Lack of LDL Receptor Enhances Amyloid Deposition and Decreases Glial Response in an Alzheimer's Disease Mouse Model

BACKGROUND: Apolipoprotein E (ApoE), a cholesterol carrier associated with atherosclerosis, is a major risk factor for Alzheimer's disease (AD). The low-density lipoprotein receptor (LDLR) regulates ApoE levels in the periphery and in the central nervous system. LDLR has been identified on astrocytes and a number of studies show that it modulates amyloid deposition in AD transgenic mice. However these findings are controversial on whether LDLR deletion is beneficial or detrimental on the AD-like phenotype of the transgenic mice. METHODOLOGY/PRINCIPAL FINDINGS: To investigate the role of LDLR in the development of the amyloid related phenotype we used an APP/PS1 transgenic mouse (5XFAD) that develops an AD-like pathology with amyloid plaques, astrocytosis and microgliosis. We found that 4 months old 5XFAD transgenic mice on the LDLR deficient background (LDLR-/-) have increased amyloid plaque deposition. This increase is associated with a significant decrease in astrocytosis and microgliosis in the 5XFAD/LDLR-/- mice. To further elucidate the role of LDLR in relation with ApoE we have generated 5XFAD transgenic mice on the ApoE deficient (ApoE-/-) or the ApoE/LDLR double deficient background (ApoE-/-/LDLR -/-). We have found that ApoE deletion in the 4 months old 5XFAD/ApoE-/- mice decreases amyloid plaque formation as expected, but has no effect on astrocytosis or microgliosis. By comparison 5XFAD/ApoE-/-LDLR -/- double deficient mice of the same age have increased amyloid deposition with decreased astrocytosis and microgliosis. CONCLUSIONS: Our analysis shows that LDL deficiency regulates astrocytosis and microgliosis in an AD mouse model. This effect is independent of ApoE, as both 5XFAD/LDLR -/- and 5XFAD/ApoE-/- LDLR -/- mice show reduction in inflammatory response and increase in amyloid deposition compared to control mice. These results demonstrate that LDLR regulates glial response in this mouse model independently of ApoE and modifies amyloid deposition

Calcium Channel CaV2.3 Subunits Regulate Hepatic Glucose Production by Modulating Leptin-Induced Excitation of Arcuate Pro-opiomelanocortin Neurons.

Leptin acts on hypothalamic pro-opiomelanocortin (POMC) neurons to regulate glucose homeostasis, but the precise mechanisms remain unclear. Here, we demonstrate that leptin-induced depolarization of POMC neurons is associated with the augmentation of a voltage-gated calcium (CaV) conductance with the properties of the "R-type" channel. Knockdown of the pore-forming subunit of the R-type (CaV2.3 or Cacna1e) conductance in hypothalamic POMC neurons prevented sustained leptin-induced depolarization. In vivo POMC-specific Cacna1e knockdown increased hepatic glucose production and insulin resistance, while body weight, feeding, or leptin-induced suppression of food intake were not changed. These findings link Cacna1e function to leptin-mediated POMC neuron excitability and glucose homeostasis and may provide a target for the treatment of diabetes

Modulation of SF1 neuron activity coordinately regulates both feeding behaviour and associated emotional states

Feeding requires the integration of homeostatic drives with emotional states relevant to food procurement in potentially hostile environments. The ventromedial hypothalamus (VMH) regulates feeding and anxiety, but how these are controlled in a concerted manner remains unclear. Using pharmacogenetic, optogenetic, and calcium imaging approaches with a battery of behavioral assays, we demonstrate that VMH steroidogenic factor 1 (SF1) neurons constitute a nutritionally sensitive switch, modulating the competing motivations of feeding and avoidance of potentially dangerous environments. Acute alteration of SF1 neuronal activity alters food intake via changes in appetite and feeding-related behaviors, including locomotion, exploration, anxiety, and valence. In turn, intrinsic SF1 neuron activity is low during feeding and increases with both feeding termination and stress. Our findings identify SF1 neurons as a key part of the neurocircuitry that controls both feeding and related affective states, giving potential insights into the relationship between disordered eating and stress-associated psychological disorders in humans

Study of the role of apolipoprotein E and cholesterol in the pathogenesis of Alzheimer's disease in transgenic mice

