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Medial ganglionic eminence (MGE)-derived parvalbumin (PV)+, somatostatin (SST)+ and Neurogliaform (NGFC)-type cortical and hippocampal interneurons, have distinct molecular, anatomical and physiological properties. However, the molecular mechanisms regulating their diversity remain poorly understood. Here, via single-cell transcriptomics, we show that the obligate NMDA-type glutamate receptor (NMDAR) subunit geneGrin1mediates subtype-specific transcriptional regulation of gene expression in MGE-derived interneurons, leading to altered subtype abundances. Notably, MGE-specific conditionalGrin1loss results in a systemic downregulation of diverse transcriptional, synaptogenic and membrane excitability regulatory programs. These widespread gene expression abnormalities mirror aberrations that are typically associated with neurodevelopmental disorders, particularly schizophrenia. Our study hence provides a road map for the systematic examination of NMDAR signaling in interneuron subtypes, revealing potential MGE-specific genetic targets that could instruct future therapies of psychiatric disorders.

Background Glioblastoma-associated macrophages and microglia (GAMs) are the predominant immune cells in the tumor microenvironment. Activation of MerTK, a receptor tyrosine kinase, polarizes GAMs to an immunosuppressive phenotype, promoting tumor growth. Here, the role of MerTK inhibition in the glioblastoma microenvironment is investigated in vitro and in vivo. Methods Effects of MRX-2843 in glioblastoma microenvironment regulation were determined in vitro by cell viability, cytokine array, in vitro tube formation, Western blotting, and wound healing assays. A syngeneic GL261 orthotopic glioblastoma mouse model was used to evaluate the survival benefit of MRX-2843 treatment. Multiplex fluorescent immunohistochemistry was used to evaluate the expression of CD206, an anti-inflammatory marker on GAMs, and angiogenesis in murine brain tumor tissues. Results MRX-2843 inhibited cell growth and induced apoptosis in human glioblastoma cells and decreased protein expression of phosphorylated MerTK, AKT, and ERK, which are essential for cell survival signaling. Interleukin-8 and C-C motif chemokine ligand 2, the pro-glioma and pro-angiogenic cytokines, were decreased by MRX-2843. Decreased vascular formation and numbers of immunosuppressive (CD206+) GAMs were observed following MRX-2843 treatment in vivo, suggesting that in addition to alleviating immunosuppression, MRX-2843 also inhibits neoangiogenesis in the glioma microenvironment. These results were supported by a prolonged survival in the syngeneic mouse orthotopic GL261 glioblastoma model following MRX-2843 treatment. Conclusion Our findings suggest that MRX-2843 has a therapeutic benefit via promoting GAM polarization away from immunosuppressive condition, inhibiting neoangiogenesis in the glioblastoma microenvironment and inducing tumor cell death.

The VPS13 proteins (VPS13A–D) are thought to mediate lipid transport between organelles and are linked to distinct neurological disorders in humans. Baldwin et al. found that, in addition to known involvement in mitochondrial morphology, VPS13D is essential for peroxisome biogenesis., The VPS13 gene family consists of VPS13A–D in mammals. Although all four genes have been linked to human diseases, their cellular functions are poorly understood, particularly those of VPS13D. We generated and characterized knockouts of each VPS13 gene in HeLa cells. Among the individual knockouts, only VPS13D-KO cells exhibit abnormal mitochondrial morphology. Additionally, VPS13D loss leads to either partial or complete peroxisome loss in several transformed cell lines and in fibroblasts derived from a VPS13D mutation–carrying patient with recessive spinocerebellar ataxia. Our data show that VPS13D regulates peroxisome biogenesis.

Agonists of the vanilloid receptor transient vanilloid potential 1 (TRPV1) are emerging as highly efficacious nonopioid analgesics in preclinical studies. These drugs selectively lesion TRPV1+ primary sensory afferents, which are responsible for the transmission of many noxious stimulus modalities. Resiniferatoxin (RTX) is a very potent and selective TRPV1 agonist and is a promising candidate for treating many types of pain. Recent work establishing intrathecal application of RTX for the treatment of pain resulting from advanced cancer has demonstrated profound analgesia in client-owned dogs with osteosarcoma. The present study uses transcriptomics and histochemistry to examine the molecular mechanism of RTX action in rats, in clinical canine subjects, and in 1 human subject with advanced cancer treated for pain using intrathecal RTX. In all 3 species, we observe a strong analgesic action, yet this was accompanied by limited transcriptional alterations at the level of the dorsal root ganglion. Functional and neuroanatomical studies demonstrated that intrathecal RTX largely spares susceptible neuronal perikarya, which remain active peripherally but unable to transmit signals to the spinal cord. The results demonstrate that central chemo-axotomy of the TRPV1+ afferents underlies RTX analgesia and refine the neurobiology underlying effective clinical use of TRPV1 agonists for pain control.

Background Standard therapy for chordoma consists of surgical resection followed by high-dose irradiation. Protein phosphatase 2A (PP2A) is a ubiquitously expressed serine/threonine phosphatase involved in signal transduction, cell cycle progression, cell differentiation, and DNA repair. LB100 is a small-molecule inhibitor of PP2A designed to sensitize cancer cells to DNA damage from irradiation and chemotherapy. A recently completed phase I trial of LB100 in solid tumors demonstrated its safety. Here, we show the therapeutic potential of LB100 in chordoma. Methods Three patient-derived chordoma cell lines were used: U-CH1, JHC7, and UM-Chor1. Cell proliferation was determined with LB100 alone and in combination with irradiation. Cell cycle progression was assessed by flow cytometry. Quantitative γ-H2AX immunofluorescence and immunoblot evaluated the effect of LB100 on radiation-induced DNA damage. Ultrastructural evidence for nuclear damage was investigated using Raman imaging and transmission electron microscopy. A xenograft model was established to determine potential clinical utility of adding LB100 to irradiation. Results PP2A inhibition in concert with irradiation demonstrated in vitro growth inhibition. The combination of LB100 and radiation also induced accumulation at the G2/M phase of the cell cycle, the stage most sensitive to radiation-induced damage. LB100 enhanced radiation-induced DNA double-strand breaks. Animals implanted with chordoma cells and treated with the combination of LB100 and radiation demonstrated tumor growth delay. Conclusions Combining LB100 and radiation enhanced DNA damage-induced cell death and delayed tumor growth in an animal model of chordoma. PP2A inhibition by LB100 treatment may improve the effectiveness of radiation therapy for chordoma.

We thank Ganley et al. for their thoughtful comment on our recent report [1]. We agree with Ganley et al. that both mito-Keima and mt-QC detect mitophagy. Our report demonstrates, however, that mit...

Uncontrolled seizures in patients with low grade gliomas have a significant impact on quality of life and morbidity, yet the mechanisms through which these tumors cause seizures remain unknown. Albeit there are multiple features that contribute to tumor related epileptogenesis, IDH mutations are determined to be an independent factor, although the pathogenesis remains poorly understood. Here, we hypothesize that the active metabolite D-2-hydroxyglutarate (D-2-HG) produced by the IDH-mutant enzyme leads to metabolic disruptions in surrounding cortical neurons that consequently promote seizures. We use a complementary study of in vitro cortical cultures and electrographically sorted human cortical tissue from patients (n=5) with IDH-mutant gliomas to test this hypothesis. We demonstrate that D-2-HG leads to increased neuronal spiking activity (p< 0.0001) and promotes a distinct metabolic profile in surrounding neurons and upregulation of mTOR signaling (p< 0.0001), which is consistent in human epileptic cortex compared to peritumoral nonepileptic cortex. Furthermore, increases in neuronal activity are induced by mTOR activation and reversed with mTOR inhibition. Together, our data suggest that metabolic disruptions and mTOR signaling upregulation in the surrounding cortex due to D-2-HG may be a driving event for epileptogenesis in patients with IDH-mutant low grade gliomas.

