Human Nucleus Research Articles

Matrix Stiffness Activating YAP/TEAD1-Cyclin B1 in Nucleus Pulposus Cells Promotes Intervertebral Disc Degeneration.

Intervertebral disc degeneration is a leading cause of disability in the elderly population. Rigid extracellular matrix is a critical pathological feature of disc degeneration, leading to aberrant nucleus pulposus cells (NPCs) proliferation. However, the underlying mechanism is unclear. Here, we hypothesize that increased matrix stiffness induces proliferation and thus degenerative phenotypes of NPCs through YAP/TEAD1 signaling pathway. We established hydrogel substrates to mimic stiffness of degenerated human nucleus pulposus tissues. RNA-sequencing identified differentially expressed genes between primary rat NPCs cultured on rigid and soft hydrogels. Dual luciferase assay and gain- and loss-function experiments evaluated the correlation between YAP/TEAD1 and Cyclin B1. Furthermore, single-cell RNA-sequencing of human NPCs was performed to identify specific cell clusters with high YAP expression. Matrix stiffness increased in severely degenerated human nucleus pulposus tissues (p < 0.05). Rigid substrate enhanced rat NPCs proliferation mainly through Cyclin B1, which was directly targeted and positively regulated by YAP/TEAD1. Depletion of YAP or Cyclin B1 arrested G2/M phase progression of rat NPCs and reduced fibrotic phenotypes including MMP13 and CTGF (p < 0.05). Fibro NPCs with high YAP expression were identified in human tissues and responsible for fibrogenesis during degeneration. Furthermore, inhibition of YAP/TEAD interaction by verteporfin suppressed cell proliferation and alleviated degeneration in the disc needle puncture model (p < 0.05). Our results demonstrate that elevated matrix stiffness stimulates fibro NPCs proliferation through YAP/TEAD1-Cyclin B1 axis, indicating a therapeutic target for disc degeneration.

Contributions of human amygdala nuclei to resting-state networks.

The amygdala is a brain region with a complex internal structure that is associated with psychiatric disease. Methodological limitations have complicated the study of the internal structure of the amygdala in humans. In the current study we examined the functional connectivity between nine amygdaloid nuclei and existing resting-state networks using a high spatial-resolution fMRI dataset. Using data-driven analysis techniques we found that there were three main clusters inside the amygdala that correlated with the somatomotor, ventral attention and default mode networks. In addition, we found that each resting-state networks depended on a specific configuration of amygdaloid nuclei. Finally, we found that co-activity in the cortical-nucleus increased with the severity of self-rated fear in participants. These results highlight the complex nature of amygdaloid connectivity that is not confined to traditional large-scale divisions, implicates specific configurations of nuclei with certain resting-state networks and highlights the potential clinical relevance of the cortical-nucleus in future studies of the human amygdala.

Post Mortem Image Analysis of Astrocytes of the Human Principal Olivary Nucleus Using Geometrical and Fractal Parameters

Based on their morphology, the most abundant cells within the nervous tissue of the central nervous system, astrocytes, can be divided into two types, protoplasmic astrocytes and fibrous astrocytes. A further analysis of the brain tissue with the preserved astrocytes from the human principal olivary nucleus, based on their morphological differences with age, is successfully performed in this paper. Moreover, the images of 294 astrocytes, 148 fibrous and 146 protoplasmic, from the principal olivary nucleus were used. Applied for the first time in astrocytes image analysis, the principal component analysis was used to find the most informative parameters among geometrical and fractal in each of the four predefined groups, i.e., categories, of the morphological measurements of astrocytes in the images. The proposed subsets representing different morphological features can be used to distinguish astrocyte subtypes and predict their changes during normal aging. The values of the adequated parameters in different subsets were compared between the fibrous and protoplasmic astrocytes and correlated with age. Significant differences (p &lt; 0.05) between the two subtypes were found in four Euclidean and four monofractal parameters. In addition, significant correlations were found between selected parameters and the age of subjects. In the upcoming iterations of this procedure, possible refinement and upgrades are expected.

