Mitochondrial impairment and rescue in riboflavin responsive neuropathy

Brown-Vialetto-Van Laere syndrome (BVVLS) represents a phenotypic spectrum of motor, sensory, and cranial nerve neuropathy, often with ataxia, optic atrophy and respiratory problems leading to ventilator-dependence. Loss-of-function mutations in two riboflavin transporter (RFVT) genes, SLC52A2 and SLC52A3, have recently been linked to BVVLS. However, the genetic frequency, neuropathology and downstream consequences of RFVT mutations have previously been undefined. By screening a large cohort of 132 patients with early-onset severe sensory, motor and cranial nerve neuropathy we confirmed the strong genetic link between RFVT mutations and BVVLS, identifying twenty-two pathogenic mutations in SLC52A2 and SLC52A3, fourteen of which were novel. Brain and spinal cord neuropathological examination of two cases with SLC52A3 mutations showed classical symmetrical brainstem lesions resembling pathology seen in mitochondrial disease, including severe neuronal loss in the lower cranial nerve nuclei, anterior horns and corresponding nerves, atrophy of the spinothalamic and spinocerebellar tracts and posterior column-medial lemniscus pathways. Mitochondrial dysfunction has previously been implicated in an array of neurodegenerative disorders. Since riboflavin metabolites are critical components of the mitochondrial electron transport chain (ETC), we hypothesized that reduced riboflavin transport would result in impaired mitochondrial activity, and confirmed this using in vitro and in vivo models. ETC complex I and complex II activity were decreased in SLC52A2 patient fibroblasts, while global knockdown of the single Drosophila RFVT homologue revealed reduced levels of riboflavin, downstream metabolites, and ETC complex I activity. RFVT knockdown in Drosophila also resulted in severely impaired locomotor activity and reduced lifespan, mirroring patient pathology, and these phenotypes could be partially rescued using a novel esterified derivative of riboflavin. Our findings indicate mitochondrial dysfunction as a downstream consequence of RFVT gene defects in BVVLS and validate riboflavin esters as a potential therapeutic strategy.


Introduction
Brown-Vialetto-Van Laere syndrome (BVVLS) is an autosomal recessive neurological disorder first described by Brown in 1894 and later by Vialetto and Van Laere (Brown, 1894;Van Laere, 1966;Vialetto, 1936). Affected patients mostly present with neuropathy, bilateral sensorineural deafness, bulbar palsy and respiratory compromise. Other cranial nerve palsies, optic atrophy, upper and lower motor neuron involvement and ataxia can occur particularly as disease progresses, mimicking conditions such as amyotrophic lateral sclerosis (ALS), Madras motor neuron disease and Nathalie syndrome (Anand et al., 2012;Manole et al., 2014). Deafness is the most common sign of this condition, with most affected individuals exhibiting hearing loss during the disease course. The time between the onset of deafness and the development of other manifestations varies but is usually in early childhood (Manole and Houlden, 2015).
Previous work has revealed strong links between mutations in two genes (SLC52A2 and SLC52A3) and BVVLS, both of which encode riboflavin transporters (RFVTs) (Foley et al., 2014;Green et al., 2010;Johnson et al., 2012). The role of another RFVT-encoding gene, SLC52A1, in BVVLS pathogenicity is still uncertain, as it was found to be defective in only one case (Ho et al., 2011).
SLC52A2 and SLC52A3 mutations include missense, nonsense, frame-shift, and splice-site alterations, but uniformly result in loss-of-function through reduced RFVT expression and/or riboflavin uptake (Foley et al., 2014;Intoh et al., 2016;Udhayabanu et al., 2016). Riboflavin (7,8-dimethyl-10-ribityl-isoalloxazine) is a water-soluble compound and acts as a precursor for flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). Both FMN and FAD function in biological redox reactions such as in the mitochondrial electron transport chain (ETC) (Powers, 2003). Since riboflavin cannot be synthesised by mammals de novo, RFVTs are indispensable for normal cellular metabolism, suggesting that reduced intracellular riboflavin is a critical pathological mediator of BVVLS. Indeed, although infants with early-onset RFVT deficiency rapidly become ventilator-dependent and usually die in the first year of life, treatment with high-dose riboflavin supplementation partially ameliorates the progression of this neurodegenerative condition, particularly if initiated soon after the onset of symptoms (Foley et al., 2014).
Motor neurons are thought to be uniquely susceptible to impaired energy metabolism because of their high metabolic rate and axonal length (Cozzolino and Carri, 2012). Furthermore, mitochondrial perturbations causing alteration of the ETC and increased oxidative stress are known to be involved in the pathomechanisms of neurodegeneration in motor neuron diseases such as ALS or spinal muscular atrophy (Bartolome et al., 2013;Cozzolino and Carri, 2012;Johnson et al., 2010). Given the critical role of riboflavin in the generation of substrates used for the ETC, we hypothesized that reduced riboflavin transport results in impaired ETC, which may in turn contribute to neurodegeneration.
Here, we expand the clinic-genetic spectrum of riboflavin transporter genes and then perform a series of experiments to identify the underlying effects of loss of RFVT function on neuronal integrity and mitochondrial function. We review clinical case histories and undertake pathological evaluation of brain and spinal cord of two patients with confirmed SLC52A3 mutations who presented either in infancy or in later childhood. Finally, we investigate the in vitro cellular effects of SLC52A2 mutations on metabolism and ETC function and also the in vivo consequences of loss of the SLC52A3 homologue in Drosophila, and test whether these can be mitigated by supplementation with a riboflavin derivative.

