SCAR32: Functional characterization and expansion of the clinical‐genetic spectrum

Abstract Objective Biallelic mutations in PRDX3 have been linked to autosomal recessive spinocerebellar ataxia type 32. In this study, which aims to contribute to the growing body of knowledge on this rare disease, we identified two unrelated patients with mutations in PRDX3. We explored the impact of PRDX3 mutation in patient skin fibroblasts and the role of the gene in neurodevelopment. Methods We performed trio exome sequencing that identified mutations in PRDX3 in two unrelated patients. We also performed functional studies in patient skin fibroblasts and generated a “crispant” zebrafish (Danio rerio) model to investigate the role of the gene during nervous system development. Results Our study reports two additional patients. Patient 1 is a 19‐year‐old male who showed a novel homozygous c.525_535delGTTAGAAGGTT (p. Leu176TrpfsTer11) mutation as the genetic cause of cerebellar ataxia. Patient 2 is a 20‐year‐old male who was found to present the known c.425C>G/p. Ala142Gly variant in compound heterozygosity with the p. Leu176TrpfsTer11 one. While the fibroblast model failed to recapitulate the pathological features associated with PRDX3 loss of function, our functional characterization of the prdx3 zebrafish model revealed motor defects, increased susceptibility to reactive oxygen species‐triggered apoptosis, and an impaired oxygen consumption rate. Conclusions We identified a new variant, thereby expanding the genetic spectrum of PRDX3‐related disease. We developed a novel zebrafish model to investigate the consequences of prdx3 depletion on neurodevelopment and thus offered a potential new tool for identifying new treatment opportunities.


Background
The term hereditary ataxia (HA) refers to a highly heterogeneous group of disorders phenotypically characterized by progressive damage to the cerebellum and/or its associated afferent tracts. 1,2Collectively, these disorders have an estimated prevalence of 26/100,000 children. 3 Although more than 200 genes have been associated with different types of HA, many patients remain undiagnosed, particularly if they are simplex cases with a silent family history.Among the key molecular "hubs" underlying HA, oxidative stress and mitochondrial dysfunction emerge as common themes.When the balance between cellular oxidants and antioxidants is shifted in favor of the former, the cells tend to overproduce reactive oxygen species (ROS).This leads to mitochondrial damage, impaired oxidative phosphorylation, altered membrane permeability, and alterations of Ca 2+ homeostasis. 4All these changes amplify motor and cognitive dysfunction and accelerate the development of neurodegeneration. 5,6The role of deregulated expression or activity of antioxidant enzymes in neurodegeneration has recently received considerable attention.Peroxiredoxins (PRDXs) are members of a conserved peroxidase family, and they maintain ª 2024 The Authors.Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association.This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.intracellular ROS homeostasis. 7In mammals, six distinct PRDXs have been identified (PRDX1-PRDX6) and all contain conserved reactive cysteine residues in the active site essential for their antioxidant activity. 8An increasing number of studies suggests that these enzymes may be involved in the process of neurodegeneration. 7Among the PRDX proteins, PRDX3, which appears to be localized only in neuronal mitochondria, 8,9 is considered a master regulator of mitochondrial quality control, regulating both ROS removal and mitophagy in mitochondria. 103][14][15][16] In vitro knockdown of PRDX3 results in reduced levels of glutathione peroxidase activity and increased susceptibility to apoptosis triggered by ROS. 11The fruit fly model of the disease showed a deficit in locomotor activity and reduced survival times upon exposure to oxidative stress, 11 whereas a knockout murine model (Prdx3 À/À ) displayed reduced mitochondrial DNA content and ATP production, as well as impaired glucose tolerance and insulin resistance, 17 suggesting that the gene product plays an important role in multiple processes.The early manifestations of impaired motricity and cognition suggest that PRDX3 plays a role in neurodevelopment.However, to date, no model for studying this aspect has been available.Danio rerio (the zebrafish) is a valuable organism for studying several human diseases including the different forms of HA, [18][19][20] but until now there has existed no zebrafish model for investigating prdx3 functions during neurodevelopment.Here, we describe a new biallelic pathogenic variant in PRDX3 occurring in two unrelated boys who presented an early onset, slowly progressive cerebellar syndrome, external ophthalmoplegia, global hypokinesia, and behavioral changes.We further explored the impact of the gene mutation in patient dermal fibroblasts, as well as the effects of depletion of the gene in zebrafish, in the latter case by designing the "crispant" model that displays stable gene downregulation 21,22 without off-target or toxic effects on embryonic development that could mask the phenotype.Our findings provide additional evidence supporting the involvement of PRDX3 in neurodevelopment.