Streptozotocin-induced Alzheimer's disease investigation by one-dimensional plasmonic grating chip

Recently, there has been significant interest in researching brain insulin resistance as it has been hypothesized that it may play a role in the progression of Alzheimer's disease. Alzheimer’s disease (AD) is brain dementia that contributes to damage to the neuron cells and then patient death. This dementia is ranked as the fifth more dangerous disease in the world. Streptozotocin (STZ) is used to induce Alzheimer’s disease experimentally. STZ is toxic to the pancreatic beta cells and induces insulin resistance. Neuroplasmonin techniques have been used to investigate the ability of STZ on the activity of cultured neuron cells. Neuroplasmonic is a novel technology that combines nanotechnology and biosensor. This technique has been used to record neuron signals in vivo and in vitro. Also, it has many facilities such as label-free detection, real-time analysis, biological compatibility, small sample, high throughput, and low detection limit. In this paper, we introduce a one-dimensional electro-plasmonic nanograting platform that consists of a straight nanorod of gold embedded in a dielectric layer of polycarbonate. The chip is connected with an externally applied voltage to induce tunable PIT and increase the sensor sensitivity. To evaluate the sensing performance of the electro-plasmonic sensor, this chip was cultured with Human Nucleus Pulposus Cells (HNPC). The first step was to measure the neuron cell activity in a healthy case. The next step was to measure the activity of neuron cells injected with different concentrations of STZ (0.5, 1, 2 mM) to induce the formation of Alzheimer’s disease in the cultured neuron cells. The results indicated that the electro-plasmonics sensor had a high sensitivity to the cells' activity and showed good results for the effecting STZ on the neuron cell’s activities.

Elevated Glucagon-like Peptide-1 Receptor Level in the Paraventricular Hypothalamic Nucleus of Type 2 Diabetes Mellitus Patients.

Glucagon-like peptide-1 (GLP-1) receptor (GLP-1R) agonists have been approved for the treatment of type 2 diabetes mellitus (T2DM); however, the brain actions of these drugs are not properly established. We used post mortem microdissected human hypothalamic samples for RT-qPCR and Western blotting. For in situ hybridization histochemistry and immunolabelling, parallel cryosections were prepared from the hypothalamus. We developed in situ hybridization probes for human GLP-1R and oxytocin. In addition, GLP-1 and oxytocin were visualized by immunohistochemistry. Radioactive in situ hybridization histochemistry revealed abundant GLP-1R labelling in the human paraventricular hypothalamic nucleus (PVN), particularly in its magnocellular subdivision (PVNmc). Quantitative analysis of the mRNA signal demonstrated increased GLP-1R expression in the PVNmc in post mortem hypothalamic samples from T2DM subjects as compared to controls, while there was no difference in the expression level of GLP-1R in the other subdivisions of the PVN, the hypothalamic dorsomedial and infundibular nuclei. Our results in the PVN were confirmed by RT-qPCR. Furthermore, we demonstrated by Western blot technique that the GLP-1R protein level was also elevated in the PVN of T2DM patients. GLP-1 fibre terminals were also observed in the PVNmc closely apposing oxytocin neurons using immunohistochemistry. The data suggest that GLP-1 activates GLP-1Rs in the PVNmc and that GLP-1R is elevated in T2DM patients, which may be related to the dysregulation of feeding behaviour and glucose homeostasis in T2DM.

