Retina-Specific Loss of Ikbkap/Elp1 Causes Mitochondrial Dysfunction

RESEARCH ARTICLE Retina-specific loss of Ikbkap/Elp1 causes mitochondrial dysfunction that leads to selective retinal ganglion cell degeneration in a mouse model of familial dysautonomia Yumi Ueki*, Veronika Shchepetkina and Frances Lefcort

ABSTRACT tissue-specific exon skipping and generates an unstable mRNA, Familial dysautonomia (FD) is an autosomal recessive disorder causing a loss-of-function phenotype predominantly in the nervous marked by developmental and progressive neuropathies. It is system (Cuajungco et al., 2003; Dietrich et al., 2012; Keren et al., caused by an intronic point-mutation in the IKBKAP/ELP1 gene, 2010). FD patients suffer from congenital and progressive neuropathies, which encodes the inhibitor of κB kinase complex-associated protein including reduced peripheral afferent sensory function, unstable (IKAP, also called ELP1), a component of the elongator complex. blood pressure, hypotonia, poor growth and spinal curvature; Owing to variation in tissue-specific splicing, the mutation primarily patients often die in early adulthood owing to sudden unexpected affects the nervous system. One of the most debilitating hallmarks of death during sleep (Axelrod, 2002; Palma et al., 2014, 2017; Riley FD that affects patients’ quality of life is progressive blindness. To et al., 1949). The complete functional repertoire of IKAP/ELP1 determine the pathophysiological mechanisms that are triggered by remains unresolved, but includes a key role as the scaffolding the absence of IKAP in the retina, we generated retina-specific subunit of the six-subunit elongator complex (ELP1-6) that modifies Ikbkap conditional knockout (CKO) mice using Pax6-Cre,which wobble uridine subunits of tRNA during translation (Bauer and abolished Ikbkap expression in all cell types of the retina. Although Hermand, 2012; Chen et al., 2009a; Huang et al., 2005). In its sensory and autonomic neuropathies in FD are known to be absence, translation of codon-biased mRNAs is impaired, resulting developmental in origin, the loss of IKAP in the retina did not affect in perturbations in levels of specific proteins (Goffena et al., 2018). its development, demonstrating that IKAP is not required for retinal Either as a direct or indirect consequence of this altered translation, Ikbkap development. The loss of IKAP caused progressive degeneration of conditional knockout (CKO) neurons exhibit impaired retinal ganglion cells (RGCs) by 1 month of age. Mitochondrial axonal transport, target innervation and cell survival (Abashidze membrane integrity was breached in RGCs, and later in other retinal et al., 2014; Chaverra et al., 2017; Close et al., 2006; George et al., neurons. In Ikbkap CKO retinas, mitochondria were depolarized, and 2013; Hunnicutt et al., 2012; Jackson et al., 2014; Johansen et al., 2008; complex I function and ATP were significantly reduced. Although Lefler et al., 2015; Naftelberg et al., 2016; Naumanen et al., 2008; mitochondrial impairment was detected in all Ikbkap-deficient retinal Tourtellotte, 2016; Ueki et al., 2016). neurons, RGCs were the only cell type to degenerate; the survival of FD is classified as a hereditary sensory and autonomic neuropathy other retinal neurons was unaffected. This retina-specific FD model (HSAN III), yet closer examination of both the patient and mouse- is a useful in vivo model for testing potential therapeutics for model phenotypes reveals central nervous system (CNS) pathology mitigating blindness in FD. Moreover, our data indicate that RGCs (Axelrod et al., 2010; Mendoza-Santiesteban et al., 2014; Ochoa, and mitochondria are promising targets. 2003). Furthermore, accumulating evidence demonstrates a vital role of the elongator complex in the CNS: variants in ELP2 are KEY WORDS: IKAP/ELP1, Familial dysautonomia, Mitochondria, associated with neurodevelopmental disability (Cohen et al., Retinal degeneration, Retinal ganglion cells 2015; Franićet al., 2015) and variants in ELP3 with amyotrophic lateral sclerosis (ALS) (Simpson et al., 2009). In addition, loss of INTRODUCTION ELP4 causes Rolandic epilepsy syndrome, autism and intellectual Familial dysautonomia (FD; MIM223900) is an autosomal recessive disability (Addis et al., 2015; Gkampeta et al., 2014; Nguyen congenital neuropathy that is caused by an intronic mutation in the et al., 2010; Reinthaler et al., 2014; Strug et al., 2009). One of the IKBKAP gene, which encodes the inhibitor of κB kinase complex- major clinical hallmarks of FD is progressive blindness, which associated protein (IKAP), also called elongator complex protein 1 starts at an early age as a result of the progressive loss of retinal (ELP1) (Anderson et al., 2001; Dong et al., 2002; Riley et al., 1949; ganglion cells (RGCs) (Mendoza-Santiesteban et al., 2014, 2012, Slaugenhaupt et al., 2001). A point-mutation in intron 20 results in 2017). Patients are often legally blind by their thirties. In the FD community, there is mounting interest in developing treatments to ameliorate the blindness by preventing the progressive RGC loss Department of Cell Biology and Neuroscience, Montana State University, Bozeman, in order to improve the quality of life of FD patients. MT 59717, USA. The pathophysiological mechanisms underlying the loss of *Author for correspondence ([email protected]) vision have not been the focus of any study until recently, however. Our recent work with Ikbkap CKO mice, which lack Ikbkap in both Y.U., 0000-0003-2898-3267; V.S., 0000-0003-0948-3689 CNS and peripheral nervous system (PNS) neurons, demonstrated This is an Open Access article distributed under the terms of the Creative Commons Attribution that loss of Ikbkap in RGCs causes their progressive death (Ueki License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. et al., 2016), recapitulating the retinal phenotype of the FD patients (Mendoza-Santiesteban et al., 2014, 2017). Unfortunately, with

Received 22 January 2018; Accepted 12 June 2018 this model we were unable to analyze the consequence of Ikbkap Disease Models & Mechanisms

