Nonneuronal cell types in the CNS are increasingly implicated as critical players in brain health and disease. While gene expression profiling of bulk brain tissue is routinely used to examine alterations in the brain under various conditions, it does not capture changes that occur within single cell types or allow interrogation of crosstalk among cell types. To this end, we have developed a concurrent brain cell type acquisition (CoBrA) methodology, enabling the isolation and profiling of microglia, astrocytes, endothelia, and oligodendrocytes from a single adult mouse forebrain. By identifying and validating anti-ACSA-2 and anti-CD49a antibodies as cell surface markers for astrocytes and vascular endothelial cells, respectively, and using established antibodies to isolate microglia and oligodendrocytes, we document that these 4 major cell types are isolated with high purity and RNA quality. We validated our procedure by performing acute peripheral LPS challenge, while highlighting the underappreciated changes occurring in astrocytes and vascular endothelia in addition to microglia. Furthermore, we assessed cell type–specific gene expression changes in response to amyloid pathology in a mouse model of Alzheimer’s disease. Our CoBrA methodology can be readily implemented to interrogate multiple CNS cell types in any mouse model at any age.
Dan B. Swartzlander, Nicholas E. Propson, Ethan R. Roy, Takashi Saito, Takaomi Saido, Baiping Wang, Hui Zheng
To what extent does the subarachnoid cerebrospinal fluid (CSF) compartment communicate directly with the extravascular compartment of human brain tissue? Interconnection between the subarachnoid CSF compartment and brain perivascular spaces is reported in some animal studies, but with controversy, and in vivo CSF tracer studies in humans are lacking. In the present work, we examined the distribution of a CSF tracer in the human brain by MRI over a prolonged time span. For this, we included a reference cohort, representing close to healthy individuals, and a cohort of patients with dementia and anticipated compromise of CSF circulation (idiopathic normal pressure hydrocephalus). The MRI contrast agent gadobutrol, which is confined to the extravascular brain compartment by the intact blood-brain barrier, was used as a CSF tracer. Standardized T1-weighted MRI scans were performed before and after intrathecal gadobutrol at defined time points, including at 24 hours, 48 hours, and 4 weeks. All MRI scans were aligned and brain regions were segmented using FreeSurfer, and changes in normalized T1 signals over time were quantified as percentage change from baseline. The study provides in vivo evidence of access to all human brain subregions of a substance administered intrathecally. Clearance of the tracer substance was delayed in the dementia cohort. These observations translate previous findings in animal studies into humans and open new prospects concerning intrathecal treatment regimens, extravascular contrast-enhanced MRI, and assessment of brain clearance function.
Geir Ringstad, Lars M. Valnes, Anders M. Dale, Are H. Pripp, Svein-Are S. Vatnehol, Kyrre E. Emblem, Kent-Andre Mardal, Per K. Eide
Loss of the NF1 tumor suppressor gene causes the autosomal dominant condition, neurofibromatosis type 1 (NF1). Children and adults with NF1 suffer from pathologies including benign and malignant tumors to cognitive deficits, seizures, growth abnormalities, and peripheral neuropathies. NF1 encodes neurofibromin, a Ras-GTPase activating protein, and NF1 mutations result in hyperactivated Ras signaling in patients. Existing NF1 mutant mice mimic individual aspects of NF1, but none comprehensively models the disease. We describe a potentially novel Yucatan miniswine model bearing a heterozygotic mutation in NF1 (exon 42 deletion) orthologous to a mutation found in NF1 patients. NF1+/ex42del miniswine phenocopy the wide range of manifestations seen in NF1 patients, including café au lait spots, neurofibromas, axillary freckling, and neurological defects in learning and memory. Molecular analyses verified reduced neurofibromin expression in swine NF1+/ex42del fibroblasts, as well as hyperactivation of Ras, as measured by increased expression of its downstream effectors, phosphorylated ERK1/2, SIAH, and the checkpoint regulators p53 and p21. Consistent with altered pain signaling in NF1, dysregulation of calcium and sodium channels was observed in dorsal root ganglia expressing mutant NF1. Thus, these NF1+/ex42del miniswine recapitulate the disease and provide a unique, much-needed tool to advance the study and treatment of NF1.
