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1.  Roles of Wnt/β-Catenin Signaling in Retinal Neuron-Like Differentiation of Bone Marrow Mesenchymal Stem Cells from Nonobese Diabetic Mice 
Recent studies have shown that mesenchymal stem cells (MSCs) are expected to become promising therapeutic agents for the treatment of diabetic retinopathy (DR); moreover, we previously demonstrated that bone marrow (BM)-MSCs from nonobese diabetic (NOD) mice (an ideal DR model) had abnormal migration and adhesion. So, we hypothesized that NOD-MSCs also have abnormal retinal neuron-like differentiation potential. MSCs were cultured with brain-derived neurotrophic factor, nerve growth factor, and basic fibroblast growth factor. Western blot analysis and immunofluorescence both showed that the level of retinal neuron-like markers, such as glial fibrillary acidic protein, neuron-specific nuclear protein, tyrosine hydroxy-lase, Thy-1, glutamine synthetase, and rhodopsin was lower in NOD-MSCs than in imprinting control region MSCs. Furthermore, we explored the precise mechanisms controlling this change in NOD-MSCs. The expression levels of some important member proteins in Wnt/β-catenin signaling were determined and suggested the downregulation of Wnt/β-catenin signaling with retinal neuron-like differentiation of NOD-MSCs. Incubation of NOD-MSCs in medium supplemented with human recombinant Wnt1 resulted in a significant upregulation of retinal neuron-like markers, and the effects of Wnt1 were dose-dependent. Taken together, our study indicated that the inhibition of Wnt/β-catenin signaling in NOD-MSCs after induction could contribute to the abnormal retinal neuron-like differentiation. These data provide important preclinical references supporting the basis for further development of autologous MSC-based therapies for DR.
doi:10.1007/s12031-012-9917-z
PMCID: PMC4242726  PMID: 23229835
BM-MSCs; Diabetic retinopathy; NOD mice; Wnt/β-catenin signaling; Differentiation
2.  Neuronal Lifespan versus Healthspan: Principles of Natural Selection at Work in the Degenerating Brain 
Impaired nutrient delivery to the brain due to decreased blood flow contributes to cognitive decline and dementia in Alzheimer’s disease (AD). Considering this, many studies have suggested that neuroprotective agents like those used in stroke could prevent AD onset or progression by promoting cell survival. However, research in the past decade suggests that the culprit behind the cognitive loss in AD models is actually the soluble tau accumulating inside of surviving neurons. In fact, tau reductions improve cognition in mouse models of AD, even those that only deposit amyloid plaques. There is emerging evidence that neuroprotection alone in these AD models may be insufficient to restore neuron function and cognition. Only when soluble tau is reduced on a neuroprotective background could memory be rescued. Thus, once a neuron begins to accumulate tau, it may survive in a malfunctioning capacity, leading to impaired electrical signaling and memory formation in the brain. These data imply that multiple drugs may be necessary to ameliorate the different disease components. In fact, strategies to preserve neurons without affecting the soluble protein burden within neurons may accelerate the disease course.
doi:10.1007/s12031-011-9540-4
PMCID: PMC4235992  PMID: 21559875
3.  Ceftriaxone treatment affects the levels of GLT1 and ENT1 as well as ethanol intake in alcohol-preferring rats 
Studies have demonstrated that deletion of equilibrative nucleoside transporter 1 (ENT1) is associated with reduced glutamate transporter 1 (GLT1) level, and consequently increased ethanol intake. In this study, we measured changes in GLT1 and ENT1 levels in prefrontal cortex (PFC), and nucleus accumbens (NAc) core and shell associated with alcohol drinking in alcohol-preferring (P) rats. We examined then whether ceftriaxone (CEF) would affect both GLT1 and ENT1 levels in these brain regions. P rats were given 24-hour concurrent access to 15% and 30% ethanol, water, and food for five weeks. On Week 6, P rats received 100 mg/kg CEF (i.p.) or a saline vehicle for five consecutive days. Ethanol intake was measured daily for 8 days starting on the first day of injections. We found a significant reduction in daily ethanol intake in CEF treated group, starting on day 2 of injections. Western blot for GLT1 and binding assay for ENT1 revealed downregulation of GLT1 level, whereas ENT1 levels were increased in the NAc core and NAc shell, respectively, but not in the PFC in saline vehicle group. Importantly, CEF treatment reversed these effects in both NAc core and shell. These findings provide evidence for potential regulatory effects of CEF on both GLT1 and ENT1 expression in reducing ethanol intake.
