Association of Mitochondrial Biogenesis With Variable Penetrance of Schizophrenia

Jianping Li; Oanh T Tran; T Blaine Crowley; Tyler M Moore; Elaine H Zackai; Beverly S Emanuel; Donna M McDonald-McGinn; Raquel E Gur; Douglas C Wallace; Stewart A Anderson

doi:10.1001/jamapsychiatry.2021.0762

. 2021 May 19;78(8):1–11. doi: 10.1001/jamapsychiatry.2021.0762

Association of Mitochondrial Biogenesis With Variable Penetrance of Schizophrenia

Jianping Li ¹, Oanh T Tran ², T Blaine Crowley ², Tyler M Moore ³, Elaine H Zackai ^2,⁴, Beverly S Emanuel ^2,⁴, Donna M McDonald-McGinn ^2,⁴, Raquel E Gur ^1,³, Douglas C Wallace ^4,⁵, Stewart A Anderson ^1,^3,^✉

¹Department of Child and Adolescent Psychiatry and Behavioral Sciences, The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania

²Division of Human Genetics, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania

³Department of Psychiatry, Perelman School of Medicine, University of Pennsylvania, Philadelphia

⁴Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia

⁵Center for Mitochondrial and Epigenomic Medicine, Children's Hospital of Philadelphia, Philadelphia

^✉

Corresponding Author: Stewart A. Anderson, MD, ARC 517, 3615 Civic Center Blvd, Philadelphia, PA 19104 (sande@pennmedicine.upenn.edu).

Accepted for Publication: March 17, 2021.

Published Online: May 19, 2021. doi:10.1001/jamapsychiatry.2021.0762

Author Contributions: Drs Li and Anderson had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.

Concept and design: Li, Moore, McDonald-McGinn, Gur, Wallace, Anderson.

Acquisition, analysis, or interpretation of data: All authors.

Drafting of the manuscript: Li, Emanuel, McDonald-McGinn, Anderson.

Critical revision of the manuscript for important intellectual content: All authors.

Statistical analysis: Li, Moore, McDonald-McGinn, Anderson.

Obtained funding: Gur, Wallace, Anderson.

Administrative, technical, or material support: Li, Tran, Crowley, Moore, Emanuel, McDonald-McGinn, Gur, Anderson.

Supervision: Moore, McDonald-McGinn, Gur, Anderson.

Conflict of Interest Disclosures: Dr Wallace reported nonfinancial support from the National Institutes of Health during the conduct of the study; being a consultant for Pano Therapeutics, other support from MitoCUREia and Mitrios; and personal fees from various lectures (all <$1000) outside the submitted work; in addition, Dr Wallace had a patent for 62/658,303 pending. No other disclosures were reported.

Funding/Support: This work was supported by National Institutes of Health grants MH110185 and MH066912 (Dr Anderson), MH108592 and MH110185 (Dr Wallace), MH191719 (Drs McDonald-McGinn, Emanuel, and Gur), MH087636 (Drs McDonald-McGinn and Emanuel), MH119738, MH119219, and MH101719 (Dr Gur), and the Penn/CHOP Lifespan Brain Institute.

Role of the Funder/Sponsor: The funding sources had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.

Additional Contributions: We thank Cecilia Giulivi, MS, PhD, University of California, Davis, for critical reading of the manuscript.

^✉

Corresponding author.

PMCID: PMC8135063 PMID: 34009292

Key Points

Question

Could variable penetrance for schizophrenia in 22q11.2 deletion syndrome (22q11.2DS) be associated with variable capacity for compensation for deficits associated with hemizygosity of mitochondrial-functioning genes in the deleted region?

Findings

In this study, in induced pluripotent stem cell–derived neurons, the presence of 22q11DS and schizophrenia was associated with reduced mitochondrial function, which was reversible by agents that enhance mitochondrial biogenesis. 22q11.2 Deletion syndrome without schizophrenia was associated with an increase in expression of genes encoding mitochondrial-functioning proteins and of regulators of these genes, relative to both 22q11DS with schizophrenia and to control individuals.

Meanings

Failure of compensatory mitochondrial biogenesis may be an actionable risk factor for schizophrenia in 22q11DS.

Abstract

Importance

Discovery of mechanisms that underlie variable penetrance for neuropsychiatric illness in the context of genetic variants that carry elevated risk can advance novel treatment approaches for these disorders.

