Bi-allelic JAM2 Variants Lead to Early-Onset Recessive Primary Familial Brain Calcification.

Primary familial brain calcification (PFBC) is a rare neurodegenerative disorder characterized by a combination of neurological, psychiatric, and cognitive decline associated with calcium deposition on brain imaging. To date, mutations in five genes have been linked to PFBC. However, more than 50% of individuals affected by PFBC have no molecular diagnosis. We report four unrelated families presenting with initial learning difficulties and seizures and later psychiatric symptoms, cerebellar ataxia, extrapyramidal signs, and extensive calcifications on brain imaging. Through a combination of homozygosity mapping and exome sequencing, we mapped this phenotype to chromosome 21q21.3 and identified bi-allelic variants in JAM2. JAM2 encodes for the junctional-adhesion-molecule-2, a key tight-junction protein in blood-brain-barrier permeability. We show that JAM2 variants lead to reduction of JAM2 mRNA expression and absence of JAM2 protein in patient's fibroblasts, consistent with a loss-of-function mechanism. We show that the human phenotype is replicated in the jam2 complete knockout mouse (jam2 KO). Furthermore, neuropathology of jam2 KO mouse showed prominent vacuolation in the cerebral cortex, thalamus, and cerebellum and particularly widespread vacuolation in the midbrain with reactive astrogliosis and neuronal density reduction. The regions of the human brain affected on neuroimaging are similar to the affected brain areas in the myorg PFBC null mouse. Along with JAM3 and OCLN, JAM2 is the third tight-junction gene in which bi-allelic variants are associated with brain calcification, suggesting that defective cell-to-cell adhesion and dysfunction of the movement of solutes through the paracellular spaces in the neurovascular unit is a key mechanism in CNS calcification.

Evidence for human transmission of amyloid-ß pathology and cerebral amyloid angiopathy.

More than two hundred individuals developed Creutzfeldt-Jakob disease (CJD) worldwide as a result of treatment, typically in childhood, with human cadaveric pituitary-derived growth hormone contaminated with prions. Although such treatment ceased in 1985, iatrogenic CJD (iCJD) continues to emerge because of the prolonged incubation periods seen in human prion infections. Unexpectedly, in an autopsy study of eight individuals with iCJD, aged 36-51 years, in four we found moderate to severe grey matter and vascular amyloid-ß (Aß) pathology. The Aß deposition in the grey matter was typical of that seen in Alzheimer's disease and Aß in the blood vessel walls was characteristic of cerebral amyloid angiopathy and did not co-localize with prion protein deposition. None of these patients had pathogenic mutations, APOE ε4 or other high-risk alleles associated with early-onset Alzheimer's disease. Examination of a series of 116 patients with other prion diseases from a prospective observational cohort study showed minimal or no Aß pathology in cases of similar age range, or a decade older, without APOE ε4 risk alleles. We also analysed pituitary glands from individuals with Aß pathology and found marked Aß deposition in multiple cases. Experimental seeding of Aß pathology has been previously demonstrated in primates and transgenic mice by central nervous system or peripheral inoculation with Alzheimer's disease brain homogenate. The marked deposition of parenchymal and vascular Aß in these relatively young patients with iCJD, in contrast with other prion disease patients and population controls, is consistent with iatrogenic transmission of Aß pathology in addition to CJD and suggests that healthy exposed individuals may also be at risk of iatrogenic Alzheimer's disease and cerebral amyloid angiopathy. These findings should also prompt investigation of whether other known iatrogenic routes of prion transmission may also be relevant to Aß and other proteopathic seeds associated with neurodegenerative and other human diseases.

Prevalence in Britain of abnormal prion protein in human appendices before and after exposure to the cattle BSE epizootic.

Widespread dietary exposure of the population of Britain to bovine spongiform encephalopathy (BSE) prions in the 1980s and 1990s led to the emergence of variant Creutzfeldt-Jakob Disease (vCJD) in humans. Two previous appendectomy sample surveys (Appendix-1 and -2) estimated the prevalence of abnormal prion protein (PrP) in the British population exposed to BSE to be 237 per million and 493 per million, respectively. The Appendix-3 survey was recommended to measure the prevalence of abnormal PrP in population groups thought to have been unexposed to BSE. Immunohistochemistry for abnormal PrP was performed on 29,516 samples from appendices removed between 1962 and 1979 from persons born between 1891 through 1965, and from those born after 1996 that had been operated on from 2000 through 2014. Seven appendices were positive for abnormal PrP, of which two were from the pre-BSE-exposure era and five from the post BSE-exposure period. None of the seven positive samples were from appendices removed before 1977, or in patients born after 2000 and none came from individuals diagnosed with vCJD. There was no statistical difference in the prevalence of abnormal PrP across birth and exposure cohorts. Two interpretations are possible. Either there is a low background prevalence of abnormal PrP in human lymphoid tissues that may not progress to vCJD. Alternatively, all positive specimens are attributable to BSE exposure, a finding that would necessitate human exposure having begun in the late 1970s and continuing through the late 1990s.

