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(dabigatran, rivaroxaban and apixaban), as well as heparin (http://www.perosphere.com/pdf/PER977_AHA-presentation.pdf). Such binding allows rapid reestablishment of normal blood coagulation. To date, PER977 has been shown to rapidly and completely reverse the anticoagulant effects of the Xa and IIa inhibitors in vitro, in a rat-tail transection bleeding model, and ex vivo, in human plasma and human whole blood. Further studies are ongoing with this agent.

New target-specific oral anticoagulants offer great advantages over traditional vitamin K antagonists, and they are poised to change the landscape of how we treat venous throm-boembolic disease. However, without specific methodologies or antidotes to treat major

bleeding episodes or to reverse their antico-agulant effect, physicians will be reluctant to use these agents. The work of Lu et al.11 and others suggests that we may be on the path to developing effective antidotes, which can hopefully maximize the therapeutic potential of next-generation oral anticoagulants.

COMPETING FINANCIAL INTERESTS The author declares competing financial interests: details are available in the online version of the paper.

1. Ansell, J.E. Arch. Intern. Med. 153, 586–596 (1993).

2. Thomas, K. A promising drug with a flaw. New York Times (3 November 2012).

3. Kaatz, S. et al. Am. J. Hematol. 87, S141–S145 (2012).

4. Budnitz, D.S., Lovegrove, M.C., Shehab, N. & Richards, C.L. N. Engl. J. Med. 365, 2002–2012 (2011).

5. QuarterWatch. http://www.ismp.org/quarterwatch/pdfs/2011Q4.pdf (2012).

6. Sarode, R. et al. Am. J. Hematol. 87, S146 (2012).

7. Bauer, K.A. Am. J. Hematol. 87, S119–S126 (2012).8. Dager, W. & Roberts, A. Crit. Care Med. 39 (suppl. 12),

243 (2011).9. Garber, S.T., Sivakumar, W. & Schmidt, R.H.

J. Neurosurg. 116, 1093–1096 (2012).10. Levi, M. Thromb. Haemost. 108, 201–202 (2012).11. Lu, G. et al. Nat. Med. 19, 446–451 (2013).12. Marketwire. http://www.marketwire.com/press-release/

portola-initiates-phase-2-study-prt4445-universal-antidote-factor-xa-inhibitor-anticoagulants-1735434.htm (2012).

13. Rusconi, C.P. et al. Nat. Biotechnol. 22, 1423–1428 (2004).

14. Povsic, T.J. et al. Euro. Heart J. published online, http://dx.doi.org/10.1093/eurheartj/ehs232 (2 August 2012).

to regulate the lysosomal and proteasomal pathways other wise inclined to reduce cell surface GluR expression, and overexpression of SNX27 led to reduced net rates of GluR internalization, suggesting that SNX27 acts to encourage recycling of GluR from endosomal compartments to the plasma membrane.

Given the evidence that endosomal abnor-malities are a feature of Down’s syndrome neuropathology1, Wang et al.4 went on to measure SNX27 mRNA and protein levels in post- mortem human cortex from a collection of samples from perinatal to young adult indi-viduals with Down’s syndrome. SNX27 levels proved markedly (~40%) reduced in Down’s syndrome cortex, and GluR1 amounts were also approximately halved; hence, reduced SNX27 and GluR1 expression in Down’s syndrome is reminiscent of that seen in the hippocampi of both Snx27-heterozygous and Ts65Dn mice. The authors went on to show that reduced SNX27 expression was not evident in post-mortem cortical tissue from patients with spontaneous Alzheimer’s disease. This is an important dissociation, given that over expression of Hsa21-encoded amyloid precursor protein gives rise to age-dependent Alzheimer’s disease pathologies in Down’s syndrome, and abnormal handling of amyloid- related peptides by the retromer may contribute to hippo campal dysfunction in

Synapses continuously demand a precise supply of protein components to maintain their activity and plasticity. An intracellular network of neuronal endosomes underpins this supply chain, regulating the degradation and recycling of synaptic proteins such as neurotransmitter receptors. Synapses are therefore unlikely to escape unscathed if endosomal mechanisms are disrupted, and endosomal abnormalities are early features of synaptopathies including Alzheimer’s disease and Down’s syndrome1–3.

