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Low back pain (LBP) is a pervasive global health concern, primarily associated with intervertebral disc (IVD) degeneration. Although oxidative stress has been shown to contribute to IVD degeneration, the underlying mechanisms remain undetermined. This study aimed to unravel the role of superoxide dismutase 2 (SOD2) in IVD pathogenesis and target oxidative stress to limit IVD degeneration. SOD2 demonstrated a dynamic regulation in surgically excised human IVD tissues, with initial upregulation in moderate degeneration and downregulation in severely degenerated IVDs. Through a comprehensive set of in vitro and in vivo experiments, we found a suggestive association between excessive mitochondrial superoxide, cellular senescence, and matrix degradation in human and mouse IVD cells. We confirmed that aging and mechanical stress, established triggers for IVD degeneration, escalated mitochondrial superoxide levels in mouse models. Critically, chondrocyte-specific Sod2 deficiency accelerated age-related and mechanical stress-induced disc degeneration in mice, and could be attenuated by β-nicotinamide mononucleotide treatment. These revelations underscore the central role of SOD2 in IVD redox balance and unveil potential therapeutic avenues, making SOD2 and mitochondrial superoxide promising targets for effective LBP interventions.

Thirty genes are involved in the biosynthesis and modification of glycosylphosphatidylinositol (GPI)-anchored proteins, and defects in these genes cause inherited GPI deficiency (IGD). PIGA is X-linked and involved in the first step of GPI biosynthesis, and only males are affected by variations in this gene. The main symptoms of IGD are neurological abnormalities, such as developmental delay and seizures. There is no effective treatment at present. We crossed Nestin-Cre mice with Piga-floxed mice to generate CNS-specific Piga knockout (KO) mice. Hemizygous KO male mice died by P10 with severely defective growth. Heterozygous Piga KO female mice are mosaic for Piga expression and showed severe defects in growth and myelination and died by P25. Using these mouse models, we evaluated the effect of gene replacement therapy with adeno-associated virus (AAV). It expressed efficacy within 6 days, and the survival of male mice was extended to up to 3 weeks, whereas 40% of female mice survived for approximately 1 year and the growth defect was improved. However, liver cancer developed in all three treated female mice at 1 year of age, which was probably caused by the AAV vector bearing a strong CAG promoter.

Summary: An imaging technique across multiple spatial scales is required for extracting structural information on neurons with processes of meter scale length and specialized nanoscale structures. Here, we present a protocol combining multi-scale light microscopy (LM) with electron microscopy (EM) in mouse brain tissue. We describe tissue slice preparation and LM/EM dual labeling with EGFP-APEX2 fusion protein. We then detail ScaleSF tissue clearing and successive LM/EM imaging. Our protocol allows for deciphering structural information across multiple spatial scales on neurons.For complete details on the use and execution of this protocol, please refer to Furuta et al. (2022). : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: The mammalian brain is organized over sizes that span several orders of magnitude, from synapses to the entire brain. Thus, a technique to visualize neural circuits across multiple spatial scales (multi-scale neuronal imaging) is vital for deciphering brain-wide connectivity. Here, we developed this technique by coupling successive light microscopy/electron microscopy (LM/EM) imaging with a glutaraldehyde-resistant tissue clearing method, ScaleSF. Our multi-scale neuronal imaging incorporates (1) brain-wide macroscopic observation, (2) mesoscopic circuit mapping, (3) microscopic subcellular imaging, and (4) EM imaging of nanoscopic structures, allowing seamless integration of structural information from the brain to synapses. We applied this technique to three neural circuits of two different species, mouse striatofugal, mouse callosal, and marmoset corticostriatal projection systems, and succeeded in simultaneous interrogation of their circuit structure and synaptic connectivity in a targeted way. Our multi-scale neuronal imaging will significantly advance the understanding of brain-wide connectivity by expanding the scales of objects.

