Normal Mouse Brain Research Articles

The radial organization of neuronal primary cilia is acutely disrupted by seizure and ischemic brain injury

Neuronal primary cilia are sensory organelles that are critically involved in the proper growth, development, and function of the central nervous system (CNS). Recent work also suggests that they signal in the context of CNS injury, and that abnormal ciliary signaling may be implicated in neurological diseases. We quantified the distribution of neuronal primary cilia alignment throughout the normal adult mouse brain by immunohistochemical staining for the primary cilia marker adenylyl cyclase III (ACIII) and measuring the angles of primary cilia with respect to global and local coordinate planes. We then introduced two different models of acute brain insult-temporal lobe seizure and cerebral ischemia, and re-examined neuronal primary cilia distribution, as well as ciliary lengths and the proportion of neurons harboring cilia. Under basal conditions, cortical cilia align themselves radially with respect to the cortical surface, while cilia in the dentate gyrus align themselves radially with respect to the granule cell layer. Cilia of neurons in the striatum and thalamus, by contrast, exhibit a wide distribution of ciliary arrangements. In both cases of acute brain insult, primary cilia alignment was significantly disrupted in a region-specific manner, with areas affected by the insult preferentially disrupted. Further, the two models promoted differential effects on ciliary lengths, while only the ischemia model decreased the proportion of ciliated cells. These findings provide evidence for the regional anatomical organization of neuronal primary cilia in the adult brain and suggest that various brain insults may disrupt this organization.

Viability of SH-SY5Y Cells is Associated with Purinergic P2 Receptor Expression Alterations

To investigate the role of metabotrophic purinergic P2Y receptors in neuroblastoma cell survival, expression of P2 receptors by normal mouse (C57BL/6) brain and human neuroblastoma SH-SY5Y cells was investigated by Western blot and real time PCR studies. Viability of SH-SY5Y cells treated with purinergic receptor antagonists suramin and pyridoxal-phosphate-6-azophenyl-2’,4’-disulfonate (PPADS) was evaluated by MTT assay and flow cytometry. In the brain samples of C57BL/6 mice, expressions of P2Y4 and P2X7 were significantly reduced, whereas that of P2Y1 was significantly elevated in an age-dependent manner. SH-SY5Y cell viability was significantly reduced and necrotic cell rates were mildly increased by 400 μM suramin and 100 μM PPADS treatment. Antagonist treatment downregulated P2Y1, P2Y2 and P2Y4 and upregulated P2Y6, P2Y12 and P2X7 mRNA levels in SH-SY5Y cells on the 24th hour. These alterations were abolished for all P2 receptors except P2Y1 in the 48th hour. P2Y receptors are expressed by both normal mouse brain and human neuroblastoma cells. Purinergic receptor antagonism interferes with neuroblastoma viability through elevation of necrotic cell death and modulation of P2 receptor expression. P2Y receptors might thus be useful targets for future anti-tumor treatment trials.

Two-Photon Ratiometric Fluorescence Probe with Enhanced Absorption Cross Section for Imaging and Biosensing of Zinc Ions in Hippocampal Tissue and Zebrafish.

Zinc ion (Zn2+) not only plays an important function in the structural, catalytic, transcription, and regulatory of proteins, but is also an essential ionic signal to regulate brain neurotransmitters pass process. In this work, we designed and synthesized an intramolecular charge transfer-based ratiometric two-photon fluorescence probe, P-Zn, for imaging and biosensing of Zn2+ in live cell, hippocampal tissue, and zebrafish. The developed probe demonstrated high two-photon absorption cross section (δ) of 516 ± 77 GM, which increased to 958 ± 144 GM after the probe was coordinated with Zn2+. Furthermore, this P-Zn probe quickly recognized Zn2+ with high selectivity, over other metal ions, amino acids, and reactive oxygen species. More interestingly, the initial emission peak of the present probe at 465 nm decreased with a new peak increased at 550 nm, leading to the ratiometric determination of Zn2+ with high accuracy. Finally, this two-photon fluorescence probe with high temporal resolution and remarkable analytical performance, as well as low-cytotoxicity, was successfully applied in imaging of live cells, hippocampal tissues, and zebrafishes. The present P-Zn probe combined with FLIM provided accurate mapping of Zn2+ distribution at single-cell level. More interestingly, the two-photon spectroscopic results demonstrated that the level of Zn2+ in hippocampal tissue of mouse with AD was higher than that in normal mouse brain.

Association of TrkA and APP Is Promoted by NGF and Reduced by Cell Death-Promoting Agents.

