GLUT1 is redundant in hypoxic and glycolytic nucleus pulposus cells of the intervertebral disc.

Glycolysis is central to homeostasis of nucleus pulposus (NP) cells in the avascular intervertebral disc. Since the glucose transporter, GLUT1, is a highly enriched phenotypic marker of NP cells, we hypothesized that it is vital for the development and postnatal maintenance of the disc. Surprisingly, primary NP cells treated with 2 well-characterized GLUT1 inhibitors maintained normal rates of glycolysis and ATP production, indicating intrinsic compensatory mechanisms. We showed in vitro that NP cells mitigated GLUT1 loss by rewiring glucose import through GLUT3. Of note, we demonstrated that substrates, such as glutamine and palmitate, did not compensate for glucose restriction resulting from dual inhibition of GLUT1/3, and inhibition compromised long-term cell viability. To investigate the redundancy of GLUT1 function in NP, we generated 2 NP-specific knockout mice: Krt19CreERT Glut1fl/fl and Foxa2Cre Glut1fl/fl. There were no apparent defects in postnatal disc health or development and maturation in mutant mice. Microarray analysis verified that GLUT1 loss did not cause transcriptomic alterations in the NP, supporting that cells are refractory to GLUT1 loss. These observations provide the first evidence to our knowledge of functional redundancy in GLUT transporters in the physiologically hypoxic intervertebral disc and underscore the importance of glucose as the indispensable substrate for NP cells.

The role of HIF proteins in maintaining the metabolic health of the intervertebral disc.

The physiologically hypoxic intervertebral disc and cartilage rely on the hypoxia-inducible factor (HIF) family of transcription factors to mediate cellular responses to changes in oxygen tension. During homeostatic development, oxygen-dependent prolyl hydroxylases, circadian clock proteins and metabolic intermediates control the activities of HIF1 and HIF2 in these tissues. Mechanistically, HIF1 is the master regulator of glycolytic metabolism and cytosolic lactate levels. In addition, HIF1 regulates mitochondrial metabolism by promoting flux through the tricarboxylic acid cycle, inhibiting downsteam oxidative phosphorylation and controlling mitochondrial health through modulation of the mitophagic pathway. Accumulation of metabolic intermediates from HIF-dependent processes contribute to intracellular pH regulation in the disc and cartilage. Namely, to prevent changes in intracellular pH that could lead to cell death, HIF1 orchestrates a bicarbonate buffering system in the disc, controlled by carbonic anhydrase 9 (CA9) and CA12, sodium bicarbonate cotransporters and an intracellular H+/lactate efflux mechanism. In contrast to HIF1, the role of HIF2 remains elusive; in disorders of the disc and cartilage, its function has been linked to both anabolic and catabolic pathways. The current knowledge of hypoxic cell metabolism and regulation of HIF1 activity provides a strong basis for the development of future therapies designed to repair the degenerative disc.

Binding and transport of SFPQ-RNA granules by KIF5A/KLC1 motors promotes axon survival.

Complex neural circuitry requires stable connections formed by lengthy axons. To maintain these functional circuits, fast transport delivers RNAs to distal axons where they undergo local translation. However, the mechanism that enables long-distance transport of RNA granules is not yet understood. Here, we demonstrate that a complex containing RNA and the RNA-binding protein (RBP) SFPQ interacts selectively with a tetrameric kinesin containing the adaptor KLC1 and the motor KIF5A. We show that the binding of SFPQ to the KIF5A/KLC1 motor complex is required for axon survival and is impacted by KIF5A mutations that cause Charcot-Marie Tooth (CMT) disease. Moreover, therapeutic approaches that bypass the need for local translation of SFPQ-bound proteins prevent axon degeneration in CMT models. Collectively, these observations indicate that KIF5A-mediated SFPQ-RNA granule transport may be a key function disrupted in KIF5A-linked neurologic diseases and that replacing axonally translated proteins serves as a therapeutic approach to axonal degenerative disorders.

Hypoxic Regulation of Mitochondrial Metabolism and Mitophagy in Nucleus Pulposus Cells Is Dependent on HIF-1α-BNIP3 Axis.

