Reelin Mobilizes a VAMP7-Dependent Synaptic Vesicle Pool and Selectively Augments Spontaneous Neurotransmission

SummaryReelin is a glycoprotein that is critical for proper layering of neocortex during development as well as dynamic regulation of glutamatergic postsynaptic signaling in mature synapses. Here, we show that Reelin also acts presynaptically, resulting in robust rapid enhancement of spontaneous neurotransmitter release without affecting properties of evoked neurotransmission. This effect of Reelin requires a modest but significant increase in presynaptic Ca2+ initiated via ApoER2 signaling. The specificity of Reelin action on spontaneous neurotransmitter release is encoded at the level of vesicular SNARE machinery as it requires VAMP7 and SNAP-25 but not synaptobrevin2, VAMP4, or vti1a. These results uncover a presynaptic regulatory pathway that utilizes the heterogeneity of synaptic vesicle-associated SNAREs and selectively augments action potential-independent neurotransmission

Cannabinoid Receptor Activation In The Nucleus Tractus Solitaries Produces Baroreflex-Like Responses In The Rat

The effects of cannabinoids on the baroreflex have been investigated in the nucleus tractus solitarii (NTS). In urethane-anesthetized rats, microinjection of the cannabinoid (CB) receptor agonist WIN 55212-2 (100 mM) into the NTS produced a short lasting decrease in arterial pressure (from 95.2 ± 2.9 to 76.2 ± 1.5, n=5, P0.05) though it inhibited the agonist-induced responses. The non-NMDA receptor antagonist, DNQX (4 mM) microinjections antagonized the actions of CB agonist WIN 55212-2. Furthermore, sinoaortic denervation attenuated the responses to CB agonists suggesting an intact baroreflex arc is necessary to elicit CB-mediated effects. Neither WIN 55212-2 nor AM 281, altered baroreceptor reflex activation by bolus phenylephrine (25 microg//kg) injections. These data suggest that cannabinoid receptors in the NTS are not involved in the tonic regulation of the arterial pressure but may have a modulatory role in the baroreceptor reflex integration.PubMe

Reelin signaling antagonizes β-amyloid at the synapse

Abnormal processing of the amyloid precursor protein (APP) and β-amyloid (Aβ) plaque accumulation are defining features of Alzheimer disease (AD), a genetically complex neurodegenerative disease that is characterized by progressive synapse loss and neuronal cell death. Aβ induces synaptic dysfunction in part by altering the endocytosis and trafficking of AMPA and NMDA receptors. Reelin is a neuromodulator that increases glutamatergic neurotransmission by signaling through the postsynaptic ApoE receptors Apoer2 and Vldlr and thereby potently enhances synaptic plasticity. Here we show that Reelin can prevent the suppression of long-term potentiation and NMDA receptors, which is induced by levels of Aβ comparable to those present in an AD-afflicted brain. This reversal is dependent upon the activation of Src family tyrosine kinases. At high concentrations of Aβ peptides, Reelin can no longer overcome the Aβ induced functional suppression and this coincides with a complete blockade of the Reelin-dependent phosphorylation of NR2 subunits. We propose a model in which Aβ, Reelin, and ApoE receptors modulate neurotransmission and thus synaptic stability as opposing regulators of synaptic gain control

Total Bilirubin Levels Predict Subclinical Atherosclerosis in Patients With Prediabetes

Vuruskan, Ertan/0000-0001-6820-3582; Durakoglugil, Emre/0000-0001-5268-4262; duman, hakan/0000-0002-1441-7320WOS: 000386036600005PubMed: 26921264Bilirubin may have important antiatherosclerotic effects. Prediabetes (PD), the intermediate stage before diabetes mellitus, is associated with increased cardiovascular morbidity and mortality. We evaluated the relationship between serum bilirubin levels and carotid intima-media thickness (cIMT), as a surrogate marker of subclinical atherosclerosis, in patients with PD. We enrolled 170 consecutive patients with PD. the patients underwent ultrasonography to evaluate cIMT. the patients were divided into groups according to cIMT values (<0.9 vs 0.9 mm). the patients with cIMT 0.9 mm had significantly higher diastolic blood pressure, neutrophil-lymphocyte ratio (NLR), and glycated hemoglobin values compared with patients having cIMT < 0.9 mm, whereas total and direct bilirubin values were significantly lower in this group. Multivariate regression analyses revealed NLR and total bilirubin as the independent predictors of subclinical atherosclerosis. the present study demonstrated that NLR and lower total bilirubin levels were independent predictors of subclinical atherosclerosis in patients with PD. Simple measures such as NRL and total bilirubin may provide predictive information regarding the risk of cardiovascular disease in patients with PD

