apoE Receptor Research Articles

Multi-functional role of apolipoprotein E in neurodegenerative diseases

Genetic diversity in the apolipoprotein E (ApoE) gene has been identified as the major susceptibility genetic risk factor for sporadic Alzheimer’s disease (SAD). Specifically, the ApoEε4 allele is a significant risk factor for SAD, while ApoEε2 allele provides protection compared to the more common ApoEε3 allele. This review discusses the role of the ApoE in AD and other neurodegenerative disorders. ApoE, a cholesterol transport protein, influences several pathways involved in neurodegeneration, particularly in AD. Beyond its established role in amyloid β-protein (Aβ) metabolism and deposition, ApoE also impacts tau pathology, neurodegeneration, and the microglial response to AD. The review aims to provide an updated overview of ApoE’s diverse roles, emphasizing its involvement in Aβ clearance through ApoE receptors. It also covers ApoE’s influence in other neurodegenerative diseases like Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD), Huntington’s disease (HD), vascular dementia (VD), and multiple sclerosis (MS). New research highlights the interaction between ApoE and presenilin (PS), suggesting connections between familial AD (FAD) and SAD. The review also explores protective effects of ApoE mutations against AD and ApoE4-induced tauopathy, neurodegeneration, and neuroinflammation. The insights from this comprehensive update could indeed lead to new therapeutic strategies for neurodegenerative diseases.

Propionate Decreases Microglial Activation but Impairs Phagocytic Capacity in Response to Aggregated Fibrillar Amyloid Beta Protein.

Microglia, the innate immune cell of the brain, are a principal player in Alzheimer's disease (AD) pathogenesis. Their surveillance of the brain leads to interaction with the protein aggregates that drive AD pathogenesis, most notably Amyloid Beta (Aβ). Microglia attempt to clear and degrade Aβ using phagocytic machinery, spurring damaging neuroinflammation in the process. Thus, modulation of the microglial response to Aβ is crucial in mitigating AD pathophysiology. SCFAs, microbial byproducts of dietary fiber fermentation, are blood-brain barrier permeable molecules that have recently been shown to modulate microglial function. It is unclear whether propionate, one representative SCFA, has beneficial or detrimental effects on microglia in AD. Thus, we investigated its impact on microglial Aβ response in vitro. Using a multiomics approach, we characterized the transcriptomic, metabolomic, and lipidomic responses of immortalized murine microglia following 1 h of Aβ stimulation, as well as characterizing Aβ phagocytosis and secretion of reactive nitrogen species. Propionate blunted the early inflammatory response driven by Aβ, downregulating the expression of many Aβ-stimulated immune genes, including those regulating inflammation, the immune complement system, and chemotaxis. Further, it reduced the expression of Apoe and inflammation-promoting Aβ-binding scavenger receptors such as Cd36 and Msr1 in favor of inflammation-dampening Lpl, although this led to impaired phagocytosis. Finally, propionate shifted microglial metabolism, altering phospholipid composition and diverting arginine metabolism, resulting in decreased nitric oxide production. Altogether, our data demonstrate a modulatory role of propionate on microglia that may dampen immune activation in response to Aβ, although at the expense of phagocytic capacity.

High-affinity agonism at the P2X7 receptor is mediated by three residues outside the orthosteric pocket

P2X receptors are trimeric ATP-gated ion channels that activate diverse signaling cascades. Due to its role in apoptotic pathways, selective activation of P2X7 is a potential experimental tool and therapeutic approach in cancer biology. However, mechanisms of high-affinity P2X7 activation have not been defined. We report high-resolution cryo-EM structures of wild-type rat P2X7 bound to the high-affinity agonist BzATP as well as significantly improved apo receptor structures in the presence and absence of sodium. Apo structures define molecular details of pore architecture and reveal how a partially hydrated Na+ ion interacts with the conductance pathway in the closed state. Structural, electrophysiological, and direct binding data of BzATP reveal that three residues just outside the orthosteric ATP-binding site are responsible for its high-affinity agonism. This work provides insights into high-affinity agonism for any P2X receptor and lays the groundwork for development of subtype-specific agonists applicable to cancer therapeutics.

Platelets as an inter-player between hyperlipidaemia and atherosclerosis.

