The brain is the most lipid-rich organ in the body and, owing to the impermeable nature of the blood–brain barrier, lipid and lipoprotein metabolism within this organ is distinct from the rest of the body. Apolipoproteins play a well-established role in the transport and metabolism of lipids within the CNS; however, evidence is emerging that they also fulfill a number of functions that extend beyond lipid transport and are critical for healthy brain function. The importance of apolipoproteins in brain physiology is highlighted by genetic studies, where apolipoprotein gene polymorphisms have been identified as risk factors for several neurological diseases. Furthermore, the expression of brain apolipoproteins is significantly altered in several brain disorders. The purpose of this article is to provide an up-to-date assessment of the major apolipoproteins found in the brain (ApoE, ApoJ, ApoD and ApoA-I), covering their proposed roles and the factors influencing their level of expression. Particular emphasis is placed on associations with neurological and psychiatric disorders.

Apolipoproteins in the brain: implications for neurological and psychiatric disorders

Macroautophagy (hereafter referred to as autophagy) is a lysosome-dependent mechanism of intracellular degradation that is used for the turnover and recycling of cytoplasmic constituents. It involves the sequestration of a fraction of cytosol and organelles within a de novo synthesized, double-membraned organelle called the autophagosome, which then fuses with the endosome and/or lysosome to create the amphisome and autolysosome, respectively. Eventually, the inner membrane of the autophagosome, together with the enclosed cargo, is degraded by the lysosomal hydrolases.1 Autophagy in mammals serves multiple purposes: it functions as a survival mechanism during periods of starvation and stress, it is directly involved in eliminating aberrant protein aggregates, removing damaged/aged organelles, and defending against pathogens, and it serves an essential role in early neonatal development.2 Evidently, autophagy also functions as type II programmed cell death (PCD), or autophagic cell death (ACD), in multi-cellular organisms.3,4 It has been reported that autophagy is involved in physiological cell death during development of Drosophila and Dictyostelium discoideum, however, ACD has not yet been observed/demonstrated in mammals in vivo.5,6

In the past two decades, yeast has been used as an experimental model in dissecting the molecular bases of autophagy. Currently 31 ATG (autophagy-related) genes have been identified in yeast.3,6 Recent identification and characterization of human orthologs/homologs of yeast ATG genes and the link between autophagy and human diseases further emphasize the importance of autophagy in health and disease. Deficiency of autophagy correlates with diseases such as cancer, cardiomyopathies, inflammatory bowel diseases and neurodegenerative diseases.5,7 In addition, autophagy involves unique membrane dynamics and biosynthesis, requiring de novo synthesis of double-membrane autophagosomes, in which both protein and lipid components are required. Regarding protein components, aside from Atg proteins, Vps34, a class III phosphatidylinositol-3-phosphate kinase (PI3K), the proteins that are part of its complex, and the Bcl-2 family members play critical roles in regulating autophagosome formation. Interestingly, Beclin 1, the mammalian ortholog of yeast Atg6 that is required for autophagy, is a BH3-only protein. In addition, other Bcl-2 family members, such as Bcl-2, Bax, Bid and BNIP3, regulate autophagy.6,8 By contrast, the lipid composition of autophagosomal membranes remains unknown because it is technically difficult to isolate pure autophagosomes. It is evident however that some lipid species, for example, phosphatitylinositol-3-phosphate or PI3P, are required for autophagosomal membranes.1,9

In an effort to identify and characterize novel BH3-only proteins in ACD, and discover molecular regulators that dictate autophagy for death or survival, we examined a novel BH3-only protein, apolipoprotein L1 (ApoL1). We show that wild-type ApoL1 induces ACD in a variety of cells, whereas a BH3 deletion allele of ApoL1 fails to induce cell death, indicating that ApoL1 is a bona fide BH3-only pro-death protein. We further demonstrate that ApoL1 is a potent and specific inducer of ACD that stimulates time-dependent accumulation and translocation of lipidated LC3-II, formation of autophagosomes and subsequently cell death. We also show that ApoL1 is inducible by p53 during p53-induced cell death. p53, a well-known tumor suppressor, regulates both apoptosis and ACD.4 Interestingly, a recent study shows that p53 plays a dual role in the control of autophagy: on the one hand, nuclear p53 can induce autophagy through transcriptional effects, whereas on the other hand, cytoplasmic p53 may act as a master repressor of autophagy.10 Our results suggest that ApoL1 is a novel BH3-only protein and is a p53 downstream effector functioning as a molecular determinant that determines whether autophagy plays a role in cell death.4 Importantly, ApoL1 has been found only in humans and African green monkeys (NCBI Entrez Gene, ApoL1), suggesting that ApoL1-induced ACD is primate-specific.

