Relationship between Brain Metabolic Disorders and Cognitive Impairment: LDL Receptor Defect

The low-density-lipoprotein receptor (LDLr) removes low-density lipoprotein (LDL), an endovascular transporter that carries cholesterol from the bloodstream to peripheral tissues. The maintenance of cholesterol content in the brain, which is important to protect brain function, is affected by LDLr. LDLr co-localizes with the insulin receptor and complements the internalization of LDL. In LDLr deficiency, LDL blood levels and insulin resistance increase, leading to abnormal cholesterol control and cognitive deficits in atherosclerosis. Defects in brain cholesterol metabolism lead to neuroinflammation and blood–brain-barrier (BBB) degradation. Moreover, interactions between endoplasmic reticulum stress (ER stress) and mitochondria are induced by ox-LDL accumulation, apolipoprotein E (ApoE) regulates the levels of amyloid beta (Aβ) in the brain, and hypoxia is induced by apoptosis induced by the LDLr defect. This review summarizes the association between neurodegenerative brain disease and typical cognitive deficits.


Introduction
Low-density-lipoprotein receptor (LDLr) is involved in the regulation of blood cholesterol. LDLr internalizes cholesterol-containing LDL ligands, and insulin receptor (IR) maintains glycemic homeostasis. Although the exact mechanism is not completely clear, diabetes is associated with dyslipidemia. Diabetes is also characterized by abnormalities ranging from IR to absolute deficiency of insulin (type 1 diabetes) or abnormalities in the receptor protein itself post-transcription/translation/translation (type 2 diabetes). The LDL-clearing activity of LDLr depends on the interaction between insulin and IR [1].
Insulin has been reported to stimulate LDLr expression, an IR-dependent signaling event [2]. The inactivation of IR and LDLr prevents the removal of extracellular LDL and promotes hyperproteinemia through intravascular LDL deposition. In insulin-deficient type 1 diabetes and insulin-resistant type 2 diabetes, IR inactivation leads to LDLr inactivation, which increases the severity of atherosclerotic complications due to the inability to remove vascular LDL.
Cholesterol in the brain builds up the membrane surfaces of large numbers of axon dendrites and synapses, including post-synaptic spines and pre-synaptic vesicles [3][4][5]. The brain comprises neurons and glial cells, which build up a large amount of membrane and occupy a high area and volume; thus, the cholesterol requirement is very high. Cholesterol metabolism is important in neurons and glial cells, which should cooperate for brain development and function, and LDLr, which interacts with IR, plays an important role in cholesterol synthesis and turnover regulation [6]. Cholesterol depletion in neurons impairs

Interaction of LDLr and IR
LDLr, a transmembrane glycoprotein, is involved in regulating blood cholesterol by binding and internalizing LDL containing cholesterol [40][41][42][43]. LDLr removes cholesterolcontaining LDL particles [41,44], and IR maintains glycemic homeostasis [45][46][47]. On the other hand, although the exact mechanism is not completely clear, diabetes is associated with dyslipidemia [48]. Diabetes mellitus is characterized by metabolic abnormalities due to insufficient insulin production due to loss of beta cells (type 1 diabetes) or abnormalities in the insulin receptor protein itself (type 2 diabetes). Insulin is known to regulate several biological activities [49][50][51], resulting in increased glucose transport and the maintenance of adequate blood glucose levels [52].
LDLr and IR co-localization in organelles has been observed via electron microscopy. Increased insulin levels increase the LDL uptake of HepG2 cells via LDLr due to the disruption of the LDLr-IR co-localized complex; they further promote the internalization of extracellular LDL particles by directly regulating insulin-mediated LDLr function [53,54]. We have demonstrated the co-localization of LDLr and IR by reducing the expression of LDLr and IR-related proteins via LDLr-specific siRNA treatment [1].
In LDL exposure, autophagosome formation is suppressed via PI3K/Akt/mTOR activation, a key autophagy regulator in HUVECs, similar to the insulin pathway, and LC3 and p62 expression in the lipidized form of the autophagosome decreases. After LDL treatment, LDLr and IR expression increases in the cytosol rather than the membrane, indicating endocytosis from the cell membrane to cytosol, opposite to GLUT1's translocation from the cell cytosol to membrane [55,56].

