J Inherit Metab Dis DOI 10.1007/s10545-014-9770-z

COMPLEX LIPIDS

Defective lipid metabolism in neurodegeneration with brain iron accumulation (NBIA) syndromes: not only a matter of iron Cristina Colombelli & Manar Aoun & Valeria Tiranti

Received: 18 July 2014 / Revised: 2 September 2014 / Accepted: 9 September 2014 # SSIEM 2014

Abstract Neurodegeneration with brain iron accumulation (NBIA) is a group of devastating and life threatening rare diseases. Adult and early-onset NBIA syndromes are inherited as X-chromosomal, autosomal dominant or recessive traits and several genes have been identified as responsible for these disorders. Among the identified disease genes, only two code for proteins directly involved in iron metabolism while the remaining NBIA genes encode proteins with a wide variety of functions ranging from fatty acid metabolism and autophagy to still unknown activities. It is becoming increasingly evident that many neurodegenerative diseases are associated with metabolic dysfunction that often involves altered lipid metabolism. This is not surprising since neurons have a peculiar and heterogeneous lipid composition critical for the development and correct functioning of the nervous system. This review will focus on specific NBIA forms, namely PKAN, CoPAN, PLAN, FAHN and MPAN, which display an interesting link between neurodegeneration and alteration of phospholipids and sphingolipids metabolism, mitochondrial morphology and membrane remodelling Abbreviations 2-hFA 2-hydroxylated fatty acids 2-nFA 2-non hydroxylated fatty acids 2-OHOA 2’-hydroxyoleic acidp AA arachidonic acid Communicated by: Jean-Marie Saudubray

ATP C19orf12 CL CL-ox CNS CoA DHA ENU EPA ER FA GalCer LPC MPAN MRI PC PE PKAN PL PLAN PNS PPARs PS PUFA ROS

Adenosine triphosphate chromosome 19 open-reading frame 12 gene cardiolipin oxidized CL central nervous system coenzyme A docosahexaenoic acid N-ethyl-N-nitrosourea eicosapentaenoic acid endoplasmic reticulum fatty acids galactosylceramide lysophosphatidylcholine mitochondrial membrane protein-associated neurodegeneration magnetic resonance imaging phosphatidylcholine phosphatidylethanolamine PANK-associated neurodegeneration with brain iron accumulation phospholipids PLA2G6-associated neurodegeneration with brain iron accumulation peripheral nervous system peroxisome proliferator-activated receptors phosphatidylserine poly-unsaturated fatty acids reactive oxygen species

Presented at the Workshop “Diagnostic approach, and classification of IEM affecting the synthesis and catabolism of complex lipids” in Paris, France, June 14-15, 2013 C. Colombelli : M. Aoun : V. Tiranti (*) Unit of Molecular Neurogenetics - Pierfranco and Luisa Mariani Centre for the Study of Mitochondrial Disorders in Children, Foundation IRCCS Neurological Institute “Carlo Besta”, Via Temolo 4, 20126 Milan, Italy e-mail: [email protected]

Introduction Neurodegeneration with brain iron accumulation (NBIA) constitutes a group of neurodegenerative disorders presenting

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with a progressive extra-pyramidal syndrome and excessive iron deposition in the brain. Most NBIA disorders are inherited in an autosomal recessive mode and generally begin in childhood or adolescence. Iron deposition occurs in defined areas of the brain, usually including the globus pallidus, but sometimes extending to other sites, such as cerebellum or substantia nigra, depending on the disease. Moreover, iron accumulation may not be apparent at the time of first symptoms and may not be documented until later stages (Gregory and Hayflick 2011). By using the power of genetics, mutations in ten genes have been identified so far: PANK2, PLA2G6, FA2H, ATP13A2, C19orf12, FTL, CP, C2orf37/ DCAF17, WDR45 (Rouault 2013) and COASY (Dusi et al 2013). Nonetheless, still in a large proportion of patients no genetic alteration has been found. Additional features such as pyramidal involvement or ataxia are frequent. Retinopathy or optic atrophy may be diagnostic clues; however, there is overlap between the disorders and it is difficult to find features that univocally identify a specific NBIA form. Among the known disease genes, only two code for proteins directly involved in iron metabolism, ceruloplasmin (CP) and ferritin light chain (FTL) (Rouault 2013). The other eight genes encode for proteins with different functions. Interestingly, neurodegeneration associated with pantothenate kinase 2 (PANK2), phospholipase A2 group VI (PLA2G6), fatty acid 2-hydroxylase (FA2H), CoA synthase (COASY) and C19orf12 are caused by mutations in genes directly or indirectly involved in fatty acid (FA) metabolism and the reason for iron accumulation in these disorders is less clear. Table 1 reports the main molecular, clinical and neuropathological features of these different NBIA sub-types. Membrane lipid repair and remodelling represent a compelling common pathway in which the protein products of these genes play critical roles. Thus, iron accumulation, though profound in some patients, is likely not the primary cause of neurodegeneration and a defect in membrane remodelling and/or in signalling pathways involving lipids may underlie the shared neuropathological signs in these diseases. We think that the evaluation of phospholipid (PL) and sphingolipid metabolism in these forms of NBIA may provide new clues to better understand their pathomechanism and will be useful to design rationalebased therapeutic interventions. In this review we will describe the potential implications of lipid metabolism in NBIA.