Platelet hyperreactivity and hyperlipidaemia contribute significantly to atherosclerosis. Thus, it is desirable to review the platelet-hyperlipidaemia interplay and its impact on atherogenesis. Native low-density lipoprotein (nLDL) and oxidized LDL (oxLDL) are the key proatherosclerotic components of hyperlipidaemia. nLDL binds to the platelet-specific LDL receptor (LDLR) ApoE-R2', whereas oxLDL binds to the platelet-expressed scavenger receptor CD36, lectin-type oxidized LDLR 1 and scavenger receptor class A 1. Ligation of nLDL/oxLDL induces mild platelet activation and may prime platelets for other platelet agonists. Platelets, in turn, can modulate lipoprotein metabolisms. Platelets contribute to LDL oxidation by enhancing the production of reactive oxygen species and LDLR degradation via proprotein convertase subtilisin/kexin type 9 release. Platelet-released platelet factor 4 and transforming growth factor β modulate LDL uptake and foam cell formation. Thus, platelet dysfunction and hyperlipidaemia work in concert to aggravate atherogenesis. Hypolipidemic drugs modulate platelet function, whereas antiplatelet drugs influence lipid metabolism. The research prospects of the platelet-hyperlipidaemia interplay in atherosclerosis are also discussed.

The Reelin receptor ApoER2 is a cargo for the adaptor protein complex AP-4: Implications for Hereditary Spastic Paraplegia

Adaptor protein complex 4 (AP-4) is a heterotetrameric complex that promotes export of selected cargo proteins from the trans-Golgi network. Mutations in each of the AP-4 subunits cause a complicated form of Hereditary Spastic Paraplegia (HSP). Herein, we report that ApoER2, a receptor in the Reelin signaling pathway, is a cargo of the AP-4 complex. We identify the motif ISSF/Y within the ApoER2 cytosolic domain as necessary for interaction with the canonical signal-binding pocket of the µ4 (AP4M1) subunit of AP-4. AP4E1- knock-out (KO) HeLa cells and hippocampal neurons from Ap4e1-KO mice display increased co-localization of ApoER2 with Golgi markers. Furthermore, hippocampal neurons from Ap4e1-KO mice and AP4M1-KO human iPSC-derived cortical i3Neurons exhibit reduced ApoER2 protein expression. Analyses of biosynthetic transport of ApoER2 reveal differential post-Golgi trafficking of the receptor, with lower axonal distribution in KO compared to wild-type neurons, indicating a role of AP-4 and the ISSF/Y motif in the axonal localization of ApoER2. Finally, analyses of Reelin signaling in mouse hippocampal and human cortical KO neurons show that AP4 deficiency causes no changes in Reelin-dependent activation of the AKT pathway and only mild changes in Reelin-induced dendritic arborization, but reduces Reelin-induced ERK phosphorylation, CREB activation, and Golgi deployment. This work thus establishes ApoER2 as a novel cargo of the AP-4 complex, suggesting that defects in the trafficking of this receptor and in the Reelin signaling pathway could contribute to the pathogenesis of HSP caused by mutations in AP-4 subunits.

The low-density lipoprotein receptor promotes infection of multiple encephalitic alphaviruses

Members of the low-density lipoprotein receptor (LDLR) family, including LDLRAD3, VLDLR, and ApoER2, were recently described as entry factors for different alphaviruses. However, based on studies with gene edited cells and knockout mice, blockade or abrogation of these receptors does not fully inhibit alphavirus infection, indicating the existence of additional uncharacterized entry factors. Here, we perform a CRISPR-Cas9 genome-wide loss-of-function screen in mouse neuronal cells with a chimeric alphavirus expressing the Eastern equine encephalitis virus (EEEV) structural proteins and identify LDLR as a candidate receptor. Expression of LDLR on the surface of neuronal or non-neuronal cells facilitates binding and infection of EEEV, Western equine encephalitis virus, and Semliki Forest virus. Domain mapping and binding studies reveal a low-affinity interaction with LA domain 3 (LA3) that can be enhanced by concatenation of LA3 repeats. Soluble decoy proteins with multiple LA3 repeats inhibit EEEV infection in cell culture and in mice. Our results establish LDLR as a low-affinity receptor for multiple alphaviruses and highlight a possible path for developing inhibitors that could mitigate infection and disease.