Neuronal and Peripheral Pentraxins Modify Glutamate Release and may Interact in Blood-Brain Barrier Failure.

Neuronal pentraxin 1 (NPTX1) has been implicated in Alzheimer's disease, being present in and around dystrophic neurons in plaques, affecting glutamatergic transmission postsynaptically and mediating effects of amyloidß. Here, we confirm the presence of NPTX1 around plaques in postmortem Alzheimer's disease brain and report that acutely applied human NPTX1 increases paired-pulse ratio at mouse CA3-CA1 hippocampal synapses, indicating a decrease in glutamate release. In contrast, chronic exposure to NPTX1, NPTX2, or NPTX receptor decreases paired-pulse ratio, mimicking some of the earliest changes in mice expressing familial Alzheimer's disease genes. The peripheral pentraxin, serum amyloid P component (SAP), causes similar synaptic effects to NPTX1. The presence of SAP on amyloid plaques in Alzheimer's disease confirms that it can enter the brain. We show that SAP and neuronal pentraxins can interact and that SAP can enter the brain if the blood-brain barrier is compromised, suggesting that peripheral pentraxins could affect central synaptic transmission via this interaction, especially in the event of blood-brain barrier breakdown.

Selective Interleukin-6 Trans-Signaling Blockade Is More Effective Than Panantagonism in Reperfused Myocardial Infarction.

Interleukin (IL)-6 is an emerging therapeutic target in myocardial infarction (MI). IL-6 has 2 distinct signaling pathways: trans-signaling, which mediates inflammation, and classic signaling, which also has anti-inflammatory effects. The novel recombinant fusion protein sgp130Fc achieves exclusive trans-signaling blockade, whereas anti-IL-6 antibodies (Abs) result in panantagonism. In a rat model of reperfused MI, sgp130Fc, but not anti-IL-6-Ab, attenuated neutrophil and macrophage infiltration into the myocardium, reduced infarct size, and preserved cardiac function 28 days after MI. These data demonstrate the efficacy of exclusive IL-6 trans-signaling blockade and support further investigation of sgp130Fc as a potential novel therapy in MI.

Amyloid ß oligomers constrict human capillaries in Alzheimer's disease via signaling to pericytes.

Cerebral blood flow is reduced early in the onset of Alzheimer's disease (AD). Because most of the vascular resistance within the brain is in capillaries, this could reflect dysfunction of contractile pericytes on capillary walls. We used live and rapidly fixed biopsied human tissue to establish disease relevance, and rodent experiments to define mechanism. We found that in humans with cognitive decline, amyloid ß (Aß) constricts brain capillaries at pericyte locations. This was caused by Aß generating reactive oxygen species, which evoked the release of endothelin-1 (ET) that activated pericyte ETA receptors. Capillary, but not arteriole, constriction also occurred in vivo in a mouse model of AD. Thus, inhibiting the capillary constriction caused by Aß could potentially reduce energy lack and neurodegeneration in AD.

Mutant IDH Sensitizes Gliomas to Endoplasmic Reticulum Stress and Triggers Apoptosis via miR-183-Mediated Inhibition of Semaphorin 3E.

