Tissue-Specific FAH Deficiency Alters Sleep–Wake Patterns and Results in Chronic Tyrosinemia in Mice

Tissue-specific FAH deficiency alters sleep–wake patterns and results in chronic tyrosinemia in mice

Shuzhang Yanga,b, Sandra M. Siepkac, Kimberly H. Coxa, Vivek Kumara,1, Marleen de Groota,b, Yogarany Chelliaha,b, Jun Chend, Benjamin Tud, and Joseph S. Takahashia,b,2

aDepartment of Neuroscience, Peter O’Donnell Jr. Brain Institute, University of Texas Southwestern Medical Center, Dallas, TX 75390-9111; bHoward Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX 75390-9111; cDepartment of Neurobiology, Northwestern University, Evanston, IL 60208; and dDepartment of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75390

Contributed by Joseph S. Takahashi, September 5, 2019 (sent for review March 20, 2019; reviewed by Richard Halberg and David W. Threadgill) Fumarylacetoacetate hydrolase (FAH) is the last enzyme in tyro- nature of random mutagenesis allows for novel gene discovery sine catabolism, and mutations in the FAH gene are associated and also causes point mutations that may alter gene function in with hereditary tyrosinemia type I (HT1 or TYRSN1) in humans. unpredictable ways (13–15). In a behavioral screen of N-ethyl-N-nitrosourea mutagenized mice Although the available models of FAH deficiency have been we identified a mutant line which we named “swingshift” (swst, useful for studying the biochemical and pathological features of MGI:3611216) with a nonsynonymous point mutation (N68S) in HT1, they are perinatally or postnatally lethal due to liver Fah that caused age-dependent disruption of sleep–wake pat- failure, making behavioral and physiological studies impossible terns. Mice homozygous for the mutation had an earlier onset of without pharmacological intervention (16, 17). Here we report activity (several hours before lights off) and a reduction in total the identification of an ENU-induced, nonsynonymous mutation in activity and body weight when compared with wild-type or het- the Fah gene. Biochemical analyses indicate that this mutation erozygous mice. Despite abnormal behavioral entrainment to promotes degradation of the FAH protein, resulting in diminished light–dark cycles, there were no differences in the period or phase FAH enzymatic activity in a tissue-specific manner. In contrast to of the central clock in mutant mice, indicating a defect down- the lethality previously reported in other HT1 mouse models, our

stream of the suprachiasmatic nucleus. Interestingly, these behav- mutant mice survived into adulthood, making it possible to exam- GENETICS ioral phenotypes became milder as the mice grew older and were ine their behavioral and physiological characteristics. Our studies completely rescued by the administration of NTBC [2-(2-nitro-4- suggest that FAH deficiency interferes with behavioral entrainment trifluoromethylbenzoyl)-1,3-cyclohexanedione], an inhibitor of 4- to light–dark cycles downstream of the suprachiasmatic nucleus hydroxyphenylpyruvate dioxygenase, which is upstream of FAH. (SCN), leading to disrupted sleep–wake patterns. Interestingly, the Mechanistically, the swst mutation had no effect on the enzymatic behavioral defects in our model are reversible and are associated activity of FAH, but rather promoted the degradation of the mu- with reduced FAH protein levels and FAH activity in the liver and tant protein. This led to reduced FAH protein levels and enzymatic kidney, resulting in chronic tyrosinemia. activity in the liver and kidney (but not the brain or fibroblasts) of homozygous mice. In addition, plasma tyrosine—but not methio- Results nine, phenylalanine, or succinylacetone—increased in homozy- Mutagenesis, Screening, and Identification of the swingshift Mutation. gous mice, suggesting that swst mutants provide a model of Using a recessive approach, we screened ENU-mutagenized mice mild, chronic HT1. for abnormal circadian behaviors. One of the mutant mouse lines was characterized by early activity onset (Fig. 1A), and we named ENU mutagenesis | tyrosinemia | sleep Significance ereditary tyrosinemia type I (HT1 or TYRSN1; OMIM:276700) His an autosomal recessive disorder caused by deficiency of We report the identification of an N-ethyl-N-nitrosourea– fumarylacetoacetate hydrolase (FAH), the last enzyme in the ty- induced, nonsynonymous mutation in the fumarylacetoacetate rosine catabolic pathway (1–3). FAH deficiency leads to the ac- hydrolase (Fah) gene in mice that results in increased plasma cumulation of toxic metabolites such as fumaryl–acetoacetate, tyrosine (chronic tyrosinemia) due to reduced FAH activity in maleyl–acetoacetate, and succinylacetone. This buildup induces the liver and kidney. Importantly, in contrast to the lethality progressive liver disease, secondary renal tubular dysfunction, previously reported with other Fah mutations, our mutant and neurologic crises that can include changes in mental status, mice survived into adulthood, making them a useful model abdominal pain, peripheral neuropathy, and/or respiratory failure for examining the long-term behavioral and physiological (1, 2, 4–6). Based on clinical features, HT1 can be classified into 2 effects of chronic tyrosinemia. major types: The acute form has symptoms occurring within the first month after birth and usually results in death within the Author contributions: S.Y., S.M.S., and J.S.T. designed research; S.Y., S.M.S., V.K., M.d.G., Y.C., J.C., and B.T. performed research; S.Y., S.M.S., K.H.C., V.K., M.d.G., J.C., and B.T. first year and the chronic form shows a slower progression of liver analyzed data; and S.Y., K.H.C., and J.S.T. wrote the paper. disease (6–8). Reviewers: R.H., University of Wisconsin; and D.W.T., Texas A&M University. Several mouse models of acute HT1 have been described (9). The authors declare no competing interest. The “albino lethal” mouse has a large deletion on chromosome 7, including the albino locus and the Fah gene (10), while an- Published under the PNAS license. “ ” other FAH-deficient mouse was generated by targeted disrup- Data deposition: Mutant mouse line swingshift and the gene Fah have been registered with the Mouse Genome Informatics (MGI) database (http://www.informatics.jax.org/ tion of the Fah gene (11). In addition, 2 N-ethyl-N-nitrosourea marker/MGI:3611216). (ENU)-induced point mutations also caused FAH deficiency: A 1Present address: Department of Genetics and Genomics, The Jackson Laboratory, Bar missense mutation in exon 6 and a splice mutation causing loss of Harbor, ME 04609. exon 7 with a subsequent frameshift in the coding region of the 2To whom correspondence may be addressed. Email: [email protected]. Fah gene (12). Phenotypic screens using random mutagenesis This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. have increased our understanding of the genetic basis of many 1073/pnas.1904485116/-/DCSupplemental. diseases. In contrast with targeted mutagenesis, the unbiased First published October 14, 2019.