The amyloid precursor protein (APP) interacts with the tropomyosin receptor kinase A (TrkA) in normal rat, mouse, and human brain tissue but not in Alzheimer’s disease (AD) brain tissue. However, it has not been reported whether the two proteins interact directly, and if so, which domains are involved. Clarifying these points will increase our understanding of the role and regulation of the TrkA/APP interaction in normal brain functioning as well as in AD. Here we addressed these questions using bimolecular fluorescence complementation (BiFC) and the proximity ligation assay (PLA). We demonstrated that exogenously expressed APP and TrkA associate through their juxtamembrane/transmembrane domains, to form a complex that localizes mainly to the plasma membrane, endoplasmic reticulum (ER) and Golgi. Formation of the complex was inhibited by p75NTR, ShcC and Mint-2. Importantly, we demonstrated that the association between endogenous APP and TrkA in primary septal neurons were modified by NGF, or by drugs that either inhibit ER-to-Golgi transport or perturb microtubules and microfilaments. Interestingly, several agents that induce cell death [amyloid β (Aβ)-peptide, staurosporine and rapamycin], albeit via different mechanisms, all caused dissociation of APP/TrkA complexes and increased production of C-terminal fragment (β-CTF) APP fragment. These findings open new perspectives for investigating the interplay between these proteins during neurodegeneration and AD.

Zika Virus Targeting in the Developing Brain

Zika virus (ZIKV), a positive-sense RNA flavivirus, has attracted considerable attention recently for its potential to cause serious neurological problems, including microcephaly, cortical thinning, and blindness during early development. Recent findings suggest that ZIKV infection of the brain can occur not only during very early stages of development, but also in later fetal/early neonatal stages of maturation. Surprisingly, after peripheral inoculation of immunocompetent mice on the day of birth, the first cells targeted throughout the brain were isolated astrocytes. At later stages, more neurons showed ZIKV immunoreactivity, in part potentially due to ZIKV release from infected astrocytes. In all developing mice studied, we detected infection of retinal neurons; in many mice, this was also associated with infection of the lateral geniculate, suprachiasmatic nuclei, and superior colliculus, suggesting a commonality for the virus to infect cells of the visual system. Interestingly, in mature mice lacking a Type 1 interferon response (IFNR-/-), after inoculation of the eye, the initial majority of infected cells in the visual system were glial cells along the optic tract. ZIKV microinjection into the somatosensory cortex on one side of the normal mouse brain resulted in mirror infection restricted to the contralateral somatosensory cortex without any infection of midline brain regions, indicating the virus can move by axonal transport to synaptically coupled brain loci. These data support the view that ZIKV shows considerable complexity in targeting the CNS and may target different cells at different stages of brain development.SIGNIFICANCE STATEMENT Zika virus (ZIKV) can cause substantial damage to the developing human brain. Here we examine a developmental mouse model of ZIKV infection in the newborn mouse in which the brain is developmentally similar to a second-trimester human fetus. After peripheral inoculation, the virus entered the CNS in all mice tested and initially targeted astrocytes throughout the brain. Infections of the retina were detected in all mice, and infection of CNS visual system nuclei in the brain was common. We find that ZIKV can be transported axonally, thereby enhancing virus spread within the brain. These data suggest that ZIKV infects multiple cell types within the brain and that astrocyte infection may play a more important role in initial infection than previously appreciated.

Increase in \u03b1-tubulin modifications in the neuronal processes of hippocampal neurons in both kainic acid-induced epileptic seizure and Alzheimer\u2019s disease

Neurodegeneration includes acute changes and slow-developing alterations, both of which partly involve common cellular machinery. During neurodegeneration, neuronal processes are impaired along with dysregulated post-translational modifications (PTMs) of cytoskeletal proteins. In neuronal processes, tubulin undergoes unique PTMs including a branched form of modification called glutamylation and loss of the C-terminal tyrosine residue and the penultimate glutamic acid residue forming Δ2-tubulin. Here, we investigated the state of two PTMs, glutamylation and Δ2 form, in both acute and slow-developing neurodegenerations, using a newly generated monoclonal antibody, DTE41, which had 2-fold higher affinity to glutamylated Δ2-tubulin, than to unmodified Δ2-tubulin. DTE41 recognised glutamylated Δ2-tubulin preferentially in immunostaining than in enzyme-linked immunosorbent assay and immunoblotting. In normal mouse brain, DTE41 stained molecular layer of the cerebellum as well as synapse-rich regions in pyramidal neurons of the cerebral cortex. In kainic acid-induced epileptic seizure, DTE41-labelled signals were increased in the hippocampal CA3 region, especially in the stratum lucidum. In the hippocampi of post-mortem patients with Alzheimer’s disease, intensities of DTE41 staining were increased in mossy fibres in the CA3 region as well as in apical dendrites of the pyramidal neurons. Our findings indicate that glutamylation on Δ2-tubulin is increased in both acute and slow-developing neurodegeneration.