Currently, the identification of PUFA targets in the brain is an active area of research in the fields of drug design and nutrition, with the double aim to treat a variety of neurodegenerative diseases and to provide general neuroprotection to the brain (Bosetti 2007; Ross et al 2007). In some pathological conditions, such as inflammation and ischemia, large quantities of unesterified FAs are liberated from brain PLs and contribute to cell dysfunction or death (Rabin et al 1997). Abnormal brain phospholipid metabolism also occurs in essential FA deficiency and Alzheimer’s disease (Ginsberg et al 1995). The activation of peroxisome proliferator-activated receptors (PPARs) by FAs has been implicated in the regulation of neuronal cell death during ischemia, neurodegenerative or neuroinflammatory diseases (Bordet et al 2006). Clinical studies indicate that low dietary consumption of n3 PUFAs or a low plasma docosahexaenoic acid (DHA) concentration are correlated with a number of brain diseases and cognitive and behavioural defects in development and aging (Conquer et al 2000), and that dietary n-3 PUFA supplementation may be beneficial in some of these conditions (Innis 2000). Studies have shown decreased plasma phosphatidylcholine (PC) DHA levels (Schaefer et al 2006), reduced lysoPC/PC ratios (Mulder et al 2003), and increased cerebrospinal fluid (CSF) PC metabolites in patients with Alzheimer’s disease (Walter et al 2004). More recently, Mapstone et al have described a lipidomic approach to detect preclinical Alzheimer’s disease in a group of cognitively normal older adults (Mapstone et al 2014). Among a large panel of molecules, ten PL biomarkers from peripheral blood, including PC and acylcarnitine species, were described as predicting phenoconversion to either amnestic mild cognitive impairment or Alzheimer’s disease. Additional validation should be considered in other cohorts to verify the reproducibility of these data. Moreover, because the composition of blood lipid FAs reflects the composition of the diet fat and life style (Hodson et al 2014) thus making blood-based biomarkers very critical, differences in blood lipid FAs between patients with different fat intakes are to be expected. As a result, the use of plasma lipidomics to find biological markers for neurologic diseases might be a useful approach, but the validation of such markers needs to take into account a detailed analysis of the patients’ nutritional habits. Moreover, initial studies on plasma should be combined with the analysis of lipid profiles in CSF to unravel potential correlations.

Impairment of fatty acid and phospholipid metabolism in neurological diseases An altered polyunsaturated fatty acid (PUFA) metabolism has been implicated in several neurological disorders such as depression, bipolar disorder and Alzheimer’s disease (Jadoon et al 2012; Evans et al 2012; Esposito et al 2008), but little is known about PUFA metabolism in NBIA disorders.

Pantothenate kinase-associated neurodegeneration (PKAN) Pantothenate kinase-associated neurodegeneration, previously known as Hallervorden-Spatz syndrome, is a rare inborn error of vitamin B5/pantothenate metabolism, characterized by iron

PANK2 (MIM # 606157)

COASY (MIM # 609855)

PLA2G6 (MIM # 603604)

FA2H (MIM # 611026)

C19orf12 (MIM # 614297)

PKAN (MIM # 234200)

CoPAN (MIM # 615643)

PLAN (MIM # 256600; 610217)

FAHN (MIM # 612319)

MPAN (MIM # 614298)

Unknown

Fatty acid 2hydroxylase (2hydroxylation of the N-acyl chain of ceramide)

Calcium-independent phospholipase A2β: hydrolysis of the sn-2 acyl-ester bond of PLs

CoA synthase: PPAT and DPCK activities

Pantothenate kinase: pantothenate phosphorylation

Enzymatic defect

Unknown. Hypothetic role in FA metabolism and aminoacid degradation.

Abnormal ceramide and sphingolipid metabolism; lack of long-term myelin maintenance

Impaired release of FAs from PLs, altered remodelling of membrane PLs

Defective de novo CoA synthesis.

Defective de novo CoA synthesis.

Effects on lipid metabolism

Cell body and synapses of neurons (dorsal root ganglia, Purkinje cells, cortex) and astrocytes Oligodendrocyte and Schwann cells; neurons.

Unknown

Cell body and dendrites of neurons (cortex, GP, nucleus basalis of Meynert, pontine nuclei)

Cellular localization in the nervous system

Mitochondria

ER membranes

Cytoplasm, mitochondria, nucleus

Mitochondria (OMM, matrix) and cytosol

Mitochondria (IMS; matrix)

Intracellular localization Dystonia, dysarthria, spasticity, parkinsonism, mental retardation, retinopathy, occasional peripheral neuropathy Dystonia, dysarthria, spastic paraparesis, cognitive impairment, obsessivecompulsive disorder, motor axonal neuropathy Hypotonia, dystonia, ataxia, cognitive decline, sensorimotor axonal neuropathy, optic atrophy Lower limb dystonia, dysarthria, ataxia, spastic quadriparesis, seizures, optic atrophy Dystonia, dysarthria, spasticity, parkinsonism, motor axonal neuropathy, optic atrophy.

Clinical features

T2 hypointensity of the GP and SN. Dystrophic white matter changes, CC thinning, pontocerebellar atrophy. T2 hypointensity of the GP and SN. Cortical and cerebellar atrophy, in some cases.

Hypointensity in the GP, SN involvement in less than 50 % cases. Cerebellar atrophy.

Hypointensity in the GP. Hyperintensity of caudate nuclei, putamina, and thalami.

‘Eye of the tiger’ sign (GP)

MRI findings on T2 scans

Abbreviations: CoA coenzyme A; CC corpus callosum; GP globus pallidus; IMS intermembrane mitochondrial space; OMM outer mitochondrial membrane; SN substantia nigra; PPAT 4′phosphopantetheine adenylyltransferase; DPCK dephospho-CoA kinase