Traumatic brain injuries (TBIs) pose a massive burden of disease and continue to be a leading cause of morbidity and mortality throughout the world. A major obstacle in developing effective treatments is the lack of comprehensive understanding of the underlying mechanisms that mediate tissue damage and recovery after TBI. As such, our work aims to highlight the development of a novel experimental platform capable of fully characterizing the underlying pathobiology that unfolds after TBI. This platform encompasses an empirically optimized multiplex immunohistochemistry staining and imaging system customized to screen for a myriad of biomarkers required to comprehensively evaluate the extent of neuroinflammation, neural tissue damage, and repair in response to TBI. Herein, we demonstrate that our multiplex biomarker screening platform is capable of evaluating changes in both the topographical location and functional states of resident and infiltrating cell types that play a role in neuropathology after controlled cortical impact injury to the brain in male Sprague-Dawley rats. Our results demonstrate that our multiplex biomarker screening platform lays the groundwork for the comprehensive characterization of changes that occur within the brain after TBI. Such work may ultimately lead to the understanding of the governing pathobiology of TBI, thereby fostering the development of novel therapeutic interventions tailored to produce optimal tissue protection, repair, and/or regeneration with minimal side effects, and may ultimately find utility in a wide variety of other neurological injuries, diseases, and disorders that share components of TBI pathobiology.

INTRODUCTION Despite multi-model therapies that include maximal surgical resection, radiation, chemotherapy, and tumor treating fields, the median survival of Glioblastoma (GBM) patients is around 15 months. WASP-family verprolin homologous protein 1 (WAVE1) is a downstream effector that receives signals from small GTPases to regulate the actin cytoskeleton. WAVE1’s interaction with arp2/3 modulates critical roles, such as cell motility and morphologic changes. Expression of WAVE1 has been implicated in leukemia, ovarian, and prostate cancer. In this study, we tested the role of WAVE1 in GBM tumor biology. METHODS Expression of WAVE1 in normal brain and GBM tumor specimens was assessed by immunohistochemistry (IHC). The relevance of targeting WAVE1 for GBM therapy was evaluated in vitro by western blot, proliferation assay, cell cycle analysis, apoptosis assay, migration assay, and neurosphere formation assay using scrambled and target specific WAVE1-siRNA in patient-derived primary GBM neurospheres (GBMNS). RESULTS IHC data shows that the expression of WAVE1 is higher in GBM tumor samples than in low-grade gliomas and normal brain tissues. WAVE1 expression is upregulated in GBMNS compared to normal human astrocytes. WAVE1 knockdown significantly decreased the proliferation, migration, and self-renewal of GBMNS without affecting its cell cycle progression. Furthermore, WAVE1 depletion did not show any alterations in the apoptotic cell population, indicating that WAVE1 knockdown has a cytostatic effect on GBMNS. CONCLUSION Expression of WAVE1 positively correlates with GBM, and its knockdown imparts antitumor efficacy through cytostasis.

The clinical application of stem cells offers great promise as a potential avenue for therapeutic use in neurodegenerative diseases. However, cell loss after transplantation remains a major challenge which currently plagues the field. Based on our previous findings that fibroblast growth factor 21 (FGF-21) protected neurons from glutamate excitotoxicity and that upregulation of FGF-21 in a rat model of ischemic stroke was associated with neuroprotection, we proposed that overexpression of FGF-21 protects bone marrow-derived mesenchymal stem cells (MSCs) from apoptosis. To test this hypothesis, we examined whether the detrimental effects of apoptosis can be mitigated by the transgenic overexpression of FGF-21 in MSCs. FGF-21 was transduced into MSCs by lentivirus and its overexpression was confirmed by quantitative PCR. Moreover, FGF-21 overexpression did not stimulate the expression of other FGF family members, suggesting it does not activate a positive feedback system. The effects of hydrogen peroxide (H2 O2 ), tumor necrosis factor-α (TNF-α), and staurosporine, known inducers of apoptosis, were evaluated in FGF-21 overexpressing MSCs and mCherry control MSCs. Caspase 3 and 7 activity was markedly and dose-dependently increased by all three stimuli in mCherry MSCs. FGF-21 overexpression robustly suppressed caspase activation induced by H2 O2 and TNF-α, but not staurosporine. Moreover, the assessment of apoptotic morphological changes confirmed the protective effects of FGF-21 overexpression. Taken together, these results provide compelling evidence that FGF-21 plays a crucial role in protecting MSCs from apoptosis induced by oxidative stress and inflammation, and merits further investigation as a strategy for enhancing the therapeutic efficacy of stem cell-based therapies. This article is protected by copyright. All rights reserved.

Preclinical models that reliably recapitulate the immunosuppressive properties of human gliomas are essential to assess immune-based therapies. Intracranially injected GL261 cells are widely used as an immunocompetent animal model of glioma, but it is common practice to transfect these with luciferase to facilitate tumor monitoring during treatment. Our group has previously shown that the luciferase-expressing GL261 Red-FLuc cells create an inflammatory response when implanted intracranially. Now, we additionally explore the inflammatory response of GL261-Luc2 cells and demonstrate a similar host immune response occurs with this model as well. In our in vivo evaluation, C57BL/6 mice underwent stereotaxic, intracranial implantation with GL261, GL261 Red-FLuc or GL261-Luc2 cells at doses of 5x104cells/5mL or 3x105cells/5uL.MRIs were performed to monitor relative tumor growth. To assess intrinsic differences between cell lines, in vitro cytokine profiles were evaluated by proteome microarray. Kaplan-Meier survival analyses demonstrated median survival for mice implanted with GL261 cells at 5x104cells was 18 to 21 days. The GL261-Red FLuc implanted mice cells did not reach median survival at either tumor dose with greater than 60% of mice termed long-term survivors. Finally, mice injected with GL261-Luc2 cells at 3x105cells reached median survival at 23 days, but median survival was significantly prolonged for mice implanted with GL261-Luc2 at a dose of 5x104cells (37 days, with 40% becoming long-term survivors) compared to GL261 implanted mice. MRIs reveal differences in tumor growth that correspond with the differences in median survival between groups. In addition, proteomic analyses revealed significantly elevated inflammatory cytokines such as IFN-gamma, IL-7 and TNF-alpha in the supernatants of the GL261 Red-FLuc cells and GL261-Luc2 cells. Further immune characterization is ongoing. Our data suggests that GL261 Red-FLuc and GL261-Luc2 murine models elicit an anti-tumor immune response by increasing pro-inflammatory modulators which stimulate the tumoricidal function of immune cells in the tumor microenvironment.

Immunotherapy with oncolytic herpes simplex virus-1 therapy offers an innovative, targeted, less-toxic approach for treating brain tumors. However, a major obstacle in maximizing oncolytic virotherapy is a lack of comprehensive understanding of the underlying mechanisms that unfold in CNS tumors/associated microenvironments after infusion of virus. We demonstrate that our multiplex biomarker screening platform comprehensively informs changes in both topographical location and functional states of resident/infiltrating immune cells that play a role in neuropathology after treatment with HSV G207 in a pediatric Phase 1 patient. Using this approach, we identified robust infiltration of CD8+ T cells suggesting activation of the immune response following virotherapy; however there was a corresponding upregulation of checkpoint proteins PD-1, PD-L1, CTLA-4, and IDO revealing a potential role for checkpoint inhibitors. Such work may ultimately lead to an understanding of the governing pathobiology of tumors, thereby fostering development of novel therapeutics tailored to produce optimal responses.

