GFAP Expression Research Articles

Circadian Clock Gene bmal1 Acts as a Tumor Suppressor Gene in a Mice Model of Human Glioblastoma.

Glioblastomas derived from malignant astrocytes are the most common primary tumors of the central nervous system in humans, exhibiting very bad prognosis. Treatment with surgery, radiotherapy, and chemotherapy (mainly using temozolomide), generates as much one-year survival. The circadian clock controls different aspects of tumor development, and its role in GBM is beginning to be explored. Here, the role of the canonic circadian clock gene bmal1 was studied in vivo in a nude mice model bearing human GBMs from LN229 cells xenografted orthotopically in the dorsal striatum. For that aim, a bmal1 knock-down was generated in LN229 cells by CRISPR/Cas9 gene editing tool, and tumor progression was followed in male mice by measuring survival, tumor growth, cell proliferation and prognosis with CD44 marker, as well as astrocyte activation in the tumor microenvironment with GFAP and nestin markers. Disruption of bmal1 in the tumor decreased survival, increased tumor growth and CD44 expression, worsened motor performance, as well as increased GFAP expression in astrocytes at tumor microenvironment. In addition, survival and tumor progression was not affected in mice bearing LN229 wild type GBM that underwent circadian disruption by constant light, as compared to mice synchronized to 12:12 light-dark cycles. These results consistently demonstrate in an in vivo orthotopic model of human GBM, that bmal1 has a key role as a tumor suppressor gene regulating GBM progression.

IPSC-derived PSEN2 (N141I) astrocytes and microglia exhibit a primed inflammatory phenotype

BackgroundWidescale evidence points to the involvement of glia and immune pathways in the progression of Alzheimer’s disease (AD). AD-associated iPSC-derived glial cells show a diverse range of AD-related phenotypic states encompassing cytokine/chemokine release, phagocytosis and morphological profiles, but to date studies are limited to cells derived from PSEN1, APOE and APP mutations or sporadic patients. The aim of the current study was to successfully differentiate iPSC-derived microglia and astrocytes from patients harbouring an AD-causative PSEN2 (N141I) mutation and characterise the inflammatory and morphological profile of these cells.MethodsiPSCs from three healthy control individuals and three familial AD patients harbouring a heterozygous PSEN2 (N141I) mutation were used to derive astrocytes and microglia-like cells and cell identity and morphology were characterised through immunofluorescent microscopy. Cellular characterisation involved the stimulation of these cells by LPS and Aβ42 and analysis of cytokine/chemokine release was conducted through ELISAs and multi-cytokine arrays. The phagocytic capacity of these cells was then indexed by the uptake of fluorescently-labelled fibrillar Aβ42.ResultsAD-derived astrocytes and microglia-like cells exhibited an atrophied and less complex morphological appearance than healthy controls. AD-derived astrocytes showed increased basal expression of GFAP, S100β and increased secretion and phagocytosis of Aβ42 while AD-derived microglia-like cells showed decreased IL-8 secretion compared to healthy controls. Upon immunological challenge AD-derived astrocytes and microglia-like cells showed exaggerated secretion of the pro-inflammatory IL-6, CXCL1, ICAM-1 and IL-8 from astrocytes and IL-18 and MIF from microglia.ConclusionOur study showed, for the first time, the differentiation and characterisation of iPSC-derived astrocytes and microglia-like cells harbouring a PSEN2 (N141I) mutation. PSEN2 (N141I)-mutant astrocytes and microglia-like cells presented with a ‘primed’ phenotype characterised by reduced morphological complexity, exaggerated pro-inflammatory cytokine secretion and altered Aβ42 production and phagocytosis.

UCH-L1 Inhibitor Alleviates Nerve Damage Caused by Moyamoya Disease.

Moyamoya disease (MMD) leads to nerve injury. Exosomes are touted as bio-shuttles for the delivery of distinct biomolecules inside the cells. Recently, UCH-L1 was shown to play a vital role in nerve injury. However, it is still unknown whether UCH-L1 can improve the nerve injury of MMD. Exosomes were isolated from the serum of patients with MMD and healthy controls. The total RNA was extracted from the exosomes, and the level of GFAP and UCH-L1 between the serum exosomes of the two groups was analyzed by a quantitative reverse transcription-polymerase chain reaction and western blot. Exosome labeling and uptake by SH-SY5Y cells were observed by confocal laser microscopy. Cell counting kit-8 assay and flow cytometry were used to determine the viability and apoptosis of SH-SY5Y cells, respectively. Exosomes were successfully isolated and identified from serum. The expression of GFAP and UCH-L1 was significantly higher in the serum-derived exosomes from MMD patients compared with the healthy controls (P < 0.05). Compared to the blank and control exosome group, serum-derived exosomes from MMD significantly suppress cellular vitality and promote apoptosis of SH-SY5Y cells, while the use of LDN-91946, a specific inhibitor of UCH-L1, could reverse the effects induced by serum-derived exosomes from MMD. UCH-L1 inhibitor could reverse MMD-induced inhibition of SH-SY5Y cell viability and promotion of apoptosis. UCH-L1 may be a therapeutic target for the treatment of nerve damage caused by MMD.

