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.

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.

Lipidomic and Transcriptomic Basis of Lysosomal Dysfunction in Progranulin Deficiency.

Defective lysosomal function defines many neurodegenerative diseases, such as neuronal ceroid lipofuscinoses (NCL) and Niemann-Pick type C (NPC), and is implicated in Alzheimer's disease (AD) and frontotemporal lobar degeneration (FTLD-TDP) with progranulin (PGRN) deficiency. Here, we show that PGRN is involved in lysosomal homeostasis and lipid metabolism. PGRN deficiency alters lysosome abundance and morphology in mouse neurons. Using an unbiased lipidomic approach, we found that brain lipid composition in humans and mice with PGRN deficiency shows disease-specific differences that distinguish them from normal and other pathologic groups. PGRN loss leads to an accumulation of polyunsaturated triacylglycerides, as well as a reduction of diacylglycerides and phosphatidylserines in fibroblast and enriched lysosome lipidomes. Transcriptomic analysis of PGRN-deficient mouse brains revealed distinct expression patterns of lysosomal, immune-related, and lipid metabolic genes. These findings have implications for the pathogenesis of FTLD-TDP due to PGRN deficiency and suggest lysosomal dysfunction as an underlying mechanism.

Functional Roles of the Interaction of APP and Lipoprotein Receptors.

The biological fates of the key initiator of Alzheimer's disease (AD), the amyloid precursor protein (APP), and a family of lipoprotein receptors, the low-density lipoprotein (LDL) receptor-related proteins (LRPs) and their molecular roles in the neurodegenerative disease process are inseparably interwoven. Not only does APP bind tightly to the extracellular domains (ECDs) of several members of the LRP group, their intracellular portions are also connected through scaffolds like the one established by FE65 proteins and through interactions with adaptor proteins such as X11/Mint and Dab1. Moreover, the ECDs of APP and LRPs share common ligands, most notably Reelin, a regulator of neuronal migration during embryonic development and modulator of synaptic transmission in the adult brain, and Agrin, another signaling protein which is essential for the formation and maintenance of the neuromuscular junction (NMJ) and which likely also has critical, though at this time less well defined, roles for the regulation of central synapses. Furthermore, the major independent risk factors for AD, Apolipoprotein (Apo) E and ApoJ/Clusterin, are lipoprotein ligands for LRPs. Receptors and ligands mutually influence their intracellular trafficking and thereby the functions and abilities of neurons and the blood-brain-barrier to turn over and remove the pathological product of APP, the amyloid-ß peptide. This article will review and summarize the molecular mechanisms that are shared by APP and LRPs and discuss their relative contributions to AD.

Reelin: Neurodevelopmental Architect and Homeostatic Regulator of Excitatory Synapses.

Over half a century ago, D. S. Falconer first reported a mouse with a reeling gate. Four decades later, the Reln gene was isolated and identified as the cause of the reeler phenotype. Initial studies found that loss of Reelin, a large, secreted glycoprotein encoded by the Reln gene, results in abnormal neuronal layering throughout several regions of the brain. In the years since, the known functions of Reelin signaling in the brain have expanded to include multiple postdevelopmental neuromodulatory roles, revealing an ever increasing body of evidence to suggest that Reelin signaling is a critical player in the modulation of synaptic function. In writing this review, we intend to highlight the most fundamental aspects of Reelin signaling and integrate how these various neuromodulatory effects shape and protect synapses.

Synaptic vesicle pool-specific modification of neurotransmitter release by intravesicular free radical generation.