Regulation of the hippocampal translatome by Apoer2-ICD release

Abstract Background ApoE4, the most significant genetic risk factor for late-onset Alzheimer’s disease (AD), sequesters a pro-synaptogenic Reelin receptor, Apoer2, in the endosomal compartment and prevents its normal recycling. In the adult brain, Reelin potentiates excitatory synapses and thereby protects against amyloid-β toxicity. Recently, a gain-of-function mutation in Reelin that is protective against early-onset AD has been described. Alternative splicing of the Apoer2 intracellular domain (Apoer2-ICD) regulates Apoer2 signaling. Splicing of juxtamembraneous exon 16 alters the γ-secretase mediated release of the Apoer2-ICD as well as synapse number and LTP, and inclusion of exon 19 ameliorates behavioral deficits in an AD mouse model. The Apoer2-ICD has also been shown to alter transcription of synaptic genes. However, the role of Apoer2-ICD release upon transcriptional regulation and its role in AD pathogenesis is unknown. Methods To assess in vivo mRNA-primed ribosomes specifically in hippocampi transduced with Apoer2-ICD splice variants, we crossed wild-type, cKO, and Apoer2 cleavage-resistant mice to a Cre-inducible translating ribosome affinity purification (TRAP) model. This allowed us to perform RNA-Seq on ribosome-loaded mRNA harvested specifically from hippocampal cells transduced with Apoer2-ICDs. Results Across all conditions, we observed ~4,700 altered translating transcripts, several of which comprise key synaptic components such as extracellular matrix and focal adhesions with concomitant perturbation of critical signaling cascades, energy metabolism, translation, and apoptosis. We further demonstrated the ability of the Apoer2-ICD to rescue many of these altered transcripts, underscoring the importance of Apoer2 splicing in synaptic homeostasis. A variety of these altered genes have been implicated in AD, demonstrating how dysregulated Apoer2 splicing may contribute to neurodegeneration. Conclusions Our findings demonstrate how alternative splicing of the APOE and Reelin receptor Apoer2 and release of the Apoer2-ICD regulates numerous translating transcripts in mouse hippocampi in vivo. These transcripts comprise a wide range of functions, and alterations in these transcripts suggest a mechanistic basis for the synaptic deficits seen in Apoer2 mutant mice and AD patients. Our findings, together with the recently reported AD-protective effects of a Reelin gain-of-function mutation in the presence of an early-onset AD mutation in Presenilin-1, implicate the Reelin/Apoer2 pathway as a target for AD therapeutics. Graphical Abstrac

Lrp4 domains differentially regulate limb/brain development and synaptic plasticity.

Apolipoprotein E (ApoE) genotype is the strongest predictor of Alzheimer's Disease (AD) risk. ApoE is a cholesterol transport protein that binds to members of the Low-Density Lipoprotein (LDL) Receptor family, which includes LDL Receptor Related Protein 4 (Lrp4). Lrp4, together with one of its ligands Agrin and its co-receptors Muscle Specific Kinase (MuSK) and Amyloid Precursor Protein (APP), regulates neuromuscular junction (NMJ) formation. All four proteins are also expressed in the adult brain, and APP, MuSK, and Agrin are required for normal synapse function in the CNS. Here, we show that Lrp4 is also required for normal hippocampal plasticity. In contrast to the closely related Lrp8/Apoer2, the intracellular domain of Lrp4 does not appear to be necessary for normal expression and maintenance of long-term potentiation at central synapses or for the formation and maintenance of peripheral NMJs. However, it does play a role in limb development

Reelin regulates neuronal excitability through STriatal Enriched Protein Tyrosine phosphatase (STEP61) and Calcium Permeable AMPARs in an NMDAR-dependent manner

Alzheimer's disease (AD) is a progressive neurodegenerative disease marked by the accumulation of amyloid-β (Aβ) plaques and neurofibrillary tangles. Aβ oligomers cause synaptic dysfunction early in AD by enhancing long-term depression (LTD; a paradigm for forgetfulness) via metabotropic glutamate receptor (mGluR)-dependent regulation of striatal-enriched tyrosine phosphatase (STEP61). Reelin is a neuromodulator that signals through ApoE (apolipoprotein E) receptors to protect the synapse against Aβ toxicity (Durakoglugil et al., 2009) Reelin signaling is impaired by ApoE4, the most important genetic risk factor for AD, and Aβ-oligomers activate metabotropic glutamate receptors (Renner et al., 2010). We therefore asked whether Reelin might also affect mGluR-LTD. To this end, we induced chemical mGluR-LTD using DHPG (Dihydroxyphenylglycine), a selective mGluR5 agonist. We found that exogenous Reelin reduces the DHPG-induced increase in STEP61, prevents the dephosphorylation of GluA2, and concomitantly blocks mGluR-mediated LTD. By contrast, Reelin deficiency increased expression of Ca2+-permeable GluA2-lacking AMPA receptors along with higher STEP61 levels, resulting in occlusion of DHPG-induced LTD in hippocampal CA1 neurons. We propose a model in which Reelin modulates local protein synthesis as well as AMPA receptor subunit composition through modulation of mGluR-mediated signaling with implications for memory consolidation or neurodegeneration.SIGNIFICANCE STATEMENT Reelin is an important neuromodulator, which in the adult brain controls synaptic plasticity and protects against neurodegeneration. Amyloid-β has been shown to use mGluRs to induce synaptic depression through endocytosis of NMDA and AMPA receptors, a mechanism referred to as LTD, a paradigm of forgetfulness. Our results show that Reelin regulates the phosphatase STEP, which plays an important role in neurodegeneration, as well as the expression of calcium-permeable AMPA receptors, which play a role in memory formation. These data suggest that Reelin uses mGluR LTD pathways to regulate memory formation as well as neurodegeneration

Limb and bone structure of different <i>Lrp4</i> KI mutants.