Electroacupuncture Suppresses Oxidative Stress and Ferroptosis by Activating the mTOR/SREBP1 Pathway in Ischemic Stroke.

Ischemic stroke (IS) is one of the leading causes of death and disability worldwide. Electroacupuncture (EA) has been shown to exert a neuroprotective effect in IS. However, its specific anti-IS mechanisms remain to be fully elucidated. By constructing a rat IS (middle cerebral artery occlusion, or MCAO) model and performing EA treatment, neurological deficit score, brain water content, and cerebral infarction were evaluated. ELISA was used to measure the levels of oxidative stress-related molecules (MDA, SOD, GSH, and CAT). Ferroptosis-related proteins (GPX4, SLC7A11, TfR1, L-ferritin, and hepcidin), neurological damage-related proteins (GFAP, Iba-1, and Nestin), α7nAChR, and mTOR pathway-related proteins (mTOR, p-mTOR, and SREBP1) in the rat brain penumbra were assessed by western blotting. Following EA treatment, neurological deficit scores, brain water content, cerebral infarction area, and GFAP, Iba-1, and Nestin expression were reduced. Additionally, EA treatment decreased MDA and increased SOD, GSH, and CAT. Moreover, the rats showed elevated GPX4 and SLC7A11 and lowered TfR1, L-ferritin, and hepcidin. In contrast, a7nAChR, mTOR, p-mTOR, and SREBP1 expression were upregulated. EA treatment inhibited OS and ferroptosis to exert a neuroprotective effect in IS, which might be realized via the activation of mTOR/SREBP1 signaling.

Porcine ex vivo retina model to study retinal neurodegenerative diseases: Potential role of mesenchymal stem cells

Aims/Purpose: Retinal neurodegenerative diseases (RND) are associated with a progressive degeneration and death of retinal cells. Recently, mesenchymal stem cells (MSCs) showed a protective role against retinal cells degeneration and apoptosis. However, due to the complexity of such diseases, there is the need to develop in vitro or ex vivo models which allow the study of RND pathogenesis and better understand the regenerative role of MSCs. Therefore, the aim of our study was to use porcine retinas to establish a possible ex vivo model of spontaneous neuroretinal degeneration and employ this model to evaluate the efficacy of human amniotic fluid stem cells (hAFSCs) on retinal cells undergoing damage.Methods: To this end, neuroretinal explants were obtained from pig eyes and placed over the porous membrane of a transwell plate. To induce a time‐dependent spontaneous retinal degeneration, explants were cultured for 24, 48, 72, 96 h, 5 and 8 days. The damage grade was assessed with qRT‐PCR and immunohistochemistry analyses for GFAP and TUNEL‐assay. Then, the explants were co‐cultured with hAFSCs up to 8 days to evaluate their effect on retinal cells death via propidium iodide (PI) staining.Results: Results demonstrated that the cultivation of retinal explants up to 8 days induced an increase in the gene expression of inflammatory (NF‐kB and TNF‐alpha), pro‐apoptotic (p21 and p53) and oxidative stress markers (NRF2). We also observed an increased expression of GFAP and CD11b, suggesting an activation of the microglia and up‐regulation of Müller cells. In addition, the cultivation time induced an increase in the number of apoptotic cells (TUNEL). However, interestingly, the cells death decreased after co‐cultivation with hAFSCs (PI staining).Conclusions: In conclusion, our data demonstrate that the prolonged cultivation of porcine ex vivo retina can induce a time‐dependent spontaneous degeneration able to simulate a model of RND. Of note, the co‐culture preliminary data suggest a protective effects of hAFSCs on retinal cells death.