Study Subjects
Patients were enrolled with informed consent from the patient and/or their parental guardian. DNA was collected from a total of 132 suspected cases (probands and their relatives) presenting with cranial neuropathies and sensorimotor neuropathy with or without respiratory insufficiency. Patients' DNA samples were collected at medical centres in England (including from patients originating from Pakistan, India, Saudi Arabia, Kuwait, Iran and Turkey) as well as from medical centres in Wales, Scotland, Northern Ireland, Ireland, France, Belgium, the Netherlands, Greece, Malta, Russia, Lebanon, Iceland, Australia and the United States, following the announcement of an on-going molecular study at the UCL Institute of Neurology, University College London (National Hospital for Neurology & Neurosurgery, Queen Square, London) of patients presenting with this phenotype. This study was ethically approved by the UCL/University College London Hospital Joint Research Office (99/N103), and written informed consent to perform a skin biopsy and fibroblasts was obtained as appropriate.

PCR and Sanger sequencing
Primer sequences, PCR and Sanger sequencing conditions for SLC52A1, SLC52A2 and SLC52A3 were as in (Foley et al., 2014). Segregation of pathogenic variants was also assessed. Where one heterozygous mutation was identified, deletions in the other allele were investigated by array CGH but no deletions or insertions were identified. Mutation positions are based on NCBI reference sequences for complementary DNA. SLC52A2 mutation positions are based on sequences NM_024531.4 for the nucleotide sequence and NP_078807.1 for the protein sequence. SLC52A3 mutation positions are based on sequences NM_033409.3 for the nucleotide sequence, and NP_212134.3 for the protein sequence.

Determination of Flavin status in fibroblasts
Riboflavin, FMN and FAD content were measured in neutralized perchloric extracts by means of High Performance Liquid Chromatography (HPLC), as previously described (Giancaspero et al., 2009).
Quantitative determination of riboflavin, FMN, and FAD was carried out using the Whole Blood Chromsystems vitamins B 1 /B 2 kit (Chromsystems, Germany) as per the manufacturer's protocol. The Bio-Rad DC protein assay (Bio-Rad Laboratories, USA) was used to normalise for protein concentration.

Assessment of ETC complex I, II, and citrate synthase activities in fibroblasts
All enzyme activities were determined at 30 o C. Prior to analysis all samples were subjected to three cycles of freeze/thawing to lyse membranes. Enzymatic activities were determined using an Uvikon 940 spectrophotometer (Kontron Instruments Ltd, Watford, UK).
Complex I activity was measured according to the method of (Ragan, 1987), which involved monitoring the oxidation of NADH at 340 nm. Complex II assay was measured according to the method of (Birch-Machin et al., 1994), which monitored the succinate-dependent 2-thenoyltriflouroacetone sensitive reduction of 2,6-dichlorophenolindophenol at 600 nm. The activity of citrate synthase was measured by the formation of the anion of thionitrobenzoate from 5,5′-dithiobis(2-nitrobenzoate) and CoA at 412 nm (Shephard, 1969). This provided an estimate of mitochondrial content and was therefore used to normalise complex I and II activities for mitochondrial enrichment (Hargreaves et al., 1999).