doi:10.1007/s12031-013-0064-y
PMCID: PMC3797852  PMID: 23893122
ENT1; GLT1; EAAT2; alcohol dependence; glutamate
4.  Hypersomnolence and reduced activity in pan-leptin receptor knockout mice 
Excessive obesity correlates with hypersomnolence and impaired cognitive function, presumably induced by metabolic factors and cytokines. Production of the adipokine leptin correlates with the amount of adiposity, and leptin has been shown to promote sleep. To determine whether leptin plays a major role in the hypersomnolence of obesity, we measured sleep architecture in pan-leptin receptor knockout (POKO) mice that do not respond to leptin because of the production of a mutant, non-signaling receptor. The obese POKO mice had more non-rapid eye movement (NREM) sleep and less waking time than their littermate controls. This was mainly seen during the light span, although increased bouts of rapid eye movement (REM) sleep were also seen in the dark span. The increase of NREM sleep correlated with the extent of obesity. The POKO mice also had decreased locomotor activity and more immobility in the open field test, but there was no increase of forced immobility nor reduction of sucrose intake as would be seen in depression. The increased NREM sleep and reduced locomotor activity in the POKO mice suggest that it was obesity, rather than leptin signaling, that played a predominant role in altering sleep architecture and activity.
doi:10.1007/s12031-013-0093-6
PMCID: PMC3797855  PMID: 23955775
Leptin; Activity; Sleep; Obesity
5.  Age and Dark Rearing Bidirectionally Regulate the Level and Laminar Pattern of Expression of Abelson Interacting Protein 2 (Abi-2), a Novel Candidate Visual Cortical Plasticity Gene 
Electrophysiological studies indicate that cat visual cortical critical period neuronal plasticity peaks around 5 weeks and largely disappears by 20 weeks. Dark rearing slows this time course. Normal cats are more plastic than dark reared cats at 5 weeks but the opposite is true at 20 weeks. Thus, a stringent criterion for identifying genes controlling neuronal plasticity is that normal and dark rearing produce opposite direction differences in expression between young and older animals. Differential display PCR identified Abelson interacting protein 2 (Abi-2) as a candidate plasticity gene regulated according to this criterion. Western blotting showed bidirectional regulation of Abi-2 protein levels in cats and mice that was specific to visual cortex and did not occur in frontal cortex. Immunohistochemistry indicated developmental changes in Abi-2 laminar expression in cat visual cortex. Dark rearing altered laminar expression such that at 5 weeks dark reared cats were similar to 1 week normally reared cats and at 20 weeks, dark reared cats were similar to 5–10 week normally reared animals. The effect of dark rearing on both Abi-2 expression levels and laminar expression patterns was to slow the normal developmental process, the same effect seen on physiologically assessed plasticity in visual cortex.
doi:10.1007/s12031-013-0037-1
PMCID: PMC3797860  PMID: 23828391
Visual Deprivation; Visual Development; differential display PCR; Neuronal Plasticity
6.  Neural cell adhesion molecule L1 modulates type I but not type II inner ear spiral ganglion neurite outgrowth in an in vitro alternate choice assay 
Journal of molecular neuroscience : MN  2013;51(3):10.1007/s12031-013-0040-6.
L1, a neural cell adhesion molecule of the immunoglobulin superfamily, is widely expressed in the nervous system and important in axonal outgrowth, guidance, synapse formation, and signaling. Gene deletion studies emphasize the significance of L1 during development of the central nervous system and L1 is crucial for the topographic targeting of retinal axons.
In contrast to the brain and retina, the role of L1 in the inner ear is largely unknown. While previous studies have localized L1 in the developing inner ear of the chicken and mouse, its function during the innervation of the cochlea still remains largely unclear. We therefore investigated the functional role of L1 in the mammalian inner ear. Our aim was to determine whether or not L1 can modulate type I and/or type II spiral ganglion neuron outgrowth using an in vitro alternate choice assay. We found that L1, presented in stripe micropatterns, provide directional cues to neonatal rodent type I but not type II inner ear spiral ganglion neurites.
The results suggest that L1 may play a role in axonal pathfinding of type I spiral ganglion dendrites toward their inner hair cell targets, but not of type II toward the outer hair cells.
doi:10.1007/s12031-013-0040-6
PMCID: PMC3825176  PMID: 23760987
axonal guidance; axonal outgrowth; inner ear; L1; spiral ganglion
7.  α-Synuclein overexpression represses 14-3-3θ transcription 
Journal of molecular neuroscience : MN  2013;51(3):10.1007/s12031-013-0086-5.