Identifying the layers in the human lateral geniculate nucleus using quantitative and functional MRI

Identifying the layers in the human lateral geniculate nucleus using quantitative and functional MRI

Microglia‐specific Alzheimer’s disease polygenic risk score is associated with amyloid‐β, tau, and microglial activation

AbstractBackgroundGenome‐wide association studies (GWAS) of Alzheimer’s disease (AD) have implicated microglia in AD pathogenesis, and microglia is a promising cellular therapeutic target in AD. However, it remains unclear when and how AD genetic risk localizing to microglia contributes to AD pathophysiology.MethodParticipants are from two community‐based cohorts (Religious Orders Study, the Rush Memory and Aging Project [ROSMAP]; n = 299 deceased; age 88.9±7.1, female 67%) and the screening phase of an AD prevention trial (the Anti‐Amyloid Treatment in Asymptomatic Alzheimer’s study [A4]; n = 2919 cognitively unimpaired; age 71.4±4.8, female 60%). We quantified post‐mortem Aβ and tau in ROSMAP, and used PET to quantify Aβ (florbetapir cortical composite standardized uptake value ratio [SUVR]) and tau (flortaucipir temporal composite SUVR; subset n = 302) in A4. In a subset of ROSMAP participants (n = 31), we evaluated the proportion of activated microglia (PAM) in post‐mortem neocortex. We combined AD GWAS summary statistics with cellular gene expression profiles from human brain single nucleus sequencing, to derive cell‐type‐specific ADPRS (excluding APOE region) in ROSMAP and A4. We assessed the association of each (standardized) cell‐type‐specific ADPRS with AD pathology (linear regression; adjusting for APOE genotypes, age, sex, and first 10 genotype principal components), and examined the correlation between the microglial ADPRS and PAM (Pearson’s correlation). We used Bonferroni‐corrected p‐value threshold, p&lt;6.25×10−3.ResultMicroglial, astrocytic, and endothelial ADPRS were associated with higher Aβ and tau in ROSMAP (Figure 1A‐B). In A4, microglial ADPRS showed the strongest association with Aβ (beta = 0.017, p = 1.6×10−7; Figure 1C), while the association of all other cell‐type‐specific ADPRS with Aβ were no longer significant once adjusted for microglial ADPRS (all p&gt;0.05). Microglial ADPRS showed a suggestive association with temporal composite tau (beta = 0.033, p = 0.012; Figure 1D), even after additionally adjusting for Aβ (beta = 0.024, p = 0.048). Microglial ADPRS was positively correlated with PAM in ROSMAP (Figure 2).ConclusionGenetic risk of AD localizing to microglial genes may contribute to Aβ and tau accumulation and microglial activation, likely starting from the preclinical (cognitively unimpaired) stage. These findings directly connect aggregate microglial AD genetic risk with Aβ and tau accumulation, and provide a genetic rationale to test microglia‐specific interventions in AD prevention trials.

Cell‐specific responses to Tau pathology in the aging brain

AbstractBackgroundGene expression profiling has emerged as a promising unbiased approach to characterize putative mechanisms of Alzheimer’s disease (AD). We previously highlighted transcriptional signatures that are driven by Tau pathology or aging in a longitudinal Drosophila model of tauopathy and human RNA‐sequencing data. However, cellular heterogeneity confounds the interpretation of bulk‐tissue gene expression. Defining cell types and characterizing cell‐specific processes that precipitate neurodegeneration is crucial for elucidating AD pathogenesis.MethodWe use a highly tractable, longitudinal, Drosophila model of tauopathy and single cell transcriptomics (scRNA‐seq) to explore cell‐specific contributions to tau‐mediated gene expression while accounting for age‐related changes. Brains of adult Drosophila pan‐neuronally expressing a mutant variant of human Tau (R406W) and respective controls were dissociated for droplet‐based scRNA‐seq at 1‐, 10‐, and 20‐ days post‐eclosion. We computed differentially expressed genes (DEGs) for each cell subpopulation and profiled gene regulatory networks (regulons), highlighting cell‐specific Tau‐ and aging‐related changes. Tau‐induced changes were compared with previously published bulk RNA‐sequencing data and gene co‐expression networks. To address cross‐species conservation of single cell transcriptional signatures, we leverage published human single nuclei RNA‐sequencing (snRNA‐seq) data.ResultWe identified 96 cell subpopulations clustered by transcriptomic similarities in the adult Drosophila brain and annotated 59 with cell identities. We mapped half of all DEGs identified in our published bulk‐tissue RNA‐sequencing to cell subpopulations. We characterized 183 regulons in our dataset and demonstrated network‐level expression changes driven by Tau. Most Tau‐induced gene expression changes were observed in glutamatergic and cholinergic excitatory neurons while fewer changes were seen in GABAergic inhibitory neurons. Likewise, we observed Tau‐induced reductions in cholinergic and glutamatergic neuron numbers. Correlation of cluster‐specific transcriptional profiles between our data and mammalian snRNA‐seq demonstrate conservation of gene expression among inferred cell identities. Strikingly, we found abundant innate immune gene expression signatures among neuronal subpopulations, and pinpointed cell autonomous Nuclear Factor Kappa B (NFkB) signaling as a modifier in Tau‐induced neurodegeneration.ConclusionOur results comprise a powerful single cell transcriptomic resource for studying tau‐mediated disruption of gene expression and dynamic age‐dependent changes at cellular resolution in a tractable model system.