Cyclodextrin has conflicting actions on autophagy flux in vivo in brains of normal and Alzheimer model mice.

2-hydroxypropyl-β-cyclodextrin (CYCLO), a modifier of cholesterol efflux from cellular membrane and endo-lysosomal compartments, reduces lysosomal lipid accumulations and has therapeutic effects in animal models of Niemann-Pick disease type C and several other neurodegenerative states. Here, we investigated CYCLO effects on autophagy in wild-type mice and TgCRND8 mice-an Alzheimer's Disease (AD) model exhibiting β-amyloidosis, neuronal autophagy deficits leading to protein and lipid accumulation within greatly enlarged autolysosomes. A 14-day intracerebroventricular administration of CYCLO to 8-month-old TgCRND8 mice that exhibit moderately advanced neuropathology markedly diminished the sizes of enlarged autolysosomes and lowered their content of GM2 ganglioside and Aβ-immunoreactivity without detectably altering amyloid precursor protein processing or extracellular Aβ/β-amyloid burden. We identified two major actions of CYCLO on autophagy underlying amelioration of lysosomal pathology. First, CYCLO stimulated lysosomal proteolytic activity by increasing cathepsin D activity, levels of cathepsins B and D and two proteins known to interact with cathepsin D, NPC1 and ABCA1. Second, CYCLO impeded autophagosome-lysosome fusion as evidenced by the accumulation of LC3, SQSTM1/p62, and ubiquitinated substrates in an expanded population of autophagosomes in the absence of greater autophagy induction. By slowing substrate delivery to lysosomes, autophagosome maturational delay, as further confirmed by our in vitro studies, may relieve lysosomal stress due to accumulated substrates. These findings provide in vivo evidence for lysosomal enhancing properties of CYCLO, but caution that prolonged interference with cellular membrane fusion/autophagosome maturation could have unfavorable consequences, which might require careful optimization of dosage and dosing schedules.

Age and Environment Influences on Mouse Prion Disease Progression: Behavioral Changes and Morphometry and Stereology of Hippocampal Astrocytes.

Because enriched environment (EE) and exercise increase and aging decreases immune response, we hypothesized that environmental enrichment and aging will, respectively, delay and increase prion disease progression. Mice dorsal striatum received bilateral stereotaxic intracerebral injections of normal or ME7 prion infected mouse brain homogenates. After behavior analysis, animals were euthanized and their brains processed for astrocyte GFAP immunolabeling. Our analysis related to the environmental influence are limited to young adult mice, whereas age influence refers to aged mice raised on standard cages. Burrowing activity began to reduce in ME7-SE two weeks before ME7-EE, while no changes were apparent in ME7 aged mice (ME7-A). Object placement recognition was impaired in ME7-SE, NBH-A, and ME7-A but normal in all other groups. Object identity recognition was impaired in ME7-A. Cluster analysis revealed two morphological families of astrocytes in NBH-SE animals, three in NBH-A and ME7-A, and four in NBH-EE, ME7-SE, and ME7-EE. As compared with control groups, astrocytes from DG and CA3 prion-diseased animals show significant numerical and morphological differences and environmental enrichment did not reverse these changes but induced different morphological changes in GFAP+ hippocampal astroglia. We suggest that environmental enrichment and aging delayed hippocampal-dependent behavioral and neuropathological signs of disease progression.

Label-Free Electrochemical Biosensor for Monitoring of Chloride Ion in an Animal Model of Alzhemier's Disease.