Cellular model
The cellular study methods are reported as Supplementary Material.
prdx3 zebrafish model Downregulation of gene function in zebrafish was carried out using a zebrafish wild-type strain (WT AB), adopting standard breeding conditions. 23 and S2).This in silico analysis revealed high percentage of identity for all prdx protein family members among fish and human, indicating that peroxiredoxins are highly evolutionary conserved across different species.In particular, we found a high conservation of the prdx3 protein with 70% identity between the human (ENST00000 298510.4)and zebrafish (ENSDART00000041700.9) sequences, suggesting that prdx3 can be considered the fish ortholog of the human counterpart with a conserved putative function (see Fig. S1).
We generated a prdx3 knockdown in zebrafish injecting single-guide RNA (sgRNAs) together with Cas9 protein in single-cell stage embryos (100 pg of sgRNAs and 100 pg of Cas9). 22The single-guide RNAs (sgRNAs) were chosen from the list of the five best targets identified by CHOP-CHOP software (https://chopchop.cbu.uib.no/),all found to have zero off-target effects, and to be complementary to prdx3 in facilitating mRNA degradation.DNA was extracted from embryos at 24 hours postfertilization (hpf), and CRISPR/Cas9 editing efficacy was confirmed by Sanger sequencing.The toxic effect after sgRNA injection was calculated by counting dead and alive embryos at 48 hpf.Behavioral evaluations focused on coiling activity, touch-evoked escape responses, and locomotor tracking, and were performed using previously described protocols. 22,24Total RNA was extracted from 20 embryos, at different stages, using Quick RNA miniprep (Zymo Research, Irvine, CA, USA) according to the manufacturer's instructions.cDNA and qRT-PCR were performed as described by Naef et al 25 Relative expression levels of each gene were calculated using the 2 ÀΔΔCt method. 26The results obtained in at least three independent experiments were normalized to the expression of the housekeeping gene, b-actin (ENSDARG00000037746).To evaluate intracellular ROS production, the in vivo carboxy-H2DCFDA fluorescent probe (#8206004, Abcam, Cambridge, MA) was used as reported by Naef et al 25 Mitochondrial respiration was analyzed in wild-type and prdx3-deficient ("crispant") larvae at 120 hpf using the XFe24 analyzer (Seahorse Bioscience, North Billerica, MA) as described by Rollwitz and Jastroch. 27Apoptotic cells from larvae at 24 hpf were detected by staining with acridine orange (#235474, Sigma-Aldrich, St. Louis, MO) as described by Xia et al 28

