Complement in the Brain: Contributions to Neuroprotection, Neuronal Plasticity, and Neuroinflammation.

The complement system is an ancient collection of proteolytic cascades with well-described roles in regulation of innate and adaptive immunity. With the convergence of a revolution in complement-directed clinical therapeutics, the discovery of specific complement-associated targetable pathways in the central nervous system, and the development of integrated multi-omic technologies that have all emerged over the last 15 years, precision therapeutic targeting in Alzheimer disease and other neurodegenerative diseases and processes appears to be within reach. As a sensor of tissue distress, the complement system protects the brain from microbial challenge as well as the accumulation of dead and/or damaged molecules and cells. Additional more recently discovered diverse functions of complement make it of paramount importance to design complement-directed neurotherapeutics such that the beneficial roles in neurodevelopment, adult neural plasticity, and neuroprotective functions of the complement system are retained.

Microglial-derived C1q integrates into neuronal ribonucleoprotein complexes and impacts protein homeostasis in the aging brain.

Neuroimmune interactions mediate intercellular communication and underlie critical brain functions. Microglia, CNS-resident macrophages, modulate the brain through direct physical interactions and the secretion of molecules. One such secreted factor, the complement protein C1q, contributes to complement-mediated synapse elimination in both developmental and disease models, yet brain C1q protein levels increase significantly throughout aging. Here, we report that C1q interacts with neuronal ribonucleoprotein (RNP) complexes in an age-dependent manner. Purified C1q protein undergoes RNA-dependent liquid-liquid phase separation (LLPS) in vitro, and the interaction of C1q with neuronal RNP complexes in vivo is dependent on RNA and endocytosis. Mice lacking C1q have age-specific alterations in neuronal protein synthesis in vivo and impaired fear memory extinction. Together, our findings reveal a biophysical property of C1q that underlies RNA- and age-dependent neuronal interactions and demonstrate a role of C1q in critical intracellular neuronal processes.

Ubiquitin Ligase COP1 Suppresses Neuroinflammation by Degrading c/EBPß in Microglia.

Dysregulated microglia are intimately involved in neurodegeneration, including Alzheimer's disease (AD) pathogenesis, but the mechanisms controlling pathogenic microglial gene expression remain poorly understood. The transcription factor CCAAT/enhancer binding protein beta (c/EBPß) regulates pro-inflammatory genes in microglia and is upregulated in AD. We show expression of c/EBPß in microglia is regulated post-translationally by the ubiquitin ligase COP1 (also called RFWD2). In the absence of COP1, c/EBPß accumulates rapidly and drives a potent pro-inflammatory and neurodegeneration-related gene program, evidenced by increased neurotoxicity in microglia-neuronal co-cultures. Antibody blocking studies reveal that neurotoxicity is almost entirely attributable to complement. Remarkably, loss of a single allele of Cebpb prevented the pro-inflammatory phenotype. COP1-deficient microglia markedly accelerated tau-mediated neurodegeneration in a mouse model where activated microglia play a deleterious role. Thus, COP1 is an important suppressor of pathogenic c/EBPß-dependent gene expression programs in microglia.

Human-Specific NOTCH2NL Genes Affect Notch Signaling and Cortical Neurogenesis.

Genetic changes causing brain size expansion in human evolution have remained elusive. Notch signaling is essential for radial glia stem cell proliferation and is a determinant of neuronal number in the mammalian cortex. We find that three paralogs of human-specific NOTCH2NL are highly expressed in radial glia. Functional analysis reveals that different alleles of NOTCH2NL have varying potencies to enhance Notch signaling by interacting directly with NOTCH receptors. Consistent with a role in Notch signaling, NOTCH2NL ectopic expression delays differentiation of neuronal progenitors, while deletion accelerates differentiation into cortical neurons. Furthermore, NOTCH2NL genes provide the breakpoints in 1q21.1 distal deletion/duplication syndrome, where duplications are associated with macrocephaly and autism and deletions with microcephaly and schizophrenia. Thus, the emergence of human-specific NOTCH2NL genes may have contributed to the rapid evolution of the larger human neocortex, accompanied by loss of genomic stability at the 1q21.1 locus and resulting recurrent neurodevelopmental disorders.

TREM2 receptor protects against complement-mediated synaptic loss by binding to complement C1q during neurodegeneration.

