Accepted Manuscript Title: Mitochondria: A crossroads for lipid metabolism defect in neurodegeneration with brain iron accumulation diseases Author: Manar Aoun Valeria Tiranti PII: DOI: Reference:

S1357-2725(15)00028-X http://dx.doi.org/doi:10.1016/j.biocel.2015.01.018 BC 4543

To appear in:

The International Journal of Biochemistry & Cell Biology

Received date: Revised date: Accepted date:

24-10-2014 15-1-2015 29-1-2015

Please cite this article as: Aoun, M., and Tiranti, V.,Mitochondria: A crossroads for lipid metabolism defect in neurodegeneration with brain iron accumulation diseases, International Journal of Biochemistry and Cell Biology (2015), http://dx.doi.org/10.1016/j.biocel.2015.01.018 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Mitochondria: a crossroads for lipid metabolism defect in neurodegeneration with brain iron accumulation diseases Manar Aoun and Valeria Tiranti

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Unit of Molecular Neurogenetics - Pierfranco and Luisa Mariani centre for the study of mitochondrial disorders in children, Foundation IRCCS Neurological Institute “Carlo Besta”,

Correspondence to:

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20126 Milan, Italy

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Valeria Tiranti ([email protected]) Unit of Molecular Neurogenetics

Foundation Neurological Institute C. Besta Via Temolo 4

20126 Milan, Italy

Phone:+390223942633 Fax: +390223942619

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Abstract: Neurodegeneration with brain iron accumulation (NBIA) comprises a group of brain iron deposition syndromes that lead to mixed extrapyramidal features and progressive dementia. Exact pathologic

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mechanism of iron deposition in NBIA remains unknown. However, it is becoming increasingly evident that many neurodegenerative diseases are hallmarked by metabolic dysfunction that often

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involves altered lipid profile. Among the identified disease genes, four encode for proteins localized in mitochondria, which are directly or indirectly implicated in lipid metabolism: PANK2, CoASY,

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PLA2G6 and C19orf12. Mutations in PANK2 and CoASY, both implicated in CoA biosynthesis that acts as a fatty acyl carrier, lead respectively to PKAN and CoPAN forms of NBIA. Mutations in PLA2G6, which plays a key role in the biosynthesis and remodeling of membrane phospholipids

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including cardiolipin, lead to PLAN. Mutations in C19orf12 lead to MPAN, a syndrome similar to that caused by mutations in PANK2 and PLA2G6. Although the function of C19orf12 is largely

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unknown, experimental data suggest its implication in mitochondrial homeostasis and lipid metabolism. Altogether, the identified mutated proteins localized in mitochondria and associated with different NBIA forms support the concept that dysfunctions in mitochondria and lipid

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metabolism play a crucial role in the pathogenesis of NBIA.

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Introduction Neurodegeneration with brain iron accumulation (NBIA) constitutes a group of neurodegenerative disorders inherited as autosomal dominant, recessive or X-linked trait in which iron accumulates in the brain resulting in progressive dystonia, spasticity, parkinsonism, neuropsychiatric

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abnormalities, and optic atrophy or retinal degeneration. The frequency of NBIA in the general population is estimated between one to three people per one million individuals. Onset of NBIA

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ranges from infancy to adulthood and progression can be rapid or slow with long periods of stability.

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NBIA comprises a heterogeneous collection of disorders that share key features and which are differentiated according to genetic, radiologic and clinical findings.

To date, ten genes causing NBIA have been identified: PANK2, PLA2G6, FA2H, ATP13A2,

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C19orf12, FTL, CP, C2orf37, WDR45 (Rouault et al., 2013), and CoASY (Dusi et al., 2014), but still in a large proportion (around 30%) of patients, no genetic alteration can be found. Among the

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mutated identified genes, two encode proteins that are specifically involved in iron metabolism – ceruloplasmin (CP) and ferritin light chain (FTL) (Rouault et al., 2013). The other NBIA disease genes encode proteins with other functions. For example, WDR45 and ATP13A2 genes are

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implicated in autophagosome and lysosomal activity respectively, while the C2orf37 gene encodes