Interaction of LDLr and IR
LDLr, a transmembrane glycoprotein, is involved in regulating blood cholesterol by binding and internalizing LDL containing cholesterol [40][41][42][43]. LDLr removes cholesterolcontaining LDL particles [41,44], and IR maintains glycemic homeostasis [45][46][47]. On the other hand, although the exact mechanism is not completely clear, diabetes is associated with dyslipidemia [48]. Diabetes mellitus is characterized by metabolic abnormalities due to insufficient insulin production due to loss of beta cells (type 1 diabetes) or abnormalities in the insulin receptor protein itself (type 2 diabetes). Insulin is known to regulate several biological activities [49][50][51], resulting in increased glucose transport and the maintenance of adequate blood glucose levels [52].
LDLr and IR co-localization in organelles has been observed via electron microscopy. Increased insulin levels increase the LDL uptake of HepG2 cells via LDLr due to the disruption of the LDLr-IR co-localized complex; they further promote the internalization of extracellular LDL particles by directly regulating insulin-mediated LDLr function [53,54]. We have demonstrated the co-localization of LDLr and IR by reducing the expression of LDLr and IR-related proteins via LDLr-specific siRNA treatment [1].
In LDL exposure, autophagosome formation is suppressed via PI3K/Akt/mTOR activation, a key autophagy regulator in HUVECs, similar to the insulin pathway, and LC3 and p62 expression in the lipidized form of the autophagosome decreases. After LDL treatment, LDLr and IR expression increases in the cytosol rather than the membrane, indicating endocytosis from the cell membrane to cytosol, opposite to GLUT1's translocation from the cell cytosol to membrane [55,56].
The impairment of IR function is associated with abnormal glucose metabolism leading to impaired glucose tolerance or diabetes, a condition of obesity and insulin resistance [57]. Insulin resistance is also associated with intravascular LDL accumulation due to poor clearance by functionally impaired LDLr. Insulin resistance results in decreased expression levels of insulin and IGF receptors, which promote neurite growth, synapse formation, and neuronal survival in the brains of Alzheimer's patients [58]. The intraventricular injection of streptozotocin, which induces diabetes in rats, or the depletion of neuronal IR results in cognitive decline [59][60][61][62][63].

Cholesterol Metabolism in the Brain
Cholesterol is a structural component of the membrane that acts as a buffer for changes in the fluidity of cell lipid membranes and is involved in membrane-intrinsic proteins and cell signal transduction [64,65]. Despite these important functions, high cholesterol levels can be toxic to cells. An overload of total cellular cholesterol in the plasma membrane triggers its migration to the ER, resulting in the depletion of calcium stores, leading to cell death [66,67]. Additionally, a loss of membrane fluidity due to increased cholesterol levels can lead to dysfunction of the integral membrane protein and damage to the membrane domain, resulting in the disruption of signaling events [68].
Consequently, sterol-sensing proteins that regulate cholesterol homeostasis, sterol synthesis and degradation by regulatory mechanisms, and LDLr are also involved [69]. The brain is the most cholesterol-rich organ in the human body and contains 25% cholesterol [70,71]. Cholesterol homeostasis may influence neuroinflammatory expression for PCSK9 and neuronal receptors. PCSK9, which can regulate the cholesterol receptor LDLr and apolipoprotein E (ApoE), maintains a certain concentration of cholesterol in the human cerebrospinal fluid under normal conditions [72,73]. Under increased BBB permeability in disease states, PCSK9 crosses the BBB and induces LDLr degradation in the brain [74,75].
High levels of LDL-C have been observed in stroke patients with mutations in LDLrrelated genes. Sequencing has been performed in stroke patients with LDLr, apolipoprotein B (ApoB), and PCSK9 gene targets, and the familial hypercholesterolemia pathogenic gene may indicate atherosclerotic phenotypes such as increased carotid intima-media thickness and ischemic attacks [76][77][78][79].

