior frontal and subvertical lobes. Despite the well‐established functional importance of this system, little is known about neuropil organization of these structures and there is to date no evidence that the five longitudinal gyri of the vertical lobe, perhaps the most distinctive morphological feature of the octopus brain, differ in their connections or molecular identities. We studied the histochemical organization of these structures in hatchling and adult Octopus bimaculoides brains with immunostaining for serotonin, octopus gonadotropin‐releasing hormone (oGNRH), and octopressin‐neurophysin (OP‐NP). Our major finding is that the five lobules forming the vertical lobe gyri have distinct neurochemical signatures. This is most prominent in the hatchling brain, where the median and mediolateral lobules are enriched in OP‐NP fibers, the lateral lobule is marked by oGNRH innervation, and serotonin immunostaining heavily labels the median and lateral lobules. A major source of input to the vertical lobe is the superior frontal lobe, which is dominated by a neuropil of interweaving fiber bundles. We have found that this neuropil also has an intrinsic neurochemical organization: it is partitioned into territories alternately enriched or impoverished in oGNRH‐containing fascicles. Our findings establish that the constituent lobes of the octopus superior frontal–vertical system have an intricate internal anatomy, one likely to reflect the presence of functional subsystems within cephalopod learning circuitry. J. Comp. Neurol. 523:1297–1317, 2015. © 2015 Wiley Periodicals, Inc.     

Brain region‐dependent differential expression of alpha‐synuclein

α‐Synuclein, the major constituent of Lewy bodies (LBs), is normally expressed in presynapses and is involved in synaptic function. Abnormal intracellular aggregation of α‐synuclein is observed as LBs and Lewy neurites in neurodegenerative disorders, such as Parkinson's disease (PD) or dementia with Lewy bodies. Accumulated evidence suggests that abundant intracellular expression of α‐synuclein is one of the risk factors for pathological aggregation. Recently, we reported differential expression patterns of α‐synuclein between excitatory and inhibitory hippocampal neurons. Here we further investigated the precise expression profile in the adult mouse brain with special reference to vulnerable regions along the progression of idiopathic PD. The results show that α‐synuclein was highly expressed in the neuronal cell bodies of some early PD‐affected brain regions, such as the olfactory bulb, dorsal motor nucleus of the vagus, and substantia nigra pars compacta. Synaptic expression of α‐synuclein was mostly accompanied by expression of vesicular glutamate transporter‐1, an excitatory presynaptic marker. In contrast, expression of α‐synuclein in the GABAergic inhibitory synapses was different among brain regions. α‐Synuclein was clearly expressed in inhibitory synapses in the external plexiform layer of the olfactory bulb, globus pallidus, and substantia nigra pars reticulata, but not in the cerebral cortex, subthalamic nucleus, or thalamus. These results suggest that some neurons in early PD‐affected human brain regions express high levels of perikaryal α‐synuclein, as happens in the mouse brain. Additionally, synaptic profiles expressing α‐synuclein are different in various brain regions. J. Comp. Neurol. 524:1236–1258, 2016. © 2015 Wiley Periodicals, Inc.     

Executive function deficits and glutamatergic protein alterations in a progressive 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine mouse model of Parkinson's disease