Nucleus pulposus (NP) cells reside in an avascular and hypoxic microenvironment of the intervertebral disc and are predominantly glycolytic due to robust HIF-1 activity. It is generally thought that NP cells contain few functional mitochondria compared with cells that rely on oxidative metabolism. Consequently, the contribution of mitochondria to NP cell metabolism and the role of hypoxia and HIF-1 in mitochondrial homeostasis is poorly understood. Using mitoQC reporter mice, we show for the first time to our knowledge that NP cell mitochondria undergo age-dependent mitophagy in vivo. Mechanistically, in vitro studies suggest that, under hypoxic conditions, mitochondria in primary NP cells undergo HIF-1α-dependent fragmentation, controlled by modulating the levels of key proteins DRP1 and OPA1 that are involved in mitochondrial fission and fusion, respectively. Seahorse assays and steady state metabolic profiling coupled with [1-2-13 C]-glucose flux analysis revealed that in hypoxia, HIF-1α regulated metabolic flux through coordinating glycolysis and the mitochondrial TCA cycle interactions, thereby controlling the overall biosynthetic capacity of NP cells. We further show that hypoxia and HIF-1α trigger mitophagy in NP cells through the mitochondrial translocation of BNIP3, an inducer of receptor-mediated mitophagy. Surprisingly, however, loss of HIF-1α in vitro and analysis of NP-specific HIF-1α null mice do not show a decrease in mitophagic flux in NP cells but a compensatory increase in NIX and PINK1-Parkin pathways with higher mitochondrial number. Taken together, our studies provide novel mechanistic insights into the complex interplay between hypoxia and HIF-1α signaling on the mitochondrial metabolism and quality control in NP cells. © 2020 American Society for Bone and Mineral Research.

Lactate Efflux From Intervertebral Disc Cells Is Required for Maintenance of Spine Health.

Maintenance of glycolytic metabolism is postulated to be required for health of the spinal column. In the hypoxic tissues of the intervertebral disc and glycolytic cells of vertebral bone, glucose is metabolized into pyruvate for ATP generation and reduced to lactate to sustain redox balance. The rise in intracellular H+ /lactate concentrations are balanced by plasma-membrane monocarboxylate transporters (MCTs). Using MCT4 null mice and human tissue samples, complemented with genetic and metabolic approaches, we determine that H+ /lactate efflux is critical for maintenance of disc and vertebral bone health. Mechanistically, MCT4 maintains glycolytic and tricarboxylic acid (TCA) cycle flux and intracellular pH homeostasis in the nucleus pulposus compartment of the disc, where hypoxia-inducible factor 1α (HIF-1α) directly activates an intronic enhancer in SLC16A3. Ultimately, our results provide support for research into lactate as a diagnostic biomarker for chronic, painful, disc degeneration. © 2019 American Society for Bone and Mineral Research.

A novel mouse model of intervertebral disc degeneration shows altered cell fate and matrix homeostasis.

Intervertebral disc degeneration and associated low back and neck pain is a ubiquitous health condition that affects millions of people world-wide, and causes high incidence of disability and enormous medical/societal costs. However, lack of appropriate small animal models with spontaneous disease onset has impeded our ability to understand the pathogenetic mechanisms that characterize and drive the degenerative process. We report, for the first time, early onset spontaneous disc degeneration in SM/J mice known for their poor regenerative capacities compared to "super-healer" LG/J mice. In SM/J mice, degenerative process was marked by decreased nucleus pulposus (NP) cellularity and changes in matrix composition at P7, 4, and 8 weeks with increased severity by 17 weeks. Distinctions between NP and annulus fibrosus (AF) or endplate cartilage were lost, and NP and AF of SM/J mice showed higher histological grades. There was increased NP cell death in SM/J mice with decreased phenotypic marker expression. Polarized microscopy and FTIR spectroscopy demonstrated replacement of glycosaminoglycan-rich NP matrix with collagenous fibrous tissue. The levels of ARGxx were increased in, indicating higher aggrecan turnover. Furthermore, an aberrant expression of collagen X and MMP13 was observed in the NP of SM/J mice, along with elevated expression of Col10a1, Ctgf, and Runx2, markers of chondrocyte hypertrophy. Likewise, expression of Enpp1 as well as Alpl was higher, suggesting NP cells of SM/J mice promote dystrophic mineralization. There was also a decrease in several pathways necessary for NP cell survival and function including Wnt and VEGF signaling. Importantly, SM/J discs were stiffer, had decreased height, and poor vertebral bone quality, suggesting compromised motion segment mechanical functionality. Taken together, our results clearly demonstrate that SM/J mouse strain recapitulates many salient features of human disc degeneration, and serves as a novel small animal model.