Identify Alzheimer’s disease subtypes and markers from multi‐omic data of human brain and blood with a subspace merging algorithm

AbstractBackgroundAlzheimer’s disease (AD) is a highly heterogeneous disease with different disease trajectory, brain pathological changes, and risk factors. Identifying subtypes of AD can have greatly impact AD diagnosis, treatment, and disease management. Our goal is to identify subtypes of AD by integrating multiple types of omics data from AD patients’ brain samples and blood samples.MethodWe applied a subspace merging algorithm on AD patient brain samples with matched transcriptomic, DNA methylation, and proteomics data collected from the ROSMAP study. This algorithm merges patient graphs generated from these three datasets on a Grassmann manifold followed by spectral clustering. Differential gene expression and pathway analysis were applied to establish the gene signatures between the subtypes (patient clusters) and to the transcriptomic data of the peripheral blood monocyte (PBMC) from the matching patients to identify possible blood markers.ResultOur analysis identified two patient clusters between which the cognitive test scores or the degree of AD pathology are significantly different. Differentially expressed genes between the two groups were identified from both brain and blood samples. Gene set enrichment analysis identified a group of genes significantly enriched with synaptic functions in the brain samples. The Ingenuity Pathway Analysis was used to examine any connections between the significantly differentially expressed genes in the brain samples with the ones identified from the PBMC samples. The low density lipoprotein receptor (LDLR) gene, which was an APOE receptor observed from the blood sample is connected with 12 genes from the brain sample. The results suggested that LDLR could be a novel blood biomarker for AD subtyping.ConclusionThe subspace merging algorithm integrated multimodal omics data from the patients and successfully identified two clusters with distinct clinical progression. Differential expression analysis identified 388 significant genes from the brain samples and 18 from the PBMC samples. The pathway analysis indicated that LDLR could be a novel blood biomarker for AD subtyping. Future work will focus on validating LDLR as a candidate blood marker for AD subtyping using other public datasets with similar study design. The clinical relevance and molecular mechanism basis for the findings will also be investigated.

Activation/Deactivation Free-Energy Profiles for the β2-Adrenergic Receptor: Ligand Modes of Action.

We use enhanced-sampling simulations with an effective collective variable to study the activation of the β2-adrenergic receptor in the presence of ligands with different efficacy. The free-energy profiles are computed for the ligand-free (apo) receptor and binary (apo-receptor + G-protein α-subunit and receptor + ligand) and ternary complexes. The results are not only compatible with available experiments but also allow unprecedented structural insight into the nature of GPCR conformations along the activation pathway and their role in the activation mechanism. In particular, the simulations reveal an unexpected mode of action of partial agonists such as salmeterol and salbutamol that arises already in the binary complex without the G-protein. Specific differences in the polar interactions with residues in TM5, which are required to stabilize an optimal TM6 conformation that facilitates G-protein binding and receptor activation, play a major role in differentiating them from full agonists.

Regulation of the hippocampal translatome by Apoer2-ICD release

BackgroundApoE4, 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.MethodsTo 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.ResultsAcross 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.ConclusionsOur 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

Different sensitivity to diet-induced hyperinsulinemia and hyperglycemia between mice with global or bone marrow-specific apoE receptor-2 deficiency.

This study explored the role of apoE receptor-2 (apoER2), a unique member of the LDL receptor family proteins with a restricted tissue expression profile, in modulating diet-induced obesity and diabetes. Unlike wild-type mice and humans in which chronic feeding of a high-fat Western-type diet leads to obesity and the prediabetic state of hyperinsulinemia before hyperglycemia onset, the Lrp8-/- mice with global apoER2 deficiency displayed lower body weight and adiposity, slower development of hyperinsulinemia, but the accelerated onset of hyperglycemia. Despite their lower adiposity, adipose tissues in Western diet-fed Lrp8-/- mice were more inflamed compared with wild-type mice. Additional experiments revealed that the hyperglycemia observed in Western diet-fed Lrp8-/- mice was due to impaired glucose-induced insulin secretion, ultimately leading to hyperglycemia, adipocyte dysfunction, and inflammation upon chronic feeding of the Western diet. Interestingly, bone marrow-specific apoER2-deficient mice were not defective in insulin secretion, exhibiting increased adiposity and hyperinsulinemia compared with wild-type mice. Analysis of bone marrow-derived macrophages revealed that apoER2 deficiency impeded inflammation resolution with lower secretion of IFN-β and IL-10 in response to LPS stimulation of IL-4 primed cells. The apoER2-deficient macrophages also showed an increased level of disabled-2 (Dab2) as well as increased cell surface TLR4, suggesting that apoER2 participates in Dab2 regulation of TLR4 signaling. Taken together, these results showed that apoER2 deficiency in macrophages sustains diet-induced tissue inflammation and accelerates obesity and diabetes onset while apoER2 deficiency in other cell types contributes to hyperglycemia and inflammation via defective insulin secretion.

