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Exenatide induces frataxin expression and improves mitochondrial function in Friedreich ataxia.

Friedreich ataxia is an autosomal recessive neurodegenerative disease associated with a high diabetes prevalence. No treatment is available to prevent or delay disease progression. Friedreich ataxia is caused by intronic GAA trinucleotide repeat expansions in the frataxin-encoding FXN gene that reduce frataxin expression, impair iron-sulfur cluster biogenesis, cause oxidative stress, and result in mitochondrial dysfunction and apoptosis. Here we examined the metabolic, neuroprotective and frataxin-inducing effects of glucagon-like-peptide 1 (GLP-1) analogs in in vivo and in vitro models and in Friedreich ataxia patients. The GLP-1 analog exenatide improved glucose homeostasis of frataxin-deficient mice through enhanced insulin content and secretion in pancreatic β-cells. Exenatide induced frataxin and iron-sulfur cluster-containing proteins in β-cells and brain, and was protective to sensory neurons in dorsal root ganglia. GLP-1 analogs also induced frataxin expression, reduced oxidative stress and improved mitochondrial function in Friedreich ataxia patients' induced pluripotent stem cell-derived β-cells and sensory neurons. The frataxin-inducing effect of exenatide was confirmed in a pilot trial in Friedreich ataxia patients, showing modest frataxin induction in platelets over a 5-week treatment course. Taken together, GLP-1 analogs improve mitochondrial function in frataxin-deficient cells and induce frataxin expression. Our findings identify incretin receptors as a therapeutic target in Friedreich ataxia.


Introduction
Friedreich ataxia (FRDA) is an autosomal recessive neurodegenerative disease with a prevalence of 1/30,000 in Caucasians. Most patients are homozygous for a GAA trinucleotide repeat expansion in the first intron of the frataxin-encoding FXN gene; a few are compound heterozygotes for an expanded GAA repeat and a FXN loss-of-function mutation (1). Most normal FXN alleles have [8][9] repeats, a few up to 30-35, while expanded alleles contain from 70 to over 1700 repeats that interfere with FXN transcription by heterochromatin silencing (2)(3)(4). Longer repeats lead to more severe repression of frataxin expression (65-95% decreased compared to healthy controls), such that most residual frataxin in FRDA patients derives from the allele with the shorter GAA repeat (GAA1), the length of which correlates inversely with age of onset and directly with disease severity (1,5,6). The mitochondrial protein frataxin is involved in iron-sulfur cluster (ISC) biogenesis, and reduced frataxin expression leads to impaired function and/or expression of ISC-containing enzymes, iron accumulation in the mitochondrial matrix, oxidative stress and mitochondrial dysfunction (7,8). Besides the neurological manifestations that include progressive gait ataxia, dysarthria, instability, oculomotor abnormalities and loss of proprioception (9,10), most FRDA patients also develop impaired glucose tolerance or diabetes (11)(12)(13) and hypertrophic cardiomyopathy, the latter being the main cause of premature death (6,10,14). Frataxin deficiency causes early loss of large dorsal root ganglia neurons followed by neuronal loss in the cerebellar dentate nucleus and other nervous system regions (15,16), and dysfunction and apoptosis of insulin-producing pancreatic β-cells (13,17). Advances in the understanding of FRDA pathophysiology have so far not translated into treatments to prevent, delay or revert disease manifestations. Antioxidants such as high dose coenzyme Q 10 plus vitamin E or idebenone (18) failed to show efficacy in clinical trials (19)(20)(21). Other approaches tried to enhance frataxin transcription or translation.
Interferon-γ (22), erythropoietin (23) or epigenetic modifiers such as histone deacetylase inhibitors (HDACis) (4,24) showed promising frataxin induction in in vitro and in vivo models, but the former 2 failed to reach endpoints in clinical trials (25,26) and the latter requires pharmacologic optimization to improve efficacy and reduce toxicity (27).
Incretins are gut hormones secreted in response to food intake. Glucagon-like-peptide 1 (GLP-1) is generated by post-translational cleavage of preproglucagon in enteroendocrine L-cells from the ileum and colon, while glucose-dependent insulinotropic polypeptide (GIP) is secreted by duodenal K-cells (28). GLP-1 is also produced by preproglucagon neurons in the brain stem (29). It can be released in response to non-nutrient stimuli including neurotransmitters, neuropeptides and hormones. GLP-1 and GIP have very short half-lives in the circulation (less than 7 min), due to dipeptidyl-peptidase IV (DPP-IV)-mediated degradation. Long-acting GLP-1 analogs, such as exenatide and liraglutide, and dual GLP-1-GIP agonists have been developed to treat type 2 diabetes (30,31). GLP-1 and GIP receptors are expressed on β-cells, as well as in brown fat, heart, kidney and brain (32). GIP and GLP-1 analogs stimulate cAMP formation through binding to their G-protein-coupled receptors, and activate intracellular signaling pathways that enhance insulin synthesis and glucoseinduced insulin secretion and prevent β-cell apoptosis (33-36). Besides these beneficial effects on β-cells, the drugs have cardiovascular (37) and neuroprotective actions (38, 39). We have previously shown that the cAMP inducer forskolin and incretin analogs [D-vitro, by decreasing oxidative stress and inhibiting the mitochondrial pathway of apoptosis (13,17). In the course of these experiments, we serendipitously found that cAMP induction enhances frataxin protein expression in vitro in clonal rat β-cells. Given the potential therapeutic relevance of these findings, the aim of this study was to evaluate the metabolic, neuroprotective and frataxin-inducing effect of exenatide in in vivo and in vitro FRDA models. We also performed an open-label pilot clinical trial with GLP-1 analogs in FRDA patients to assess if the frataxin-inducing effect could be safely obtained in vivo.