Disease gene

Disorder

Table 1 Molecular, clinical and neuroradiological features of NBIA sub-types

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accumulation in the basal ganglia. It is the most prevalent form of NBIA, accounting for approximately 50 % of all cases, with an estimated prevalence of 1-3 per million. There are two distinct manifestations of this disease: classical and atypical. The majority of PKAN patients present combinations of dystonia, parkinsonism, dysarthria, spasticity, mental retardation and pigmentary retinopathy (Hayflick 2006). Classic PKAN patients develop the disease in the first decade of life and die before the age of 20. Atypical patients do not present overt pathology before the second or third decade, have a slower progression compared to patients with classic PKAN, and generally develop neuropsychiatric symptoms. The latter include obsessive-compulsive disorder, schizophrenia-like psychosis and depression. The main diagnostic criterion for both phenotypes is the observation of the typical magnetic resonance imaging (MRI) pattern known as ‘eye of tiger’ on T2 scans: this is a region of hyperintensity surrounded by an area of hypointensity in the medial globus pallidus. PKAN patients carry mutations in the pantothenate kinase 2 (PANK2) gene, which maps to chromosome 20p13 (OMIM # 606157). Pantothenate kinase catalyzes the ATP-dependent conversion of pantothenate to 4′-phosphopantothenate, the first committed step in coenzyme A (CoA) biosynthesis. 4′phosphopantothenate normally condenses with cysteine in the second step of the pathway. Then, decarboxylation, conjugation to an adenosyl group and phosphorylation lead to the synthesis of CoA. This high-energy carrier of acetyl and FA groups is important in multiple metabolic pathways, including citric acid cycle, FA synthesis and oxidation, and the synthesis of cholesterol, PLs and sphingolipids (Fig. 1). Four human isoforms of this enzyme have been identified: PANK1, PANK2, PANK3 and PANK4. It is unclear whether PANK4 is functional, but PANK1 and PANK3 are active in the cytosol, while PANK2 is localized and active in the mitochondrial inter-membrane space in humans (Kotzbauer et al 2005; Johnson et al 2004). A 1.85 kb transcript of human PANK2 is reported to be expressed in most tissues, with the highest levels found in liver and brain (Zhou et al 2001). Redundancy of PANK enzymes may explain why PKAN patients can survive to the first or second decade of life. Probably, the different isoforms can compensate each other to maintain adequate CoA levels. This is supported by the finding that simultaneous knockout of two different Pank genes in mice lead to embryonic lethality (Pank2/Pank3 or Pank1/Pank3), or early postnatal death (Pank1/Pank2) (Garcia et al 2012). The reason why iron accumulates in the brain of PKAN patients remains unknown. An initial hypothesis postulated that iron aggregates with free cysteine, which has ironchelating properties, and subsequently these aggregates mediate neurotoxic oxidative stress (Perry et al 1985). Nonetheless, after the discovery of forms of NBIA with iron deposits similar

to PKAN but without increased cysteine levels, other theories based on the association between iron and lipid metabolism were proposed instead (Schneider and Bhatia 2010). Mutations in PANK2 are expected to result in defective CoA biosynthesis, which in turn could provoke a variety of metabolic alterations and may affect membrane lipid synthesis and lipid β-oxidation. A deficit of coenzyme A is supposed to be mostly apparent in cells with the highest energy demand, like retinal photoreceptor cells or neurons in the globus pallidus, or for myelin maintenance. In 2005, the group of Paul Kotzbauer suggested that alterations of mitochondrial and lipid metabolism might play a crucial role in the pathogenesis of PKAN, and possibly in other forms of NBIA (Kotzbauer et al 2005). In agreement with the idea that low CoA disrupts lipid homeostasis, lipid deregulation was also observed in Drosophila CoA mutants, including dPANK/fbl (Bosveld et al 2008). Moreover, iron plays a pivotal role in cellular redox chemistry and, if inappropriately managed, it is capable of generating neurotoxic reactive oxygen species (ROS), thus contributing to neuronal death of ironoverloaded cells (Sian-Hülsmann et al 2011). Furthermore, the central nervous system is particularly vulnerable to redox damage due to excessive generation of ROS by defective mitochondria and the susceptibility to lipid peroxidation accruing from brain high cholesterol and unsaturated FAs content. After the discovery of PANK2 mutations in PKAN patients, a Pank2 knockout mouse has been generated to get insights into the pathogenesis of the disease (Kuo et al 2005). Although this mouse exhibits retinal degeneration and azoospermia, it fails to model PKAN as it does not recapitulate the neurological phenotype of the disease and lacks brain iron accumulation. A subsequent study showed that the neurological symptoms become manifest upon pantothenic acid deprivation (Kuo et al 2007). Moreover, Brunetti et al reported that neurons derived from Pank2 knockout mice have an altered mitochondrial membrane potential and a defective respiration, and proved that the neurological phenotype can be elicited when knockout mice were fed a ketogenic diet (Brunetti et al 2012 and 2014). More recently, a global metabolic profiling approach on plasma samples and follow-up studies in PKAN patientderived fibroblasts revealed defects in lipid metabolism, mainly reduced lipid and cholesterol biosynthesis and impaired bile acid metabolism, in patients compared to healthy controls (Leoni et al 2012). The elevation of lactate is suggestive of possible mitochondrial dysfunction. Furthermore, the in vitro approach on fibroblasts derived from PKAN patients showed that several FAs were significantly reduced. These FAs, including myristic (C14:0), palmitic (C16:0) and oleic (C18:1 n9) acid, are mainly present in the sn-1 position of all membrane PLs. Stearic acid (C18:0), abundant in cerebroside, was also slightly reduced in PKAN patient fibroblasts. In

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combination with the observed reduced plasma levels of several triglycerides and PLs in PKAN patients, these data suggest an impairment of FA synthesis that might cause deficiencies in several classes of neutral lipids, like triglycerides and cholesteryl esters, and PLs of plasma and mitochondrial membranes. In addition, this metabolomic study demonstrated reduced levels of sphingomyelin species in plasma. Sphingomyelins are the principal component of the myelin sheath wrapping the axons of neuronal cells, and together with cholesterol are co-localized in membrane’s domains known as ‘lipid rafts’, which are abundant in both neurons and astrocytes and serve as signalling platforms. Alterations of lipid composition can lead to abnormal lipid raft organization and consequent deregulation of lipid raft-dependent signalling is often associated with neurodegenerative diseases (Alzheimer’s disease and Parkinson’s disease) (Sonnino et al 2013). Further studies on PL metabolism are needed in PKAN patients. However, the severity and rarity of this disorder pose a challenge for patient enrolment and sample collection. Another difficulty is represented by the obvious unavailability of brain biopsies in clinical trials. The biochemical investigation of plasma and red blood cells is definitely a non-invasive approach, but, as stated above, should be associated with analysis of diet intake. The complexity of the human plasma lipid profiles establishes it as a rich source of molecules that can be evaluated for the clues that they provide about human physiology, nutrition and disease. Thus, as an emerging ‘omics’ field, analysis of patient’s plasma or red blood cell ‘lipidome’ could provide a powerful approach in the future of clinical medicine to understand lipid biology in PKAN patients and to monitor NBIA diseases and their treatment efficacy (Quehenberger et al 2010; Brown and Murphy 2009).