On the potential therapeutic role of taurine in retinal degenerative diseases

Taurine, considered a non‐essential amino acid that differs from other amino acids in that it contains a sulfonic acid instead of a carboxyl group, is the most abundant amino acid in the retina. Taurine plays a critical role in maintaining retinal health and the survival of retinal neurons, although its exact function in the retina is not yet fully understood. Interestingly, taurine shares structural similarities with β‐alanine, another non‐proteinogenic amino acid that can act as a competitive inhibitor of taurine receptors, allowing β‐alanine to be used to induce taurine depletion. β‐alanine induced taurine depletion results in the activation and migration of microglial cells in the retina, oxidative stress in the inner and outer nuclear and ganglion cell layers, loss of retinal synaptic connectivity, and impaired phagocytic capacity of retinal pigment epithelial cells. In addition, taurine depletion leads to photoreceptor and retinal ganglion cell loss. Most of these detrimental events are exacerbated by light exposure. These findings suggest that taurine levels are associated with retinal sensitivity to light.Given the important role of taurine in the retina, it has been proposed as a promising therapeutic candidate for retinal degenerative diseases. These diseases, which can be inherited or acquired, are a leading cause of irreversible blindness worldwide. Age‐related macular degeneration is the most common acquired cause of photoreceptor degeneration and a major public health concern due to the aging population. Retinitis pigmentosa is the most common inherited retinal degenerative disease, which is a heterogeneous group of sight‐threatening disorders. These diseases share photoreceptor degeneration as a common feature, typically due to genetic defects affecting the photoreceptors or the retinal pigment epithelium but can also be influenced by environmental factors. Additionally, inflammation and oxidative stress contribute to their development and progression. These diseases represent a major economic burden to healthcare systems and society. Therefore, it is imperative to develop preventive and/or therapeutic strategies that can impede or halt their progression.Taurine supplementation is a promising therapeutic approach for these diseases due to its antioxidant, anti‐apoptotic, and anti‐inflammatory properties. Our group has investigated the effects of taurine supplementation in the Royal College of Surgeons (RCS) rat, an established model of inherited retinal degeneration that manifests an impaired phagocytic capacity of the retinal pigment epithelium due to a MERKT gene mutation. Our findings demonstrate that taurine supplementation improves functional photoreceptor survival, decreases glial activation (including decreased GFAP expression in Müller cells and inhibition of microglial activation and migration), reduces oxidative stress in the outer and inner nuclear layers, suppresses apoptosis, and preserves synaptic connections in the RCS rat retina. Therefore, taurine is essential for retinal health and may provide neuroprotection against retinal degeneration thanks to its multifaceted properties.

Therapeutic effects of green-formulated gold nanoparticles by Origanum majorana on spinal cord injury in rats

Abstract In a recent study, we evaluated the remedial properties of gold nanoparticles (gold NPs) green-formulated by Origanum majorana on spinal cord injury in rats. UV-Vis, FE-SEM, FT-IR, and TEM tests were applied to characterize the recent gold NPs. The IC50 values of O. majorana, gold NPs, and butylated hydroxytoluene were 382, 250, and 165 µg/mL, respectively, against DPPH. In in vivo tests, 40 male rats were grouped into four groups: the sham, intact, control, and gold NP (50 µg/kg) groups. Hematoxylin and eosin staining was done to test the lesions post-injury. To determine the neural conduction recovery, somatosensory evoked potential experiments were carried out. For determining the astrogliosis, the GFAP expression was evaluated. All rats were assessed for behavior outcomes using the Basso, Beattie, and Bresnahan (BBB) scores every week after spinal cord injury onset. The neuroprotective effects of gold NPs caused improvement in the spinal cord injury contusive model in Wistar rats. Delayed responses were reduced significantly and BBB scores were raised significantly in the gold NP group on sensory tests. The highest reduction in GFAP was evident in the gold NP group. The ventral motor neuron number raised significantly and cavity areas reduced significantly in the gold NP group. Electromyography findings indicated notable improvement of hindlimbs in the gold NP (50 µg/kg) group.

DIETARY IMPACT ON GUT MICROBIOTA, COGNITIVE PERFORMANCE, AND HIPPOCAMPAL MARKERS IN AGING RATS

Abstract Many patients with mild cognitive impairment have alterations in gut microbiota composition and dietary consumption directly modulates gut microbiota; therefore, the quality of a diet may impact cognitive function. Twelve-month-old male Fischer 344 rats (n=10/group) were assigned to a Mediterranean diet (MeDi), Western diet (WD), or Chow for three months to determine the effect of diet in aged animals. Animals underwent neurobehavioral assessments including the Morris water maze (MWM), and radial arm water maze (RAWM). Fecal samples were analyzed via 16S rRNA sequencing. Markers of barrier and immune function in the hippocampus were measured via qPCR. The MeDi (p=0.06) and Chow (p=0.003) groups had a trend to or swam a shorter distance compared to the WD in the MWM. There was no difference in RAWM total errors between Diets (p=0.43). Microbiota analyses exhibited differences in alpha- and beta-diversity. Chow diet had a higher Simpson and Shannon index compared to MeDi (p&amp;lt; 0.02) and WD (p&amp;lt; 0.001). The MeDi (p=0.03) and Chow (p&amp;lt; 0.001) groups had a higher Chao1 index compared to the WD group. Beta-diversity differed by Diet for weighted UniFrac (p&amp;lt; 0.03), and unweighted UniFrac (p&amp;lt; 0.001). For barrier integrity, there was a trend for Occludin expression to differ by Diet (p=0.07) but Claudin-5 did not differ (p=0.81). For immune function, the WD group had a trend to have a higher expression of GFAP compared to the MeDi (p=0.05) but a lower Iba-1 expression (p=0.009). A short-term diet had a subtle effect on cognitive performance but modulated microbiota compositions and hippocampal immune marker expression.