www.pnas.org/cgi/doi/10.1073/pnas.1904485116 PNAS | October 29, 2019 | vol. 116 | no. 44 | 22229–22236 Downloaded by guest on October 2, 2021 this mutant “swingshift” (swst, MGI:3611216). Using C57BR/sdJ heterozygous; r = 0.171, P = 0.088 for homozygous mutant). In and C57L/J strains, we mapped swst to a 33.7-Mb interval on contrast, the % Light activity was correlated with age in all 3 chromosome 7 between rs31304724 (4 recombinants/76 meioses) genotypes. However, this correlation was positive in WT (Fig. 4A; and rs13479454 (6 recombinants/78 meioses) (Fig. 1B). This region r = 0.3191, P = 0.005) and heterozygous mice (Fig. 4B; r = 0.3351, contains 511 Refgenes according to the mouse genome assembly P < 0.0001) and negative in homozygous mice (Fig. 4C; r = −0.3377, GRCm38/mm10. To identify a candidate gene, we performed P = 0.0006). The negative correlation in homozygous mutant exome sequencing using genomic DNA from one homozygous mice was likely due to the higher light-phase activity levels in mouse and found a nonsynonymous point mutation within younger animals, which were comparable to heterozygous and WT the coding region of Fah. There was a single base transition from littermates when they were older. A to G at chromosome 7:84,601,144 in exon 3 of Fah in swst We also found that, starting at 5 wk of age, body weights of mice, which was confirmed by sequencing the flanking region homozygous mutant mice were less than that of heterozygous using DNA samples from 4 homozygous and 3 wild-type (WT) and WT mice in both males (Fig. 4D; P < 0.01) and females (Fig. mice (Fig. 1C). The point mutation converts the highly con- 4E; P < 0.01). This decrease in body weight was associated with served amino acid residue 68 from an asparagine to a serine (N68S; lower body fat in homozygous males, but not in females (Fig. 4F; Fig. 1 C and D). P < 0.0001). Moreover, when challenged with a high-fat diet (HFD) the homozygous males were resistant to weight gain (Fig. swst Mutant Mice Have Disrupted Sleep–Wake Patterns but a Normal 4G; P < 0.0001). Interestingly, the body weights of homozygous Circadian Clock. In order to investigate genotype–phenotype as- mutant mice gradually caught up with WT and heterozygous sociations, we quantified the swst behavioral phenotype using mice after 21 wk of age (Fig. 4 D and E). These results suggest total activity (wheel revolutions per day) from the 6th to the that the swst mutation in Fah causes developmental defects that 15th day and the percentage of activity during the light phase (% improve with aging. Light) in 12:12 light–dark (LD) cycles. When compared with heterozygous and WT mice, homozygous mutant mice had lower The swst Mutation Reduces FAH Protein Stability in a Tissue-Specific total activity (Fig. 2 A and B; P < 0.0001) even though they had Manner, Resulting in a Mild, Chronic HT1 Phenotype. Fah is highly increased activity during the light phase (Fig. 2C; P < 0.01). expressed in the liver, and its deficiency causes HT1 due to a lack Moreover, homozygous mutants had more activity bouts per day of enzymatic activity (16). To examine if the swst mutation al- (Fig. 2D; P < 0.0001), while bout length and activity level per tered FAH enzymatic activity, we measured FAH activity in liver bout were reduced (Fig. 2 E and F; P < 0.0001). Taken together, lysates. Heterozygous livers had FAH activity about half of that these results point to a failure of entrainment of sleep–wake in WT livers, while the homozygous livers showed no detectable rhythms with light–dark cycles in swst mutant mice, while the lack FAH activity (Fig. 5 A and B; P < 0.0001). However, when we of phenotype in the heterozygotes confirms the autosomal re- examined FAH activity of purified recombinant proteins cessive inheritance of the swst mutation. expressed in Escherichia coli, we were surprised to find that the To further verify that the identified swst mutation was causative for the observed behavioral changes, we next sought to rescue the phenotype (18). Hereditary tyrosinemia is commonly treated with 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione A C (NTBC), an inhibitor of 4-hydroxyphenylpyruvate dioxygenase WT upstream of FAH (19, 20) that has also been used to successfully rescue lethality in Fah knockout mice (16, 17). When swst mutant WT Day mice were given NTBC in their drinking water, their behavioral TLNNF phenotype was completely rescued (see SI Appendix,Fig.S1for swst representative actograms). Both the percentage of activity during the light phase (% Light, assessed from days 9 to 13 in 12:12 LD Time (h)

cycles) and the total activity in NTBC-treated swst homozygous T LSNF mice were restored to levels similar to heterozygous mice (Fig. 3 A and B; swst homozygous [s/s] untreated different from all other < swst D