Objective

To test the hypothesis that mitochondrial compensation is associated with the variable penetrance of schizophrenia in the 22q11.2 deletion syndrome (22q11DS).

Design, Setting, and Participants

This case-control study compared measures of mitochondrial function and the expression of related genes in 14 induced pluripotent stem cell–derived neurons from typically developing control individuals (6 lines) and from adults with 22q11DS (8 lines). The individuals with 22q11DS included 2 groups, those carrying a diagnosis of schizophrenia and those without this diagnosis (4 lines each). Similar measures were made of lymphoblastic cells lines (LCLs) from a separate group of adults with 22q11DS with (10 lines) or without (8 lines) schizophrenia. The study included samples derived from a clinical setting. The induced pluripotent stem cell lines were derived from individuals with 22q11DS with or without a diagnosis of schizophrenia at Stanford University. The LCLs were from adults within the 22q and You Center at the Children’s Hospital of Philadelphia. Data were analyzed between July 1, 2019, and January 24, 2021.

Main Outcomes and Measures

Total adenosine triphosphate (ATP), oxidative phosphorylation (OXPHOS) complex activity, and messenger RNA expression via reverse transcription–polymerase chain reaction of selected genes encoding for mitochondrial proteins.

Results

Study participants included men and women aged 18 to 37 years. Of 32 participants, the mean (SD) age of men was 27 (1.9) years and of women was 29 (1.2) years. Replicating a previous study, neurons from the 22q11DS and schizophrenia (22q+Sz) group had reduced ATP levels (mean [SD], 15.6 [1.5] vs 21.9 [1.4]; P = .02) and reduced OXPHOS activity (ie, complex I; 1.51 [0.1] vs 1.89 [0.1]; P = .01). These deficits were not present in neurons from individuals with 22q11DS without schizophrenia (22q[−]Sz). In this group, the expression of multiple genes encoding OXPHOS subunits was significantly upregulated. For example, compared with control individuals, NDUFV2 expression was increased by 50% in the 22q(−)Sz group (P < .001) but not significantly changed in the 22q+Sz group. Expression of genes driving mitochondrial biogenesis, including PGC1α, showed a similar pattern of upregulation in the 22q(−)Sz group compared with the control and the 22q+Sz groups. Stimulation of mitochondrial biogenesis normalizes the ATP deficit seen in 22q+Sz neurons. Finally, using LCLs from a separate group of adults with 22q11DS, evidence for enhanced mitochondrial biogenesis was again found in the 22q(−)Sz group.

Conclusions and Relevance

In this study, an increase in mitochondrial biogenesis and function was associated with the absence of schizophrenia in neurons and LCLs from individuals with 22q11DS, but the deficit in the 22q+Sz group was reversible by agents that enhance mitochondrial biogenesis. Enhancement of mitochondrial biogenesis may provide a targetable opportunity for treatment or prevention of this disorder in individuals with 22q11DS.

This study tests the hypothesis that mitochondrial compensation is associated with the variable penetrance of schizophrenia in the 22q11.2 deletion syndrome.

Introduction

Schizophrenia is a heterogeneous disorder generally characterized by adolescent onset of psychosis together with deteriorations of social and cognitive functioning.^1,2 Metabolic compromise, including mitochondrial dysfunction, has long been posited to contribute to the development of symptoms in schizophrenia.^3,4 Studies published in the last 6 years have further strengthened this notion. For example, gene expression alterations in postmortem neocortical pyramidal neurons suggest disruption of mitochondrial function,^5,6 and other studies reported mitochondrial defects in neural progenitors, cerebral organoids, and cortical interneurons derived from induced pluripotent stem cells (iPSCs) from patients with schizophrenia compared with healthy control individuals.^7,8,9 However, despite the potential importance to the discovery of novel treatments for schizophrenia, evidence for mitochondrial dysfunction in schizophrenia in living, human neurons with a defined genetic alteration has been lacking.