Summary: A short form of cellular FLICE-inhibitory protein encoded by CFLARs promotes necroptosis. Although necroptosis is involved in various pathological conditions, the detailed mechanisms are not fully understood. Here we generated transgenic mice wherein CFLARs was integrated onto the X chromosome. All male CFLARs Tg mice died perinatally due to severe ileitis. Although necroptosis was observed in various tissues of CFLARs Tg mice, large numbers of intestinal epithelial cells (IECs) died by apoptosis. Deletion of Ripk3 or Mlkl, essential genes of necroptosis, prevented both necroptosis and apoptosis, and rescued lethality of CFLARs Tg mice. Type 3 innate lymphoid cells (ILC3s) were activated and recruited to the small intestine along with upregulation of interleukin-22 (Il22) in CFLARs Tg mice. Deletion of ILC3s or Il22 rescued lethality of CFLARs Tg mice by preventing apoptosis, but not necroptosis of IECs. Together, necroptosis-dependent activation of ILC3s induces lethal ileitis in an IL-22-dependent manner. : Immunology; Immune Response; Cell Biology; Functional Aspects of Cell Biology Subject Areas: Immunology, Immune Response, Cell Biology, Functional Aspects of Cell Biology

Summary: We developed an adeno-associated virus (AAV) vector-based technique to label mouse neostriatal neurons comprising direct and indirect pathways with different fluorescent proteins and analyze their axonal projections. The AAV vector expresses GFP or RFP in the presence or absence of Cre recombinase and should be useful for labeling two cell populations exclusively dependent on its expression. Here, we describe the AAV vector design, stereotaxic injection of the AAV vector, and a highly sensitive immunoperoxidase method for axon visualization.For complete details on the use and execution of this protocol, please refer to Okamoto et al. (2020).

Summary: Indirect pathway medium-sized spiny neurons (iMSNs) in the neostriatum are well known to project to the external segment of the globus pallidus (GPe). Although direct MSNs (dMSNs) also send axon collaterals to the GPe, it remains unclear how dMSNs and iMSNs converge within the GPe. Here, we selectively labeled neighboring dMSNs and iMSNs with green and red fluorescent proteins using an adeno-associated virus vector and examined axonal projections of dMSNs and iMSNs to the GPe in mice. Both dMSNs and iMSNs formed two axonal arborizations displaying topographical projections in the dorsoventral and mediolateral planes. iMSNs displayed a wider and denser axon distribution, which included that of dMSNs. Density peaks of dMSN and iMSN axons almost overlapped, revealing convergence of dMSN axons in the center of iMSN projection fields. These overlapping projections suggest that dMSNs and iMSNs may work cooperatively via interactions within the GPe.

Objectives: Distal radius fractures occur due to reflex clasp when falling. Recently, attention has been focused on the strong relationship between sagittal spine alignment and falls. Therefore, we investigated the parameters of sagittal spinal alignment in distal radius fractures in female patients. Patients and methods: The subjects were group D: 28 female patients with distal radius fractures aged 50 years or older (mean age: 69.3 years), and group C: 26 healthy female patients without a history of fragility fractures (mean age: 70.5 years). Height, body weight, and body mass index (BMI) were measured as physical indices. As parameters of sagittal spinal alignment, the sagittal vertical axis (SVA), pelvic tilt (PT), pelvic incidence (PI), sacral slope (SS), lumber lordosis (LL), and thoracic kyphosis (TK) were measured on lateral whole-spine plain radiographs in a standing position. The measured physical indices and sagittal spinal alignment parameters were compared between groups. Results: Height, weight, and BMI did not differ significantly between the two groups. Among the sagittal spinal alignment parameters, PT, PI, SS, LL, and TK did not differ significantly between groups, whereas SVA was significantly higher in group D than in group C (P < 0.05). Conclusion: In this study, SVA was significantly higher in group D than in group C. As SVA increased, the center of gravity of the body shifts forward, which can cause the body to lose balance and fall. This study suggested that an increase in SVA is associated with distal radius fractures.