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a nucleolar protein of unknown function. Pantothenate kinase 2 (PANK2) and CoA Synthase (CoASY) are implicated in biosynthesis of CoA, which acts as an acyl group carrier in several

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biochemical reactions particularly in fatty acids metabolism. Phospholipase A2 Group VI (PLA2G6), fatty acid-2-hydroxylase (FA2H), and C19orf12 genes seem to be involved in lipid metabolism, membrane integrity and mitochondrial function. Iron accumulates in the brain in numerous neurodegenerative disorders such as Parkinson, Alzheimer and Friedreich Ataxia. Nevertheless, in NBIA, iron accumulation occurs in defined areas of the brain, usually including the globus pallidus (GP) but sometimes extending to other areas such as cerebellum or substantia nigra.

However, the magnetic resonance imaging (MRI) itself

effectively differentiates between PANK2-associated neurodegeneration (PKAN), which accounts for most NBIA cases, and non-PKAN forms of NBIA. Therefore, the absence of brain iron does not exclude this diagnosis since it may not be apparent at the time of first symptoms and may not be documented until later stages (Gregory and Hayflick, 2011). For example, some patients with neurologic impairment and PLA2G6 mutations did not show MRI evidence of brain iron accumulation (Morgan et al., 2006) and DNA testing was essential in establishing the diagnosis. Clearly, the significant progress made in identifying new NBIA genes helped recognizing the expanding phenotypes within this category. However, the reason for iron accumulation in these Page 3 of 21

disorders is less clear and many questions concerning their pathogenesis remain unanswered. Iron accumulation, though profound in some patients, is likely not the primary cause of neurodegeneration in NBIA and perhaps defects in mitochondrial function and lipid metabolism underline shared pathological mechanisms in these diseases. Although conflicting reports exist as to the cellular localization of the mutated proteins identified in

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NBIA diseases, they are mainly cytosolic except for PANK2, PLA2G6, CoASY and C19orf12 genes (Table 1), which encode for proteins localized in mitochondria (Johnson et al., 2004; Seleznev et

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al., 2006; Rhee et al., 2013; Dusi et al., 2014; Hartig et al., 2011). The NBIA forms associated to altered mitochondrial proteins are thus believed to be mitochondrial diseases with no overt

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deficiency of the oxidative phosphorylation (OXPHOS) system. This would point to mitochondria as central actors in the development of such disorders due to the massive presence of these organelles, crucial for energy generation and lipid metabolism, in vulnerable tissues such as brain,

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retina and skeletal muscles.

In this review, we will try to highlight the available evidences for a potential role of mitochondria in PANK2, CoASY, PLA2G6 and C19orf12.

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NBIA diseases subforms caused by dysfunctions in genes coding for mitochondrial proteins:

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Coenzyme A-associated Neurodegeneration with brain iron accumulation

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1- Pantothenate kinase-associated neurodegeneration (PKAN) Approximately half of the NBIA cases can be explained by pantothenate kinase 2 (PANK2) gene

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mutations causing pantothenate kinase-associated neurodegenration (PKAN) (Gregory A, Hayflick S 2014). PKAN is a rare, inborn error of coenzyme A metabolism characterized by iron accumulation in the globus pallidus (GP) in a perivascular distribution, both as ferric ion (Fe3+) and to a lesser degree as ferrous iron (Fe2+) (Schneider et al., 2013). There are two distinct manifestations of this disease: classical and atypical. The majority of PKAN patients present a combination 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 years. Atypical patients do not present the disease until the second or third decade of life and progress more slowly than classic PKAN with a predominant neuropsychiatric syndrome characterized by obsessive-compulsive disorder, schizophrenia-like psychosis and depression. The main diagnostic criterion for both phenotypes is the presence of the “eye of tiger” pattern in the medial GP on MRI. Mutations in Pantothenate kinase 2 (PANK2) gene mapped to chromosome 20p13 (OMIM 234200) represent the most common genetic cause of this disorder. PANK2 encodes for an essential