In this issue of Nature Medicine, Wang et al.4 show that overexpression of a human chromo-some 21 (Hsa21)-encoded miRNA—miR-155— results in decreased expression of the sorting nexin 27 (SNX27) gene, disrupting endosomal protein sorting and culminating in diminished glutamate receptor expression at hippocam-pal synapses. Normalizing Snx27 expres-sion in adult hippocampus rectifies learning and memory impairments in the Ts65Dn mouse model of Down’s syndrome. Although miR-155– and SNX27-dependent mechanisms certainly affect more than only hippocampal glutamate receptors, this study highlights the potential of targeting endosomal function to get Down’s syndrome neurons back on track.

A subset of sorting nexin proteins is able to form cargo-specific retromer complexes that support recycling from the endosome back to the plasma membrane5. The authors demonstrate the crucial nature of this recy-cling, showing that mice lacking SNX27 died ~1 month postnatally and had cortical and hippocampal neurons reduced in number and dendritic length. Snx27-heterozygous mice, however, had a normal lifespan and suffered subtler central nervous system dysfunction characterized by hippocampus-dependent learning deficits, compromised excitatory synaptic transmission at CA3-CA1 hippo-campal synapses and reduced cell-surface expression of glutamate receptor (GluR) sub-units GluR1/2 and NR1/2A/2B (ref. 4).

SNX27 has previously been shown to inter-act with a number of neuronal transmitter receptors and ion channels, but Wang et al.4 highlighted several clues suggestive of its par-ticular influence on glutamatergic transmis-sion: surface levels of a1/b2 GABAA receptor subunits were unaffected in Snx27-knockout mice; GluR mRNA levels were normal in Snx27-heterozygous mice, indicating post- translational problems; and SNX27 seemed to bind directly to GluR subunits via its PDZ domain, a distinguishing feature rela-tive to other sorting nexins. In cultured cells, normal SNX27 expression levels were required

Matt W. Jones is at the School of Physiology &

Pharmacology, University of Bristol, Bristol, UK.

e-mail: matt.jones@bristol.ac.uk

Sorting receptors at Down’s syndrome synapsesMatt W Jones

Trisomy 21 triggers multiple potential routes to intellectual disability in Down’s syndrome. A new study suggests that aberrant endosomal function may contribute to the neuronal deficits behind learning and memory impairments in affected individuals (pages 473–480).

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Alzheimer’s disease6. So if amyloid-dependent mechanisms are not solely to blame for the SNX27 and GluR phenotypes in Down’s syndrome, what connects trisomy 21 to under-expression SNX27?

MiRNAs are thought to regulate the expres-sion of up to 60% of human genes, and recent work has implicated the overexpression of miRNAs encoded on Hsa21 in triggering altered gene expression in Down’s syndrome7. Wang et al.4 confirmed increased levels of several of Hsa21’s 19 currently recognized miRNAs in Down’s syndrome cortical samples. Of these, over expression of miR-155 is known to trigger dysregulated gene expression in a range of Down’s syndrome tissues, potentially contributing to carcinogenesis, cardiovascu-lar, immune and brain phenotypes8. In fact, an oligonucleotide antagonist of miR-155 has been shown to rescue the expression of methyl-CpG–binding protein (MeCP2) and its target genes in the hippocampus of Ts65Dn mice9.

Wang et al.4 followed this lead to show that only miR-155 levels significantly (negatively) correlated with SNX27 mRNA levels across their collection of Down’s syndrome and control brain samples (implying that SNX27 transcription may also be a metric of synap-tic function in non–Down’s syndrome brains) and that overexpression of an miR-155 mimic in a neuroblastoma cell line decreased SNX27 expression. However, as miR-155 did not bind SNX27 directly, the modulation is prob-ably indirect, which led the authors to study the transcription factor C/EBPb, which is known to be regulated by miR-155 (ref. 10). They found several potential binding sites for C/EBPb on the SNX27 promoter and showed that the active isoform of C/EBPb can augment SNX27 expression in vitro and that SNX27 expression was low in C/EBPb-deficient mice. Wang et al.4 therefore mapped a route from tri-somy of Hsa21 to downregulation of SNX27 via increased miR-155 levels triggering reduced C/EBPb expression.