BACKGROUNDCerebral malaria (CM) accounts for nearly 400,000 deaths annually in African children. Current dogma suggests that CM results from infected RBC (iRBC) sequestration in the brain microvasculature and resulting sequelae. Therapies targeting these events have been unsuccessful; findings in experimental models suggest that CD8+ T cells drive disease pathogenesis. However, these data have largely been ignored because corroborating evidence in humans is lacking. This work fills a critical gap in our understanding of CM pathogenesis that is impeding development of therapeutics.METHODSUsing multiplex immunohistochemistry, we characterized cerebrovascular immune cells in brain sections from 34 children who died from CM or other causes. Children were grouped by clinical diagnosis (CM+ or CM-), iRBC sequestration (Seqhi, Seqlo, Seq0) and HIV status (HIV+ or HIV-).RESULTSWe identified effector CD3+CD8+ T cells engaged on the cerebrovasculature in 69% of CM+ HIV- children. The number of intravascular CD3+CD8+ T cells was influenced by CM status (CM+ > CM-, P = 0.004) and sequestration level (Seqhi > Seqlo, P = 0.010). HIV coinfection significantly increased T cell numbers (P = 0.017) and shifted cells from an intravascular (P = 0.004) to perivascular (P < 0.0001) distribution.CONCLUSIONWithin the studied cohort, CM is associated with cerebrovascular engagement of CD3+CD8+ T cells, which is exacerbated by HIV coinfection. Thus, CD3+CD8+ T cells are highly promising targets for CM adjunctive therapy, opening new avenues for the treatment of this deadly disease.FUNDINGThis research was supported by the Intramural Research Program of the National Institutes of Health.

Advances in antiretroviral therapy have resulted in significantly decreased HIV-related mortality. HIV-associated neurocognitive disorders, however, continue to be a major problem in infected patients. The neuropathology underlying HIV-associated neurocognitive disorders has not been well characterized, and evidence suggests different contributing mechanisms. One potential mechanism is the induction of oxidative stress. Using the HIV-1 transgenic (Tg) rat model of HIV, we found increased striatal NADPH oxidase-4 and neuronal nitric oxide synthase expression in the adult (7- to 9-month-old) Tg rat compared with control rats but not in the young (1-month-old) Tg rats. This was accompanied by increased 3-nitrotyrosine (3-NT) immunostaining in the adult Tg rats, which worsened significantly in the old Tg rats (18 to 20 months old). There was, however, no concurrent induction of the antioxidant systems because there was no change in the expression of the nuclear factor-erythroid 2-related factor 2 and its downstream targets (thioredoxin and glutathione antioxidant systems). Colocalization of 3-NT staining with neurofilament proteins and evidence of decreased tyrosine hydroxylase and dopamine transporter expression in the old rats support dopaminergic involvement. We conclude that the HIV-1 Tg rat brain shows evidence of nitrosative stress without appropriate oxidation-reduction adaptation, whereas 3-NT modification of striatal neurofilament proteins likely points to the ensuing dopaminergic neuronal loss and dysfunction in the aging HIV-1 Tg rat.

Neurological and cognitive problems are a common complication of HIV infection and are prevalent even in treated individuals. Although the molecular processes underlying brain involvement with HIV are not completely understood, inflammation is suspected to play a significant role. Our work presents an in vivo assessment of neuroinflammation in an animal model of HIV, the simian immunodeficiency virus (SIV)-infected rhesus macaque. Using positron emission tomography (PET) imaging, we identified changes in brain inflammation after inoculation with SIV over time. Interestingly, we found decreased binding of the PET ligand in the presence of very high cerebrospinal fluid (CSF) viral loads. These findings were supported by immunostaining which showed marked glial loss instead of inflammation. This study provides insight into glial and neuronal changes associated with very high CSF viral load and could reflect similar changes occurring in HIV-infected patients., The exact cause of neurocognitive dysfunction in HIV-positive patients despite successful control of the infection in the periphery is not completely understood. One suggested mechanism is a vicious cycle of microglial activation and release of proinflammatory chemokines/cytokines that eventually leads to neuronal loss and dysfunction. However, the exact role of microglial activation in the earliest stages of the infection with high cerebrospinal fluid (CSF) viral loads (VL) is unclear. In this study, we imaged the translocator protein (TSPO), a mitochondrial membrane receptor known to be upregulated in activated microglia and macrophages, in rhesus macaques before and multiple times after inoculation with a neurotropic simian immunodeficiency virus (SIV) strain (SIVsm804E), using 18F-DPA714 positron emission tomography (PET). The whole-brain standardized uptake values of TSPO at equilibrium reflecting total binding (SUVT) and binding potentials (BPND) were calculated and correlated with CSF and serum markers of disease, and a corresponding postmortem immunostaining analysis was also performed. SUVT was found to be inversely correlated with both CSF VL and monocyte chemoattractant protein 1 (MCP-1) levels. In SIV-infected macaques with very high CSF VL at necropsy (>106 copies/ml), we found decreased TSPO binding by PET, and this was supported by immunostaining which showed glial and neuronal apoptosis rather than microglial activation. On the other hand, with only moderately elevated CSF VL (∼104 copies/ml), we found increased TSPO binding as well as focal and diffuse microglial activation on immunostaining. Our results in the SIV-infected macaque model provide insights into the relationship between HIV neuropathology and CSF VL at various stages of the disease.

In MRI-negative cases Cushing's disease (CD), surgeons perform a more extensive exploration of the pituitary gland, with fewer instances of hormonal remission.Subjects with a likely diagnosis of CD (n = 27, 20 females) each underwent twoThe mean-SUV of adenomas was significantly increased from baseline (3.6 ± 1.5) with oCRH administration (3.9 ± 1.7; one-tailed p = 0.003). Neuroradiologists agreed that adenomas were visible on 21 scans, not visible on 26 scans (disagreed about 7, kappa = 0.7). oCRH-stimulation led to the detection of additional adenomas (n = 6) not visible on baseline-PET study. Of the MRI-negative adenomas (n = 5), two were detected on PET imaging (one only after oCRH-stimulation). USP8 mutations or glycolytic pathway proteins were not associated with SUV in corticotropinomas.The results of the current study suggest that oCRH-stimulation may lead to increased

Introduction: The imaging characteristics of ischemic lesions seen on MRI aid in the interpretation of the lesion pathogenesis. Early ischemic lesions are seen on diffusion weighted imaging (DWI) with low apparent diffusion coefficient (ADC), later evolve on T2-FLAIR, develop elevated ADC, then disappear on DWI. Chronic white matter lesions (WML) seen on T2-FLAIR but not DWI may or may not be of ischemic origin. Here we present an MRI targeted pathology approach to better describe in-vivo MRI based on cellular changes. Methods: The brain of a patient was obtained for pathological evaluation early after stroke recurrence. As part of clinical care and study procedures, 3T MRI was obtained at baseline (within 2-hours), multiple follow-up visits, and during second admission. Radiological-pathological methods previously validated in trauma was employed. Whole brain 7T MRI was performed after formalin fixation, and MRI targeted pathology was performed, co-localizing three lesions of different ages based on history and presumed imaging characteristics; i) acute, ii) subacute, and iii) chronic vs WML. Homologous contralateral tissue was used as control. Results: Lesions seen in-vivo were observed in post mortem MRI, and reconfirmed in histology. An example of in-vivo and post-mortem MRI, along with corresponding tissue sections and H&E are shown in figure. Qualitative delineation of the boundaries of the chronic lesion on H&E was challenging. A large number of small vessels with enlarged perivascular spaces dominated. The subacute lesion, was characterized by loss of white mater, infiltration of macrophages, and astrocytosis. The acute lesion had a text-book appearance. Conclusion: Dramatic differences are seen in the volume fraction of free water, which likely a major contributor to the appearance on DWI/ADC and T2 FLAIR. Work continues with immunohistochemistry to better delineate the boundaries of the lesions and cellular morphology relevant to in-vivo MRI.