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mitochondrial regulatory enzyme, which catalyzes the ATP-dependent conversion of pantothenate, to 4’-phosphopantothenate, the first committed step in Coenzyme A (CoA) biosynthesis (see figure 1). Phosphopantothenate, the product of the PANK reaction normally condenses with cysteine in the next step of the pathway. Then, decarboxylation, conjugation to an adenosyl group and phosphorylation lead to the synthesis of CoA, an essential cofactor in mammalian cells. Together

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with its thioester derivatives (acetyl-CoA, malonyl-CoA, 3-hydroxy-3-methylglutaryl-CoA…), CoA participates in anabolic and catabolic pathways, allosteric regulatory interactions and

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regulation of gene expression (Davaapil et al., 2014). Among diverse cell biochemical reactions, CoA is critical to the fatty acid metabolism and the synthesis of cholesterol and membrane

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phospholipids. However, the exact role of CoA in the pathogenesis of NBIA is not well understood. Four PANK genes are present in the human genome, which code for PANK enzymes (PANK1a and b, PANK2, PANK3 and PANK4). PANK2 is localized and active in the

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mitochondrial inter-membrane space in human (Kotzbauer et al., 2005) and in mouse (Johnson et al., 2004; Brunetti et al., 2012) and is reported to be present in most tissues. The enzymatic activity

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was inhibited by CoA (IC50=50 µM) and more strongly by a CoA ester (IC50=1 µM). This block could be overcome by palmitoylcarnitine, which works as a potent activator of PANK2 function (Leonardi et al., 2007). This explain how PANK2 can function in vivo with the physiological

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concentration of CoA and acyl-CoA esters and suggests that the presence of enzyme within

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mitochondria inner space could be important to sense levels of palmitoylcarnitine and modulate CoA biosynthesis according to requirement for beta-oxidation.

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The reason for iron accumulation in PKAN remains unknown. Original hypothesis postulated that iron aggregates with cysteine due to its iron-chelating properties and these aggregates mediate neurotoxic oxidative stress in affected patients (Perry et al., 1985). After the discovery of other NBIAs with similar pattern of iron accumulation but without increased cysteine levels, other theories based on the link between iron, CoA and lipid metabolism were proposed instead (Schneider et al., 2013). For example, specific siRNA silencing of PANK2 in different human cell lines induced a mark reduction in cell proliferation together with unexpected signs of iron deficiency, with decrease of ferritin and increase of transferrin receptor 1 levels (Poli et al., 2010). Interestingly, the reduced level of PANK2 was associated to a remarkable increase of ferroportin expression, the sole cellular iron exporter, thus suggesting a possible linkage between PANK2 function and iron transport to the brain. Mutations in PANK2 are expected to result in defective mitochondrial CoA biosynthesis, which could lead to a variety of metabolic defects associated to lipid metabolism, which is necessary for membrane remodeling. Kotzbauer et al (2005) first suggested that alterations of mitochondrial and

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lipid metabolism could play a crucial role in the pathogenesis of PKAN, and possibly in other forms of NBIA. In agreement with the idea that low CoA disrupts lipid homeostasis, lipid dysregulation was also observed in Drosophila CoA mutants, including dPANK/fbl (Bosveld et al., 2008). Another typical pathologic finding in PKAN is the presence of axonal spheroids likely representing swollen and dystrophic axons. Defects in the axonal transport or membrane integrity possibly arising from

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insufficient neuronal energy metabolism are supposedly the cause of their formation (Gregory et al., 2009). Even though the precise role of PANK2 in CoA biosynthesis is not fully understood, the

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strong connection between this metabolic pathway, iron deposition and neurodegenerative processes is confirmed by the recent identification of mutations in COASY in two subjects with

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clinical and MRI features of NBIA (Dusi et al., 2014). As a matter of fact, no definitive evidence is available showing decreased level of CoA in human PANK2 deficient cells or tissues, suggesting that the expression of the other PANK isoforms may partly compensate for the absence of PANK2

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function. Signs of oxidative stress were detected in cells from PKAN patients already in basal conditions, and reactive oxygen species (ROS) production was increased in these cells after

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exposure to iron (Campanella et al., 2012). If inappropriately managed, iron play a pivotal role in cellular redox chemistry and is capable of generating neurotoxic ROS thus generating the condition for further ROS production in a vicious cycle and contributing to neuronal death in iron-overloaded