Case description
Patient 1 is a 19-year-old male admitted to our neurology department with imbalance and clumsiness present since the age of 10 years, when he also started to manifest introversion, social isolation, and academic decline.With the exception of a mild language delay, his developmental milestones were reached on time.The family history was unremarkable for neurological disease.Neurological examination showed a wide-based gait with positive Romberg's test, slurred speech, and global hypokinesia.Tandem gait was not possible.He also presented upper and lower limb dysmetria, slight upper limb postural tremor, and hyporeflexia.Saccades were limited in the horizontal and vertical planes and associated with gaze-evoked nystagmus.No pyramidal signs were detected.His Scale for the Assessment and Rating of Ataxia (SARA) score was 7/40.Extensive biochemical and neurometabolic investigations were all negative.Motor and sensory conduction studies were also normal.Brain magnetic resonance imaging (MRI) showed diffuse cerebellar atrophy associated with T2-weighted hyperintensity of the middle cerebellar peduncles.An extensive neuropsychological evaluation revealed global cognitive functioning within normal limits.
Patient 2 is 23-year-old male born at term to healthy, unrelated parents.The family history was remarkable due to the patient's elder brother having intellectual disability and absence epilepsy since the age of 5 and psychiatric manifestations since his late teens.The proband had delayed developmental milestones with a mild language delay and learning difficulties in childhood.In his early teens, he presented gait ataxia and exercise intolerance.Shortly afterward he began experiencing frequent falls and difficulty climbing stairs, and brain MRI showed cerebellar atrophy.When examined by us at age 20, Patient 2 showed clumsy gait and was found to have a mood disorder.He could walk unsupported but unsteadily and with a wide gait.Tandem gait was not possible.He also presented upper limb dysmetria, slow saccades, and dysarthria.His SARA score was 10/40.According to the caregiver, he had recently developed sluggish hypophonic speech, upper limb rigidity, and occasional difficulty swallowing both solids and liquids.
Having ruled out the most common forms of spinocerebellar ataxia (SCA) and autosomal recessive cerebellar ataxia (ARCA), we performed trio exome sequencing (ES), as reported elsewhere, 29 in both of the cases.In Patient 1, ES revealed a homozygous variant (c.525_535delGTTAGAAGGTT/ p. Leu176TrpfsTer11) in PRDX3 (NM_006793.5)located in the PRX_Tyrp2cys domain of the protein.The parents were healthy carriers of the gene variant not previously reported in gnomAD and classified as "Likely Pathogenic" (PVS1very strong and PM2moderate) according to the American College of Medical Genetics and Genomics (ACMG) guidelines.Patient 2 harbored the c.425C>G/p.Ala142Gly on the paternal allele in compound heterozygosity with the c.525_535delGTTAGAAGGTT on the maternal one.The c.425C>G variant is classified as "Uncertain significance" (PM2-moderate, PP5-supporting) according to ACMG guidelines with a low frequency in gnomAD database (<0.01%), and it predicts a deleterious effect on protein (CADD = 25.6,REVEL = 0.45 scores).This variant has been reported in literature (Rebelo et al. 2021).
A cultured skin fibroblast cell line was available only for Patient 1. RT-PCR amplification study demonstrated low mRNA expression levels as compared to a normal control.Sanger sequencing of cDNA from skin cells showed the c.525_535delGTTAGAAGGTT mutation and also a weakly expressed transcript possibly skipping exon 6 (not shown).These data do not exclude the occurrence of a nonsense-mediated mRNA decay (NMD) mechanism, that still remains a possibility (Rebelo et al. 2021).
Biallelic PRDX3 mutations have recently been described in SCAR32 (OMIM #619862) and associated with impaired oxidative stress defense and mitochondrial dysfunction.Functional analyses in cultured skin fibroblasts derived from Patient 1 did not display bioenergetic changes when compared with results obtained in healthy controls.Calculation of the key parameters of mitochondrial function, performed using the Seahorse XF Cell Mito Stress Test (Fig. 1), showed normal levels of basal respiration, ATP production, maximal respiration, and spare respiratory capacity, as well as normal proton leak and nonmitochondrial oxygen consumption.No significant changes in mitochondrial ROS levels were observed when ROS were measured using the H2DCFDA probe both in regular medium and in the presence of hydrogen peroxide to evaluate the cellular free radical scavenging capacity (Fig. 1).Our data suggest that the presumptive neuronal functions of PRDX3 are not seen in peripheral cell tissues.