COASY protein-associated neurodegeneration (CoPAN) The bifunctional 4′-phosphopantetheine adenylyltransferase/ dephospho-CoA kinase (PPAT/DPCK), also known as CoA synthase (COASY), is found on human chromosome 17q21. Mutations in COASY have been recently reported as the second inborn error of CoA synthesis leading to a neurodegenerative disorder termed CoPAN (Dusi et al 2013) (OMIM # 615643). Whole-exome sequencing and Sanger sequence analyses revealed the presence of recessive mutations in COASY in two distinct NBIA-affected subjects. Reported clinical signs were: gait abnormalities, dystonia, dysarthria, spastic paraparesis, cognitive impairment, behavioural disturbances and motor axonal neuropathy. Hypointensity in the globus pallidus was found on T2-weighted images by MRI analysis (Dusi et al 2013).

COASY codes for three splicing variants of which only two, COASY-α and COASY-β, are protein-coding (Nemazanyy et al 2006). COASY-α has ubiquitous expression, whereas the β isoform, which possesses a 29 aa-long Nterminal extension, is primarily expressed in the brain. COASY α and β are anchored to the outer mitochondrial membrane through the N-terminal region (Zhyvoloup et al 2001 and 2003), or localized within the mitochondrial matrix (Rhee et al 2013; Dusi et al 2013). Unlike prokaryotes that use two separate enzymes with phospho-pantetheine adenylyltransferase (PPAT) and dephospho-CoA kinase (DPCK) activities, in mammals a unique enzyme catalyzes the last two steps of de novo CoA biosynthesis: the coupling of 4′-phosphopantetheine with ATP to give dephospho-CoA and the phosphorylation of the 3′-hydroxyl group to generate CoA. Interestingly, it has been shown that, at least in vitro, the activity of COASY is regulated by membrane PLs, like phosphatidylcholine (PC) and phosphatidylethanolamine (PE), which are major components of the mitochondrial outer membrane (Zhyvoloup et al 2003). This may link neurodegenerative diseases often characterized by altered mitochondria with defects in CoA synthesis. Fibroblasts from the two reported CoPAN patients showed decreased levels of the mutant protein, as well as reduced amounts of newly synthetized acetyl-CoA compared to controls. In Drosophila, it has been demonstrated that abolishing the different genes of CoA biosynthetic pathway, including the dPANK/fbl and dPPAT-DPCK, causes a neurological phenotype characterized by brain vacuolization without iron accumulation (Bosveld et al 2008). Moreover, Drosophila CoA mutants exhibit progressive abnormalities in locomotor function and have an altered lipid homeostasis. Impaired lipid metabolism has also been implicated in the pathogenesis of PKAN, as previously discussed. These findings indicate that a defect in CoA biosynthesis can cause NBIA. As coenzyme A is a key molecule required for FA metabolism, hence implicated in PL biosynthesis, COASY mutations and alterations of its activity may also influence membranes’ integrity and homeostasis as suggested in PANK2-related NBIA. The identification of new CoPAN cases and, possibly, the generation of a mouse model may allow a deeper characterization of potential modifications of the neuronal lipid profile and the neuropathological features of the disease.

PLA2G6-associated neurodegeneration (PLAN) PLAN is a heterogeneous group of related neurodegenerative conditions comprising infantile neuroaxonal dystrophy (INAD, OMIM # 256600) and atypical neuroaxonal dystrophy (ANAD, OMIM # 610217), both due to mutations in PLA2G6 (Morgan et al 2006; Khateeb et al 2006), but each one characterized by distinct evolutions. In classical

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neuroaxonal dystrophy (NAD) progressive motor, mental and visual deterioration begin between 6 months and 2 years of age, lead to loss of ambulation by 5 years and death within the first decade. Major clinical signs are bilateral pyramidal tract signs, truncal hypotonia, cerebellar ataxia, spastic tetraplegia, peripheral neuropathy and optic atrophy. Instead, atypical NAD presents with a milder phenotype and a slower neurodegenerative progression in adolescence or adulthood (Lamari et al 2013). Contrary to PKAN, iron accumulation is not a universal feature of PLAN. Half of INAD patients may lack signs of iron deposition early in the disease course, and they usually develop hypointensity of the globus pallidus only later, without the ‘eye of the tiger’ sign (Kurian et al 2008; Schneider et al 2013). The major neuropathological hallmark shared by typical and atypical NAD is the presence of axonal spheroids throughout the nervous system, with predominant accumulation at axonal endings (Kimura et al 1991; Liu et al 1974). Similarly to what found in patient-derived post-mortem tissues, ultrastructural analyses of two different knockouts and an N-ethyl-N-nitrosourea (ENU)-induced mouse model of the disease have shown that the axonal swellings contain degenerating membranes with tubulovesicular structures (Malik et al 2008; Shinzawa et al 2008; Wada et al 2009). These findings hint at a prominent role of the enzyme encoded by PLA2G6, calcium-independent phospholipase A 2 β (iPLA2β or iPLA2VI), in the remodelling of PLs in neuronal cellular and subcellular membranes. The densely compacted tubulovesicular elements were also found to contain remnants of degenerating mitochondrial inner membranes (Beck et al 2011), which suggests that impairment of mitochondria function might be involved in disease development and/or progression. The accumulation of degenerating plasma, ER, mitochondrial and presynaptic membranes may then causes the formation of axonal swellings. PLA2G6 maps to human chromosome 22q13.1 and iPLA2β hydrolyzes the sn-2 acyl-ester bond in PLs including glycerophospholipids such as PC, PE, phosphatidylserine (PS) and cardiolipin (CL), to yield free FAs and lysophospholipids (Burke and Dennis 2009) (Fig. 1). A functional genotype-phenotype study showed that PLAN is associated with a nearly complete loss of iPLA2β phospholipase/ lysophospholipase activity. Thus, it is predicted that the primary pathogenic event of PLAN may be related to either decreased turnover and membrane accumulation of iPLA2β substrates, or reduced release of FAs and lysophospholipids that either serve as signalling molecules or undergo FA oxidation (Fig. 1). Indeed, it is hypothesized that loss of iPLA2β leads to a limited ability to modify PL FA composition, which in turn affects membrane fluidity and permeability, and reduces the capability to repair oxidative damage and to respond to different availability of FAs in the diet.