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cells (Sian-Hülsmann et al., 2011). Central nervous system is particularly vulnerable to redox

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damages due to excessive generation of free radicals by defective mitochondria, one of the major sources of ROS production, and the susceptibility to lipid peroxidation accruing from brain high

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cholesterol, ceramide and membranes unsaturated fatty acids content. Moreover, the discovery of PANK2 mutations in patients has also led to the development of a Pank2 null mouse (Kuo et al., 2005). This model followed for more than one year, showed a 20% growth reduction and was infertile. No sign of motor dysfunction nor of iron deposition in the brain were evident, as assessed both by MRI and by histochemistry, even after 16 months of age or after backcrossing into the C57/Bl6J strain. A subsequent study showed that neurological symptoms occurred in Pank2-KO mice upon dietary pantothenic acid deprivation (Kuo et al., 2007). Brunetti et al (2012 and 2014) showed further that PANK2-defective neurons derived from Pank2-KO mice have a profound alteration of mitochondria. These organelles were swollen with altered cristae and membrane potential, which could explain the significantly reduced oxygen consumption, albeit the function of each single respiratory chain complex appeared to be normal (Brunetti et al., 2012). When Pank2-KO mice were fed a ketogenic diet for 2 months, they developed clear signs of motor and neurological impairment with feet clasping, tail rigidity and dystonic limb positioning. The ketogenic diet consists of a low glucose and high lipid content, stimulating lipid use by

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mitochondrial beta-oxidation. Under ketogenic diet, the 3-months-old Pank2-KO mice exacerbated mitochondrial alterations, which were also present in the brain of 12-month-old Pank2-KO mice on a standard diet (Brunetti et al., 2012). The electron microscopy analysis confirmed the presence of altered mitochondria with swollen cristae in the basal ganglia of Pank2-KO mice under a standard diet. The mitochondrial morphology was worsened with focal loss of cristae by exposure to the

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ketogenic diet. Thus, these data showing mitochondrial bioenergetics failure due to the absence of PANK2 protein could result from defects in mitochondrial membrane integrity. In fact, PANK2 by

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synthesizing CoA to activate fatty acids required for membrane phospholipids remodeling and repair, indirectly contributes to the synthesis of membrane phospholipids, particularly

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mitochondrial cardiolipin, which is implicated both in the formation of the cristae and in the stabilization of respiratory chain supercomplexes..

Very few clinical data demonstrated defects in mitochondrial integrity and lipid metabolism. The

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skeletal muscle of a 6-year-old patient with PKAN showed giant mitochondria with a mild alteration of the cristae, which was also observed in the muscle of Pank2-KO mouse model

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(Brunetti et al., 2014). Moreover, a global metabolic profiling approach on plasma samples and follow-up studies in PKAN patient-derived fibroblasts revealed defects in lipid metabolism, mainly reduced lipid and cholesterol biosynthesis and impaired bile acid metabolism (Leoni et al., 2012).

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More studies are needed to understand the link between mitochondria and lipid metabolism

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alterations in order to better explore the implication of these defects in PKAN patients. Given that the severity and rarity of this disorder pose a challenge for patient enrollment and sample

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collection, in vitro tools like patients’ fibroblasts could be a useful approach to investigate functional and structural analysis on mitochondria. Moreover, despite the absence of an animal model that develops overt brain iron overload, the Pank2-KO mouse model and the drosophila dPANK/fbl mutants are promising tools for investigations into PKAN molecular pathogenesis as concerning mitochondrial dysfunctions, lipid metabolism and membranes alterations. 2- CoA synthase protein-associated neurodegeneration (CoPAN)

A distinct form of NBIA, termed CoPAN (CoA synthase protein-associated neurodegeneration), is caused by mutations in the CoASY (CoA synthase) gene coding for a bifunctional mitochondrial enzyme PPAT/DPCK (4’-phosphopantetheine adenylyltransferase/dephospho-CoA kinase), which converts 4’-phosphopantetheine into dephospho-CoA and then CoA. CoASY is found on human chromosome 17q21 (MIM 615643) and has been reported to code for three transcript variants resulting in tissue-specific isoforms: CoASY alpha, beta and gamma (Nemazanyy et al., 2006). The activity of CoASY is ubiquitous and seems to be regulated by membrane phospholipids, mainly