One of the most common genetic risk factors for schizophrenia is the 22q11.2 deletion syndrome (22q11DS),¹⁰ occurring in approximately 1 in 4000 births and in 1 in 1000 pregnancies.^11,12 Roughly one-quarter of people with 22q11DS develop schizophrenia in a manner that is similar to nonsyndromic schizophrenia,^13,14 reflecting a 25-fold increase in schizophrenia risk compared with the general population. In 22q11DS, 6 of roughly 46 deleted genes encode for proteins that are mitochondrial localizing, and 4 others strongly influence mitochondrial function.^15,16 One study has found evidence for mitochondrial dysfunction in lymphocytes and plasma samples from children with 22q11DS,¹⁶ while another used proteomic analyses in fibroblasts from patients with 22q11DS, together with analyses of 22q model transgenic mice and flies to conclude that mitochondrial SLC25A1/A4 and TXNRD2 influence synaptic function.¹⁷ The importance of TXNRD2 in long-range cortical connectivity and psychosis-related cognitive deficits was further established in transgenic mice.¹⁸ Finally, MRPL40 was identified as a candidate schizophrenia risk gene,¹⁹ and transgenic mice lacking 1 copy of Mrpl40 show alterations in mitochondrial calcium as well as psychosis-related cognitive deficits.²⁰

We found mitochondrial dysfunction in iPSC-derived neurons from individuals with 22q11DS with schizophrenia (22q+Sz) vs healthy control individuals.²¹ Patient-derived neurons had reduced adenosine triphosphate (ATP) levels and reduced activity of complexes I and IV of the electron transport chain (ETC). In addition, the levels of multiple mitochondrial-translated proteins were reduced, and these findings were duplicated in an iPSC line that we edited to be heterozygous for MRPL40. Together with the previous literature, these results suggested that mitochondrial deficits may contribute to the development of schizophrenia in the context of 22q11DS, but the more important comparison is between 22q+Sz and 22q(−)Sz. In fact, a study of serum samples suggested that mitochondrial dysfunction in 22q11.2 is exacerbated in those with psychosis.²²

Here, using a different set of iPSC lines, we replicated the core findings in our initial article²¹ of reduced ATP levels and reduced complex I and IV activities in neurons from 22q+Sz vs control individuals. Our new results suggest that variable penetrance to the development of schizophrenia is influenced by compensatory mitochondrial biogenesis that occurs in patients with 22q11DS without schizophrenia. Stimulation of mitochondrial biogenesis may provide a novel treatment or prevention opportunity for the development of schizophrenia in 22q11DS.

Methods

Human iPSCS and LCLs

Approval for this study was obtained from the Stanford and Children’s Hospital of Philadelphia institutional review board/ethics committee, and informed consent was signed by all participants. Human iPSCs were contributed by Sergiu P. Pasca, MD (Stanford University; eTable 1 in the Supplement).²³ All lines had SALSA MLPA KIT P250-A1 DiGeorge (MRC Holland) testing for the 22q11.2 deletion to confirm genotype. Lines were tested for mycoplasma infection monthly and at each freezing.

Lymphoblast lines (LCLs; eTable 2 in the Supplement) were grown in RPMI-1640 medium supplemented with 15% fetal bovine serum and 2nM L-glutamine. They were testing for mycoplasma at initial expansion and then at freezing.

Lentiviral Vector Generation

Plasmid of VSVG.HIV.SIN.cPPT.CMV.mNgn2. WPRE and VSVG.HIV.SIN. cPPT.CMV.rTTA. WPRE were provided by Marius Wernig, MD (Stanford University); the plasmid was packaged into lentivirus by the CHOP vector core.

Neuronal Differentiation of iPSCs

Neuronal differentiation was performed using established methods with minor modification.^21,24 The iPSC lines were infected with lentiviral vectors using lentiblast (OZ Bioscience): TetO-mNgn2-T2A-PuroR and Ubiq-rTTA. Differentiation was initiated with doxycycline (2 μg/mL; Sigma), followed by puromycin (5 μg/mL; Sigma) selection, then cells replated into Neurobasal medium (Gibco) with B27 (Life Technologies) and doxycycline until differentiation day (DD) 10.

RNA Extraction, Reverse Transcription, and Quantitative Polymerase Chain Reaction

Trizol (Ambion), chloroform, isopropanol, and ethanol were used to extract and purify total RNA. This was reverse transcript using transcript IV VILO Master Mix (ThermoFisher). Complementary DNA abundance was measured by quantitative polymerase chain reaction using TaqMan Gene Expression Master Mix (Applied Biosystems) for primer pairs of ACTIN, GAPDH, and MT-CO1 purchased from ThermoFisher Scientific and SYBR Green PCR Mix (Applied Biosystems). Primer sequences are listed in eTable 3 in the Supplement.