Triggering receptor expressed on myeloid cells 2 (TREM2) is strongly linked to Alzheimer's disease (AD) risk, but its functions are not fully understood. Here, we found that TREM2 specifically attenuated the activation of classical complement cascade via high-affinity binding to its initiator C1q. In the human AD brains, the formation of TREM2-C1q complexes was detected, and the increased density of the complexes was associated with lower deposition of C3 but higher amounts of synaptic proteins. In mice expressing mutant human tau, Trem2 haploinsufficiency increased complement-mediated microglial engulfment of synapses and accelerated synaptic loss. Administration of a 41-amino-acid TREM2 peptide, which we identified to be responsible for TREM2 binding to C1q, rescued synaptic impairments in AD mouse models. We thus demonstrate a critical role for microglial TREM2 in restricting complement-mediated synaptic elimination during neurodegeneration, providing mechanistic insights into the protective roles of TREM2 against AD pathogenesis.

COQ4 is required for the oxidative decarboxylation of the C1 carbon of coenzyme Q in eukaryotic cells.

Coenzyme Q (CoQ) is a redox lipid that fulfills critical functions in cellular bioenergetics and homeostasis. CoQ is synthesized by a multi-step pathway that involves several COQ proteins. Two steps of the eukaryotic pathway, the decarboxylation and hydroxylation of position C1, have remained uncharacterized. Here, we provide evidence that these two reactions occur in a single oxidative decarboxylation step catalyzed by COQ4. We demonstrate that COQ4 complements an Escherichia coli strain deficient for C1 decarboxylation and hydroxylation and that COQ4 displays oxidative decarboxylation activity in the non-CoQ producer Corynebacterium glutamicum. Overall, our results substantiate that COQ4 contributes to CoQ biosynthesis, not only via its previously proposed structural role but also via the oxidative decarboxylation of CoQ precursors. These findings fill a major gap in the knowledge of eukaryotic CoQ biosynthesis and shed light on the pathophysiology of human primary CoQ deficiency due to COQ4 mutations.

Polysome collapse and RNA condensation fluidize the cytoplasm.

The cell interior is packed with macromolecules of mesoscale size, and this crowded milieu significantly influences cellular physiology. Cellular stress responses almost universally lead to inhibition of translation, resulting in polysome collapse and release of mRNA. The released mRNA molecules condense with RNA-binding proteins to form ribonucleoprotein (RNP) condensates known as processing bodies and stress granules. Here, we show that polysome collapse and condensation of RNA transiently fluidize the cytoplasm, and coarse-grained molecular dynamic simulations support this as a minimal mechanism for the observed biophysical changes. Increased mesoscale diffusivity correlates with the efficient formation of quality control bodies (Q-bodies), membraneless organelles that compartmentalize misfolded peptides during stress. Synthetic, light-induced RNA condensation also fluidizes the cytoplasm. Together, our study reveals a functional role for stress-induced translation inhibition and formation of RNP condensates in modulating the physical properties of the cytoplasm to enable efficient response of cells to stress conditions.

Understanding coenzyme Q.

Coenzyme Q (CoQ), also known as ubiquinone, comprises a benzoquinone head group and a long isoprenoid side chain. It is thus extremely hydrophobic and resides in membranes. It is best known for its complex function as an electron transporter in the mitochondrial electron transport chain (ETC) but is also required for several other crucial cellular processes. In fact, CoQ appears to be central to the entire redox balance of the cell. Remarkably, its structure and therefore its properties have not changed from bacteria to vertebrates. In metazoans, it is synthesized in all cells and is found in most, and maybe all, biological membranes. CoQ is also known as a nutritional supplement, mostly because of its involvement with antioxidant defenses. However, whether there is any health benefit from oral consumption of CoQ is not well established. Here we review the function of CoQ as a redox-active molecule in the ETC and other enzymatic systems, its role as a prooxidant in reactive oxygen species generation, and its separate involvement in antioxidant mechanisms. We also review CoQ biosynthesis, which is particularly complex because of its extreme hydrophobicity, as well as the biological consequences of primary and secondary CoQ deficiency, including in human patients. Primary CoQ deficiency is a rare inborn condition due to mutation in CoQ biosynthetic genes. Secondary CoQ deficiency is much more common, as it accompanies a variety of pathological conditions, including mitochondrial disorders as well as aging. In this context, we discuss the importance, but also the great difficulty, of alleviating CoQ deficiency by CoQ supplementation.

GAGA-associated factor fosters loop formation in the Drosophila genome.