What might this mean for Down’s syn-drome therapy? Wang et al.4 used viral vectors to drive SNX27 expression in hippocampal CA1 of adult Ts65Dn mice, rescuing SNX27 protein levels and normalizing several layers of the Ts65Dn neuronal phenotype, including GluR expression, synaptic plasticity and per-formance of a learning and memory task—even in adult Ts65Dn mice that had developed and matured with reduced GluR levels. These results suggest SNX27 as something of a pana-cea, though of course Wang et al.4 specifically targeted the hippocampal dysfunction and learning and memory impairment central to particular aspects of intellectual disability in Down’s syndrome11. Nevertheless, comparing

SNX27 knockouts and the Ts65Dn model with genetically altered mice lacking GluR selectively from hippocampus may provide further insights into mechanisms underlying intellectual disability in Down’s syndrome.

Relatively high levels of SNX27 expression (see www.brainatlas.org) may render area CA1 of the hippo campus preferentially vulnerable to trisomy 21, but SNX27-based therapeutic strategies for Down’s syndrome may not be so relevant to other brain regions (and cognitive symptoms). For example, the dentate gyrus of the mouse hippocampal formation is also involved in learning and memory but expresses lower levels of SNX27 than CA1. Some phar-macological studies targeting overactive GABAergic neurotransmission (that is, not targeting endosomal signaling) in the dentate gyrus of Ts65Dn mice report full reversal of cognitive and synaptic plasticity phenotypes12. Others studies using the Ts65Dn model suggest targeting the widespread neuromodulatory adrenergic projections from locus coeruleus13. It remains to be seen whether these ostensibly distinct mechanisms converge, whether they simply reflect multiple, overlapping routes to similar and coarse behavioral endpoints in mice, or whether they can counter the multiple developmental, physiological and degenerative challenges that afflict neurons in individuals with Down’s syndrome.

The full interactome of Hsa21 is vast, and each step of the pathway mapped out in this study allows for considerable divergence

(Fig. 1): trisomy 21 means potential over-expression of 19 miRNAs amongst the ~550 genes found in Hsa21; miR-155 has potential to regulate about 140 genes—one of them being the one encoding C/EBPb, which then regulates multiple promoters; and SNX27 itself, through interactions with retromer-mediated endosomal transport proteins, affects cell sur-face expression of over 100 proteins, includ-ing a diverse subset of integral membrane proteins14. Wang et al.4 acknowledged that the link from SNX27 to retromer signaling opens pathways to other receptors and proteins, any number of which may contribute to neuronal dysfunction in Down’s syndrome. Targeting the SNX27 pathway closer to the Hsa21 source—for example using the anti–miR-155 strategy previously tested in the brains of Ts65Dn mice9—may therefore prove a more compre-hensive therapy against the neuronal impact of trisomy 21 and may also alleviate miR-155– triggered pathologies in non-nervous cells and organs. Nevertheless, it is clear that the endo-some marks an important beginning.

COMPETING FINANCIAL INTERESTS The author declares no competing financial interests.

1. Cataldo, A.M. et al. Am. J. Pathol. 157, 277–286 (2000).

2. Ginsberg, S.D. et al. Biol. Psychiatry 68, 885–893 (2010).

3. Siegenthaler, B.M. & Rajendran, L. Neurodegener. Dis. 10, 116–121 (2012).

4. Wang, X. et al. Nat. Med. 19, 473–480 (2013).5. Cullen, P.J. & Korswagen, H.C. Nat. Cell Biol. 14,

29–37 (2011).

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Figure 1 A route from trisomy 21 to synaptic dysfunction in Down’s syndrome. A trisomic copy of Hsa21 leads to overexpression of the microRNA miR-155, which in turn suppresses expression of the transcription factor C/EBPb. C/EBPb would normally act to upregulate expression of the sorting nexin protein SNX27, which promotes endosome-based recycling of GluR to the synaptic plasma membranes of pyramidal neurons in CA1 of the hippocampus. Reduced C/EBPb in Down’s syndrome hippocampal neurons therefore culminates in reduced synaptic GluR expression, impaired synaptic function, and learning and memory deficits.

Hsa21

Other genesand miRNAs

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(hippocampus)

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receptors andchannels?

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the investigators to probe the identity of the immune factors involved in FX-mediated liver tropism. By examining Ad5 liver transduction in different strains of immunodeficient mice, they determined that the presence of B cells was crucial for FX-mediated transduction. In particular, they found that naturally occur-ring IgM is essential, as reconstitution of B cell–deficient mice with purified natural IgM showed a requirement for FX-mediated liver transduction.