G3BP1 coordinates lysophagy activity to protect against compression-induced cell ferroptosis during intervertebral disc degeneration.

Lysophagy is a form of selective autophagy to remove unwanted lysosomes. However, its role in the pathogenesis of intervertebral disc degeneration (IDD) remains unclear. We intended to investigate the relationship between lysophagy and ferroptosis, as well as the potential involved molecules during IDD. Human nucleus pulposus (NP) cells were obtained from clinical patients. The protein levels, protein colocalization and cellular reactive oxygen species levels were assessed by western blotting, immunofluorescence analysis, immunoprecipitation and flow cytometry, respectively. The in vivo experiments were conducted based on the needle puncture-induced IDD model in rats. Compression pressure induces the lysophagy inactivation and lysosomal damage, resulting in iron overload and ferroptosis in human NP cells. Notably, Ras GTPase-activating protein-binding proteins 1 (G3BP1) resides at lysosomes to coordinate lysophagy activity mainly via the function of G3BP1/TSC2 complex. Dysfunction of G3BP1/TSC2 complex accelerates the lysosomal damage and ferroptosis in NP cells. Besides, inhibition of mTOR signalling ameliorates lysosomal damage and protects against cell ferroptosis. The in vivo experiments also demonstrate that the G3BP1/mTOR signalling is involved in the progression of IDD. These findings illustrate the relationship between lysophagy and compression-induced cell ferroptosis. It also indicates the positive role of G3BP1 and may provide potential targets for IDD treatment.

Long non-coding RNAs LINC00689 inhibits the apoptosis of human nucleus pulposus cells via miR-3127-5p/ATG7 axis-mediated autophagy.

This study aimed to explore the effects of long non-coding RNAs LINC00689 (LINC00689) in human nucleus pulposus cells (NPCs). NPCs were isolated and their morphology was observed. The proliferation and apoptosis of NPCs, and the levels of LINC00689, miR-3127-5p, Bax, Bcl-2, Cleaved caspase-3, ATG5, ATG7, p62, and LC3Ⅱ/LC3I were detected. Interrelations of LINC00689, miR-3127-5p, and ATG7 were analyzed. LINC00689 was down-regulated yet miR-3127-5p was up-regulated in NPCs. LINC00689 could competitively bind with miR-3127-5p, and ATG7 was targeted by miR-3127-5p in NPCs. Overexpressed LINC00689 promoted proliferation yet inhibited apoptosis of NPCs, whereas LINC00689 silencing did the opposite. Overexpressed LINC00689 raised ATG7 level and LC3Ⅱ/LC3I value yet reduced that of p62 level, but the depletion of LINC00689 did the contrary. ATG7 silencing abolished the effects of overexpressed LINC00689 in NPCs, and likewise, up-regulation of miR-3127-5p overturned the effects of overexpressed LINC00689 in NPCs. Collectively, the up-regulation of LINC00689 inhibits the apoptosis of NPCs via miR-3127-5p/ATG7 axis-mediated autophagy.