Understanding molecular alterations associated with peripheral inflammation is a critical factor in selectively controlling acute and persistent pain. The present report employs in situ hybridization of the 2 opioid precursor mRNAs coupled with quantitative measurements of 2 peptides derived from the prodynorphin and proenkephalin precursor proteins: dynorphin A 1-8 and [Met5]-enkephalin-Arg6-Gly7-Leu8. In dorsal spinal cord ipsilateral to the inflammation, dynorphin A 1-8 was elevated after inflammation, and persisted as long as the inflammation was sustained. Qualitative identification by high performance liquid chromatography and gel permeation chromatography revealed the major immunoreactive species in control and inflamed extracts to be dynorphin A 1-8. In situ hybridization in spinal cord after administration of the inflammatory agent, carrageenan, showed increased expression of prodynorphin (Pdyn) mRNA somatotopically in medial superficial dorsal horn neurons. The fold increase in preproenkephalin mRNA (Penk) was comparatively lower, although the basal expression is substantially higher than Pdyn. While Pdyn is not expressed in the dorsal root ganglion (DRG) in basal conditions, it can be induced by nerve injury, but not by inflammation alone. A bioinformatic meta-analysis of multiple nerve injury datasets confirmed Pdyn upregulation in DRG across different nerve injury models. These data support the idea that activation of endogenous opioids, notably dynorphin, is a dynamic indicator of persistent pain states in spinal cord and of nerve injury in DRG. PERSPECTIVE: This is a systematic, quantitative assessment of dynorphin and enkephalin peptides and mRNA in dorsal spinal cord and DRG neurons in response to peripheral inflammation and axotomy. These studies form the foundational framework for understanding how endogenous spinal opioid peptides are involved in nociceptive circuit modulation.

Background Neural stem cell (NSC)-based therapies hold great promise for treating diseases of the central nervous system (CNS). However, several fundamental problems still need to be overcome to fully exploit the clinical potential of NSC therapeutics. Chief among them is the limited survival of NSC grafts within hostile microenvironments. Methods Herein, we sought to engineer NSCs in an effort to increase graft survival within ischemic brain lesions via upregulation of global SUMOylation, a post-translational modification critically involved in mediating tolerance to ischemia/reperfusion. Findings NSCs overexpressing the SUMO E2-conjugase Ubc9 displayed resistance to oxygen-glucose-deprivation/restoration of oxygen/glucose (OGD/ROG) and enhanced neuronal differentiation in vitro, as well as increased survival and neuronal differentiation when transplanted in mice with transient middle cerebral artery occlusion in vivo. Interpretation Our work highlights a critical role for SUMOylation in NSC biology and identifies a biological pathway that can be targeted to increase the effectiveness of exogenous stem cell medicines in ischemic stroke. Fund Intramural Research Program of the NINDS/NIH, the Italian Multiple Sclerosis Foundation (FISM), the Bascule Charitable Trust, NIH-IRTA-OxCam and Wellcome Trust Research Training Fellowships.

The deadliest complication of Plasmodium falciparum infection is cerebral malaria (CM), with a case fatality rate of 15 to 25% in African children despite effective antimalarial chemotherapy. No adjunctive treatments are yet available for this devastating disease. We previously reported that the glutamine antagonist 6-diazo-5-oxo-l-norleucine (DON) rescued mice from experimental CM (ECM) when administered late in the infection, a time by which mice had already suffered blood–brain barrier (BBB) dysfunction, brain swelling, and hemorrhaging. Herein, we used longitudinal MR imaging to visualize brain pathology in ECM and the impact of a new DON prodrug, JHU-083, on disease progression in mice. We demonstrate in vivo the reversal of disease markers in symptomatic, infected mice following treatment, including the resolution of edema and BBB disruption, findings usually associated with a fatal outcome in children and adults with CM. Our results support the premise that JHU-083 is a potential adjunctive treatment that could rescue children and adults from fatal CM.

INTRODUCTION Epileptic seizures in patients with low-grade, isocitrate dehydrogenase (IDH) mutated gliomas reach 90%, a major source of morbidity for these patients. Albeit there are multiple features that contribute to tumor related epileptogenesis, IDH mutations are determined to be an independent factor, although the pathogenesis remains poorly understood. We demonstrate IDH-mutated tumors promote epileptogenesis through D-2-hydroxyglutarate (D-2-HG) dependent mTOR hyperactivation and metabolic reprogramming. METHODS Human epileptic and nonepileptic cortex were identified via subdural electrodes in patients with IDH-mutated gliomas (n=5). An in vitro rat cortical neuronal model on microelectrode arrays were utilized to investigate the role of D-2-HG on neuronal excitability. mTOR and lysine demethylase (KDM) modulators were applied to elucidate the epileptogenic mechanism. Tetrodotoxin was utilized to evaluate the contribution of neuronal activity to mTOR signaling and metabolism. mTOR signaling was evaluated through western blot analysis and multiplex immunofluorescence. Metabolic function were analyzed via Seahorse assays and metabolomic analysis. RESULTS D-2-HG increased normalized bursting rate in the neuronal cultures (p CONCLUSION We demonstrate IDH-mutated gliomas promote epileptogenesis through a D-2-HG dependent mTOR hyperactivation via KDM inhibition, a putative mechanism and potential therapeutic targets. Furthermore, we argue mTOR hyperactivation results in metabolic reprogramming, independent of neuronal firing, which may contribute to epileptogenesis, a heretofore unrecognized aspect of pathologic mTOR signaling in neurological diseases.

Huntington's disease (HD) is a fatal neurodegenerative disorder caused by CAG repeat expansions in the huntingtin gene. Although, stem cell-based therapy has emerged as a potential treatment for neurodegenerative diseases, limitations remain, including optimizing delivery to the brain and donor cell loss after transplantation. One strategy to boost cell survival and efficacy is to precondition cells before transplantation. Because the neuroprotective actions of the mood stabilizers lithium and valproic acid (VPA) induce multiple pro-survival signaling pathways, we hypothesized that preconditioning bone marrow-derived mesenchymal stem cells (MSCs) with lithium and VPA prior to intranasal delivery to the brain would enhance their therapeutic efficacy, and thereby facilitate functional recovery in N171-82Q HD transgenic mice. MSCs were treated in the presence or absence of combined lithium and VPA, and were then delivered by brain-targeted single intranasal administration to eight-week old HD mice. Histological analysis confirmed the presence of MSCs in the brain. Open-field test revealed that ambulatory distance and mean velocity were significantly improved in HD mice that received preconditioned MSCs, compared to HD vehicle-control and HD mice transplanted with non-preconditioned MSCs. Greater benefits on motor function were observed in HD mice given preconditioned MSCs, while HD mice treated with non-preconditioned MSCs showed no functional benefits. Moreover, preconditioned MSCs reduced striatal neuronal loss and huntingtin aggregates in HD mice. Gene expression profiling of preconditioned MSCs revealed a robust increase in expression of genes involved in trophic effects, antioxidant, anti-apoptosis, cytokine/chemokine receptor, migration, mitochondrial energy metabolism, and stress response signaling pathways. Consistent with this finding, preconditioned MSCs demonstrated increased survival after transplantation into the brain compared to non-preconditioned cells. Our results suggest that preconditioning stem cells with the mood stabilizers lithium and VPA before transplantation may serve as an effective strategy for enhancing the therapeutic efficacy of stem cell-based therapies.

The transgenic HIV-1 rat (Tg) is a commonly used neuroHIV model with documented neurologic/behavioral deficits. Using immunofluorescent staining of the Tg brain, we found astrocytic dysfunction/damage, as well as dopaminergic neuronal loss/dysfunction, both of which worsening significantly in the striatum with age. We saw mild microglial activation in young Tg brains, but this decreased with age. There were no differences in neurogenesis potential suggesting a neurodegenerative rather than a neurodevelopmental process. Gp120 CSF levels exceeded serum gp120 levels in some animals, suggesting local viral protein production in the brain. Further probing of the pathophysiology underlying astrocytic injury in this model is warranted.