Expression of Carbonic Anhydrase III, a Nucleus Pulposus Phenotypic Marker, is Hypoxia-responsive and Confers Protection from Oxidative Stress-induced Cell Death.

The integrity of the avascular nucleus pulposus (NP) phenotype plays a crucial role in the maintenance of intervertebral disc health. While advances have been made to define the molecular phenotype of healthy NP cells, the functional relevance of several of these markers remains unknown. In this study, we test the hypothesis that expression of Carbonic Anhydrase III (CAIII), a marker of the notochordal NP, is hypoxia-responsive and functions as a potent antioxidant without a significant contribution to pH homeostasis. NP, but not annulus fibrosus or end-plate cells, robustly expressed CAIII protein in skeletally mature animals. Although CAIII expression was hypoxia-inducible, we did not observe binding of HIF-1α to select hypoxia-responsive-elements on Car3 promoter using genomic chromatin-immunoprecipitation. Similarly, analysis of discs from NP-specific HIF-1α null mice suggested that CAIII expression was independent of HIF-1α. Noteworthy, silencing CAIII in NP cells had no effect on extracellular acidification rate, CO2 oxidation rate, or intracellular pH, but rather sensitized cells to oxidative stress-induced death mediated through caspase-3. Our data clearly suggests that CAIII serves as an important antioxidant critical in protecting NP cells against oxidative stress-induced injury.

Glycosaminoglycan synthesis in the nucleus pulposus: Dysregulation and the pathogenesis of disc degeneration.

Few human tissues have functions as closely linked to the composition of their extracellular matrices as the intervertebral disc. In fact, the hallmark of intervertebral disc degeneration, commonly accompanying low back and neck pain, is the progressive loss of extracellular matrix molecules - specifically the GAG-substituted proteoglycans. While this loss is often associated with increased extracellular catabolism via metalloproteinases and pro-inflammatory cytokines, there is strong evidence that disc degeneration is related to dysregulation of the enzymes involved in GAG biosynthesis. In this review, we discuss those environmental factors, unique to the disc, that control expression and function of XT-1, GlcAT-I, and ChSy/ChPF in the healthy and degenerative state. Additionally, we address the pathophysiology of aberrant GAG biosynthesis and highlight therapeutic strategies designed to augment the loss of extracellular matrix molecules that afflict the degenerative state.

Bicarbonate Recycling by HIF-1-Dependent Carbonic Anhydrase Isoforms 9 and 12 Is Critical in Maintaining Intracellular pH and Viability of Nucleus Pulposus Cells.

Intervertebral disc degeneration is a ubiquitous condition closely linked to chronic low-back pain. The health of the avascular nucleus pulposus (NP) plays a crucial role in the development of this pathology. We tested the hypothesis that a network comprising HIF-1α, carbonic anhydrase (CA) 9 and 12 isoforms, and sodium-coupled bicarbonate cotransporters (NBCs) buffer intracellular pH through coordinated bicarbonate recycling. Contrary to the current understanding of NP cell metabolism, analysis of metabolic-flux data from Seahorse XF analyzer showed that CO2 hydration contributes a significant source of extracellular proton production in NP cells, with a smaller input from glycolysis. Because enzymatic hydration of CO2 is catalyzed by plasma membrane-associated CAs we measured their expression and function in NP tissue. NP cells robustly expressed isoforms CA9/12, which were hypoxia-inducible. In addition to increased mRNA stability under hypoxia, we observed binding of HIF-1α to select hypoxia-responsive elements on CA9/12 promoters using genomic chromatin immunoprecipitation. Importantly, in vitro loss of function studies and analysis of discs from NP-specific HIF-1α null mice confirmed the dependency of CA9/12 expression on HIF-1α. As expected, inhibition of CA activity decreased extracellular acidification rate independent of changes in HIF activity or lactate/H+ efflux. Surprisingly, CA inhibition resulted in a concomitant decrease in intracellular pH that was mirrored by inhibition of sodium-bicarbonate importers. These results suggested that extracellular bicarbonate generated by CA9/12 is recycled to buffer cytosolic pH fluctuations. Importantly, long-term intracellular acidification from CA inhibition lead to compromised cell viability, suggesting that plasma-membrane proton extrusion pathways alone are not sufficient to maintain homeostatic pH in NP cells. Taken together, our studies show for the first time that bicarbonate buffering through the HIF-1α-CA axis is critical for NP cell survival in the hypoxic niche of the intervertebral disc. © 2017 American Society for Bone and Mineral Research.