The role of the miR-4306/PAK6 axis in degenerative nucleus pulposus cells in human intervertebral disc degeneration

Intervertebral disc degeneration (IDD), characterized by degenerative changes that occur in intervertebral discs due to aging or structural injury, is thought to be the most common cause of lower back pain. Recent studies have shown that microRNAs (miRNAs) have a critical role in the etiopathogenesis of IDD. In the current study, we aimed to determine the role of miRNAs in mediating the underlying mechanisms associated with IDD. First, differentially expressed miRNAs (DEmiRNAs) were identified using the GEO database, and subsequently confirmed by RT-qPCR and in situ hybridization. We found that miR-4306 expression was significantly decreased in human nucleus pulposus (NP) tissues compared with healthy controls, and was negatively correlated with the patients' Pfirrmann grade. To determine the mechanism by which miR-4306 was involved in IDD pathogenesis, we examined the effects of overexpressing or silencing miR-4306 on extracellular matrix (ECM) synthesis/degradation, proliferation, autophagy and apoptosis of human degenerated NP cells isolated from IDD patients. Next, we used dual-luciferase reporter assays to demonstrate that miR-4306 interacted with the 3′-untranslated regions of p21-activated kinase 6 (PAK6) mRNA, resulting in significant suppression of PAK6 expression. This effect was abolished by miR-4306 binding site mutations. Using miR-4306/PAK6 gain-of-function and loss-of-function studies in human degenerated NP cells, we demonstrated that miR-4306 promoted NP cell proliferation, ECM synthesis and autophagy, while inhibiting apoptosis and ECM degradation via PAK6. Thus, our findings indicate that miR-4306, acting via PAK6, has an important role in IDD and can be used as a promising therapeutic target for the treatment of patients with IDD.

Collagen Niches Affect Direct Transcriptional Conversion toward Human Nucleus Pulposus Cells via Actomyosin Contractility.

Cellular niches play fundamental roles in regulating cellular behaviors. However, the effect of niches on direct converted cells remains unexplored. In the present study, the specific combination of transcription factors is first identified to directly acquire induced nucleus pulposus-like cells (iNPLCs). Next, tunable physical properties of collagen niches are fabricated based on various crosslinking degrees. Collagen niches significantly affect actomyosin cytoskeleton and then influence the maturation of iNPLCs. Using gain- and loss of function approaches, the appropriate physical states of collagen niches are found to significantly enhance the maturation of iNPLCs through actomyosin contractility. Moreover, in a rat model of degenerative disc diseases, iNPLCs with collagen niches are transplanted into the lesion to achieve significant improvements. As a result, overexpression of transcription factors in human dermal fibroblasts are efficiently converted into iNPLCs and the optimal collagen niches affect cellular cytoskeleton and then facilitate iNPLCs maturation toward human nucleus pulposus cells. These findings encourage more in-depth studies toward the interactions of niches and direct conversion, which would contribute to the development of direct conversion.

Hypothalamic interaction with reward-related regions during subjective evaluation of foods

The reward system implemented in the midbrain, ventral striatum, orbitofrontal cortex, and ventromedial prefrontal cortex evaluates and compares various types of rewards given to the organisms. It has been suggested that autonomic factors influence reward-related processing via the hypothalamus, but how the hypothalamus modulates the reward system remains elusive. In this functional magnetic resonance imaging study, the hypothalamus was parcellated into individual hypothalamic nuclei performing different autonomic functions using boundary mapping parcellation analyses. The effective interaction during subjective evaluation of foods in a reward task was then investigated between the human hypothalamic nuclei and the reward-related regions. We found significant brain activity decrease in the paraventricular nucleus (PVH) and lateral nucleus in the hypothalamus in food evaluation compared with monetary evaluation. A psychophysiological interaction analysis revealed dual interactions between the PVH and (1) midbrain region and (2) ventromedial prefrontal cortex, with the former correlated with the stronger tendency of participants toward food-seeking. A dynamic causal modeling analysis further revealed unidirectional interactions from the PVH to the midbrain and ventromedial prefrontal cortex. These results suggest that the PVH in the human hypothalamus interacts with the reward-related regions in the cerebral cortex via multiple pathways (i.e., the midbrain pathway and ventromedial prefrontal pathway) to evaluate rewards for subsequent decision-making.