Glucose homeostasis in KIKO mice
The frataxin-deficient knockin-knockout (KIKO) mouse has a 230 GAA repeat expansion in one Fxn allele and a Fxn knockout in the other (40). Because this mouse has a mild phenotype (41, 42), we exposed it to metabolic stress of high fat feeding. Wild type (WT) and KIKO mice were randomized to receive regular (10 kcal% fat) or high fat diet (60 kcal% fat). After 15 weeks, mice were again randomized to receive vehicle (white and grey boxes in Overall, the metabolic phenotype of KIKO mice is mild compared to the more severe phenotype of FRDA patients, who are insulin resistant and develop impaired glucose tolerance and diabetes as a consequence of β-cell failure (13).

Exenatide improves glucose tolerance and β-cell function in KIKO mice
We next examined the metabolic impact of 12-week exenatide treatment in KIKO and WT mice (red boxes in Figure

Exenatide enhances insulin secretion and content of frataxin-deficient islets
Mouse islets were isolated at the end of the experiment to examine insulin content and ex vivo insulin secretion. Exenatide tended to enhance basal insulin secretion and insulin content of KIKO mouse islets ( Figure 1L

Exenatide induces frataxin expression in cerebrum and cerebellum of KIKO mice
We previously demonstrated that exenatide, [D-Ala 2 ]-GIP and forskolin protect β-cells from apoptosis (13). In the course of these studies, we unexpectedly observed that these agents upregulate frataxin protein in frataxin-competent clonal rat β-cells