An altered phospholipid content, with reduced concentrations of several FAs esterified in ethanolamine- and serineglyceropho spholip ids, and a 40 % redu ction in lysophosphatidylcholine, was found in the brain of knockout mice at 4-5 months, long before the manifestation of obvious motor symptoms (Cheon et al 2012). Moreover, at this stage a reduced incorporation of plasma DHA and a decreased DHA signalling were observed in the brain of Pla2g6 knockout mice either under resting conditions or upon agonist stimulation (Basselin et al 2010). These findings suggest that deficiency in the turnover of sn-2-esterified PUFAs and disturbances in whole brain lipid composition and metabolism may be causative of INAD. Furthermore, a quantitative lipid profiling performed by liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) revealed differences in the content of PLs and FAs in the spinal cord of Pla2g6 null mice at 56 weeks of age, when symptoms become evident (Beck et al 2011). Another emerging point regarding the pathogenesis of PLAN is the potential involvement of mitochondria. This hypothesis fits well with at least three lines of evidence. First, it has been suggested that iPLA2β plays a protective role against ROS-mediated mitochondrial damage caused by staurosporine-induced apoptosis (Seleznev et al 2006). Second, not only patient specimens (Itoh et al 1993; Mahadevan et al 2000), but also mice lacking Pla2g6 exhibit degenerating mitochondria with tubular and branching cristae throughout the nervous system (Beck et al 2011). Third, the mitochondrial signature lipid cardiolipin, which is enriched in esterified PUFAs, hence highly susceptible to lipid peroxidation (Schlame 2008; Pope et al 2008), undergoes Pla2-dependent remodelling and repair (Zhao et al 2010). Although yet to be demonstrated, the hypothesis of lipid peroxidation is strengthened by the evidence of an agedependent accumulation of 4-hydroxy-2-nonenal (4-HNE) in white matter and distal parts of axons of the spinal cord of Pla2g6 null mice (Beck et al 2011). 4-HNE is a major α,βunsaturated aldehyde product which is generated when PUFA-containing lipids are subjected to oxidative stress (for reviews see Pizzimenti et al 2013 and Reed 2011). Being highly reactive, 4-HNE can form protein adducts, propagate oxidative stress, disrupt Ca2+ homeostasis and glutamate transport and induce apoptosis, thereby accelerating axonal degeneration (Dalleau et al 2013). Moreover, it is well established that cytochrome c-catalyzed peroxidation of CL and accumulation of oxidized CL (CL-ox) are required for the completion of the cell death program (Kagan et al 2009). It is tempting to speculate that loss of the repairing activity of iPLA2β accelerates CL-ox accumulation, thus favouring neuronal cell loss in PLAN, similarly to what happens in other pathological conditions like traumatic brain injury (Bayir et al 2007; Ji et al 2012). In the future, global lipidomics based on two-dimensional liquid

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Fig. 1 Schematic representation of brain metabolic pathways implicated in CoA, and PUFA utilization and membrane phospholipid remodelling. Enzymes associated to NBIA disorders are shown in red. Polyunsaturated fatty acids (PUFAs) enter the brain crossing the blood–brain barrier (BBB). In the endoplasmic reticulum (ER), they are activated to polyunsaturated fatty acyl-CoAs (PUFA-CoAs) by acyl-CoA synthetases. Five enzymatic reactions (blue arrows) synthesize CoA from pantothenate. The first step is catalyzed by pantothenate kinase (PANK), whereas COASY catalyzes the last two steps. Human PANK2, whose mutations lead to PKAN, is present in mitochondria, while the localization of COASY, responsible for CoPAN, remains unclear (outer mitochondrial membrane or matrix). Ninety per cent of PUFA-CoAs are esterified into lysophospholipids by an acyltransferase, thus participating to the remodelling of mitochondrial and plasma membrane phospholipids. PUFACoAs can also undergo β-oxidation in mitochondria or peroxysomes (not shown). Membrane phospholipids (mbPLs) containing sn-2esterified PUFAs can be released by phospholipases A2, like PLA2G6, which is mutated in PLAN. Under oxidative stress attack, PLA2G6

eliminates peroxidized PUFAs. A fraction of free PUFAs, mainly arachidonic acid (AA) and docosahexaenoic acid (DHA), is converted into bioactive molecules, eicosanoids and docosanoids respectively, by cyclooxygenase (COX), lipoxygenase (LOX), or cytochrome P450 (cytP450) enzymes. In the ER the FAHN-associated enzyme FA2H catalyzes the formation of 2-hydroxylated fatty acids (2-OH FAs) that are precursors of sphingolipids, major components of lipid rafts and myelin sheath. Note that CoA is also implicated in sphingolipids biosynthesis (green arrows). C19orf12 (mutated in MPAN) codes for a mitochondrial membraneassociated protein whose role is still unknown. The biosynthesis of phospholipids (PLs) is mainly located in ER/mitochondria-associated membranes (MAM) and to a lesser extent in mitochondria. PLs are synthesized from phosphatidic acid (PA), which relies on two acyl-CoA for its synthesis. CDP-DAG, cytidine diphosphate diacylglycerol; Cds1, cytidine diphosphate diacylglycerol synthase; CPT1, carnitine palmitoyltransferase I; Pem1/Pem2, phosphatidylethanolamine methyltransferases 1/2; PSd1, phosphatidylserine decarboxylase 1; PG, phosphatidylglycerol; PGP, phosphatidylglycerol phosphate

chromatography-mass spectrometry (LC-MS) could address this point in INAD animal models. Another argument supporting that PLAN is a disease related to PL dys-homeostasis is the potential lack of lipid mediators that are normally released by iPLA2β enzymatic activity. For instance, iPLA2β-derived DHA is involved directly, as well as through its oxygenated derivatives, in a plethora of well-known biological activities like neurotransmission, anti-inflammatory and anti-oxidant responses, signal transduction and modulation of ionic channels. A portion of released 22:6n-3 can be converted into

anti-inflammatory neuroprotectin D1 (PD1) or resolvin E1 (RvE1), two molecules involved in brain cell survival and the resolution of inflammation (Hong et al 2003; Schwab et al 2007). iPLA2β is also responsible for the release of lysophosphatidylcholine (LPC), an ‘eat me’ signal for macrophages (Lauber et al 2003). Notably, Pla2g6 genetic ablation in mice causes neuroinflammation and Purkinje cell loss, ultimately leading to cerebellar atrophy (Zhao et al 2011). It has been proposed that loss of functional iPLA2β in vivo may lead to both increased Purkinje cell apoptosis and decreased apoptotic cell clearance; nonetheless,