Objective Inaccessibility of the inflammation compartmentalized to the central nervous system (CNS) may underlie the lack of efficacy of immunomodulatory treatments in progressive multiple sclerosis (MS). The double blind combination of Rituximab by IntraVenous and IntraThecAl injection versus placebo in patients with Low-Inflammatory SEcondary progressive MS (RIVITALISE; NCT01212094) trial was designed to answer: (1) Whether an induction dose of intravenous and intrathecal rituximab efficiently depletes CNS B cells? and (2) If so, whether this leads to global inhibition of CNS inflammation and slowing of CNS tissue destruction? Methods Patients aged 18–65 years were randomly assigned to rituximab or placebo. Protocol-stipulated interim analysis quantified the efficacy of B-cell depletion. Results The efficacy on cerebrospinal fluid (CSF) biomarkers failed to reach criteria for continuation of the trial. B-cell-related CSF biomarkers (sCD21 and B-cell activating factor) changed only in the active-treatment arm. While CSF B cells were killed robustly (median −79.71%, P = 0.0176), B cells in CNS tissue were depleted inadequately (~−10–20%, P

The protein phosphatase 2A (PP2A) inhibitor, LB100, has been shown in pre-clinical studies to be an effective chemo- and radio-sensitizer for treatment of various cancers. We investigated effects associated with LB100 treatment alone and in combination with cisplatin for medulloblastoma (MB) in vitro and in vivo in an intracranial xenograft model. We demonstrated that LB100 had a potent effect on MB cells. By itself, LB100 inhibited proliferation and induced significant apoptosis in a range of pediatric MB cell lines. It also attenuated MB cell migration, a pre-requirement for invasion. When used in combination, LB100 enhanced cisplatin-mediated cytotoxic effects. Cell viability in the presence of 1 uM cisplatin alone was 61% (DAOY), 100% (D341), and 58% (D283), but decreased with the addition of 2 μM of LB100 to 26% (DAOY), 67% (D341), and 27% (D283), (p < 0.005). LB100 suppressed phosphorylation of the STAT3 protein and several STAT3 downstream targets. Also, LB100 directly increased cisplatin uptake and overcame cisplatin-resistance in vitro. Finally, LB100 exhibited potent in vivo anti-neoplastic activity in combination with cisplatin in an intracranial xenograft model.

INTRODUCTION Glioblastoma (GBM) is the most common malignant primary brain tumor. With limited effective therapeutic strategies, prognosis for GBM is very poor. Our previous study shows that the expression of Protein Arginine Methyltransferase 5 (PRMT5) is upregulated in GBM; its inhibition promotes anti-GBM effect through apoptosis and senescence of mature and immature tumor cells, respectively. In GBM, RAS-RAF- MEK-ERK signaling is aberrantly activated and promotes tumor growth. Therefore, MEK inhibitors, including trametinib are currently under investigation for GBM therapy. In this study, we tested whether inhibition of PRMT5 can enhance the anti-tumor efficacy of trametinib in GBM. METHODS Patient-derived primary GBM neurospheres (GBMNS) with transient PRMT5 knockdown were treated with trametinib and cell viability, proliferation, cell cycle progression, and western blot analysis were conducted. In vivo, PRMT5-intact and -depleted GBMNS were intracranially implanted in NSG mice and treated with trametinib by daily oral gavage, and tumor progression and mice survival rate were analyzed by MRI and Kaplan-Meier survival curve, respectively. RESULTS Trametinib treatment upregulated the expression of PRMT5 in GBMNS. Depletion of PRMT5 increased the cytotoxic effect of trametinib in GBMNS. In concurrence with the trametinib-induced PRMT5 upregulation, trametinib treatment increased the activity of AKT that was blocked with PRMT5 knockdown. In vivo, PRMT5-depletion extended the survival of the tumor bearing mice that further increased in combination with trametinib treatment. Interestingly, trametinib treatment alone had no survival benefit. CONCLUSION Trametinib treatment induces PRMT5 expression. Depletion of trametinib-induced PRMT5 expression sensitizes GBMNS for trametinib by inhibiting AKT activity.

Background Glioblastoma is the most common primary malignancy of the brain, with a dismal prognosis. Immunomodulation via checkpoint inhibition has provided encouraging results in non-CNS malignancies, but prediction of responders has proven to be challenging in glioblastoma patients. Objective To determine the proportion of patients who have a measurable increase of interferon gamma levels in brain tumor tissue after their first dose of nivolumab, and to evaluate the safety of using brain tumor microdialysis to monitor for immune response while evaluating the safety of the combination of anti-programmed death 1 (PD-1) and anti-lymphocyte activation gene 3 (LAG-3) checkpoint inhibition. Methods The study design is a single-center, nonrandomized phase 1 clinical trial. Up to 15 adult patients with recurrent glioblastoma will be enrolled with the goal of 10 patients completing the trial over an anticipated 18 mo. Patients will undergo biopsy; placement of microdialysis catheters and lumbar drains; treatment with anti-PD-1 checkpoint inhibition; comprehensive immune biomarker collection; tumor resection; and then treatment with anti-PD-1 and anti-LAG-3 checkpoint inhibition until progression. Expected outcomes We expect interferon gamma levels to increase in the brain as measured via microdialysis in treated patients. Based on published reports, microdialysis in this patient population is expected to be safe, and anti-LAG-3 and anti-PD-1 combined will likely have a similar side effect profile to other checkpoint inhibitor combinations. Discussion The failure of recent trials of immune therapies in glioblastoma underscores the need to appropriately measure response in the treated tissue. This trial may provide insight on indicators of which patients will respond to immune therapy.

There is tremendous interest in transplanting neural precursor cells for brain tissue regeneration. However, it remains unclear whether a vascularized and integrated complex neural tissue can be generated within the brain through transplantation of cells. Here, we report that early stage neural precursor cells recapitulate their seminal properties and develop into large brain-like tissue when implanted into the rat brain ventricle. Whereas the implanted cells predominantly differentiated into glutamatergic neurons and astrocytes, the host brain supplied the intact vasculature, oligodendrocytes, GABAergic interneurons, and microglia that seamlessly integrated into the new tissue. Furthermore, local and long-range axonal connections formed mature synapses between the host brain and the graft. Implantation of precursor cells into the CSF-filled cavity also led to a formation of brain-like tissue that integrated into the host cortex. These results may constitute the basis of future brain tissue replacement strategies. Nikorn Pothayee et al. show that early neural precursor cells (NPCs) derived from the embryonic telencephalon or midbrain can develop into brain-like tissue when implanted into the rat brain ventricle. Telencephalon-derived NPCs also form brain tissue in the host cortex when implanted into a CSF-filled cavity formed by cortical ablation.

Neocortical circuits consist of stereotypical motifs that must self-assemble during development. Recent evidence suggests that the subtype identity of both excitatory projection neurons (PNs) and inhibitory interneurons (INs) is important for this process. We knocked out the transcription factor Satb2 in PNs to induce those of the intratelencephalic (IT) type to adopt a pyramidal tract (PT)-type identity. Loss of IT-type PNs selectively disrupted the lamination and circuit integration of INs derived from the caudal ganglionic eminence (CGE). Strikingly, reprogrammed PNs demonstrated reduced synaptic targeting of CGE-derived INs relative to controls. In control mice, IT-type PNs targeted neighboring CGE INs, while PT-type PNs did not in deep layers, confirming this lineage-dependent motif. Finally, single-cell RNA sequencing revealed that major CGE IN subtypes were conserved after loss of IT PNs, but with differential transcription of synaptic proteins and signaling molecules. Thus, IT-type PNs influence CGE-derived INs in a non-cell-autonomous manner during cortical development.