Previous gene microarray studies have shown that expression of 14-3-3θ is significantly decreased in an α-synuclein transgenic mouse model. In this study, we tested whether α-synuclein can regulate 14-3-3θ transcription. We demonstrate that 14-3-3θ mRNA level is decreased in SH-SY5Y cells overexpressing α-synuclein. Luciferase activity under control of the 14-3-3θ promoter is reduced both in stable SH-SY5Y cells constitutively overexpressing α-synuclein and in doxycycline-inducible SH-SY5Y cells upon α-synuclein induction, suggesting that the regulation of 14-3-3θ by α-synuclein occurs at the transcriptional level. Knockdown of α-synuclein by RNA interference does not increase the 14-3-3θ mRNA level. These findings suggest that α-synuclein represses 14-3-3θ transcription under pathologic conditions but that regulation of 14-3-3θ expression is not a function of endogenous α-synuclein at baseline.
doi:10.1007/s12031-013-0086-5
PMCID: PMC3809755  PMID: 23912650
α-synuclein; 14-3-3s; transcription regulation; histone deacetylation
8.  Diagnostic and Therapeutic Potential of Tetanus Toxin-Derivatives in Neurological Diseases 
Journal of molecular neuroscience : MN  2013;51(3):10.1007/s12031-013-0065-x.
We assessed the ex vivo reactivity of peptidic constructs of Tet1 (analog of tetanus toxin non-virulent C fragment) with sequence homology to the cysteine-active site of thioredoxin (Tet1THO) or tetralysine (Tet1PLYS) with oxidative species or axonopathic sodium cyanate (NaOCN), respectively. We then assessed their neuronal uptake in vivo in laboratory animals. The reactivity of Tet1PLYS with NaOCN (1:2.5 to 1:37.5 molar ratios) or Tet1THO with hydrogen peroxide (1:0.4 to 1:6.2 molar ratios) was assessed by mass spectrometry. Green fluorescence protein (GFP)-tagged Tet1-derivatives (3-mg/ml in artificial cerebrospinal fluid) were administered daily to rats by intramuscular injection in latissimus dorsi at lumborum at the dose of 1 µl/g of body weight, for 3 days. Motor neuron uptake was assessed after double immunolabeling for GFP and choline acetyltransferase. Mass spectrometry analysis successfully demonstrated the ex vivo reactivity of Tet1-derivatives in a concentration-dependent manner. Confocal microscopy revealed the localization of Tet1-derivatives in axons and motor neuron cell bodies. Intramuscular delivery of Tet1-derivatives appears to be a practical approach to circumvent the blood nerve barrier and selectively deliver small molecules to the nervous system, for diagnostic and/or treatment purposes.
doi:10.1007/s12031-013-0065-x
PMCID: PMC3838929  PMID: 23842888
Axonal transport; Blood nerve barrier; Drug delivery; Tetanus toxin; Neurotoxicity; Neurodegeneration
9.  Saturable Leptin Transport Across the BBB Persists in EAE Mice 
We have shown that mice with experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis, have upregulated leptin receptor expression in reactive astrocytes of the hippocampus, a region involved in sickness behavior. Leptin can exacerbate EAE when its serum concentration is high. Although leptin receptors in astrocytes modulate leptin transport across cultured endothelial cell monolayers, it is not known how leptin transport in EAE mice is regulated. Here, we determined brain and cervical spinal cord uptake of leptin in early and recovery stages of EAE, after either intravenous delivery or in situ brain perfusion of 125I-leptin and the vascular marker 131I-albumin. While increased vascular space and general blood–brain barrier (BBB) permeability after EAE were expected, the specific saturable transport system for leptin crossing the BBB also persisted. Moreover, there was upregulation of leptin transport in hippocampus and cervical spinal cord in the early stage of EAE, shown by higher leptin uptake in these regions and by competitive inhibition with coadministered excess unlabeled leptin. We conclude that EAE induced a time- and region-specific increase of leptin transport. The results provide a link between circulating leptin and enhanced leptin signaling that may play a crucial role in disease progression.
doi:10.1007/s12031-013-9993-8
PMCID: PMC3726568  PMID: 23504255
Leptin; EAE; Blood—brain barrier; Transport; Hippocampus
10.  INCREASED TRPV4 EXPRESSION IN URINARY BLADDER AND LUMBOSACRAL DORSAL ROOT GANGLIA IN MICE WITH CHRONIC OVEREXPRESSION OF NGF IN UROTHELIUM 
Transient receptor potential vanilloid (TRPV) family member 4 (TRPV4) expression has been demonstrated in urothelial cells and dorsal root ganglion (DRG) neurons and roles in normal micturition reflexes as well as micturition dysfunction have been suggested. TRP channel expression and function is dependent upon target tissue expression of growth factors. These studies expand upon the target tissue dependence of TRPV4 expression in the urinary bladder and lumbosacral DRG using a recently characterized transgenic mouse model with chronic overexpression of nerve growth factor (NGF-OE) in the urothelium. Immunohistochemistry with image analyses, real-time quantitative polymerase chain reaction (Q-PCR) and western blotting were used to determine TRPV4 protein and transcript expression in the urinary bladder (urothelium + suburothelium, detrusor) and lumbosacral DRG from littermate wildtype (WT) and NGF-OE mice. Antibody specificity controls were performed in TRPV4-/- mice. TRPV4 transcript and protein expression was significantly (p ≤ 0.001) increased in the urothelium + suburothelium and suburothelial nerve plexus of the urinary bladder and in small- and medium-sized lumbosacral (L1, L2, L6-S1) DRG cells from NGF-OE mice compared to littermate WT mice. NGF-OE mice exhibit significant (p ≤ 0.001) increases in NGF transcript and protein in the urothelium + suburothelium and lumbosacral DRG. These studies demonstrate regulation of TRPV4 expression by NGF in lower urinary tract tissues. Ongoing studies are characterizing the functional roles of TRPV4 expression in the sensory limb (DRG, urothelium) of the micturition reflex.