In vivo studies in zebrafish
In zebrafish, six peroxiredoxins (prxd1-6) have been identified. 30However, as far we know only prdx1 has fully been characterized in zebrafish sharing the 80% of similar identity with the human protein. 30,31Therefore, we performed multi-alignment analysis of the amino acid sequence of all prdx genes in zebrafish compared to human proteins and observed that all prdx members in zebrafish are the fish orthologs of the human counterpart (Figs.S1 and S2).
Thus, we characterized the prdx3 "crispant" mutant F0 line (Fig. S1) and observed no toxic effects or morphological defects at 48 hpf (Fig. S1).Due to the lack of a reliable specific antibody for zebrafish, it was not possible to test protein abundance in depleted embryos through western blotting.However, qRT-PCR analysis showed a significant decrease in the level of zebrafish prdx3 transcript (Fig. S1).Analysis of tail flicks at 30 hpf in mutant F0 embryos revealed a significant decrease in burst activity (i.e., the percentage of time an embryo is moving) compared with controls (Fig. 2A).Touch-evoked response assay at 48 hpf showed a slight reduction in responses to tail tap in F0 embryos compared with control siblings (Fig. 2B).At 120 hpf, video tracking data revealed significantly reduced locomotor activity in F0 larvae compared with controls, in terms of both velocity and distance covered (Fig. 2C,C 0 ).Fluorescence measurements in mutant zebrafish F0 embryos showed a significant increase in susceptibility to oxidative stress (Fig. 2D,D 0 ), and oxygen consumption rate studies revealed impaired mitochondrial bioenergetics in 120-hpf larvae compared with controls, with significant reductions in ATP production and maximal respiration (Fig. 2E,E 0 ).As already seen in cerebellar medulloblastoma cells, 11 we observed a likely ROS-triggered increment of acridine orange-positive cells (Fig. 2F,F 0 ), as confirmed by the significant increase in transcription levels of typical apoptosis-associated genes 32 (Fig. 2G).

Discussion
In this work, we expanded the allelic heterogeneity associated with PRDX3 pathogenic variants and highlighted the role of prdx3 in zebrafish development.PRDX3 is a mitochondrial peroxiredoxin that plays a crucial role in energy metabolism by detoxifying ROS. 7Several neurological disorders, including different forms of ataxia, are associated with increased levels of free radicals 33,34 since the central nervous system, given its high oxygen demand, is particularly vulnerable to oxidative stress. 35OS are signaling molecules important in maintaining normal tissue homeostasis. 36However, when intracellular ROS concentrations are not tightly controlled and exceed the cellular antioxidant capacity, they can damage all macromolecules, including lipids, proteins, and nucleic acids, leading to defects in their physiological functions. 37,38For this reason, considerable attention has been paid to the role of deregulated expression and activity of antioxidant enzymes in neurodegeneration, 7 and many studies have been conducted to validate the therapeutic roles of antioxidants in ARCA.Biallelic mutations in PRDX3 have recently been reported to cause SCAR32, [11][12][13]39 a rare form of ARCA to date reported in only 15 patients whose clinical features and MRI findings were similar to those of our patient. Ourcase supports the evidence that oculomotor abnormalities occur in SCAR32, and represent, together with cerebellar ataxia, a possible red flag of the condition.To analyze the effect of stable downregulation of prdx3 during neurodevelopment, we developed a new "crispant" prdx3 zebrafish model that showed locomotor deficits, higher ROS production, and bioenergetic defects probably due to alterations in mitochondrial functionality.Altering mitochondrial membrane permeability and structure, membrane potential, and the respiratory chain has the effect of altering apoptosis in a caspase-dependent manner.40 Although apoptosis occurs naturally during neural development, 41 we observed that mutant "crispant" prdx3 embryos displayed an increase in cell death, particularly in the anterior region of the central nervous system.As reported in the prdx3-knockdown fruit fly model, which shows a decreased lifespan, we found that F0 mutant larvae showed increased susceptibility to ROS-triggered apoptosis 11 and significantly enhanced expression of several apoptosis-associated genes.All these findings suggest that the endogenous apoptotic signaling pathway is activated by loss of prdx3-induced oxidative stress in zebrafish larvae.Overall, our study provides novel insights into the consequences of impaired PRDX3.The novel prdx3deficient zebrafish model could help to pave the way for future drug discovery and optimization in mammalian models.