groups, P 0.0001). These findings indicate that swst is allelic with Day Homo Fah and further support the hypothesis that the behavioral defects Pan observed in swst mice are due to the mutation that we found in the Macaca Canis Fah gene. Bos Altered sleep–wake rhythms are commonly associated with Time (h) Mus Chr7 Rattus malfunctions of the circadian master clock located in the SCN. B 74,788,642 Gallus rs31304724 Danio Therefore, to investigate whether the swst mutation causes dis- (4/76) Drosophila rupted circadian behavior by altering SCN function, we crossed swingshift (swst) Anopheles Caenorhabditis the swst mice to PER2::Luciferase reporter mice and cultured Magnaporthe Neurospora SCN explants to monitor their central clock (Fig. 3C). Surpris- Arabidopsis ingly, we failed to detect any differences in period (Fig. 3D)or rs13479454 Oryza (6/78) Xenopus phase (Fig. 3E) of the central clock of mutant mice. Thus, the 108,465,371 swst mutation in Fah alters sleep–wake patterns without affecting the function of the SCN. Fig. 1. Identification of the swst mutation in the Fah gene in mice. (A)Rep- resentative wheel-running actograms for WT (Upper) and mutant littermates Behavioral and Metabolic Alterations in swst Mutant Mice Improve (swst, Lower). Recordings were started with 12:12 LD cycles (lights on indicated – with large arrows) followed by a period of constant darkness (indicated with with Age. Despite strong genotype phenotype associations, we – found that some older mice did not show a swst phenotype, sug- small arrows). The swst mutant mice had disrupted sleep wake patterns. (B) The swst mutation maps to a 33.7-Mb region on chromosome 7. (C)Se- gesting an age-dependent effect of the mutation (see SI Appendix, quencing of genomic DNA confirmed a point mutation in the Fah gene. An A- Fig. S2 for representative actograms). When we applied regression to-G transition converts amino acid residue 68 from asparagine to serine (N68S) analysis to the wheel-running data, total activity was not correlated in the mouse FAH protein. (D) Alignment of FAH protein indicates that as- with age (r = −0.065, P = 0.577 for WT; r = −0.017, P = 0.840 for paragine 68 is highly conserved.

22230 | www.pnas.org/cgi/doi/10.1073/pnas.1904485116 Yang et al. Downloaded by guest on October 2, 2021 ABC 60000 40 15 * 30 10 40000 ** ** 20 5

20000 % Light 10 0 Avg. Counts Avg. (Revolutions/min) Total Activity (revs) Total 0 0 -5

+/+ s/+ s/s +/+ s/+ s/s +/+ s/+ s/s

D E F 15 500 30000 ** 400 10 20000 300 ** 200 ** 5 10000 Bouts/day

100 Activity/bout Bout Length (min) 0 0 0

+/+ s/+ s/s +/+ s/+ s/s +/+ s/+ s/s

Fig. 2. The swst mutant mice have altered activity levels. (A) Homozygous mutant mice (s/s, n = 71) exhibited decreased total activity (wheel revolutions per GENETICS day) as compared to heterozygous (s/+, n = 102) and WT littermates (+/+, n = 49). (B) This decrease in total activity in s/s mice was reflected in their reduced overall average activity per minute, although s/s mice had increased activity during the light phase (C). (D) The average number of activity bouts per day was increased in s/s mice, while bout length (E) and average activity per bout (F) were decreased. There were no differences between s/+ and +/+ littermates for any measure. *P < 0.01; **P < 0.0001.

swst mutation did not disrupt FAH activity. Rather, the mutant FAH To test this hypothesis, we next examined FAH protein ex- showed slightly higher activity than WT (Fig. 5 C and D; P < 0.001). pression in various tissues from mutant mice and their littermates. These results indicate that the lack of FAH activity in homozygous Western blots of lysates from mutant and WT livers derived from livers might instead be caused by reduced FAH protein levels. young (10 d old, Fig. 5E)oradultmice(7moold,SI Appendix,

150 150 AC* +/+ 100 s/+ 100 s/s 50 50

%Light 0 Amplitude 0 -50

-50 -100 s/s s/s s/+ s/+ 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 NTBC NTBC Time (days) B 60000 DE * 26 22 40000 25 20

24 20000 18

23 Phase Period (h) 16 22 Total activity (revs) Total 0 21 14 +/+ s/+ s/s +/+ s/+ s/s s/s s/s s/+ s/+ NTBC NTBC

Fig. 3. Behavioral alterations in swst mice can be reversed with NTBC treatment but are not due to defects in the SCN. (A) NTBC treatment completely rescued the percentage of light activity in homozygous mutant mice (s/s, n = 11 to 14) without altering behavior in heterozygous mice (s/+, n = 9to16).(B) NTBC treatment in s/s mice increased total activity to a level comparable to that of s/+ mice, while NTBC treatment in s/+ mice had no effect on total activity. (C) Real-time bio- luminescence recording of SCN explants from s/s (red, n = 11), s/+ (green, n = 6), and +/+ (blue, n = 8) littermates heterozygous for the mPer2-Luc knock-in allele. (D and E) Circadian period (D) and phase (E). No differences were detected among the different genotypes. *P < 0.0001, untreated s/s mice different from all other groups.

