Autophagy Induction in C12orf65 Mutation-related Autosomal Recessive Hereditary Spastic Paraplegia

Background: Spastic paraplegia type 55 (SPG55) is an autosomal recessive complicated hereditary spastic paraplegia. Here we report an SPG55 case with typical neurological phenotypes including optic atrophy, lower extremity spasticity and peripheral neuropathy. Methods: The present study involved one patient in a Chinese family. Neurological examination including the ophthalmology related examinations as well as nerve conduction velocity and a sural nerve biopsy were performed for the patient. We performed a genetic analysis of genes associated with peripheral neuropathy and spastic paraplegia using a multigene next-generation sequencing(NGS) panel and sanger sequencing. Furthermore, we cultured patient’s primary skin broblasts, then we examined the cytoplasmic LC3B immunouorescence in the patient’s primary broblast and after two drugs (butylphthalide sodium chloride and idebenone) were used to the target the mitochondria function of the broblast. Results: Here we reported an SPG55 case with typical neurological phenotypes including optic atrophy, lower extremity spasticity and peripheral neuropathy. We identied a homozygous C12orf65 nonsense mutation (c.394C>T, p. R132*) in the affected patient. The mutation was associated with active autophagosome formation with increased LC3B puncta in the patient’s broblasts compared with an age-matched healthy individual, while the latter phenotype was normalized by Dl-3-N-butylphthalide treatment. Conclusions: This is the rst pilot study to characterize the SPG55 mutation in the Chinese population; it will contribute to further research revealing the role of C12orf65 mutations in regulation of mitochondria function and autophagy.


Background
Hereditary spastic paraplegia is a clinically and genetically heterogeneous group of inherited neurodegenerative disorders in which the main neurological symptoms comprise progressive spasticity and weakness of the lower limbs [1,2]. So far, 79 genes have been linked to spastic paraplegia (SPG1-SPG79, in the order of discovery) with X-linked, autosomal dominant or recessive inheritance, with or without maternal imprinting [3]. Spastic paraplegia type 55 (SPG55) is an autosomal recessive pathology caused by homozygous mutation in the Chromosome 12 open reading frame 65 (C12orf65) gene mapped to chromosome 12q24.31 [4]. SPG55 varies in disease phenotypes including early onset slowly progressive spastic paraplegia, progressive visual loss with optic atrophy, distal axonal motor and sensory neuropathy, delayed psychomotor development with mental retardation, pes equinovarus, and arthrogryposis. Neuroimaging studies demonstrated hypoplastic corpus callosum in some patients [2,5].
C12orf65 is a nuclear gene encoding a mitochondrial matrix protein which is critical for the release of newly synthesized proteins from mitochondrial ribosomes [6]. Shimazak et al. identi ed a homozygous truncating mutation in C12orf65 causing reduced synthesis of most mitochondrial proteins and reduced activities of some respiratory complex enzymes [2]. These ndings indicated that defects in mitochondrial translation may be a mechanism underlying degeneration of the corticospinal tracts and spastic paraplegia.
Importantly, reduced ATP levels due to decreased activity of ATP synthase may cause autophagy through the Amp-activated protein kinase (AMPK) pathway [7]. AMPK is a major metabolic energy sensor that regulates energy homeostasis by controlling several homeostatic mechanisms, including autophagy [8,9]. AMPK serves as a positive regulator of autophagy mainly via inhibiting the mammalian target of rapamycin (mTOR) complex and phosphorylating unc-51-like kinase 1 (ULK1, the ortholog of Atg1 in mammals) [10,11]. Furthermore, the defective mitochondrial function in patients with hereditary spastic paraplegia might cause PINK1 (PTEN-induced putative kinase 1) and the E3 ligase Parkin1-dependent activation of mitophagy, a variant of autophagy associated with mitochondria recycling [12][13][14]. Indeed, Chang et al. identi ed a depletion of free lysosomes and an accumulation of autolysosomes due to an impaired lysosome formation upon the loss of function of SPG11/15 [15].
The precise pathogenesis mechanism of SPG55 is still unclear. In the current work, we present a patient with a unique combination of early onset optic atrophy, progressive distal lower extremity spasticity and sensorimotor peripheral neuropathy. We demonstrate that a nonsense mutation in C12orf65 is associated with an increased number of microtubule-associated proteins 1A/1B LC3B-positive puncta detected in broblasts from SPG55 patient.