As IgM antibodies often work in concert with components of the complement system to inactivate microbial agents, the investigators

More than a decade ago, Jesse Gelsinger, a young patient in a gene therapy trial, died after receiving a high dose of an Ad5 vector1. Although the exact cause of this unfortunate incident remains uncertain, we now know more about the complex interplay between the host and Ad5 that dictates the efficacy and safety of Ad5-mediated gene transfer. This increased knowledge is exemplified in a report in this issue of Nature Medicine by Xu et al.2 that shows that systemically administered Ad5 vectors are protected from antibody- and complement-mediated neutralization by asso-ciation with coagulation factor X (FX; Fig. 1).

Ad5 receptors for attachment (the coxsackie and adenovirus receptor) and entry (av integ-rins) have key roles in virus infection in vitro3; however, associations with plasma proteins can also affect the tissue distribution of the virus in vivo4–6. In fact, Ad5 transduction of liver hepatocytes in mice occurs independently of viral association with CAR and integrins7. Instead, FX was previously reported to facili-tate Ad5 invasion of liver hepatocytes, thereby circumventing the use of the normal cell recep-tors. In particular, the N-terminal γ-carboxy glutamic acid domain of FX was shown to bind the major outer capsid protein (hexon) of Ad5, thus positioning the C-terminal serine protease domain of FX away from the virus so that it can associate with cell-membrane heparan-sulfated molecules5. This virus–cell bridging mechanism by FX was thought to be the major route of virus entry into the liver. In a new twist, however, the paper by

Xu et al.2 reveals an alternative mechanism for FX-mediated Ad5 liver tropism.

An important clue to this new mechanism appeared when the authors examined the role of FX in Ad5 liver transduction in Rag1−/− mice, which are devoid of T and B cells. In contrast to normal mice, in these immune-deficient mice liver transduction by Ad5 did not show a requirement for FX, as determined by warfarin-mediated depletion of this coagu-lation factor. Moreover, a hexon-modified Ad5 vector that lacks FX-binding activity showed similar levels of liver transduction as an unmodified Ad5 vector. These findings led

Glen R. Nemerow is in the Department of

Immunology and Microbial Science, The Scripps

Research Institute, La Jolla, California, USA.

e-mail: gnemerow@scripps.edu

Coagulation factor defends adenovirus from immune attackGlen R Nemerow

Adenovirus type 5 (Ad5) has been intensively studied as a viral vector for gene therapy, and understanding host–Ad5 interactions will be key to the safe and effective use of this vector. A recent study in mice provides new insights in this area by showing that Ad5 enlists a host coagulation factor to overcome complement-mediated blockade and infect the liver (pages 452–457).

Figure 1 Adenovirus liver tropism is regulated by interactions with FX. (a) Xu et al.2 now show that association of Ad5 with FX (purple) present in normal plasma restricts subsequent blockade of the virus by IgM and complement, thereby allowing productive viral–host cell interactions that lead to infection. (b) In contrast, in the absence of FX (warfarin-treated plasma), the virus is capable of binding to natural IgM and the early-acting classical complement pathway components C1q and C4 (yellow). Coating of the virus particle with these complement components prevents its interactions with host cells and productive infection.

Liver hepatocyte

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6. Muhammad, A. et al. Proc. Natl. Acad. Sci. USA 105, 7327–7332 (2008).

7. Elton, T.S., Sansom, S.E. & Martin, M.M. RNA Biol. 7, 540–547 (2010).

8. Elton, T.S., Selemon, H., Elton, S.M. & Parinandi, N.L. Gene published online, http://dx.doi.org/10.1016/j.gene.2012.12.009 (13 December 2012).

9. Kuhn, D.E. et al. J. Biol. Chem. 285, 1529–1543 (2010).

10. He, M., Xu, Z., Ding, T., Kuang, D.M. & Zheng, L. Cell Mol. Immunol. 6, 343–352 (2009).

11. Edgin, J.O., Mason, G.M., Spano, G., Fernandez, A. & Nadel, L. Prog. Brain Res. 197, 123–151 (2012).

12. Kleschevnikov, A.M. et al. J. Neurosci. 32, 9217–9227 (2012).

13. Salehi, A. et al. Restoration of norepinephrine- modulated contextual memory in a mouse model of Down syndrome. Sci. Transl. Med. 1, 7ra17 (2009).

14. Steinberg, F. et al. Nat. Cell Biol. (in the press).

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