Regulation of the hippocampal translatome by Apoer2-ICD release.

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-ß 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.

Apoer2-ICD-dependent regulation of hippocampal ribosome mRNA loading.

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-ß 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 g-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 splicing for 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 ribosome-associated 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 ribosome-associated 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.

NHE6 depletion corrects ApoE4-mediated synaptic impairments and reduces amyloid plaque load.

Apolipoprotein E4 (ApoE4) is the most important and prevalent risk factor for late-onset Alzheimer's disease (AD). The isoelectric point of ApoE4 matches the pH of the early endosome (EE), causing its delayed dissociation from ApoE receptors and hence impaired endolysosomal trafficking, disruption of synaptic homeostasis, and reduced amyloid clearance. We have shown that enhancing endosomal acidification by inhibiting the EE-specific sodium-hydrogen exchanger 6 (NHE6) restores vesicular trafficking and normalizes synaptic homeostasis. Remarkably and unexpectedly, loss of NHE6 (encoded by the gene Slc9a6) in mice effectively suppressed amyloid deposition even in the absence of ApoE4, suggesting that accelerated acidification of EEs caused by the absence of NHE6 occludes the effect of ApoE on amyloid plaque formation. NHE6 suppression or inhibition may thus be a universal, ApoE-independent approach to prevent amyloid buildup in the brain. These findings suggest a novel therapeutic approach for the prevention of AD by which partial NHE6 inhibition reverses the ApoE4-induced endolysosomal trafficking defect and reduces plaque load.

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-ß (Aß) plaques and neurofibrillary tangles. Aß 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 Aß toxicity (Durakoglugil et al., 2009) Reelin signaling is impaired by ApoE4, the most important genetic risk factor for AD, and Aß-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-ß 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.

NGP 555, a γ-secretase modulator, shows a beneficial shift in the ratio of amyloid biomarkers in human cerebrospinal fluid at safe doses.

INTRODUCTION: Currently, there is no cure for Alzheimer's disease (AD), and it is widely accepted that AD is a complex disease with multiple approaches necessary to prevent and treat the disease. METHODS: Using amyloid biomarkers in human cerebrospinal fluid, pharmacokinetic, safety, and metabolism studies, we investigate the properties of NGP 555, γ-secretase modulator, for the first time in human clinical trials. RESULTS: Our preclinical and clinical studies combined show beneficial effects with NGP 555 on synaptic response and amyloid cerebrospinal fluid biomarkers while avoiding negative side effects. Importantly, pharmacokinetic and pharmacodynamic parameters combined with safety outcomes indicate that NGP 555 penetrates the blood-brain barrier and increases the ratio of amyloid-ß peptide Aß37 and Aß38 compared with that of Aß42, establishing a proof of target engagement in humans in a 14 day, once-daily oral dosing trial. DISCUSSION: In humans, NGP 555 has demonstrated a beneficial shift in the production of Aß37 and Aß38 versus Aß42 biomarker levels in the cerebrospinal fluid while maintaining an adequate safety profile. The overall clinical goal is to achieve an optimal balance of efficacy for altering amyloid-ß peptide (Aß) biomarkers while maintaining a safe profile so that NGP 555 can be given early in AD to prevent production of Aß42 and accumulation of amyloid plaques, in an effort to prevent aggregation of tau and destruction of neurons and synapses resulting in cognitive decline.

Reversal of ApoE4-induced recycling block as a novel prevention approach for Alzheimer's disease.

ApoE4 genotype is the most prevalent and also clinically most important risk factor for late-onset Alzheimer's disease (AD). Available evidence suggests that the root cause for this increased risk is a trafficking defect at the level of the early endosome. ApoE4 differs from the most common ApoE3 isoform by a single amino acid that increases its isoelectric point and promotes unfolding of ApoE4 upon endosomal vesicle acidification. We found that pharmacological and genetic inhibition of NHE6, the primary proton leak channel in the early endosome, in rodents completely reverses the ApoE4-induced recycling block of the ApoE receptor Apoer2/Lrp8 and the AMPA- and NMDA-type glutamate receptors that are regulated by, and co-endocytosed in a complex with, Apoer2. Moreover, NHE6 inhibition restores the Reelin-mediated modulation of excitatory synapses that is impaired by ApoE4. Our findings suggest a novel potential approach for the prevention of late-onset AD.

Genetic Restoration of Plasma ApoE Improves Cognition and Partially Restores Synaptic Defects in ApoE-Deficient Mice.