Yang et al. PNAS | October 29, 2019 | vol. 116 | no. 44 | 22231 Downloaded by guest on October 2, 2021 100 100 100 A Y = 0.02395*X - 1.782 B Y = -0.1255*X + 30.08 C Y = -0.1255*X + 30.08 r = 0.3191, p=0.005 r = 0.3351, p<0.0001 r = -0.3377, p=0.0006

50 +/+ 50 s/+ 50 s/s %Light %Light %Light

0 0 0 0 100 200 300 0 100 200 300 0 100 200 300 Age (d) Age (d) Age (d)

DE40 40 Males Females

30 30

20 20

10 +/+ 10 +/+ Body Weight (g) Body Weight Body Weight (g) Body Weight s/+ s/+ s/s s/s 0 0 3 4 5 6 7 8 9 1011121314151617181920212223242526 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 Age (Weeks) Age (Weeks) FG 100 * Fat Males Females 50 30 Lean ** * * Control +/+ 40 ** * * Control s/s 20 HFD +/+ 30 * **

% BW 50 HFD s/s 20 10 10 Body Weight (g) Body Weight (g) Body Weight 0 0 0 6101418 6101418 Age (Weeks) Age (Weeks) +/+ M s/s M +/+ F s/s F

Fig. 4. Age-dependent phenotypes in swst mice. (A–C) Activity during the light phase was significantly correlated with age. The percentage of light activity (% Light) tended to increase with age in WT (+/+, n = 76) and heterozygous (s/+, n = 147) mice but decrease with age in homozygous mutant (s/s, n = 101) mice. (D and E) Body weights of male (D) and female (E) s/s mice. Blue bars indicate significant differences (P < 0.01) between the s/s mice (n = 22 males, 18 females) and the +/+ (n = 12 males, 16 females) and s/+ (n = 35 males, 30 females) mice. (F) Body compositions of male (n = 8 +/+, 10 s/s) and female (n = 2 +/+, 6 s/s) at 10 to 12 mo of age (*P < 0.0001 as compared to +/+ males). (G) Effects of HFD on body weights in male and female +/+ and s/s mice (n = 10 to 12/ group). *P < 0.0001 +/+ different from s/s; **P < 0.0001 control +/+ different from all other groups).

Fig. S3A) revealed decreased FAH protein in homozygous livers. impaired transcription or mRNA stability, we isolated total RNA In addition to the liver, the mouse kidney also expresses Fah (21, from livers and measured pre-mRNA and mature mRNA using 22). Therefore, we also examined the effects of the swst mutation qRT-PCR. Neither pre-mRNA nor mature mRNA levels were FAH protein levels in the kidney. Similar to the results in the liver, altered in heterozygous livers, but in homozygous livers both pre- FAH protein is undetectable in the homozygous kidney, but not mRNA and mature mRNA levels were reduced (Fig. 6A; P < in heterozygous or WT kidneys (SI Appendix, Fig. S3B). The 0.0001). These findings argue against any deleterious effect of behavioral defects in mutant mice prompted us to also examine the swst mutation on Fah gene transcription or mRNA stability FAH protein expression in the brain. In contrast to the mutant because otherwise we would expect about half the levels of pre- livers and kidneys, FAH protein is expressed at similar levels in mRNA or mature mRNA in heterozygous mice compared to the brains of homozygous, heterozygous, and WT mice (SI Ap- WT. Therefore, to further explore the effect of the swst mu- pendix, Fig. S3C). We also established fibroblasts from the ears tation on mRNA stability, we established HEK293T cells stably of the mutant mice and examined FAH protein levels in these expressing Myc-tagged mutant and WT FAH proteins. Using cultures. Similar to the brain tissue, fibroblasts derived from ho- primer pairs specifically targeting the plasmid DNA and its derived mozygous mutants express FAH protein at a level comparable to mRNA, but not the endogenous FAH gene, we found that ec- those fibroblasts derived from heterozygous and WT mice (SI topically expressed FAH mRNA was proportional to the copy Appendix,Fig.S3D). Taken together, these findings show that the numbers of integrated plasmid DNA for both WT and swst mutant reduced FAH enzymatic activity in swst homozygous mutant livers cells (Fig. 6B), indicating that neither transcription nor mRNA is due to the loss of FAH protein and that there is a tissue-specific stability is altered by the swst mutation. Indeed, the mRNA half- mechanism regulating the expression or degradation of FAH. life for WT and mutant FAH mRNAs did not differ between the Marked elevation of succinylacetone in urine or plasma is stable cell lines (Fig. 6 C and D). However, as in the mouse liver considered pathognomonic for HT1 (23–25). Therefore, to as- lysates (Fig. 5E), there were reduced levels of mutant FAH protein sess whether succinylacetone levels are altered in swst mutants, in the stable cells (Fig. 6 B and E). We therefore examined the we collected plasma from mutant mice and their littermates. stability of the mutant protein and found that mutant FAH was Liquid chromatography followed by mass spectrometry failed to degraded 5-fold faster than WT FAH, with half-lives of 33.4 and detect succinylacetone in any sample. However, we did detect an 165.2 h, respectively (Fig. 6F; P < 0.001 after 24 h). These results elevation of tyrosine in mutant plasma (Fig. 5F; P < 0.05). In- suggest that faster degradation of mutant FAH protein contributes terestingly, although HT1 is commonly associated with increased to its reduced protein expression and enzymatic activity in ho- methionine and phenylalanine in addition to tyrosine, the mu- mozygous swst livers. tant mice did not have altered plasma methionine or phenylalanine (Fig. 5F). These results suggest that the swst mutant may Discussion represent a model for a mild form of chronic HT1. HT1 is a genetic disease resulting from a deficiency in FAH. To date, more than 150 mutations in the FAH gene are associated The swst Mutation Enhances Degradation of the FAH Protein. To with HT1 in humans, including missense and nonsense mutations examine whether the reduction in FAH protein was due to as well as other variants that cause frameshifts or disrupt splicing

22232 | www.pnas.org/cgi/doi/10.1073/pnas.1904485116 Yang et al. Downloaded by guest on October 2, 2021 A C E +/+ s/+ s/s 0.5 WT 0.00 swst FAH +/+ 0.0 -0.05 s/+ Tubulin -0.10 s/s -0.5 1.2 1.0 OD330nm OD330nm -0.15 -1.0 0.8 -0.20 0.6 **** -0.25 -1.5 0.4 0 100 200 300 400 500 0 153045607590 Relative Quantity Time (sec) 0.2 Time (sec) **** 0.0 +/+ +/s s/s B D 1.0 F 0.08 *** 100 +/+ * 0.8 0.06 M) 80 s/+