Measurement of Mitochondria Oxidative Phosphorylation Activity and ATP Levels

Mitochondrial ETC enzyme activity were measured using complex I enzyme activity microplate assay kit (colorimetric) (ab109721; Abcam), complex IV human enzyme activity microplate assay kit (ab109909; Abcam), and ATP synthase enzyme activity microplate assay kit (ab109714; Abcam) following the manufacturer’s instruction. The ATP levels were quantified on a Luminescence plate reader using an ATP Bioluminescence Assay Kit (Abcam).

Mitochondrial DNA Copy Number

All reactions were performed in fast optical 96-well reaction plates with Barcode (Applied Biosystems) on an SDS7900HT system; each sample was analyzed in triplicate. Relative mitochondrial DNA (mtDNA) copy number (mtDNA amount/nDNA amount) was calculated by a comparative threshold cycle method, using the following equation: mtDNA/nDNA = 2^−ΔCt. Primer sequences are listed in eTable 3 in the Supplement.

Treatment of Neurons With Bezafibrate and miR-181a/b Inhibitor

For bezafibrate (5μM; Sigma 72516), iPSCs-derived neurons (iNrns) were treated at DD 21 and again at DD 24, then samples were collected at DD 26. For miR-181a/b antagomir, lipofectamine RNAiMAX Transfection Reagent (ThermoFisher Scientific, 13778075) was used to deliver 100nM siRNA into iNrns following the manufacturer’s recommendations. Either miRIDIAN microRNA Hairpin Inhibitor Negative Control #1 (IN-001005-01-05; Horizon) or a mix of -miR-181a-5p inhibitor (IH-300552-07) and miR-181b-5p inhibitor (IH-300554-08) was applied at DD 21. Cells were harvested at DD 24.

Statistical Analysis

Experimental N refers to the number of iPSCs lines from different individuals of each group (control n = 6; 22q[−]Sz n = 4; 22q+Sz n = 4) used in each experiment and number of LCL lines from individuals of each group (22q-Sz n = 8; 22q+Sz n = 10). Each data point is the mean of at least 2 technical replicates.

Data were presented as mean (SEM). Unpaired 2-tailed t test was applied for data analysis. A 2-sided P value of less than .05 was considered a significant difference. False discovery rate corrections are included in the figure legends and in eTable 4 in the Supplement.

Results

Lack of Mitochondrial ETC Deficits in iPSC-Derived Neurons From Individuals With 22q11DS(-)Sz

Our previous study uncovered deficits of ATP production and reduced ETC (complex I/IV) enzyme activity in iNrns from patients with 22q+Sz compared with those from healthy control individuals.²¹ To determine whether these associations were associated with the presence or absence of schizophrenia, we obtained additional iPSC lines (eTable 1 in the Supplement), including those from typically developing controls (n = 6), from individuals with 22q(−)Sz (n = 4), and from individuals with 22q+Sz (n = 4). Excitatory neurons were generated via transient expression of Neurogenin 2,^21,24 then evaluated after 3 weeks of differentiation.

Total ATP levels were reduced in the 22q+Sz group compared with the control group (Figure 1A; mean [SEM], 15.6 [1.5] vs 21.9 [1.4]; P = .02), replicating our previous study in a separate group of iPSC lines.²¹ This difference was also present when the 22q+Sz group was compared with the 22q(−)Sz group (Figure 1A; 15.6 [1.5] vs 21.8 [1.0]; P = .02), while the 22q(−)Sz and control groups did not differ. Complex I enzyme activity was also significantly reduced in the 22q+Sz group compared with the 22q(−)Sz group (Figure 1B; 1.51 [0.08] vs 1.89 [0.07]; P = .01), and complex IV enzyme activity showed a similar trend (Figure 1C; 2.05 [0.30] vs 2.96 [0.46]; P = .15). In addition, ATP synthase activity (complex V) was reduced in the 22q+Sz iNrns compared with the 22q(−)Sz group (Figure 1D; 3.58 [0.18] vs 4.86 [0.42]; P = .02), although not compared with the control group. These results suggest ETC deficits in iNrns from individuals with 22q11DS appear to be selectively present in those individuals who are also diagnosed with schizophrenia.