Katherine A. White, Vicki J. Swier, Jacob T. Cain, Jordan L. Kohlmeyer, David K. Meyerholz, Munir R. Tanas, Johanna Uthoff, Emily Hammond, Hua Li, Frank A. Rohret, Adam Goeken, Chun-Hung Chan, Mariah R. Leidinger, Shaikamjad Umesalma, Margaret R. Wallace, Rebecca D. Dodd, Karin Panzer, Amy H. Tang, Benjamin W. Darbro, Aubin Moutal, Song Cai, Wennan Li, Shreya S. Bellampalli, Rajesh Khanna, Christopher S. Rogers, Jessica C. Sieren, Dawn E. Quelle, Jill M. Weimer
BACKGROUND. Prader-Willi syndrome (PWS) is a genetic neurodevelopmental disorder of life-threatening hyperphagia, obesity, intellectual deficits, compulsivity, and other behavioral problems. The efficacy and safety of i.n. carbetocin, an oxytocin analog, was evaluated in a prospective, randomized, double-blinded trial in adolescents with PWS. METHODS. Eligible patients aged 10–18 years with genetically confirmed PWS were randomized (1:1) to i.n. carbetocin or placebo 3 times daily for 14 days. The primary efficacy endpoint was change in parent/caregiver-rated Hyperphagia in PWS Questionnaire–Responsiveness (HPWSQ-R) total score. Secondary efficacy endpoints included HPWSQ-R behavior, drive, and severity domains; clinician-rated HPWSQ; Children’s Yale-Brown Obsessive-Compulsive Severity Scale; food domain of the Reiss Profile; and Clinical Global Impression–Improvement scale. Endpoints were assessed using analysis of covariance. Relationship between primary and secondary endpoints was assessed using Pearson correlation coefficients. Safety was assessed throughout the study. RESULTS. Demographics and clinical characteristics were similar between treatment groups (carbetocin, n = 17; placebo, n = 20). Patients receiving carbetocin had statistically significant reductions in HPWSQ-R total score at study end (–15.6) versus patients receiving placebo (–8.9; P = 0.029); several secondary efficacy endpoints also demonstrated significant differences (P < 0.05). Treatment effects for the primary and secondary endpoints were highly correlated (P ≤ 0.0001). Incidence of adverse events (AEs) was similar between treatment groups. CONCLUSION. I.n. carbetocin was well tolerated and improved hyperphagia and behavioral symptoms of PWS. TRIAL REGISTRATION. ClinicalTrials.gov: NCT01968187 FUNDING. The study was funded by Ferring Pharmaceuticals. Recruitment was aided by ongoing work in PWS performed through Eunice Kennedy Shriver National Institute of Child Health and Human Development grant U54 HD083211.
Elisabeth M. Dykens, Jennifer Miller, Moris Angulo, Elizabeth Roof, Michael Reidy, Hind T. Hatoum, Richard Willey, Guy Bolton, Paul Korner
Functional bowel disorder patients can suffer from chronic abdominal pain, likely due to visceral hypersensitivity to mechanical stimuli. As there is only a limited understanding of the basis of chronic visceral hypersensitivity (CVH), drug-based management strategies are ill defined, vary considerably, and include NSAIDs, opioids, and even anticonvulsants. We previously reported that the 1.1 subtype of the voltage-gated sodium (NaV; NaV1.1) channel family regulates the excitability of sensory nerve fibers that transmit a mechanical pain message to the spinal cord. Herein, we investigated whether this channel subtype also underlies the abdominal pain that occurs with CVH. We demonstrate that NaV1.1 is functionally upregulated under CVH conditions and that inhibiting channel function reduces mechanical pain in 3 mechanistically distinct mouse models of chronic pain. In particular, we use a small molecule to show that selective NaV1.1 inhibition (a) decreases sodium currents in colon-innervating dorsal root ganglion neurons, (b) reduces colonic nociceptor mechanical responses, and (c) normalizes the enhanced visceromotor response to distension observed in 2 mouse models of irritable bowel syndrome. These results provide support for a relationship between NaV1.1 and chronic abdominal pain associated with functional bowel disorders.