Human astrocytomas and oligodendrogliomas are defined by mutations of the metabolic enzymes isocitrate dehydrogenase (IDH) 1 or 2, resulting in the production of the abnormal metabolite D-2 hydroxyglutarate. Here, we studied the effect of mutant IDH on cell proliferation and apoptosis in a glioma mouse model. Tumors were generated by inactivating Pten and p53 in forebrain progenitors and compared with tumors additionally expressing the Idh1 R132H mutation. Idh-mutant cells proliferated less in vitro and mice with Idh-mutant tumors survived significantly longer compared with Idh-wildtype mice. Comparison of miRNA and RNA expression profiles of Idh-wildtype and Idh-mutant cells and tumors revealed miR-183 was significantly upregulated in IDH-mutant cells. Idh-mutant cells were more sensitive to endoplasmic reticulum (ER) stress, resulting in increased apoptosis and thus reduced cell proliferation and survival. This was mediated by the interaction of miR-183 with the 5' untranslated region of semaphorin 3E, downregulating its function as an apoptosis suppressor. In conclusion, we show that mutant Idh1 delays tumorigenesis and sensitizes tumor cells to ER stress and apoptosis. This may open opportunities for drug treatments targeting the miR-183-semaphorin axis. SIGNIFICANCE: The pathologic metabolite 2-hydroxyglutarate, generated by IDH-mutant astrocytomas, sensitizes tumor cells to ER stress and delays tumorigenesis. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/79/19/4994/F1.large.jpg.

Pro-resolving mediators promote resolution in a human skin model of UV-killed Escherichia coli-driven acute inflammation.

While the treatment of inflammatory disorders is generally based on inhibiting factors that drive onset of inflammation, these therapies can compromise healing (NSAIDs) or dampen immunity against infections (biologics). In search of new antiinflammatories, efforts have focused on harnessing endogenous pathways that drive resolution of inflammation for therapeutic gain. Identification of specialized pro-resolving mediators (SPMs) (lipoxins, resolvins, protectins, maresins) as effector molecules of resolution has shown promise in this regard. However, their action on inflammatory resolution in humans is unknown. Here, we demonstrate using a model of UV-killed Escherichia coli-triggered skin inflammation that SPMs are biosynthesized at the local site at the start of resolution, coinciding with the expression of receptors that transduce their actions. These include receptors for lipoxin A4 (ALX/FPR2), resolvin E1 (ChemR23), resolvin D2 (GPR18), and resolvin D1 (GPR32) that were differentially expressed on the endothelium and infiltrating leukocytes. Administering SPMs into the inflamed site 4 hours after bacterial injection caused a reduction in PMN numbers over the ensuing 6 hours, the phase of active resolution in this model. These results indicate that in humans, the appearance of SPMs and their receptors is associated with the beginning of inflammatory resolution and that their therapeutic supplementation enhanced the resolution response.

Inhibition of GPR158 by microRNA-449a suppresses neural lineage of glioma stem/progenitor cells and correlates with higher glioma grades.

To identify biomarkers for glioma growth, invasion and progression, we used a candidate gene approach in mouse models with two complementary brain tumour phenotypes, developing either slow-growing, diffusely infiltrating gliomas or highly proliferative, non-invasive primitive neural tumours. In a microRNA screen we first identified microRNA-449a as most significantly differentially expressed between these two tumour types. miR-449a has a target dependent effect, inhibiting cell growth and migration by downregulation of CCND1 and suppressing neural phenotypes by inhibition of G protein coupled-receptor (GPR) 158. GPR158 promotes glioma stem cell differentiation and induces apoptosis and is highest expressed in the cerebral cortex and in oligodendrogliomas, lower in IDH mutant astrocytomas and lowest in the most malignant form of glioma, IDH wild-type glioblastoma. The correlation of GPR158 expression with molecular subtypes, patient survival and therapy response suggests a possible role of GPR158 as prognostic biomarker in human gliomas.

Prolonged immune alteration following resolution of acute inflammation in humans.

Acute inflammation is an immediate response to infection and injury characterised by the influx of granulocytes followed by phagocytosing mononuclear phagocytes. Provided the antigen is cleared and the immune system of the host is fully functional, the acute inflammatory response will resolve. Until now it is considered that resolution then leads back to homeostasis, the physiological state tissues experienced before inflammation occurred. Using a human model of acute inflammation driven by intradermal UV killed Escherichia coli, we found that bacteria and granulocyte clearance as well as pro-inflammatory cytokine catabolism occurred by 72h. However, following a lag phase of about 4 days there was an increase in numbers of memory T cells and CD163+ macrophage at the post-resolution site up to day 17 as well as increased biosynthesis of cyclooxygenase-derived prostanoids and DHA-derived D series resolvins. Inhibiting post-resolution prostanoids using naproxen showed that numbers of tissue memory CD4 cells were under the endogenous control of PGE2, which exerts its suppressive effects on T cell proliferation via the EP4 receptor. In addition, we re-challenged the post-resolution site with a second injection of E. coli, which when compared to saline controls resulted in primarily a macrophage-driven response with comparatively fewer PMNs; the macrophage-dominated response was reversed by cyclooxygenase inhibition. Re-challenge experiments were also carried out in mice where we obtained similar results as in humans. Therefore, we report that acute inflammatory responses in both humans and rodents do not revert back to homeostasis, but trigger a hitherto unappreciated sequence of immunological events that dictate subsequent immune response to infection.