Evidence for Enhanced Mitochondrial Biogenesis in 22q(−)Sz iNrns Compared With Control and 22q+Sz Groups

To explore a potential mechanism for the lack of an ETC phenotype in the 22q(−)Sz iNrns, we next evaluated the expression of both nuclear and mitochondrial genes that encode components of the ETC. Both NDUFV1 (Figure 2A) and NDUFV2 (Figure 2B), nuclear components of complex I associated with schizophrenia,^25,26,27 were significantly elevated in the 22q(−)Sz group compared with both controls and the 22q+Sz group, which were unchanged compared with each other. Ubiquinone oxidoreductase core subunit S1 was not changed across the 3 groups (Figure 2C).

To determine whether mitochondrial DNA (mtDNA)–encoded genes were also differentially upregulated in the 22q(−)Sz group, we measured mRNA expression of complex I components MT-ND1 (Figure 2D), MT-ND2 (Figure 2E), MT-ND4 (Figure 2F), complex III component MT-CYB (Figure 2G), complex IV component MT-CO1 (Figure 2H), and complex V components MT-ATP6 and MT-ATP8 (Figure 2I and J). Strikingly, most of the genes tested that encode for ETC-component proteins were significantly upregulated in the 22q(−)Sz group compared with both the control and the 22q+Sz groups, which were consistently unchanged compared with each other.

Enhanced expression of nuclear DNA- and mtDNA-encoded ETC component genes in the 22q(−)Sz group (Figure 2) is suggestive of associated enhanced mitochondrial biogenesis that normalizes total ATP and complex I, IV, and V activity to control levels (Figure 1). Indeed, mtDNA content is elevated in this group compared with that from the 22q+Sz group (Figure 3A). To determine whether regulators of mitochondrial biogenesis are driving these associations, we examined PGC1α (Figure 3B) and PGC1β (Figure 3C), transcription factors that directly activate both nDNA and mtDNA ETC component genes, and their cofactor PPARα (Figure 3E). All 3 of these factors were upregulated in the 22q(−)Sz group compared with the control and 22q+Sz groups. PRKN was also selectively upregulated in the 22q(−)Sz group (Figure 3F). PRKN can activate PGC1α to support mitochondrial biogenesis.²⁸ The cofactor ERRα was not changed in the 22q(−)Sz group compared with the 22q+Sz group (Figure 3D). These results suggest that the absence of schizophrenia-related symptoms is associated, in iPSC-derived neurons, with normalized ETC function relative to 22q+Sz, and with enhanced mitochondrial biogenesis relative to both individuals with 22q+Sz and to typically developing control individuals.

Figure 3. — Dots show averaged data from duplicate experimental runs of independent induced pluripotent stem cell lines differentiated to induced pluripotent stem cell–derived neurons.^20,23 P values derive from 2-tailed t test. A, Elevated mitochondrial DNA copy number in the 22q11.2 deletion syndrome (22q11DS) without schizophrenia (22q[−]Sz) group compared with 22q11DS and schizophrenia (22q+Sz) but not controls. B-F, The mitochondrial biogenesis-related transcription factors *PGC1α*, *PGC1β*, and *PPARα* are also increased in the 22q(−)Sz group as is *PRKN* (F). D, A cofactor with *PGC1α*, *ERRα* was elevated in both 22q11DS groups compared with controls, albeit with a trend towards higher expression in the 22q(−)Sz group. All other potential modulators of mitochondrial biogenesis that were tested are shown in eFigure 1 in the Supplement.

Lymphoblastoid Cell Lines From Adults With 22q11DS Show Similar Differences Between Groups With or Without Schizophrenia-Related Symptoms as Seen in the iPSC-Derived Neurons

The pattern of mitochondrial compensation in the 22q(−)Sz iNrns suggests that the penetrance for schizophrenia-related phenotypes in the 22q11DS context may be influenced by an individual’s capacity for this compensation. In Leber hereditary optic neuropathy, a blindness developing by early adulthood caused by mutations in MT-ND4,²⁹ the unaffected carrier state is associated with elevated mtDNA copy number and expression of PGC1α in blood and enhanced mitochondrial function in cultured fibroblasts.^30,31 To determine whether mitochondria from a nonneural, more accessible source of cells from patients with 22q11DS can be used to differentiate between those individuals with and without schizophrenia-related symptoms, we studied LCLs from these 2 groups (eTable 2 in the Supplement). Groups were nearly matched for sex, although the 22q+Sz group was younger in terms of the age at which lymphocytes for generating the LCLs were collected (mean [SEM] 22q+Sz, 26 [2.5]; 22q(−)Sz, 34 [2.0]; P = .02). Importantly, because age is associated with reduced mitochondrial function, an age effect would be expected to reduce the differences between the groups. In fact, when adjusted for age, the group differences became greater.