The impact of genome organization on the control of gene expression persists as a major challenge in regulatory biology. Most efforts have focused on the role of CTCF-enriched boundary elements and TADs, which enable long-range DNA-DNA associations via loop extrusion processes. However, there is increasing evidence for long-range chromatin loops between promoters and distal enhancers formed through specific DNA sequences, including tethering elements, which bind the GAGA-associated factor (GAF). Previous studies showed that GAF possesses amyloid properties in vitro, bridging separate DNA molecules. In this study, we investigated whether GAF functions as a looping factor in Drosophila development. We employed Micro-C assays to examine the impact of defined GAF mutants on genome topology. These studies suggest that the N-terminal POZ/BTB oligomerization domain is important for long-range associations of distant GAGA-rich tethering elements, particularly those responsible for promoter-promoter interactions that coordinate the activities of distant paralogous genes.

PAX3-FOXO1 coordinates enhancer architecture, eRNA transcription, and RNA polymerase pause release at select gene targets.

Transcriptional control is a highly dynamic process that changes rapidly in response to various cellular and extracellular cues, making it difficult to define the mechanism of transcription factor function using slow genetic methods. We used a chemical-genetic approach to rapidly degrade a canonical transcriptional activator, PAX3-FOXO1, to define the mechanism by which it regulates gene expression programs. By coupling rapid protein degradation with the analysis of nascent transcription over short time courses and integrating CUT&RUN, ATAC-seq, and eRNA analysis with deep proteomic analysis, we defined PAX3-FOXO1 function at a small network of direct transcriptional targets. PAX3-FOXO1 degradation impaired RNA polymerase pause release and transcription elongation at most regulated gene targets. Moreover, the activity of PAX3-FOXO1 at enhancers controlling this core network was surprisingly selective, affecting single elements in super-enhancers. This combinatorial analysis indicated that PAX3-FOXO1 was continuously required to maintain chromatin accessibility and enhancer architecture at regulated enhancers.

Structure and functionality of a multimeric human COQ7:COQ9 complex.

Coenzyme Q (CoQ) is a redox-active lipid essential for core metabolic pathways and antioxidant defense. CoQ is synthesized upon the mitochondrial inner membrane by an ill-defined "complex Q" metabolon. Here, we present structure-function analyses of a lipid-, substrate-, and NADH-bound complex comprising two complex Q subunits: the hydroxylase COQ7 and the lipid-binding protein COQ9. We reveal that COQ7 adopts a ferritin-like fold with a hydrophobic channel whose substrate-binding capacity is enhanced by COQ9. Using molecular dynamics, we further show that two COQ7:COQ9 heterodimers form a curved tetramer that deforms the membrane, potentially opening a pathway for the CoQ intermediates to translocate from the bilayer to the proteins' lipid-binding sites. Two such tetramers assemble into a soluble octamer with a pseudo-bilayer of lipids captured within. Together, these observations indicate that COQ7 and COQ9 cooperate to access hydrophobic precursors within the membrane and coordinate subsequent synthesis steps toward producing CoQ.

The 10q26 Risk Haplotype of Age-Related Macular Degeneration Aggravates Subretinal Inflammation by Impairing Monocyte Elimination.

A minor haplotype of the 10q26 locus conveys the strongest genetic risk for age-related macular degeneration (AMD). Here, we examined the mechanisms underlying this susceptibility. We found that monocytes from homozygous carriers of the 10q26 AMD-risk haplotype expressed high amounts of the serine peptidase HTRA1, and HTRA1 located to mononuclear phagocytes (MPs) in eyes of non-carriers with AMD. HTRA1 induced the persistence of monocytes in the subretinal space and exacerbated pathogenic inflammation by hydrolyzing thrombospondin 1 (TSP1), which separated the two CD47-binding sites within TSP1 that are necessary for efficient CD47 activation. This HTRA1-induced inhibition of CD47 signaling induced the expression of pro-inflammatory osteopontin (OPN). OPN expression increased in early monocyte-derived macrophages in 10q26 risk carriers. In models of subretinal inflammation and AMD, OPN deletion or pharmacological inhibition reversed HTRA1-induced pathogenic MP persistence. Our findings argue for the therapeutic potential of CD47 agonists and OPN inhibitors for the treatment of AMD.

Loss of LUC7L2 and U1 snRNP subunits shifts energy metabolism from glycolysis to OXPHOS.