Drosophila stocks and culture conditions
Fly strains were obtained from the Bloomington Drosophila Stock Center (Indiana, USA) and Vienna Drosophila Resource Center (Austria). All transgenic insertions used in this study were outcrossed at least 5 times into an isogenic (iso31) background. These were: cg11576 UAS-RNAi ((VDRC 7578) (Dietzl et al., 2007) and HMC04813 (Perkins et al., 2015)), and daughterless-GAL4 (Bloomington stock 55850 representing the product formation was made for both primer pairs and cycles 26 and 29 were chosen for rp49 gene and drift respectively so that amplification rates were in the linear range for semi-quantitative comparisons.

Drosophila
Riboflavin, FMN and FAD content, and ETC complex I, II redox activity and citrate synthase activities were measured in flies (approximately 10/genotype) as described above for patient fibroblasts. Prior to analysis all samples were subjected to three cycles of freeze/thawing to lyse membranes. A Lowry assay was used to normalize for protein concentration (Frolund et al., 1995). Complex II/III activity was determined at 30°C using the method of (King, 1967) which followed the succinate-dependent antimycin A sensitive reduction of cytochrome c at 550 nm.

Larval behaviour
Larval locomotion was tested by placing individual third instar larvae in the center of petri dishes (8.5 cm diameter, 1.4 cm height) coated with 10 ml of 4% agar. On average, 30 larvae were tested per strain. The number of grid squares (1 cm) entered per min by the larva was analysed using Kruskal-Wallis tests and subsequently Dunn's post-hoc tests.

Adult behaviour
Adult flies were kept as groups of males and females in a 12h:12h light-dark (LD) cycle at 25°C for 1 day prior to testing. Single virgin females (approx. 1 day old) were loaded into glass tubes (with 2% agar and 4% sucrose food) and monitored using the Drosophila Activity Monitoring System (Trikinetics) in LD at 25°C with an approximate intensity of 700-1000 Lux during the L condition. For experiments involving supplementation, riboflavin or RLAM was added to the above food. Fly activities were deduced from the number of times flies broke beams of infrared light passing through the middle of the tube. Locomotor activity was recorded in 30 min bins and an analysis was performed on the second day after loading. Data were pooled from at least two independent experiments. The relative locomotor activities per 30 min bin for individual flies were averaged for each genotype and also the average locomotor activity per day was calculated. Locomotion graphs were generated using GraphPad Prism 6 and Microsoft Excel.

Life span
Adult female flies were collected from eclosion and transferred to fresh food tubes (10 flies/tube) with or without RLAM supplementation. Each day, death events were scored and viable flies were transferred to fresh tubes. Survival proportions were plotted as percentage of live flies against days. Approximately 100 flies were tested for each genotype.

Other statistics
Statistics were performed using GraphPad Prism 6. The significance between the variables was shown based on the p-value obtained (ns indicates p > 0.05, * indicates p < 0.05, ** indicates p < 0.005, *** indicates p < 0.0005). Data are presented as box plots illustrating 80% of the data distribution, together with the median and 10 th , 25 th , 75 th and 90 th percentiles.