doi:10.1007/s12031-013-0033-5
PMCID: PMC3779511  PMID: 23690258
micturition; sensory transducer; growth factor; Q-PCR; western blot
11.  Generation of human induced pluripotent stem cells to model spinocerebellar ataxia type 2 in vitro 
Spinocerebellar ataxia type 2 (SCA 2) is caused by triple nucleotide repeat (CAG) expansion in the coding region of the ATAXN2 gene on chromosome 12, which produces an elongated, toxic polyglutamine tract, leading to Purkinje cell loss. There is currently no effective therapy. One of the main obstacles that hamper therapeutic development is lack of an ideal disease model. In this study, we have generated and characterized SCA2 induced pluripotent stem (iPS) cell lines as an in vitro cell model. Dermal fibroblasts (FBs) were harvested from primary culture of skin explants obtained from a SCA2 subject and a healthy subject. For reprogramming, hOct4, hSox2, hKlf4, and hc-Myc were transduced to passage-3 FBs by retroviral infection. Both SCA2 iPS and control iPS cells were successfully generated and showed typical stem cell growth patterns with normal karyotype. All iPS cell lines expressed stem cell markers and differentiated in vitro into cells from three embryonic germ layers. Upon in vitro neural differentiation, SCA2 iPS cells showed abnormality in neural rosette formation but successfully differentiated into neural stem cells (NSCs) and subsequent neural cells. SCA2 and normal FBs showed a comparable level of ataxin-2 expression; whereas SCA2 NSCs showed less ataxin-2 expression than normal NSCs and SCA2 FBs. Within neural lineage, neurons have the most abundant expression of ataxin-2. Time-lapsed neural growth assay indicated terminally differentiated SCA2 neural cells were short-lived compared to control neural cells. The expanded CAG repeats of SCA2 were stable throughout reprogramming and neural differentiation. In conclusion, we have established the first disease-specific human SCA2 iPS cell line. These mutant iPS cells have the potential for neural differentiation. The differentiated neural cells harboring mutations are invaluable for the study of SCA2 pathogenesis and therapeutic drug development.
doi:10.1007/s12031-012-9930-2
PMCID: PMC3608734  PMID: 23224816
Induced pluripotent stem cells; spinocerebellar ataxia; ataxin- polyglutamine
12.  Is PACAP the Major Neurotransmitter for Stress Transduction at the Adrenomedullary Synapse? 
It has been known for more than a decade that the neuropeptide PACAP (pituitary adenylate cyclase-activating polypeptide) is co-stored with acetylcholine in the splanchnic nerve terminals innervating the adrenal medulla. Both transmitters are robust secretagogues for catecholamine release from chromaffin cells. Here, we review the unique contribution of PACAP to the functioning of the splanchnic–adrenal synapse in stress. While acetylcholine is released across a wide range of firing frequencies, PACAP is released only at high frequencies of stimulation, and its role in the regulation of epinephrine secretion and biosynthesis is highly specialized. PACAP is responsible for long-term catecholamine secretion using secretory mechanisms different from the rapidly desensitizing depolarization evoked by acetylcholine through nicotinic receptor activation. PACAP signaling also maintains catecholamine synthesis required for sustained secretion during prolonged stress via induction of the enzymes TH and PNMT, and enhances transcription of additional secreted molecules found in chromaffin cells that alter further secretion through both autocrine and paracrine mechanisms. PACAP thus mediates chromaffin cell plasticity via functional encoding of cellular experience. These features of PACAP action at the splanchnic–adrenal synapse may be paradigmatic for the general actions of neuropeptides as effectors of stimulus–secretion–synthesis coupling in stress.