Changes in executive function are at the root of most cognitive problems associated with Parkinson's disease. Because dopaminergic treatment does not necessarily alleviate deficits in executive function, it has been hypothesized that dysfunction of neurotransmitters/systems other than dopamine (DA) may be associated with this decrease in cognitive function. We have reported decreases in motor function and dopaminergic/glutamatergic biomarkers in a progressive 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) Parkinson's mouse model. Assessment of executive function and dopaminergic/glutamatergic biomarkers within the limbic circuit has not previously been explored in our model. Our results show progressive behavioral decline in a cued response task (a rodent model for frontal cortex cognitive function) with increasing weekly doses of MPTP. Although within the dorsolateral (DL) striatum mice that had been given MPTP showed a 63% and 83% loss of tyrosine hydroxylase and dopamine transporter expression, respectively, there were no changes in the nucleus accumbens or medial prefrontal cortex (mPFC). Furthermore, dopamine‐1 receptor and vesicular glutamate transporter (VGLUT)?1 expression increased in the mPFC following DA loss. There were significant MPTP‐induced decreases and increases in VGLUT‐1 and VGLUT‐2 expression, respectively, within the DL striatum. We propose that the behavioral decline following MPTP treatment may be associated with a change not only in cortical–cortical (VGLUT‐1) glutamate function but also in striatal DA and glutamate (VGLUT‐1/VGLUT‐2) input. © 2015 Wiley Periodicals, Inc.     

Cell‐specific and developmental expression of lectican‐cleaving proteases in mouse hippocampus and neocortex

Mounting evidence has demonstrated that a specialized extracellular matrix exists in the mammalian brain and that this glycoprotein‐rich matrix contributes to many aspects of brain development and function. The most prominent supramolecular assemblies of these extracellular matrix glycoproteins are perineuronal nets, specialized lattice‐like structures that surround the cell bodies and proximal neurites of select classes of interneurons. Perineuronal nets are composed of lecticans, a family of chondroitin sulfate proteoglycans that includes aggrecan, brevican, neurocan, and versican. These lattice‐like structures emerge late in postnatal brain development, coinciding with the ending of critical periods of brain development. Despite our knowledge of the presence of lecticans in perineuronal nets and their importance in regulating synaptic plasticity, we know little about the development or distribution of the extracellular proteases that are responsible for their cleavage and turnover. A subset of a large family of extracellular proteases (called a disintegrin and metalloproteinase with thrombospondin motifs [ADAMTS]) is responsible for endogenously cleaving lecticans. We therefore explored the expression pattern of two aggrecan‐degrading ADAMTS family members, ADAMTS15 and ADAMTS4, in the hippocampus and neocortex. Here, we show that both lectican‐degrading metalloproteases are present in these brain regions and that each exhibits a distinct temporal and spatial expression pattern. Adamts15 mRNA is expressed exclusively by parvalbumin‐expressing interneurons during synaptogenesis, whereas Adamts4 mRNA is exclusively generated by telencephalic oligodendrocytes during myelination. Thus, ADAMTS15 and ADAMTS4 not only exhibit unique cellular expression patterns but their developmental upregulation by these cell types coincides with critical aspects of neural development. J. Comp. Neurol. 523:629–648, 2015. © 2014 Wiley Periodicals, Inc.     

A comparative examination of neural circuit and brain patterning between the lamprey and amphioxus reveals the evolutionary origin of the vertebrate visual center

Vertebrates are equipped with so‐called camera eyes, which provide them with image‐forming vision. Vertebrate image‐forming vision evolved independently from that of other animals and is regarded as a key innovation for enhancing predatory ability and ecological success. Evolutionary changes in the neural circuits, particularly the visual center, were central for the acquisition of image‐forming vision. However, the evolutionary steps, from protochordates to jaw‐less primitive vertebrates and then to jawed vertebrates, remain largely unknown. To bridge this gap, we present the detailed development of retinofugal projections in the lamprey, the neuroarchitecture in amphioxus, and the brain patterning in both animals. Both the lateral eye in larval lamprey and the frontal eye in amphioxus project to a light‐detecting visual center in the caudal prosencephalic region marked by Pax6, which possibly represents the ancestral state of the chordate visual system. Our results indicate that the visual system of the larval lamprey represents an evolutionarily primitive state, forming a link from protochordates to vertebrates and providing a new perspective of brain evolution based on developmental mechanisms and neural functions. J. Comp. Neurol. 523:251–261, 2015. © 2014 Wiley Periodicals, Inc.     