Clinical and genetic analysis of BVVLS patients
We screened 132 patients with phenotypes suggestive of BVVLS and identified twenty patients (15%) with RFVT mutations. Genetic and clinical details of the twenty individuals carrying mutations in SLC52A2 or SLC52A3 are summarised in Table 1. Thirteen of the probands were males. Age of symptom onset was available for 15 patients (mean: 8.2 years; range: 7 months -21 years). First symptoms usually occurred during childhood (11 patients) and less frequently during teenage years (four patients) or adulthood (one patient), and were mostly secondary to cranial nerve involvement (Table 1). Only one patient presented with symptoms not related to cranial nerve involvement (limb weakness). Sensorineural hearing loss was both a common presenting symptom (eight patients) and a common clinical feature during follow-up (17 patients). Other frequent clinical features include optic atrophy (14 patients), weakness of facial and bulbar muscles (13 patients) and sensorimotor peripheral axonal neuropathy (16 patients). Limb weakness was more severe in the upper than in the lower limbs in nine patients. Ten patients developed some degree of respiratory involvement with three requiring assisted ventilation, and ten patients had dysphagia and/or chewing difficulty, six of them requiring nasogastric tube or gastrostomy feeding.
Of the twenty-two mutations identified, eight were found in the SLC52A2 locus and 14 in SLC52A3, of which five SLC52A2 and nine SLC52A3 variants were novel (Table 1). In contrast, no SLC52A1 mutations were observed. None of the variants were found in the homozygous state in the ExAC database of over 100,000 controls, suggesting pathogenicity (Table 1). Consistent with this hypothesis, all except one mutation (p.Arg212Cys in SLC52A3) were predicted to be at least probably damaging by SIFT and Polyphen-2 algorithms (scores ranging between 0.55 and 1). The mutations reside in transmembrane helices and in the intracellular and extracellular loops. Although three homozygous and seven compound heterozygous mutations were identified, five mutations (all in SLC52A3) were identified on only one allele. These heterozygous individuals did not differ substantially in phenotype including age of presentation from the rest of the cohort of mutation-positive cases.
There was no correlation between the nature of pathogenic variant and phenotype severity, although in the case of patient AM2, nonsense mutations on both alleles resulted in a truncated protein, and this genotype correlated with rapid progression of symptoms and death at 2 years of age.

Neuropathological analysis of BVVLS patients
To characterise in detail the neuropathological symptoms of BVVLS, we undertook a comprehensive pathological examination of two patients carrying compound heterozygous SLC52A3 mutations (AM2 and AM4; Table 1). These patients represent two ends of the spectrum of severity of BVVLS.
Patient AM2 had a normal birth at term. His motor skills were mildly delayed and he never acquired the ability to roll over completely front to back. He achieved the ability to sit with minimal support at age 7 months. From about 8 months of age he began to exhibit more clear signs of the condition such as ptosis and neck weakness. He was admitted at the age of 9 months for investigations but no diagnosis was made at that time. His condition quickly progressed to include respiratory muscle weakness, and ventilator dependence at the age of 1 year. He also developed severe weakness in his shoulder girdle areas and proximal upper limbs. Weakness subsequently developed in forearm muscles, distal lower limb muscles and thighs, trunk and face, and progressed to the point that he could only weakly move his eyelids and had very limited sideways movements of his eyes. Nerve conduction studies (NCV) and electromyography showed a severe neuropathy. He died at two years of age of respiratory failure.
The cerebral cortex, hippocampus and cerebral white matter were unremarkable. The deep grey nuclei were not available for assessment. The neuronal density in the substantia nigra was normal for the patient's age, but the 3 rd and 4 th cranial nerve nuclei showed severe neuronal loss, gliosis and microglial activation ( Supplementary Fig. 1). In the pons there were two symmetrical sharply demarcated lesions surrounding both 5 th cranial nerves ( Fig. 1A, A1, A2 -D, D1, D2). In these lesions we observed prominent neovascularisation, dense infiltration of macrophages and widespread myelin loss, with relative preservation of axons. The 5 th cranial nerve was vacuolated and its nucleus showed severe neuronal loss and gliosis ( Supplementary Fig. 1). In the medial lemniscus at the level of the midbrain and pons there was prominent gliosis, microglial activation and vacuolation of the neuropil, which was also seen in the central tegmental tract. In the medulla, the 9 th , 10 th and 12 th cranial nerve nuclei showed severe neuronal depletion and gliosis with pale corresponding nerve tracts. The neuronal loss in the 8 th cranial nerve was moderate ( Supplementary Fig. 1). The medial lemniscus, spinocerebellar tract and medullary reticular formation were all gliotic. Inferior olivary nuclei, in particular the dorsal and ventral parts, showed severe neuronal loss and gliosis. In the cerebellum, there was no significant cortical atrophy Patient AM4 also had a normal birth at term. He had hearing problems from the age of 8 years and was diagnosed with sensorineural hearing loss at the age of 11. He developed optic atrophy and difficulty walking at the age of 16 years. At age 17 years he presented with dysarthria and subsequently developed swallowing difficulties. Electromyography showed widespread denervation and NCS studies were consistent with an axonal motor-neuropathy. He died at 19 years of respiratory insufficiency.
The cerebral neocortex, hippocampus, amygdala, caudate nucleus putamen, globus pallidus, thalamus and cerebral deep white matter showed no apparent abnormality. Mild gliosis was evident in the dorsal part of the optic tract. In the midbrain, the substantia nigra was densely populated by lightly pigmented neurons in keeping with the patient's age. The corticospinal tracts were unremarkable in the cerebral peduncles of the midbrain. The 3 rd cranial nerve nucleus was not available for the assessment and the 4 th cranial nerve nuclei showed a mild degree of neuronal loss and accompanying mild gliosis ( Supplementary Fig. 3). In the pons there was prominent neuronal loss in the loci coerulei with free pigment deposits in the neuropil and gliosis ( Supplementary Fig. 3)  Fig. 3). There was a severe, slightly asymmetrical atrophy of the uncrossed anterior corticospinal tracts, whilst the lateral corticospinal tracts were densely populated by myelinated fibres with only mild vacuolation ( Supplementary Fig. 3 and Supplementary Fig. 4L1). Severe symmetrical atrophy with prominent pallor, macrophage infiltration and vacuolation of the anterior and posterior spinocerebellar tracts ( Supplementary Fig. 4 K1), spinothalamic tracts and gracile and to a lesser extent cuneate fasciculi ( Supplementary Fig. 3) was also observed.
Whilst the posterior nerve roots were densely populated by myelinated fibres with only mild macrophage activation, in the anterior nerve roots there was moderately severe loss of myelinated fibres and prominent infiltration of macrophages (Fig. 2F, F1, F2 -J, J1, J2).
Neither in the case AM2 nor AM4 were any amyloid-β, hyper-phosphorylated tau, α -synuclein, TDP-43, p62 or ubiquitin positive inclusions observed. In both cases the inflammatory reaction was restricted to microglial activation and macrophage infiltrates with no significant lymphocytic inflammation.