doi:10.1007/s12031-012-9749-x
PMCID: PMC4180436  PMID: 22610912
Chromaffin cell; Neurotransmission; PACAP; Splanchnic–adrenal synapse; Stress
13.  Sexually dimorphic response of TRPM2 inhibition following cardiac arrest-induced global cerebral ischemia in mice 
Transient global cerebral ischemia due to cardiac arrest followed by resuscitation (CA/CPR) causes significant neurological damage in vulnerable neuron populations within the brain, such as hippocampal CA1 neurons. In recent years, we have implicated the transient receptor potential M2 (TRPM2) channel as a mediator of ischemic injury to neurons. We previously demonstrated that genetic and pharmacological strategies that reduce TRPM2 function preferentially protect male neurons in vitro and reduce infarct volume following experimental stroke. Due to the narrow therapeutic window for intervention following ischemic stroke, it is important to assess the role of TRPM2 in other models of cerebral ischemia. Therefore, this study utilized a modified mouse model of cardiac arrest and cardiopulmonary resuscitation (CA/CPR) to mimic more accurately the clinical condition by maintaining body and head temperatures near the physiological range throughout. Here we report that inhibition of TRPM2 activity with clotrimazole (CTZ) reduces hippocampal CA1 neuronal injury when administered 30 minutes after resuscitation from cardiac arrest. Consistent with our previous observations, neuroprotection was observed in male mice and no effect on injury was observed in the female. These findings provide further evidence for TRPM2 as a target for protection against cerebral ischemia in the male brain.
doi:10.1007/s12031-013-0005-9
PMCID: PMC3728170  PMID: 23532768
Brain; ischemia; TRPM2; cardiac arrest
14.  A New Generation Fatty Acid Amide Hydrolase Inhibitor Protects Against Kainate-Induced Excitotoxicity 
Endocannabinoids, including anandamide (AEA), have been implicated in neuroprotective on-demand responses. Related to such a response to injury, an excitotoxic kainic acid (KA) injection (i.p.) was found to increase AEA levels in the brain. To modulate the endocannabinoid response during events of excitotoxicity in vitro and in vivo, we utilized a new generation compound (AM5206) that selectively inhibits the AEA deactivating enzyme fatty acid amide hydrolase (FAAH). KA caused calpain-mediated spectrin breakdown, declines in synaptic markers, and disruption of neuronal integrity in cultured hippocampal slices. FAAH inhibition with AM5206 protected against the neurodegenerative cascade assessed in the slice model 24 h postinsult. In vivo, KA administration induced seizures and the same neurodegenerative events exhibited in vitro. When AM5206 was injected immediately after KA in rats, the seizure scores were markedly reduced as were levels of cytoskeletal damage and synaptic protein decline. The pre- and postsynaptic proteins were protected by the FAAH inhibitor to levels comparable to those found in healthy control brains. These data support the idea that endocannabinoids are released and converge on pro-survival pathways that prevent excitotoxic progression.
doi:10.1007/s12031-010-9472-4
PMCID: PMC4124033  PMID: 21069475
AM5206; Excitotoxicity; Endocannabinoid system; Hippocampus; Neuroprotection
15.  Glia Maturation Factor Deficiency Suppresses 1-Methyl-4-Phenylpyridinium-Induced Oxidative Stress in Astrocytes 
Inflammation is closely intertwined with pathogenesis of Parkinson's disease (PD). Increasing evidence suggests that inhibition of glia-mediated inflammation might represent a promising therapeutic target for PD. Glia maturation factor (GMF), an inflammatory protein, predominantly localized in astrocytes is previously isolated, sequenced and cloned in our laboratory. In the present investigation, we demonstrate that GMF-deficiency in astrocytes upregulates the antioxidant status and limit the extent of lipid peroxidation and production of reactive oxygen species (ROS) along with diminished nuclear factor-κB-mediated inflammatory responses in 1-methyl-4-phenylpyridinium (MPP+)-induced toxicity. Primary astrocytes obtained from wild-type (Wt) and GMF-deficient (GMF-KO) mice were treated with 5, 10, and 20 μM MPP+ for 24, 48, and 72 h in vitro. Our results show decreased release of ROS and increased level of glutathione in astrocytes obtained from GMF-KO mice when compared to astrocytes derived from Wt mice following MPP+ treatment. Additionally, we found decreased activity of NF-κB, and reduced levels of proinflammatory tumor necrosis factor- α, interleukin-1β (IL-1β), IL-17, IL-33, and chemokine (C–C motif) ligand 2 (CCL2) in GMF-KO astrocytes when compared to Wt astrocytes. Our overall results suggest that GMF-KO astrocytes are significantly resistant to MPP+ toxicity when compared to Wt astrocytes.