cells
To assess the effect of incretin analogs in a patient-relevant model, we generated and differentiated FRDA patient's induced pluripotent stem cells (iPSCs) into pancreatic βcells using a 7-stage protocol and neurons (see section below). Fibroblasts were obtained from a 26-year-old male FRDA patient with 1180/980 FXN GAA repeats. His symptoms started before age 10 years with gait instability, followed by progressive trunk and limb ataxia and dysarthria. At onset, tendon reflexes were absent and he had bilateral Babinski sign. He was wheelchair bound before age 20; dysarthria, upper limb ataxia and weakness continued to worsen. He developed optic atrophy and severe hearing loss. He had hypertrophic cardiomyopathy but no heart failure or diabetes.  Figure 3A-B). Key differentiation markers followed a pattern consistent with pancreatic, endocrine and β-cell development, reaching mRNA levels that are overall comparable to clonal insulin-producing EndoC-βH1 β-cells and primary human islets (Supplementary Figure 5) and similar to that of control iPSCs differentiating into βcells (data not shown). These iPSC-β-cells had frataxin protein expression levels of around 35% of healthy control cell line HEL115.6 ( Figure 3C). Exenatide treatment mildly induced frataxin expression after 72 hours ( Figure 3C). We next measured NAD(P)H autofluorescence in FRDA iPSC-β-cells, as an indicator of production of metabolic coupling factors by glucose metabolism. Glucose increased NAD(P)H autofluorescence with a larger effect from 0 to 36 mg/dl than from 36 to 360 mg/dl glucose and the mitochondrial uncoupler FCCP (that reoxidizes mitochondrial NADH and NADPH) rapidly reduced NAD(P)H autofluorescence in the aggregates ( Figure 3D), indicating that the iPSC-β-cell mitochondria were metabolically active. NAD(P)H autofluorescence was modestly increased by exenatide ( Figure 3D). High glucose induced insulin secretion in iPSC-β-cells and a more potent induction was seen with glucose+forskolin; insulin secretory function was comparable after exenatide treatment ( Figure 3E).

Incretin analogs induce frataxin protein in FRDA patients' iPSC-derived neurons
We next examined the effects of incretin analogs on frataxin in patient neurons. We used previously characterized iPSCs from 2 FRDA patients, FA135 and FA141 (2 clones of each) that carry 800/600 and 900/400 GAA repeats, respectively (43). The iPSCs were differentiated using a protocol that maintains and expands neural progenitors in the form of neurospheres and generates a mixture of neurons and astrocytes (17,43,44).
At the end of differentiation the cells expressed low but detectable GLP-1 receptor levels (Supplementary Figure 6A). Differentiated cells were then exposed to vehicle, exenatide, The expression of ISC-containing proteins aconitase and NADH dehydrogenase ubiquinone oxidoreductase Fe-S 3 (NDUFS3) is reduced in iPSC-derived neurons from FRDA patients (44). Similar to HDACi 109 (44), exenatide treatment induced aconitase and NDUSF3 expression in FRDA iPSC-neurons ( Figure 4C). Lipoic acid synthase is another ISC-containing enzyme, required for the activity and stabilization of pyruvate dehydrogenase (PDH), oxaloglutarate dehydrogenase (OGDH) and branched-chain ketoacid dehydrogenase, thereby being an essential cofactor for mitochondrial metabolism (45). The lipoic acid-bound forms of PDH and OGDH are reduced in FRDA iPSC-derived neurons (44). Interestingly, exenatide enhanced lipoic acid-containing OGDH and PDH expression ( Figure 4C). Collectively, these results show that exenatide induces frataxin expression and ISC availability in patient neurons.

Exenatide induces frataxin and alleviates oxidative stress and mitochondrial dysfunction in FRDA patients' iPSC-derived sensory neurons
We have previously shown that forskolin, [D-Ala 2 ]-GIP and exenatide protect frataxindeficient β-cells and neurons from apoptosis by decreasing oxidative stress (13,17).
Recent developments in iPSC differentiation make it possible to generate functional sensory neurons using a one-step protocol (46). We implemented this technology to differentiate sensory neurons from FRDA patients' iPSCs FA135 and HEL135, and also used 2 previously characterized control iPSCs, HEL115.6 and HEL46.11 (47,48). At the end of the 8-day differentiation, most cells expressed sensory neuron-specific markers Brn3a, peripherin and ISL1, and neuron-specific cytoskeletal marker β-tubulin III, confirming sensory neuron lineage; expression of these markers was confirmed at the mRNA level ( Figure Figure 7F). The patients' sensory neurons had enhanced oxidative stress compared to control cells, and this was reverted by exenatide treatment ( Figure 5C). By Seahorse assessment of mitochondrial function, patient iPSC-derived sensory neurons had impaired basal respiration, ATP production and maximal respiratory capacity compared to control cells ( Figure 5D-G). Exenatide improved these parameters, pointing to improved mitochondrial function ( Figure 5D-G).
Taken together, we demonstrate that exenatide induces frataxin, reduces oxidative stress and improves mitochondrial function in FRDA iPSC-sensory neurons, supporting the therapeutic potential of GLP-1 analogs in this neurodegenerative disease.