Fibroblasts biochemical studies
Mitochondrial dysfunction has long been documented in neurodegenerative diseases (Palomo and Manfredi, 2015). For example, SOD1 mutations in familial ALS have been shown to lead to abnormalities in mitochondrial morphology, both in biopsies and post-mortem tissues of human patients (Sasaki and Iwata, 2007;Sasaki, 2010) and in cellular and mouse models of the disease (Magrane et al., 2009;Magrane et al., 2014;Palomo and Manfredi, 2015;Vinsant et al., 2013). However, whether mitochondrial function is perturbed by BVVLS-linked mutations has yet to be examined. We hypothesized that lower levels of intracellular riboflavin as a result of mutated RFVTs would lead to reduced levels of FMN and FAD, which in turn would lead to impairments at the level of the ETC complex I and complex II. Using fibroblasts derived from BVVLS patients with RFVT mutations and healthy age-matched controls, we found a significant reduction in the intracellular levels of FMN and FAD in patient fibroblasts when grown in low extracellular riboflavin conditions (Fig. 3A, B). Levels of intracellular riboflavin in patient fibroblasts frequently fell below the threshold of detection under these conditions (but not in control fibroblasts; data not shown), consistent with defective riboflavin transport.
Furthermore, we observed a significant reduction in ETC complex I and complex II activity in patient fibroblasts compared to controls (Fig. 3C, D).