Employing a protein-lipid overlay assay, we demonstrate that ApoL1 is an intracellular lipid-binding protein that binds strongly with phosphatidic acid (PA) and cardiolipin (CL), and with less affinity with different species of phosphoinositides (PIPs) in the following order PI(3,5)P2>PI4P>PI5P>PI3P>PI(4,5)P2>PI(3,4,5)P3>PI(3,4)P2.4 PIPs are phosphorylated derivatives of the membrane phospholipids phosphatidylinositols and play critical roles in regulating receptor signaling, cytoskeleton function and membrane trafficking. They are highly concentrated and localized in distinct pools in the plasma membrane, endosomes, nucleus and autophagosomal membranes, where they function as ligands, adaptors or docking sites for PIP-binding proteins. In addition, the homeostatic equilibrium of PIPs determines cellular proliferation, differentiation and PCD.11,12 Vps34 is required for autophagy, implying an essential role of its product PI3P in this process.13 Recently, Obara and colleagues9 showed that PI3P is highly enriched and delivered to autophagosomal membranes but not as a cargo enclosed in autophagosomes, implying direct involvement of PI3P in autophagosome formation. They also report a possible enrichment of PI3P on the inner autophagosomal membranes compared to the outer membrane.9 Regarding binding with PIPs, we show that ApoL1 possesses higher affinity for PI(3,5)P2, PI4P, PI5P, and PI3P, implying that intracellular accumulation of ApoL1 may alter the homeostasis of PIPs leading to ACD. In addition, we conducted a protein domain homology search and found no homology between ApoL1 and any known PIP-binding domains, including FYVE (Fab1, YOTB, Vac1 and EEA1), PX (Phox homology), PH (Pleckstrin homology), ENTH (Epsin N-terminal homology), FERM (Band4.1, Ezrin, Radixin, and Moesin), GRAM (Glucosyltransferase, Rab-like GTPase Activator, and Myotubularins), ANTH (AP180/CALM), and WD-repeats of WIPIs, human orthologs of yeast Atg18 and/or Atg20.7,14,15 In fact, no specific binding domain for PI(3,5)P2 has been identified thus far. Taken together, ApoL1 may represent a novel class of PIP-binding protein. Importantly, in autophagy, at least 2 Atg proteins are involved in PIP synthesis (Atg6 and Atg14) and 5 Atg proteins bind PIP (Atg18, Atg20, Atg21, Atg24 and Atg27).1,7 Evidently, yeast mutant strains deficient in PIP binding will be useful for functional complementation and biochemical analysis of human ApoL1.

Interestingly, ApoL1 also binds PA and CL. PA is a mitogenic messenger that serves as a critical component of mTOR signaling. PA directly interacts with the domain in mTOR that is targeted by rapamycin, and this interaction positively correlates with mTOR's ability to activate downstream effectors. mTOR is a negative regulator of autophagy;16,17 thus, binding of PA by ApoL1 might inhibit mTOR activation and subsequently induce autophagy. In addition, CL, a negatively charged phospholipid abundant in the mitochondrial inner membrane, is important in maintaining protein (for example cytochrome c) and membrane integrity of mitochondria. During apoptosis, CL functions as the docking site for t-Bid and Bad and induces mitochondrial membrane permeability and cytochrome c release.18,19 In addition, CL relocates to the plasma membrane and other subcellular organelles during cell death.20 Sequestering of PA and CL with ApoL1 may alter the homeostasis between survival and death leading to ACD. To our knowledge, this is the first BH3-only protein with lipid binding activity that, when overproduced intracellularly, induces ACD.

ApoL1, a BH3-only lipid-binding protein, induces autophagic cell death.

APOL1 Kidney Disease Risk Variants: An Evolving Landscape

Autophagy is a vital lysosomal degradation and recycling pathway in the eukaryotic cell, responsible for maintaining an intricate balance between cell survival and cell death, necessary for neuronal survival and function. This dual role played by autophagy raises the question whether this process is a protective or a destructive pathway, the contributor of neuronal cell death or a failed attempt to repair aberrant processes? Deregulated autophagy at different steps of the pathway, whether excessive or downregulated, has been proposed to be associated with neurodegenerative disorders such as Alzheimer's-, Huntington's-, and Parkinson's-disease, known for their intracellular accumulation of protein aggregates. Recent observations of impaired autophagy also appeared in psychiatric disorders such as schizophrenia and bipolar disorder suggesting an additional contribution to the pathophysiology of mental illness. Here we review the current understanding of autophagy's role in various neuropsychiatric disorders and, hitherto, the prevailing new potential autophagy-related therapeutic strategies for their treatment.

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Dual Role of Autophagy in Diseases of the Central Nervous System.

Probing molecular brain mechanisms related to increased suicide risk is an important issue in biological psychiatry research Gene expression studies on post mortem brains indicate extensive changes prior to a successful suicide attempt; however, proteomic studies are scarce Thus, we performed a DIGE proteomic analysis of post mortem tissue samples from the prefrontal cortex and amygdala of suicide victims to identify protein changes and biomarker candidates of suicide Among our matched spots we found 46 and 16 significant differences in the prefrontal cortex and amygdala, respectively; by using the industry standard t test and 13 fold change as cut off for significance Because of the risk of false discoveries (FDR) in these data, we also made FDR adjustment by calculating the q-values for all the t tests performed and by using 006 and 04 as alpha thresholds we reduced the number of significant spots to 27 and 9 respectively From these we identified 59 proteins in the cortex and 11 proteins in the amygdala These proteins are related to biological functions and structures such as metabolism, the redox system, the cytoskeleton, synaptic function, and proteolysis Thirteen of these proteins (CBR1, DPYSL2, EFHD2, FKBP4, GFAP, GLUL, HSPA8, NEFL, NEFM, PGAM1, PRDX6, SELENBP1 and VIM,) have already been suggested to be biomarkers of psychiatric disorders at protein or genome level We also pointed out 9 proteins that changed in both the amygdala and the cortex, and from these, GFAP, INA, NEFL, NEFM and TUBA1 are interacting cytoskeletal proteins that have a functional connection to glutamate, GABA, and serotonin receptors Moreover, ACTB, CTSD and GFAP displayed opposite changes in the two examined brain structures that might be a suitable characteristic for brain imaging studies The opposite changes of ACTB, CTSD and GFAP in the two brain structures were validated by western blot analysis

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Brian Galloway

Papers

Association of SNPs and Haplotypes in APOL1, 2 and 4 with schizophrenia