Human subjects
The proband was a patient observed in Department of Neurology

Next-generation Sequencing Analysis And Sanger Sequencing
Genomic DNA was extracted using QIAamp DNA extraction kit (QIAGEN), and the concentration was measured. DNA was then fragmented by DNase and puri ed by magnetic bead method, followed by PCR ampli cation and ligation of the adapter sequences. Further, it was captured and puri ed twice by a custom Panel probe (Illumina Inc, USA), and then ampli ed by PCR. The nal library obtained after puri cation was used to sequence exon regions of Panel-related genes on a NextSeq500 sequencer (Illumina Inc, USA). All data were compared to the reference sequence (UCSC hg19) using the BWA algorithm with the instrument's default settings [16] and data reporting methods for annotation [17]. By implementing clinical data and the prediction results of bioinformatics software including PolyPhen2, LRT, Mutation Taster, the functional, mutation and genetic patterns of each gene were screened to obtain the list of candidate mutations. PCR primers were designed for the sites of candidate mutations in the patient's parents for ampli cation and Sanger sequencing veri cation.

Primary Fibroblast Culture
Brie y, after local sterilization and anesthesia, full-thickness skin biopsies (∼ 5 mm 3 ) were taken from the patient and a 25-year-old healthy volunteer. The isolates were placed into 50-ml conical tubes lled with biopsy culture medium containing 400 ml minimum essential medium, 100 ml fetal bovine serum, 5 ml penicillin/streptomycin solution (10,000 U/ml penicillin G and 10,000 µg/ml streptomycin) and stored in culture medium for 12 hours at 4 °C before dissection for primary culture. After removing the biopsy culture medium, the biopsy samples were washed three times with 10 ml DPBS without calcium and magnesium. Further, they were placed to a sterile 6-cm tissue culture dish containing ∼ 7 ml primary culture medium containing 400 ml minimum essential medium, 100 ml fetal bovine serum, 5 ml penicillin/streptomycin/Fungizone solution (10,000 U/ml penicillin G, 10,000 µg/ml streptomycin, 25 µg/ml amphotericin B) and anti-mycoplasma reagent. The biopsies were rst cut into small pieces, then re ned to pieces with the size of a pinhead. Further, we placed each 10 pinhead-sized explants into one 25-cm 2 tissue culture-treated ask (Nunc™ EasYFlask™ 156340), waited for 20 minutes for the explants to adhere and added 12 ml primary culture medium. The asks were placed into a dedicated 37 °C, 5% CO 2 incubator for 5 days, followed by replacement of the medium by fresh primary culture medium. The broblast cultures were further maintained, checked daily for growth, con uence and contamination, with medium replacements every 3-5 days according to Villegas et al [18]. When the con uence was ~ 95%, the passage 2 broblasts from patient and healthy volunteer were seeded into two 96-well plates with the densities 3-9 × 10 3 cells/ml. Drug treatment was started when the con uence reached 60%~70%. Two drugs were used to target the mitochondria function of the broblasts.

Statistical analysis
For the in vitro experiment's analyses, we used ordinary two-way ANOVA followed by Holm-Sidak's multiple comparisons test vs. each healthy individual's broblasts in every treatment group and ordinary one-way ANOVA followed by Holm-Sidak's multiple comparisons test at all treated patient groups vs. the untreated patient group. The levels of signi cance were set at *P < 0.05 and **P < 0.001. Values were presented as mean ± standard error of mean (SEM).