1 RESEARCH ARTICLE Disease Models & Mechanisms (2018) 11, dmm033746. doi:10.1242/dmm.033746 loss in cell types other than RGCs, as the Ikbkap deletion was RESULTS primarily restricted to RGCs in the retina. Moreover, Ikbkap was IKAP expression is significantly reduced in the Ikbkap CKO deletedinthemajorityofcelltypesinboththePNSandCNS,sowe retina were unable to determine whether the loss of RGCs was the direct We previously generated Ikbkap CKO mice using a TUBA1a or indirect consequence of loss of Ikbkap throughout the nervous promoter-driven Cre (Tuba1α-Cre), which targets ∼90% of system. These mice had a severe progressive peripheral neuropathy postmitotic RGCs but not other cell types in the retina (Ueki and CNS impairments (Chaverra et al., 2017). To overcome these et al., 2016). We demonstrated that loss of IKAP in the RGCs complications, in this study we have generated and characterized a caused a slow, progressive RGC degeneration most severely in new mouse FD retina model by conditional deletion of Ikbkap the temporal retina, later followed by indirect photoreceptor loss solely in the retina, using a retina-specific Pax6-Cre,whichis and complete retinal disorganization. In this model, however, expressed in the retinal progenitors; thus, all retinal cell types are Ikbkap was deleted from the majority of CNS neurons and affected (Marquardt et al., 2001). This retina-specific FD model approximately 40% of PNS neurons; thus, whether all or some system allowed us to directly interrogate the consequence of loss of of these phenotypes were directly due to autonomous loss of Ikbkap in all retinal cell types in the context of an otherwise healthy Ikbkap in retinal cells could not be determined. In this current nervous system. Our data demonstrate that loss of Ikbkap solely in the study, we have generated a retinal-specific Ikbkap CKO mouse retina causes RGC degeneration, yet the survival of other retinal cell using Pax6-Cre. Pax6-Cre is expressed in retinal progenitors, types is unaffected. In addition, there was no developmental phenotype affecting all six types of retinal neurons and Müller glia, and its in the FD retinas, establishing for the first time that the loss of RGCs expression is restricted solely to the retina (Marquardt et al., occurs postnatally and is a progressive neurodegeneration as in the 2001). Therefore, we can characterize the effect of autonomous human disease. loss of Ikbkap not only in RGCs, but in all retinal neurons. To Defects in mitochondria have been implicated in many, if not identify appropriate therapeutic targets, it is important to all, neurodegenerative disorders (Schon and Przedborski, 2011). understand the consequence of IKAP loss in all retinal neurons Similar to FD, two other optic neuropathies that are considered and not only in RGCs. mitochondrial diseases, Leber’s hereditary optic neuropathy Western blot analysis showed a significant reduction in IKAP (LHON) and dominant optic atrophy (DOA), are also characterized by protein in the Pax6-Cre Ikbkap CKO retinas at 1 month compared loss of vision owing to slow, progressive RGC degeneration with littermate control retinas (Fig. 1A); on average, there was (Carelli et al., 2009; Chevrollier et al., 2008; Kirches, 2011; over 70% reduction in IKAP expression in the CKO retina Newman and Biousse, 2004; Yu-Wai-Man et al., 2011; Zanna (Fig. 1B). Previous studies show that Pax6-Cre is expressed in the et al., 2008). In this study, we hypothesized that the progressive peripheral retina, but not in the central retina (Georgi and Reh, demise of retinal neurons in FD results from mitochondrial 2010; La Torre et al., 2013; Marquardt et al., 2001). We also dysfunction. Our data demonstrate perturbation of mitochondrial observed this peripheral bias when we crossed Pax6-Cre mice membrane integrity and function, as well as decreased ATP with Rosa-EGFP Cre reporter mice (Fig. 1C,D). In a typical Cre+ content in the Ikbkap CKO retinas. Interestingly, although retina, 50-85% retinal regions were GFP+ (Fig. 1C), which mitochondria of all Ikbkap-deficient retinal neurons seem to be corresponds with ∼70% reduction in IKAP expression (Fig. 1A, affected, RGCs are the only cell type that degenerate. Together, B). When we analyzed Cre reporter retinal cross-sections at these results suggest that RGCs and mitochondria are promising 1 month, all the retinal cell types including RGCs [identified using targets for future therapeutics to mitigate the progressive loss of the retinal markers Brn transcription factor (Brn3+) and/or RNA- vision in FD patients. binding protein with multiple splicing (RBPMS+)] expressed GFP

Fig. 1. Generation of a retinal model of FD. Pax6-Cre+;IkbkapFlox/Flox mice were generated to delete Ikbkap selectively from the retina. (A) Representative IKAP western blot at 1 month. (B) Pax6-Cre+;IkbkapFlox/Flox (CKO) retinas show a 70% decrease in IKAP expression compared with Pax6-Cre−;IkbkapFlox/Flox (Control; Ctrl) retinas at 1 month. *P=0.000016 with Student’s t-test (n=5). (C-E) Pax6-Cre expression was analyzed on retinal (C) flatmounts and (D) cross-sections using Rosa-EGFP Cre reporter mice at 1 month. Cre expression was detected in the mid-peripheral retinas and, typically, 50-85% of the retina was affected in each eye. Images in C and D are composites. Multiple images were taken and reconstructed to show whole retinal (C) flatmount and (D) cross sections. (E) Representative images of retinal cross-sections in the Cre-expressing region are shown. All cell types of the retina expressed Cre (GFP+), including RGCs (Brn3+ and/or RBPMS+). GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Disease Models & Mechanisms

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(Fig. 1E), as reported in previous studies (Marquardt et al., 2001). in order to identify the retinal regions that were deficient in We used Pax6-Cre Ikbkap CKO that had been crossed with the IKAP. In summary, we have established a useful mouse model of Rosa-EGFP Cre reporter strain for immunohistological experiments, FD blindness.