Postsynaptic gephyrin clustering controls the development of adult‐born granule cells in the olfactory bulb

In adult rodent olfactory bulb, GABAergic signaling regulates migration, differentiation, and synaptic integration of newborn granule cells (GCs), migrating from the subventricular zone. Here we show that these effects depend on the formation of a postsynaptic scaffold organized by gephyrin—the main scaffolding protein of GABAergic synapses, which anchors receptors and signaling molecules to the postsynaptic density—and are regulated by the phosphorylation status of gephyrin. Using lentiviral vectors to selectively transfect adult‐born GCs, we observed that overexpression of the phospho‐deficient gephyrin mutant eGFP‐gephyrin(S270A), which facilitates the formation of supernumerary GABAergic synapses in vitro, favors dendritic branching and the formation of transient GABAergic synapses on spines, identified by the presence of α2‐GABA_ARs. In contrast, overexpression of the dominant‐negative eGFP‐gephyrin(L2B) (a chimera that is enzymatically active but clustering defective), curtailed dendritic growth, spine formation, and long‐term survival of GCs, pointing to the essential role of gephyrin cluster formation for its function. We could exclude any gephyrin overexpression artifacts, as GCs infected with eGFP‐gephyrin were comparable to those infected with eGFP alone. The opposite effects induced by the two gephyrin mutant constructs indicate that the gephyrin scaffold at GABAergic synapses orchestrates signaling cascades acting on the cytoskeleton to regulate neuronal growth and synapse formation. Specifically, gephyrin phosphorylation emerges as a novel mechanism regulating morphological differentiation and long‐term survival of adult‐born olfactory bulb neurons. J. Comp. Neurol. 523:1998–2016, 2015 © 2015 Wiley Periodicals, Inc.     

Neuroanatomical details of the lateral neurons of Drosophila melanogaster support their functional role in the circadian system

Drosophila melanogaster is a long‐standing model organism in the circadian clock research. A major advantage is the relative small number of about 150 neurons, which built the circadian clock in Drosophila. In our recent work, we focused on the neuroanatomical properties of the lateral neurons of the clock network. By applying the multicolor‐labeling technique Flybow we were able to identify the anatomical similarity of the previously described E2 subunit of the evening oscillator of the clock, which is built by the 5th small ventrolateral neuron (5th s‐LN_v) and one ITP positive dorsolateral neuron (LN_d). These two clock neurons share the same spatial and functional properties. We found both neurons innervating the same brain areas with similar pre‐ and postsynaptic sites in the brain. Here the anatomical findings support their shared function as a main evening oscillator in the clock network like also found in previous studies. A second quite surprising finding addresses the large lateral ventral PDF‐neurons (l‐LN_vs). We could show that the four hardly distinguishable l‐LN_vs consist of two subgroups with different innervation patterns. While three of the neurons reflect the well‐known branching pattern reproduced by PDF immunohistochemistry, one neuron per brain hemisphere has a distinguished innervation profile and is restricted only to the proximal part of the medulla‐surface. We named this neuron “extra” l‐LN_v (l‐LN_vx). We suggest the anatomical findings reflect different functional properties of the two l‐LN_v subgroups.     

mfat‐1 transgene protects cultured adult neural stem cells against cobalt chloride‐mediated hypoxic injury by activating Nrf2/ARE pathways