Abstract 677: Short Hydrocarbon-Stapled Apolipoprotein E Mimetic Peptides Enhance Lipoprotein Uptake

ApoE facilitates the clearance of apoB-containing lipoproteins by binding to heparan sulfate proteoglycans (HSPG), low density lipoprotein receptor (LDLR) and LDLR-related protein 1 (LRP1). ApoE has a receptor binding and a lipid binding domain, which are both needed for its biological function. Several apoE-based peptides have been developed that lower plasma lipid levels in animal models and in early-stage clinical trials. However, these peptides are long (28-61 residues), costly to synthesize and are potentially immunogenic as they often contain many amino acid substitutions that vary from the native apoE sequence. We developed novel short (10-12 residues) apoE mimetic peptides based on the apoE receptor binding region (residues 141-150) containing only 2 amino acid substitutions. We used hydrocarbon stapling to stabilize the native alpha helical structure of this region and to increase the lipid binding affinity of the peptides. In addition, octanoic or myristic acid was attached to the N-terminus of the peptides to further increase lipid binding affinity. Helical conformation and lipid binding of the peptides were confirmed by circular dichroism spectroscopy and phospholipid vesicle solubilization assay. Our peptides increased the uptake of fluorescent tagged VLDL and LDL in a concentration dependent manner (0.625-20 μM peptides) in both wild-type (WT) and LDLR knockout (KO) HepG2 cells as measured by flow cytometry. In addition, an all D-amino acid version of one of the peptides had a similar effect on LDL uptake as its all L-amino acid version. Altogether, this suggests that peptide-mediated uptake is LDLR independent. Treatment of WT HepG2 cells with heparinases diminished the low-dose effect (up to 2.5 μM) of the peptides on LDL uptake, indicating that HSPGs plays a role in peptide-mediated LDL uptake. Finally, the peptides (4.6 mg/kg, IV) lowered plasma total cholesterol (37% at 3h; 29% at 6h) and triglycerides (33% at 1h) in apoE-KO mice on a standard chow diet. In summary, the novel apoE peptides are shorter than previously reported peptides, are more similar to the native sequence of apoE, and are likely resistant to proteolysis, because of hydrocarbon stapling and, therefore, could potentially be developed into an oral therapy.

Data-Driven Genome-Wide Association Study of Pain Trajectories in Persons with Parkinson’s Disease (P7-11.012)