Clinical details
The proband was a 30-year-old man, who visited our hospital because of decreased visual acuity and slowly progressive weakness of the lower extremities. His delivery was normal, with normal motor and mental development in infancy. Retrospectively, the patient's progressive loss of visual acuity at the age of nine was the rst reported symptom. One year later, he developed muscle atrophy and fatigue of both lower limbs, foot drop leading to a steppage gait and slowly developing weakness of the lower extremities. No history of learning di culties or seizures had been detected.
Neurological examination on admission at the age of 30 revealed loss of visual acuity with bilateral optic atrophy (Fig. 1A): the naked eye vision was scored as 0.2/1.0 and 0.05/1.0 (left and right eyes, respectively) without any macular morphology abnormalities on both sides, as revealed by in vivo optical coherence tomography (Fig. 1B).
The A sural nerve biopsy demonstrated a predominantly chronic demyelinating neuropathy (Fig. 1D-G). Electron microscopy imaging revealed a slight loss of myelinated bers and multiple bers with extremely thin myelin sheaths, indicating a defective myelination. Schwann cell degeneration was apparent in the patient as demonstrated by increased perinuclear π particles in Schwann cells, and shingled Schwann cell processes around the unmyelinated bers.

Identi cation of a C12orf65 mutation
The C12orf65 gene (c.394C > T) [NM_152269.4] homozygous point mutation in the spastic paraplegia patient was identi ed using next-generation sequencing ( Fig. 2A). The variant causes an arginine 132 substitution by a stop codon (p.R132*) in the release factor-1 (RF-1) domain (Fig. 2B). Next, we sequenced this locus in every volunteer in this pedigree revealing an asymptomatic carriers of the heterozygous c.394C > T mutation indicated in Fig. 2C as IV2, IV6, IV7, IV10 and V4.

Increased LC3B signal in primary broblasts from the SPG55 patient
Consistent with other studies [2,20], we discovered no morphological changes in mitochondria such as increased fragmentation, ssion or fusion in the SPG55 patient primary broblasts (Fig. 3A). In order to study whether the defective mitochondrial translation in SPG55 could trigger reactive autophagy, we examined the abundance of cytoplasmic puncta positive for LC3B, a common marker for autophagic structures. Indeed, we detected a signi cant increase in cytoplasmic LC3B immuno uorescence (Fig. 3B) in primary broblast cultures from the SPG55 patient compared to an age-matched healthy individual. Butylphthalide and idebenone are promising candidate drugs for mitochondrial protection. Butylphthalide has been reported to restore mitochondrial membrane potential and prevent reactive oxygen species (ROS) generation [21]. Idebenone is a potent antioxidant and inhibitor of lipid peroxidation, interacting with the mitochondrial electron transport chain and facilitating mitochondrial electron ux in bypassing complex [22]. As such, we sought to explore whether these drugs could normalize the autophagy-related phenotype detected in SPG55 primary broblasts. Indeed, we observed a tendency towards attenuation of the patient broblasts' LC3B signal upon idebenone or butylphthalide treatment (with the latter reaching a statistical signi cance at the concentration of 26 µM), supporting a key role of mitochondrial defects in the observed autophagy induction (Fig. 3).