BBB Breakdown
The BBB is formed and maintained by cerebral capillaries, pericytes surrounding the capillaries, and endothelial cells surrounding astrocyte ends that surround these two layers. The BBB, composed of adherens junctions (AJs) and tight junctions (TJs), selectively regulates the transport of molecules and cells in and out of the brain, thereby regulating the brain microenvironment [80,81]. It is composed of TJs comprising transmembrane proteins such as occludin and claudins and AJs comprising VE-cadherin, which plays an important role in maintaining brain endothelial junctions. The BBB maintains the homeostasis of neurovascular units, including blood vessels and nerve cells [82].
Many studies have demonstrated the potential for BBB disruption to alter brain regions and dissociation states, allowing neurotoxic plasma components, blood cells, and pathogens to enter the brain and cause neuroinflammation [83,84]. LDLr −/− mice exposed to a high-cholesterol diet and accompanied by a decreased gene expression of claudin-5 and occludin and increased BBB permeability to stimulate neuroinflammation and cognition are more susceptible to the disorder, and BBB disruption is associated with brain changes due to hypercholesterolemia [75]. In LDLr −/− mice fed a high-cholesterol diet, aquaporin-4 (AQP-4) expression increases [85,86], occludin and claudin-5 gene expression decreases, BBB permeability increases, and GFAP-derived neuroinflammation increases in the hippocampus [75].

Neuroinflammation
LDLr is important for regulating LDL homeostasis. LDL is oxidized, producing oxLDL. Although LDL-C levels decrease in the sera of patients with rheumatoid arthritis, a chronic inflammatory disease, high levels of oxLDL [87] produce cytokines such as TNF-a and IL-6 [88,89]. After cerebral I/R injury, the downregulation of LDLr expression in neurons, astrocytes, and oligodendrocytes [90] leads to neurological deficits, infarction, and edema in the CNS. Additionally, LDLr KO activates pattern recognition receptors (PRRs) present in innate immune cells with damage-associated molecular patterns (DAMPs) generated in damaged cells or tissues ( Figure 2) [91], and in the middle cerebral artery occlusion (MCAO) model under ischemic conditions. Additionally, LDL accumulation was proven to be inflammatory by inducing TLR activation via pathogen-associated molecular patterns (PAMPs) [92,93], and oxLDL, acting as a DAMP and PAMP, forms the NLRP3 inflammasome complex [94,95]. The complex induces gasdermin D (GSDMD)-mediated lytic apoptosis (pyroptosis) by increasing the levels of active caspase-1. Pyroptosis, a programmed cell death process, is mediated by a pore formed by the binding of GSDMD N-terminal fragments cleaved by active caspase-1 to the plasma membrane, inducing the release of inflammatory cytokines such as IL-1β and IL-18, leading to neuronal pyroptosis [96][97][98][99][100]. Additionally, interleukin-10 (IL-10), an anti-inflammatory cytokine, inhibits the activity of inflammatory cytokines by inhibiting TLR induction [93].