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phosphatidylcholine (PC) and phosphatidylethanolamine (PE) (Zhyvoloup et al., 2003). In contrast to previous report showing localization of CoASY at the outer mitochondrial membrane (Zhyvoloup et al., 2002), two different groups found the enzyme in the mitochondrial matrix, probably anchored to the inner membrane (Dusi et al., 2014; Rhee et al., 2013). Together with PANK2 localized in the mitochondria inter membrane space, these two inborn errors of CoA

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metabolism further support the concept that dysfunctions in CoA synthesis process inside the mitochondria may play a crucial role in the pathogenesis of NBIA (Venco et al., 2014).

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Whole-exome sequencing and Sanger sequence analysis revealed the presence of recessive mutations in CoASY in two distinct NBIA-affected subjects (Dusi et al., 2014). Patients’ fibroblasts

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showed decreased levels of the mutant protein as well as acetyl CoA amount compared to controls, suggesting that the mutant protein was unstable. However, it remains unexplained why only the brain is affected and other organs are preserved in these patients, since CoASY is expressed in all

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tissues.

It is still unknown how mutations in CoASY cause neurodegeneration with brain iron accumulation.

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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

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mutants exhibit progressive abnormal locomotor functions and have altered lipid homeostasis.

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Impaired lipid metabolism has also been implicated in PKAN disease pathogenesis, as previously discussed. As Coenzyme A is a key molecule required for fatty acids metabolism and thus

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implicated in phospholipids biosynthesis, CoASY mutations and activity alterations may also influence membranes integrity and homeostasis as suggested in PANK2-related NBIA.

PLA2G6-associated neurodegeneration (PLAN) The second core NBIA syndrome is due to PLA2G6 gene mutations (PLAN). Similar to PKAN, there appears to be an age-dependent phenotype but abnormal iron accumulation in the GP is observed only in approximately 50% of PLAN cases (Gregory and Hayflick, 2011). 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), but each characterized by distinct evolutions. Early-onset cases, within the first 2 years of life, have INAD characterized by progressive psychomotor development, followed by cerebellar ataxia, gait instability, pyramidal signs and early visual disturbances due to optic atrophy. Late-onset cases described as ANAD usually begins on average at 4-5 years of age. ANAD is characterized by a slower progression of

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dystonia with pyramidal signs and neurobehavioral disturbances. Cerebellar signs and sensory abnormalities that are often prominent in the early childhood variant of PLAN were absent. In line with this, neuroimaging shows cerebellar atrophy occurring in early stages of INAD, which is not a usual feature of the late-onset form (Paisán-Ruiz et al., 2012). However, the major neuropathological hallmark shared by INAD and ANAD is the presence of axonal spheroids

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composed of accumulated aberrant phospholipid membrane (Kimura et al., 1991; Liu et al., 1974) although patients without spheroids found in autopsied brains have been described (Morgan et al., proportion of patients (Khateeb et al., 2006; Kurian et al., 2008).

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2006). Moreover, white matter changes suggestive of demyelination were also described in a large

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The PLA2G6 gene is located on chromosome 22q. The gene is ubiquitously expressed but seems to have a particularly relevant role in the central nervous system. It can be found in all regions of the mammalian brain and its activity is the most prominent among PLA2s in the rat brain (Balboa et

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al., 2002). The protein is usually considered to reside in the cytosol, but translocation to membrane Gottlieb, 2002; Seleznev et al., 2006).