The potential damage of Alzheimer's disease (AD) in brain function has attracted extensive attention. As the most common anion, Cl- has been indicated to play significant roles in brain diseases, particularly in the pathological process of AD. In this work, a label-free selective and accurate electrochemical biosensor was first developed for real-time monitoring of Cl- levels in a mouse brain model of AD and rat brain upon global cerebral ischemia. Silver nanoparticles (AgNPs) were designed and synthesized as selective recognition element for Cl-, while 5'-MB-GGCGCGATTTT-SH-3' (SH-DNA-MB, MB = methylene blue) was selected as an inner reference molecule for a built-in correction to avoid the effects from the complicated brain. The electrochemical biosensor showed high accuracy and remarkable selectivity for determination of Cl- over other anions, metal ions, amino acids, and other biomolecules. Furthermore, three-dimensional nanostructures composed of single-walled carbon nanotubes (SWNTs) and Au nanoleaves were assembled on the carbon fiber microelectrode (CFME) surface to enhance the response signal. Finally, the developed biosensor with high analytical performance, as well as the unique characteristic of CFME itself including inertness in live brain and good biocompatibility, was successfully applied to in vivo determination of Cl- levels in three brain regions: striatum, hippocampus, and cortex of live mouse and rat brains. The comparison of average levels of Cl- in normal striatum, hippocampus, and cortex of normal mouse brains and those in the mouse model brains of AD was reported. In addition, the results in rat brains followed by cerebral ischemia demonstrated that the concentrations of Cl- decreased by 19.8 ± 0.5% (n = 5) in the striatum and 27.2 ± 0.3% (n = 5) in hippocampus after cerebral ischemia for 30 min, but that negligible change in Cl- concentration was observed in cortex.

Fluselenamyl: A Novel Benzoselenazole Derivative for PET Detection of Amyloid Plaques (Aβ) in Alzheimer's Disease.

Fluselenamyl (5), a novel planar benzoselenazole shows traits desirable of enabling noninvasive imaging of Aβ pathophysiology in vivo; labeling of both diffuse (an earlier manifestation of neuritic plaques) and fibrillar plaques in Alzheimer’s disease (AD) brain sections, and remarkable specificity for mapping Aβ compared with biomarker proteins of other neurodegenerative diseases. Employing AD homogenates, [18F]-9, a PET tracer demonstrates superior (2–10 fold higher) binding affinity than approved FDA tracers, while also indicating binding to high affinity site on Aβ plaques. Pharmacokinetic studies indicate high initial influx of [18F]-9 in normal mice brains accompanied by rapid clearance in the absence of targeted plaques. Following incubation in human serum, [18F]-9 indicates presence of parental compound up to 3h thus indicating its stability. Furthermore, in vitro autoradiography studies of [18F]-9 with AD brain tissue sections and ex vivo autoradiography studies in transgenic mouse brain sections show cortical Aβ binding, and a fair correlation with Aβ immunostaining. Finally, multiphoton- and microPET/CT imaging indicate its ability to penetrate brain and label parenchymal plaques in transgenic mice. Following further validation of its performance in other AD rodent models and nonhuman primates, Fluselenamyl could offer a platform technology for monitoring earliest stages of Aβ pathophysiology in vivo.

Disrupted hippocampal network physiology following PTEN deletion from newborn dentate granule cells

Abnormal hippocampal granule cells are present in patients with temporal lobe epilepsy, and are a prominent feature of most animal models of the disease. These abnormal cells are hypothesized to contribute to epileptogenesis. Isolating the specific effects of abnormal granule cells on hippocampal physiology, however, has been difficult in traditional temporal lobe epilepsy models. While epilepsy induction in these models consistently produces abnormal granule cells, the causative insults also induce widespread cell death among hippocampal, cortical and subcortical structures. Recently, we demonstrated that introducing morphologically abnormal granule cells into an otherwise normal mouse brain – by selectively deleting the mTOR pathway inhibitor PTEN from postnatally-generated granule cells – produced hippocampal and cortical seizures. Here, we conducted acute slice field potential recordings to assess the impact of these cells on hippocampal function. PTEN deletion from a subset of granule cells reproduced aberrant responses present in traditional epilepsy models, including enhanced excitatory post-synaptic potentials (fEPSPs) and multiple, rather than single, population spikes in response to perforant path stimulation. These findings provide new evidence that abnormal granule cells initiate a process of epileptogenesis – in the absence of widespread cell death – which culminates in an abnormal dentate network similar to other models of temporal lobe epilepsy. Findings are consistent with the hypothesis that accumulation of abnormal granule cells is a common mechanism of temporal lobe epileptogenesis.