doi:10.1007/s12031-013-0225-z
PMCID: PMC4101074  PMID: 24430624
Glia maturation factor; Astrocytes; MPP+; Cytotoxicity; NF-κB; Cytokines
16.  Genomic Organization and Identification of Promoter Regions for the BDNF Gene in the Pond Turtle Trachemys scripta elegans 
Brain-derived neurotrophic factor (BDNF) is an important regulator of neuronal development and synaptic function. The BDNF gene undergoes significant activity-dependent regulation during learning. Here, we identified the BDNF promoter regions, transcription start sites, and potential regulatory sequences for BDNF exons I–III that may contribute to activity-dependent gene and protein expression in the pond turtle Trachemys scripta elegans (tBDNF). By using transfection of BDNF promoter/luciferase plasmid constructs into human neuroblastoma SHSY5Y cells and mouse embryonic fibroblast NIH3T3 cells, we identified the basal regulatory activity of promoter sequences located upstream of each tBDNF exon, designated as pBDNFI–III. Further, through chromatin immunoprecipitation (ChIP) assays, we detected CREB binding directly to exon I and exon III promoters, while BHLHB2, but not CREB, binds within the exon II promoter. Elucidation of the promoter regions and regulatory protein binding sites in the tBDNF gene is essential for understanding the regulatory mechanisms that control tBDNF gene expression.
doi:10.1007/s12031-014-0229-3
PMCID: PMC4103967  PMID: 24443176
BDNF; Promoter; ChIP assay; CREB; Turtle
17.  Different MicroRNA Profiles in Chronic Epilepsy Versus Acute Seizure Mouse Models 
Epilepsy affects around 50 million people worldwide, and in about 65 % of patients, the etiology of disease is unknown. MicroRNAs are small non-coding RNAs that have been suggested to play a role in the pathophysiology of epilepsy. Here, we compared microRNA expression patterns in the hippocampus using two chronic models of epilepsy characterised by recurrent spontaneous seizures (pilocarpine and self-sustained status epilepticus (SSSE)) and an acute 6-Hz seizure model. The vast majority of microRNAs deregulated in the acute model exhibited increased expression with 146 microRNAs up-regulated within 6 h after a single seizure. In contrast, in the chronic models, the number of up-regulated microRNAs was similar to the number of down-regulated microRNAs. Three microRNAs—miR-142-5p, miR-331-3p and miR-30a-5p—were commonly deregulated in all three models. However, there is a clear overlap of differentially expressed microRNAs within the chronic models with 36 and 15 microRNAs co-regulated at 24 h and at 28 days following status epilepticus, respectively. Pathway analysis revealed that the altered microRNAs are associated with inflammation, innate immunity and cell cycle regulation. Taken together, the identified microRNAs and the pathways they modulate might represent candidates for novel molecular approaches for the treatment of patients with epilepsy.
Electronic supplementary material
The online version of this article (doi:10.1007/s12031-014-0368-6) contains supplementary material, which is available to authorized users.
doi:10.1007/s12031-014-0368-6
PMCID: PMC4303710  PMID: 25078263
Epilepsy; miRNA; Status epilepticus; Microarray; Hippocampus
18.  Inhibition of Vasoactive Intestinal Polypeptide (VIP) Induces Resistance to Dextran Sodium Sulfate (DSS)-Induced Colitis in Mice 
VIP is highly expressed in the colon and regulates motility, vasodilatation, and sphincter relaxation. However, its role in the development and progress of colitis is still controversial. Our aim was to determine the participation of VIP on dextran sodium sulfate (DSS)-induced colonic mucosal inflammation using VIP−/− and WT mice treated with VIP antagonists. Colitis was induced in 32 adult VIP−/− and 14 age-matched WT litter-mates by giving 2.5 % DSS in the drinking water. DSS-treated WT mice were injected daily with VIP antagonists, VIPHyb (n=22), PG 97–269 (n=9), or vehicle (n=31). After euthanasia, colons were examined; colonic cytokines mRNA were quantified. VIP−/− mice were remarkably resistant to DSS-induced colitis compared to WT. Similarly, DSS-treated WT mice injected with VIPHyb (1 μM) or PG 97–269 (1 nM) had significantly reduced clinical signs of colitis. Furthermore, colonic expression of IL-1, TNF-α, and IL-6 was significantly lower in VIP−/− and VIPHyb or PG 97–269 compared to vehicle-treated WT. Genetic deletion of VIP or pharmacological inhibition of VIP receptors resulted in resistance to colitis. These data demonstrate a pro-inflammatory role for VIP in murine colitis and suggest that VIP antagonists may be an effective clinical treatment for human inflammatory bowel diseases.