Purpose: Glioblastoma (GBM) is highly resistant to treatment, largely due to disease heterogeneity and resistance mechanisms. We sought to investigate a promising drug that can inhibit multiple aspects of cancer cell survival mechanisms and become an effective therapeutic for GBM patients. Experimental Design: To investigate TG02, an agent with known penetration of the blood–brain barrier, we examined the effects as single agent and in combination with temozolomide, a commonly used chemotherapy in GBM. We used human GBM cells and a syngeneic mouse orthotopic GBM model, evaluating survival and the pharmacodynamics of TG02. Mechanistic studies included TG02-induced transcriptional regulation, apoptosis, and RNA sequencing in treated GBM cells as well as the investigation of mitochondrial and glycolytic function assays. Results: We demonstrated that TG02 inhibited cell proliferation, induced cell death, and synergized with temozolomide in GBM cells with different genetic background but not in astrocytes. TG02-induced cytotoxicity was blocked by the overexpression of phosphorylated CDK9, suggesting a CDK9-dependent cell killing. TG02 suppressed transcriptional progression of antiapoptotic proteins and induced apoptosis in GBM cells. We further demonstrated that TG02 caused mitochondrial dysfunction and glycolytic suppression and ultimately ATP depletion in GBM. A prolonged survival was observed in GBM mice receiving combined treatment of TG02 and temozolomide. The TG02-induced decrease of CDK9 phosphorylation was confirmed in the brain tumor tissue. Conclusions: TG02 inhibits multiple survival mechanisms and synergistically decreases energy production with temozolomide, representing a promising therapeutic strategy in GBM, currently under investigation in an ongoing clinical trial. Clin Cancer Res; 24(5); 1124–37. ©2017 AACR.

Disorders of pain neural systems are frequently chronic and, when recalcitrant to treatment, can severely degrade the quality of life. The pain pathway begins with sensory neurons in dorsal root or trigeminal ganglia, and the neuronal subpopulations that express the transient receptor potential cation channel, subfamily V, member 1 (TRPV1) ion channel transduce sensations of painful heat and inflammation and play a fundamental role in clinical pain arising from cancer and arthritis. In the present study, we elucidate the complete transcriptomes of neurons from the TRPV1 lineage and a non-TRPV1 neuroglial population in sensory ganglia through the combined application of next-gen deep RNA-Seq, genetic neuronal labeling with fluorescence-activated cell sorting, or neuron-selective chemoablation. RNA-Seq accurately quantitates gene expression, a difficult parameter to determine with most other methods, especially for very low and very high expressed genes. Differentially expressed genes are present at every level of cellular function from the nucleus to the plasma membrane. We identified many ligand receptor pairs in the TRPV1 population, suggesting that autonomous presynaptic regulation may be a major regulatory mechanism in nociceptive neurons. The data define, in a quantitative, cell population–specific fashion, the molecular signature of a distinct and clinically important group of pain-sensing neurons and provide an overall framework for understanding the transcriptome of TRPV1 nociceptive neurons. Perspective Next-gen RNA-Seq, combined with molecular genetics, provides a comprehensive and quantitative measurement of transcripts in TRPV1 lineage neurons and a contrasting transcriptome from non-TRPV1 neurons and cells. The transcriptome highlights previously unrecognized protein families, identifies multiple molecular circuits for excitatory or inhibitory autocrine and paracrine signaling, and suggests new combinatorial approaches to pain control.

Atypical and anaplastic meningiomas (AAM) represent 20% of all meningiomas. They are associated with poor outcomes due to their tendency to recur. While surgery and radiation (RT) are first line therapy, no effective systemic medical treatment has been identified. Protein phosphatase 2A (PP2A) is a ubiquitously expressed serine/threonine phosphatase involved in cell cycle regulation and DNA repair. Here, we examined radiosensitizing effects of LB-100, a novel inhibitor of PP2A against AAM as a novel treatment strategy. Three human-derived immortalized meningioma cell lines, IOMM-LEE, GAR, and CH-157, were used to investigate the radio-sensitizing potential of LB-100 in AAM. Survival fraction by clonogenic assay, immunofluorescence, cell cycle analysis and protein expression were evaluated in vitro. The antitumor effects of combining LB-100 with RT were verified in vivo by using intracranial orthotopic xenograft mouse model. Pharmacologic PP2A inhibition with LB-100 prior to RT enhanced the radiosensitivity of meningioma cells and reduced survival fraction in clonogenic assays. LB-100 increased DNA double-strand breakage (measured by γ-H2AX), mitotic catastrophe cell death, and G2/M cell cycle arrest in irradiated meningioma cells. Also, LB-100 decreased activation of STAT3 and expression of its downstream proteins. In vivo, LB-100 and RT combined treatment prolonged the survival of mice with xenografts compared to RT alone. Taken together, these results provide convincing preclinical data to support the use of LB-100 as a radiosensitizing agent for treatment of malignant meningioma. Its potential for clinical application deserves further investigation.

Introduction In vivo imaging biomarkers of various HIV neuropathologies, including dopaminergic dysfunction, are still lacking. Towards developing dopaminergic biomarkers of brain involvement in HIV, we assessed the pre and postsynaptic components of the dopaminergic system in the HIV-1 transgenic rat (Tg), a well-characterized model of treated HIV + patients, using small-animal PET imaging. Methods Fifteen to 18 month-old Tg and wild type (WT) rats were imaged with both [18F]-FP-CMT, a dopamine transporter (DAT) ligand (n = 16), and [18F]-Fallypride, a D2/D3 dopamine receptor (D2/D3DR) ligand (n = 16). Five to 8 month-old Tg and WT rats (n = 18) were also imaged with [18F]-FP-CMT. A subset of animals was imaged longitudinally at 7 and 17 months of age. Multiplex immunohistochemistry staining for DAT, tyrosine hydroxylase, D2DR, D3DR, GFAP, Iba1 and NeuN was performed on a subgroup of the scanned animals. Results [18F]-FP-CMT and [18F]-Fallypride binding potential (BPND) values were significantly lower in 15–18 month-old Tg compared to age-matched WT rats (p Conclusions We found presynaptic and postsynaptic dopaminergic dysfunction/loss in older Tg compared to WT rats. We believe this to be related to neurotoxicity of viral proteins present in the Tg rats’ serum and brain. Advances in knowledge Our findings confirm prior reports of neurobehavioral abnormalities suggestive of dopaminergic dysfunction in this model. They also suggest similarities between the Tg rat and HIV + patients as far as dopaminergic dysfunction. Implications for patient care The Tg rat, along with the above-described quantitative PET imaging biomarkers, can have a role in the evaluation of HIV neuroprotective therapies prior to human translation.