 ** s/s 0.04 **** 0.6 60

0.02 0.4 40 OD330nm/min) OD330nm/min) ****

0.00 (- (- 0.2 20 Concentration ( Relative Fah Activity Relative Fah Relative Fah Activity Relative Fah GENETICS -0.02 0.0 0 +/+ s/+ s/s WT swst Met Phe Tyr

Fig. 5. The swst mutation causes reduced FAH protein in the liver but does not alter enzymatic activity. (A) Representative spectrometric FAH assay results with liver lysates from homozygous mutant (s/s, n = 5), heterozygous mutant (s/+, n = 5), and WT littermates (+/+, n = 4). (B) Relative enzymatic activity quantified from FAH assays in A.(C) Representative FAH assay results using purified WT and mutant FAH proteins. (D) Relative enzymatic activity quantified from FAH assays in C. Two separate experiments were performed with 2 replicates each (n = 4/group). (E) Representative immunoblots of FAH in liver lysates and relative FAH levels quantified from immunoblotting (n = 3 samples/group, average of 3 separate assays). (F) Plasma concentrations of methionine (Met), phenylalanine (Phe), and tyrosine (Tyr) in s/s (n = 5), s/+ (n = 4), and +/+ (n = 4) mice. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

(26, 27). In addition, 4 mutations have been described in mouse trafficking or inducing conformational changes that interfere with models (10–12). Several of the known missense mutations in hu- substrate binding (28). In this study, we report a missense muta- mans decrease FAH enzymatic activity by altering protein folding/ tion (swst) that causes an HT1 phenotype in mice, including the

1.5 A +/+ C E +/- WT 1.0 WT SWST 1.0 s/s SWST 0.8 03 6122403 61224 0.6 FAH-myc 0.5 *** 0.4 Tubulin

Relative Quantity *** 0.2 Relative Fah mRNA 0.0 0.0 preFAH FAH 02468 Time (h) 1.2 B F * * 1.2 D 1.0 ** 293T/FAH 3 1.0 293T/SWST 0.8 0.8 293T 2 0.6 0.6 0.4 0.4 1 WT 0.2 Relative Quantity Relative Quantity 0.2 SWST

Fah mRNA Half life (h) 0.0 0.0 0 Protein mRNA DNA WT SWST 0 6 12 18 24 Time (h)

Fig. 6. The swst mutation reduces protein stability but not transcription or mRNA stability. (A) Relative amounts of pre-mRNA and mature mRNA in the livers of WT (+/+, n = 4), heterozygous mutant (+/s, n = 5), and homozygous mutant (s/s, n = 5) mice as measured by RT-qPCR. (B) Relative amounts of protein, mRNA, and DNA construct in HEK293T cells stably expressing WT (293T/FAHWT) or mutant (293T/FAHSwst) FAH vs. untransfected controls (293T, n = 3/group). (C) Representative results of mRNA stability in HEK293T cells stably expressing WT (FAHWT) or mutant (FAHSwst) FAH. Actinomycin D was added at time 0. (D) mRNA half-life calculated from mRNA stability assays (n = 4 per group). (E) Representative immunoblotting for protein stability assay in HEK293T cells stably expressing WT or mutant (SWST) FAH. Time 0 indicates the addition of cyclohexamide. (F) Quantified protein stability from 4 independent assays. *P < 0.05, **P < 0.001, ***P < 0.0001.