Exenatide increases frataxin protein expression in FRDA patients
To evaluate whether GLP-1 analogs induce frataxin expression in FRDA patients, we performed an open-label pilot trial of 5-week liraglutide or exenatide treatment followed by a 4-week washout ( Figure 6A). The primary objective was to assess the frataxininducing effect of GLP-1 analogs in peripheral blood mononuclear cells (PBMCs) and platelets, as well as drug safety and tolerability; the secondary objective was to evaluate changes in FRDA clinical rating scales. Sixteen participants, none of them known to have diabetes, were enrolled (baseline characteristics in Table 1).
PBMC and platelet frataxin expression has previously been used as an FRDA biomarker No serious adverse events occurred during the study. The most common side effects were nausea and decreased appetite (Table 2), as described for exenatide or liraglutide therapy at the rapidly titrated doses. The GLP-1 analogs led to an average 1.5 kg body weight loss, which was regained after washout (Table 3). In this non-diabetic population of FRDA patients, HbA1c dropped by 0.2% over the 5-week treatment period (Table 3).
There was no change in the neurologic deficit of patients after 5-week exenatide or liraglutide treatment, as assessed by the Scale for the Assessment and Rating of Ataxia, Inventory of Non-Ataxic Symptoms and Activities of Daily Living scales (Table 3).