<h3>Objective:</h3> To conduct the first genome-wide association study (GWAS) of pain in persons with Parkinson’s disease (PWP). <h3>Background:</h3> Pain is a common and complex non-motor symptom experienced by ~60% of PWP. Little is known about the genetic and non-genetic determinants of pain in PWP. <h3>Design/Methods:</h3> Using data spanning ~4.5 years from 8,612 PWP who are early in their disease course (&lt;3 years), and available through the Fox Insight Data Exploration Network, we empirically discerned 5 subgroups of PWP with similar longitudinal patterns of pain using latent class growth analysis. 4,411 PWP were also genotyped across two customized Illumina platforms that had significant overlap. Quality control criteria (minor allele frequency &lt;1%, Hardy Weinberg Equilibrium p&lt;5×10⁻⁵) were imposed prior imputation using the TOPMed reference panel. After post-imputation quality control (same as above + imputation R²&gt;70%), there were 7.7×10⁶ genetic variants. We retained 4,159 PWP who self-identified as White and who genetically clustered with other European samples. For this analysis, we focused on a GWAS of the most severe pain subgroup (N=97) to the least impaired subgroup (N=934). Gene-based and pathway enrichment analyses were also conducted. <h3>Results:</h3> For the GWAS of extreme pain phenotypes, top genic SNP association were <i>MAPK8</i>-rs72794357 (OR=4.6; p=1×10⁻⁶) and <i>VLDLR-</i>rs4741753 (OR=2.2; p=1.5×10⁻⁶) and top gene-based results included <i>CTNNB1</i> (p=3.5×10⁻⁵) and <i>KLK7</i> (p=7.7×10⁻⁵). These results are intriguing since <i>MAPK8</i> is involved in opioid receptor pathways, <i>VLDLR</i> is a ApoE receptor involved in brain development through Reelin signaling, <i>CTNNB1</i> promotes neurogenesis and de novo mutations have been associated with polyneuropathy in lower limbs, and <i>KLK7</i> is an astrocyte-derived amyloid-β degrading enzyme. Overall there was an enrichment of genes associated with oxytocin signaling (p=0.14), opioid signaling (p=0.045), and Wnt signaling pathway (p=0.021). <h3>Conclusions:</h3> The findings are consistent with known neuropathic mechanisms, thus therapeutic targets for these mechanisms may be relevant for PWP. <b>Disclosure:</b> The institution of Miss Liu has received research support from NIH/NIGMS. The institution of Miss Liu has received research support from Diana Jacobs Kalman/AFAR Scholarships for Research in the Biology of Aging The Program. The institution of Miss Liu has received research support from Michael J. Fox Foundation. Doug Gunzler has nothing to disclose. Dr. Gunzler has received research support from NIH/NINDS. The institution of Dr. Gunzler has received research support from Biogen. The institution of Dr. Gunzler has received research support from Amneal. Dr. Gunzler has received research support from Michael J Fox Foundation. Dr. Crawford has received personal compensation in the range of $5,000-$9,999 for serving as an Editor, Associate Editor, or Editorial Advisory Board Member for Frontiers in Genetics. The institution of Dr. Crawford has received research support from NIH/NIDDK. The institution of Dr. Crawford has received research support from NIH/NCATS. The institution of Dr. Crawford has received research support from VA. The institution of Dr. Crawford has received research support from NIH/NIGMS. The institution of Dr. Crawford has received research support from NHGRI. The institution of Dr. Crawford has received research support from NIH/NIA. Dr. Crawford has received personal compensation in the range of $500-$4,999 for serving as a Member with NIH Study Section. The institution of Prof. Briggs has received research support from NIH. The institution of Prof. Briggs has received research support from Michael J. Fox Foundation.

TRIM40 ameliorates diabetic retinopathy through suppressing inflammation via Reelin/DAB1 signaling disruption: A mechanism by proteasomal degradation of DAB1

Diabetic retinopathy (DR) is a common microvascular complication of diabetes mellitus. Reelin, an extracellular matrix protein, and its effector protein Disabled1 (DAB1) have been linked to cellular events and retinal development. However, whether and how Reelin/DAB1 signaling causes DR remains to be investigated. In our study, significantly increased expression of Reelin, very low density lipoprotein receptor (VLDLR), ApoE receptor 2 (ApoER2) and phosphorylated DAB1 in retinas of streptozotocin (STZ)-induced DR mouse model was observed, along with enhanced expression of proinflammatory factors. Similar results are confirmed in high glucose (HG)-treated human retinal pigment epithelium cell line ARPE-19. Surprisingly, dysregulated tripartite motif-containing 40 (TRIM40), an E3 ubiquitin ligase, is found to be involved in DR progression by bioinformatic analysis. We observe a negative correlation between TRIM40 and p-DAB1 protein expression levels under HG conditions. Importantly, we find that TRIM40 over-expression markedly ameliorates HG-induced p-DAB1, PI3K, p-protein B kinase (AKT) and inflammatory response in HG-treated cells, but dose not affect Reelin expression. Of note, Co-IP and double immunofluorescence identify an interaction between TRIM40 and DAB1. Furthermore, we show that TRIM40 enhances K48-linked polyubiquitination of DAB1, thereby promoting DAB1 degradation. Finally, promoting TRIM40 expression by intravenous injection of the constructed adeno-associated virus (AAV-TRIM40) markedly ameliorates DR phenotypes in STZ-treated mice, as indicated by the decreased blood glucose and glycosylated hemoglobin (HbAlc) levels, and increased hemoglobin contents. Additionally, diabetes-related elevation of acellular capillaries was also meliorated in mice over-expressing TRIM40. The electroretinogram (ERG) deficits were strongly rescued in mice receiving AAV-TRIM40 injection. Moreover, AAV-TRIM40 attenuates the inflammation and p-DAB1 expression in retinal tissues of STZ-treated mice. Collectively, our findings disclose a mechanism through which TRIM40 limits DAB1 stability under physiological conditions and reveals TRIM40 as a potential therapeutic target for the intervention of Reelin/DAB1 signaling, contributing to DR treatment.