Alzheimer’s disease (AD) is a neurodegenerative disease that is affecting the higher cognitive functions. It is the most common form of senile dementia and is mainly affecting people after the age of 65. Inheritance of the ε4 allele of Apolipoprotein E (ApoE4) is the major risk factor for the development of the sporadic form of AD. The LDLR is one of the main APOE receptors both in the circulation and in the CNS. In the present study the role of the LDLR was studied in a transgenic mouse model with Alzheimer’s-like pathology (5xFAD). In addition, the role of the peripheral-derived (non-CNS) APOE4 was analysed. To study the role of the LDLR in the pathogenesis of Alzheimer’s disease we have generated transgenic mice that develop an Alzheimer’s like phenotype (5xFAD transgenic mice) and lack the endogenous Ldlr gene (Ldlr-/-). The 5XFAD;Ldlr-/- mice were analysed for amyloid deposition and APOE levels and it was shown that amyloid plaques as well as amyloid β (Αβ) brain levels were increased irrespectively of the presence or the absence of APOE. The guanidine levels of APOE (plaque-associated fraction) were also increased in brain homogenates of 5XFAD;Ldlr-/- mice. There was no difference in levels of the total amyloid precursor protein (APP) or its carboxy-terminal fragments (CTF) indicating that the increase in the Aβ levels is probably due to defective clearance of amyloid. Furthermore, analysis of the inflammatory response showed a significant decrease in astrocytosis and microgliosis in the absence of LDLR independently of APOE. To study the role of peripheral-derived APOE in the pathogenesis of Alzheimer’s disease we generated transgenic mice that express the genomic human allele ε4 of ApoE under the transthyretin (TTRI) promoter (TTRI-huApoE4 transgenic mouse model). These mice express the transgene only in the liver and macrophages and not in the brain. The TTRI-huApoE4 transgenic mice were crossed with a mouse model with Alzheimer-like pathology (5xFAD). Pathogenesis of the AD-like phenotype was analysed in mice of the genotypes 5xFAD, 5xFAD;ApoE-/- and 5xFAD;TTRI-huApoE4;ApoE-/- with immunoblotting, ELISA and immunohistochemistry methods. Histological analysis showed that 5xFAD;TTRI-huApoE4;ApoE-/- had more hippocampal and cortical amyloid plaques than the 5xFAD;ApoE-/- mice. The 5xFAD mice that have the endogenous ApoE, had the most abundant load of Aβ and amyloid plaques compared to the other genotypes. Immunohistochemistry for the Aβ peptide revealed that the 5xFAD mice had the most intense and dense pattern of amyloid and the 5xFAD;ApoE-/- the most diffused. The 5xFAD;TTRI-huApoE4;ApoE-/- mice that express APOE4 only in the periphery and they don’t have the endogenous ApoE had a diffused pattern like the 5xFAD;ApoE-/- mice but the spatial distribution resembled the pattern of the 5xFAD’s Aβ deposition. Astrocytosis and microgliosis were also analysed in the aforementioned groups of mice. It was shown that microgliosis in the 5xFAD;TTRI-huApoE4;ApoE-/- was decreased compared to both the 5xFAD and the 5xFAD;ApoE-/- mice. In addition the microglia/macrophages (IBA1-positive cells) surrounding the amyloid plaques were also positive for huAPOE4 in the 5xFAD;TTRI-huApoE4;ApoE-/- mice. In order to establish if these cells were blood-derived infiltrating microglia/macrophages, 5xFAD;ApoE-/- mice received bone-marrow transplantation from Tg(CAG-EGFP)10sb/J;TTRI-huApoE4;ApoE mice which express the green fluorescent protein (GFP) in all cell types. We found that GFP-positive cells from the periphery infiltrated the blood-brain barrier and expressed APOE4. Also, the anti-inflammatory cytokine IL-10 was increased in the brains of 5xFAD;TTRI-huApoE4;ApoE-/- compared to the 5xFAD;ApoE-/-. Our data demonstrate the importance of LDLR in the amyloid plaque formation and the inflammatory response in the brain with Alzheimer-like pathology. Moreover, this study showed that peripheral expression of huApoE4 in the 5xFAD;TTRI-huApoE4 mice results in increased amyloid deposition and reduced microglial activation compared to the 5xFAD;ApoE-/- mice. Our proposed mechanism is that peripheral APOE promotes Aβ aggregation by modulating the activation of microglial cells through infiltration of huAPOE4-expressing macrophages in the brain, thus providing an accessible target to regulate in AD pathogenesis.