Microglia specific ablation of C1q gene definitively demonstrates microglia as the dominant source of C1q in both normal mouse brain and in models of Alzheimer's disease

Microglia specific ablation of C1q gene definitively demonstrates microglia as the dominant source of C1q in both normal mouse brain and in models of Alzheimer's disease

Abstract 1950: Whole genome microRNA profiling in functionally validated invasive GBM cells identifies a novel set of microRNAs driving GBM invasion

Abstract Glioblastoma multiforme (GBM) is the most malignant brain tumor that occurs in both adults and children. One of the key features that makes GBM particularly difficult to treat is the diffuse invasion of tumor cells into surrounding normal brain tissue. We hypothesize that direct comparison of matched invasive (GBMINV) and tumor core GBM cells (GBMTC) would facilitate the discovery of drivers of GBM invasion. However, GBMINV cells that have migrated deep into normal brain tissues are extremely difficult to obtain from patients as resection of normal human brain tissue can result in debilitating morbidities. To overcome this barrier, we utilized a panel of 6 pediatric patient tumor-derived orthotopic xenograft (PDOX) mouse models to isolate matched pairs of GBMTC cells from the visible tumor mass and GBMINV cells from the “normal” mouse brains. Global profiling of 768 human microRNA using a real-time PCR-based Taqman system identified 23 microRNAs were upregulated in the GBMINV cells in at least 5 of the 6 GBM models as compared with the matching GBMTC cells. Functional validation was performed by lentivirus-mediated silencing of miR-126, miR-487b and miR-369-5p, the top three overexpressed microRNA in the GBMINV cells. Compared with the untreated parental cells and tumor cells transduced with non-target control lentiviruses, silencing of miR-126, miR-487b or miR-369-5p suppressed GBMINV cell (both as neurosphere and monolayer) migration in vitro without affecting tumor proliferation and significantly inhibited the in vivo invasive growth of GBMINV cells, which were pre-enriched through 3 rounds of in vivo selections, into normal mouse brains. The depth of the invasive fronts were remarkably decreased/eliminated, exhibiting sharp, well-demarcated margins between tumor and normal mouse brain. To identify target genes of the 3 microRNAs, whole genome gene expression profiling from the same pairs of GBMINV and GBMTC cells was performed and identified a subset of genes suppressed by miR-126 (n = 74), miR-487b (n = 46) and miR-369-5p (n = 2) that occurred in at least 4 of the 6 pairs. Among them, RDX was inhibited by all three microRNAs, MANEA by miR-126 and miR-369, and a 32 genes (including PDK1, CREBZF, PRKAA2, TMF1 and HSPA13) by miR-126 and miR-487b. In conclusion, our novel strategy of utilizing functionally validated pairs of GBMINV and GBMTC cells has identified a set of microRNAs that are selectively over-expressed in GBMINV cells and allowed functionally confirmation of miR-126, miR-487b and miR-369-5p as drivers of GBM invasion. Citation Format: Lin Qi, Yu-lun Huang, Hua Mao, Mari Kogiso, Xiumei Zhao, Yuchen Du, Holly Lindsay, Xiaojun Yuan, Patricia Baxter, Jack Su, Chris Man, Laszlo Perlaky, Zhong Wang, You-Xing Zhou, Xiao-Nan Li. Whole genome microRNA profiling in functionally validated invasive GBM cells identifies a novel set of microRNAs driving GBM invasion. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1950.

Abstract 2455: Development of patient-derived orthotopic xenograft mouse models of pediatric low grade gliomas