Generation of a novel Drosophila model of BVVLS
We next sought to confirm the link between RFVT dysfunction and reduced mitochondrial activity in vivo. Previous work has utilised knock-out of the mouse SLC52A3 orthologue to model BVVLS (Intoh et al., 2016;Yoshimatsu et al., 2016 RFVTs are also conserved ( Fig. 4A and Supplementary Fig. 5A) (Russ and Engelman, 2000;Zhang et al., 2010). The L1 loop is a region of the protein shown to recognize riboflavin through both hydrogen bonds and van der Waals interactions (Zhang et al., 2010), while the GXXXG motif is required for dimerization (Russ and Engelman, 2000). DRIFT is also highly homologous to the hRFVT3 paralogues hRFVT1 and hRFVT2 (Fig. 4A). Furthermore, 10/19 and 11/25 residues in hRFVT2 and hRFVT3 respectively that are altered by BVVLS-linked mutations are either identical or functionally similar in DRIFT. Mammalian RFVTs exhibit wide domains of expression, including the nervous system, intestine, testes, and placenta (Yonezawa and Inui, 2013). Similarly, drift is transcribed in several adult Drosophila tissues, including the head, gut, abdomen and thorax ( Supplementary Fig. 5B). This broad expression pattern is in agreement with RNAseq data from the Drosophila ModEncode Project (Graveley et al., 2011).
To examine the biochemical and phenotypic consequences of loss of DRIFT, we disrupted drift  5C). However, this knockdown did not result in early lethality (suggesting stronger RNAi expression by actin-relative to da-Gal4), facilitating analysis of drift knockdown flies at later developmental stages.
Using whole-body tissue from drift knockdown adults and associated control lines (heterozygotes for da-Gal4 and the drift RNAi transgene), we found that drift knockdown resulted in a substantial reduction of in vivo riboflavin levels as well as the riboflavin metabolites FMN and FAD (Fig. 4B-D). These results, combined with the high homology of DRIFT to hRFVT1-3, strongly suggest that DRIFT is a bona fide riboflavin transporter. We next asked whether drift knockdown resulted in reduced mitochondrial activity.
Similarly to BVVLS patient fibroblasts, ETC complex I activity was profoundly reduced by drift knockdown (Fig. 4E), while ETC complex II and II-III activity exhibit a trend towards lower levels, albeit non-significant (Fig. 4F, G). Thus, in Drosophila, complex I activity appears particularly sensitive to DRIFT expression.

drift knockdown results in reduced locomotion and lifespan in Drosophila
The viability resulting from drift knockdown via da-Gal4 allowed us to assess whether RFVT knockdown impacts post-embryonic organismal phenotypes in a manner consistent with BVVLS pathology (Foley et al., 2014;Manole et al., 2014). We found that drift knockdown resulted in profound locomotor defects in both larval and adult Drosophila. drift knockdown 3 rd instar larvae exhibit significantly reduced locomotion, as measured by the number of grid crosses per minute on an agar plate ( Supplementary Fig.   5D). We also used the Drosophila activity monitor (DAM) system to perform automated recordings of adult locomotion, measured as the number of infra-red beam breaks across a 24 h day/night cycle. Under 12 h light: 12 h dark conditions, control 1-2 day old adult female Drosophila exhibit peaks of activity at dawn and dusk, and relative quiescence during the afternoon and night (Fig. 5A, B). In contrast, peak activity and total beam breaks in drift knockdown adults were substantially reduced (Fig. 5C, D).
Furthermore, drift knockdown resulted in greatly reduced lifespan, with 99% mortality within four days post-eclosion (Fig. 5E). These phenotypes mimic motor problems and early mortality observed in BVVLS patients, suggesting a conserved link between RFVT dysfunction, locomotor strength and lifespan.

An esterified riboflavin derivative partially rescues drift knockdown phenotypes
Since BVVLS pathology can be partially ameliorated by riboflavin treatment, we asked whether locomotor defects in drift knockdown flies could be rescued by supplementing Drosophila culture medium with riboflavin (0.1 mg/ml). However, we found no enhancement of locomotor activity following riboflavin supplementation ( Supplementary Fig. 5E). Riboflavin is a water-soluble vitamin that is easily excreted, leading to low bioavailability and short half-life. Furthermore, since RFVT expression is very low in drift knockdown flies ( Supplementary Fig. 5C), riboflavin in the Drosophila haemolymph may fail to be transported into relevant cell-types. We sought to circumvent these issues using an esterified derivative of riboflavin (riboflavin-5'-lauric acid monoester; RLAM; 0.1 mg/ml) that could act as a pro-drug, likely diffuse into the intracellular space independently of RFVT function and be cleaved by esterases to release active riboflavin (Fig. 6A). As predicted, food supplementation with RLAM dramatically increased complex I activity (~ 3-fold), and critically, resulted in heightened total locomotion (Fig. 6B, C), increased peak levels of daily activity (Fig. 6D, E), and a partial extension of lifespan ( Fig. 6F) in drift knockdown flies. We speculate that RLAM may represent a more efficient treatment method for BVVLS patients since cellular uptake of RLAM may still robustly occur in the absence of functional endogenous RFVTs.