Η νόσος του Alzheimer (AD) είναι μια νευροεκφυλιστική νόσος που έχει ως αποτέλεσμα την προοδευτική απώλεια των ανώτερων γνωστικών λειτουργιών. Το αλληλόμορφο ε4 της Απολιποπρωτεΐνης Ε (Apolipoprotein E4, ApoE4) είναι ο κυριότερος παράγοντας κινδύνου για την εμφάνιση της σποραδικής μορφής της νόσου. Ο LDLR, το αρχέγονο μέλος αυτής της υπεροικογένειας, είναι ο κυριότερος υποδοχέας της ΑΡΟΕ στον εγκέφαλο. Στην παρούσα μελέτη εξετάστηκε ο ρόλος του LDLR και της προερχόμενης από την περιφέρεια ΑΡΟΕ4 στην παθογένεση της νόσου σε ένα διαγονιδιακό μοντέλο ποντικού με παθολογία τύπου Alzheimer. Για τη μελέτη του ρόλου του LDLR στη νόσο του Alzheimer αναλύθηκαν διαγονιδιακά ποντίκια με τη συγκεκριμένη παθολογία (ποντίκια 5xFAD) στα οποία είχε αποσιωποιηθεί το γονίδιο του Ldlr (5xFAD;Ldlr-/-). Στα ποντίκια 5xFAD;Ldlr-/- επισημάνθηκε αύξηση της ΑΡΟΕ στο συσχετιζόμενο με τις πλάκες κλάσμα από εκχύλισμα εγκεφάλων σε σχέση με τα ποντίκια 5xFAD που έχουν τον ενδογενή LDLR. Για να διασαφηνιστεί ο ρόλος της ΑΡΟΕ σε σχέση με τον LDLR, χρησιμοποιήθηκαν επίσης ποντίκια 5xFAD και 5xFAD;Ldlr-/- με αδρανοποιημένο το γονίδιο της ApoE (5xFAD;ApoE-/- και 5xFAD;ApoE-/-;Ldlr-/- αντίστοιχα). Η ανάλυση έδειξε ότι στα ποντίκια με αδρανοποιημένο τον LDLR υπήρξε αύξηση του σχηματισμού αμυλοειδών πλακών καθώς και των επιπέδων του β-αμυλοειδούς (Αβ), ανεξάρτητα από την παρουσία της ΑΡΟΕ. Δεν υπήρξε καμιά διαφορά στα πρωτεϊνικά επίπεδα των καρβοξυτελικών πρωτεολυτικών θραυσμάτων (CTF) της πρόδρομης πρωτεΐνης του αμυλοειδούς (ΑΡΡ) γεγονός που υποδηλώνει ότι μειώνεται η απομάκρυνση και όχι η παραγωγή του Αβ. Ανάλυση της φλεγμονώδους αντίδρασης στα ποντίκια αυτά έδειξε μείωση στην αστροκυττάρωση και τη μικρογλοίωση απουσία του LDLR ανεξάρτητα από την παρουσία της ΑΡΟΕ. Για τη μελέτη του ρόλου της προερχόμενης από την περιφέρεια Απολιποπρωτεΐνης Ε στην παθογένεση της νόσου του Alzheimer, κατασκευάστηκαν τα διαγονιδιακά ποντίκια TTRI-huApoE4. Τα ποντίκια αυτά εκφράζουν το γενωμικό αλληλόμορφο ε4 της ανθρώπινης ΑΡΟΕ υπό τον υποκινητή της τρανσθυρετίνης (TTRI), o οποίος εξασφαλίζει έκφραση του διαγονιδίου στο ήπαρ και τα μακροφάγα. Τα TTRI-huApoE4 διασταυρώθηκαν αρχικά με ποντίκια ApoE-/- και στη συνέχεια με το διαγονιδιακό μοντέλο 5xFAD. H ανάλυση έδειξε ότι τα 5xFAD;TTRI-huApoE4;ApoE-/- σχηματίζουν περισσότερες αμυλοειδείς πλάκες από τα 5xFAD;ApoE-/- τόσο στο φλοιό όσο και στον ιππόκαμπο. Από ανοσοϊστοχημεία για το Αβ προέκυψε ότι τα 5xFAD;TTRI-huApoE4;ApoE-/- ποντίκια, έχουν όμοιο πρότυπο με τα 5xFAD;ApoE-/- ως προς το σχήμα των εναποθέσεων. Αντιθέτως, ως προς τις περιοχές στις οποίες το Αβ εναποτίθεται στα 5xFAD;TTRI-huApoE4;ApoE-/-, το πρότυπο μοιάζει με αυτό των ποντικών 5xFAD που έχουν την ενδογενή ΑΡΟΕ. Ανάλυση της φλεγμονώδους αντίδρασης έδειξε ότι η μικρογλοίωση είναι μειωμένη στα ποντίκια 5xFAD;TTRI-huApoE4;ApoE-/- σε σχέση με τις ομάδες ελέγχου. Επίσης βρέθηκε ότι κύτταρα θετικά για IBA1 (μικρογλοιακά/μακροφάγα) συνεντοπίζονται με την ευρισκόμενη πάνω στις αμυλοειδείς πλάκες ΑΡΟΕ4. Για να διαπιστωθεί εάν αυτά τα κύτταρα προέρχονται από την περιφέρεια, ποντίκια 5xFAD;ApoE-/- δέχτηκαν μεταμόσχευση μυελού των οστών από ποντίκια Tg(CAG-EGFP)10sb/J;TTRI-huApoE4;ApoE που εκφράζουν σε όλα τους τα κύτταρα την πράσινη φθορίζουσα πρωτεΐνη (GFP). Ανοσοϊστοχημεία για GFP έδειξε ότι στα ποντίκια που υπέστησαν μεταμόσχευση, κύτταρα από την περιφέρεια διαπερνούν τον αιματο-εγκεφαλικό φραγμό και εκφράζουν ΑΡΟΕ4. Επίσης βρέθηκε αύξηση της αντιφλεγμονώδους κυτοκίνης ιντερλευκίνης-10 (IL-10) στα ποντίκια 5xFAD;TTRI-huApoE4;ApoE-/- σε σχέση με τα ποντίκια 5xFAD;ApoE-/-. Στην παρούσα μελέτη, παρουσιάζεται η σημαντικότητα του ρόλου του LDLR στην εναπόθεση του Αβ και στο σχηματισμό των αμυλοειδών πλακών. Καταδεικνύεται, ακόμη, ότι ο LDLR ρυθμίζει τη φλεγμονώδη αντίδραση στον εγκέφαλο των ποντικών με παθολογία τύπου Alzheimer. Επιπρόσθετα, δείχνεται ότι η περιφερική έκφραση της ΑΡΟΕ4 επηρεάζει τόσο το σχηματισμό αμυλοειδών πλακών όσο και τη φλεγμονώδη απόκριση στο διαγονιδιακό μοντέλο με παθολογία τύπου Alzheimer κι έτσι υποδεικνύεται ένας στόχος πιο προσβάσιμος στις φαρμακευτικές αγωγές

