SORL1 is strongly associated with both sporadic and familial forms of Alzheimer's disease (AD), but a lack of information about alternatively spliced transcripts currently limits our understanding of the role of SORL1 in AD. Here, we describe a SORL1 transcript (SORL1-38b) characterized by inclusion of a novel exon (E38b) that encodes a truncated protein. We identified E38b-containing transcripts in several brain regions, with the highest expression in the cerebellum and showed that SORL1-38b is largely located in neuronal dendrites, which is in contrast to the somatic distribution of transcripts encoding the full-length SORLA protein (SORL1-fl). SORL1-38b transcript levels were significantly reduced in AD cerebellum in three independent cohorts of postmortem brains, whereas no changes were observed for SORL1-fl. A trend of lower 38b transcript level in cerebellum was found for individuals carrying the risk variant at rs2282649 (known as SNP24), although not reaching statistical significance. These findings suggest synaptic functions for SORL1-38b in the brain, uncovering novel aspects of SORL1 that can be further explored in AD research.

The ADAMTS9 rs4607103 C allele is one of the few gene variants proposed to increase the risk of type 2 diabetes through an impairment of insulin sensitivity. We show that the variant is associated with increased expression of the secreted ADAMTS9 and decreased insulin sensitivity and signaling in human skeletal muscle. In line with this, mice lacking Adamts9 selectively in skeletal muscle have improved insulin sensitivity. The molecular link between ADAMTS9 and insulin signaling was characterized further in a model where ADAMTS9 was overexpressed in skeletal muscle. This selective overexpression resulted in decreased insulin signaling presumably mediated through alterations of the integrin β1 signaling pathway and disruption of the intracellular cytoskeletal organization. Furthermore, this led to impaired mitochondrial function in mouse muscle-an observation found to be of translational character because humans carrying the ADAMTS9 risk allele have decreased expression of mitochondrial markers. Finally, we found that the link between ADAMTS9 overexpression and impaired insulin signaling could be due to accumulation of harmful lipid intermediates. Our findings contribute to the understanding of the molecular mechanisms underlying insulin resistance and type 2 diabetes and point to inhibition of ADAMTS9 as a potential novel mode of treating insulin resistance.

Coronary artery disease is the main cause of death worldwide and accelerated by increased plasma levels of cholesterol-rich low-density lipoprotein particles (LDL). Circulating PCSK9 contributes to coronary artery disease by inducing lysosomal degradation of the LDL receptor (LDLR) in the liver and thereby reducing LDL clearance. Here, we show that liver heparan sulfate proteoglycans are PCSK9 receptors and essential for PCSK9-induced LDLR degradation. The heparan sulfate-binding site is located in the PCSK9 prodomain and formed by surface-exposed basic residues interacting with trisulfated heparan sulfate disaccharide repeats. Accordingly, heparan sulfate mimetics and monoclonal antibodies directed against the heparan sulfate-binding site are potent PCSK9 inhibitors. We propose that heparan sulfate proteoglycans lining the hepatocyte surface capture PCSK9 and facilitates subsequent PCSK9:LDLR complex formation. Our findings provide new insights into LDL biology and show that targeting PCSK9 using heparan sulfate mimetics is a potential therapeutic strategy in coronary artery disease.PCSK9 interacts with LDL receptor, causing its degradation, and consequently reduces the clearance of LDL. Here, Gustafsen et al. show that PCSK9 interacts with heparan sulfate proteoglycans and this binding favors LDLR degradation. Pharmacological inhibition of this binding can be exploited as therapeutic intervention to lower LDL levels.

This article describes an immunocytochemistry (ICC) method for staining against phosphorylated forms of the kinases Akt (pAkt) and MAPK (pMAPK). Phosphorylation is induced upon their activation by a number stimuli including insulin and brain-derived neurotrophic factor (BDNF), and is prerequisite for a number of cellular processes including cell proliferation and survival [1], [2], [3], [4], [5], [6]. ICC using antibodies raised against specific phosphorylation sites allows cell-type specific and subcellular monitoring of kinase activation. Here, we test how four different antibodies against pAkt and pMAPK, respectively perform in different cell types following insulin or BDNF stimulation using different protocol conditions. We find that phospho-specific-antibodies generally perform better when using Triton X-100 as a permeabilization agent compared to Saponin. In addition, two antibodies against pAkt and two against pMAPK gave a clear increase in signal in cells stimulated with insulin or BDNF compared to the signal obtained in unstimulated cells. These antibodies also performed well when tested with western blotting. Our results illustrate that both the choice of antibody as well as protocol details are critical parameters for successful detection of phosphorylated forms of kinases by ICC. This article includes: •A protocol for subcellular detection of phosphorylated Akt and MAPK.•Validation of 8 antibodies by immunocytochemistry.•Confirmation by western blotting.