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compartments has been documented as well as localization in the mitochondria (Williams and The PLA2G6 gene encodes iPLA2β, a group VIA Calcium-independent phospholipase that catalyzes the hydrolysis of ester bonds at the sn-2 position of glycerophospholipids, thus releasing

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free polyunsaturated fatty acids (PUFA) and lysophospholipids. The best recognized function of

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the enzyme is the homeostatic regulation of membranes fatty acids composition and phospholipids amount. The protein is also implicated in a variety of other cellular processes as signal

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transduction, cell proliferation and apoptosis (Balboa et al., 2008; Pérez et al., 2004), mainly associated to release of arachidonic (AA) and docosahexaenoic (DHA) acids from brain membrane phospholipids. Altered lipid composition of membranes may have far-reaching consequences thus affecting neuronal membranes fluidity and integrity. The direct connection between PLA2G6 and the neurodegenerative process is not understood yet. However, considering PLA2G6 physiological activity, two features may be involved: membranes phospholipid turnover and mass and cell cycle regulation. Pla2g6-KO mouse model displays slowly progressing predominantly cerebellar neurodegeneration with age-dependent accumulation of spheroids but without iron accumulation (Malik I et al., 2008; Zhao et al., 2011). Interestingly, another mouse model with point mutation of Pla2g6 gene causing its deactivation led to an anticipate disease onset with widespread formation of spheroids containing tubulovesicular membranes similar to human INAD (Wada et al., 2009). Pathophysiology is unclear but according to these structure abnormalities in murine models and to axonal spheroids formations in patients cells, it is possible to speculate that defects in the calcium-independent phospholipase A2β may

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lead to perturbation of membrane lipid homeostasis in neuronal cellular and subcellular membranes. Another emerging point regarding the pathogenesis of PLAN is the potential involvement of mitochondria. Biochemical and ultrastructure analysis of the spinal cords and sciatic nerves from Pla26-KO mice at different ages showed many abnormal mitochondria with branched and tubular

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cristae (Beck et al., 2011), which are distinctive because they contain large amounts of cardiolipin (CL). This suggests that impairment of mitochondria function might be involved in disease

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development and/or progression. The lipid content of these tissues was analyzed and a severe perturbation in the relative amount of different types of phospholipids mainly phosphatidylcholines

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(PC) containing AA and DHA, and CL was documented. These data suggest that the absence of PLA2G6 enzymatic activity may lead to insufficient remodeling of the mitochondrial inner membranes mainly CLs with abnormal accumulation of DHA- and AA-containing phospholipids

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(Beck et al., 2011). In fact, PLA2G6 is implicated in mature CL species synthesis by removing saturated fatty acid side-chains from newly synthesized CL so they can be replaced by the PUFA

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linoleic (LA) (Schlame, 2008). Moreover, due to its high PUFA content, CL is prone to lipid peroxidation (Schlame, 2008; Pope et al., 2008) and consequently undergoes PLA2-dependent repair (Zhao et al., 2010). PLA2-dependent membrane repair is important in restoring the

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membrane damaged by peroxidation by removing oxidized fatty acids from PL and in preventing

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apoptosis induction in brain. Although yet to be demonstrated, the hypothesis of lipid peroxidation is strengthened by the evidence of an age-dependent accumulation of 4-hydroxy-2-nonenal (4-

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HNE) in white matter and distal parts of axons of the spinal cord of Pla2g6-KO mice (Beck et al., 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). In brain aging, it was suggested that oxidation/depletion of CL could be responsible, at least in part, for the decline of cytochrome c oxidase and mitochondrial dysfunctions (Petrosillo et al., 2013). Thus, the loss of the repairing activity of PLA2G6 could accelerate CL-ox accumulation and favor 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). Altogether, these perturbations would cause a progressive degeneration of mitochondria and plasma membranes at synaptic terminals and ultimately determining the formation of tubulovesicular structures and swollen axons, the main pathological features of PLAN. More

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studies on PLAN animal models could provide important information as to the possible connection between mitochondrial membrane homeostasis and iron metabolism in neurodegenerative disorders.

C19orf12-associated neurodegeneration (MPAN)

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Mutations in the C19orf12 gene are associated with MPAN (mitochondrial-membrane protein associated neurodegeneration) (OMIM 624298), an autosomal recessive disorder that

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represents between 5 and 30 % of NBIA cases (Hartig et al., 2011; Panteghini et al., 2012; Hogarth et al., 2013). Hartig et al (2011) firstly described the gene in a group of patients with childhood-

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onset dysarthria and gait difficulty, followed by the development of spastic paraparesis, extrapyramidal features (dystonia and parkinsonism), neuropathy, optic atrophy and psychiatric symptoms. Similarly to other forms of NBIA and more common neurodegenerative disorders,

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postmortem brain examination from 2 patients revealed iron-containing deposits in the GP and the substantia nigra, axonal spheroids and neuronal inclusions containing hyper-phosphorylated tau.

