The 1.6-megabase deletion at chromosome 3q29 (3q29Del) is the strongest identified genetic risk factor for schizophrenia, but the effects of this variant on neurodevelopment are not well understood. We interrogated the developing neural transcriptome in two experimental model systems with complementary advantages: isogenic human cortical organoids and isocortex from the 3q29Del mouse model.We profiled transcriptomes from isogenic cortical organoids that were aged for 2 and 12 months, as well as perinatal mouse isocortex, all at single-cell resolution. Systematic pathway analysis implicated dysregulation of mitochondrial function and energy metabolism. These molecular signatures were supported by analysis of oxidative phosphorylation protein complex expression in mouse brain and assays of mitochondrial function in engineered cell lines, which revealed a lack of metabolic flexibility and a contribution of the 3q29 gene PAK2. Together, these data indicate that metabolic disruption is associated with 3q29Del and is conserved across species.

Neurodevelopmental disorders offer insight into synaptic mechanisms. To unbiasedly uncover these mechanisms, we studied the 22q11.2 syndrome, a recurrent copy number variant, which is the highest schizophrenia genetic risk factor. We quantified the proteomes of 22q11.2 mutant human fibroblasts from both sexes and mouse brains carrying a 22q11.2-like defect, Df(16)A+/-. Molecular ontologies defined mitochondrial compartments and pathways as some of top ranked categories. In particular, we identified perturbations in the SLC25A1-SLC25A4 mitochondrial transporter interactome as associated with the 22q11.2 genetic defect. Expression of SLC25A1-SLC25A4 interactome components was affected in neuronal cells from schizophrenia patients. Furthermore, hemideficiency of the Drosophila SLC25A1 or SLC25A4 orthologues, dSLC25A1-sea and dSLC25A4-sesB, affected synapse morphology, neurotransmission, plasticity, and sleep patterns. Our findings indicate that synapses are sensitive to partial loss of function of mitochondrial solute transporters. We propose that mitoproteomes regulate synapse development and function in normal and pathological conditions in a cell-specific manner.

Rare neurological diseases shed light onto universal neurobiological processes. However, molecular mechanisms connecting genetic defects to their disease phenotypes are elusive. Here, we obtain mechanistic information by comparing proteomes of cells from individuals with rare disorders with proteomes from their disease-free consanguineous relatives. We use triple-SILAC mass spectrometry to quantify proteomes from human pedigrees affected by mutations in ATP7A, which cause Menkes disease, a rare neurodegenerative and neurodevelopmental disorder stemming from systemic copper depletion. We identified 214 proteins whose expression was altered in ATP7A −/y fibroblasts. Bioinformatic analysis of ATP7A-mutant proteomes identified known phenotypes and processes affected in rare genetic diseases causing copper dyshomeostasis, including altered mitochondrial function. We found connections between copper dyshomeostasis and the UCHL1/PARK5 pathway of Parkinson disease, which we validated with mitochondrial respiration and Drosophila genetics assays. We propose that our genealogical “omics” strategy can be broadly applied to identify mechanisms linking a genomic locus to its phenotypes. Rare genetic diseases provide fundamental insight into important biological questions and common diseases. Zlatic et al. present a strategy, termed genealogical proteomics, to investigate the molecular manifestations and mechanism of disease by comparing within a family samples from normal and affected subjects. Using this approach, the authors study a rare copper metabolism genetic disorder and find tantalizing connections with molecules and mechanisms known in Parkinson disease. Thus, genealogical proteomics is a promising approach for biological discovery and precision medicine.