The activity of excitatory neurons is controlled by a highly diverse population of inhibitory interneurons. These cells show a high level of physiological, morphological and neurochemical heterogeneity, and play highly specific roles in neuronal circuits. In the mammalian hippocampus, these are divided into 21 different subtypes of GABAergic interneurons based on their expression of different markers, morphology and their electrophysiological properties. Ideally, all can be marked using an antibody directed against the inhibitory neurotransmitter GABA, but parvalbumin, calbindin, somatostatin, and calretinin are also commonly used as markers to narrow down the specific interneuron subtype. Here, we describe a journey to find the necessary immunological reagents for studying GABAergic interneurons of the mouse hippocampus. Based on web searches there are several hundreds of different antibodies on the market directed against these four markers. Searches in the literature databases allowed us to narrow it down to a subset of antibodies most commonly used in publications. However, in our hands the most cited ones did not work for immunofluorescence stainings of formaldehyde fixed tissue sections and cultured hippocampal neurons, and we had to immunostain our way through thirteen different commercial antibodies before finally finding a suitable antibody for each of the four markers. The antibodies were evaluated based on signal-to-noise ratios as well as if positive cells were found in layers of the hippocampus where they have previously been described. Additionally, the antibodies were also tested on sections from mouse spinal cord with similar criteria for specificity of the antibodies. Using the antibodies with a high rating on pAbmAbs, an antibody review database, stainings with high signal-to-noise ratios and location of the immunostained cells in accordance with the literature could be obtained, making these antibodies suitable choices for studying the GABAergic system.

Schwann cell reprogramming and differentiation are crucial prerequisites for neuronal regeneration and re-myelination to occur following injury to peripheral nerves. The neurotrophin receptor p75NTR has been identified as a positive modulator for Schwann cell myelination during development and implicated in promoting nerve regeneration after injury. However, most studies base this conclusion on results obtained from complete p75NTR knockout mouse models and cannot dissect the specific role of p75NTR expressed by Schwann cells. In this present study, a conditional knockout model selectively deleting p75NTR expression in Schwann cells was generated, where p75NTR expression is replaced with that of an mCherry reporter. Silencing of Schwann cell p75NTR expression was confirmed in the sciatic nerve in vivo and in vitro, without altering axonal expression of p75NTR. No difference in sciatic nerve myelination during development or following sciatic nerve crush injury was observed, as determined by quantification of both myelinated and unmyelinated nerve fiber densities, myelinated axonal diameter and myelin thickness. However, the absence of Schwann cell p75NTR reduced motor nerve conduction velocity after crush injury. Our data indicate that the absence of Schwann cell p75NTR expression in vivo is not critical for axonal regrowth or remyelination following sciatic nerve crush injury, but does play a key role in functional recovery. Overall, this represents the first step in redefining the role of p75NTR in the peripheral nervous system, suggesting that the Schwann cell-axon unit functions as a syncytium, with the previous published involvement of p75NTR in remyelination most likely depending on axonal/neuronal p75NTR and/or mutual glial-axonal interactions.

Schwann cells (SCs) are the main glial cells of the peripheral nervous system (PNS) and are known to be involved in various pathophysiological processes, such as diabetic neuropathy and nerve regeneration, through neurotrophin signaling. Such glial trophic support to axons, as well as neuronal survival/death signaling, has previously been linked to the p75 neurotrophin receptor (p75NTR) and its co-receptor Sortilin. Recently, SC-derived extracellular vesicles (EVs) were shown to be important for axon growth and nerve regeneration, but cargo of these glial cell-derived EVs has not yet been well-characterized. In this study, we aimed to characterize signatures of small RNAs in EVs derived from wild-type (WT) SCs and define differentially expressed small RNAs in EVs derived from SCs with genetic deletions of p75NTR (Ngfr-/-) or Sortilin (Sort1-/-). Using RNA sequencing, we identified a total of 366 miRNAs in EVs derived from WT SCs of which the most highly expressed are linked to the regulation of axonogenesis, axon guidance and axon extension, suggesting an involvement of SC EVs in axonal homeostasis. Signaling of SC EVs to non-neuronal cells was also suggested by the presence of several miRNAs important for regulation of the endothelial cell apoptotic process. Ablated p75NTR or sortilin expression in SCs translated into a set of differentially regulated tRNAs and miRNAs, with impact in autophagy and several cellular signaling pathways such as the phosphatidylinositol signaling system. With this work, we identified the global expression profile of small RNAs present in SC-derived EVs and provided evidence for a regulatory function of these vesicles on the homeostasis of other cell types of the PNS. Differentially identified miRNAs can pave the way to a better understanding of p75NTR and sortilin roles regarding PNS homeostasis and disease.