Yang et al. PNAS | October 29, 2019 | vol. 116 | no. 44 | 22233 Downloaded by guest on October 2, 2021 absence of FAH enzymatic activity in the liver and an increase in for HT1 patients, those will be interesting questions to address in plasma tyrosine. In contrast with the previously reported mouse future studies. models that display lethality shortly after birth, these swst mutants The original purpose of our study was to perform a behavioral survive into adulthood. Therefore, the swst mutation represents a screen to find novel genes involved in regulating sleep and cir- mouse model for the chronic form of HT1, allowing for behavioral cadian behavior; thus, we were surprised to uncover a gene im- and physiological studies throughout the life span without phar- plicated in HT1. The clinical and pathological manifestations in macological intervention. Interestingly, the swst mutation is in a HT1 involve mainly the liver, kidney, and peripheral nerves (7). region of the FAH gene (exon 3) deficient in missense mutations in However, our studies in the swst mutant mice have the potential humans. Since the mouse and human genes share 84% sequence to open up new research areas, as our results suggest a potential identity and 89% amino acid identity, additional targeted muta- defect in the central nervous system. The swst homozygous mice genesisofthisgeneinmicemayallowforthediscoveryofgeno- show disrupted sleep–wake patterns characterized by advanced type–phenotype correlations as they relate to HT1. activity onset and fragmented sleep. These behavioral pheno- There are several features of the swst mutation that distinguish types can be rescued with NTBC treatment and also seem to this model of HT1 from other human/murine mutations in the improve with aging. Even though we did not detect the recovery FAH/Fah gene. First, the swst mutation does not impair enzy- of FAH protein levels in livers of older swst mice at an age (7 mo) matic activity by itself; instead, it causes faster degradation of when the behavioral and body-weight phenotypes largely dis- FAH protein. It is worth noting that the half-lives of the mutant appear, we cannot rule out the possibility that the demand for and WT FAH proteins as measured in HEK293T cells (33.4 vs. tyrosine metabolism decreases with aging; thus, the deleterious 165.2 h) do not necessarily represent the true values in mouse effects of FAH deficiency may be ameliorated. Although the livers or kidneys. Nonetheless, faster degradation could poten- mechanism is still elusive, circuits downstream from the central tially diminish FAH to undetectable levels in these tissues, circadian clock might be involved, given that the SCN remains leading to elevated plasma tyrosine. The elevation in tyrosine intact in terms of period and phase. While these neurobehavioral due to FAH deficiency is thought to be due to reduced inhibition findings are unusual, there is some indication that HT1 patients of other steps in the tyrosine catabolic process (11, 29). While we suffer from neurological and neuropsychological problems (39), did not assess the function of other enzymes in this pathway, such although these studies are complicated by the fact that NTBC as tyrosine amino transferase, the swst model could be a useful treatment may contribute to behavioral and cognitive pheno- tool to better understand the complex roles that FAH plays in types in humans (40). Thus, the swst mouse may allow for more the regulation of tyrosine catabolism. in-depth modeling of the direct effects of tyrosinemia on brain A second distinct feature of the swst model is the apparent development and behavioral abnormalities. tissue specificity of its degradation that could be due to the nature Conclusions of the point mutation, which converts an arginine to a serine. We In summary, we have identified a mutation in the Fah gene that speculate that such a change could introduce a novel, tissue- causes a chronic HT1 phenotype in mice through tissue-specific specific phosphorylation site, due either to tissue-specific motif disruption of FAH expression. The mild defects result in a much recognition or kinase expression (30–32). The liver and kidney longer life span for these mutant mice as compared to other HT1 have more FAH than any other tissues in mice (21, 22), suggesting models, which allowed us to reveal not only metabolic anomalies that these tissues are major sites of tyrosine catabolism. However, but also potential dysfunction in the central nervous system our findings indicate that the presence of functional FAH in other controlling sleep–wake rhythms. tissues may compensate for the defects in the liver and kidney, resulting in a less severe phenotype than what has been reported Methods previously (10, 12, 16). While we did not assess every possible Mutagenesis, Behavioral Screening, and Body-Weight Phenotyping. ENU (Sigma tissue in detail, our results call for caution when interpreting catalog #N3385) was prepared as described previously (41). Six-week-old screening/diagnosing data from succinylacetone assays using plasma male C57BL/6J mice were injected with 250 mg per 1 kg of body weight of samples or from enzymatic assays using tissues or cells other than ENU. After a 6-wk recovery period, the ENU-treated mice were mated with liver and kidney, as these results may not reflect tissue-specific loss WT C57BL/6J females to generate generation 1 (G1) males. G1 males were of FAH. mated with WT C57BL/6J females to produce G2 females. Four G2 females Third, while tyrosine levels were elevated in the swst model, were backcrossed to their G1 fathers to produce G3 mice for phenotyping. the other metabolites that we measured were not. We also ob- Five G3 mice from every G2 backcross (20 mice per G1 pedigree) were phenotyped to ensure an 85% probability of detecting a recessive mutation. served a reduction in body weight in our mouse model that im- Mice (8 to 12 wk of age) were placed in individual running wheel cages and proved with age. Presumably, the reduction in body weight is a activity was recorded using the ClockLab data collection system (Actimetrics). direct effect of the tyrosinemia, rather than the abnormalities in After at least 2 wk on LD 12:12, some of the mice were put into constant sleep–wake cycles. Humans and mice with altered sleep tend to darkness (DD) for an additional 3 wk. Total activity and percentage of activity weigh more, not less; however, there are other mouse models of during the light phase were analyzed using data from the 9th to the 13th day disrupted sleep that exhibit a reduced body weight phenotype in LD. The free-running period was measured by linear regression analysis of under certain conditions (33); therefore, we cannot rule out a activity onsets from data collected during the DD portion of the assay using circadian-metabolic connection in this model. Nonetheless, to- ClockLab software (42). To rescue the behavioral phenotypes, NTBC (Nitisinone, Sigma, SML0269) was prepared as described (17) and was given in drinking gether these findings suggest a potentially milder form of tyrosinemia water (7.5 μg/mL) to heterozygous mating pairs to make sure the pups were that is more phenotypically consistent, perhaps, with tyrosinemia treated from the beginning of their lives. The pups were subjected to run- type III, even though that form of tyrosinemia is usually the result ning wheel cages from 8 to 12 wk of age. of mutations in the HPD (4-hydroxyphenylpyruvic dioxygenase) A separate group of mice were weighed weekly from 3 to 28 wk, while gene (29, 34, 35). It is unclear why we were unable to detect another cohort had body composition assessed by NMR at 10 mo of age. An succinylacetone in our samples, but there are cases of HT1 in the additional group of mice was given either a HFD (D12451, Research Diets) or a literature with normal or undetectable succinylacetone levels (36, control diet (7012, Harlan Teklad) for 3 to 4 wk starting at 6 to 8 wk of age. At 37). Thus, some FAH mutations may be incompletely penetrant or the end of the altered diet period, all mice were weighed. The mice were then continued on their same diet and placed on running wheels for an additional there may be variable expressivity of phenotypes among mutation 3 to 4 wk to determine if there was a corresponding change in wheel-running carriers. While we did not examine the effects of the swst mutation behavior and subsequently weighed again. For all experiments, food and or NTBC treatment on liver and kidney function or the develop- water were provided ad libitum. All animal care and experimental procedures ment of hepatocellular carcinoma (16, 17, 38), 2 of the hallmarks were performed in accordance with the recommendations in the Guide for