Pressure-Related Effects on Homeostatic Müller Cell Proteins in the Adult Porcine in Vitro Retina

PurposeTo explore early pressure-related effects on Müller cell homeostatic proteins in the in vitro adult porcine retina.MethodsRetinal explants were subjected to 0-, 10-, 30-, or 60-mmHg of pressure for 24 or 48 h in culture. Retinal explants fixed immediately after enucleation were used as controls. Müller cell proteins were evaluated by GFAP, GS, CRALBP, and bFGF immunohistochemistry.ResultsGFAP-labeling revealed no differences in fluorescence intensity after 24 or 48 h in any of the pressure groups compared with control retinas. However, a higher intensity was found in the 30- and 60-mmHg groups compared with 0-mmHg counterparts after 24 and 48 h. A higher intensity in GS-labeled sections was found in the 10-and 60-mmHg groups compared with controls and remaining pressure groups after 48 h. Compared with control retinas, CRALBP labeling revealed a higher intensity in the 60-mmHg group after 24 h and in the 10-, 30-, and 60-mmHg groups after 48 h. After 24 and 48 h, a lower intensity was found in bFGF-labeled cells in the 0-, 10-, and 30-mmHg groups compared with controls, while no difference was seen for the 60-mmHg group.ConclusionsMüller cells in the cultured porcine adult retina respond early to pressure by altering the expression of GFAP as well as the homeostatic proteins GS, CRALBP, and bFGF.

Entopallium Lost GFAP Immunoreactivity during Avian Evolution: Is GFAP a “Condition Sine Qua Non”?

Introduction: The present study demonstrates that in the same brain area the astroglia can express GFAP (the main cytoskeletal protein of astroglia) in some species but not in the others of the same vertebrate class. It contrasts the former opinions that the distribution of GFAP found in a species is characteristic of the entire class. The present study investigated birds in different phylogenetic positions: duck (Cairina moschata domestica), chicken (Gallus gallus domesticus), and quails (Coturnix japonica and Excalfactoria chinensis) of Galloanserae; pigeon (Columba livia domestica) of a group of Neoaves, in comparison with representatives of other Neoaves lineages, which emerged more recently in evolution: finches (Taeniopygia guttata and Erythrura gouldiae), magpie (Pica pica), and parrots (Melopsittacus undulatus and Nymphicus hollandicus). Methods: Following a perfusion with 4% buffered paraformaldehyde, immunoperoxidase reactions were performed with two types of anti-GFAP: monoclonal and polyclonal, on floating sections. Results: The entopallium (formerly “ectostriatum,” a telencephalic area in birds) was GFAP-immunopositive in pigeon and in the representatives of Galloanserae but not in songbirds and parrots, which emerged more recently in evolution. The lack of GFAP expression of a brain area, however, does not mean the lack of astroglia. Lesions induced GFAP expression in the territory of GFAP-immunonegative entopallia. It proved that the GFAP immunonegativity is not due to the lack of capability, but rather the suppression of GFAP production of the astrocytes in this territory. In the other areas investigated besides the entopallium (optic tectum and cerebellum), no considerable interspecific differences of GFAP immunopositivity were found. It proved that the immunonegativity of entopallium is due to neither the general lack of GFAP expression nor the incapability of our reagents to detect GFAP in these species. Conclusion: The data are congruent with our proposal that a lack of GFAP expression has evolved in different brain areas in vertebrate evolution, typically in lineages that emerged more recently. Comparative studies on GFAP-immunopositive and GFAP-immunonegative entopallia may promote understanding the role of GFAP in neural networks.

Comparison of the Effects of Botulinum Toxin Doses on Nerve Regeneration in Rats with Experimentally Induced Sciatic Nerve Injury.