In comparing the 22q(−)Sz and 22q+Sz groups, there was a similarity between data sets generated from iNrns and LCLs. Expression of all 7 mitochondrial-encoded genes tested was higher in the 22q(−) group (Figure 4A and D-I), as was 1 of 2 nuclear-encoded components of the ETC tested (NDUFV1, Figure 4B; NDUFV2, Figure 4C, was unchanged). This was not because of higher mitochondrial DNA content, which was unchanged between the groups (eFigure 3 in the Supplement). Complex I and complex IV activities were also higher in the 22q(−)Sz LCLs (Figure 4J and K). Complex V activity was not changed (Figure 4L). Positive modulators of mitochondrial biogenesis were also increased in the 22q(−)Sz group, including PPARα and PRKN, and NRF2 with a strong trend and PGC1α with a weaker trend (Figure 4M-P). All gene expression studies conducted on the LCLs are shown in eFigure 2 in the Supplement with the exception of miR-181a/b, and generally display trends for the same relative increase in the 22q(−)Sz group. Comparisons of P values, standardized by sex, with and without correction for false discovery rate, are included in eTable 4 in the Supplement.

Figure 4. — Data are from 18 LCL lines from individual patients with 22q11DS (eTable 2 in the Supplement). A-I, Messenger RNA reverse transcription–polymerase chain reaction data. B and C, Expression of the nuclear-encoded complex I gene *NDUFV1* is upregulated in the 22q(−)Sz group, and *NDUFV2* shows a strong trend for increase. C-I, Expressions of all tested mitochondrially encoded genes are increased in the 22(−)Sz group, including *MT-ND4* (A), *MT-ND1* (D), *MT-ND2* (E), *MT-CYB* (F), *MT-CO1* (*COX1*) (G), *MT-ATP6* (H), and *MT-ATP8* (I). J-L, Increased complex I and complex IV activity in the 22q(−)Sz group. Adenosine triphosphate synthase activity (L, complex V) was not changed. M-P, The mitochondrial biogenesis-related transcription factors *PGC1α* (trend only, P = .13), *PPARα*, and *NRF2* are also increased in the 22q(−)Sz group, as is *PRKN* (O).

Association of Activation of PGC1α With an Increase in ATP Production by iNrns From 22q+Sz

The association of enhanced mitochondrial biogenesis, in both iNrns and LCLs, with the absence of schizophrenia-related symptoms in 22q11DS, suggests that enhanced mitochondrial biogenesis may be a target for treatment or prevention of this disorder. To test this concept, we exposed iNrns from individuals with 22q+Sz to 2 conditions. First, we used an antagomir pool targeting miR-181a/b. miR-181a/b represses the expression of PGC1α along with multiple additional regulators of mitochondrial biogenesis.^32,33 Application of the miR-181a/b antagomir to DD 21 cultures significantly enhanced ATP levels and complex I activity by iNrns from the 22q+Sz group (Figure 5A and B). This increase was associated with enhanced expression of PGC1α, ND2, cytochrome B, and COX1 (Figure 5C-F). Similar associations on the same measures were seen with application of the FDA-approved PGC1α activator Bezafibrate (5 μm; Figure 5G-L; eFigures 4 and 5 in the Supplement). These results indicate that, in iNrns from the 22q+Sz group, the relatively reduced total ATP and the reduced expression of both gene-encoding proteins that function in the ETC as well as upstream regulators of those genes were reversible by activating PGC1α and other factors associated with mitochondrial biogenesis.