Abstract Background: Low grade gliomas (LGGs) account for 1/3 of pediatric brain tumors. Despite favorable outcomes with current therapeutic strategies, some tumors still recur following total resection. New clinically-relevant pediatric LGG (PLGG) animal models that replicate this subset of aggressive tumors are therefore needed. Methods: Fresh tumor tissues from 36 PLGGs were collected. Self-renewal capacity was examined in vitro in 11 tumors with a neurosphere assay. Putative glioma stem cells (CD133+ and/or CD15+ cells) were analyzed in 23 PLGGs by flow cytometry (FCM). Formation of orthotopic xenograft tumors was examined by direct implantation of 25 PLGG tumor cells into the matched locations in the brains of SCID mice followed by serial tumor sub-transplantations. In addition to histo-pathological characterizations, BRAF V600E mutation was validated with pyrosequencing, and CDKN2A deletion with FISH. Results: The yield of viable tumor cells was low. Neurosphere formation was absent in 10/11 PLGGs after 37.6 ± 15.2 days. FCM analysis of 20 pilocytic astrocytomas and 2 gangliogliomas confirmed the presence of low levels (<1.5%) of CD133+ (CD133+/CD15− and CD133+/CD15+) cells, and relatively high fractions of CD133−/CD15+ cells (12.9 ± 20.1% in pilocytic astrocytomas and 67.9 ± 26.1% in gangliogliomas). Unlike 21 pilocytic astrocytomas, 1 grade II astrocytoma and 2 gangliogliomas that did not formed xenograft tumors after 219.9 ± 70.4 days of observations, a pleomorphic xanthoastrocytoma (PXA) (WHO grade II) formed intra-cerebral (IC) xenografts that have been serially subtransplanted 3 times in vivo. Similar to the patient tumor, xenografts from this model (IC-3635PXA) exhibited low cell proliferation (Ki67+ cells <10%), medium microvessel density, homozygous CDKN2A deletion and relatively low abundance of CD133+ (<2%). The GFAP+ and CD15+ cells in xenografts, however, were slightly reduced while the vimentin positive cells dramatically increased from 50% to 100% compared to patient tumor. The allele frequency of BRAF V600E mutation increased from 28% in patient tumor to 33%, 70% and 67% in passage I, II and III xenografts, respectively, and to 69% and 67% in cells grown in FBS-based media and serum-free media, respectively. All these biological changes were accompanied by extensive single cell invasion along blood vessels into normal mouse brains. Conclusion: This study demonstrated that the tumor take rate of PLGGs was low despite the presence of putative glioma stem cells. Nonetheless, we have successfully established a PXA xenograft mouse model and xenograft-derived 3635PXA cell lines which replicate key features of the original patient tumor and demonstrate both enrichment of the BRAF V600E mutant allele frequency and active invasive growth in mouse brains. This novel model system should facilitate biological studies and pre-clinical drug screenings targeting the BRAF V600E mutation in pediatric PXA. Citation Format: Mari Kogiso, Lin Qi, Frank K. Braun, Xiumei Zhao, Zhigang Liu, Holly B. Lindsay, Yuchen Du, Huiyuan Zhang, Sibo Zhao, Ching-Hua Liu, Vidya P. Mehta, Xiaoyun Shen, Jeffrey Murray, Laszlo Perlaky, Jack Su, Patricia Baxter, Adekunle Adesina, Donald W. Parsons, Xinyan Lu, Murali Chintagumpala, Xiao-Nan Li. Development of patient-derived orthotopic xenograft mouse models of pediatric low grade gliomas. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2455.

Abstract 4390: SVV-001 prolongs animal survival in PDOX adult GBM models

Abstract Background: Glioblastoma (GBM) is the most malignant and aggressive brain tumor, and survival of patients affected by GBM has remained virtually unchanged over numerous years. GBM is only minimally responsive to aggressive standard therapies including radical surgery and concurrent chemo-radiation treatment with temozolomide (TMZ). Seneca Valley virus (SVV-001) is a non-pathogenic oncolytic virus that can be systemically administered and can pass through the blood-brain barrier. In this study, we developed a new panel of patient derived orthotopic xenograft (PDOX) models to examine the therapeutic efficacy of SVV-001 combined with irradiation. In addition, we explored the mechanism of tumor cell infection with SVV-001 in malignant gliomas. Material and Methods: Surgical GBM tumor samples were obtained from 17 patients and directly implanted into the right cerebrum of NOD/SCID mice (1×105 cells suspended in 2 uL growth medium). Once tumor formation was confirmed, we performed H&E staining and IHC to determine the pathologic characteristics of the PDOX models over serial in vivo passages and compared with the matched patient tumors. In vitro antitumor activities of SVV-001 were examined in primary cultures, pre-formed neurospheres, and monolayer cells derived from PDOX models. In vivo therapeutic efficacy was examined by single I.V. injection of SVV-001 alone or administered in combination with radiation treatment of pre-formed xenograft tumors in permissive models. Results: 8 out from 17 samples had confirmed tumor formation in mouse brains; 4 of these samples have since been serially sub-transplanted for a total of 5 generations, while 4 intracerebral xenografts are in generation 2. The tumorigenicity of 8 additional samples is pending. These xenograft models precisely replicated histopathologic characteristics of their parental human tumors. SVV-001 at a multiplicity of infection of 0.5 to 25 replicated in and effectively killed primary cultures, pre-formed neurospheres, and monolayer glioma cells derived from adult glioma xenograft models in vitro. A single I.V. injection of SVV-001 (1 ×1011 viral particles/kg) administered immediately after radiation or 1 week after radiation led to the infection of orthotopic xenografts without harming normal mouse brain cells and resulted in significantly prolonged survival in permissive mouse models. Additionally, SVV-001 injected immediately after radiation significantly improved the overall survival of animals compared to administration of the virus 1 week after radiation, indicating that dose timing was crucial for full efficacy of the combinatory therapy. Conclusion: Our results demonstrated that SVV-001 possesses potent anti-tumor activity against adult malignant high-grade gliomas. Because this study was performed in a panel of patient tumor-derived orthotopic xenograft models, it provides a pre-clinical rationale that supports the consideration of SVV-001 for clinical trials against adult gliomas. Citation Format: Huiyuan Zhang, Lin Qi, Yuchen Du, Mari Kogiso, Frank K. Braun, Sibo Zhao, Holly Lindsay, Nabil Ahmed, Akash J. Patel, Patricia A. Baxter, Jack M. Su, Xiao-nan Li. SVV-001 prolongs animal survival in PDOX adult GBM models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4390.