doi:10.1007/s12031-013-0205-3
PMCID: PMC4114158  PMID: 24395090
VIP; Colitis; VIP antagonist: IBD
19.  Inhibition of Neuronal p38α, but not p38β MAPK, Provides Neuroprotection Against Three Different Neurotoxic Insults 
The p38 mitogen-activated protein kinase (MAPK) pathway plays a key role in pathological glial activation and neuroinflammatory responses. Our previous studies demonstrated that microglial p38α and not the p38β isoform is an important contributor to stressor-induced proinflammatory cytokine upregulation and glia-dependent neurotoxicity. However, the contribution of neuronal p38α and p38β isoforms in responses to neurotoxic agents is less well understood. In the current study, we used cortical neurons from wild-type or p38β knockout mice, and wild-type neurons treated with two highly selective inhibitors of p38α MAPK. Neurons were treated with one of three neurotoxic insults (L-glutamate, sodium nitroprusside, and oxygen-glucose deprivation), and neurotoxicity was assessed. All three stimuli led to neuronal death and neurite degeneration, and the degree of neurotoxicity induced in wild-type and p38β knockout neurons was not significantly different. In contrast, selective inhibition of neuronal p38α was neuroprotective. Our results show that neuronal p38β is not required for neurotoxicity induced by multiple toxic insults, but that p38α in the neuron contributes quantitatively to the neuronal dysfunction responses. These data are consistent with our previous findings of the critical importance of microglia p38α compared to p38β, and continue to support selective targeting of the p38α isoform as a potential therapeutic strategy.
doi:10.1007/s12031-014-0372-x
PMCID: PMC4303701  PMID: 25012593
p38 MAPK; Glutamate; Sodium nitroprusside; Oxygen-glucose deprivation; Neuroprotection; Kinase inhibitor
20.  [No title available] 
PMCID: PMC4289935  PMID: 24985705
21.  PACAP Protects Against Salsolinol-Induced Toxicity in Dopaminergic SH-SY5Y Cells: Implication for Parkinson’s Disease 
Pituitary adenylate cyclase-activating polypeptide (PACAP) is an endogenous 38 amino acid containing neuropeptide with various cytoprotective functions including neuroprotection. Administration of PACAP has been shown to reduce damage induced by ischemia, trauma or exogenous toxic substances. Moreover, mice deficient in PACAP are more vulnerable to damaging insults. In this study we sought to determine whether PACAP may also be protective against salsolinol-induced toxicity in SH-SY5Y cells and if so, elucidate its mechanism(s) of action. Salsolinol (SALS) is an endogenous dopamine metabolite with selective toxicity to nigral dopaminergic neurons, which are directly implicated in Parkinson’s disease (PD). SH-SY5Y cells, derived from human neuroblastoma cells express high levels of dopaminergic activity and are used extensively as a model to study these neurons. Exposure of SH-SY5Y cells to 400uM SALS for 24 h resulted in approximately 50% cell death that was mediated by apoptosis as determined by cell flow cyotmetry and increases in caspase 3 levels. Cellular toxicity was also associated with reductions in brain-derived neurotrophic factor (BDNF) and phosphorylated cyclic AMP response element-binding (p-CREB) protein. Pretreatment with PACAP dose-dependently attenuated SALS-induced toxicity and the associated apoptosis and the chemical changes. PACAP receptor antagonist PACAP 6-38 in turn, dose-dependently blocked the effects of PACAP. Neither PACAP nor PACAP antagonist had any effect of its own on cellular viability. These results suggest protective effects of PACAP in a cellular model of PD. Hence, PACAP or its agonists could be of therapeutic benefit in PD.
doi:10.1007/s12031-013-0015-7
PMCID: PMC3676705  PMID: 23625270
PACAP; Salsolinol; SH-SY5Y cell line; Neuroprotection; Apoptosis; BDNF; p-CREB
22.  Insufficient Resolution Response in the Hippocampus of a Senescence-Accelerated Mouse Model — SAMP8 
Aging is the primary risk factor for Alzheimer’s disease (AD), and it is known that inflammation is associated with both aging and AD. To resolve inflammation, biosynthesis of the specialized pro-resolving mediators (SPMs) is enhanced in a programmed and active manner. We investigated the effect of age on resolution by analyzing hippocampal tissue from 2- and 9-month-old senescence-accelerated mouse prone 8 (SAMP8), as well as age-matched senescence-accelerated mouse resistant 1 (SAMR1). Pro-inflammatory markers increased upon age in SAMP8 mice and were also higher than those in age-matched SAMR1 mice. However, neither SPMs nor their receptors were enhanced upon age in SAMP8 mice compared to age-matched SAMR1 mice. Analysis of SPM biosynthetic enzymes revealed elevated levels of leukocyte type 12-lipoxygenase (L12-LOX) and decreased 5-LOX levels upon age in SAMR1 mice, whereas they remained unchanged in SAMP8 mice. Moreover, we found partial co-localization of L12-LOX and amyloid beta (Aβ) staining, as well as correlation between L12-LOX and phosphorylated tau levels in SAMP8, but not SAMR1 mice. Thus, we conclude that the resolution response in SAMP8 mice is insufficient to counteract the increased inflammation with age, and this may have a role in the development of AD-like pathologies.