We report the functional and temporal relationship between cellular phenotypes such as oxidative stress, p38MAPK-dependent responses and genomic instability persisting in the progeny of cells exposed to sparsely ionizing low-Linear Energy Transfer (LET) radiation such as X-rays or high-charge and high-energy (HZE) particle high-LET radiation such as 56Fe ions. We found that exposure to low and high-LET radiation increased reactive oxygen species (ROS) levels as a thresholdlike response induced independently of radiation quality and dose. This response was sustained for two weeks, which is the period of time when genomic instability is evidenced by increased micronucleus formation frequency and DNA damage associated foci. Indicators for another persisting response sharing phenotypes with stress-induced senescence, including beta galactosidase induction, increased nuclear size, p38MAPK activation and IL-8 production, were induced in the absence of cell proliferation arrest during the first, but not the second week following exposure to high-LET radiation. This response was driven by a p38MAPK-dependent mechanism and was affected by radiation quality and dose. This stress response and elevation of ROS affected genomic instability by distinct pathways. Through interference with p38MAPK activity, we show that radiation-induced stress phenotypes promote genomic instability. In contrast, exposure to physiologically relevant doses of hydrogen peroxide or increasing endogenous ROS levels with a catalase inhibitor reduced the level of genomic instability. Our results implicate persistently elevated ROS following exposure to radiation as a factor contributing to genome stabilization.

Base excision repair (BER), which is initiated by DNA N-glycosylase proteins, is the frontline for repairing potentially mutagenic DNA base damage. The NTHL1 glycosylase, which excises DNA base damage caused by reactive oxygen species, is thought to be a tumor suppressor. However, in addition to NTHL1 loss-of-function mutations, our analysis of cancer genomic datasets reveals that NTHL1 frequently undergoes amplification or upregulation in some cancers. Whether NTHL1 overexpression could contribute to cancer phenotypes has not yet been explored. To address the functional consequences of NTHL1 overexpression, we employed transient overexpression. Both NTHL1 and a catalytically-dead NTHL1 (CATmut) induce DNA damage and genomic instability in non-transformed human bronchial epithelial cells (HBEC) when overexpressed. Strikingly, overexpression of either NTHL1 or CATmut causes replication stress signaling and a decrease in homologous recombination (HR). HBEC cells that overexpress NTHL1 or CATmut acquire the ability to grow in soft agar and exhibit loss of contact inhibition, suggesting that a mechanism independent of NTHL1 catalytic activity contributes to acquisition of cancer-related cellular phenotypes. We provide evidence that NTHL1 interacts with the multifunctional DNA repair protein XPG suggesting that interference with HR is a possible mechanism that contributes to acquisition of early cellular hallmarks of cancer.

Genetic and environmental factors, such as metals, interact to determine neurological traits. We reasoned that interactomes of molecules handling metals in neurons should include novel metal homeostasis pathways. We focused on copper and its transporter ATP7A because ATP7A null mutations cause neurodegeneration. We performed ATP7A immunoaffinity chromatography and identified 541 proteins co-isolating with ATP7A. The ATP7A interactome concentrated gene products implicated in neurodegeneration and neurodevelopmental disorders, including subunits of the Golgi-localized conserved oligomeric Golgi (COG) complex. COG null cells possess altered content and subcellular localization of ATP7A and CTR1 (SLC31A1), the transporter required for copper uptake, as well as decreased total cellular copper, and impaired copper-dependent metabolic responses. Changes in the expression of ATP7A and COG subunits in Drosophila neurons altered synapse development in larvae and copper-induced mortality of adult flies. We conclude that the ATP7A interactome encompasses a novel COG-dependent mechanism to specify neuronal development and survival.

The Biogenesis of Lysosome-Related Organelles Complex 1 (BLOC-1) is a protein complex containing the schizophrenia susceptibility factor dysbindin, which is encoded by the gene DTNBP1. However, mechanisms engaged by dysbindin defining schizophrenia susceptibility pathways have not been quantitatively elucidated. Here, we discovered prevalent and novel cellular roles of the BLOC-1 complex in neuronal cells by performing large-scale Stable Isotopic Labeling of Cells in Culture quantitative proteomics (SILAC) combined with genetic analyses in dysbindin-null mice (Mus musculus) and the genome of schizophrenia patients. We identified 24 proteins that associate with the BLOC-1 complex many of which were altered in content/distribution in cells or tissues deficient in BLOC-1. New findings include BLOC-1 interactions with the COG complex, a Golgi apparatus tether, and antioxidant enzymes peroxiredoxins 1-2. Importantly, loci encoding eight of the 24 proteins are affected by genomic copy number variation in schizophrenia patients. Thus, our quantitative proteomic studies expand the functional repertoire of the BLOC-1 complex and provide insight into putative molecular pathways of schizophrenia susceptibility.