Figure 5. — Dots show data from independent 22q+Sz induced pluripotent stem cell lines differentiated to induced pluripotent stem cell–derived neurons (iNrn). A-F, miR-181 antagomir added to the iNrn culture media from differentiation day 21 to 23. A and B, Adenosine triphosphate (ATP) levels and complex I activity are increased in the 22q+Sz iNrns that received the miR-181 antagomir compared with the control antagomir. C-F, Reverse transcription–polymerase chain reaction measurements of expression levels of the indicated genes. C, *PGC1α* is elevated by the miR-181a/b antagomir. *MT-ND2* (D), *MT-CYB* (E), and *MT-CO1* (*COX1*) (F) are also elevated by the miR-181a/b antagomir. G-L, Bezafibrate (5 μm) or dimethyl sulfoxide only was added to the iNrn culture media on differentiation day 21 to 26. The same measures elevated by the miR-181a/b antagomir were elevated by bezafibrate.

Discussion

We reported that iPSC-derived neurons from patients with 22q11DS and schizophrenia have mitochondrial deficits relative to typically developing control individuals.²¹ Here, we replicated that finding in iNrns derived from a separate set of iPSCs but found that the deficiencies in the 22q+Sz group were not present in iNrns from patients with 22q11DS without schizophrenia (Figure 1). In this group, expression of nuclear genes encoding ETC subunits, and of multiple mitochondrially encoded genes, were upregulated, as was mitochondrial DNA content (Figure 2 and Figure 3). Expression of genes affecting mitochondrial biogenesis and function, such as PGC1α and PPARα, was also upregulated in iNrns from the 22q(−)Sz group relative to both the control and the 22q+Sz groups (Figure 3). Enhanced complex I and IV activity and an increase in expression of mRNAs associated with mitochondrial biogenesis were also found in LCLs from a separate group of adults with 22q11DS without schizophrenia vs those with schizophrenia (Figure 4). Finally, stimulation of mitochondrial biogenesis in iPSC-derived neurons with an antagomir for miR-181a/b, a repressor of PGC1α, and with application of bezafibrate that activates PGC1α, normalizes the ATP deficit seen in 22q+Sz neurons (Figure 5). We conclude that enhanced mitochondrial biogenesis and function were associated with the absence of schizophrenia in iPSC-derived neurons and in LCLs from patients with 22q11DS and that deficits in mitochondrial function in the 22q+Sz group were reversible by activating PGC1α and mitochondrial biogenesis.

Bioenergetics and mitochondrial function have long been posited to influence the development and course of schizophrenia,^34,35 but the heterogeneity of this disorder has complicated the rigorous study of this concept. Here we sought to reduce the associations of heterogeneity in multiple ways. First, we limited our analysis to the study of schizophrenia in the context of the 22q11DS, in which 6 of more than 40 directly affected genes encode for mitochondrial-localizing proteins,¹⁵ and in which there is a 25-fold increase in risk of schizophrenia.^10,36 In addition, studies of 22q11DS model mice and other experimental systems have implicated mitochondrial dysfunction in schizophrenia-related phenotypes,^17,18,20 and a study of RNA transcripts in blood suggested that schizophrenia in 22q11DS is associated with mitochondrial stress.²² Second, we limited our analysis to neurons induced by the transient expression of neurogenin 2 and to LCLs immortalized by the Epstein-Barr virus. While there are weaknesses associated with each method, both approaches greatly limit variability that can be especially problematic in the case of human iPSC-derived cells.