KEY POINTS: Synaptic transmission is mediated by the release of neurotransmitters from synaptic vesicles in response to stimulation or through the spontaneous fusion of a synaptic vesicle with the presynaptic plasma membrane. There is growing evidence that synaptic vesicles undergoing spontaneous fusion versus those fusing in response to stimuli are functionally distinct. In this study, we acutely probe the effects of intravesicular free radical generation on synaptic vesicles that fuse spontaneously or in response to stimuli. By targeting vesicles that preferentially release spontaneously, we can dissociate the effects of intravesicular free radical generation on spontaneous neurotransmission from evoked neurotransmission and vice versa. Taken together, these results further advance our knowledge of the synapse and the nature of the different synaptic vesicle pools mediating neurotransmission. ABSTRACT: Earlier studies suggest that spontaneous and evoked neurotransmitter release processes are maintained by synaptic vesicles which are segregated into functionally distinct pools. However, direct interrogation of the link between this putative synaptic vesicle pool heterogeneity and neurotransmission has been difficult. To examine this link, we tagged vesicles with horseradish peroxidase (HRP) - a haem-containing plant enzyme - or antibodies against synaptotagmin-1 (syt1). Filling recycling vesicles in hippocampal neurons with HRP and subsequent treatment with hydrogen peroxide (H2 O2 ) modified the properties of neurotransmitter release depending on the route of HRP uptake. While strong depolarization-induced uptake of HRP suppressed evoked release and augmented spontaneous release, HRP uptake during mild activity selectively impaired evoked release, whereas HRP uptake at rest solely potentiated spontaneous release. Expression of a luminal HRP-tagged syt1 construct and subsequent H2 O2 application resulted in a similar increase in spontaneous release and suppression as well as desynchronization of evoked release, recapitulating the canonical syt1 loss-of-function phenotype. An antibody targeting the luminal domain of syt1, on the other hand, showed that augmentation of spontaneous release and suppression of evoked release phenotypes are dissociable depending on whether the antibody uptake occurred at rest or during depolarization. Taken together, these findings indicate that vesicles that maintain spontaneous and evoked neurotransmitter release preserve their identity during recycling and syt1 function in suppression of spontaneous neurotransmission can be acutely dissociated from syt1 function to synchronize synaptic vesicle exocytosis upon stimulation.

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.

Loss of Reelin protects against atherosclerosis by reducing leukocyte-endothelial cell adhesion and lesion macrophage accumulation.

The multimodular glycoprotein Reelin controls neuronal migration and synaptic transmission by binding to apolipoprotein E receptor 2 (Apoer2) and very low density lipoprotein receptor (Vldlr) on neurons. In the periphery, Reelin is produced by the liver, circulates in blood, and promotes thrombosis and hemostasis. To investigate if Reelin influences atherogenesis, we studied atherosclerosis-prone low-density lipoprotein receptor-deficient (Ldlr(-/-)) mice in which we inducibly deleted Reelin either ubiquitously or only in the liver, thus preventing the production of circulating Reelin. In both types of Reelin-deficient mice, atherosclerosis progression was markedly attenuated, and macrophage content and endothelial cell staining for vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) were reduced at the sites of atherosclerotic lesions. Intravital microscopy revealed decreased leukocyte-endothelial adhesion in the Reelin-deficient mice. In cultured human endothelial cells, Reelin enhanced monocyte adhesion and increased ICAM1, VCAM1, and E-selectin expression by suppressing endothelial nitric oxide synthase (eNOS) activity and increasing nuclear factor κB (NF-κB) activity in an Apoer2-dependent manner. These findings suggest that circulating Reelin promotes atherosclerosis by increasing vascular inflammation, and that reducing or inhibiting circulating Reelin may present a novel approach for the prevention of cardiovascular disease.

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.

Cholesterol-dependent balance between evoked and spontaneous synaptic vesicle recycling.

Cholesterol is a prominent component of nerve terminals. To examine cholesterol's role in central neurotransmission, we treated hippocampal cultures with methyl-beta-cyclodextrin, which reversibly binds cholesterol, or mevastatin, an inhibitor of cholesterol biosynthesis, to deplete cholesterol. We also used hippocampal cultures from Niemann-Pick type C1-deficient mice defective in intracellular cholesterol trafficking. These conditions revealed an augmentation in spontaneous neurotransmission detected electrically and an increase in spontaneous vesicle endocytosis judged by horseradish peroxidase uptake after cholesterol depletion by methyl-beta-cyclodextrin. In contrast, responses evoked by action potentials and hypertonicity were severely impaired after the same treatments. The increase in spontaneous vesicle recycling and the decrease in evoked neurotransmission were reversible upon cholesterol addition. Cholesterol removal did not impact on the low level of evoked neurotransmission seen in the absence of synaptic vesicle SNARE protein synaptobrevin-2 whereas the increase in spontaneous fusion remained. These results suggest that synaptic cholesterol balances evoked and spontaneous neurotransmission by hindering spontaneous synaptic vesicle turnover and sustaining evoked exo-endocytosis.