Discussion
In previous studies, all clinically relevant C12orf65 mutations were truncating, including nonsense mutations (p.L94*, p.V116*, p.R132* and p.R139*) and frameshift mutations (p.T2Rfs*54, p.P34Ifs*25, p.V83Gfs*2, p.G72Afs*13 and p.K138Rfs*17) ( Fig. 2A, B) with the mutation sites located in the release factor-1 (RF-1) domain, with the exception of p.K138Rfs*17 [2,26,23,24]. Disease severity has been previously reported to depend on the residual length of the truncated C12orf65 protein. Indeed, patients with truncating mutations close to the N-terminal demonstrate severe phenotypes, including neonatal death, optical atrophy and severe cognitive dysfunction in addition to the main symptoms. In contrast, patients with truncations close to the C-terminus showed only mild intellectual disability associated with the main symptoms [25]. In the current study, homozygous p.R132* mutation close to the C-terminus resulted in an optic atrophy with reduced visual acuity, lower limb spasticity with peripheral neuropathy.
Recently, a Japanese group has reported the same mutation site resulting in almost the same clinical manifestations as in our patient (Shimazaki, Takiyama et al. 2012). They detected that the c.394C > T mutation was associated with optic atrophy and peripheral neuropathy in addition to the main symptoms [2]. Thus, it can be inferred that the above three signs may be characteristic for this nonsense mutation.
Consistent with the critical role of C12orf65 in releasing newly synthesized mitochondrial proteins, SPG55 cases were found to be associated with decreases in respiration, in ATP synthase activity and in mitochondrial membrane potential [26]. As mentined above, Shimazaki et al. described an SPG55 patient with the identical c.394C > T mutation [2]. In that study, patient-derived broblasts were found to have a decrease in expression of most mitochondrial proteins and reduced activities of respiratory complex enzymes including complexes I, III and IV indicating a profound mitochondrial defect associated with this mutation [2]. Balanced mitochondrial biogenesis and mitochondrial quality control helps remain mitochondria function in neurons [27]. In our study, we sought to analyze the morphology of mitochondria and autophagosomes in patient-derived primary broblasts. There, we detected a prominent enrichment of cytoplasmic LC3B-positive puncta compared to an age-matched healthy individual (Fig. 3A). These data suggest a reactive over-activation of autophagosome formation in response to the mitochondria impairment, as previously described [28]. The mechanism of selective tagging of damaged mitochondria by such control mechanism has been largely attributed to the coordinated action of PTEN-induced putative kinase (PINK1) and Parkin [29]. As such, defective mitochondria normally trigger PINK1/Parkin-dependent activation of mitophagy [30]. Furthermore, mutations in autophagy-related genes have been reported in spastic paraplegia patients [31,32].
Interestingly, tectonin beta-propeller repeat-containing protein 2 (TECPR2), the causative gene in SPG 49, has been previously found to interact with autophagy-related 8 orthologues [31,33]. Moreover, since the activity of ATP synthase has been reported to be reduced in patients with C12orf65 inactivation (see above) [26], reduced ATP levels could trigger the AMPK-mediated mTOR pathway inhibition and ULK1 phosphorylation and thus induction of autophagy [34,35]. Indeed, our study showed that application of butylphthalide can attenuate the LC3B phenotype in the SPG55 patient-derived broblasts. Therefore, this autophagy induction could represent a compensatory reaction on the accumulation of defective mitochondria. Butylphthalide is a synthetic compound that had been approved by the State Food and Drug Administration of China for the treatment of ischemic stroke in 2002. Interestingly, previous in vivo and in vitro studies have reported that this drug can attenuate neuronal autophagy [36,37]. Although the positive effects of butylphthalide on brain vascular and neurodegenerative diseases has been veri ed in many experiments, its effects on SPG-related pathologies remain to be addressed in detail.

Conclusions
In summary, in the present study we described a typical case of complicated hereditary spastic paraplegia manifesting an early onset of optic atrophy, progressive lower extremity spasticity, and distal peripheral neuropathy, resulting from a nonsense c.394C > T, p.R132* mutation in C12orf65. Active autophagosome formation with increased LC3B puncta was found in the patient's broblasts, and its level was normalized when treated by Dl-3-N-butylphthalide. Thus our study deepens the clinical and molecular characterization of SPG55 and suggests a potential therapeutic approach to treat spastic paraplegia.

Declarations
Ethics approval and consent to participate The study was approved by the Ethics Committee of Shanghai Ninth People's Hospital, School of Medicine, Shanghai Jiao Tong University. Written informed consent was obtained from the parents to take part in this study and for possible publication. A copy of the written consent is available for review by the Editor of this journal.

Consent for publication
Written informed consents for publication of clinical details and clinical images were obtained from participants Availability of data and material The dataset used and analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors report no con icts of interest.