Phosphoglycerate mutase family member 5 (PGAM5) is a mitochondrial protein phosphatase that has been reported to be involved in various stress responses from mitochondrial quality control to cell death. However, its roles in vivo are largely unknown. Here, we show that Pgam5-deficient mice are resistant to several metabolic insults. Under cold stress combined with fasting, Pgam5-deficient mice better maintained body temperature than wild-type mice and showed an extended survival rate. Serum triglycerides and lipid content in brown adipose tissue (BAT), a center of adaptive thermogenesis, were severely reduced in Pgam5-deficient mice. Moreover, although Pgam5 deficiency failed to maintain proper mitochondrial integrity in BAT, it reciprocally resulted in the dramatic induction of fibroblast growth factor 21 (FGF21) that activates various functions of BAT including thermogenesis. Thus, the enhancement of lipid metabolism and FGF21 may contribute to the cold resistance of Pgam5-deficient mice under fasting condition. Finally, we also found that Pgam5-deficient mice are resistant to high-fat-diet-induced obesity. Our study uncovered that PGAM5 is involved in the whole-body metabolism in response to stresses that impose metabolic challenges on mitochondria.

Previously, we described the safety and therapeutic potential of neurospheres (NSs) derived from a human induced pluripotent stem cell (iPSC) clone, 201B7, in a spinal cord injury (SCI) mouse model. However, several safety issues concerning iPSC-based cell therapy remain unresolved. Here, we investigated another iPSC clone, 253G1, that we established by transducing OCT4, SOX2, and KLF4 into adult human dermal fibroblasts collected from the same donor who provided the 201B7 clone. The grafted 253G1-NSs survived, differentiated into three neural lineages, and promoted functional recovery accompanied by stimulated synapse formation 47 days after transplantation. However, long-term observation (for up to 103 days) revealed deteriorated motor function accompanied by tumor formation. The tumors consisted of Nestin+ undifferentiated neural cells and exhibited activation of the OCT4 transgene. Transcriptome analysis revealed that a heightened mesenchymal transition may have contributed to the progression of tumors derived from grafted cells.

The genus Cordyceps and its specific ingredient, cordycepin, have attracted much attention for multiple health benefits and expectations for lifespan extension. We analyzed whether Cordyceps militaris (CM), which contains large amounts of cordycepin, can extend the survival of Dahl salt-sensitive rats, whose survival was reduced to ∼3 months via a high-salt diet. The survival of these life-shortened rats was extended significantly when supplemented with CM, possibly due to a minimization of the effects of stroke. Next, we analyzed the effect of CM on hypertension-sensitive organs, the central nervous systems (CNS), heart, kidney and liver of these rats. We attempted to ascertain how the organs were improved by CM, and we paid particular attention to mitochondria and autophagy functions. The following results were from CM-treated rats in comparison with control rats. Microscopically, CNS neurons, cardiomyocytes, glomerular podocytes, renal epithelial cells, and hepatocytes all were improved. However, immunoblot and immunohistochemical analysis showed that the expressions of mitochondria-related proteins, ATP synthase β subunit, SIRT3 and SOD2, and autophagy-related proteins, LC3-II/LC3-I ratio and cathepsin D all were reduced significantly in the CNS neurons, but increased significantly in the cells of the other three organs, although p62 was decreased in its expression in all the organs tested. Activity of Akt and mTOR was enhanced but that of AMPK was reduced in the CNS, while such kinase activity was completely the opposite in the other organs. Together, the influence of CM may differ between mitochondria and autophagy functioned between the two organ groups, as mitochondria and autophagy seemed to be repressed and promoted, respectively, in the CNS, while both mitochondria and autophagy were activated in the others. This could possibly be related to the steady or improved cellular activity in both the organs, which might result in the life extension of these rats.