This study was designed to compare the effects of various doses of botulinum neurotoxin A (BoNT/A) on nerve regeneration. Sixty-five six-week-old rats with sciatic nerve injury were randomly allocated to three experimental groups, a control group, and a sham group. The experimental groups received a single session of intraneural BoNT/A (3.5, 7.0, or 14 U/kg) injection immediately after nerve-crushing injury. The control group received normal intraneural saline injections after sciatic nerve injury. At three, six, and nine weeks after nerve damage, immunofluorescence staining, an ELISA, and toluidine blue staining was used to evaluate the regenerated nerves. Serial sciatic functional index analyses and electrophysiological tests were performed every week for nine weeks. A higher expression of GFAP, S100β, GAP43, NF200, BDNF, and NGF was seen in the 3.5 U/kg and 7.0 U/kg BoNT/A groups. The average area and myelin thickness were significantly greater in the 3.5 U/kg and 7.0 U/kg BoNT/A groups. The sciatic functional index and compound muscle action potential amplitudes exhibited similar trends. These findings indicate that the 3.5 U/kg and 7.0 U/kg BoNT/A groups exhibited better nerve regeneration than the 14 U/kg BoNT/A and control group. As the 3.5 U/kg and the 7.0 U/kg BoNT/A groups exhibited no statistical difference, we recommend using 3.5 U/kg BoNT/A for its cost-effectiveness.

Butyrate suppresses experimental necrotizing enterocolitis-induced brain injury in mice.

Necrotizing enterocolitis (NEC) is a devastating disease in premature infants, and 50% of infants with surgical NEC develop neurodevelopmental defects. The mechanisms by which NEC-induced cytokine release and activation of inflammatory cells in the brain mediate neuronal injury, and whether enteral immunotherapy attenuates NEC-associated brain injury remain understudied. Based on our prior work, which demonstrated that experimental NEC-like intestinal injury is attenuated by the short-chain fatty acid, butyrate, in this study, we hypothesize that NEC-induced brain injury would be suppressed by enteral butyrate supplementation. A standardized NEC mouse model [enteral formula feeding, lipopolysaccharide (LPS), and hypoxia] was used. Mice were randomized into the following groups: control, NEC, butyrate pretreated NEC, and butyrate control. NEC scoring (1-4 with 4 representing severe injury) was performed on ileal sections using a validated scoring system. Intestinal and brain lysates were used to assess inflammation, proinflammatory signaling, and apoptosis. NEC-induced intestinal injury was attenuated by butyrate supplementation. NEC-induced microglial activation in the cerebral cortex and hippocampus was suppressed with butyrate. NEC increased the number of activated microglial cells but decreased the number of oligodendrocytes. Butyrate pretreatment attenuated these changes. Increased activation of proinflammatory Toll-like receptor signaling, cytokine expression, and induction of GFAP and IBA1 in the cerebral cortex observed with NEC was suppressed with butyrate. Experimental NEC induced inflammation and activation of microglia in several regions of the brain, most prominently in the cortex. NEC-induced neuroinflammation was suppressed with butyrate pretreatment. The addition of short-chain fatty acids to diet may be used to attenuate NEC-induced intestinal injury and neuroinflammation in preterm infants.

Long-term exposure to cadmium disrupts neurodevelopment in mature cerebral organoids

Cadmium (Cd) is a pervasive environmental pollutant. Increasing evidence suggests that Cd exposure during pregnancy can induce adverse neurodevelopmental outcomes. However, due to the limitations of neural cell and animal models, it is challenging to study the developmental neurotoxicity and underlying toxicity mechanism of long-term exposure to environmental pollutants during human brain development. In this study, chronic Cd exposure was performed in human mature cerebral organoids for 49 or 77 days. Our study found that prolonged exposure to Cd resulted in the inhibition of cerebral organoid growth and the disruption of neural differentiation and cortical layer organization. These potential consequences of chronic Cd exposure may include impaired GFAP expression, a reduction in SOX2+ neuronal progenitor cells, an increase in TUJ1+ immature neurons, as well as an initial increase and a subsequent decrease in both TBR2+ intermediate progenitors and CTIP2+ deep layer cortical neurons. Transcriptomic analyses revealed that long-term exposure to Cd disrupted zinc and copper ion homeostasis through excessive synthesis of metallothionein and disturbed synaptogenesis, as evidenced by inhibited postsynaptic protein. Our study employed mature cerebral organoids to evaluate the developmental neurotoxicity induced by long-term Cd exposure.

Nuclear Glycoprotein A Repetitions Predominant (GARP) Is a Common Trait of Glioblastoma Stem-like Cells and Correlates with Poor Survival in Glioblastoma Patients.