Generation of brain tumours in mice by Cre-mediated recombination of neural progenitors in situ with the tamoxifen metabolite endoxifen.

Targeted cell- or region-specific gene recombination is widely used in the functional analysis of genes implicated in development and disease. In the brain, targeted gene recombination has become a mainstream approach to study neurodegeneration or tumorigenesis. The use of the Cre-loxP system to study tumorigenesis in the adult central nervous system (CNS) can be limited, when the promoter (such as GFAP) is also transiently expressed during development, which can result in the recombination of progenies of different lineages. Engineering of transgenic mice expressing Cre recombinase fused to a mutant of the human oestrogen receptor (ER) allows the circumvention of transient developmental Cre expression by inducing recombination in the adult organism. The recombination of loxP sequences occurs only in the presence of tamoxifen. Systemic administration of tamoxifen can, however, exhibit toxicity and might also recombine unwanted cell populations if the promoter driving Cre expression is active at the time of tamoxifen administration. Here, we report that a single site-specific injection of an active derivative of tamoxifen successfully activates Cre recombinase and selectively recombines tumour suppressor genes in neural progenitor cells of the subventricular zone in mice, and we demonstrate its application in a model for the generation of intrinsic brain tumours.

Comparative expression analysis reveals lineage relationships between human and murine gliomas and a dominance of glial signatures during tumor propagation in vitro.

Brain tumors are thought to originate from stem/progenitor cell populations that acquire specific genetic mutations. Although current preclinical models have relevance to human pathogenesis, most do not recapitulate the histogenesis of the human disease. Recently, a large series of human gliomas and medulloblastomas were analyzed for genetic signatures of prognosis and therapeutic response. Using a mouse model system that generates three distinct types of intrinsic brain tumors, we correlated RNA and protein expression levels with human brain tumors. A combination of genetic mutations and cellular environment during tumor propagation defined the incidence and phenotype of intrinsic murine tumors. Importantly, in vitro passage of cancer stem cells uniformly promoted a glial expression profile in culture and in brain tumors. Gene expression profiling revealed that experimental gliomas corresponded to distinct subclasses of human glioblastoma, whereas experimental supratentorial primitive neuroectodermal tumors (sPNET) correspond to atypical teratoid/rhabdoid tumor (AT/RT), a rare childhood tumor.

Prevalent abnormal prion protein in human appendixes after bovine spongiform encephalopathy epizootic: large scale survey.

OBJECTIVES: To carry out a further survey of archived appendix samples to understand better the differences between existing estimates of the prevalence of subclinical infection with prions after the bovine spongiform encephalopathy epizootic and to see whether a broader birth cohort was affected, and to understand better the implications for the management of blood and blood products and for the handling of surgical instruments. DESIGN: Irreversibly unlinked and anonymised large scale survey of archived appendix samples. SETTING: Archived appendix samples from the pathology departments of 41 UK hospitals participating in the earlier survey, and additional hospitals in regions with lower levels of participation in that survey. SAMPLE: 32,441 archived appendix samples fixed in formalin and embedded in paraffin and tested for the presence of abnormal prion protein (PrP). RESULTS: Of the 32,441 appendix samples 16 were positive for abnormal PrP, indicating an overall prevalence of 493 per million population (95% confidence interval 282 to 801 per million). The prevalence in those born in 1941-60 (733 per million, 269 to 1596 per million) did not differ significantly from those born between 1961 and 1985 (412 per million, 198 to 758 per million) and was similar in both sexes and across the three broad geographical areas sampled. Genetic testing of the positive specimens for the genotype at PRNP codon 129 revealed a high proportion that were valine homozygous compared with the frequency in the normal population, and in stark contrast with confirmed clinical cases of vCJD, all of which were methionine homozygous at PRNP codon 129. CONCLUSIONS: This study corroborates previous studies and suggests a high prevalence of infection with abnormal PrP, indicating vCJD carrier status in the population compared with the 177 vCJD cases to date. These findings have important implications for the management of blood and blood products and for the handling of surgical instruments.