22234 | www.pnas.org/cgi/doi/10.1073/pnas.1904485116 Yang et al. Downloaded by guest on October 2, 2021 the Care and Use of Laboratory Animals of the National Institutes of Health onto polyvinylidene difluoride membrane. Immunoblotting was carried out (43) and approved by the University of Texas Southwestern Institutional using the fumarylacetoacetase antibody (C-20, sc-66223, Santa Cruz Bio- Animal Care and Use Committee. technology) that targets the C terminus of FAH, thus recognizing WT and the swst mutant FAH equally well. α-Tubulin antibody (TU-02, sc-8035, Santa Identification of the swingshift Gene. Genetic mapping was carried out using Cruz Biotechnology) or β-actin antibody (sc-1616, Santa Cruz Biotechnology) C57BR/sdJ and C57L/J strains as described previously (41). Briefly, homozygous were used as loading controls. swst mice were outcrossed to C57BR/sdJ and C57L/J for F1 mice, which were then intercrossed to create F2 mice for mapping. Wheel-running behavior of Protein and mRNA Stability Assays. HEK293T cells stably expressing WT and all mapping mice was collected and analyzed as described above. Only mutant FAH were seeded onto 12-well plates until they reached confluency. presumptive homozygous mice were used for genetic mapping. For the mRNA stability assay, actinomycin D (5 μg/mL final concentration) was Exome sequencing was performed using DNA extracted from the livers of added to the culture media. At various time points the cells were washed one presumptive homozygous and one WT mouse. Briefly, livers were har- twice with PBS and lysed in 500 μL TRIzol reagent for total RNA isolation and vested and quick-frozen in liquid nitrogen, tissue was pulverized using a qRT-PCR as described above. For the protein stability assay, cycloheximide frozen mortar and pestle, and DNA was extracted and quantified using a (50 μg/mL, final concentration) was added to the media, and the cells were NanoDrop spectrophotometer. Only one homozygous mutation in the Fah lysed in 150 μL RIPA buffer at different time points. The lysates were then gene was identified within the mapping locus. subjected to Western blotting. To validate the exome sequencing results, exon 3 of the mouse Fah gene was PCR-amplified from genomic DNA samples of 4 homozygous mutant Primary Fibroblast Cell Culture. Primary fibroblasts were isolated from the ears and 3 WT mice with the following primers: forward primer (5′-cagctc- of WT and swst mutant mice. Briefly, minced ear tissue was incubated with tatgccggaggac-3′) and reverse primer (5′-gaggaagttggcctgactgtac-3′). The collagenase III (1 mg/mL) and trypsin (0.05%). After 10 min of incubation at PCR product was purified using a Qiagen gel extraction kit and sequenced 37 °C, minced tissue was allowed to adhere onto a 35-mm culture dish. using the above primers. For distribution, swingshift mutant mice have been Approximately 5 d after culture in Dulbecco’s Modified Eagle Media (DMEM) donated to the Jackson Laboratories (JAX #030622). (Mediatech) supplemented with 10% FBS, ear fragments were removed, and outgrowing fibroblasts were replated into 100-mm dishes by trypsinization. swst Genotyping. Mice were genotyped for the swst mutation using qRT-PCR to detect single-nucleotide polymorphisms. PCR reactions were carried out in FAH Assay. The enzymatic activity of swst mutant and WT FAH was assessed 10-μL volumes using 10 to 25 ng genomic DNA with 1× SYBR Green Master as described (44), using either purified E. coli expressed proteins or liver Mix (part #4309155, Applied Biosystems, Foster City, CA), 5 pM of forward homogenates. Briefly, since the FAH substrate FAA is not commercially (WT forward primer: 5′-ctgttttggtcttagacaactctGaa-3′ or swst forward primer: available, we constructed an FAA-generating system by incubating Homo-

5′-ctgttttggtcttagacaactctcTg-3′) and 5 pM of reverse primer (5′-ctggcaga- gentisic acid (Sigma, H0751) with homogentisate 1, 2-dioxygenase (Hgd) and GENETICS cagtaagttctgtaagg-3′) in MicroAmp 384-well Optical Reaction Plates (Applied glutathione transferase zeta 1 (Gstz1), which were cloned from mouse liver Biosystems). Primer pairs (forward: 5′-GACTTCGACAACCGGCTG-3′; reverse: 5′- cDNA and expressed and purified from BL21(DE3) cells as described above. CGCTTGTTCTGGCTGATGTC-3′) targeting Heat Shock Protein 70 were used as a The assay buffer contained 50 mM potassium phosphate buffer, pH 7.4, normalization control. Thermocycling reaction conditions were as follows: 40 100 mM KCl, 5 mM ascorbate (omitted for tissue homogenate assay), 50 μM