Relay and higher-order thalamic nuclei show an intertwined functional association with cortical-networks

Almost all functional processing in the cortex strongly depends on thalamic interactions. However, in terms of functional interactions with the cerebral cortex, the human thalamus nuclei still partly constitute a terra incognita. Hence, for a deeper understanding of thalamic-cortical cooperation, it is essential to know how the different thalamic nuclei are associated with cortical networks. The present work examines network-specific connectivity and task-related topical mapping of cortical areas with the thalamus. The study finds that the relay and higher-order thalamic nuclei show an intertwined functional association with different cortical networks. In addition, the study indicates that relay-specific thalamic nuclei are not only involved with relay-specific behavior but also in higher-order functions. The study enriches our understanding of interactions between large-scale cortical networks and the thalamus, which may interest a broader audience in neuroscience and clinical research.

TRPV4 mRNA is elevated in the caudate nucleus with NPH but not in Alzheimer's disease.

Symptoms of normal pressure hydrocephalus (NPH) and Alzheimer's disease (AD) are somewhat similar, and it is common to misdiagnose these two conditions. Although there are fluid markers detectable in humans with NPH and AD, determining which biomarker is optimal in representing genetic characteristics consistent throughout species is poorly understood. Here, we hypothesize that NPH can be differentiated from AD with mRNA biomarkers of unvaried proximity to telomeres. We examined human caudate nucleus tissue samples for the expression of transient receptor potential cation channel subfamily V member 4 (TRPV4) and amyloid precursor protein (APP). Using the genome data viewer, we analyzed the mutability of TRPV4 and other genes in mice, rats, and humans through matching nucleotides of six genes of interest and one house keeping gene with two factors associated with high mutation rate: 1) proximity to telomeres or 2) high adenine and thymine (A + T) content. We found that TRPV4 and microtubule associated protein tau (MAPT) mRNA were elevated in NPH. In AD, mRNA expression of TRPV4 was unaltered unlike APP and other genes. In mice, rats, and humans, the nucleotide size of TRPV4 did not vary, while in other genes, the sizes were inconsistent. Proximity to telomeres in TRPV4 was <50Mb across species. Our analyses reveal that TRPV4 gene size and mutability are conserved across three species, suggesting that TRPV4 can be a potential link in the pathophysiology of chronic hydrocephalus in aged humans (>65years) and laboratory rodents at comparable ages.

Hydroxytyrosol Ameliorates Intervertebral Disc Degeneration and Neuropathic Pain by Reducing Oxidative Stress and Inflammation.