Context Remission failure following transsphenoidal surgery in Cushing disease (CD) from pituitary corticotroph tumors (CtTs) remains clinically challenging. Histone deacetylase inhibitors (HDACis) are antitumor drugs approved for clinical use, with the potential to affect adrenocorticotropin hormone (ACTH) hypersecretion by inhibiting pro-opiomelanocortin (POMC) transcription. Objective Testing the efficacy of suberoylanilide hydroxamic acid (SAHA) on human and murine ACTH-secreting tumor (AtT-20) cells. Design Cell viability, ACTH secretion (enzyme-linked immunosorbent assay), apoptosis, and gene expression profile were investigated on AtT-20 cells. In vivo efficacy was examined in an athymic nude mouse AtT-20 xenograft model. SAHA efficacy against human-derived corticotroph tumor (hCtT) (n = 8) was tested in vitro. Setting National Institutes of Health. Intervention SAHA (0.5 to 8 µM). Main Outcome Measures AtT-20 and hCtT cell survival, in vitro/invivo ACTH measurements. Results SAHA (1 µM) reduced AtT-20 viability to 75% at 24 hours, 43% at 48 hours (analysis of variance; P = 0.002). Apoptosis was confirmed with elevated BAX/Bcl2 ratio and FACS. Intriguingly, early (3-hour) significant decline (70%; P < 0.0001) of secreted ACTH and diminished POMC transcription was observed with SAHA (1 µM). Microarray analysis revealed a direct association between liver X receptor alpha (LXRα) and POMC expression. Accordingly, SAHA reduced LXRα in AtT-20 cells but not in normal murine corticotrophs. Xenografted nude-mice tumor involution (126 ± 33/160 ± 35 vs 337 ± 49 mm3; P = 0.0005) was observed with 5-day intraperitoneal SAHA, with reversal of elevated ACTH (P < 0.0001). SAHA did not affect serum ACTH in nontumor mice. Lastly, we confirmed that SAHA (1 µM/24 h) decreased hCtT survival (78.92%; P = 0.0007) and ACTH secretion (83.64%; P = 0.03). Conclusion Our findings demonstrate SAHA’s efficacy in reducing survival and ACTH secretion in AtT-20 and hCtT cells, providing a potential intervention for recurrent/unremitting CD.

Ischemic stroke continues to be a leading cause of morbidity and mortality throughout the world. To protect and/or repair the ischemic brain, a multitiered approach may be centered on neural stem cell (NSC) transplantation. Transplanted NSCs exert beneficial effects not only via structural replacement, but also via immunomodulatory and/or neurotrophic actions. Unfortunately, the clinical translation of such promising therapies remains elusive, in part due to their limited persistence/survivability within the hostile ischemic microenvironment. Herein, we discuss current approaches for the development of NSCs more amenable to survival within the ischemic brain as a tool for future cellular therapies in stroke.

Von Hippel-Lindau (VHL)-associated hemangioblastomas (VHL-HB) arise in the central nervous system (CNS), and are a leading cause of morbidity and mortality in VHL disease. Currently, surgical resection is the most effective way to manage symptomatic VHL-HBs. Surgically unresectable VHL-HBs or those in frail patients are challenging problems. Therapies targeting oncologic and vascular endothelial growth factor (VEGF) pathways have failed to demonstrate tumor control. Our experience and previous reports on VHL-HB avidity to somatostatin analogues suggested somatostatin receptor (SSTR) expression in VHL-HBs, offering an alternative therapeutic strategy. We explored this possibility by demonstrating consistent histologic expression of SSTR1, 2a, 4, and 5 in VHL-HBs. We found that somatostatin analogue octreotide induces apoptosis in VHL-HB stromal cells in a dose-dependent fashion by BAX – caspase-3 pathway unrelated to canonical VHL pathway. When administered to a patient with unresectable symptomatic suprasellar hemangioblastoma, octreotide resulted in tumor volume reduction, symptom stabilization, and tumor cytopenia on repeat 68Ga-DOTA-TATE positron emission tomography (PET) within 6 months, suggesting tumor infarction. We conclude that VHL-HBs harbor multiple SSTR subtypes that offer actionable chemo-therapeutic strategy for management of symptomatic, unresectable tumors by somatostatin analogue therapy.

Myeloid cells that orchestrate malignant progression in the tumor microenvironment offer targets for a generalized strategy to attack solid tumors. Through an analysis of tumor microenvironments, we explored an experimental model of lung cancer that uncovered a network of Dll4/Notch/TGF-β1 signals that links myeloid cells to cancer progression. Myeloid cells attracted to the tumor microenvironment by the tumor-derived cytokines CCL2 and M-CSF expressed increased levels of the Notch ligand Dll4, thereby activating Notch signaling in the tumor cells and amplifying tumor-intrinsic Notch activation. Heightened Dll4/Notch signaling in tumor cells magnified TGF-β–induced pSMAD2/3 signaling and was required to sustain TGF-β–induced tumor cell growth. Conversely, Notch blockade reduced TGF-β signaling and limited lung carcinoma tumor progression. Corroborating these findings, by interrogating RNAseq results from tumor and adjacent normal tissue in clinical specimens of human head and neck squamous carcinoma, we found evidence that TGF-β/Notch crosstalk contributed to progression. In summary, the myeloid cell-carcinoma signaling network we describe uncovers novel mechanistic links between the tumor microenvironment and tumor growth, highlighting new opportunities to target tumors where this network is active. Cancer Res; 74(7); 2038–49. ©2014 AACR.

Barrier-to-autointegration factor (BAF or BANF1) is highly conserved in multicellular eukaryotes and was first identified for its role in retroviral DNA integration. Homozygous BAF mutants are lethal and depletion of BAF results in defects in chromatin segregation during mitosis and subsequent nuclear envelope assembly. BAF exists both in phosphorylated and unphosphorylated forms with phosphorylation sites Thr-2, Thr-3, and Ser-4, near the N terminus. Vaccinia-related kinase 1 is the major kinase responsible for phosphorylation of BAF. We have identified the major phosphatase responsible for dephosphorylation of Ser-4 to be protein phosphatase 4 catalytic subunit. By examining the cellular distribution of phosphorylated BAF (pBAF) and total BAF (tBAF) through the cell cycle, we found that pBAF is associated with the core region of telophase chromosomes. Depletion of BAF or perturbing its phosphorylation state results not only in nuclear envelope defects, including mislocalization of LEM domain proteins and extensive invaginations into the nuclear interior, but also impaired cell cycle progression. This phenotype is strikingly similar to that seen in cells from patients with progeroid syndrome resulting from a point mutation in BAF.

BACKGROUND Glioblastoma-associated macrophages and microglia (GAMs) are the predominant immune cells in the tumor microenvironment. Activation of MerTK, a receptor tyrosine kinase, triggers efferocytosis and polarizes GAMs to an immunosuppressive phenotype, promoting glioma growth. Our previous findings showed that UNC2371, a small-molecule inhibitor of MerTK, induced a less immunosuppressive phenotype of GAMs. Here, we investigate the role of MerTK inhibition on glioblastoma cells in the tumor microenvironment in vitro and in vivo. METHODS Cytotoxicity of UNC2371 in glioblastoma cells was determined by cell viability and colony formation assays. The protein expression of MerTK, AKT, and Erk were quantified by Western blotting in UNC2371-treated glioblastoma cells. A syngeneic GL261 mouse orthotopic glioblastoma model was used to evaluate the survival benefit of UNC2371 treatment. Fluorescent multiplex immunohistochemistry (IHC) was used to evaluate the expression of CD206, an anti-inflammatory marker on GAMs in murine brain tumor tissues. RESULTS UNC2371 inhibited GBM cell growth with an EC50 < 100 nM in both human U251 and mouse GL261 glioma cells, but not in GAMs. UNC2371-induced cell death and decreased cell proliferation were demonstrated by colony formation assays. UNC2371 decreased protein expression of phosphorylated MerTK, AKT, and Erk, which are essential for cell survival signaling, in U251 and GL261 cells. Furthermore, UNC2371 treatment prolonged survival in the mouse orthotopic GL261 glioblastoma model, suggesting that UNC2371 induces glioma cell death. A decreased of CD206+ GAMs was found in mice glioma tissues by fluorescent multiplex IHC, consistent with our previous findings in the in vitro cell-based assays. These data suggest that in addition to alleviate immunosuppression in the glioma microenvironment, UNC2371 directly inhibits GBM cell growth in vitro and in vivo. CONCLUSION Our findings suggest that UNC2371 has a therapeutic benefit via promoting GAM polarization towards proinflammatory status in the glioblastoma microenvironment and unexpectedly, inducing tumor cell death.