Results of Student's t-test for Aβ immunostaining in the cortices of the analyzed groups.

<p>Results of Student's t-test for Aβ immunostaining in the cortices of the analyzed groups.</p

Lack of LDLR increases brain ApoE levels in the 5XFAD/<i>LDLR</i>-/- mice and has no effect on the APP processing.

<p>A. Western blot for ApoE of protein extracts of 5XFAD and 5XFAD/<i>LDLR</i>-/- mouse brains. Tubulin was used as a loading control. 30 µg from the guanidine fraction of brain homogenates was loaded in each lane. In the absence of LDLR the levels of brain ApoE are increased. B. Densitometry of western blots shows a significant increase in the levels of ApoE in the guanidine fraction of total brain extracts. Statistical analysis was performed with the Student's <i>t-test</i> (** P = 0.0041). C. 10 µg of protein from the lysis fraction of total brain homogenates were loaded in each lane and immunoblotted for full length APP and CTFs. GAPDH was used as a loading control. No difference was observed in the full length APP or the CTFs when normalised to the control indicating that LDLR deficiency has no effect on the steady-state levels of full-length APP or on α- and β-secretase activity (5-7 animals per genotype were analysed and the experiments were repeated 3 times).</p

Astrocytosis is decreased in the brains of 5XFAD/<i>LDLR</i>-/- and 5XFAD/<i>ApoE-/- LDLR</i>-/- mice.