Conclusions
In conclusion, a novel mutation in PRDX3 is here reported as the genetic cause of SCAR32, a disorder whose red flags seem to include cerebellar atrophy, delayed neurodevelopment, and impaired oculomotor function.In addition, we generated a novel vertebrate (zebrafish) model to investigate the consequences of prdx3 depletion on neurodevelopment and thus provided a new tool for investigating, in vivo, the early stages of locomotor impairment.We believe that prdx3-deficient zebrafish embryos might offer a suitable platform for in vivo drug discovery studies and therefore allow preliminary testing of new treatment avenues ahead of more costly studies in mice.

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2024 The Authors.Annals of Clinical and Translational Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association.

Figure 1 .
Figure 1.(A) Kinetic micro-oxygraphy graph depicting mean AE SD of OCR values generated using the XFe24 analyzer (Seahorse Biosciences) under basal conditions and in response to the indicated drugs.Metabolic parameters obtained from OCR traces did not show significant changes in mitochondrial function between the patient and the controls.Data (Ctrls: five replicates from three different control cell lines; Pt PRDX3: 10 replicates from the patient cell line; Ctrl group: data (obtained from 31 previously tested control lines) were expressed as pmol O 2 /minute and normalized post assay to Hoechst 33342 intensity, as a function of the number of cells.Micro-oxygraph traces were further normalized as percent of control baseline.Oligo: 2-lM oligomycin; FCCP: 2-lM carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone; Rot/aA: 0.5/0.5-lMrotenone/antimycin A. OCR, oxygen consumption rate.(B) Evaluation of susceptibility to oxidative stress in patient fibroblasts under both regular medium (RM) and stress conditions.Data represent mean AE SD of controls (n = 3) and the patient analyzed in technical triplicate.Statistical analysis performed by ordinary ANOVA test (one-way ANOVA) did not show significant differences between patient and controls.

Figure 2 .
Figure 2. (A) The tail-coiling test results showed a significant decrease in burst activity in prdx3-F0 animals compared with controls (N = 50 per group) (****p ≤ 0.0001, statistics were calculated using the Mann-Whitney U test).(B) Touch-evoked response test analysis showed slightly decreased movement in response to touch stimulus in morphants at 48 hpf compared with controls (statistics were calculated using chi-square tests).(C and C 0 ) Automated analysis of spontaneous motor activity revealed a reduction in swim distance and velocity in mutant F0 larvae at 120 hpf compared with control siblings (N = 200 per group in three independent experiments) (****p < 0.001 and **p < 0.01, statistics were calculated using the Mann-Whitney U test).(D) Representative fluorescence images of ROS generation in zebrafish larvae at 30 hpf (N = 30 per group).(D 0 ) Graphs show the quantitative analysis of fluorescent signals.(*p < 0.05, statistics were calculated using the Mann-Whitney U test).(E and E 0 ) Mitochondrial respiratory analysis of controls (N = 20) and mutant F0 larvae (N = 20) at 120 hpf (*p < 0.05 and **p < 0.01, statistics were calculated using the Mann-Whitney U test).(F and F 0 ) Representative fluorescence images showing the presence, visible to the naked eye, of AO-positive cells in controls and mutant F0 embryos at 30 hpf (lateral views).AO-positive cells were counted in the area defined by the yellow line (****p < 0.001, statistics were calculated using the Mann-Whitney U test).(G) qRT-PCR analysis revealed increases in apoptosis-related gene expression, once the mRNA expression levels had been normalized to b-Actin.Three independent RNA samples from controls and prdx3-F0 larvae at 120 hpf were analyzed (*p ≤ 0.05, statistics were calculated using Student's t-test).AO, acridine orange; N, number of evaluated embryos in total; n.s., not significant; OCR, oxygen consumption rate; ROS, reactive oxygen species.Error bars indicate standard error of the mean.
All data reported in the manuscript come from three or more independent experiments giving similar results.Data were analyzed by applying either parametric or nonparametric methods, depending on the distribution of the response variable in question, as shown by the Shapiro-Wilk test.Statistical analysis used GraphPad Prism 6 software; data were plotted as the mean AE standard error of the means.Statistical significance is reported as follows: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, or ****p ≤ 0.0001.