Oxidative phosphorylation (OXPHOS) and glycolysis are the two major pathways for ATP production. The reliance on each varies across tissues and cell states, and can influence susceptibility to disease. At present, the full set of molecular mechanisms governing the relative expression and balance of these two pathways is unknown. Here, we focus on genes whose loss leads to an increase in OXPHOS activity. Unexpectedly, this class of genes is enriched for components of the pre-mRNA splicing machinery, in particular for subunits of the U1 snRNP. Among them, we show that LUC7L2 represses OXPHOS and promotes glycolysis by multiple mechanisms, including (1) splicing of the glycolytic enzyme PFKM to suppress glycogen synthesis, (2) splicing of the cystine/glutamate antiporter SLC7A11 (xCT) to suppress glutamate oxidation, and (3) secondary repression of mitochondrial respiratory supercomplex formation. Our results connect LUC7L2 expression and, more generally, the U1 snRNP to cellular energy metabolism.

Brown adipose tissue CoQ deficiency activates the integrated stress response and FGF21-dependent mitohormesis.

Coenzyme Q (CoQ) is essential for mitochondrial respiration and required for thermogenic activity in brown adipose tissues (BAT). CoQ deficiency leads to a wide range of pathological manifestations, but mechanistic consequences of CoQ deficiency in specific tissues, such as BAT, remain poorly understood. Here, we show that pharmacological or genetic CoQ deficiency in BAT leads to stress signals causing accumulation of cytosolic mitochondrial RNAs and activation of the eIF2α kinase PKR, resulting in activation of the integrated stress response (ISR) with suppression of UCP1 but induction of FGF21 expression. Strikingly, despite diminished UCP1 levels, BAT CoQ deficiency displays increased whole-body metabolic rates at room temperature and thermoneutrality resulting in decreased weight gain on high-fat diets (HFD). In line with enhanced metabolic rates, BAT and inguinal white adipose tissue (iWAT) interorgan crosstalk caused increased browning of iWAT in BAT-specific CoQ deficient animals. This mitohormesis-like effect depends on the ATF4-FGF21 axis and BAT-secreted FGF21, revealing an unexpected role for CoQ in the modulation of whole-body energy expenditure with wide-ranging implications for primary and secondary CoQ deficiencies.

Coenzyme Q biochemistry and biosynthesis.

Coenzyme Q (CoQ) is a remarkably hydrophobic, redox-active lipid that empowers diverse cellular processes. Although most known for shuttling electrons between mitochondrial electron transport chain (ETC) complexes, the roles for CoQ are far more wide-reaching and ever-expanding. CoQ serves as a conduit for electrons from myriad pathways to enter the ETC, acts as a cofactor for biosynthetic and catabolic reactions, detoxifies damaging lipid species, and engages in cellular signaling and oxygen sensing. Many open questions remain regarding the biosynthesis, transport, and metabolism of CoQ, which hinders our ability to treat human CoQ deficiency. Here, we recount progress in filling these knowledge gaps, highlight unanswered questions, and underscore the need for novel tools to enable discoveries and improve the treatment of CoQ-related diseases.

Trials and tribulations of statistical significance in biochemistry and omics.

Over recent years many statisticians and researchers have highlighted that statistical inference would benefit from a better use and understanding of hypothesis testing, p-values, and statistical significance. We highlight three recommendations in the context of biochemical sciences. First recommendation: to improve the biological interpretation of biochemical data, do not use p-values (or similar test statistics) as thresholded values to select biomolecules. Second recommendation: to improve comparison among studies and to achieve robust knowledge, perform complete reporting of data. Third recommendation: statistical analyses should be reported completely with exact numbers (not as asterisks or inequalities). Owing to the high number of variables, a better use of statistics is of special importance in omic studies.

Bi-allelic variants in COQ8B, a gene involved in the biosynthesis of coenzyme Q10, lead to non-syndromic retinitis pigmentosa.

Retinitis pigmentosa (RP) is a Mendelian disease characterized by gradual loss of vision, due to the progressive degeneration of retinal cells. Genetically, it is highly heterogeneous, with pathogenic variants identified in more than 100 genes so far. Following a large-scale sequencing screening, we identified five individuals (four families) with recessive and non-syndromic RP, carrying as well bi-allelic DNA changes in COQ8B, a gene involved in the biosynthesis of coenzyme Q10. Specifically, we detected compound heterozygous assortments of five disease-causing variants (c.187C>T [p.Arg63Trp], c.566G>A [p.Trp189Ter], c.1156G>A [p.Asp386Asn], c.1324G>A [p.Val442Met], and c.1560G>A [p.Trp520Ter]), all segregating with disease according to a recessive pattern of inheritance. Cell-based analysis of recombinant proteins deriving from these genotypes, performed by target engagement assays, showed in all cases a significant decrease in ligand-protein interaction compared to the wild type. Our results indicate that variants in COQ8B lead to recessive non-syndromic RP, possibly by impairing the biosynthesis of coenzyme Q10, a key component of oxidative phosphorylation in the mitochondria.