<p>Immunohistochemistry for GFAP (red) and Thioflavine-S (green) in the hippocampi (A) and the cortices (B) of mouse brains. Representative pictures are shown for each genotype. In both areas examined the absence of LDLR results in reduction of GFAP immunoreactivity. Astrocytosis in both the cortex and the hippocampus is more intense in the 5XFAD and the 5XFAD/<i>ApoE</i>-/- compared to the 5XFAD/<i>LDLR</i>-/- and the 5XFAD/<i>ApoE</i>-/-<i>LDLR</i>-/- sections respectively. Scale bar 250 µm. The pictures were taken under the same conditions of intensity and the experiment was repeated 3 times. (n = 5−7, 3−4 independent sections per animal were analyzed). C. Quantitation of GFAP positive staining in the hippocampi and cortices showing a decrease in the 5XFAD/<i>LDL</i>-/- and 5XFAD/<i>ApoE</i>-/-<i>LDLR</i>-/- mice compared to the control mice. P-values in the hippocampi (left graph) ** P = 0.0040 for 5XFAD vs. 5XFAD/LDLR-/- and P = 0.0031 for 5XFAD/ApoE-/- vs. 5XFAD/ApoE-/-LDLR-/-. In the cortices (right graph) * P = 0.0399 for 5XFAD vs. 5XFAD/LDLR-/- and P = 0.0205 for 5XFAD/ApoE-/- vs. 5XFAD/ApoE-/-LDLR-/-.</p

LDLR deletion increases Aβ deposition in the mouse hippocampus.

<p>A. Immunohistochemistry for total Aβ (6E10) in the brains of female mice. Representative pictures are shown for each genotype. The Aβ deposition is increased in the subiculum of the 5XFAD/<i>LDLR</i>-/- mice compared to the 5XFAD mice. The same effect is observed in the 5XFAD/<i>ApoE</i>-/-<i>LDLR</i>-/- mice where Aβ deposition is more intense in the subiculum. Aβ deposition in the 5XFAD/<i>ApoE</i>-/-<i>LDLR</i>-/- cortices appears more ‘dense’ and “compact” compared to the 5XFAD/<i>ApoE</i>-/- mice where Aβ deposition is more “diffuse”. Scale bar 0.5 mm (n = 5−7, 6–7 sections per animal, 240 mm apart). B. Quantitation of Aβ immunoreactivity in the hippocampi (left) of the analysed groups shows a significant increase in the Aβ deposition in the 5XFAD/<i>LDLR</i>-/- and the 5XFAD/<i>ApoE</i>-/-<i>LDLR</i>-/- mice. In the cortices (right) there is no significant difference between 5XFAD and 5XFAD/<i>LDLR</i>-/- as well as between 5XFAD/<i>ApoE</i>-/- and 5XFAD/<i>ApoE</i>-/-/<i>LDLR</i>-/-.<i>*P<0.05.</i> P-values among all groups are analysed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021880#pone-0021880-t002" target="_blank">Table 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021880#pone-0021880-t003" target="_blank">Table 3</a> for hippocampi and cortices respectively. C. Quantitation of Aβ₄₂ and Aβ₄₀ levels by ELISA in the 5XFAD and the 5XFAD/<i>LDLR</i>-/- mouse brain extracts showed an increase both in the guanidine and lysis fraction in the 5XFAD/<i>LDLR</i>-/- mice. A similar increase was also observed in the guanidine fraction of the 5XFAD/<i>ApoE</i>-/-<i>LDLR</i>-/- mice compared to the 5XFAD/<i>ApoE</i>-/- but not in the lysis fraction (n = 5−7) <i>*P<0.05, **P<0.001.</i></p

Results of Student's t-test for Aβ immunostaining in the hippocampi of the analyzed groups.

<p>Results of Student's t-test for Aβ immunostaining in the hippocampi of the analyzed groups.</p