Discussion
There are currently no disease-modifying therapies for FRDA, and treatment remains symptomatic and supportive. We have previously demonstrated that cAMP induction prevents apoptosis and reduces oxidative stress in frataxin-deficient β-cells and neurons (13,17). In this study, we set out to investigate the therapeutic potential of GLP-1 analogs for FRDA using in vitro and in vivo disease models. The data show that 12-week exenatide treatment in vivo improves β-cell insulin secretory function in KIKO mice, mildly induces frataxin and ISC-containing protein expression in cerebrum and cerebellum and confers sensory neuroprotection in dorsal root ganglia. In vitro in FRDA patients' iPSC-derived neurons and sensory neurons, generated using 2 differentiation protocols, exenatide induces frataxin, ISC availability, reduces oxidative stress and improves mitochondrial function. Based on these promising preclinical findings, we assessed the frataxin-inducing effect in a pilot study in FRDA patients, and showed that 5-week exenatide treatment induces frataxin expression in patients' platelets. We propose that 2 main mechanisms of action underlie the putative beneficial effects of GLP-1 analogs in FRDA, namely improvement of oxidative stress and frataxin induction.
The insulinotropic effect of GLP-1 analogs relies on cAMP-mediated protein kinase A activation that increases insulin biosynthesis and secretion. GLP-1 analogs also have cytoprotective effects that may be beneficial for frataxin-deficient cells: they improve mitochondrial function, reduce expression of the pro-apoptotic thioredoxin-interacting protein Txnip, inhibit c-Jun N-terminal kinase, activate protein kinase A and exchange protein activated by cAMP-dependent signaling, leading to enhanced expression of the antioxidant enzymes glutathione peroxidase, manganese superoxide dismutase and catalase and thereby reducing oxidative stress (52)(53)(54)(55)(56)(57). The metabolic studies in KIKO and WT mice showed that frataxin-deficient animals have greater responses to GLP-1 analogs, with clearer improvement in β-cell function (Figure 1 and Supplementary Figure   1). Frataxin-deficient mice might have a better response to GLP-1 analogs through alleviation of oxidative stress, which is present in FRDA patients and frataxin-deficient βcells and neurons, and contributes to cellular dysfunction and death (17,58,59).
Prolonged oxidative stress reduces insulin gene expression and insulin content and causes β-cell dysfunction (60,61). Our data indicate that high fat feeding reduces insulin content, and this was partially restored by exenatide in KIKO mice (Supplementary Figure 1M). We have previously demonstrated that forskolin improves mitochondrial oxidative status in frataxin-deficient β-cells (17) and show here that exenatide alleviates oxidative stress in FRDA patients' iPSC-derived sensory neurons ( Figure 5).
A beneficial impact of GLP-1 analogs was reported in a model of Wolfram syndrome 2, an autosomal recessive disease caused by mitochondrial membrane protein NAF-1 deficiency (62). NAF-1 is involved in ISC transfer into apoproteins. Its deficiency in βcells leads to mitochondrial iron accumulation, mitochondrial dysfunction, oxidative stress, and reduced insulin secretion and content, defects that were partially corrected by exenatide (62). Mitochondrial iron accumulation is a hallmark of FRDA (63), but whether GLP-1 analogs improve this remains to be determined. GLP-1 analogs increase β-cell antioxidant capacity by inducing NF-E2-related factor (NRF2), a master regulator of redox homeostasis, and its target glutathione reductase (56). NRF2 signaling is defective in frataxin deficiency models and in FRDA patients (64)(65)(66), and NRF2 inducers have been proposed as neuroprotective drugs for FRDA (67). GLP-1 analogmediated NRF2 induction may be beneficial for frataxin-deficient β-cells and neurons.
The present data also show that cAMP induction by exenatide, forskolin and GIP induces frataxin protein expression in rat, mouse and human cells without affecting frataxin mRNA (Figures 2-6 and Supplementary Figures 3, 6-7). Exenatide has been shown to enhance selected protein synthesis in mouse pancreas without modifying gene transcription (68); the mechanism(s) remains to be elucidated. The frataxin induction by cAMP inducers, even if modest, may be sufficient to beneficially impact FRDA pathophysiology and hence be clinically relevant. Indeed, in FRDA patients higher frataxin protein levels associate with less severe phenotype, and heterozygous carriers of a FXN GAA repeat expansion, who have half of normal frataxin levels (range 30-80%), remain asymptomatic (69). In in vitro and in vivo rodent models, GLP-1 analogs induced expression of frataxin and its downstream targets. The same effects were seen in FRDA patient iPSC-derived sensory neurons, together with improved mitochondrial function. Exenatide administration to FRDA patients led to modest platelet frataxin protein induction ( Figure 6). The response in this pilot study was heterogeneous, with a hint to better responses occurring in patients with larger GAA1 repeat expansions (i.e. less frataxin expression and more severe disease). The study was designed nor powered to assess this, but future incretin analog trials could evaluate whether GAA repeat size predicts therapeutic response, as this might guide patient stratification.
Some of the variability in the data may be related to the sensitivity of frataxin protein assay methods. The study was not designed to assess beneficial effects of GLP-1 analogs on neurological manifestations of FRDA, as it was very short and did not include a placebo group. Besides the well-known gastrointestinal side effects, there were no adverse events, suggesting that these molecules can safely be tested in larger trials.
The choice of an incretin analog to treat FRDA should take into consideration the drug's crossing of the blood-brain-barrier. Several reports describe that exenatide and GLP-1 analogs with higher molecular weight (through linkage with fatty acids and/or carrier proteins) can directly access the brain, especially the hypothalamus (70,71). The magnitude seems, however, limited from liraglutide levels measured in cerebrospinal fluid (72). More data are needed to assess this for different GLP-1 analogs. Through inhibitory effects on inflammation and apoptosis, GLP-1 and/or GIP analogs have been shown to confer neuroprotection in traumatic and neurodegenerative Parkinson's and Alzheimer's disease (73,74).
Altogether, our preclinical and clinical data indicate that GLP-1 analogs are beneficial for frataxin-deficient β-cells and neurons. They improve β-cell function, reduce oxidative stress and improve mitochondrial function of FRDA patient sensory neurons, possibly, but not exclusively, through a frataxin-inducing effect. These data provide a strong rationale for the design of a long-term clinical trial to assess the disease-modifying effect of GLP-1 analogs in FRDA patients.