Cell-type-specific effects of autism-associated 15q duplication syndrome in the human brain.

Recurrent copy-number variation represents one of the most well-established genetic drivers in neurodevelopmental disorders, including autism spectrum disorder. Duplication of 15q11-q13 (dup15q) is a well-described neurodevelopmental syndrome that increases the risk of autism more than 40-fold. However, the effects of this duplication on gene expression and chromatin accessibility in specific cell types in the human brain remain unknown. To identify the cell-type-specific transcriptional and epigenetic effects of dup15q in the human frontal cortex, we conducted single-nucleus RNA sequencing and multi-omic sequencing on dup15q-affected individuals (n = 6) as well as individuals with non-dup15q autism (n = 7) and neurotypical control individuals (n = 7). Cell-type-specific differential expression analysis identified significantly regulated genes, critical biological pathways, and differentially accessible genomic regions. Although there was overall increased gene expression across the duplicated genomic region, cellular identity represented an important factor mediating gene-expression changes. As compared to other cell types, neuronal subtypes showed greater upregulation of gene expression across a critical region within the duplication. Genes that fell within the duplicated region and had high baseline expression in control individuals showed only modest changes in dup15q, regardless of cell type. Of note, dup15q and autism had largely distinct signatures of chromatin accessibility but shared the majority of transcriptional regulatory motifs, suggesting convergent biological pathways. However, the transcriptional binding-factor motifs implicated in each condition implicated distinct biological mechanisms: neuronal JUN and FOS networks in autism vs. an inflammatory transcriptional network in dup15q microglia. This work provides a cell-type-specific analysis of how dup15q changes gene expression and chromatin accessibility in the human brain, and it finds evidence of marked cell-type-specific effects of this genetic driver. These findings have implications for guiding therapeutic development in dup15q syndrome, as well as understanding the functional effects of copy-number variants more broadly in neurodevelopmental disorders.

A regulatory variant impacting TBX1 expression contributes to basicranial morphology in Homo sapiens.

Changes in gene regulatory elements play critical roles in human phenotypic divergence. However, identifying the base-pair changes responsible for the distinctive morphology of Homo sapiens remains challenging. Here, we report a noncoding single-nucleotide polymorphism (SNP), rs41298798, as a potential causal variant contributing to the morphology of the skull base and vertebral structures found in Homo sapiens. Screening for differentially regulated genes between Homo sapiens and extinct relatives revealed 13 candidate genes associated with basicranial development, with TBX1, implicated in DiGeorge syndrome, playing a pivotal role. Epigenetic markers and in silico analyses prioritized rs41298798 within a TBX1 intron for functional validation. CRISPR editing revealed that the 41-base-pair region surrounding rs41298798 modulates gene expression at 22q11.21. The derived allele of rs41298798 acts as an allele-specific enhancer mediated by E2F1, resulting in increased TBX1 expression levels compared to the ancestral allele. Tbx1-knockout mice exhibited skull base and vertebral abnormalities similar to those seen in DiGeorge syndrome. Phenotypic differences associated with TBX1 deficiency are observed between Homo sapiens and Neanderthals (Homo neanderthalensis). In conclusion, the regulatory divergence of TBX1 contributes to the formation of skull base and vertebral structures found in Homo sapiens.

An Isoprene Lipid-Binding Protein Promotes Eukaryotic Coenzyme Q Biosynthesis.

The biosynthesis of coenzyme Q presents a paradigm for how cells surmount hydrophobic barriers in lipid biology. In eukaryotes, CoQ precursors-among nature's most hydrophobic molecules-must somehow be presented to a series of enzymes peripherally associated with the mitochondrial inner membrane. Here, we reveal that this process relies on custom lipid-binding properties of COQ9. We show that COQ9 repurposes the bacterial TetR fold to bind aromatic isoprenes with high specificity, including CoQ intermediates that likely reside entirely within the bilayer. We reveal a process by which COQ9 associates with cardiolipin-rich membranes and warps the membrane surface to access this cargo. Finally, we identify a molecular interface between COQ9 and the hydroxylase COQ7, motivating a model whereby COQ9 presents intermediates directly to CoQ enzymes. Overall, our results provide a mechanism for how a lipid-binding protein might access, select, and deliver specific cargo from a membrane to promote biosynthesis.