PHD3 is a transcriptional coactivator of HIF-1α in nucleus pulposus cells independent of the PKM2-JMJD5 axis.

The role of prolyl hydroxylase (PHD)-3 as a hypoxia inducible factor (HIF)-1α cofactor is controversial and remains unknown in skeletal tissues. We investigated whether PHD3 controls HIF-1 transcriptional activity in nucleus pulposus (NP) cells through the pyruvate kinase muscle (PKM)-2-Jumonji domain--containing protein (JMJD5) axis. PHD3-/- mice (12.5 mo old) showed increased incidence of intervertebral disc degeneration with a concomitant decrease in expression of the HIF-1α targets VEGF-A, glucose transporter-1, and lactate dehydrogenase A. PHD3 silencing decreased hypoxic activation of HIF-1α C-terminal transactivation domain (C-TAD), but not HIF-1α-N-terminal-(N)-TAD or HIF-2α-TAD. Moreover, PHD3 suppression in NP cells resulted in decreased HIF-1α enrichment on target promoters and lower expression of select HIF-1 targets. Contrary to other cell types, manipulation of PKM2 and JMJD5 levels had no effect on HIF-1 activity in NP cells. Likewise, stabilization of tetrameric PKM2 by a chemical approach had no effect on PHD3-dependent HIF-1 activity. Coimmunoprecipitation assays showed lack of association between HIF-1α and PKM2 in NP cells. Results support the role of the PHD3 as a cofactor for HIF-1, independent of PKM2-JMJD5.-Schoepflin, Z. R., Silagi, E. S., Shapiro, I. M., Risbud, M. V. PHD3 is a transcriptional coactivator of HIF-1α in nucleus pulposus cells independent of the PKM2-JMJD5 axis.

Circadian factors BMAL1 and RORα control HIF-1α transcriptional activity in nucleus pulposus cells: implications in maintenance of intervertebral disc health.

BMAL1 and RORα are major regulators of the circadian molecular oscillator. Since previous work in other cell types has shown cross talk between circadian rhythm genes and hypoxic signaling, we investigated the role of BMAL1 and RORα in controlling HIF-1-dependent transcriptional responses in NP cells that exist in the physiologically hypoxic intervertebral disc. HIF-1-dependent HRE reporter activity was further promoted by co-transfection with either BMAL1 or RORα. In addition, stable silencing of BMAL1 or inhibition of RORα activity resulted in decreased HRE activation. Inhibition of RORα also modulated HIF1α-TAD activity. Interestingly, immunoprecipitation studies showed no evidence of BMAL1, CLOCK or RORα binding to HIF-1α in NP cells. Noteworthy, stable silencing of BMAL1 as well as inhibition of RORα decreased expression of select HIF-1 target genes including VEGF, PFKFB3 and Eno1. To delineate if BMAL1 plays a role in maintenance of disc health, we studied the spinal phenotype of BMAL1-null mice. The lumbar discs of null mice evidenced decreased height, and several parameters associated with vertebral trabecular bone quality were also affected in nulls. In addition, null animals showed a higher ratio of cells to matrix in NP tissue and hyperplasia of the annulus fibrosus. Taken together, our results indicate that BMAL1 and RORα form a regulatory loop in the NP and control HIF-1 activity without direct interaction. Importantly, activities of these circadian rhythm molecules may play a role in the adaptation of NP cells to their unique niche.