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whether lack of PD1 and RvE1 or absence of LPC caused an inefficient phagocyte removal has yet to be tested. A similar scenario has been proposed to explain the reduction in the clearance of myelin and axonal debris after sciatic nerve injury in the absence of both iPLA 2 VI and calciumdependent group IVA phospholipase (cPLA2 GIVA) (Lopés-Vales et al 2008). In both excitable and non-excitable cells iPLA2β is also involved in the regulation of various ion channels, thereby altering their capacity to respond to changes in intra- or extracellular ion concentrations. This activity can be mediated by membrane-derived lipids through either specific membrane receptors or by direct interaction with ion channels (Vanden Abeele et al 2006; Beech 2007). For instance, several studies have shown a role for non-esterified AA and 2lysophospholipids in the regulation of voltage-operated Ca2+ channels, plasma membrane Na/K-ATPase, ATP-sensitive (KATP) and voltage-gated Kv1.2 potassium channels (Vacher et al 1989; Owada et al 1999; Eddlestone et al 1995; Bao et al 2008). Furthermore, recent demonstrations that second messenger lipids like AA are able to tune synaptic transmission by direct modulation of presynaptic voltage-gated potassium channels (Carta et al 2014) open the possibility that a perturbation of such a mechanism of synaptic regulation may occur in affected PLAN patients as well. Notably, another relevant role of iPLA2β is the modulation of capacitative Ca2+ entry (CCE), a type of Ca2+ influx into the cells that is activated when ER Ca2+ stores are depleted. It has been demonstrated that iPLA2β, the ER calcium sensor STIM1 and the plasma membrane pore-forming subunit Orai1 are equally required for CCE (Smani et al 2004; Bolotina 2008), and a very recent study has shown that PUFAs, like linoleic acid, inhibit STIM1-Orai1 coupling by a mechanism that involves perturbation of ER membrane structure (Holowka et al 2014). Relevant to INAD pathology, using two different mouse models of the disease, the group of Georg Reiser demonstrated a severe disturbance in astrocyte ATPstimulated Ca2+ signalling in vivo (Strokin et al 2012). Hence, impairment of Ca2+ homeostasis is likely implicated in the pathogenesis of the disease. Consistent with this, it is interesting to note that a gene coding for a non selective sodium leak channel, NALCN, has been recently associated with an atypical form of INAD with facial dysmorphism (Köroğlu et al 2013). NALCN is highly expressed in the nervous system where it regulates neuronal excitability, and studies on C. elegans, D. melanogaster and mice have shown that it controls rhythmic behaviours (Ren 2011). Intriguingly, aberrant circadian locomotor rhythms were recently reported in a new fly model of PKAN (Pandey et al 2013), envisioning potential connections between the two forms of NBIA. Nonetheless, an obvious functional link between NALCN, iPLA 2 β and PANK2 is still

lacking; future studies may unveil common pathomechanims for mutations in these three different genes.

Fatty acid hydroxylase-associated neurodegeneration (FAHN) FAHN (OMIM # 612319) is another subtype of autosomal recessive neurodegeneration with brain iron accumulation characterized by an inherited defect of lipid metabolism. Specifically, for the first time in 2010 mutations in the gene coding for fatty acid hydroxylase-2 (FA2H; fatty acid α-hydroxylase) associated NBIA to abnormalities in ceramide and sphingolipid metabolism (Kruer et al 2010). Similarly to PLA2G6, FA2H mutations can give rise to a wide spectrum of distinct neurological conditions ranging from FAHN and hereditary spastic paraplegia (SPG35, OMIM # 612319) (Dick et al 2010) to progressive familial leukodystrophy with spastic paraparesis and dystonia (Edvardson et al 2008). FAHN patients exhibit features that are similar to those of NAD-affected individuals, like profound ataxia, dystonia, dysarthria, spastic quadriplegia, axial hypotonia and neuroophtalmologic features that culminate in optic atrophy. The disease typically begins in childhood and is slowly progressive with gradual gait disturbances, loss of visual acuity and episodic intellectual decline. T2 scans MRI show hypointensity in the globus pallidus and to a lesser extent in the substantia nigra, dystrophic white matter changes, thinning of the corpus callosum, and ponto-cerebellar atrophy (Kruer et al 2010). FA2H maps to chromosome 16q23.1 and codes for a 43 kDa-NADPH-dependent mono-oxygenase residing in ER membranes (Fig. 1). During de novo synthesis of ceramide, FA2H catalyzes the 2′-hydroxylation of the N-acyl chain of the ceramide moiety, which is then incorporated into complex sphingolipids. Sphingolipids containing 2′-hydroxy fatty acids (hFA) are present in several tissues, but they are uniquely abundant in the nervous system and in the epidermis of the skin. Of note, hFA-galactosylceramides (GalCer) and sulfatides (3-sulfate esters of GalCer) are enriched in the myelin sheath of central and peripheral nervous system and it is estimated that approximately 25 % of the outer leaflet lipids in myelin are hydroxylated (Kishimoto and Radin 1963; Alderson et al 2004). In addition, FA2H is the unique 2hydroxylase expressed by myelin-forming oligodendrocytes and Schwann cells and its expression is upregulated with myelination (Alderson et al 2006; Maldonado et al 2008; Hama 2012). Thus, it is not surprising that the major alterations caused by malfunctioning of FA2H lead to white matter defects. Three main pathogenetic mechanisms can be hypothesized: first, 2-hydroxyl groups in myelin sphingolipids may have a