Interaction between ER Stress and Mitochondria
LDLr deficiency can lead to cognitive impairment due to interactions between ER stress and mitochondria. In particular, the brain is vulnerable to oxidative damage and apoptosis induction by abnormal calcium and ATP levels due to its high energy metabolism rate, high oxygen consumption, and high ratio of polyunsaturated fatty acids [105][106][107][108][109][110]. Oxidative stress is attracting attention as a cause of several neurodegenerative diseases [111][112][113]. The blockade of NLRP3 delays neuronal pyroptosis in LDLr −/− mice and cultured LDLr −/− neurons after experimental stroke [91]. Oxidative stress and mitochondrial DNA (mtDNA) damage have been observed in atherosclerotic plaques in LDLr −/− mice, and the number of atherosclerotic plaques increase due to an additional deficiency of Ogg1 promoting NLRP3 inflammasome activation in LDLr −/− mice. The mitochondria and mtDNA, which are sites for reactive oxygen species (ROS) production, are vulnerable to oxidative stress due to a lack of protective histones. Ogg1, a glycosylase responsible for the cleavage of 7,8-dihydro-8-oxo-2'-deoxyguanosine, a byproduct of ROS, and AMPK, an upstream regulator, are targets of miR-33, a proatherogenic microRNA. As a result, the levels of Ogg1 and AMPK decrease in atherosclerotic plaques [101].
LDLr overexpression is associated with inflammation relief. Lipopolysaccharides (LPS), which are made up of lipids and polysaccharides, can cause inflammation. They have the highest binding affinity for high-density lipoprotein (HDL), and HDL-binding LPS is redistributed to LDL and VLDL [102]. Pathogen lipids such as LPS are integrated and transduced into lipoprotein particles such as HDL, LDL, and VLDL, triggering an immuneinflammatory response through Toll-like receptors (TLRs). TLRs are mammalian PRRs that recognize structural pathogen-associated molecular patterns shared by microorganisms in innate immunity. The inhibition of PCSK9, which promotes the degradation of LDLr lysosomes, increases the ability of LDLr to remove pathogenic lipids, thereby reducing the inflammatory response [103,104].

Interaction between ER Stress and Mitochondria
LDLr deficiency can lead to cognitive impairment due to interactions between ER stress and mitochondria. In particular, the brain is vulnerable to oxidative damage and apoptosis induction by abnormal calcium and ATP levels due to its high energy metabolism rate, high oxygen consumption, and high ratio of polyunsaturated fatty acids [105][106][107][108][109][110]. Oxidative stress is attracting attention as a cause of several neurodegenerative diseases [111][112][113].
Abnormally folded proteins in the ER lumen are increased in atherosclerosis resulting from cholesterol accumulation. The three major ER-stress sensor proteins of the UPR that remove these abnormally folded proteins are inositol-requiring enzyme 1 (IRE1), activating transcription factor 6 (ATF6) and protein kinase RNA-like ER kinase (PERK). In the steady state, the three sensor proteins remain inactive due to their binding to the ER chaperone, glucose response protein-78 (BiP/GRP78). Stress releases BiP from the ER sensor and induces the phosphorylation and dimerization of IRE1 and PERK in cancers, whereby active PERK promotes the phosphorylation of eukaryotic initiation factor 2α (eIF2α) and inhibits protein translation by the activation of activating transcription factor 4 (ATF4) [123,124], and X-box binding protein 1 (XBP1) expression is achieved through ATF6 activation [125]. However, prolonged cellular stress induces CHOP expression, and a CHOP-mediated imbalance in the Bcl-2 family activates proapoptotic proteins in the mitochondrial membrane, inducing the release of cytochrome c, leading to subsequent mitochondrion-dependent apoptosis [126,127].

Figure 3.
Interaction between ER stress and mitochondria. The process by which ER stress and mitochondria interact due to oxLDL accumulation due to LDLr deficiency affects cognitive impairment.