<p><b>A</b>: Illustration of the different Lrp4 protein products of all KI mutants. Panels are aligned to paw images in B and C to indicate genotypes. <b>B</b>: Ventral view of fore and hind limbs of <i>Lrp4</i> KI mutants. Homozygous mutant mice for each allelic variant (<i>KI/KI</i>) and compound mutant mice that carry one allelic variant and one KO allele (<i>KO/KI</i>) are shown. Note that there are strong defects in the limb pattering of <i>Lrp4</i>^{<i>ECD/ECD</i>}, intermediate defects in <i>Lrp4</i>^{<i>ΔICD/ΔIC</i>}, and only mild defects in <i>Lrp4</i>^{<i>LDLR-ICD/LDLR-ICD</i>} (red arrows). <b>C</b>: Ventral view of alizarin red (stains bones) and alcian blue (stains cartilage) of different <i>Lrp4</i> KI mutants. A WT-KI allele (2^nd panel in A and B) was generated to control for the lack of introns in the ICD-cassette in the other KI mutants. Black arrowheads: ectopic bone or bony fusion; red arrowheads: soft-tissue fusion. (modified from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116701#pone.0116701.ref022" target="_blank">22</a>]).</p

LTP is impaired in <i>Lrp4</i>^{<i>ECD/ECD</i>} but not in <i>Lrp4</i>^{<i>ΔICD/ΔICD</i>} mice.

<p><b>A</b>: <i>Upper Panel</i>, Sample traces before and 40 min after theta-burst stimulation (TBS); <i>Lower Panel</i>, Results of experiments from <i>Lrp4</i>^{<i>ECD/ECD</i>} mice compared to their <i>Lrp4</i>^{<i>WT-KI/WT-KI</i>} controls. TBS induced on average a 37.63 ± 7.88% increase in <i>Lrp4</i>^{<i>WT-KI/WT-KI</i>} control slices (open squares, n = 16, N = 5), but only 14.83 ± 3.39% LTP in slices from the <i>Lrp4</i>^{<i>ECD/ECD</i>} mice (black triangles, n = 10, N = 3). <b>B</b>: Unpaired t-test was used to compare each sample for LTP calculated 40–60 min after theta-burst. Values are the means of the normalized fEPSP slopes. * denotes significance, p = 0.0387. <b>C:</b><i>Upper Panel</i>, Sample traces before and 40 min after TBS. <i>Lower Panel</i>, Results of experiments from <i>Lrp4</i>^{<i>ΔICD/ΔICD</i>} mice compared to their <i>Lrp4</i>^{<i>WT-KI/WT-KI</i>} controls. <i>Lrp4</i>^{<i>WT-KI/WT-KI</i>} slices were recorded on consecutive days and used as internal controls and pooled together. TBS induced a 37.63 ± 7.88% LTP in <i>Lrp4</i>^{<i>WT-KI/WT-KI</i>} control slices (open squares, n = 16, N = 5), and 32.42 ± 6.27% LTP in slices from the <i>Lrp4</i>^{<i>ΔICD/ΔICD</i>} mice (gray filled rhombus, n = 11, N = 5). <b>D</b>: There was no significant difference in LTP between <i>Lrp4</i>^{<i>WT-KI/WT-KI</i>}<i>and Lrp4</i>^{<i>ΔICD/ΔICD</i>} mice (p = 0.63). <b>E.</b> Input-output curves calculated as a function of fiber volley amplitude to the slopes of fEPSP’s. Average peak amplitudes for <i>Lrp4</i>^{<i>WT-KI/WT-KI</i>}, <i>Lrp4</i>^{<i>ECD/ECD</i>} and <i>Lrp4</i>^{<i>ΔICD/ΔICD</i>} slices used in the experiments were 1.68 ± 0.15 mV, 1.27 ± 0.25 mV, and 1.50 ± 0.20 mV), respectively, and were not significantly different from each other. (One-way ANOVA, F = 1.124, p = 0.33.) <b>F:</b> Theta-burst analysis or <b>G:</b> paired pulse ratios (n = 8, N = 3 for each) did not reveal any significant differences between <i>Lrp4</i>^{<i>WT-KI/WT-KI</i>} and <i>Lrp4</i>^{<i>ECD/ECD</i>} (two-way ANOVA, F(3,56) = 0.46, p = 0.71). N = number of animals.</p