Cardiolipin (CL), also known as diphosphatidyglycerol, is localized and synthesized exclusively in the mitochondria. This glycerophospholipid was first characterized by Mary Pangborn and McFarlane in 1941. Presently, CL is considered a potential therapeutic target for several neurodegenerative diseases (NDDs). Recent developments in the field of lipidomics indicate that the ratio of monolysocardiolipin-to-native CL is a valuable biomarker for diagnosing NDDs such as Barth Syndrome (BTHS). Studies have reported that protein–lipid interactions are associated with the function and organization of the oxidative phosphorylation (OXPHOS) system. CL constitutes 15% of the inner mitochondrial membrane (IMM) lipids. It is localized, synthesized, and deacylated exclusively in the mitochondria. Neuronal and mitochondrial dysfunctions have been attributed to abnormalities in the concentration and changes in the intracellular localization of CL. Understanding the significance of CL in NDDs requires knowledge of two main areas, which include the role of IMM structure in NDDs and the significance of NDD biomarkers and protein signatures. These two areas can be better understood by applying lipidomics, which is a field of study in which lipid profiles are identified, quantified, and characterized to understand their role in biological systems. Recently, NDD research has had a focal point around the mitochondria’s role in disease development and diagnosis. Broadly, mitochondria have been at the forefront of biochemical research, being a focal point for numerous Nobel prizes in Chemistry, owing to its crucial role in cellular respiration, cardiovascular disease, and NDDs. In terms of function, mitochondria perform oxidative phosphorylation (OXPHOS), an oxidative process in the IMM that synthesizes ATP. The exergonic flow of electrons in the IMM fuels the endergonic pumping of protons across the proteins in the respirasome super-complex (RSC), which drives the phosphorylation of ADP to ATP via ATP synthase. OXPHOS is a process that involves five protein complexes that constitute the electron transport chain (ETC). The ETC specifically has three complexes - Complex 1 (NADH Dehydrogenase), Complex 3 (Ubiquinol-ferricytochrome-c oxidoreductase) and Complex 4 (Cytochrome-c oxidase) - which form the RSC. Also, in the IMM is ATP synthase, otherwise known as Complex V, which functions to synthesize ATP.

OXPHOS is important within the context of CL’s role because CL is involved in maintaining the structure of the RSC. CL has a conical shape due to its four acyl chains. Also, CL operates as an anchorage and docking component for the RSC in the IMM. Being situated in the IMM, an understanding of CL and mapping out its relevance to NDDs necessitates the use of different analytical fields of study such as lipidomics. This research is centered around supporting lipidomics as a field of study to provide an understanding of CL in the context of NDD progression. Lipidomics is a field of study that includes analyzing the biosynthetic, degradative, and regulatory pathways of all lipids. It aids in the systematic analysis and quantification of the overall lipid profile in an organism, organ, or cell. The lipid profile consists of different types of lipids such as prenols, sphingolipids, phospholipids, fatty acids, and sterols. In addition to lipid profiling, analyses of lipid structures such as the acyl chains of CL can enhance mapping out the role lipids have in the development or diagnoses of generally idiopathic NDDs. With NDDs, the classic paradigm of “form follows function” is evident. Likewise, the loss of function and abnormalities with it can be understood in terms of lipid structure via lipidomics. The loss of function can pinpoint a disease’s genetic and metabolic origins. In the same vein, the loss of function with knockout cell lines in mice and the subsequent analyses of those cell lines via lipidomics have served as models for specific aspects of a NDDs’ phenotypes in NDD research.

The knockout cell lines for specific genes expressing typical proteins in NDD progression such as α-synuclein (SNCA) and amyloid-β (Aβ) with lipidomics allows for relationships between key protein signatures and CL to be established. Typically, loss of function analyses compare wild-type (WT) cell lines and transgenic cell lines. From these types of analyses via lipidomics, how CL and its aberrations relate to characteristic protein signatures in NDDs (e.g., SNCA in Parkinson’s Disease (PD) and Aβ in Alzheimer’s Disease (AD)) can be elucidated. However, it is important to note that transgenic knockout cell lines are useful in simulating a specific aspect of a NDD’s phenotype such as cognitive impairment or motor deficits, but not sufficient to replicate the entire suite of complex abnormalities and co-morbidities associated with a NDD.