Glioblastoma (GB) is notoriously resistant to therapy. GB genesis and progression are driven by glioblastoma stem-like cells (GSCs). One goal for improving treatment efficacy and patient outcomes is targeting GSCs. Currently, there are no universal markers for GSCs. Glycoprotein A repetitions predominant (GARP), an anti-inflammatory protein expressed by activated regulatory T cells, was identified as a possible marker for GSCs. This study evaluated GARP for the detection of human GSCs utilizing a multidimensional experimental design that replicated several features of GB: (1) intratumoral heterogeneity, (2) cellular hierarchy (GSCs with varied degrees of self-renewal and differentiation), and (3) longitudinal GSC evolution during GB recurrence (GSCs from patient-matched newly diagnosed and recurrent GB). Our results indicate that GARP is expressed by GSCs across various cellular states and disease stages. GSCs with an increased GARP expression had reduced self-renewal but no alterations in proliferative capacity or differentiation commitment. Rather, GARP correlated inversely with the expression of GFAP and PDGFR-α, markers of astrocyte or oligodendrocyte differentiation. GARP had an abnormal nuclear localization (GARPNU+) in GSCs and was negatively associated with patient survival. The uniformity of GARP/GARPNU+ expression across different types of GSCs suggests a potential use of GARP as a marker to identify GSCs.

Association between brain [18F]FDG‐PET signal and genetic cell markers in the brain tissue

AbstractBackgroundBrain glucose hypometabolism, indexed by [18F]FDG‐PET, is considered a biomarker of neurodegeneration in Alzheimer’s disease (AD). However, other brain cells, such as astrocytes and microglia, also consume considerable amounts of glucose and may contribute to the [18F]FDG‐PET signal in the brain. Thus, the cellular source of the [18F]FDG‐PET signal remains controversial. Here, we aimed to evaluate whether canonical markers of different brain cell types (neuron, oligodendrocyte, astrocyte, and microglia) associate with brain [18F]FDG‐PET signal in humans and a rat model of human amyloidosis.Method[18F]FDG‐PET images were acquired in ten‐month‐old APP/PS1 (TgF344‐AD, n = 8) and wild‐type (WT, n = 6) rats. Next, we evaluated the gene expression of NeuN, Olig2, GFAP, S100B, and IBA1 in the frontal and temporoparietal cortices and cerebellum. Association maps integrating mRNA with brain [18F]FDG‐PET signal were conducted at the voxel level using RMINC. [18F]FDG‐PET and brain tissue mRNA (GFAP, ACSA2, S100B, GLAST, IBA1, CSF1R, TREM2, Olig2, Cldn11, NeuN) of cognitively unimpaired patients were obtained from ADNI and Allen Brain Atlas database, respectively. In humans, mean regional [18F]FDG‐PET SUVr (reference region: pons) and mRNA expression were used to evaluate their association using a gaussian model. Differences were considered statistically significant at p&lt;0.05 (t&gt;2).ResultGFAP and S100B mRNA levels in the temporoparietal and frontal cortices positively correlated with brain [18F]FDG‐PET signal (local maxima, GFAPt(13) = 9.4, S100Bt(13) = 6.6; Fig1B‐C). No associations were found with TREM2, Olig2 and NeuN (t(13)&lt;2; Fig1D‐F). We expanded our analysis by comparing gene astrocyte markers and astrocyte gene cell markers in humans. At the regional level, we found that FDG‐PET SUVr and astrocyte mRNA were positively associated: S100B (R‐squared = 0.34), GFAP (R‐squared = 0.33), GLAST (R‐squared = 0.31) and ACSA‐2 (R‐squared = 0.31; Fig1G).ConclusionOur findings suggest astrocyte mRNA markers are more closely associated with brain [18F]FDG‐PET signal than neuronal, oligodendrocyte, and microglial mRNA markers.

A preliminary in vitro study on Adult Neural Stem cells from 3xTg mice

AbstractBackgroundIn AD, abnormalities in brain wiring and variations of the brain energetic metabolism are considered a key pathological framework of cognitive decline (Heininger, 2000). Neural stem cells (NSCs) can contribute to brain regeneration and, their transplantation improves the behaviour of neurodegenerative mice models (De Gioia et.al., 2021) although, the molecular mechanisms involved are still unidentified. Our preliminary study aims to explore the potential differences between NSCs‐derived neurons from 6‐months‐old healthy and AD mice in terms of neuronal network morphology. Furthermore, the study wants to provide a tool to investigate whether cell energy metabolism is modified, considering that bioenergetic failure can influence the regenerative potential of adult AD brain, an issue that is still ill defined.MethodNSCs were isolated from subventricular zone of healthy (H) and 3xTg (AD) mice. NSCs selection was based on their ability to proliferate and to form 3D floating neurospheres. Isolated cells were amplified and characterised for their expression of stem‐related markers. Subsequently, neuron and astrocyte differentiation protocols were set up. Morphological analyses were performed on H and AD NSC‐derived neurons and the expressions of glutamate transporters and sodium/calcium exchangers (NCXs) were investigated.ResultH and AD NSCs were strongly positive for Nestin and Sox2 stem markers and a faint expression of NeuN and Gfap, markers related to neuron and astrocyte mature phenotype respectively, was detected. These data confirmed the purity of the cell cultures as well as the reliability of the protocols. Morphological investigations highlighted a different distribution of the neurite network between NSCs‐derived neurons isolated from H and AD mice. Furthermore, H and AD mature cells expressed glutamate transporters essential not only for cellular transmission but also for energy production by their interplay with NCXs.ConclusionPreliminary data suggest that the pathological features of adult neurons from AD brain can be traced back to “stem level” and continue during differentiation. The different morphological organization of neurite network observed in NSCs‐derive AD neurons could be involved in the alteration of the impulse transmission and plasticity. Further studies will be necessary to better clarify the consequence of dysregulated glutamate transporters as well the role of astrocytes.