doi:10.1007/s12031-014-0346-z
PMCID: PMC4303707  PMID: 24913689
Aging; Alzheimer; Lipoxygenase; LXA4; Resolution of inflammation; RvD1; Tau
23.  Origins and Neurochemical Complexity of Preganglionic Neurons Supplying the Superior Cervical Ganglion in the Domestic Pig 
The superior cervical ganglion (SCG) is a center of sympathetic innervation of all head and neck organs. SCG sympathetic preganglionic neurons (SPN) were found in the nucleus intermediolateralis pars principalis (IMLpp), the nucleus intermediolateralis pars funicularis (IMLpf), the nucleus intercalatus spinalis (IC), and the nucleus intercalatus spinalis pars paraependymalis (ICpe). Despite its importance, little is known of SCG innervation and chemical coding in the laboratory pig, a model that is physiologically and anatomically representative of humans. Here in our study, we established the distribution and chemical coding of Fast Blue (FB) retrogradely labelled SPN innervating porcine SCG. After unilateral injection of FB retrograde tracer into the left SCG, labeled neurons were found solely on the ipsilateral side with approximately 98 % located in Th1–Th3 segments and predominantly distributed in the IMLpp and IMLpf. Neurochemical analysis revealed that approximately 80 % of SPN were positive both to choline acetyltransferase (ChAT) and nitric oxide synthase (NOS) and were surrounded by a plethora of opioidergic and peptiergic nerve terminals. The results of our study provide a detailed description of the porcine preganglionic neuroarchitecture of neurons controlling the SCG, setting the stage for further studies concerning SPN plasticity under experimental/pathological conditions.
doi:10.1007/s12031-014-0321-8
PMCID: PMC4303702  PMID: 24854048
Sympathetic preganglionic neurons; Superior cervical ganglion; Domestic pig; Retrograde tracing; Neurochemical coding
24.  Cloning and Characterization of Glutamate Receptor Subunit 4 (Glua4) and Its Alternatively Spliced Isoforms in Turtle Brain 
Ionotropic glutamate receptors sensitive to α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), GluAs, play an important role in neural development, synaptic plasticity, and neurodegeneration. Previous studies using an in vitro model of eyeblink classical conditioning in pond turtles suggested that acquisition of conditioning is associated with synaptic delivery of AMPA receptors containing GluA4 subunits. However, sequences of the GluA4 subunit, expression profile, and its alternatively spliced isoforms in turtle brain have not been previously determined. The sequence and domain structure of turtle GluA4 (tGluA4) and its splice variants was characterized. We found ten isoforms of tGluA4 including several previously unidentified truncated variants. Analysis of the nucleotide sequences of tGluA4 flip/flop, tGluA4c flip/flop and tGluA4s showed they are highly similar to known isoforms of the GluA4 subunit identified in chick. Examination of the relative abundance of mRNA expression for the tGluA4 variants showed that the flip and flop versions of tGluA4 and tGluA4c, and a novel truncated variant, tGluA4trc1, which is also expressed as protein, are major forms in the adult turtle brain. Identification of these alternatively spliced isoforms of tGluA4 will provide a unique opportunity to assess their role in synaptic plasticity through the application of short interfering RNAs (siRNAs).
doi:10.1007/s12031-010-9405-2
PMCID: PMC4022150  PMID: 20549383
AMPA receptor; GluA4; turtle; flip/flop splice variants; alternative splicing
25.  Cocaine sensitization increases Ih current channel subunit 2 (HCN2) protein expression in structures of the Mesocorticolimbic System 
Alteration of the biological activity among neuronal components of the Mesocorticolimbic (MCL) system has been implicated in the pathophysiology of drug abuse. Changes in the electrophysiological properties of neurons involved in the reward circuit seem to be of utmost importance in addiction. The Hyperpolarization-Activated Cyclic-Nucleotide Current, Ih, is a prominent mixed cation current present in neurons. The biophysical properties of the Ih and its potential modulatory role in cell excitability depend on the expression profile of the Hyperpolarization-activated cyclic nucleotide gated channel (HCN) subunits. We investigated whether cocaine-induced behavioral sensitization, an animal model of drug addiction, elicits region-specific changes in the expression of the HCN2 channel’s subunit in the MCL system. Tissue samples from the ventral tegmental area, prefrontal cortex, nucleus accumbens and hippocampus were analyzed using Western Blot. Our findings demonstrate that cocaine treatment induced a significant increase in the expression profile of the HCN2 subunit in both, its glycosylated and non-glycosylated protein isoforms, in all areas tested. The increase in the glycosylated isoform was only observed in the ventral tegmental area. Together, these data suggest that the observed changes in MCL excitability during cocaine addiction might be associated to alterations in the subunit composition of their HCN channels.
doi:10.1007/s12031-012-9920-4
PMCID: PMC3742011  PMID: 23203153
addiction; cocaine sensitization; ventral tegmental area; accumbens; Ih current; HCN channels

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