Fig. 2. Loss of IKAP in the retina causes progressive loss of RGCs. (A) At 9 months, Pax6-Cre Ikbkap CKO retinas show a decrease in the number of Brn3+ RGCs at 1 mm from the optic nerve. (B) Significant loss of Brn3+ RGCs in CKO retinas was detected in all quadrants (temporal, superior, nasal and inferior) as early as 1 month, and the loss was progressive. Rapid RGC loss occurs between 1 month and 3 months. There was no significant difference in the number of RGCs at P14, when retinal development has completed. P14 (n=4), 1 month (n=6), 3 months (n=4), 6 months (n=6) and 9 months (n=3). The number of RGCs in the control and CKO retinas at the same region and age were compared with Student’s t-test (*P<0.05). (C) Cross-sections of 6 month temporal retinas at 1.5 mm from the optic nerve. There was an apparent reduction in the number of Brn3+ RGCs and total RGCs (RBPMS+, pan-RGC marker). (D) Cross-sections of optic nerves at 1 month (top) and 9 months (bottom) were visualized with DAPI staining. (E) There was no difference in control and CKO optic nerve circumference at 1 month. P=0.41 (not significant) with Student’s t-test (n=3). (F) By 9 months, there is a 45% decrease in CKO optic nerve circumference compared with controls. *P=0.018 with Student’s t-test (n=3). (G,H) There was no loss of melanopsin+ intrinsically photosensitive RGCs at 18 months. Melanopsin+ cells were counted at 1 mm from the optic nerve in each quadrant. P=0.2 (temporal), P=0.3 (superior), P=0.3 (nasal) and P=0.1 (inferior) with

Student’s t-test (n=5). Scale bars: 100 µm (A,D,G) and 50 µm (C). Disease Models & Mechanisms

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Loss of IKAP induces progressive RGC degeneration, Mitochondrial membrane integrity and function is impaired whereas other retinal cell types are unaffected in the absence of IKAP As expected from our previous study (Ueki et al., 2016), we Our recent study revealed that mitochondria of embryonic Ikbkap observed progressive loss of RGCs owing to loss of IKAP in the CKO dorsal root ganglia neurons are depolarized, produce elevated retina. Staining of 9 month retinal flatmounts and 6 month retinal levels of reactive oxygen species (ROS), are fragmented, and do not cross-sections with RGC markers Brn3 (Fig. 2A) and RBPMS aggregate normally at axonal branch points (Ohlen et al., 2017). In (Fig. 2C), respectively, showed an apparent reduction in the RGC support of a mitochondrial deficit in FD, FD patients have a temporal number in all four retinal quadrants. Quantification of RGC number optic nerve degeneration that is reminiscent of LHON and DOA, both reveals a significant loss of RGCs at 1 month, followed by a rapid of which are caused by mutations that affect mitochondrial function reduction in the RGC number over the subsequent 2 months (Carelli et al., 2009; Chevrollier et al., 2008; Kirches, 2011; Newman (Fig. 2B). By 6 months, there was a 40-60% loss of RGCs in CKO and Biousse, 2004; Yu-Wai-Man et al., 2011; Zanna et al., 2008). retinas compared with controls. This progressive loss of RGCs is not Therefore, we investigated the health of mitochondria in our CKO due to Pax6-Cre expression itself or a result of the loss of one Ikbkap retinas. We performed transmission electron microscopy (TEM) on allele, as there was no significant difference in RGC counts in Pax6- control and CKO retinas at 1 month and 2.5 months (Fig. 4 and Fig. S3). Cre+; IkbkapFlox/+ or Pax6-Cre−; IkbkapFlox/Flox (control) retinas at We observed a breakdown in the mitochondrial double-membrane 18 months (Fig. S1). To determine whether IKAP is required in the integrity in CKO RGCs as early as 1 month, which is during the developing retina, we quantified RGC number at postnatal day 14 period of rapid RGC degeneration (Fig. 4). By contrast, although (P14), the age when retinal development has completed. mitochondria of 2.5 month CKO amacrine cells show a similar loss Importantly, there was no significant difference in RGC number of membrane integrity, mitochondria of 1 month CKO amacrine cells between control and CKO retinas at P14, indicating that RGC display no difference compared with controls (Fig. S3). Moreover, development does not require IKAP (Fig. 2B). Thinning of the CKO despite this slower progressive loss of membrane integrity, amacrine optic nerves (RGC axon bundles) appeared to temporally follow cells do not die in the absence of IKAP (Fig. S2A and Fig. 3D,E). RGC degeneration (Fig. 2D). Although there was no difference in This finding suggests that loss of IKAP affects different cell types at the thickness of the optic nerve at 1 month (Fig. 2E), CKO optic different rates, and that RGCs are more vulnerable to the loss of nerves at 9 months were significantly thinner compared with those IKAP than are other retinal cell types. of the controls (Fig. 2F). As observed in FD patients (Mendoza- Given the morphological disruption to mitochondrial membrane Santiesteban et al., 2017), intrinsically photosensitive, melanopsin+ integrity detected in TEM, we sought to characterize membrane RGCs were preserved even at 18 months, despite extensive integrity at the molecular level. We collected retinas at 3 months and degeneration of conventional RGCs (Fig. 2G,H). This observation used an antibody cocktail that measures levels of mitochondrial suggests that loss of IKAP differentially affects RGC subtypes. proteins in four different mitochondrial compartments (outer and We have previously shown that IKAP is expressed in RGCs, inner membrane, intermembrane space and matrix) (Fig. 5). photoreceptors, amacrine cells and a subset of bipolar cells in adult Significant decreases in the mitochondrial proteins cyclophilin D mouse retinas (Ueki et al., 2016). Although the loss of RGCs was and cytochrome c were detected in CKO retinas at 3 months significant by 1 month in the CKO retinas (Fig. 2), histological (12 weeks), supporting the loss of membrane integrity seen in TEM analyses of 1, 3, 6, 9, 12 and 15 month CKO retinas did not show any analyses (Fig. 4 and Fig. S3). Loss of mitochondrial integrity in the additional retinal phenotype nor were any other retinal cell types CKO retinas was progressive: at 2 weeks (P14), the time when retinal affected by IKAP loss (Fig. 3 and Fig. S2). Immunohistochemistry development is completed, there was no difference in cyclophilin D (IHC) with various cell-type markers showed no significant and cytochrome c expression, but both proteins did progressively difference in photoreceptor (Otx2), bipolar cell (Otx2), amacrine decline in expression in older CKO retinas (Fig. 5B,C). These data cell (Pax6) or Müller glial (Sox9, Sox2, GFAP) number, suggest that loss of Ikbkap during development does not affect morphology or organization at 9 months (Fig. S2A). There was no retinal cell mitochondria. Although RGCs showed morphological abnormal morphology in the CKO retinas at 12 months compared mitochondrial abnormality and cell death as early as 1 month with the controls (Fig. S2B). At 15 months, although retinal cross- (Figs 2B and 4), we did not see a significant difference in the sections of the Cre-affected area (middle-peripheral) show decreased mitochondrial protein levels by western blot at 1 month (4 weeks). RGC number, the overall retinal morphology remains intact (Fig. 3A This is probably because RGCs only represent 0.5% of the total and Fig. S2C). When the number of rows of photoreceptors were retinal population (Jeon et al., 1998) so that RGC changes are not counted at 0.25 mm intervals from the optic nerve head, there was no reflected in the total retinal lysates. As retinas aged, however, we did difference in the number of photoreceptors between control and measure a significant difference in whole retinal lysates after 7- CKO retinas (Fig. 3B). In our previous study, Ikbkap−/− CNS 12 weeks. This decrease in mitochondrial proteins in CKO retinas neurons showed disrupted cilia morphology (Chaverra et al., 2017). corresponds well with the morphological changes in mitochondria Therefore, we analyzed photoreceptor cilia, located in between inner observed in other cell types of the retina (Fig. S3). Our western blot and outer segments. A cilia marker, PKD2L-1(red), showed no analysis suggests that the number of total mitochondria in the CKO abnormality in photoreceptor cilia structure in CKO photoreceptors retinas was unchanged, as the expression of mitochondrial loading at 9 months, further indicating that photoreceptors are not affected by control porin/voltage-dependent anion-selective channel protein 1 the absence of Ikbkap. As we also observed loss of cholinergic (VDAC1) was the same between control and CKO retinas (Fig. 5A). neurons in our other FD model (Chaverra et al., 2017), the number of In support of this, IHC analysis of the RGC layer shows comparable ChAT+ cholinergic amacrine cells was counted in both the inner levels of VDAC expression between control and CKO retinas at nuclear layer (INL) and ganglion cell layer (GCL) at 6 months 3 months (Fig. S4), also suggesting that the total retinal mitochondrial (Fig. 3D,E). However, we did not observe any reduction in ChAT+ number is unaffected by the loss of IKAP. amacrine cells in the CKO retina. In summary, we determined that To assess changes in the mitochondrial membrane potential, we RGCs are the only cell type to require IKAP for their survival, even briefly treated freshly isolated 3 month retinal explants with though Pax6-Cre is expressed in all retinal cell types. MitoTracker Red CMXRos, a dye retained by healthy mitochondria Disease Models & Mechanisms