Attention deficit hyperactivity disorder (ADHD) is the most frequently diagnosed neurodevelopmental disorder worldwide. Affected individuals present with hyperactivity, inattention, and cognitive deficits and display a characteristic paradoxical response to drugs affecting the dopaminergic system. However, the underlying pathophysiology of ADHD and how this relates to dopaminergic transmission remains to be fully understood. Sorcs2-/- mice uniquely recapitulate symptoms reminiscent of ADHD in humans. Here, we show that lack of SorCS2 in mice results in lower sucrose intake, indicating general reward deficits. Using in-vivo recordings, we further find that dopaminergic transmission in the ventral tegmental area (VTA) is shifted towards a more regular firing pattern with marked reductions in the relative occurrence of irregular firing in Sorcs2-/- mice. This was paralleled by abnormal acute behavioral responses to dopamine receptor agonists, suggesting fundamental differences in dopaminergic circuits and indicating a perturbation in the balance between the activities of the postsynaptic dopamine receptor DRD1 and the presynaptic inhibitory autoreceptor DRD2. Interestingly, the hyperactivity and drug response of Sorcs2-/- mice were markedly affected by novelty. Taken together, our findings show how loss of a candidate ADHD-risk gene has marked effects on dopaminergic circuit function and the behavioral response to the environment.

Peripheral nerve regeneration relies on the ability of Schwann cells to support the regrowth of damaged axons. Schwann cells re-differentiate when reestablishing contact with the sprouting axons, with large fibers becoming remyelinated and small nociceptive fibers ensheathed and collected into Remak bundles. We have previously described how the receptor sortilin facilitates neurotrophin signaling in peripheral neurons via regulated trafficking of Trk receptors. This study aims to characterize the effects of sortilin deletion on nerve regeneration following sciatic crush injury. We found that Sort1 - / - mice displayed functional motor recovery like that of WT mice, with no detectable differences in relation to nerve conduction velocities and morphological aspects of myelinated fibers. In contrast, we found abnormal ensheathment of regenerated C-fibers in injured Sort1 - / - mice, demonstrating a role of sortilin for Remak bundle formation following injury. Further studies on Schwann cell signaling pathways showed a significant reduction of MAPK/ERK, RSK, and CREB phosphorylation in Sort1 - / - Schwann cells after stimulation with neurotrophin-3 (NT-3), while Schwann cell migration and myelination remained unaffected. In conclusion, our results demonstrate that loss of sortilin blunts NT-3 signaling in Schwann cells which might contribute to the impaired Remak bundle regeneration after sciatic nerve injury.

Foodborne Enteropathogenic Escherichia coli (EPEC) infections of the small intestine cause diarrhea especially in children and are a major cause of childhood death in developing countries. EPEC infects the apical membrane of the epithelium of the small intestine by attaching, effacing the microvilli under the bacteria and then forming microcolonies on the cell surface. We first asked the question where on epithelial cells EPEC attaches and grows. Using models of polarized epithelial monolayers, we evaluated the sites of initial EPEC attachment to the apical membrane and found that EPEC preferentially attached over the cell-cell junctions and formed microcolonies preferentially where three cells come together at tricellular tight junctions. The ability of EPEC to adhere increased when host cell polarity was compromised yielding EPEC access to basolateral proteins. EPEC pedestals contain basolateral cytoskeletal proteins. Thus, we asked if attached EPEC causes reorganization the protein composition of the host cell plasma membrane at sites of microcolony formation. We found that EPEC microcolony growth at the apical membrane resulted in a local accumulation of basolateral plasma membrane proteins surrounding the microcolony. Basolateral marker protein aquaporin-3 localized to forming EPEC microcolonies. Components of the basolateral vesicle targeting machinery were re-routed. The Exocyst (Exo70) was recruited to individual EPEC as was the basolateral vesicle SNARE VAMP-3. Moreover, several Rab variants were also recruited to the infection site, and their dominant-negative equivalents were not. To quantitatively study the recruitment of basolateral proteins, we created a pulse of the temperature sensitive basolateral VSVG, VSVG3-SP-GFP, from the trans-Golgi Network. We found that after release from the TGN, significantly more VSVG3-SP-GFP accumulated at the site of microcolony growth than on equivalent membrane regions of uninfected cells. This suggests that trafficking of vesicles destined for the basolateral membrane are redirected to the apical site of microcolony growth. Thus, in addition to disrupting host cell fence function, local host cell plasma membrane protein composition is changed by altered protein trafficking and recruitment of basolateral proteins to the apical microcolony. This may aid EPEC attachment and subsequent microcolony growth.

Neuropathic pain is a major incurable clinical problem resulting from peripheral nerve trauma or disease. A central mechanism is the reduced expression of the potassium chloride cotransporter 2 (KCC2) in dorsal horn neurons induced by brain-derived neurotrophic factor (BDNF), causing neuronal disinhibition within spinal nociceptive pathways. Here, we demonstrate how neurotensin receptor 2 (NTSR2) signaling impairs BDNF-induced spinal KCC2 down-regulation, showing how these two pathways converge to control the abnormal sensory response following peripheral nerve injury. We establish how sortilin regulates this convergence by scavenging neurotensin from binding to NTSR2, thus modulating its inhibitory effect on BDNF-mediated mechanical allodynia. Using sortilin-deficient mice or receptor inhibition by antibodies or a small-molecule antagonist, we lastly demonstrate that we are able to fully block BDNF-induced pain and alleviate injury-induced neuropathic pain, validating sortilin as a clinically relevant target.