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stabilizing role on the myelin sheath, hence their absence may cause lack of myelin stability; second, 2-hydroxylation could also affect the size and the number of membrane domains formed by GalCer (lipid rafts), which in turn can influence signalling between myelinating cells and neurons; third, hFAsphingolipids per se may function as signalling molecules that regulate neuronal and glial cells. In the last few years the generation of two different Fa2h null mice and a Cnp1-Cre//Fa2hflox/flox conditional knockout — lacking Fa2h only in myelinating glia — shed light on the pathomechanism of FAHN and its allelic diseases (Zöller et al 2008; Potter et al 2011). These murine models confirmed the first pathogenic hypothesis, corroborating the idea that hFAgalactolipids are required for long-term myelin maintenance, as absence of hFA-GalCer lead to demyelination and profound axonal loss in diverse brain areas of aged mice. This evidence goes along with results coming from many biophysical studies, which show that the 2′-hydroxyl groups enhance tight lipid-lipid packing by increasing hydrogen bonds and complexation of ions (for review see Kota and Hama 2014). Notwithstanding, the second and the third pathogenic hypotheses are now gaining strength as it is becoming evident that in the brain FA2H has additional roles that are myelin-independent. In fact, Potter and co-workers found that complete deletion of Fa2h, but not its selective elimination from oligodendrocytes, produces significant deficits in spatial learning and memory (Potter et al 2011). This finding suggests that FA2H may have a function in the establishment of proper neural connectivity in the hippocampus and this could be mediated by FA2H-derived metabolites involved in neural cell signalling. Consistent with this, it is interesting to observe that, besides a merely structural function, hFA-sphingolipids are emerging to have roles in cell signalling and differentiation. For instance, FA2H-knockdown from a schwannoma cell line causes enhanced migratory capacity and impaired cAMP-induced cell cycle exit via effects on the cyclin-dependent kinase (cdk) inhibitors p21 and p27 (Alderson and Hama 2009). Consistently, it has been suggested that mutations in FA2H could lead to premature apoptosis of neurons through alteration of cdk inhibitor expression (Kruer et al 2010). Another example is represented by adipocytes, in which the absence of FA2H correlates with diminished expression of adipocyte markers, blocked triacylglycerol accumulation, increased endocytosis of GLUT4 glucose transporter and impaired glucose uptake and lipogenesis (Guo et al 2010). These non-canonical biological effects of FA2H may be mediated by either an alteration of lipid rafts and membrane fluidity, or by 2′-hydroxy lipids functioning as second messengers. Consistently, some recent studies have focused on 2′-hydroxy ceramide as a potent inducer of apoptosis, and 2′-hydroxyoleic acid (2OHOA) as a mediator of several biological activities, such as differentiation, ER stress and autophagy induction in tumor cells, and modulation of sphingomyelin metabolism through

the activation of sphingomyelin synthase (for reviews see Kota and Hama 2014). Interestingly, 2-hydroxylated sphingomyelin profiles were decreased to a different extent in fibroblasts from patients carrying mutations in FA2H (Dan et al 2011). Another insight into the pathomechanism of FAHN comes from the potential perturbation of the neuronal ceramide pool, which was shown to regulate the formation of Lewy bodies, one of the neuropathological hallmark of Parkinson’s disease, found in some cases of PKAN, PLAN and MPAN as well (Sharon et al 2001; Bras et al 2008). The neuropathological analysis of FAHN patients would clarify the potential role of FA2H in ceramide-dependent α-synuclein accumulation and may unravel unexpected connections between genetically distinct neurodegenerative disorders. Finally, concerning the link between iron accumulation and the decreased 2′-hydroxylation of sphingolipids, it is worth noting that FA2H contains an N-terminal cytochrome b5-like heme binding domain and a fatty acid hydroxylase domain including a non-heme di-iron active site. It can be predicted that a direct interaction of these domains with iron-containing moieties may play a role in the iron accumulation observed in FAHN. Alternatively, it has been speculated that the impairment of CNS iron homeostasis could be a direct consequence of myelin disruption (Kruer et al 2010). Definitely further studies are needed to unveil the intricate relationship between sphingolipid metabolism and iron accumulation in FAHN.

Mitochondrial membrane protein-associated neurodegeneration (MPAN) MPAN (OMIM # 614298) is a newly discovered form of NBIA named after the mitochondrial localization of the protein encoded by the orphan gene C19orf12. In 2011, this open reading frame located on human chromosome 19 was found to carry mutations in a cohort of patients with a central European descent (Hartig et al 2011). The onset of MPAN is typically between 4 and 20 years of age, and the progression is generally slower than PKAN and INAD. The clinical presentation comprises dysarthria and gait difficulties, followed by spasticity, dystonia, parkinsonism, psychiatric symptoms, motor axonal neuropathy and optic atrophy. MRI detects T2 hypointensities suggestive of iron accumulation in the globus pallidus and in the substantia nigra; only two cases presented with the PKAN-typical pathognomonic sign of the ‘eye of the tiger’ (Hartig et al 2011; Horvath et al 2012; Schulte et al 2013). Some patients with MPAN also display cortical and/or cerebellar atrophy (Hartig et al 2011; Horvath et al 2012). Autopsy from two patients allowed the neuropathological evaluation of MPAN. Similarly to other forms of NBIA and more common neurodegenerative disorders, MPAN is

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characterized by the presence of ubiquitin-positive axonal spheroids, α-synuclein-positive Lewy bodies, ironcontaining deposits, and neuronal inclusions containing hyper-phosphorylated tau. Neuroaxonal spheroids can also be appreciated in skin or peripheral nerve biopsies from MPAN patients. Moreover, myelin loss was found in the pyramidal tract of the spinal cord and in the optic tract (Hartig et al 2011). C19orf12 is highly conserved in evolution. In humans, chimps and chickens it encodes for two protein isoforms, originating from two alternative first exons, each with two predicted transmembrane domains. Experiments of co-localization, subcellular fractionation and in vitro import have shown that, similarly to PANK2 and iPLA2β, the product of C19orf12 is localized to mitochondria. Whether it is inserted in the inner or the outer mitochondrial membrane is still not known (Hartig et al 2011). Despite its mitochondrial localization, MPAN-associated mutations do not affect the mitochondrial bioenergetics in fibroblasts under basal conditions (Hartig et al 2011). Although the function of C19orf12 has not been determined yet, transcriptional analyses have shown that this protein is ubiquitously expressed, with higher expression levels observed in the brain, blood cells and fat tissue, where it is upregulated during adipocyte differentiation. Moreover, C19orf12 is co-regulated with genes that are involved in fatty acid metabolism and aminoacid degradation, two pathways that are related to mitochondrial processes involved in CoA metabolism. Thus, it is possible that C19orf12 is implied in a metabolic pathway in which PANK2 and COASY play a role. Recently, transgenic Drosophila models of MPAN have been reported. Flies in which both CG3740 and CG11671 — C19orf12 Drosophila orthologs — were knocked down exhibit defective climbing and bang sensitivity, which are signs of neurological defects. Indeed, despite absence of iron accumulation, a neurodegenerative phenotype was confirmed by the neuropathological finding of vacuoles in the brain and in the optical lobe of transgenic flies (Iuso et al 2014). Accordingly, this first in vivo model of MPAN represents a unique tool to study the pathomechanism of the disease and may be used to screen potential treatments.