Our Phase I trials of experimental virotherapy for recurrent glioblastoma (GBM) have shown that inoculation with a conditionally replication-competent early generation oncolytic herpes simplex virus (oHSV), G207, is safe. However, while 17 of 37 subjects experienced objective clinical responses, the highly attenuated oHSV did not uniformly improve survival. We sought to identify predictors that would identify mechanisms contributing to survival and improve future trial design, by studying accrued samples. We analyzed pre-treatment biopsy and post-G207-treatment tumor samples (collected D2-5 post injection) banked from the patients enrolled in the phase IB G207 trial. The key findings from these patients suggest that productive G207 infection and G207-induced changes in gene expression were predictive of oHSV therapeutic success. RNAseq-based transcriptome analysis of these samples revealed that both the intrinsic IFN mediated antiviral response and adaptive immune functional response in patients correlated significantly with improved survival following G207 inoculation. Further, GBM tissue stained using multiplex fluorescent immunohistochemistry supported differences in the tumor microenvironments that were identified from RNAseq data analysis. Our data indicate that both viral gene expression and the resulting intrinsic anti-viral and recruited adaptive response were critical for survival after G207 inoculation and predict survival with this early generation oHSV in patients with recurrent malignant glioma.

Summary Selective autophagy recycles damaged organelles and clears intracellular pathogens to prevent their aberrant accumulation. How ULK1 kinase is targeted and activated during selective autophagic events remains to be elucidated. In this study, we used chemically inducible dimerization (CID) assays in tandem with CRISPR KO lines to systematically analyze the molecular basis of selective autophagosome biogenesis. We demonstrate that ectopic placement of NDP52 on mitochondria or peroxisomes is sufficient to initiate selective autophagy by focally localizing and activating the ULK1 complex. The capability of NDP52 to induce mitophagy is dependent on its interaction with the FIP200/ULK1 complex, which is facilitated by TBK1. Ectopically tethering ULK1 to cargo bypasses the requirement for autophagy receptors and TBK1. Focal activation of ULK1 occurs independently of AMPK and mTOR. Our findings provide a parsimonious model of selective autophagy, which highlights the coordination of ULK1 complex localization by autophagy receptors and TBK1 as principal drivers of targeted autophagosome biogenesis., Graphical Abstract, Highlights • NDP52 associates with the ULK1 complex through FIP200, facilitated by TBK1 • NDP52/TBK1 targets ULK1 to cargo to initiate autophagy in the absence of LC3 • ULK1 is activated on cargo independently of AMPK and mTOR activity • Ectopic recruitment of FIP200-binding peptide is sufficient to degrade cargo, The targeting and activation mechanism of ULK1 complex during selective autophagy has been unclear. Here, Vargas et al. show that NDP52/TBK1 target the ULK1 complex to cargo in an LC3-independent manner and that ULK1 kinase activation is coupled to cargo localization, circumventing energy-sensing pathways.

Chronic immune activation is a major complication of antiretroviral therapy (ART) for HIV infection and can cause a devastating immune reconstitution inflammatory syndrome (IRIS) in the brain. The mechanism of T-cell activation in this population is not well understood. We found HIV-Tat protein and IL-17–expressing mononuclear cells in the brain of an individual with IRIS. Tat was also present in the CSF of individuals virologically controlled on ART. Hence we examined if Tat protein could directly activate T cells. Tat transcriptionally dysregulated 94 genes and induced secretion of 11 cytokines particularly activation of IL-17 signaling pathways supporting the development of a proinflammatory state. Tat increased IL-17 transcription and secretion in T cells. Tat entered the T cells rapidly by clathrin-mediated endocytosis and localized to both the cytoplasm and the nucleus. Tat activated T cells through a nonclassical pathway dependent upon vascular endothelial growth factor receptor-2 and downstream secondary signaling pathways but independent of the T-cell receptor. However, Tat stimulation of T cells did not induce T-cell proliferation but increased viral infectivity. This study demonstrates Tat’s role as a virulence factor, by driving T-cell activation and contributing to IRIS pathophysiology. This supports the necessity of an anti-Tat therapy in conjunction with ART and identifies multiple targetable pathways to prevent Tat-mediated T-cell activation.

Background: Several lines of evidence suggest a dichotomy between immune active and quiescent cancers, with the former associated with a good prognostic phenotype and better responsiveness to immunotherapy. Central to such dichotomy is the master regulator of the acute inflammatory process interferon regulatory factor (IRF)-1. However, it remains unknown whether the responsiveness of IRF-1 to cytokines is able to differentiate cancer immune phenotypes. Methods: IRF-1 activation was measured in 15 melanoma cell lines at basal level and after treatment with IFN-γ, TNF-α and a combination of both. Microarray analysis was used to compare transcriptional patterns between cell lines characterised by high or low IRF-1 activation. Results: We observed a strong positive correlation between IRF-1 activation at basal level and after IFN-γ and TNF-α treatment. Microarray demonstrated that three cell lines with low and three with high IRF-1 inducible translocation scores differed in the expression of 597 transcripts. Functional interpretation analysis showed mTOR and Wnt/β-cathenin as the top downregulated pathways in the cell lines with low inducible IRF-1 activation, suggesting that a low IRF-1 inducibility recapitulates a cancer phenotype already described in literature characterised by poor prognosis. Conclusion: Our findings support the central role of IRF-1 in influencing different tumour phenotypes.

O1 IL-15 primes an mTOR-regulated gene-expression program to prolong anti-tumor capacity of human natural killer cells #### Andreas Lundqvist1, Vincent van Hoef1, Xiaonan Zhang1, Erik Wennerberg2, Julie Lorent1, Kristina Witt1, Laia Masvidal Sanz1, Shuo Liang1, Shannon Murray3, Ola Larsson1

Arginine vasopressin (AVP) made by hypothalamic neurons is released into the circulation to stimulate water resorption by the kidneys and restore water balance after blood loss. Patients who lack this antidiuretic hormone suffer from central diabetes insipidus. We observed that many of these patients were anemic and asked whether AVP might play a role in red blood cell (RBC) production. We found that all three AVP receptors are expressed in human and mouse hematopoietic stem and progenitor cells. The AVPR1B appears to play the most important role in regulating erythropoiesis in both human and mouse cells. AVP increases phosphorylation of signal transducer and activator of transcription 5, as erythropoietin (EPO) does. After sublethal irradiation, AVP-deficient Brattleboro rats showed delayed recovery of RBC numbers compared to control rats. In mouse models of anemia (induced by bleeding, irradiation, or increased destruction of circulating RBCs), AVP increased the number of circulating RBCs independently of EPO. In these models, AVP appears to jump-start peripheral blood cell replenishment until EPO can take over. We suggest that specific AVPR1B agonists might be used to induce fast RBC production after bleeding, drug toxicity, or chemotherapy.

Polyglutamine expansion in the androgen receptor (AR) causes spinal and bulbar muscular atrophy (SBMA), an X-linked neuromuscular disease that is fully manifest only in males. It has been suggested that proteins with expanded polyglutamine tracts impair ubiquitin-dependent proteolysis due to their propensity to aggregate, but recent studies indicate that the overall activity of the ubiquitin-proteasome system is preserved in SBMA models. Here we report that AR selectively interferes with the function of the ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C), which, together with its substrate adaptor Cdh1, is critical for cell cycle arrest and neuronal architecture. We show that both wild-type and mutant AR physically interact with the APC/CCdh1 complex in a ligand-dependent fashion without being targeted for proteasomal degradation. Inhibition of APC/CCdh1 by mutant but not wild-type AR in PC12 cells results in enhanced neurite outgrowth which is typically followed by rapid neurite retraction and mitotic entry. Our data indicate a role of AR in neuronal differentiation through regulation of APC/CCdh1 and suggest abnormal cell cycle reactivation as a pathogenic mechanism in SBMA.