Ischemic stroke is a devastating neurological disorder and one of the leading causes of death and serious disability in adults. Adult neural stem cell (NSC) replacement therapy is a promising treatment for both structural and functional neurological recovery. However, for the treatment to work, adult NSCs must be protected against hypoxic‐ischemic damage in the ischemic penumbra. In the present study, we aimed to investigate the neuroprotective effects of the mfat‐1 transgene on cobalt chloride (CoCl₂)‐induced hypoxic‐ischemic injury in cultured adult NSCs as well as its underlying mechanisms. The results show that in the CoCl₂‐induced hypoxic‐ischemic injury model, the mfat‐1 transgene enhanced the viability of adult NSCs and suppressed CoCl₂‐mediated apoptosis of adult NSCs. Additionally, the mfat‐1 transgene promoted the proliferation of NSCs as shown by increased bromodeoxyuridine labeling of adult NSCs. This process was related to the reduction of reactive oxygen species. Quantitative real‐time polymerase chain reaction and Western blot analysis revealed a much higher expression of nuclear factor erythroid 2‐related factor 2 (Nrf2) and its downstream genes (HO‐1, NQO‐1, GCLC). Taken together, our findings show that the mfat‐1 transgene restored the CoCl₂‐inhibited viability and proliferation of NSCs by activating nuclear factor erythroid 2‐related factor 2 (Nrf2)/antioxidant response elements (ARE) signal pathway to inhibit oxidative stress injury. Further investigation of the function of the mfat‐1 transgene in adult protective mechanisms may accelerate the development of adult NSC replacement therapy for ischemic stroke.     

Expression profile of the STAND protein Nwd1 in the developing and mature mouse central nervous system

The orchestrated events required during brain development, as well as the maintenance of adult neuronal plasticity, highly depend on the accurate responses of neuronal cells to various cellular stress or environmental stimuli. Recent studies have defined a previously unrecognized, broad class of multidomain proteins, designated as signal transduction ATPases with numerous domains (STAND), which comprises a large number of proteins, including the apoptotic peptidase activating factor 1 (Apaf1) and nucleotide‐binding oligomerization domain‐like receptors (NLRs), central players in cell death and innate immune responses, respectively. Although the involvement of STANDs in the central nervous system (CNS) has been postulated in terms of neuronal development and function, it remains largely unclear. Here, we identified Nwd1 (NACHT and WD repeat domain‐containing protein 1), as a novel STAND protein, expressed in neural stem/progenitor cells (NSPCs). Structurally, Nwd1 was most analogous to the apoptosis regulator Apaf1, also involved in mitosis and axonal outgrowth regulation in the CNS. Using a specific antibody, we show that, during the embryonic and postnatal period, Nwd1 is expressed in nestin‐positive NSPCs in vivo and in vitro, while postnatally it is found in terminally differentiated neurons and blood vessels. At the subcellular level, we demonstrate that Nwd1 is preferentially located in the cytosolic compartment of cultured NSPCs, partially overlapping with cytochrome c. These observations imply that Nwd1 might be involved in the neuronal lineage as a new STAND gene, including having a pro‐apoptotic or nonapoptotic role, similar to Apaf1.     

Pigment epithelium‐derived factor (PEDF) and PEDF‐receptor in the adult mouse brain: Differential spatial/temporal localization pattern

Pigment epithelium‐derived factor (PEDF) is a multifunctional protein which was initially described in the retina, although it is also present in other tissues. It functions as an antioxidant agent promoting neuronal survival. Recently, a PEDF receptor has shown an elevated binding affinity for PEDF. There are no relevant data regarding the distribution of both proteins in the brain, therefore the main goal of this work was to investigate the spatiotemporal presence of PEDF and PEDFR in the adult mouse brain, and to determine the PEDF blood level in mouse and human. The localization of both proteins was analyzed by different experimental methods such as immunohistochemistry, western‐blotting, and also by enzyme‐linked immunosorbent assay. Differential expression was found in some telencephalic structures and positive signals for both proteins were detected in the cerebellum. The magnitude of the PEDFR labeling pattern was higher than PEDF and included some cortical and subventricular areas. Age‐dependent changes in intensity of both protein immunoreactions were found in the cortical and hippocampal areas with greater reactivity between 4 and 8 months of age, whilst others, like the subventricular zones, these differences were more evident for PEDFR. Although ubiquitous presence was not found in the brain for these two proteins, their relevant functions must not be underestimated. It has been described that PEDF plays an important role in neuroprotection and data provided in the present work represents the first extensive study to understand the relevance of these two proteins in specific brain areas.