LRP1 in vascular mural cells modulates cerebrovascular integrity and function in the presence of APOE4.

Cerebrovasculature is critical in maintaining brain homeostasis; its dysregulation often leads to vascular cognitive impairment and dementia (VCID) during aging. VCID is the second most prevalent cause of dementia in the elderly, after Alzheimer's disease (AD), with frequent cooccurrence of VCID and AD. While multiple factors are involved in the pathogenesis of AD and VCID, APOE4 increases the risk for both diseases. A major apolipoprotein E (apoE) receptor, the low-density lipoprotein receptor-related protein 1 (LRP1), is abundantly expressed in vascular mural cells (pericytes and smooth muscle cells). Here, we investigated how deficiency of vascular mural cell LRP1 affects the cerebrovascular system and cognitive performance using vascular mural cell-specific Lrp1-KO mice (smLrp1-/-) in a human APOE3 or APOE4 background. We found that spatial memory was impaired in the 13- to 16-month-old APOE4 smLrp1-/- mice but not in the APOE3 smLrp1-/- mice, compared with their respective littermate control mice. These disruptions in the APOE4 smLrp1-/- mice were accompanied with excess paravascular glial activation and reduced cerebrovascular collagen IV. In addition, blood-brain barrier (BBB) integrity was disrupted in the APOE4 smLrp1-/- mice. Together, our results suggest that vascular mural cell LRP1 modulates cerebrovasculature integrity and function in an APOE genotype-dependent manner.

Effect of histidine covalent modification on strigolactone receptor activation and selectivity

The parasitic weed Striga has led to billions of dollars' worth of agricultural productivity loss worldwide. Striga detects host plants using compounds of the strigolactone class of phytohormones. Early steps in the strigolactone signaling pathway involve substrate binding and hydrolysis followed by a conformational change to an "active" or "closed" state, after which it associates with a MAX2-family downstream signaling partner. The structures of the inactive and active states of strigolactone receptors are known through X-ray crystallography, and the transition pathway from the inactive to active state in apo receptors has previously been characterized using molecular dynamics simulations. However, it also has been suggested that a covalent butenolide modification of the receptor on the catalytic histidine through substrate hydrolysis promotes formation of the active state. Using molecular dynamics simulations, we show that the presence of the covalent butenolide enhances activation in both AtD14, a receptor found in Arabidopsis, and ShHTL7, a receptor found in Striga, but the enhancement is ∼50 times greater in ShHTL7. We also show that several conserved interactions with the covalent butenolide modification promote transition to the active state in both AtD14 (non-parasite) and ShHTL7 (parasite). Finally, we demonstrate that the enhanced activation of ShHTL7 likely results from disruption of ShHTL7-specific histidine interactions that inhibited activation in the apo case. These results provide a possible explanation for difference in strigolactone sensitivity seen between different strigolactone-sensitive proteins and can be used to aid the design of selective modulators to control Striga parasites.

Association between the LRP1B and APOE loci in the development of Parkinson's disease dementia.