Optically Pure Diphenoxy Derivatives as More Flexible Probes for β-Amyloid Plaques.

The highly rigid and planar scaffold with π-conjugated systems has been widely considered to be indispensable for Aβ binding probes. However, the flexible benzyloxybenzene derivative [(125)I]BOB-4 represents an excellent lead candidate for targeting Aβ in AD brains. Based on that, we designed two pairs of more flexible and optically pure diphenoxy derivatives with a chiral center as novel Aβ probes. These compounds possessed high affinity (Ki = 15.8-45.0 nM) for Aβ1-42 aggregates, and (R)-enantiomers showed slightly better binding ability than (S)-enantiomers. In addition, the competition binding assay implied that the optically pure diphenoxy derivatives with more flexible geometry shared the same binding site as IMPY, a classical rigid and planar Aβ probe. For (125)I-radiolabeled enantiomers, (S)-[(125)I]5 and (R)-[(125)I]5, specific plaque labeling on brain sections of Tg mice and AD patients were observed in in vitro autoradiography, persuasively proving the excellent affinity of the probes. In biodistribution, (S)-[(125)I]5 and (R)-[(125)I]5 with relatively low lipophilicity exhibited moderate initial brain uptake (4.37% and 3.72% ID/g at 2 min, respectively) and extremely fast washout from normal mice brain (brain2min/brain60min = 19.0 and 17.7, respectively). In summary, the separate enantiomers displayed similar properties in vitro and in vivo, and (S/R)-[(123)I]5 may be potential SPECT probes for recognizing Aβ plaques in AD brains.

Lentivirus‐Mediated Overexpression of MicroRNA‐210 Improves Long‐Term Outcomes after Focal Cerebral Ischemia in Mice

MicroRNAs play an important role in the pathogenesis of ischemic brain injury and in the repair process during postischemic condition. However, the key miRNAs and their function in these processes remain unclear. Circulating blood MicroRNAs profiles were examined in the ischemic stroke patients. The predicted network of difference was analyzed by ingenuity pathway analysis. The key MicroRNAs were selected, and the function was further studied in a mouse ischemia model. The predicted downstream target was confirmed. We found that 24 MicroRNAs were differently expressed in stroke patients compared to the control (P < 0.05). Bioinformatic analysis showed a MicroRNAs regulated network with the highest score in the stroke cascade, which was consisted of 10 MicroRNAs including key hypoxia-related miR-210 and its predicted downstream target brain derived neurotrophic factor (BDNF). Lentivirus-mediated miR-210 overexpression enhanced the microvessel density and the number of neural progenitor cells in the ischemic mouse brain (P < 0.05) and improved neurobehavioral outcomes in the ischemic mouse (P < 0.05). MiR-210 upregulation increased mBDNF/proBDNF protein expression in the normal and ischemic mouse brain. The dual-luciferase reporter assay identified that BDNF was the direct target of miR-210. MiR-210 is a crucial ischemic stroke-associated MicroRNAs and a potential target for the stroke therapy.

Systemic effects of AGEs in ER stress induction in vivo.