Low back pain (LBP) seriously affects human quality of life. Intervertebral disc degeneration (IVDD) is the main pathological factor that leads to LBP, but the pathological mechanism underlying IVDD has not been fully elucidated. Neuropathic pain caused by IVDD is an important pathological factor affecting people's daily lives. Therefore, it is very important to identify therapeutic drugs to ameliorate IVDD and secondary neuropathic pain. Hydroxytyrosol (HT) is a natural compound derived from olive leaves and oil and has anti-inflammatory, antioxidant, and antitumor activities and other properties. In this study, TNF-α-stimulated human nucleus pulposus cells (HNPCs) were used to simulate the local inflammatory microenvironment observed in IVDD in vitro to explore the role of HT in alleviating various pathological processes associated with IVDD. A rat needle puncture model was used to further explore the role of HT in alleviating IVDD. Lipopolysaccharide (LPS) was used to stimulate microglia in vitro to comprehensively explore the role of HT in alleviating neuropathic pain, and a rat model involving chronic compression of the dorsal root ganglion (CCD) was established to simulate the neuropathic pain caused by IVDD. This study suggests that HT reduces the expression of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), the NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome, a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4) and matrix metalloproteinase-13 (MMP-13); inhibits the production of mitochondrial reactive oxygen species (ROS); and maintains mitochondrial homeostasis. Thus, HT appears to reduce the rate of apoptosis and mitigate the loss of major intervertebral disc components by inhibiting the nuclear factor kappa-B (NF-κB) signaling pathway. Moreover, HT inhibited the secretion of COX-2, tumor necrosis factor-α (TNF-α), interleukin (IL)-6, IL-1β, and iNOS and activation of the NLRP3 inflammasome in microglia by inhibiting the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) and extracellular regulated protein kinase (ERK) signaling pathways. In conclusion, HT plays a protective role against IVDD and secondary neuropathic pain by inhibiting the NF-κB, PI3K/AKT, and ERK signaling pathways.

Exploration of microRNA-106b-5p as a therapeutic target in intervertebral disc degeneration: a preclinical study.

MicroRNA (miRNA) has emerge as a vital regulator in the pathogenesis of intervertebral disc degeneration (IDD). However, miR-106b-5p expression in the human nucleus pulposus (NP) and potential mechanisms remain to be elucidated. In this study, the aim was to verify the potential therapeutic mechanisms of miR-106b-5p for IDD. Key miRNAs were screened for in degenerative and normal human intervertebral disc samples. qRT-PCR and fluorescence in situ hybridization (FISH) were used to verify the miR-106b-5p differential expression. The targeting link between miR-106b-5p and Sirtuin 2 (SIRT2) was identified using the luciferase reporter assay and bioinformatics. Flow cytometry, EdU method, and cell scratching were all performed to determine the NP cell function and IDD models were constructed for in vivo experiments. SIRT2, MMP13, ADAMTS5, Col II, Aggrecan, Ras, ERK1/2, and p-ERK1/2 protein levels were assayed by western blotting. Overexpression of miR-106b-5p in NP cells decreased cell growth, induced apoptosis, hindered extracellular matrix formation, and increased the expression of matrix-degrading enzymes through the SIRT2/MAPK/ERK signaling pathway. Importantly, intradiscal delivery of antagomiR-106b-5p significantly attenuated IDD development. Our findings demonstrate that targeting miR-106b-5p in intervertebral disc has therapeutic effects on IDD.

In Vitro Characterization of a Tissue Renin-Angiotensin System in Human Nucleus Pulposus Cells.

Low back pain is a clinically highly relevant musculoskeletal burden and is associated with inflammatory as well as degenerative processes of the intervertebral disc. However, the pathophysiology and cellular pathways contributing to this devastating condition are still poorly understood. Based on previous evidence, we hypothesize that tissue renin-angiotensin system (tRAS) components, including the SARS-CoV-2 entry receptor angiotensin-converting enzyme 2 (ACE2), are present in human nucleus pulposus (NP) cells and associated with inflammatory and degenerative processes. Experiments were performed with NP cells from four human donors. The existence of angiotensin II, angiotensin II type 1 receptor (AGTR1), AGTR2, MAS-receptor (MasR), and ACE2 in human NP cells was validated with immunofluorescent staining and gene expression analysis. Hereafter, the cell viability was assessed after adding agonists and antagonists of the target receptors as well as angiotensin II in different concentrations for up to 48 h of exposure. A TNF-α-induced inflammatory in vitro model was employed to assess the impact of angiotensin II addition and the stimulation or inhibition of the tRAS receptors on inflammation, tissue remodeling, expression of tRAS markers, and the release of nitric oxide (NO) into the medium. Furthermore, protein levels of IL-6, IL-8, IL-10, and intracellular as well as secreted angiotensin II were assessed after exposing the cells to the substances, and inducible nitric oxide synthase (iNOS) levels were evaluated by utilizing Western blot. The existence of tRAS receptors and angiotensin II were validated in human NP cells. The addition of angiotensin II only showed a mild impact on gene expression markers. However, there was a significant increase in NO secreted by the cells. The gene expression ratios of pro-inflammatory/anti-inflammatory cytokines IL-6/IL-10, IL-8/IL-10, and TNF-α/IL-10 were positively correlated with the AGTR1/AGTR2 and AGTR1/MAS1 ratios, respectively. The stimulation of the AGTR2 MAS-receptor and the inhibition of the AGTR1 receptor revealed beneficial effects on the gene expression of inflammatory and tissue remodeling markers. This finding was also present at the protein level. The current data showed that tRAS components are expressed in human NP cells and are associated with inflammatory and degenerative processes. Further characterization of the associated pathways is warranted. The findings indicate that tRAS modulation might be a novel therapeutic approach to intervertebral disc disease.