Association between brain [18F]FDG‐PET signal and genetic cell markers in the brain tissue

AbstractBackgroundBrain glucose hypometabolism, indexed by [18F]FDG‐PET, is considered a biomarker of neurodegeneration in Alzheimer’s disease (AD). However, other brain cells, such as astrocytes and microglia, also consume considerable amounts of glucose and may contribute to the [18F]FDG‐PET signal in the brain. Thus, the cellular source of the [18F]FDG‐PET signal remains controversial. Here, we aimed to evaluate whether canonical markers of different brain cell types (neuron, oligodendrocyte, astrocyte, and microglia) associate with brain [18F]FDG‐PET signal in humans and a rat model of human amyloidosis.Method[18F]FDG‐PET images were acquired in ten‐month‐old APP/PS1 (TgF344‐AD, n = 8) and wild‐type (WT, n = 6) rats. Next, we evaluated the gene expression of NeuN, Olig2, GFAP, S100B, and IBA1 in the frontal and temporoparietal cortices and cerebellum. Association maps integrating mRNA with brain [18F]FDG‐PET signal were conducted at the voxel level using RMINC. [18F]FDG‐PET and brain tissue mRNA (GFAP, ACSA2, S100B, GLAST, IBA1, CSF1R, TREM2, Olig2, Cldn11, NeuN) of cognitively unimpaired patients were obtained from ADNI and Allen Brain Atlas database, respectively. In humans, mean regional [18F]FDG‐PET SUVr (reference region: pons) and mRNA expression were used to evaluate their association using a gaussian model. Differences were considered statistically significant at p&lt;0.05 (t&gt;2).ResultGFAP and S100B mRNA levels in the temporoparietal and frontal cortices positively correlated with brain [18F]FDG‐PET signal (local maxima, GFAPt(13) = 9.4, S100Bt(13) = 6.6; Fig1B‐C). No associations were found with TREM2, Olig2 and NeuN (t(13)&lt;2; Fig1D‐F). We expanded our analysis by comparing gene astrocyte markers and astrocyte gene cell markers in humans. At the regional level, we found that FDG‐PET SUVr and astrocyte mRNA were positively associated: S100B (R‐squared = 0.34), GFAP (R‐squared = 0.33), GLAST (R‐squared = 0.31) and ACSA‐2 (R‐squared = 0.31; Fig1G).ConclusionOur findings suggest astrocyte mRNA markers are more closely associated with brain [18F]FDG‐PET signal than neuronal, oligodendrocyte, and microglial mRNA markers.

APOE4 impairs Microglia ‐ Astrocyte Crosstalk in Alzheimer Disease

AbstractBackgroundAPOE4 is the major genetic risk factor for late‐onset Alzheimer Disease (AD). APOE signaling is critical for the transition of homeostatic microglia to the neurodegenerative phenotype (MGnD). The intrinsic role of APOE4 in microglia and its contribution to AD is unclear.Method Generation of Cx3cr1‐CREERT2 mice crossed to APOE‐KI(APOE3 and APOE4)fl/fl injected with labeled apoptotic neurons (MGnD‐Pardigm) on WT or APP/PS1 background. Microglia and astrocytes were isolated and sequenced using either bulk RNA‐seq (MGnD‐Paradigm) or single‐cell RNA‐seq (APP/PS1). Crosstalk between cells was determined using the NichenetR database and validated using 1) Recombinant Lgals3 intracranial injection into APP/PS1 mice and 2) Adoptive transfer of microglia, from APOE3‐KI, APOE4‐KI and APOE4‐cKO mice challenged with labeled apoptotic neurons, into WT mice and isolation of astrocytes.ResultExpression of APOE4 in microglia impaired MGnD‐transition in response to neurodegeneration and in APP/PS1 mice. Conditional deletion of APOE4 in microglia restored MGnD phenotype and reduced plaque pathology. Furthermore, increased astrocytic recruitment towards plaques and their activation by increased Gfap and Serpina3n expression was identified. Microglial Lgals3 was determined as critical crosstalk effector to promote astrocyte response to plaque pathology and acute neurodegeneration. AD female APOE4 carriers showed impaired induction of MGnD signature genes and astrocyte activation, including LGALS3 and GFAP respectively, supporting a conserved disease mechanism in a sex‐dependent manner.ConclusionAPOE4 impairs MGnD‐microglia in AD and prevents their crosstalk with astrocytes. Lgals3 is identified as important positive regulator for microglia–astrocyte crosstalk in response to AD pathology and provides a potential therapeutic target for treatment of AD.