Emerging evidence indicates that accumulation of advanced glycation end products (AGEs) in human tissues may contribute to cell injury, inflammation and apoptosis through induction of endoplasmic reticulum (ER) stress. Human metabolism relies on ER homeostasis for the coordinated response of all metabolic organs by controlling the synthesis and catabolism of various nutrients. In vitro studies have demonstrated AGE-induced enhancement of unfolded protein response (UPR) in different cell types including endothelial, neuronal, pancreatic cells and podocytes, suggesting this crosstalk as an underlying pathological mechanism that contributes to metabolic diseases. In this minireview, we describe in vivo studies undertaken by our group and others that demonstrate the diverse systemic effects of AGEs in ER stress induction in major metabolic tissues such as brain, kidney, liver and pancreas of normal mice. Administration of high-AGEs content diet to normal mice for the period of 4weeks upergulates the mRNA and protein levels of ER chaperone Bip (GRP78) indicative of UPR initiation in all major metabolic organs and induces activation of the pivotal transcription factor XBP1 that regulates glucose and lipid metabolism. Furthermore, animals with genetic ablation of UPR-activated transcription factor C/EBP homologous protein CHOP allocated in high-AGEs diet, exhibited relative resistance to UPR induction (BiP levels) and XBP1 activation in major metabolic organs. Since CHOP presents a critical mediator that links accumulation and aggregation of unfolded proteins with induction of oxidative stress and ER stress-related apoptosis, it is revealed as an important molecular target for the management of metabolic diseases.

A case of mistaken identity: CD11c-eYFP(+) cells in the normal mouse brain parenchyma and neural retina display the phenotype of microglia, not dendritic cells.

Under steady-state conditions the central nervous system (CNS) is traditionally thought to be devoid of antigen presenting cells; however, putative dendritic cells (DCs) expressing enhanced yellow fluorescent protein (eYFP) are present in the retina and brain parenchyma of CD11c-eYFP mice. We previously showed that these mice carry the Crb1(rd8) mutation, which causes retinal dystrophic lesions; therefore we hypothesized that the presence of CD11c-eYFP(+) cells within the CNS may be due to pathology associated with the Crb1(rd8) mutation. We generated CD11c-eYFP Crb1(wt/wt) mice and compared the distribution and immunophenotype of CD11c-eYFP(+) cells in CD11c-eYFP mice with and without the Crb1(rd8) mutation. The number and distribution of CD11c-eYFP(+) cells in the CNS was similar between CD11c-eYFP Crb1(wt/wt) and CD11c-eYFP Crb1(rd8/rd8) mice. CD11c-eYFP(+) cells were distributed throughout the inner retina, and clustered in brain regions that receive input from the external environment or lack a blood-brain barrier. CD11c-eYFP(+) cells within the retina and cerebral cortex of CD11c-eYFP Crb1(wt/wt) mice expressed CD11b, F4/80, CD115 and Iba-1, but not DC or antigen presentation markers, whereas CD11c-eYFP(+) cells within the choroid plexus and pia mater expressed CD11c, I-A/I-E, CD80, CD86, CD103, DEC205, CD8α and CD135. The immunophenotype of CD11c-eYFP(+) cells and microglia within the CNS was similar between CD11c-eYFP Crb1(wt/wt) and CD11c-eYFP Crb1(rd8/rd8) mice; however, CD11c and I-A/I-E expression was significantly increased in CD11c-eYFP Crb1(rd8/rd8) mice. This study demonstrates that the overwhelming majority of CNS CD11c-eYFP(+) cells do not display the phenotype of DCs or their precursors and are most likely a subpopulation of microglia. GLIA 2016. GLIA 2016;64:1331-1349.

Tumour-specific triple-regulated oncolytic herpes virus to target glioma.

Oncolytic herpes simplex virus type 1 (oHSV-1) therapy is an emerging treatment modality that selectively destroys cancer. Here we report use of a glioma specific HSV-1 amplicon virus (SU4-124 HSV-1) to selectively target tumour cells. To achieve transcriptional regulation of the SU4-124 HSV-1 virus, the promoter for the essential HSV-1 gene ICP4 was replaced with a tumour specific survivin promoter. Translational regulation was achieved by incorporating 5 copies of microRNA 124 target sequences into the 3′UTR of the ICP4 gene. Additionally, a 5′UTR of rat fibroblast growth factor -2 was added in front of the viral ICP4 gene open reading frame. Our results confirmed enhanced expression of survivin and eIF4E in different glioma cells and increased micro-RNA124 expression in normal human and mouse brain tissue. SU4-124 HSV-1 had an increased ICP4 expression and virus replication in different glioma cells compared to normal neuronal cells. SU4-124 HSV-1 exerted a strong antitumour effect against a panel of glioma cell lines. Intracranial injection of SU4-124 HSV-1 did not reveal any sign of toxicity on day 15 after the injection. Moreover, a significantly enhanced antitumour effect with the intratumourally injected SU4-124 HSV-1 virus was demonstrated in mice bearing human glioma U87 tumours, whereas viral DNA was almost undetectable in normal organs. Our study indicates that incorporation of multiple cancer-specific regulators in an HSV-1 system significantly enhances both cancer specificity and oncolytic activity.