The lipid profile of the central nervous system (CNS) plays a crucial role in nerve cell functioning, particularly in the OXPHOS process in the mitochondria. The entire lipid profile of a cell is called the lipidome. Since the CNS comprises 50% lipids by dry weight and any aberrations in its lipid content can affect its physiology, knowing the lipidome is of utmost significance. CL, also known as diphosphatidylglycerol, is an unusual member of the lipidome because it is localized in the mitochondria during the entire lifetime of the cell, unlike other members of the lipidome. In a cellular context, decreased levels of CL contribute to abnormalities in cellular respiration and production of reactive oxygen species (ROS). CL can also serve as a mitophagic and apoptotic signaling factor when oxidized. Mitophagy and apoptosis are defined as the breakdown and destruction of the mitochondria and cell, respectively. In general, CL plays a role in the docking and anchoring of the ribosomes of the IMM and protein complexes of the ETC. The ETC is situated in the IMM, and CL biogenesis occurs in the IMM. Further research into CL biogenesis is warranted because of its importance in the understanding of the function of abnormal proteins in NDDs such as BTHS. Interestingly, when the enzymes that biosynthesize CL are aberrant, they can contribute to NDD progression as has been seen in BTHS.

The first step in CL synthesis is the synthesis of phosphatidate, a common intermediate for the synthesis of phospholipids and triacylglycerols. Many of these reactions with phosphatidate, which synthesize CL are driven forward by the hydrolysis of pyrophosphate. Phosphatidate, in mammalian cells, is synthesized in the endoplasmic reticulum and the OMM. In the beginning of this anabolic pathway, glycerol-3-phosphate either from glycolysis or the phosphorylation of glycerol is used. Then, glycerol-3-phosphate, with the addition of the fatty acid, results in phosphatidate. Within this anabolic pathway, there are numerous acylations with the common intermediate, phosphatidate. In these acylation reactions, the fatty acid chain is attached to the C-1 atom and is typically saturated. However, the acyl chains attached to the C-2 atom are typically unsaturated. Pathways diverge at phosphatidate, with some membrane-lipid synthesis occurring in the endoplasmic reticulum or in the OMM. In this anabolic pathway, one of the reactants – either phosphatidic acid (PA) or the alcohol – has to be activated and is substrate dependent. Specifically, for the activated reactant PA, the pathway starts with the reaction of phosphatidate with cytidine-triphosphate (CTP) that forms an activated CDP-DAG, which is known as cytidine diphosphate diacylglycerol (CDP-DAG). Then, the activated phosphatidyl unit in CDP-DAG reacts with a hydroxyl group of phosphatidylglycerol (PG), via CL synthase, to form a phosphodiester linkage, and the resulting product is CL.

Lipidomics examines the total lipid profile of a given sample, which is also known as the lipidome. The lipidome is a subset of the metabolome comprising subclasses of lipids, including fatty acids, prenols, sphingolipids, sterols, and glycerophospholipids. Lipidomic analysis provides information regarding the variation of lipids, which facilitates the study of different disease classes, such as NDDs. For example, PD has been associated with aberrations in a spectrum of lipid pathways in the nervous system, some of which may be related to CL dysfunction. Hence, the study of CL dysfunction via lipidomics improves the ability of researchers to further understand the future outcomes of specific phenotypes in NDD diagnosis and development.

In the NDDs that are reviewed, namely AD, PD, and BTHS, structural or concentration changes in CL are associated with specific simulated NDD phenotypes. Whether through chemical inhibition or gene knockout experiments, the NDD phenotypes are simulated in murine models in many of the studies. These simulated NDD phenotypes provide an empirical basis to relate CL changes to NDDs as potential risk factors for NDD development and diagnosis. AD is a NDD that progresses gradually with worsening states of cognitive function over time. There are three features of AD that are relevant. First, AD is a primary form of dementia with the World Health Organization stating that 60-70% of dementia cases are contributed to by AD. Dementia is a syndrome, which presents with deteriorations in cognitive functions such as memory decline, poor judgement and confusion, which is atypical when compared to the normal consequences of aging. Second, AD is associated with bilateral parietal hypometabolism in posterior cingulate neurons. In terms of AD subtypes, there are two subtypes of AD based on age of onset. The two types of AD are early-onset AD and late-onset AD. Both early onset-AD and late-onset AD are associated with Aβ. Aβ is a characteristic protein hallmark that is derived from the proteolysis of amyloid-β precursor protein (APP), which is a type-1 integral membrane protein.