Discussion
We expanded the spectrum of genetic defects in RFVTs identifying SLC52A2 and SLC52A3 mutations in 15% of cases. It was previously noted that the distinct phenotype of upper limb and axial weakness, hearing loss and optic atrophy could be attributed only to patients harbouring SLC52A2 mutations (Foley et al., 2014). However, in our cohort of 20 patients, mutations in both SLC52A2 and SLC52A3 resulted in similar phenotypes. SLC52A3 mutations were more frequent than mutations in SLC52A2 in our cohort.
This is in agreement with previous reports where BVVLS-linked mutations were predominantly found in SLC52A3 (Bosch et al., 2011;Manole and Houlden, 2015). The high proportion of patients who were found to be negative for mutations in the known RFVTs, indicates that novel genetic causes are yet to be found.
The neuropathology of BVVLS has not been fully characterised in the past (Malafronte et al., 2013).  Figs. 1 and 3), the morphology of the lesions was identical in both cases and was similar to the pathology seen in mitochondrial encephalopathies (Filosto et al., 2007;Tanji et al., 2001). To the best of our knowledge such lesions have not been documented in any of the previous published cases of BVVL clinical syndrome and link with the mitochondrial abnormalities in flies and patient fibroblasts.
Although fibroblasts are non-neural cells and not particularly vulnerable as a tissue, metabolic and mitochondrial abnormalities are commonly studied in this cell-type (Distelmaier et al., 2009). We identified clear deficiencies in the activities of ETC complex I and complex II in patient-derived fibroblasts relative to fibroblasts from healthy controls. Interestingly, previous results have only shown evidence of marginally decreased ETC complex I activity in muscle cells from some SLC52A2 patients sensitive to reduced riboflavin uptake (Foley et al., 2014). Mitochondrial activity in different tissues and cell types may thus be differentially sensitive to reduced riboflavin uptake.
Finally, we examined the in vivo consequences of knockdown of the single SLC52A3 homologue drift in Drosophila. Biochemical analysis of drift knockdown tissue is consistent with data derived from patient fibroblasts, showing diminished levels of riboflavin, FMN and FAD, and reduced complex I activity. At the whole-organismal level, there was impairment in locomotion at both the larval and adult stages, reminiscent of the limb weakness and movement impairment of the BVVLS patients. Moreover, drift knockdown flies had severely reduced life span, similar to untreated patients (Bosch et al., 2011). It is interesting to note that these lifespan and locomotor defects are rescued by ingestion of an esterified derivative of riboflavin, since this provides further evidence that the phenotypic signatures of drift knockdown flies are linked to riboflavin deficiency and consequent downstream metabolic defects. We hypothesize that lack of RLAM during pupation, a critical neurodevelopmental stage during which RLAM-supplemented food will not be consumed, may contribute to the partial nature of the observed RLAM rescue. Nonetheless, RLAM fulfils the criteria for being a treatment targeting energy metabolism, a fundamental mitochondrial process in addition to circumventing the disease-related protein, features generally believed to be of great promise (Foley et al., 2014;Lin and Beal, 2006;Xuan et al., 2013).
It was previously thought that defective mitochondria occur secondary to primary disease mechanism, but current research suggests that mitochondrial dysfunction may play a role in both onset and development of neurodegenerative diseases such as Alzheimer's, Parkinson's, Huntington's and ALS, where decreases in one or more mitochondrial complexes and corresponding oxidative stress have been reported in cell or animal models (Fukui and Moraes, 2007;Golpich et al., 2016;Hoglinger et al., 2003;McInnes, 2013;Menzies et al., 2002;Yamada et al., 2014). Our results from in vitro and in vivo models of a childhood neuropathy are in line with these findings, highlight a contribution of mitochondrial dysfunction to BVVLS pathology, and suggest future therapeutic strategies based on