Lei Liu

Background: Studies using peripheral blood mononuclear cells (PBMCs) from individuals with Alzheimer’s disease (AD) biomarker information has the potential to clarify the role of peripheral immune systems in AD. However, PBMC collection often requires immediate labor‐intensive processing and large‐volume collection, making it difficult to implement in clinical AD research. Here, we present our optimized protocol to collect, cryopreserve, and process PBMC samples for monocyte and lymphocyte RNA‐sequencing (RNA‐Seq) studies in large clinical AD research programs.Method: We used 8 mL Cell Preparation Tubes (CPT) to collect blood samples, and each CPT was processed and cryopreserved into two aliquots per the manufacturer’s protocol. Later, one PBMC vial ( = 4 mL blood) was thawed and washed in RPMI (+10% FBS, +1% PS). After recording cell numbers and viability, 2.0×10⁶ cells from each sample were stained with anti‐CD4, CD8, CD14, CD20 antibodies, and Sytox blue (viability) in PBS (+2% FBS). The cells were washed and resuspended in 500 uL PBS (+2% FBS; target concentration 4.0×10⁶/mL) for flow cytometry. We sorted and collected CD4+ T cells, CD8+ T cells, CD14+ monocytes, and CD20+ B cells into RNA lysis‐buffer (TCL + 1% BME), aiming for 5,000 or 10,000 cells/cell type/donor. We used the RNeasy micro kit for RNA extraction and Fragment Analyzer for quality control.Result: We collected and cryopreserved PBMC samples from 436 Memory and Aging Cohort (MAC) participants. We used 28 samples for this pilot study (mean age = 68.2 ± 8.1, 21 females). From one cryopreserved aliquot ( = 4 mL of blood), we recovered an average of 3.40×10⁶ ± 1.43×10⁶ viable cells (mean viability 90.4% ± 5.8). Average quantity of RNA was 12.2 ± 4.95 ng/10⁴ cells. The RNA integrity (RIN) score was 8.89 ± 1.7, consistent with excellent RNA quality (Fig. 1).Conclusion: Our low‐volume protocol (4 mL of blood per participant) deployed in a large clinical AD study yielded excellent‐quality RNA for cell‐type‐specific bulk RNA‐Seq (using SMART‐Seq2 library). This protocol can be readily adapted for cell‐type‐specific proteomics or single‐cell multiome projects and is a promising approach to deeply phenotype the peripheral immune system in the context of aging and AD.

Background: Adropin is a peptide encoded by the energy homeostasis-associated gene ( Enho ) that is highly expressed in the brain. Aging and stroke are associated with reduced adropin levels in the brain and plasma. We showed that treatment with synthetic adropin provides long-lasting neuroprotection in permanent ischemic stroke. However, it is unknown whether the protective effects of adropin are observed in aged animals following cerebral ischemia/reperfusion. We hypothesized that adropin provides neuroprotection in aged mice subjected to transient middle cerebral artery occlusion. Methods: Aged (18–24 months old) male mice were subjected to 30 minutes of middle cerebral artery occlusion followed by 48 hours or 14 days of reperfusion. Sensorimotor (weight grip test and open field) and cognitive tests (Y-maze and novel object recognition) were performed at defined time points. Infarct volume was quantified by 2,3,5-triphenyltetrazolium chloride staining at 48 hours or Cresyl violet staining at 14 days post–middle cerebral artery occlusion. Blood-brain barrier damage, tight junction proteins, and MMP-9 (matrix metalloproteinase-9) were assessed 48 hours after middle cerebral artery occlusion by ELISA and Western blots. Results: Genetic deletion of Enho significantly increased infarct volume and worsened neurological function, whereas overexpression of adropin dramatically reduced stroke volume compared to wild-type controls. Postischemic treatment with synthetic adropin peptide given at the onset of reperfusion markedly reduced infarct volume, brain edema, and significantly improved locomotor function and muscular strength at 48 hours. Delayed adropin treatment (4 hours after the stroke onset) reduced body weight loss, infarct volume, and muscular strength dysfunction, and improved long-term cognitive function. Postischemic adropin treatment significantly reduced blood-brain barrier damage. This effect was associated with reduced MMP-9 and preservation of tight junction proteins by adropin treatment. Conclusions: These data unveil a promising neuroprotective role of adropin in the aged brain after transient ischemic stroke via reducing neurovascular damage. These findings suggest that poststroke adropin therapy is a potential strategy to minimize brain injury and improve functional recovery in ischemic stroke patients.