Cobalt exposure triggers impairments in cognitive and anxiety‐like behaviors, brain oxidative stress and inflammation, and hippocampo‐amygdala histomorphological alterations: Protective role of aqueous Prosopis africana seed extract

AbstractBackgroundCobalt toxicity has become a health concern in recent years, due to overexposure resulting in neurological impairments like dementia. With a growing interest in the therapeutic roles of herbs, in toxicity research, it’s worth looking into the curative effects of aqueous Prosopis africana seed extract, a plant rich in flavonoids on cobalt‐induced dementia‐like and anxiogenic behaviour.MethodWe treated rats with Cobalt (CoCl2) or CoCl2 in combination with aqueous Prosopis africana seed extract (PAE) orally for 14 days. Control rats received distilled water for the same period. Following treatments, behavioral experiments for cognition and anxiety, analysis for oxidative stress, inflammation, histological and immunohistochemical analysis were performed.ResultResults revealed that CoCl2 reduced the exploration time, recognition index in the novel object recognition test, percentage spontaneous alternation in the Y‐maze tests, and reduced open arm entry and duration in elevated plus‐maze. However, treatment with PAE improved these parameters to levels comparable with those of the control group. Furthermore, PAE therapy reduced CoCl2‐induced surge in hydrogen peroxide, malondialdehyde, TNF‐α and IL‐1β levels in brain homogenate, while also increasing superoxide dismutase and reduced reduced‐glutathione activities. CoCl2 exposure resulted in obvious features of neurodegeneration like nuclear disintegration, nuclear shrinkage, and cytoplasmic vacuolations of the cells of the hippocampus and amygdala, with an increased expression of GFAP. The hippocampal and amygdala histology improved after PAE administration, while exacerbated GFAP expressions were attenuated.ConclusionThese findings imply that PAE may be anxiolytic and can help reduce cognitive impairments and hippocampal damage caused by CoCl2 neurotoxicity, via mechanisms that involve attenuation of oxidative stress and neuroinflammation.

Mechanistic insights into the therapeutic potential of Angiotensin type 2 receptors (AT2R) in shaping behavior of astrocytes and microglia during hypertensive state

AbstractBackgroundAstrocytes are the major glial cell type in the central nervous system (CNS) and play a critical role in the pathogenesis of neuroinflammatory and neurodegenerative diseases. In particular, astrocytic activation during neuroinflammatory conditions perpetuates the inflammatory response and brain damage and neurodegeneration. Conversely, Angiotensin type 2 receptor (AT2R) activation is known to promote neuroprotection.MethodTherefore, the present study evaluated the role of AT2R in astrocytic activation in vitro in primary astrocytes and in vivo in hypertensive rats.ResultHere, we demonstrated that Ang II‐induced astrogliosis in primary astrocytes as apparent from increased expression of GFAP and iNOS, ROS generation, inflammatory imbalance and mitochondrial dysfunction in the primary astrocytes. On the contrary, AT2R activation by CGP42112A (CGP) abrogated Ang II induced astrocytic activation by inhibition of ROS production, mitochondrial dysfunction and inflammatory imbalance. However, this anti‐inflammatory action of AT2R activation by CGP was reversed by AT2R antagonist, PD123319 in both in vitro and in vivo conditions. Mechanistically, we found that AT2R via protein phosphatase‐2A (PP2A) activation abrogated the Ang II induced NFкB activation, ROS generation and consequent pro‐inflammatory activation of astrocytes. Importantly, PP2A antagonist, okadaic acid, reversed the anti‐inflammatory action of CGP to the comparable level as that by AT2R antagonist PD123319.ConclusionThus, the present study suggests that AT2R by the activation of PP2A abrogates the NFкB inflammatory signalling and inflammatory activation of astrocytes. Therefore, the therapeutic strategies targeting AT2R might be advantageous for neuroinflammatory or degenerative diseases perpetuated by astrocytic activation.