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Fig. 3. Loss of IKAP does not cause degeneration of retinal neurons other than RGCs. (A) H&E staining of 15 month retinal cross-sections at central (0.5 mm from the optic nerve head, ONH), middle (1 mm from ONH) and peripheral (1.5 mm from ONH) retina. Central retinas, where Pax6-Cre is not expressed, do not show any morphological differences between control and CKO mice. In the middle-peripheral retinas of CKO mice, where Pax6-Cre is active, reduction of RGCs in the ganglion cell layer (GCL) is apparent, whereas other cell types of the retina do not display any abnormal number and morphology. (B) The number of rows of photoreceptor nuclei in the outer nuclear layer was counted at 0.25 mm from the ONH in the temporal and nasal retinas at 15 months. There was no difference in the photoreceptor number between control and CKO retinas. The number was compared at the same distance with Student’s t-test (n=6 for control; n=5 for CKO). For all points, P>0.05 (not significant). (C) IHC of cilia marker PKD2L-1(red) shows no abnormality in photoreceptor cilia structure between the inner segment and outer segment at 9 months. (D,E) The number of ChAT+ cholinergic amacrine cells in 6 month retinas was counted at 1 mm from the ONH in each quadrant. Representative images of the temporal inner nuclear layer (INL) and GCL ChAT+ cells (red) are shown (D). There was no difference in the number of ChAT+ cholinergic amacrine cells in either INL or GCL (E). P>0.05 with Student’s t-test for all points (n=5). T, temporal; S, superior; N, nasal; I, inferior. Scale bars: 50 µm (A,C) and 100 µm (D). that have a normal membrane potential. MitoTracker images of the mitochondrial depolarization, we measured mitochondrial complex I CKO RGC layer show a dramatic reduction in fluorescence signal function at 2.5 months. Complex I was chosen because its activity (Fig. 6A,B), indicating a significant loss of mitochondrial membrane was decreased significantly in other mitochondrial optic neuropathies, potential in these cells. To identify the mechanisms underlying the such as LHON (Brown et al., 2000; Carelli et al., 1997). We measured Disease Models & Mechanisms

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Fig. 4. Integrity of the mitochondrial membrane was lost in CKO RGCs. Mitochondria of RGCs were visualized using TEM at (A-C) 1 month and (D-F) 2.5 months. At both 1 and 2.5 months, CKO mitochondria (B,C,E,F) show a disrupted double- membrane structure (arrows) compared with controls (A,D), indicating that loss of IKAP led to morphological impairment of mitochondria in RGCs. Representative images are shown. Asterisks indicate mitochondria. Squared regions in A-F are shown enlarged in A′-F′.

a 22% decrease in complex I function in the CKO retinal mitochondria progressive blindness owing to the loss of RGCs is one of the most compared with controls (Fig. 6C). This would be an underestimate debilitating phenotypes suffered by patients as they age. To determine of complex I reduction in an affected CKO cell, as the central half whether autonomous loss of IKAP function in the retina would of the CKO retinas were not affected by Pax6-Cre-mediated recapitulate the human FD optic neuropathy, we generated a new recombination of Ikbkap and were essentially the same as control mouse model in which the targeted deletion of Ikbkap was restricted retinas (Fig. 1). In support of this loss in complex I activity, the total solely to the retina with Cre expression in all retinal cell types (Fig. 1) cellular ATP concentration per retina was significantly decreased (Marquardt et al., 2001). Pax6-Cre was able to abolish 70% of IKAP (60% decrease) in CKO retinas compared with controls (Fig. 6D). expression in the retina (Fig. 1). Interestingly, Ikbkap CKO retinas As most cellular ATP is generated in mitochondria, the results showed normal retinal development but demonstrated progressive indicate impaired mitochondrial function in the CKO retinas. In RGC degeneration and optic nerve atrophy starting at 1 month of age summary, we demonstrate that there are significant reductions in (Fig. 2), whereas the survival of other retinal neurons was unaffected mitochondrial integrity, membrane potential and function in the (Fig. 3). These findings indicate that not only is IKAP not required for retina in the absence of IKAP. retinal development, but in adulthood only RGCs, and not other retinal cell types, depend on its function for survival. Finally, we DISCUSSION demonstrate here that Ikbkap-deficient retinal neurons, including FD is caused by a point-mutation in IKBKAP/ELP1, a gene that RGCs, exhibit a loss of mitochondrial integrity and function and encodes an elongator complex subunit, IKAP/ELP1. Although FD decreased levels of ATP (Figs 4-6), suggesting that mitochondrial patients suffer from many congenital and progressive neuropathies, dysfunction is one of the causes of RGC death in FD. Disease Models & Mechanisms