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Moreover, myelin loss was found in the pyramidal tract of the spinal cord and in the optic tract (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.

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C19orf12 encodes for two protein isoforms, originating from two alternative first exons, each with

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two predicted transmembrane domains. The longer isoform is localized in the mitochondria (Hartig et 2011) and possibly in the endoplasmic reticulum (Landouré et al., 2013). Whether it is inserted

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in the inner or the outer mitochondrial membrane is still not known (Hartig et al., 2011). Transcriptional analyses have shown that this protein is ubiquitously expressed with higher expression level in the brain, in blood cells and adipocytes and it appears to be co-regulated with genes involved in fatty acid biogenesis and valine, leucine, isoleucine degradation, thus indicating a connection with CoA, lipid metabolism and mitochondria. Thus, it is possible that C19orf12 maps to the same metabolic pathway as PANK2 and COASY. Despite its mitochondrial localization, MPAN-associated mutations do not affect the mitochondrial bioenergetics in fibroblasts under basal conditions (Hartig et al., 2011). No other information is available to explain the connection between the protein function, the neurodegenerative process and iron accumulation in the brain. Future studies and research tools should be directed towards elucidation of this protein’s function. Recently, transgenic Drosophila models of MPAN have been reported with impaired expression of the two orthologs of human C19orf12 (CG3740 and CG11671). Those flies showed 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

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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 underlining the disease and may be used to screen potential treatments. This could help understanding the mitochondrial

contribution

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the

mechanisms

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in iron accumulation and

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neurodegeneration. Therapeutic approaches

Treatment for NBIA disorders remains symptomatic but promising researches offer new

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perspectives for treatment. Deep brain stimulation (DBS) of the internal globus pallidus may produce some benefit, in particular, in patients with PKAN (Timmermann et al., 2010). Moreover,

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oral or intrathecal baclofen and stereotactic pallidotomy may provide symptomatic relief but have no known disease-modifying effects (Schipper, 2012). Recently, a new therapeutic chelating agent deferiprone (3-hydroxy-1,2-dimethylpyridin-4-one, DFP) with the potential to cross blood-brain

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barrier have been developed in an attempt to slow down the progression of the disease (Zorzi et al., 2011; Cossu et al., 2014). This clinical trial showed a consistent reduction of brain iron levels in

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the GP. Nonetheless, the clinical benefit was absent in one case (Zorzi et al., 2011) and partial in another ones (Abbruzzese et al., 2011; Cossu et al., 2014). A study on a larger cohort of PKAN TIRCON (http://tircon.eu/).

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patients and for long period of treatment is undergoing within the frame of the European project

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In view of molecular and pathophysiological studies, supplementing pantethine, a vitamin B5 analog, was explored in the Drosophila model of PKAN, which led to improved mitochondrial

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function, enhanced locomotor abilities, and increased lifespan (Rana et al., 2010). Furthermore, chronic administration of pantethine proved to be effective in counteracting the disease phenotype elicited by a ketogenic diet in Pank2 knockout mice, particularly improving mitochondrial cristae morphology (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 mitochondrial function has been implicated.

Conclusions While each of the NBIA types described above is associated to mutations in different genes, it is possible to underline common features that may point to common pathogenic pathways linked to mitochondria as crucial organelle. The involvement of lipid metabolism and mitochondria seems

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evident. In fact, cristae shape determines the assembly and stability of respiratory chain supercomplexes and hence mitochondrial respiratory and bioenergetics efficiency. This connection is clear for PKAN and PLAN in which mitochondrial structural and functional abnormalities are well documented and are probably linked to perturbation in the synthesis (PANK2) or remodeling (PLA2G6) of membrane lipids and mitochondrial cardiolipin. It would be interesting to analyze