Metabolic studies in mice
IPGTTs and ITTs were done as described (75). For the IPGTT, after a 16-hour fast 2 mg glucose/g body weight was administered and glycemia measured (glucometer Accu-Chek Aviva Nano, Roche) before and after 15, 30, 60, 90 and 120 min. Blood was collected from the tail vein and plasma insulin measured using ultrasensitive mouse insulin ELISA (Crystal Chem). Glucose tolerance was calculated as the area under the glycemic curve (AUC) from 0-120 min. For the ITT, 4-hour fasted mice were given insulin IP (0.75 mU Actrapid/g weight, Novo Nordisk) and glycemia was measured before and after 15, 30, 60, 90 and 120 min. Insulin sensitivity was calculated as the area over the glycemic curve (AOC) from 0-30 min in the ITT and by HOMA-IR (fasting glucose (mg/dl) x fasting insulin (µU/ml)/405). Insulinogenic index was calculated as incremental insulin (ΔI) over incremental glucose (ΔG) in the first 15 min of IPGTT. β-cell function was calculated as insulinogenic index x insulin sensitivity measure ITT AOC.

Mouse islet isolation and ex vivo glucose-induced insulin secretion
Islets were isolated by injecting Medium 199 (Gibco) containing 1 mg/ml collagenase from Clostridium histolyticum (Sigma-Aldrich) into the bile duct.  (47) with small modifications. Briefly, iPSCs were plated at high density (3x10 6
The differentiation was continued in microwells. Between day 0 and 15 of differentiation medium was freshly prepared and changed daily; from day 16 until the end of differentiation medium was refreshed every second day.

Protein extraction
Total protein was extracted from mouse tissues and iPSC-neurons in RIPA buffer (Sigma) containing protease inhibitors (cOmplete, EDTA-free protease inhibitor cocktail, Roche). Tissues were homogenized using T10 basic Ultra-Turrax disperser (IKA).
Mouse islets and iPSC-neurons were sonicated in Bioruptor NGS (Diagenode). Total extracts were clarified by centrifugation (14,000 g for 10 min at 4°C) and protein quantified by BCA method (ThermoFisher).  Table 1. Treatment: Participants were assigned to liraglutide or exenatide. There was no formal randomization process. During the first week, the liraglutide group received a starting dose of 0.6 mg liraglutide once daily. This dose was increased to 1.2 mg/kg during the following two weeks, and 1.8 mg/kg for the last 2 treatment weeks. The exenatide group received 5 µg exenatide twice daily during the first week of treatment and the standard dose of 10 µg twice daily for the following 4 weeks.

PBMC and platelet sample preparation: Blood from FRDA patients and 2 control
individuals was collected in sodium citrate tubes (BD Biosciences) and PBMCs and platelets were purified by differential centrifugation and Ficoll gradient. For protein extraction, cells were lysed by sonication in ice-cold MitoSciences extraction buffer (Abcam) with protease inhibitors (cOmplete). Cell extracts were cleared by centrifugation at 14,000 g for 20 min at 4°C. Supernatant was stored at -80°C until frataxin protein quantification.

Frataxin protein quantification
MitoSciences dipstick assay (Abcam) was used to measure frataxin protein in PBMCs and platelets. 10 µg total protein from PBMCs and 15 µg from platelets were used; dipsticks were quantified with Hamamatsu immunochromato MS1000 Dipstick reader.
Human and mouse frataxin ELISA (Abcam) was performed according to manufacturer's instructions. Samples were diluted in lysis buffer provided with the kit, and equal sample volumes used in the assay. Frataxin protein was normalized to total protein.

Immunofluorescence
Cells were fixed in 4% formaldehyde for 15-20 min, permeabilized with 0.5% triton-X100 for 10 min, blocked with UltraV block (ThermoFisher) for 8-10 min and incubated with primary antibodies (Supplementary Table 1) diluted in 0.1% Tween in PBS overnight at 4°C. Following incubation with secondary antibodies for 30 min at room temperature samples were mounted with Vectashield with DAPI (Vector Laboratories) and covered with glass coverslips.

Detection of intracellular reactive oxygen species
Reactive oxygen species were measured with the oxidation-sensitive fluorescent probe hydroxyphenyl fluorescein (HPF) (ThermoFisher) in cells cultured in black-bottom plates.
Cells were incubated with 10 µM HPF for 30 min, washed twice with PBS and fluorescence was measured in VICTOR multilabel plate reader (PerkinElmer) using excitation and emission spectra of 485 and 535 nm.