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revealed that loss of the RGCs and optic nerve fiber, especially in the maculopapillary region (temporal retina), is the cause of the progressive blindness in FD (Mendoza-Santiesteban et al., 2014, 2017). As also seen in our CKO mouse retinas, survival of retinal neurons other than RGCs is not compromised in the retinas of FD patients. Interestingly, a recent report on postmortem human FD retinas showed degenerating mitochondria in the temporal portion of the optic nerve head pre-laminar region (Mendoza-Santiesteban et al., 2017). This observation corresponds well with our finding that mitochondrial morphology and function are impaired in retinal neurons in the absence of Ikbkap (Figs 4-6). In addition, RGC degeneration was subtype-specific, with melanopsin+ RGCs being resistant to death even in the absence of Ikbkap (Fig. 2), confirming our previous finding in a mouse model in which Ikbkap was deleted from the majority of the CNS and PNS (Ueki et al., 2016). Melanopsin+ RGCs are also preserved in the retinas of human FD patients (Mendoza-Santiesteban et al., 2017). Our previous analysis conducted in an FD mouse model, in which Ikbkap was deleted in the CNS and PNS using a Tuba1α-Cre, also showed progressive RGC degeneration (Chaverra et al., 2017; Ueki et al., 2016). With that model, however, we were unable to ascertain whether the demise of RGC was the direct or indirect consequence of loss of RGC Ikbkap, given that the Tuba1α-Cre Ikbkap CKO model exhibited widespread PNS and CNS neurodegeneration, which could indirectly affect the integrity of the neural retina (Chaverra et al., 2017; Ueki et al., 2016). To overcome these complications, we generated a retina-specific Ikbkap CKO and demonstrate here that RGCs autonomously require Ikbkap for their survival. In summary, our retina-specific Ikbkap CKO is an excellent FD model to study mechanisms of RGC degeneration and to test potential therapeutics in otherwise healthy mice. Why neurons die in the absence of IKAP is not fully resolved, nor have we thoroughly identified its function(s) in neurons. In other systems, loss of IKAP also impairs target innervation, exocytosis, cytoskeletal organization and axonal transport, which might directly Fig. 5. Progressive loss of mitochondrial membrane integrity in the CKO and/or indirectly lead to cell death (Abashidze et al., 2014; Chaverra retinas. Retinas of the indicated ages (2, 4, 7 and 12 weeks) were collected et al., 2017; Close et al., 2006; George et al., 2013; Hunnicutt et al., and western blot analysis performed using the mitochondrial membrane 2012; Jackson et al., 2014; Johansen et al., 2008; Lefler et al., 2015; integrity antibody cocktail. The cocktail included protein markers of the four mitochondrial compartments. (A) Representative blot. There was no difference Naftelberg et al., 2016; Naumanen et al., 2008; Tourtellotte, 2016). in the expression of protein markers of the four mitochondrial compartments at IKAP/ELP1 is the scaffolding subunit of the six-subunit elongator 2 weeks (P14, at the completion of retinal development) between control and complex, which is required for the translation of codon-biased CKO retinas. A significant reduction in cyclophilin D and cytochrome c in CKO genes. The wobble uridine (U34) of tRNAs that recognize both AA- retinas was detected as the retina ages, suggesting progressive disruption in and AG-ending codons is modified by the addition of both a mitochondrial structure. IM, inner membrane; OM, outer membrane; IMS, thiol (s2) and a methoxy-carbonyl-methyl (mcm5). This double intermembrane space. Equal amounts of retinal lysate proteins were loaded. modification enhances the translational efficiency of AA-ending (B) Western blot quantitation for cyclophilin D. There was a progressive decrease in cyclophlin D and a significant decrease in its expression was codons (Bauer and Hermand, 2012; Chen et al., 2009b; Huang et al., detected in CKO retinas at 7 weeks. *P<0.05 with Student’s t-test; 2 weeks 2005). Importantly, these specific wobble uridine modifications in (P=0.26; n=5), 7 weeks (P=0.031; n=3 for control; n=6 for CKO) and 12 weeks tRNA are reduced in FD patients (Karlsborn et al., 2014) and FD (P=0.018; n=4). (C) Significant reduction in cytochrome c expression was mice (Goffena et al., 2018). One important class of mRNAs that we observed in CKO retinas compared with controls by 12 weeks. *P<0.05 with discovered were codon-biased, and hence their proteins were ’ Student s t-test; 2 weeks (P=0.31; n=5), 7 weeks (P=0.080; n=3 for control; expressed at lower levels in Ikbkap CKO peripheral neurons, are n=6 for CKO) and 12 weeks (P=0.016; n=4). those that repair DNA damage (Goffena et al., 2018). Interestingly, mitochondrial DNA can also be damaged in neurodegenerative The retinal phenotype observed in our CKO mice recapitulates disease (Van Houten et al., 2016) and, if unrepaired, can lead to an the human FD pathology. Although in the PNS, IKAP deficiency elevation in ROS and trigger apoptosis. Furthermore, in the clearly affects neuronal development (Abashidze et al., 2014; developing PNS, we have shown that Ikbkap−/− neurons die George et al., 2013; Hunnicutt et al., 2012; Jackson et al., 2014), we through a p53/activated caspase-3-mediated apoptosis (George did not observe any developmental defects in the CKO retinas. This et al., 2013). Whether this death results from the direct or indirect finding suggests that PNS and CNS neurons have different effects of impaired tRNA modification is still unresolved, however. susceptibility to the loss of IKAP, and that there are clearly two Finally, a study in yeast shows that wobble uridine modification by distinct stages of neuronal loss in the disease: developmental versus the elongator complex (Elp1-6) is required for mitochondrial adult progressive degeneration. Analysis of the vision of FD patients function under stress conditions (Karlsborn et al., 2014; Tigano Disease Models & Mechanisms