UNLABELLED: Alzheimer's disease (AD) is the most common form of dementia in individuals over the age of 65 years. The most prevalent genetic risk factor for AD is the ε4 allele of apolipoprotein E (ApoE4), and novel AD treatments that target ApoE are being considered. One unresolved question in ApoE biology is whether ApoE is necessary for healthy brain function. ApoE knock-out (KO) mice have synaptic loss and cognitive dysfunction; however, these findings are complicated by the fact that ApoE knock-out mice have highly elevated plasma lipid levels, which may independently affect brain function. To bypass the effect of ApoE loss on plasma lipids, we generated a novel mouse model that expresses ApoE normally in peripheral tissues, but has severely reduced ApoE in the brain, allowing us to study brain ApoE loss in the context of a normal plasma lipid profile. We found that these brain ApoE knock-out (bEKO) mice had synaptic loss and dysfunction similar to that of ApoE KO mice; however, the bEKO mice did not have the learning and memory impairment observed in ApoE KO mice. Moreover, we found that the memory deficit in the ApoE KO mice was specific to female mice and was fully rescued in female bEKO mice. Furthermore, while the AMPA/NMDA ratio was reduced in ApoE KO mice, it was unchanged in bEKO mice compared with controls. These findings suggest that plasma lipid levels can influence cognition and synaptic function independent of ApoE expression in the brain. SIGNIFICANCE STATEMENT: One proposed treatment strategy for Alzheimer's disease (AD) is the reduction of ApoE, whose ε4 isoform is the most common genetic risk factor for the disease. A major concern of this strategy is that an animal model of ApoE deficiency, the ApoE knock-out (KO) mouse, has reduced synapses and cognitive impairment; however, these mice also develop dyslipidemia and severe atherosclerosis. Here, we have shown that genetic restoration of plasma ApoE to wild-type levels normalizes plasma lipids in ApoE KO mice. While this does not rescue synaptic loss, it does completely restore learning and memory in the mice, suggesting that both CNS and plasma ApoE are independent parameters that affect brain health.

Reelin protects against amyloid ß toxicity in vivo.

Alzheimer's disease (AD) is a currently incurable neurodegenerative disorder and is the most common form of dementia in people over the age of 65 years. The predominant genetic risk factor for AD is the ε4 allele encoding apolipoprotein E (ApoE4). The secreted glycoprotein Reelin enhances synaptic plasticity by binding to the multifunctional ApoE receptors apolipoprotein E receptor 2 (Apoer2) and very low density lipoprotein receptor (Vldlr). We have previously shown that the presence of ApoE4 renders neurons unresponsive to Reelin by impairing the recycling of the receptors, thereby decreasing its protective effects against amyloid ß (Aß) oligomer-induced synaptic toxicity in vitro. We showed that when Reelin was knocked out in adult mice, these mice behaved normally without overt learning or memory deficits. However, they were strikingly sensitive to amyloid-induced synaptic suppression and had profound memory and learning disabilities with very low amounts of amyloid deposition. Our findings highlight the physiological importance of Reelin in protecting the brain against Aß-induced synaptic dysfunction and memory impairment.

Differential splicing and glycosylation of Apoer2 alters synaptic plasticity and fear learning.

Apoer2 is an essential receptor in the central nervous system that binds to the apolipoprotein ApoE. Various splice variants of Apoer2 are produced. We showed that Apoer2 lacking exon 16, which encodes the O-linked sugar (OLS) domain, altered the proteolytic processing and abundance of Apoer2 in cells and synapse number and function in mice. In cultured cells expressing this splice variant, extracellular cleavage of OLS-deficient Apoer2 was reduced, consequently preventing γ-secretase-dependent release of the intracellular domain of Apoer2. Mice expressing Apoer2 lacking the OLS domain had increased Apoer2 abundance in the brain, hippocampal spine density, and glutamate receptor abundance, but decreased synaptic efficacy. Mice expressing a form of Apoer2 lacking the OLS domain and containing an alternatively spliced cytoplasmic tail region that promotes glutamate receptor signaling showed enhanced hippocampal long-term potentiation (LTP), a phenomenon associated with learning and memory. However, these mice did not display enhanced spatial learning in the Morris water maze, and cued fear conditioning was reduced. Reducing the expression of the mutant Apoer2 allele so that the abundance of the protein was similar to that of Apoer2 in wild-type mice normalized spine density, hippocampal LTP, and cued fear learning. These findings demonstrated a role for ApoE receptors as regulators of synaptic glutamate receptor activity and established differential receptor glycosylation as a potential regulator of synaptic function and memory.