Given these efforts to limit genetic risk-related and experimental variability, a critical question is whether our rather striking results have important implications for understanding, treating, or preventing schizophrenia in the 22q11DS context, and in the context of the other 99% of people affected by schizophrenia who do not carry this deletion. On the first point, one finding that deserves particular emphasis was that both nuclear and mitochondrial DNA-encoded transcripts for ETC genes (Figure 2) as well as key factors in the transcriptional regulation of these genes (Figure 3) were significantly elevated in the 22q(−)Sz iNrns relative to both the 22q+Sz and the control groups. This scenario supports the notion that the 22q(−)Sz group is characterized by effective compensation for the various mitochondrial abnormalities that have long been documented in 22q-related models, rather than the alternative conclusion that having schizophrenia and also psychosis-related treatments results in metabolic abnormalities that are carried through the iPSC reprogramming and iNrn differentiation processes. Interestingly, a similar situation occurs in Leber hereditary optic neuropathy, an inherited blindness of young adults wherein unaffected carriers of the mutation in the ETC complex I gene MT-ND4 have enhanced mitochondrial biogenesis that is evident in peripheral tissues including monocytes and fibroblasts.^30,31,32 In light of our findings that enhancement of mitochondrial biogenesis via activation of PGC1α and related factors can improve ETC function in iNrns from the 22q+Sz group (Figure 5), the results of this study may profoundly affect future efforts to treat or prevent schizophrenia in the 22q11DS context. Importantly, iNrns are glutamatergic, projectionlike neurons that share some transcriptional similarities with layer III neocortical pyramidal neurons²⁴ that have been implicated in postmortem studies of nonsyndromic schizophrenia at the level of mitochondrial as well as synaptic deficits.^5,17,37 Mitochondrial abnormalities are also found in layer III pyramidal neurons of 22q11DS model mice.¹⁸ However, these studies should be extended to other brain cell types that have enhanced metabolic requirements and hence greater potential vulnerability to mitochondrial defects in 22q11DS, including the fast-spiking subclass of cortical interneurons, microglia, and the vascular endothelium of the blood-brain barrier. Abnormalities in each of these cell types have also been suggested to influence the development of schizophrenia.^{38,39,40,41,42}

On the second point of whether the implications of this study extend to the 99% of people affected by schizophrenia who do not carry the chromosome 22q11.2 deletion, evidence for mitochondrial abnormalities in postmortem neocortex and in iPSC-derived cortical neurons has been identified previously in nonsyndromic schizophrenia.^{5,7,8,9,17,43} Mitochondrial abnormalities have also been reported in both neural progenitors and inhibitory neurons derived from iPSCs from people with schizophrenia,^7,8 and bioenergetics-related genetic variants may increase risk of this disorder.⁴⁴ In addition, because the polygenic risk score based on nonsyndromic schizophrenia is elevated in 22q+Sz compared with 22q(−)Sz,⁴⁵ there may be mechanistic overlap in the etiologies of 22q11DS-related and nonsyndromic schizophrenia. Given the similar results we found with iNrns and LCLs as well as the widespread banking of LCLs from patients with neuropsychiatric disorders, our study provides the rationale for examining linkage between the presence of schizophrenia-related symptoms, elevated mitochondria-related polygenic risk score, and mitochondrial measures in LCLs. Such a trimodal approach could lead to the personalized rationale for prevention and treatment of some instances of nonsyndromic schizophrenia.

Limitations

The low number of iPSC lines and relatively low number of LCLs available for this study limit the capacity to test sex as a variable as well as medical comorbidities in relation to mitochondrial measures. In addition, the 22q(−)Sz iPSC group includes individuals aged 22, 25, and 26 years. Although the mean age at onset of schizophrenia-related symptoms in 22qDS is around 19 years (and unaffected by sex),⁴⁶ some of them may have subsequently developed these symptoms. Our conclusions may therefore apply more firmly to earlier-developing instances of schizophrenia.

Conclusions

These results suggest that in the context of the 22q11DS, in which schizophrenia occurs in roughly 25% of affected individuals, variable penetrance to the development of schizophrenia was associated with the capacity for compensatory mitochondrial biogenesis that occurs in those who carry this copy number variation but do not develop schizophrenia. Stimulation of mitochondrial biogenesis may provide a novel treatment or prevention opportunity for the development of schizophrenia in 22q11DS.

Supplement.

eTable 1. Human iPSC lines used in this study.

eTable 2. Lymphoblastoid cell lines (LCLs) from individuals with 22q11.2DS.

eTable 3. PCR Primers for RT-PCR experiments and mitochondrial DNA measurement

eTable 4. Comparison of T-test p values with and without correction by False Discovery Rate

eFigure 1. Additonal studies of mRNA expression of potential modulators of mitochondrial biogenesis.

eFigure 2. Lack of differences across groups in transcript levels of genes encoding for synaptic proteins.

eFigure 3. Additional studies in lymphoblastic cell lines (LCLs).

eFigure 4. Additional evidence that antagonism of miR-181a/b enhance mitochondrial biogenesis in iNrns from 22q(+)Sz.

eFigure 5. Additional evidence that activation of PGC1α by bezafibrate enhances mitochondrial biogenesis in iNrns from 22q(+)Sz.

Click here for additional data file.^{(805KB, pdf)}

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