Parkinson's disease is one of the most common age-related neurodegenerative disorders. Although predominantly a motor disorder, cognitive impairment and dementia are important features of Parkinson's disease, particularly in the later stages of the disease. However, the rate of cognitive decline varies among Parkinson's disease patients, and the genetic basis for this heterogeneity is incompletely understood. To explore the genetic factors associated with rate of progression to Parkinson's disease dementia, we performed a genome-wide survival meta-analysis of 3,923 clinically diagnosed Parkinson's disease cases of European ancestry from four longitudinal cohorts. In total, 6.7% of individuals with Parkinson's disease developed dementia during study follow-up, on average 4.4 ± 2.4 years from disease diagnosis. We have identified the APOE ε4 allele as a major risk factor for the conversion to Parkinson's disease dementia [hazards ratio = 2.41 (1.94-3.00), P = 2.32 × 10-15], as well as a new locus within the ApoE and APP receptor LRP1B gene [hazards ratio = 3.23 (2.17-4.81), P = 7.07 × 10-09]. In a candidate gene analysis, GBA variants were also identified to be associated with higher risk of progression to dementia [hazards ratio = 2.02 (1.21-3.32), P = 0.007]. CSF biomarker analysis also implicated the amyloid pathway in Parkinson's disease dementia, with significantly reduced levels of amyloid β42 (P = 0.0012) in Parkinson's disease dementia compared to Parkinson's disease without dementia. These results identify a new candidate gene associated with faster conversion to dementia in Parkinson's disease and suggest that amyloid-targeting therapy may have a role in preventing Parkinson's disease dementia.

PCSK9 Affects Astrocyte Cholesterol Metabolism and Reduces Neuron Cholesterol Supplying In Vitro: Potential Implications in Alzheimer's Disease.

The Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) involvement in Alzheimer’s disease (AD) is poorly investigated. We evaluated the in vitro PCSK9 modulation of astrocyte cholesterol metabolism and neuronal cholesterol supplying, which is fundamental for neuronal functions. Moreover, we investigated PCSK9 neurotoxic effects. In human astrocytoma cells, PCSK9 reduced cholesterol content (−20%; p < 0.05), with a greater effect in presence of beta amyloid peptide (Aβ) (−37%; p < 0.01). PCSK9 increased cholesterol synthesis and reduced the uptake of apoE-HDL-derived cholesterol (−36%; p < 0.0001), as well as the LDL receptor (LDLR) and the apoE receptor 2 (ApoER2) expression (−66% and −31%, respectively; p < 0.01). PCSK9 did not modulate ABCA1- and ABCG1-cholesterol efflux, ABCA1 levels, or membrane cholesterol. Conversely, ABCA1 expression and activity, as well as membrane cholesterol, were reduced by Aβ (p < 0.05). In human neuronal cells, PCSK9 reduced apoE-HDL-derived cholesterol uptake (−41%; p < 0.001) and LDLR/apoER2 expression (p < 0.05). Reduced cholesterol internalization occurred also in PCSK9-overexpressing neurons exposed to an astrocyte-conditioned medium (−39%; p < 0.001). PCSK9 reduced neuronal cholesterol content overall (−29%; p < 0.05) and increased the Aβ-induced neurotoxicity (p < 0.0001). Our data revealed an interfering effect of PCSK9, in cooperation with Aβ, on brain cholesterol metabolism leading to neuronal cholesterol reduction, a potentially deleterious effect. PCSK9 also exerted a neurotoxic effect, and thus represents a potential pharmacological target in AD.

Astrocyte-secreted glypican-4 drives APOE4-dependent tau hyperphosphorylation

Tau protein aggregates are a major driver of neurodegeneration and behavioral impairments in tauopathies, including in Alzheimer's disease (AD). Apolipoprotein E4 (APOE4), the highest genetic risk factor for late-onset AD, has been shown to exacerbate tau hyperphosphorylation in mouse models. However, the exact mechanisms through which APOE4 induces tau hyperphosphorylation remains unknown. Here, we report that the astrocyte-secreted protein glypican-4 (GPC-4), which we identify as a binding partner of APOE4, drives tau hyperphosphorylation. We discovered that first, GPC-4 preferentially interacts with APOE4 in comparison to APOE2, considered to be a protective allele to AD, and second, that postmortem APOE4-carrying AD brains highly express GPC-4 in neurotoxic astrocytes. Furthermore, the astrocyte-secreted GPC-4 induced both tau accumulation and propagation invitro. CRISPR/dCas9-mediated activation of GPC-4 in a tauopathy mouse model robustly induced tau hyperphosphorylation. In the absence of GPC4, APOE4-induced tau hyperphosphorylation was largely diminished using invitro tau fluorescence resonance energy transfer-biosensor cells, in human-induced pluripotent stem cell-derived astrocytes and in an invivo mouse model. We further show that APOE4-mediated surface trafficking of APOE receptor low-density lipoprotein receptor-related protein 1 through GPC-4 can be a gateway to tau spreading. Collectively, these data support that APOE4-induced tau hyperphosphorylation is directly mediated by GPC-4.