Juan Salvatierra, Joel Castro, Andelain Erickson, Qian Li, Joao Braz, John Gilchrist, Luke Grundy, Grigori Y. Rychkov, Annemie Deiteren, Rana Rais, Glenn F. King, Barbara S. Slusher, Allan Basbaum, Pankaj J. Pasricha, Stuart M. Brierley, Frank Bosmans
Multiple sclerosis (MS) is an autoimmune disease characterized by CNS inflammation leading to demyelination and axonal damage. IFN-β is an established treatment for MS; however, up to 30% of IFN-β–treated MS patients develop neutralizing antidrug antibodies (nADA), leading to reduced drug bioactivity and efficacy. Mechanisms driving antidrug immunogenicity remain uncertain, and reliable biomarkers to predict immunogenicity development are lacking. Using high-throughput flow cytometry, NOTCH2 expression on CD14+ monocytes and increased frequency of proinflammatory monocyte subsets were identified as baseline predictors of nADA development in MS patients treated with IFN-β. The association of this monocyte profile with nADA development was validated in 2 independent cross-sectional MS patient cohorts and a prospective cohort followed before and after IFN-β administration. Reduced monocyte NOTCH2 expression in nADA+ MS patients was associated with NOTCH2 activation measured by increased expression of Notch-responsive genes, polarization of monocytes toward a nonclassical phenotype, and increased proinflammatory IL-6 production. NOTCH2 activation was T cell dependent and was only triggered in the presence of serum from nADA+ patients. Thus, nADA development was driven by a proinflammatory environment that triggered activation of the NOTCH2 signaling pathway prior to first IFN-β administration.
Marsilio Adriani, Petra Nytrova, Cyprien Mbogning, Signe Hässler, Karel Medek, Poul Erik H. Jensen, Paul Creeke, Clemens Warnke, Kathleen Ingenhoven, Bernhard Hemmer, Claudia Sievers, Raija L.P. Lindberg Gasser, Nicolas Fissolo, Florian Deisenhammer, Zsolt Bocskei, Vincent Mikol, Anna Fogdell-Hahn, Eva Kubala Havrdova, Philippe Broët, Pierre Dönnes, Claudia Mauri, Elizabeth C. Jury, The ABIRISK Consortium
Elevated levels of brain natriuretic peptide (BNP) are regarded as an early compensatory response to cardiac myocyte hypertrophy, although exogenously administered BNP shows poor clinical efficacy in heart failure and hypertension. We tested whether phosphodiesterase 2A (PDE2A), which regulates the action of BNP-activated cyclic guanosine monophosphate (cGMP), was directly involved in modulating Ca2+ handling from stellate ganglia (SG) neurons and cardiac norepinephrine (NE) release in rats and humans with an enhanced sympathetic phenotype. SG were also isolated from patients with sympathetic hyperactivity and healthy donor patients. PDE2A activity of the SG was greater in both spontaneously hypertensive rats (SHRs) and patients compared with their respective controls, whereas PDE2A mRNA was only high in SHR SG. BNP significantly reduced the magnitude of the calcium transients and ICaN in normal Wistar Kyoto (WKY) SG neurons, but not in the SHRs. cGMP levels stimulated by BNP were also attenuated in SHR SG neurons. Overexpression of PDE2A in WKY neurons recapitulated the calcium phenotype seen in SHR neurons. Functionally, BNP significantly reduced [3H]-NE release in the WKY rats, but not in the SHRs. Blockade of overexpressed PDE2A with Bay 60-7550 or overexpression of catalytically inactive PDE2A reestablished the modulatory action of BNP in SHR SG neurons. This suggests that PDE2A may be a key target in modulating the action of BNP to reduce sympathetic hyperactivity.