Effect of regulating macrophage polarization phenotype on intervertebral disc degeneration.

BackgroundMacrophages are the only inflammatory cells that can penetrate the closed nucleus pulposus and their polarization plays an important role in intervertebral disc degeneration (IVDD). This paper attempted to investigate the pathogenesis of IVDD by altering the polarization state of macrophages.MethodsMacrophage RAW264.7 cells were induced by interferonγ (IFN‐γ) and lipopolysaccharide (LPS). The polarization of RAW264.7 cells was estimated by western blot and immunofluorescence. The expressions of inflammatory factors were detected by ELISA. Subsequently, RAW264.7 cells were treated with different concentrations of minocycline (Mino) and sinomenine (Sino), followed by the assessment of cell viability with cell counting kit‐8 kit. Then, RAW264.7 cell culture medium was collected for the culture of human nucleus pulposus cells (NPCs). Toluidine blue staining and type II collagen staining were applied to assay the level of type II collagen. The cell apoptosis, oxidative stress, and nitric oxide (NO) level were appraised by TUNEL, oxidative stress kits and NO kit, respectively. Western blot was employed to test the levels of apoptosis‐ and oxidative stress‐related proteins.ResultsIFN‐γ and LPS could induce M1 polarization of RAW264.7 cells. Mino and Sino could reduce the polarization of RAW264.7 cells toward M1. M1‐polarized medium inhibited LPS‐induced activity, inflammation, and damage of NPCs, which were enhanced by Mino and Sino in medium.ConclusionM1 polarization of macrophages promoted LPS‐induced inflammation and damage of NPCs.

Big contributions of the little brain for precision psychiatry.

Our previous work using 3T functional Magnetic Resonance Imaging (fMRI) parcellated the human dentate nuclei (DN), the primary output of the cerebellum, to three distinct functional zones each contributing uniquely to default-mode, salience-motor, and visual brain networks. In this perspective piece, we highlight the possibility to target specific functional territories within the cerebellum using non-invasive brain stimulation, potentially leading to the refinement of cerebellar-based therapeutics for precision psychiatry. Significant knowledge gap exists in our functional understanding of cerebellar systems. Intervening early, gauging severity of illness, developing intervention strategies and assessing treatment response, are all dependent on our understanding of the cerebello-cerebral networks underlying the pathology of psychotic disorders. A promising yet under-examined avenue for biomarker discovery is disruptions in cerebellar output circuitry. This is primarily because most 3T MRI studies in the past had to exclude cerebellum from the field of view due to limitations in spatiotemporal resolutions. Using recent technological advances in 7T MRI (e.g., parallel transmit head coils) to identify functional territories of the DN, with a focus on dentato-cerebello-thalamo-cortical (CTC) circuitry can lead to better characterization of brain-behavioral correlations and assessments of co-morbidities. Such an improved mechanistic understanding of psychiatric illnesses can reveal aspects of CTC circuitry that can aid in neuroprognosis, identification of subtypes, and generate testable hypothesis for future studies.