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Fig. 6. Loss of IKAP causes mitochondrial membrane depolarization, impairs complex I function and decreases cellular ATP levels. (A) Freshly isolated 3-month old retinas were treated briefly with MitoTracker Red CMXRos and Hoechst (nuclear marker), fixed and flatmounted for confocal imaging. Representative images of the RGC layer are shown. The signal intensity of MitoTracker Red CMXRos correlates with healthy mitochondrial membrane potential. Scale bar: 50 µm. (B) ImageJ analysis revealed a 50% decrease in MitoTracker Red CMXRos signal intensity in the CKO RGC layer compared with controls, indicating that there was significant loss of mitochondrial membrane potential in the cells of the RGC layer in CKO mice. *P=0.00026 with Student’s t-test (n=5). (C) Mitochondria of 2.5 month control and CKO retinas were isolated and mitochondrial complex I function was measured. CKO retinas had a 22% reduction in complex I activity compared with controls. *P=0.0025 with Student’s t-test (n=5). (D) Total ATP of the control and CKO retinas was measured from the total retinal lysates at 2.5 months. Total ATP content of the CKO retina was 40% that of the control. *P=0.0053 with Student’s t-test (n=5). et al., 2015). Thus, although it is clear that the loss of IKAP and were not reduced in the Ikbkap CKO retina, ATP levels were reduced elongator function cause intracellular stress, including severe by 60% which is commensurate with the 70% loss of IKAP protein. impairments in mitochondrial morphology and function, What is surprising, however, is that despite Cre being expressed in elucidating the exact cellular and molecular pathways connecting approximately 90% of retinal cells in the peripheral retina, only 40- the loss of elongator function to mitochondrial damage and 60% of the RGCs die and none of the other retinal cell types die. This neuronal death in FD requires future study. is similar to observations made in the embryonic dorsal root ganglia Recently, we demonstrated that mitochondria of Ikbkap CKO where approximately half of the tropomyosin receptor kinase A embryonic PNS neurons were fragmented, had disrupted membrane (TrkA+) nociceptors and thermoreceptor subpopulation die in the potential and had increased ROS (Ohlen et al., 2017). In this new absence of IKAP, yet the TrkC+ subset (which comprise the study, we show that mitochondria of Ikbkap-deficient retinal proprioceptors) do not die. Thus, for reasons we do not currently neurons are also morphologically and functionally impaired understand, different neuronal populations have a differential (Figs 4-6), demonstrating that mitochondrial dysfunction occurs dependency on the IKAP protein for their survival. Although these in both developing and adult neurons in the PNS and CNS in the resilient neurons can survive, they might not be as healthy as Ikbkap+/+ absence of IKAP. Our TEM analysis revealed disruption of cells, as evidenced by the breached mitochondrial morphology in mitochondrial membranes in Ikbkap-deficient retinal neurons several amacrine cells (Fig. S3 and Fig. 3D,E); thus, these neurons (Fig. 4 and Fig. S3). Mitochondrial translocation of the p53 tumor might produce less ATP, which explains the 60% reduction in ATP suppressor protein has been shown to disrupt inner and outer levels observed in the CKO retina. Given the normal cellular mitochondrial membrane integrity (Wolff et al., 2008). In line with variability in ATP production, our measurement of total retinal ATP this, given the elevated p53 activity in PNS neurons (George et al., content would not accurately reflect any changes in ATP 2013), it is possible that p53 might contribute to the breakdown in concentration per single cell. What is intriguing, however, is that mitochondrial membrane integrity in Ikbkap CKO retinal neurons. recent work has demonstrated that, although ATP concentrations in a Furthermore, we demonstrate here that Ikbkap CKO retinas had healthy cell are in the millimolar range, only micromolar decreased complex I activity and ATP levels compared with control concentrations of ATP are required for bioenergetics. By contrast, retinas, which might explain the poor growth, rhabdomyolysis and an intracellular concentration of ATP that is 1000-fold higher than reduced muscle tone observed in FD patients (Axelrod, 2002; Riley required enables it to function as a hydrotrope to keep proteins in et al., 1949). Intriguingly, a muscle biopsy conducted on an FD solution and dissolve protein aggregates that form within the cytosol patient revealed a severe impairment of mitochondrial complex I, III (Patel et al., 2017). As protein aggregation is a common hallmark of and IV activity (A.S., personal communication). Decreased ATP all major neurodegenerative diseases, and has been shown to occur in levels have also been detected in the optic nerve of a mouse glaucoma other elongator mutant systems (Laguesse et al., 2015), it is model, which had experienced RGC degeneration owing to elevated conceivable that the reduction in ATP we demonstrate in Ikbkap−/− intraocular pressure (Baltan et al., 2010). Interestingly, reduced levels neurons might exacerbate intracellular stress by triggering protein of ATP are a common hallmark of the major neurodegenerative aggregation. Hence, these reduced ATP levels might contribute to the disorders, including Alzheimer’s disease, Parkinson’s disease and progressive degeneration of several cell types in FD as patients age, a

ALS (Pathaket al., 2013). Although total mitochondrial protein levels topic to be pursued in future studies. Disease Models & Mechanisms