ApoE
Low-density-lipoprotein receptor (LDLR) and apolipoprotein E (ApoE) are responsible for the transport of cholesterol-rich lipoproteins (Figure 4). Deficiencies in LDLR and ApoE are associated with increased plasma total cholesterol and, consequently, a higher risk of hypercholesterolemia, atherosclerosis, and coronary artery disease [140][141][142]. ApoE is mainly produced by astrocytes and is a protein that plays a role in lipid transport in the CNS [143]. Unlike the LDLR-binding molecule ApoB100, small fat-soluble molecules such as ApoE can cross the BBB-forming membrane and affect BBB stability [144]. ApoE was found to co-localize with amyloid plaque [145,146]. ApoE exists in three isoforms: ApoE2, ApoE3, and ApoE4. ApoE4 induces brain damage as traumatic brain injury (TBI) [147], Alzheimer's disease [148], and conditions leading to impaired cognition [149] do.
LDLR is an important receptor for ApoE in the central nervous system. [10.1016/j.nbd.2004.01.015] (accessed on 26 July 2022) Amyloid beta (Aβ), known to cause Alzheimer's disease, is hypothesized to accumulate in the brain as plaques and is regulated by receptors for ApoE [150][151][152]. LDLr is expressed in astrocytes and induces the uptake of ApoE and Aβ [153]. It prevents Aβ deposition by reducing ApoE levels in LDLroverexpressing mice [154]. LDLr is associated with the major central nervous system ApoE receptor regulating amyloid deposition in a distinct mouse model of β-amyloidosis. The acute intraventricular injection of aggregated Aβ  peptides was used to show increased susceptibility to Aβ-induced neurotoxicity, with intrahippocampal oxidative stress, neuroinflammation, nerve membrane damage, memory deficits, and increased blood-brain-barrier permeability [155].
ApoE also affects the immune response, and in mice, ApoE KO increased the expression of Toll-like receptor 4 (TLR4) and LOX-1, suggesting the formation of foam cells and promoting the onset of arteriosclerosis [156]. Additionally, ApoE promotes phagocytosis by binding to triggering receptor expressed on myeloid cells 2 (TREM2) in microglia [157]. LDLr −/− mice exhibit reduced numbers of synaptophysin-immunoreactive presynaptic Figure 3. Interaction between ER stress and mitochondria. The process by which ER stress and mitochondria interact due to oxLDL accumulation due to LDLr deficiency affects cognitive impairment.
In pathological conditions such as ischemia and reperfusion, the depolarization of the mitochondrial membrane potential increases, causing Ca 2+ overload in the outer membrane and Ca 2+ conduction through the calcium uniporter (MCU), which is locally present in the inner mitochondrial membrane. Increased Ca 2+ is produced due to a bidirectional interaction with ROS [128][129][130][131]. The mitochondrial outer membrane channel is formed by the opening of the mitochondrial permeability transition pore (mPTP), which induces mitochondrial membrane permeability and the insertion of proapoptotic BH3-domain-containing proteins such as Bcl-2-associated X protein (Bax). In addition, due to the depletion of cardiolipin, a lipid constituting the inner membrane of mitochondria, cytochrome c is released from the inner membrane of mitochondria into the cytoplasm [132,133]. Cytochrome c forms an apoptosome and induces apoptosis by forming a complex with apoptosis-protease activating factor 1, which is required for the proteolysis of caspase-9 and caspase-3 [134,135]. In the mitochondria, ROS are formed as a byproduct of oxidative phosphorylation that induces ATP production. Imbalances in ROS, ATP, and Ca 2+ that appear in mitochondrial dysfunction increase the expression of antioxidant enzymes, such as superoxide dismutase (SOD), glutathione (GSH), and glutathione peroxidase (GPx), and the Bcl-2-mediated apoptosis mechanism [136]. Furthermore, oxidative stress also affects endothelial cells.
Hypercholesterolemia-induced LDLr −/− mice have shown a decreased activity of mitochondrial complex I and II in the cerebral cortex; decreased GSH; an approximately 40% increase in complexes formed via thiobarbituric acid's reaction with malondialdehyde (MDA), a product of lipid peroxidation; and enzymes related to peroxide removal. An imbalance in phosphorus GPx/glutathione reductase activity results in mitochondrial dysfunction and oxidative stress, leading to cognitive impairment [137,138]. In 14-monthold LDLr −/− mice, antioxidant imbalance and glutathione metabolism increase due to brain oxidative stress because of lipid peroxidation, and the acetylcholinesterase activity, which degrades the neurotransmitter acetylcholine, also increases [139]. The memory deficit observed in LDLr −/− mice is not related to Aβ-level changes in the prefrontal cortex and hippocampus; however, Bcl-2 expression and caspase-3 activation decrease, while Bax expression increases.