Mitochondrial respiration
Oxygen consumption rates of sensory neurons were measured using XFp Extracellular Flux Analyzer (Seahorse Bioscience). Cells were preincubated in Seahorse medium at pH 7.4 containing 7 mg/ml glucose and 2 mM Glutamax for 1 hour at 37°C in a non-CO 2 incubator. Mitochondrial respiration was measured basally and after injection of 1.5 µM oligomycin, 2 µM carbonyl cyanide-p-trifluoromethoxy-phenylhydrazone (FCCP), and 1 µM rotenone plus 1 µM antimycin A. Data were normalized to total protein. Basal respiration was calculated by subtracting non-mitochondrial respiration from the last measurement before oligomycin injection, ATP production by subtracting the minimum measurement after oligomycin injection from the last baseline measurement, and maximal respiration by subtracting non-mitochondrial respiration from maximum measurement after FCCP injection.

mRNA extraction and real-time PCR
Total RNA from tissues and cells was extracted using RNeasy Plus Micro Kit (Qiagen). mRNA was reverse transcribed as described (17) and assessed by real-time PCR using Rotor-Gene SyBR Green on Rotor-Gene Q cycler (Qiagen), or Q SYBR Green Supermix (BIO-RAD) on MyiQ2 instrument Single Color (BIO-RAD). Gene expression was calculated as copies/µl using the standard curve approach. Standards were prepared in conventional PCR. Expression was corrected for the geometric mean of reference genes GAPDH and β-actin. Primer sequences are described in Supplementary Tables 2 and 3.

Insulin secretion and NAD(P)H autofluorescence in iPSC-derived β-cells
For insulin secretion, 40 iPSC-β-cell aggregates per condition were washed in glucosefree Krebs buffer (Univercell-Biosolutions), 30-min pre-incubated in 30 mg/dl glucose and then incubated at low (30 mg/dl), high glucose (300 mg/dl) and high glucose plus forskolin (10 µM) for 1 hour. Insulin released to supernatant and insulin content were analyzed by human insulin ELISA (Mercodia) and normalized to total protein (Bio-Rad protein assay). For NAD(P)H measurements, aggregates were incubated for 2 hours in glucose-free bicarbonate-buffered Krebs and then perifused (flow rate ~1 ml/min) with Krebs containing 0-360 mg/dl glucose. NAD(P)H autofluorescence was recorded by microspectrofluorometry in 6 perifused aggregates as described (79). Data were normalized to fluorescence levels measured 15 min after addition of 10 µM FCCP.

Statistical analysis
Data are shown using boxplots, with the median represented by horizontal line and 25 th and 75 th percentiles at the bottom and top of the box. Comparisons between groups were performed by Kruskal-Wallis test followed by Benjamini, Krieger and Yekutieli correction for multiple comparisons, unpaired t-test followed by Holm-Sidak correction for multiple comparisons, or ANOVA followed by two-sided unpaired or paired t-test with Bonferroni or Tukey correction for multiple comparisons. p<0.05 or q<0.05 were considered statistically significant. Human pancreata not suitable for clinical purposes were collected from non-diabetic brain-dead organ donors after obtaining written informed consent from next-of-kin, and handled as described (80) with the approval of the Ethical Committee in Pisa.         Participants 01-09 were in the liraglutide arm and participants 11-17 in the exenatide arm. F denotes female, M male; GAA indicates the GAA repeat expansion number of the smaller and larger FXN allele; BP blood pressure, chol cholesterol.   Participants 01-09 were in the liraglutide arm and participants 11-17 in the exenatide arm. BMI body mass index, C-pep C-peptide, SARA Scale for the Assessment and Rating of Ataxia, INAS Inventory of Non-Ataxic Symptoms, ADL Activities of Daily Living scales, NA data not available due to patient dropout. *p<0.05, **p<0.01 vs baseline, ## p<0.01 vs end of treatment.