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Two retinal disorders, LHON and DOA, and FD share remarkable et al., 2014, 2017; Wakakura et al., 2009; Yu-Wai-Man et al., 2014). similarity in their disease progression: all are characterized by loss of In addition, among all the retinal neurons, IKAP expression is vision owing to slow, progressive RGC degeneration (Fig. 2) (Carelli highest in the RGCs (Ueki et al., 2016), suggesting that RGCs might et al., 2009; Chevrollier et al., 2008; Kirches, 2011; Mendoza- be more dependent on normal IKAP function than other retinal Santiesteban et al., 2017; Newman and Biousse, 2004; Ueki et al., neurons. In summary, our data clearly indicate that RGCs are the 2016; Yu-Wai-Man et al., 2011; Zanna et al., 2008). Both LHON cell type to target with potential treatments for FD blindness. and DOA are mitochondrial diseases: the genes that are mutated Whether the loss of mitochondrial integrity and function observed function in mitochondria. LHON is generally caused by a point- in our CKO retinas is a direct cause of RGC death or not remains mutation in one of three mitochondrial genes, all three of which currently unresolved. However, our work on Ikbkap-deficient dorsal encode mitochondrial complex I subunits (Howell et al., 1991; Johns root ganglia (DRG) neurons demonstrates that improvement of et al., 1992; Wallace et al., 1988); the LHON retina has decreased mitochondrial function in FD DRG neurons leads to increased complex I activity and reduced ATP synthesis that results in neuronal survival (Ohlen et al., 2017), suggesting that mitochondria progressive RGC degeneration. In DOA, mitochondrial fusion is are promising targets for preventing RGC loss in FD. Given the impaired. Interestingly, in both LHON and DON, melanopsin- remarkably similar phenotypes between FD, LHON and DOA, containing RGCs are spared, despite the extensive degeneration of understanding the mechanisms of RGC loss in FD, and identifying conventional RGCs (La Morgia et al., 2011, 2010; Moura et al., potential therapeutics, might help mitigate progressive RGC loss not 2013) as seen in FD mouse retinas (Fig. 2) (Ueki et al., 2016) and only in FD, but also in other mitochondrial optic neuropathies. FD patients (Mendoza-Santiesteban et al., 2017). Mitochondrial dysfunction is also implicated in glaucoma, which is characterized MATERIALS AND METHODS by RGC death (Kim et al., 2015; Osborne and del Olmo-Aguado, Mice 2013). Together, we conclude that mitochondrial dysfunction is one All mice were housed in the Animal Resource Center at Montana State of the causes of RGC degeneration in FD. University and protocols were approved by the Montana State University Institutional Animal Care and Use Committee. Retina-specific Ikbkap CKO To our surprise, despite their mitochondrial integrity and function Ikbkap also being impaired due to loss of Ikbkap, cell types other than mice were generated by crossing floxed (International Knockout Mouse Consortium) and αPax6 promoter-driven Cre (Pax6-Cre) mice RGCs were spared from death in the CKO retina (Fig. 2). Our TEM (Marquardt et al., 2001), which were a gift from the original founders (Drs analysis revealed that the mitochondrial membrane integrity of Peter Gruss and Ruth Ashery-Padan). Pax6-Cre is expressed in the retinal amacrine cells was also progressively disrupted, but at a reduced progenitors, allowing Ikbkap deletion from all the retinal neurons and pace and scale compared with that of RGCs (Fig. S3). Significant Müller glia (Marquardt et al., 2001). Both male and female mice were used reduction in mitochondrial membrane proteins, complex I function at the age indicated, and littermate Pax6-Cre−;Ikbkapflox/flox mice were used and ATP content was observed from total retinal lysates or as controls. To analyze Cre expression in the retina and to determine mitochondria of CKOs, compared with controls (Figs 5 and 6). affected retinal regions, Pax6-Cre mice were crossed to Rosa-EGFP Cre This observation suggests that mitochondria of all retinal cells that reporter mice (Jackson Laboratory, stock #004077). express Cre (50-85%) in the CKO retina (Fig. 1) are affected, as RGCs only account for 0.5% of retinal cell populations (Jeon et al., Western blots 1998). In support of this, mutations in the mitochondrial gene/ Retinas were collected at the age indicated and standard western blot procedures were performed. An equal amount of total protein was protein in LHON and DOA also cause degeneration of RGCs, but loaded in each well. Primary antibodies used were as detailed: anti-IKAP not of the other retinal neurons (Yu-Wai-Man et al., 2011). Why (1:2000; Anaspec #AS-54494), mitochondrial membrane integrity cocktail RGCs of all retinal cell types are the most vulnerable to loss of (1:1000; Abcam #ab110414) and anti-β-actin (1:10,000; Santa Cruz Ikbkap is unknown. One explanation is that the RGCs have a high Biotechnology #sc-47778). Quantitation of blots was performed using energy demand and are vulnerable to mitochondrial dysfunction ImageJ and values were normalized using a loading control. Statistical owing to their unique anatomy: RGC axons are only myelinated analysis was performed using Student’s t-test. Data are considered beyond the lamina cribosa (where the optic nerve starts), and significant when P<0.05. therefore propagation of action potentials is less efficient and requires more energy in the unmyelinated prelaminar region of the Immunohistochemistry, RGC counts and H&E staining RGC axons (Morgan, 2004). The brain consumes 20% of the basal Mice were euthanized with CO2 and the eyes were marked with a green metabolic rate (Clarke and Sokoloff, 1999), with the visual system tattoo dye on the temporal surface. IHC and confocal imaging were performed as described (Ueki et al. 2016). Primary antibodies used were as being one of the most energy-consuming systems. Moreover, the detailed: anti-GFP (1:5000; Abcam #ab13970), anti-Brn3 (1:250; Santa retina is one of the tissues with the highest oxygen demand (Ames Cruz Biotechnology #sc-6026), anti-RBPMS (1:500; PhosphoSolutions et al., 1992; Anderson and Saltzman, 1964; Niven and Laughlin, #1832-RBPMS), anti-melanopsin (1:5000; Advanced Targeting Systems 2008). Cytochrome c oxidase (COX) is a terminal enzyme of the AB-N38), anti-PKD2L-1 (1:500; Millipore #AB9084) and anti-ChAT mitochondrial electron-transport chain and is necessary for ATP (1:200; Millipore #AB114P). For Brn3+ or melanopsin+ RGC counts on production (Wikstrom, 1977). Therefore, the level and activity of flatmounts, confocal images were taken at 1 mm from the optic nerve head COX reflects energy demand. In retina, strong COX expression and in temporal, nasal, superior and inferior retinas, and the number of Brn3+ activity is detected in the nerve fiber layer, the unmyelinated RGC cells in each image was counted manually. The number of cells in 1 mm2 of axon bundle within the retina, and in the prelaminar and laminar the retina was calculated and plotted. The optic nerve was carefully cut away regions of the optic nerve (Andrews et al., 1999). These from the eyecup after fixation, rinsed in phosphate-buffered saline (PBS) and cryoprotected overnight in PBS plus 20% sucrose. Optic nerves were unmyelinated regions of RGC axons make them especially then embedded in OCT and cryostat-sectioned transversely at 16 μm. DAPI vulnerable to energy deprivation (Andrews et al., 1999; Morgan, staining was performed to visualize the optic nerve, the circumference of 2004), causing RGCs to degenerate under metabolic dysfunction. In each optic nerve was measured and plotted. Statistical analysis was fact, in FD, LHON and DOA patients, RGCs of the more performed using Student’s t-test. Data are considered significant when metabolically taxed temporal retinas are the first to die, before P<0.05. Hematoxylin and Eosin (H&E) staining and photoreceptor pan-retinal RGC degeneration is observed (Mendoza-Santiesteban counting were performed according to Ueki et al. (2016). Disease Models & Mechanisms