ApoE
Low-density-lipoprotein receptor (LDLR) and apolipoprotein E (ApoE) are responsible for the transport of cholesterol-rich lipoproteins (Figure 4). Deficiencies in LDLR and ApoE are associated with increased plasma total cholesterol and, consequently, a higher risk of hypercholesterolemia, atherosclerosis, and coronary artery disease [140][141][142]. ApoE is mainly produced by astrocytes and is a protein that plays a role in lipid transport in the CNS [143]. Unlike the LDLR-binding molecule ApoB100, small fat-soluble molecules such as ApoE can cross the BBB-forming membrane and affect BBB stability [144]. ApoE was found to co-localize with amyloid plaque [145,146]. ApoE exists in three isoforms: ApoE2, ApoE3, and ApoE4. ApoE4 induces brain damage as traumatic brain injury (TBI) [147], Alzheimer's disease [148], and conditions leading to impaired cognition [149] do.

Conclusions
Our analysis revealed that LDLr defects might lead to IR interactions, BBB bre down, neuroinflammatory responses, interactions between ER stress and mitochond and hypoxia. There are various cells in the brain that make up a complex cellular netw Each cell exhibits a lipid membrane structure as an essential element for cell maintena and signal transduction, and lipids are then delivered by LDL, which is regulated by LD However, studies on LDLr defects have mainly been conducted in the liver, whic the main organ for lipid synthesis; thus, there is a lack of studies on changes in hormo such as cortisol, insulin, and leptin secreted into the brain signaling system and the me anism of LDLr regulation specifically for brain cells.
In conclusion, this review summarizes the relationship between LDLr defects brain metabolism. Further detailed studies are required to elucidate the mechanisms volved in LDLr.  .015] (accessed on 26 July 2022) Amyloid beta (Aβ), known to cause Alzheimer's disease, is hypothesized to accumulate in the brain as plaques and is regulated by receptors for ApoE [150][151][152]. LDLr is expressed in astrocytes and induces the uptake of ApoE and Aβ [153]. It prevents Aβ deposition by reducing ApoE levels in LDLr-overexpressing mice [154]. LDLr is associated with the major central nervous system ApoE receptor regulating amyloid deposition in a distinct mouse model of β-amyloidosis. The acute intraventricular injection of aggregated Aβ  peptides was used to show increased susceptibility to Aβ-induced neurotoxicity, with intrahippocampal oxidative stress, neuroinflammation, nerve membrane damage, memory deficits, and increased blood-brain-barrier permeability [155].
ApoE also affects the immune response, and in mice, ApoE KO increased the expression of Toll-like receptor 4 (TLR4) and LOX-1, suggesting the formation of foam cells and promoting the onset of arteriosclerosis [156]. Additionally, ApoE promotes phagocytosis by binding to triggering receptor expressed on myeloid cells 2 (TREM2) in microglia [157]. LDLr −/− mice exhibit reduced numbers of synaptophysin-immunoreactive presynaptic boutons in hippocampus CA1 compared to LDLr +/+ mice, resulting in hippocampusdependent memory function impairment [0].

Conclusions
Our analysis revealed that LDLr defects might lead to IR interactions, BBB breakdown, neuroinflammatory responses, interactions between ER stress and mitochondria, and hypoxia. There are various cells in the brain that make up a complex cellular network. Each cell exhibits a lipid membrane structure as an essential element for cell maintenance and signal transduction, and lipids are then delivered by LDL, which is regulated by LDLr.
However, studies on LDLr defects have mainly been conducted in the liver, which is the main organ for lipid synthesis; thus, there is a lack of studies on changes in hormones such as cortisol, insulin, and leptin secreted into the brain signaling system and the mechanism of LDLr regulation specifically for brain cells.
In conclusion, this review summarizes the relationship between LDLr defects and brain metabolism. Further detailed studies are required to elucidate the mechanisms involved in LDLr.