cycles of 95 °C for 15 s and 60 °C for 30 s. Data were collected and analyzed FeSO4,50μM glutathione (GSH) reduced, and 200 μM homogentisic acid with an ABI 7900HT Fast Real-Time PCR System (Applied Biosystems). (HGA). To generate FAA, purified His-HGD (50 μg/mL final) and GST-GSTZ1 (50 μg/mL final) were added to the assay buffer, and the reaction mix was RNA Isolation and qRT-PCR. Total RNA was isolated from frozen tissues or cell incubated for 1 h at room temperature. One milliliter of the reaction mix cultures with TRIzol reagent (Invitrogen) and then cleaned up using an RNeasy was transferred to a cuvette, and the FAH assay started by addition of purified Plus Mini Kit (Qiagen catalog #74134) after DNase treatment (Ambion). A total FAH protein or liver homogenate. The enzymatic activity was monitored at of 500 ng of DNase-treated total RNA was reverse-transcribed using Taqman OD 330 nm every 15 s for 10 min on a NanoDrop 2000c (Thermo Scientific). reverse transcription reagent (Roche). The cDNA was diluted 100 times, and the equivalent of 2 ng of total RNA was applied to each qRT-PCR with a total Liquid Chromatography–Mass Spectrometry. A minimum of 300 μL of whole volume of 10 μL. RNA expression was quantified by using SYBR green qRT-PCR blood was drawn from tail veins and then centrifuged for 10 min at 10,000 × g analysis. Primer sequences are described in SI Appendix,TableS1. to collect plasma. One volume of methanol was added to the plasma to pre- cipitate protein, vortexed vigorously for 15 s, and then spun at 13,000 × g for Cloning and Expression of WT and swst Mutant FAH. The cDNA collected as 5 min in a microcentrifuge to pellet the protein. Supernatants were removed described above was used as template to PCR-amplify WT and swst mutant FAH and recentrifuged. Both absolute signal intensity and percentage of recovery as coding sequences. The PCR product was gel-extracted using a Gel Extraction compared to spiked samples were optimized. Formic acid was added to the Kit (Qiagen) and cloned into EcoRI-digested pcDNA3.1 (for mammalian cell solution for a final concentration of 0.1%, and internal standard compounds expression) or pGST-Parallel-1 [for BL21(DE3) expression] vectors using the In- (tyrosine, methionine, phenylalanine, and succinylacetone) were added for a Fusion HD Cloning System (Clontech, catalog #639645), resulting in pcDNA- final concentration of 25 to 50 ng/mL. Supernatants were directly analyzed FAHw (WT) and pcDNA-FAHs (swst mutant) and pGST-FAHw and pGST-FAHs. using High Performance Liquid Chromatography (Prominence LC20/SIL-20AC; For mammalian expression, the pcDNA-FAH plasmids were transfected into Shimadzu) coupled to a triple quadruple mass spectrometer (3200 QTRAP; AB HEK293T/17 cells (American Type Culture Collection CRL11268) using Lip- SCIEX). Metabolites were separated chromatographically on a C18-based col- ofectamine 2000 (Thermo Fisher, 11668027). Stable cells were selected in the umn with polar embedded groups (150 × 2.0 mm at 4 μm; Synergi Fusion; presence of 500 μg/mL Geneticin (Gibco 11811–031) for 2 wk. For BL21(DE3) Phenomenex) using a tributylammonium acetate–methanol or formic acid– expression, DNA was transformed into BL21(DE3) cells, grown in Luria–Bertani methanol gradient and subjected to tandem mass spectrometry analysis. The medium with 100 μg/mL ampicillin. Cells were induced with 0.4 mM isopropyl- flow rate was set at 0.5 mL/min using the following method: buffer A (10 mM

β-D-thiogalactoside at a cell density of 0.6 (OD600), and protein was expressed tributylamine adjusted with 15 mM acetic acid to pH 5.0 or 0.1% formic acid) at 16 °C for 20 h. The expressed proteins were purified with glutathione and buffer B (100% methanol or 0.1% formic acid in methanol). Concentra- agarose beads (Sigma #G4510). tions were as follows: t = 0 min, 0% buffer B; t = 2 min, 0% buffer B; t = 13 min, 100% buffer B; t = 15 min, 100% buffer B; t = 16 min, 0% buffer B, t = 20 min, Western Blotting. Mouse tissues were dissected and snap-frozen in liquid 0% buffer B; and t = 20.1 min, stop. The best multiple reaction monitoring nitrogen before storing at −80 °C. Approximately 100 mg of tissue was transitions for each compound were identified through quantitative optimi- homogenized using a pellet mixer in 1 mL RIPA buffer [50 mM Tris–Cl, zation. Peaks were quantitated and normalized against estimates of the total 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxylcholate, 0.1% SDS, ion count using Analyst 1.4 software (AB SCIEX). 1 mM ethylenediaminetetraacetic acid, 10 mM NaF, 1× proteinase inhibitor mixture (Sigma), pH 7.5]. Fibroblast or HEK293T cells cultured on 100-mm Organotypic Cultures and Luminescence Recording. The swst heterozygous dishes were collected by trypsinization and homogenized in 0.2 mL RIPA mice were crossed to mPer2Luc knock-in mice to introduce the reporter al- buffer using pellet mixer. Tissue or cell debris was removed by centrifuga- lele into the swst background. The littermates heterozygous for mPer2Luc tion at 14,000 × g for 10 min at 4 °C. Bradford assays (Thermo Scientific were used to monitor the central clock in vitro as previously described (45).

23200) or a Nanodrop were used to determine protein concentrations in the Briefly, 1 h before lights off, mice were anesthetized with CO2 and decapi- supernatants. After denaturation, the samples were resolved on sodium tated, and their brains were rapidly removed. Coronal sections of the brain dodecyl sulfate/polyacrylamide gel electrophoresis and then transferred (300-μm thickness) were sliced with a Vibratome in ice-cold Hank’s Balanced

Yang et al. PNAS | October 29, 2019 | vol. 116 | no. 44 | 22235 Downloaded by guest on October 2, 2021 Salt Solution (Invitrogen). Slices containing the SCN were examined under a Student’s t tests were used to identify genotype effects. For body-weight dissection microscope, and the dissected SCNs were cultured on Millicell culture data, repeated measures 1-way ANOVAs were used to identify differences membranes (PICM ORG 50, Millipore) with 1.2 mL DMEM (Invitrogen), between groups over time. For aging data, linear regression was performed, supplemented with 10 mM Hepes (pH 7.2), 0.035% NaHCO3,2%B27 and r was calculated using Pearson’s correlations. For all statistical tests, μ (Invitrogen), 25 units/mL penicillin, 25 g/mL streptomycin, and 0.1 mM significance was set at P < 0.05. luciferin (Promega) in 35-mm petri dishes. Luminescence signals were recorded using a Lumicycle machine (Actimetrics). Period and phase were ACKNOWLEDGMENTS. We thank Dr. Guocun Huang, Lisa Thomas, Delali calculated using the LumiCycle data-analysis program (Actimetrics). Bassowou, and Izabela Kornblum for technical assistance. This work was funded by the National Institute of Mental Health grants U01 MH61915 and Statistical Analyses. All statistics were carried out using Graphpad Prism. For R01 MH078024 (J.S.T.). J.S.T. is an investigator at the Howard Hughes behavioral, body composition, and biochemical data, one-way ANOVA or Medical Institute.

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