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Transmission electron microscopy References Eyes were collected and fixed in Karnovsky’s fixative overnight at 4°C, Abashidze, A., Gold, V., Anavi, Y., Greenspan, H. and Weil, M. (2014). washed in Ringer’s saline and treated with 2% osmium tetroxide in 0.1 M Involvement of IKAP in peripheral target innervation and in specific JNK and NGF signaling in developing PNS neurons. PLoS ONE 9, e113428. potassium-sodium phosphate buffer (PSPB) for 4 h at room temperature. Addis, L., Ahn, J. W., Dobson, R., Dixit, A., Ogilvie, C. M., Pinto, D., Vaags, A. K., Eyes were rinsed three times with PSPB and the cornea and lens were Coon, H., Chaste, P., Wilson, S. et al. (2015). Microdeletions of ELP4 are carefully removed. The remaining eyecups were cut into four equal pieces associated with language impairment, autism spectrum disorder, and mental under a dissecting microscope. Eyecups were dehydrated in a graded retardation. Hum. 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TEM mutations of the IKAP gene. Am. J. Hum. Genet. 68, 753-758. Andrews, R. M., Griffiths, P. G., Johnson, M. A. and Turnbull, D. M. (1999). imaging was carried out using a LEO 912 (Zeiss) operating at 100 kV Histochemical localisation of mitochondrial enzyme activity in human optic nerve accelerating voltage. Sections from the peripheral retina were imaged and and retina. Br. J. Ophthalmol. 83, 231-235. retinal cell types were identified by morphology, location and nuclear size Axelrod, F. B. (2002). Hereditary sensory and autonomic neuropathies. Familial (Jeon et al., 1998). dysautonomia and other HSANs. Clin. Auton. Res. 12 Suppl. 1, I2-I14. Axelrod, F. B., Hilz, M. J., Berlin, D., Yau, P. L., Javier, D., Sweat, V., Bruehl, H. and Convit, A. (2010). Neuroimaging supports central pathology in familial Mitotracker treatment dysautonomia. J. Neurol. 257, 198-206. Retinas were isolated and incubated with 200 nM MitoTracker Red Baltan, S., Inman, D. M., Danilov, C. A., Morrison, R. 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Images (single Functional analysis of lymphoblast and cybrid mitochondria containing the 3460, focal plane) of the RGC layer at 1.75 mm from the optic nerve head were 11778, or 14484 Leber’s hereditary optic neuropathy mitochondrial DNA mutation. captured and the intensity of MitoTracker Red CMXRos quantified using J. Biol. Chem. 275, 39831-39836. Carelli, V., Ghelli, A., Ratta, M., Bacchilega, E., Sangiorgi, S., Mancini, R., ImageJ. Leuzzi, V., Cortelli, P., Montagna, P., Lugaresi, E. et al. (1997). Leber’s hereditary optic neuropathy: biochemical effect of 11778/ND4 and 3460/ND1 Complex I activity mutations and correlation with the mitochondrial genotype. Neurology 48, Retinas were pooled and gently lysed with a Dounce homogenizer (20- 1623-1632. Carelli, V., La Morgia, C., Valentino, M. L., Barboni, P., Ross-Cisneros, F. N. and 25 strokes). The mitochondrial fraction was isolated according to Sadun, A. A. (2009). 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D., Lu, H.-M., Zeng, W., Lu, H., Chao, E. C. and Fatemi, A. (2015). ELP2 is a novel gene implicated in Author contributions neurodevelopmental disabilities. Am. J. Med. Genet. A 167, 1391-1395. Conceptualization: Y.U., F.L.; Methodology: Y.U.; Formal analysis: Y.U., V.S.; Cuajungco, M. P., Leyne, M., Mull, J., Gill, S. P., Lu, W., Zagzag, D., Axelrod, Investigation: Y.U.; Writing - original draft: Y.U.; Writing - review & editing: Y.U., F.L.; F. B., Maayan, C., Gusella, J. F. and Slaugenhaupt, S. A. (2003). Tissue- Supervision: F.L.; Funding acquisition: F.L. specific reduction in splicing efficiency of IKBKAP due to the major mutation associated with familial dysautonomia. Am. J. Hum. Genet. 72, 749-758. Funding Dietrich, P., Alli, S., Shanmugasundaram, R. and Dragatsis, I. (2012). IKAP This work was supported by the National Institutes of Health (R01NS086796 and expression levels modulate disease severity in a mouse model of familial R43 EY025458) and the Dysautonomia Foundation (F.L.). dysautonomia. Hum. Mol. Genet. 21, 5078-5090. Dimauro, I., Pearson, T., Caporossi, D. and Jackson, M. J. (2012). A simple protocol for the subcellular fractionation of skeletal muscle cells and tissue. BMC Supplementary information Res. Notes 5, 513. Supplementary information available online at Dong, J., Edelmann, L., Bajwa, A. M., Kornreich, R. and Desnick, R. J. (2002). http://dmm.biologists.org/lookup/doi/10.1242/dmm.033746.supplemental Familial dysautonomia: detection of the IKBKAP IVS20(+6T –> C) and R696P Disease Models & Mechanisms

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