Kun Liu, Dan Li, Guoliang Hao, David McCaffary, Oliver Neely, Lavinia Woodward, Demetris Ioannides, Chieh-Ju Lu, Marcella Brescia, Manuela Zaccolo, Harikrishna Tandri, Olujimi A. Ajijola, Jeffrey L. Ardell, Kalyanam Shivkumar, David J. Paterson
Sepsis-associated encephalopathy manifesting as delirium is a common problem in critical care medicine. In this study, patients that had delirium due to sepsis had significant cognitive impairments at 12–18 months after hospital discharge when compared with controls and Cambridge Neuropsychological Automated Test Battery–standardized scores in spatial recognition memory, pattern recognition memory, and delayed-matching-to-sample tests but not other cognitive functions. A mouse model of S. pneumoniae pneumonia-induced sepsis, which modeled numerous aspects of the human sepsis-associated multiorgan dysfunction, including encephalopathy, also revealed similar deficits in spatial memory but not new task learning. Both humans and mice had large increases in chemokines for myeloid cell recruitment. Intravital imaging of the brains of septic mice revealed increased neutrophil and CCR2+ inflammatory monocyte recruitment (the latter being far more robust), accompanied by subtle microglial activation. Prevention of CCR2+ inflammatory monocyte recruitment, but not neutrophil recruitment, reduced microglial activation and other signs of neuroinflammation and prevented all signs of cognitive impairment after infection. Therefore, therapeutically targeting CCR2+ inflammatory monocytes at the time of sepsis may provide a novel neuroprotective clinical intervention to prevent the development of persistent cognitive impairments.
Graciela Andonegui, Erin L. Zelinski, Courtney L. Schubert, Derrice Knight, Laura A. Craig, Brent W. Winston, Simon C. Spanswick, Björn Petri, Craig N. Jenne, Janice C. Sutherland, Rita Nguyen, Natalie Jayawardena, Margaret M. Kelly, Christopher J. Doig, Robert J. Sutherland, Paul Kubes
Androgen excess is a hallmark of polycystic ovary syndrome (PCOS), a prevalent yet poorly understood endocrine disorder. Evidence from women and preclinical animal models suggests that elevated perinatal androgens can elicit PCOS onset in adulthood, implying androgen actions in both PCOS ontogeny and adult pathophysiology. Prenatally androgenized (PNA) mice exhibit a robust increase of progesterone-sensitive GABAergic inputs to gonadotropin-releasing hormone (GnRH) neurons implicated in the pathogenesis of PCOS. It is unclear when altered GABAergic wiring develops in the brain, and whether these central abnormalities are dependent upon adult androgen excess. Using GnRH-GFP–transgenic mice, we determined that increased GABA input to GnRH neurons occurs prior to androgen excess and the manifestation of reproductive impairments in PNA mice. These data suggest that brain circuit abnormalities precede the postpubertal development of PCOS traits. Despite the apparent developmental programming of circuit abnormalities, long-term blockade of androgen receptor signaling from early adulthood rescued normal GABAergic wiring onto GnRH neurons, improved ovarian morphology, and restored reproductive cycles in PNA mice. Therefore, androgen excess maintains changes in female brain wiring linked to PCOS features and the blockade of androgen receptor signaling reverses both the central and peripheral PNA-induced PCOS phenotype.
Mauro S.B. Silva, Melanie Prescott, Rebecca E. Campbell
Pain is the predominant symptom of osteoarthritis, but the connection between joint damage and the genesis of pain is not well understood. Loss of articular cartilage is a hallmark of osteoarthritis, and it occurs through enzymatic degradation of aggrecan by cleavage mediated by a disintegrin and metalloproteinase with thrombospondin motif 4 (ADAMTS-4) or ADAMTS-5 in the interglobular domain (E373–374A). Further cleavage by MMPs (N341–342F) releases a 32-amino-acid aggrecan fragment (32-mer). We investigated the role of this 32-mer in driving joint pain. We found that the 32-mer excites dorsal root ganglion nociceptive neurons, both in culture and in intact explants. Treatment of cultured sensory neurons with the 32-mer induced expression of the proalgesic chemokine CCL2. These effects were mediated through TLR2, which we demonstrated was expressed by nociceptive neurons. In addition, intra-articular injection of the 32-mer fragment provoked knee hyperalgesia in WT but not Tlr2-null mice. Blocking the production or action of the 32-mer in transgenic mice prevented the development of knee hyperalgesia in a murine model of osteoarthritis. These findings suggest that the aggrecan 32-mer fragment directly activates TLR2 on joint nociceptors and is an important mediator of the development of osteoarthritis-associated joint pain.
Rachel E. Miller, Shingo Ishihara, Phuong B. Tran, Suzanne B. Golub, Karena Last, Richard J. Miller, Amanda J. Fosang, Anne-Marie Malfait
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