ApoE4 reduces glutamate receptor function and synaptic plasticity by selectively impairing ApoE receptor recycling.

Apolipoprotein E (ApoE) genotype is a powerful genetic modifier of Alzheimer's disease (AD). The ApoE4 isoform significantly reduces the mean age-of-onset of dementia through unknown mechanisms. Here, we show that ApoE4 selectively impairs synaptic plasticity and NMDA receptor phosphorylation by Reelin, a regulator of brain development and modulator of synaptic strength. ApoE4 reduces neuronal surface expression of Apoer2, a dual function receptor for ApoE and for Reelin, as well as NMDA and AMPA receptors by sequestration in intracellular compartments, thereby critically reducing the ability of Reelin to enhance synaptic glutamate receptor activity. As a result, the ability of Reelin to prevent LTP suppression by extracts from AD-afflicted human brains in hippocampal slices from knockin mice expressing the human ApoE4 isoform is severely impaired. These findings show an isoform-specific role of ApoE in the localization and intracellular trafficking of lipoprotein and glutamate receptors and thereby reveal an alternative mechanism by which ApoE4 may accelerate onset of dementia and neuronal degeneration by differentially impairing the maintenance of synaptic stability.

Reelin signaling antagonizes beta-amyloid at the synapse.

Abnormal processing of the amyloid precursor protein (APP) and beta-amyloid (Abeta) 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. Abeta 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 Abeta 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 Abeta peptides, Reelin can no longer overcome the Abeta induced functional suppression and this coincides with a complete blockade of the Reelin-dependent phosphorylation of NR2 subunits. We propose a model in which Abeta, Reelin, and ApoE receptors modulate neurotransmission and thus synaptic stability as opposing regulators of synaptic gain control.

Developmental maturation of synaptic and extrasynaptic GABAA receptors in mouse thalamic ventrobasal neurones.

Thalamic ventrobasal (VB) relay neurones express multiple GABA(A) receptor subtypes mediating phasic and tonic inhibition. During postnatal development, marked changes in subunit expression occur, presumably reflecting changes in functional properties of neuronal networks. The aims of this study were to characterize the properties of synaptic and extrasynaptic GABA(A) receptors of developing VB neurones and investigate the role of the alpha(1) subunit during maturation of GABA-ergic transmission, using electrophysiology and immunohistochemistry in wild type (WT) and alpha(1)(0/0) mice and mice engineered to express diazepam-insensitive receptors (alpha(1H101R), alpha(2H101R)). In immature brain, rapid (P8/9-P10/11) developmental change to mIPSC kinetics and increased expression of extrasynaptic receptors (P8-27) formed by the alpha(4) and delta subunit occurred independently of the alpha(1) subunit. Subsequently (> or = P15), synaptic alpha(2) subunit/gephyrin clusters of WT VB neurones were replaced by those containing the alpha(1) subunit. Surprisingly, in alpha(1)(0/0) VB neurones the frequency of mIPSCs decreased between P12 and P27, because the alpha(2) subunit also disappeared from these cells. The loss of synaptic GABA(A) receptors led to a delayed disruption of gephyrin clusters. Despite these alterations, GABA-ergic terminals were preserved, perhaps maintaining tonic inhibition. These results demonstrate that maturation of synaptic and extrasynaptic GABA(A) receptors in VB follows a developmental programme independent of the alpha(1) subunit. Changes to synaptic GABA(A) receptor function and the increased expression of extrasynaptic GABA(A) receptors represent two distinct mechanisms for fine-tuning GABA-ergic control of thalamic relay neurone activity during development.

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, P<0.05) but no change in the heart rate. Another cannabinoid agonist, CP 55940 (100 mM) also caused hypotensive responses (from 90.2 ± 11.3 to 66.4 ± 12.3 mmHg, n=5, P<0.05). Simultaneous sympathetic nerve discharge recordings showed suppression prior to the arterial pressure lowering effect of these agonists. Microinjection of the cannabinoid receptor antagonist, AM 281 (70 mM) did not cause any significant change in arterial pressure (from 100.8 ± 12 mmHg to 108.1 ± 12.8 mmHg, n=5, P>0.05) 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.