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mitochondria from patients with CoASY mutations, which affect the same metabolic pathway of PANK2, and with C19orf12 mutations, which display a link with mitochondrial membrane and

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lipid metabolism. A summary showing the central role of mitochondria in these NBIA disorders is provided in figure 1. This diagram demonstrates the involvement of phospholipids metabolism as a

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common pathway for NBIA mitochondrial proteins. The generation of appropriate animal models and the availability of patients fibroblasts are crucial to research progress in order to elucidate fundamental cellular functions affected by malfunctioning of these genes. Further investigations

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are needed to understand the link between mitochondrial functions, membrane homeostasis and establishment of therapeutic approaches. Acknowledgements:

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iron metabolism in the development of different types of NBIA, thus encouraging the

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The support of Mitochondrial European Educational Training, MEET, ITN MARIE CURIE

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PEOPLE (grant agreement No. 317433) to MA, of Telethon GGP11088 and TIRCON project of the European Commission’s Seventh Framework Programme (FP7/2007-2013, HEALTH-F2-2011,

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grant agreement No. 277984) to VT are gratefully acknowledged.

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Legend to figure

Figure 1: Schematic representation of the implication of mitochondria in NBIA diseases associated to mitochondrial proteins: PANK2, CoASY, PLA2G6 and C19orf12. PANK2 and CoASY catalyze, respectively, the first and the last two steps of CoA synthesis from pantothenate. CoA plays a role as fatty acyl carrier. Fatty acyl-CoA species are esterified into available lysophospholipids by acyltransferases, thus participating to membrane phospholipids (and cardiolipin) remodeling. Fatty acids esterified at the sn-2 position of membrane phospholipids can be released by the activation of PLA2G6. Under oxidative stress attack, PLA2G6 eliminates also peroxidized fatty acids from membrane phospholipids. C19orf12 gene encodes for a mitochondrial membrane protein, which specific role has not yet been elucidated. It was suggested to be coregulated with genes involved in fatty acids biogenesis. The pathogenesis of NBIA diseases is not

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well understood but mitochondrial structural and functional abnormalities, iron accumulation and axonal swelling were documented in these neurodegenerative disorders.

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Abbreviations: AA: arachidonic acid CoA: Coenzyme A CoASY: CoA Synthase CoPAN: CoA synthase protein-associated neurodegeneration CL: cardiolipin DHA: docosahexaenoic acid FA2H: fatty acid-2-hydroxylase GP: Globus Pallidus NBIA: Neurodegeneration with brain iron accumulation PANK2: Pantothenate kinase 2 PLA2G6: phospholipase A2 Group VI PC: phosphatidylcholine PE: phosphatidylethanolamine ROS: reactive oxygen species

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X: polar group (ethanolamine, inositol, serine, choline)

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Figure 1

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Table 1

Gene

Pantothenate kinaseassociated neurodegeneration (PKAN)

Pantothenate kinase-2 (PANK2)

Mitochondrial membrane protein-associated neurodegeneration (MPAN)

Mitochondria (inter membrane space)

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Phospholipase 2, group VIassociated neurodegeneration (PLAN)

pantothenate phosphorylation / Coenzyme A synthesis (fatty acid metabolism) CoA synthase Coenzyme A synthesis (fatty (COASY) acid metabolism) / 4′-PP adenyltransferase and dephospho-CoA kinase activities Phospholipas Hydrolysis of ester bonds at e A2, Group the sn-2 position of VI (PLA2G6; phospholipids / Membrane iPLA2β) phospholipids turnover C19orf12 Unknown/lipid metabolism?

Cellular location

Brain regions interested by iron deposition Globus pallidus (eye of tiger)

Mitochondria Globus pallidus and (Outer mitochondrial substantia nigra membrane, matrix), cytosol

Clinical features

Dystonia, dysarthria, spasticity, parkinsonism, mental retardation, retinopathy, occasional peripheral neuropathy Dystonia, dysarthria, spastic paraparesis, cognitive impairment, obsessive compulsive disorder, motor axonal neuropathy

Mitochondria and cytosol

Globus pallidus and substantia nigra (in