Therapeutic approaches Unfortunately, no effective therapy is now available to cure NBIA disorders. Currently, the therapeutic options are only symptomatic, with the general aim to lessen the debilitating motor and cognitive symptoms. In this direction, recent promising research works offer new perspectives. Deep brain stimulation (DBS) of the internal globus pallidus has emerged as an effective

treatment in PKAN patients, as it proved to ameliorate some of the symptoms of the disease (Timmermann et al 2010). On the other hand, oral or intrathecal baclofen and stereotactic pallidotomy, though supposed to provide symptomatic relief, did not produce any known diseasemodifying effect (Schipper 2012). Another option is represented by the use of chelating agents able to cross the blood–brain barrier, such as deferiprone (3-hydroxy-1,2dimethylpyridin-4-one, DFP). Nonetheless, despite a significant reduction in globus pallidus iron content after a 6month administration in nine PKAN patients, no demonstrable clinical benefit was reported in a first clinical trial (Zorzi et al 2011). More recently, a 4-year follow-up study demonstrated long-term safety and tolerability of DFP and the clinical stabilization of five out of six PKAN patients (Cossu et al 2014). However, the authors of this study suggested that the treatment duration was relatively short and neuronal damage was too advanced to allow a complete rescue of neurological functions. Even though iron overload probably does not represent the initiating factor triggering neurodegeneration, it still remains one of the effectively targetable factors and the use of iron chelators may possibly reduce harmful iron-dependent oxidation of DNA, proteins and lipids. Nevertheless, iron chelators should be considered with caution, as they expose the high risk of altering the fragile equilibrium between the cytosolic and the mitochondrial pool of cellular iron. On the other hand, on the basis of recent molecular and pathophysiological studies, a rationale-based treatment might be more efficacious in curing or delaying neurodegeneration in some NBIA disorders. Notably, supplementing pantethine, a vitamin B5 analog, was explored in Drosophila models of PKAN, and found to restore CoA levels, improve mitochondrial function, enhance locomotor abilities, and increase lifespan (Rana et al 2010). The idea behind this treatment is that pantethine can bypass the block due to PANK2 absence or malfunctioning thus functioning as a substrate for the enzyme pantetheine phosphate adenylyltransferase activity, performing one of the last step in CoA biosynthesis. Furthermore, chronic administration of pantethine proved to be effective in counteracting the disease phenotype elicited by a ketogenic diet in Pank2 knockout mice (Brunetti et al 2014), suggesting that providing this compound in the diet may be a potential nutritional approach for the treatment of human subjects. Although pantethine is not able to cross the blood–brain barrier (Bousquet et al 2010), it is likely that its neuroprotective effect is mediated by its hydrolysis products cysteamine and pantothenate, which can easily enter the brain instead. It would be interesting to determine if pantethine has the same therapeutic effects in other forms of NBIA in which altered lipid metabolism has been implicated.

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Currently, no conclusive statements can be made regarding NBIA treatments, but many possible approaches are taken into consideration and larger follow-up controlled studies on phenotype rescue and quality of life improvements need to be performed in more patients to generate statistically meaningful results.

ER, which are major sites of PL biosynthesis and trafficking, have also been implicated in common neurodegenerative diseases (Tatsuta et al 2014; Schon and Area-Gomez 2013; Vance 2014). Understanding the link between lipid metabolism, mitochondrial bioenergetics and iron accumulation and determining how to modify this delicate equilibrium is of primary importance to find new therapeutic targets for NBIA.

Concluding remarks In the last few years, the identification of mutations in a group of genes implicated in CoA biosynthetic pathway (PANK2 and COASY), fatty acid metabolism (C19orf12), phospholipid remodelling (PLA2G6) and sphingolipid synthesis (FA2H) shed light on the essential role of lipid metabolism, membrane phospholipid integrity, and lipid second messenger signalling in the development and functioning of the nervous system. Although a common feature of NBIA disorders is represented by T2 hypointensities on MRI, suggestive of iron accumulation, studies on animal models and analyses of affected individuals are corroborating the idea that iron deposition may be only secondary to a major metabolic impairment of neural cells, which may underlie the pathogenesis of diverse forms of NBIA. The identification of common alterations in phospholipids and/or sphingolipids in the aforementioned NBIA conditions may be of value to determine shared therapeutic targets. It is worth noting that similar neuropathological features (i.e. Lewy bodies, axonal spheroids, iron deposits, altered mitochondria) may imply that mutations in different enzymes impinge on the same structural and functional lipid-mediated processes. For example similarities between PKAN, CoPAN and PLAN may be related to the possibility that the affected enzymes contribute to the synthesis of phospholipids, either by synthesizing CoA to activate fatty acids required for membrane phospholipids (PKAN and CoPAN), or by facilitating deacylation and remodelling of mature PLs (PLAN). Moreover, alterations of membrane PLs reported in INAD may alter the complex regulation of CoA synthesis by acting directly or indirectly on PANK2 or COASY enzymes. It is becoming clear that the pathological alterations of PLs and sphingolipids found in NBIA may impact the neurological function via two main ways: by altering the properties of cellular membranes and by modifying cellular signalling involved in inflammation, neuronal excitability and survival, and myelin maintenance. In addition, the finding that the majority of the enzymes implicated in NBIA disorders are located in ER or mitochondria entails a fundamental role of these organelles and of their proper metabolic coupling for the correct functioning of neural cells. In this respect it is relevant that the mitochondria-associated membranes (MAM) of the

Acknowledgments The support of Telethon GGP11088 and TIRCON project of the European Commission’s Seventh Framework Programme (FP7/2007-2013, HEALTH-F2-2011, grant agreement No. 277984) to VT are gratefully acknowledged. MA is supported by the Mitochondrial European Educational Training, MEET, ITN MARIE CURIE PEOPLE (grant agreement No. 317433). Conflict of interest None.

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