Reactive oxygen species (ROS) are involved in amplifying the radiation-induced damage to macromolecules as well as in downstream early responses leading to DNA repair or cell death and in delayed responses leading to tissue damage and/or tumorigenesis. We found in immortalized normal human bronchial epithelial cells (HBEC-3KT) following exposure to 1 Gy low (X-rays) or high (Fe ions) LET radiation that ROS levels persist elevated for up to eight population doublings (2 weeks) in the progeny of cells that repaired the radiation-induced damage and survived. To evaluate the function of persistently elevated ROS levels, we interrogated cells over the same period for genomic instability, proliferation and senescence biomarkers and evaluated whether ROS are functionally related to any of these phenotypes. We found that increased ROS production overlapped temporally with the persistence of reporters for genomic instability, increased micronucleus frequency and the presence of γH2AX-53BP1 positive ionizing radiation-induced foci. Although low LET radiation at high doses and high LET radiation induced a senescence-like phenotype dependent on ATM and p38 MAPK activity, this pathway was not a causative factor in ROS generation and did not affect cell proliferation rates at day 7 post-irradiation. On the contrary, when this pathway was inhibited, ROS levels increased further and micronucleus formation frequency was reduced. Because these results suggest a positive effect for increased ROS levels in the resolution of persistent micronucleus formation, their role was further confirmed by exposure to exogenous hydrogen peroxide, which resulted in a reduced micronucleus frequency. These results implicate ROS as an effector in the resolution of genomic instability and suggest that the senescence phenotype induced by high LET radiation or high low LET radiation dose promotes genomic instability by interfering with the cellular mechanism regulating ROS levels or the DNA repair machinery.

DNA double-strand break (DSB) repair by homologous recombination (HR) is initiated by CtIP/MRN-mediated DNA end resection to maintain genome integrity. SAMHD1 is a dNTP triphosphohydrolase, which restricts HIV-1 infection, and mutations are associated with Aicardi-Goutières syndrome and cancer. We show that SAMHD1 has a dNTPase-independent function in promoting DNA end resection to facilitate DSB repair by HR. SAMHD1 deficiency or Vpx-mediated degradation causes hypersensitivity to DSB-inducing agents, and SAMHD1 is recruited to DSBs. SAMHD1 complexes with CtIP via a conserved C-terminal domain and recruits CtIP to DSBs to facilitate end resection and HR. Significantly, a cancer-associated mutant with impaired CtIP interaction, but not dNTPase-inactive SAMHD1, fails to rescue the end resection impairment of SAMHD1 depletion. Our findings define a dNTPase-independent function for SAMHD1 in HR-mediated DSB repair by facilitating CtIP accrual to promote DNA end resection, providing insight into how SAMHD1 promotes genome integrity and prevents disease, including cancer.

Genes mutated in monogenic neurodevelopmental disorders are broadly expressed. This observation supports the concept that monogenic neurodevelopmental disorders are systemic diseases that profoundly impact neurodevelopment. We tested the systemic disease model focusing on Rett syndrome, which is caused by mutations in MECP2 . Transcriptomes and proteomes of organs and brain regions from Mecp2 -null mice as well as diverse MECP2 -null human cells were assessed. Widespread changes in the steady-state transcriptome and proteome were identified in brain regions and organs of presymptomatic Mecp2 -null male mice as well as mutant human cell lines. The extent of these transcriptome and proteome modifications was similar in cortex, liver, kidney, and skeletal muscle and more pronounced than in the hippocampus and striatum. In particular, Mecp2 - and MECP2 -sensitive proteomes were enriched in synaptic and metabolic annotated gene products, the latter encompassing lipid metabolism and mitochondrial pathways. MECP2 mutations altered pyruvate-dependent mitochondrial respiration while maintaining the capacity to use glutamine as a mitochondrial carbon source. We conclude that mutations in Mecp2 / MECP2 perturb lipid and mitochondrial metabolism systemically limiting cellular flexibility to utilize mitochondrial fuels.