Altered Balance of Reelin Proteolytic Fragments in the Cerebrospinal Fluid of Alzheimer's Disease Patients.

Reelin binds to the apolipoprotein E receptor apoER2 to activate an intracellular signaling cascade. The proteolytic cleavage of reelin follows receptor binding but can also occur independently of its binding to receptors. This study assesses whether reelin proteolytic fragments are differentially affected in the cerebrospinal fluid (CSF) of Alzheimer’s disease (AD) subjects. CSF reelin species were analyzed by Western blotting, employing antibodies against the N- and C-terminal domains. In AD patients, we found a decrease in the 420 kDa full-length reelin compared with controls. In these patients, we also found an increase in the N-terminal 310 kDa fragment resulting from the cleavage at the so-called C-t site, whereas the 180 kDa fragment originated from the N-t site remained unchanged. Regarding the C-terminal proteolytic fragments, the 100 kDa fragment resulting from the cleavage at the C-t site also displayed increased levels, whilst the one resulting from the N-t site, the 250 kDa fragment, decreased. We also detected the presence of an aberrant reelin species with a molecular mass of around 500 kDa present in AD samples (34 of 43 cases), while it was absent in the 14 control cases analyzed. These 500 kDa species were only immunoreactive to N-terminal antibodies. We validated the occurrence of these aberrant reelin species in an Aβ42-treated reelin-overexpressing cell model. When we compared the AD samples from APOE genotype subgroups, we only found minor differences in the levels of reelin fragments associated to the APOE genotype, but interestingly, the levels of fragments of apoER2 were lower in APOE ε4 carriers with regards to APOE ε3/ε3. The altered proportion of reelin/apoER2 fragments and the occurrence of reelin aberrant species suggest a complex regulation of the reelin signaling pathway, which results impaired in AD subjects.

Lipid Peroxidation Induced ApoE Receptor-Ligand Disruption as a Unifying Hypothesis Underlying Sporadic Alzheimer's Disease in Humans.

Sporadic Alzheimer's disease (sAD) lacks a unifying hypothesis that can account for the lipid peroxidation observed early in the disease, enrichment of ApoE in the core of neuritic plaques, hallmark plaques and tangles, and selective vulnerability of entorhinal-hippocampal structures. We hypothesized that 1) high expression of ApoER2 (receptor for ApoE and Reelin) helps explain this anatomical vulnerability; 2) lipid peroxidation of ApoE and ApoER2 contributes to sAD pathogenesis, by disrupting neuronal ApoE delivery and Reelin-ApoER2-Dab1 signaling cascades. In vitro biochemical experiments; Single-marker and multiplex fluorescence-immunohistochemistry (IHC) in postmortem specimens from 26 individuals who died cognitively normal, with mild cognitive impairment or with sAD. ApoE and ApoER2 peptides and proteins were susceptible to attack by reactive lipid aldehydes, generating lipid-protein adducts and crosslinked ApoE-ApoER2 complexes. Using insitu hybridization alongside IHC, we observed that: 1) ApoER2 is strongly expressed in terminal zones of the entorhinal-hippocampal 'perforant path' projections that underlie memory; 2) ApoE, lipid aldehyde-modified ApoE, Reelin, ApoER2, and the downstream Reelin-ApoER2 cascade components Dab1 and Thr19-phosphorylated PSD95 accumulated in the vicinity of neuritic plaques in perforant path terminal zones in sAD cases; 3) several ApoE/Reelin-ApoER2-Dab1 pathway markers were higher in sAD cases and positively correlated with histological progression and cognitive deficits. Results demonstrate derangements in multiple ApoE/Reelin-ApoER2-Dab1 axis components in perforant path terminal zones in sAD and provide proof-of-concept that ApoE and ApoER2 are vulnerable to aldehyde-induced adduction and crosslinking. Findings provide the foundation for a unifying hypothesis implicating lipid peroxidation of ApoE and ApoE receptors in sAD.