Poststroke infections are common complications of stroke and are highly associated with poor outcomes for patients. Stroke induces profound immunodepression coupled with alterations to autonomic signaling, which together render the body more susceptible to infection from without (nosocomial/community-acquired infection) and from within (commensal bacterial infection). Critical to the hypothesis of commensal infection is the phenomenon of poststroke gut permeability and gut dysbiosis. Few studies have provided adequate explanations for the mechanisms underlying the molecular alterations that produce a more permeable gut and perturbed gut microbiota after stroke. A dysregulation in the production of matrix MMP-7 (metalloproteinase-7) may play a critical role in the progression of gut permeability after stroke. By cleaving junctional and extracellular matrix proteins, MMP-7 is capable of compromising gut barrier integrity. Because of MMP-7’s unique abundance in the small intestine and its capacity to be induced in states of bacterial invasion and inflammation, along with its unique degradative capability, MMP-7 may be crucially important to the progression of gut permeability after ischemic stroke.

The transcription factor Nrf2 is a central regulator of anti-inflammatory and antioxidant mechanisms that contribute to the development and progression of various neurological disorders. Although the direct and indirect Nrf2 regulatory roles on inflammation have been reviewed in recent years, the in vivo evidence of Nrf2 function on lipopolysaccharide (LPS)-induced cognitive decline and characteristic alterations of reactive microglia and astrocytes remains incomplete. During the 3–5 days after LPS or saline injection, 5–6-month-old wildtype (WT) and Nrf2^−/− C57BL/6 mice were subjected to the novel object recognition task. Immunohistochemistry staining was employed for analyses of brain cells. The Nrf2^−/− mice displayed exacerbated LPS-induced cognition impairment (28.1 ± 9.6% in the discrimination index of the novel object recognition task), enhanced hippocampal reactive microgliosis and astrogliosis, and an increased expression level of the water channel transmembrane protein aquaporin 4 when compared with WT controls. In addition, similar overt effects of Nrf2 deficiency on LPS-induced characteristic alterations of brain cells were observed in the cortex and striatum regions of mice. In summary, this transgenic loss-of-function study provides direct in vivo evidence that highlights the functional importance of Nrf2 activation in regulating LPS-induced cognitive alteration, glial responses, and aquaporin 4 expression. This finding provides a better understanding of the complex nature of Nrf2 signaling and neuroprotection.

Cerebral ischemia is a devastating disease with a high incidence of death and disability; however, effective therapeutics remain limited. The transcriptional factor Nrf2 has been shown to play a pivotal role in the endogenous defense against brain oxidative stress and inflammation and therefore represents a promising target for stroke intervention. However, the long-term effects of Nrf2 and the standardized Korean red ginseng (ginseng), a potent Nrf2 natural inducer, on permanent cerebral ischemic damage have not yet been reported. Wildtype (WT) and Nrf2^−/− adult mice were pretreated with either vehicle or ginseng and were subjected to permanent distal middle cerebral artery occlusion (pdMCAO). The infarct volume, the reactive astrocytes and microglia, and the water regulatory protein aquaporin 4 (AQP4) were examined at 28 days after stroke. When compared with the WT matched controls, the Nrf2 disruption significantly enlarged the infarct volume (40.4 ± 10.1%) and exacerbated the progression of reactive gliosis and AQP4 protein levels after pdMCAO. In contrast, ginseng significantly reduced the infarct volume and attenuated the reactive gliosis and AQP4 in the ischemic WT mice (47.3 ± 6.9%), but not in the Nrf2^−/− mice (25.5 ± 5.6%). In conclusion, Nrf2 plays an important role in the long-term recovery of permanent cerebral ischemic damage and the neuroprotection of ginseng.

Background: Reprogramming of fast-to-slow myofiber switch can improve endurance capacity and alleviate fatigue. Accumulating evidence suggests that a muscle-specific microRNA, miR-499 plays a crucial role in myofiber type transition. In this study, we assessed the effects of natural flavonoid myricetin on exercise endurance and muscle fiber constitution, and further investigated the underlying mechanism of myricetin in vivo and in vitro. Methods: A total of 66 six-week-old male Sprague Dawley rats were divided into non-exercise or exercise groups with/without orally administered myricetin (50 or 150 mg/kg) for 2 or 4 weeks. Time-to-exhaustion, blood biochemical parameters, muscle fiber type proportion, the expression of muscle type decision related genes were measured. Mimic/ inhibitor of miR-499 were transfected into cultured L6 myotubes, the expressions of muscle type decision related genes and mitochondrial respiration capacity were investigated. Results: Myricetin treatment significantly improved the time-to-exhaustion in trained rats. The enhancement of endurance capacity was associated with an increase of the proportion of slow-twitch myofiber in both soleus and gastrocnemius muscles. Importantly, myricetin treatment amplified the expression of miR-499 and suppressed the expression of Sox6, the down-stream target gene of miR-499, both in vivo and in vitro. Furthermore, inhibition of miR-499 overturned the effects of myricetin on down-regulating Sox6. Conclusions: Myricetin promoted the reprogramming of fast-to-slow muscle fiber type switch and reinforced the exercise endurance capacity. The precise mechanisms responsible for the effects of myricetin are not resolved but likely involve regulating miR-499/Sox6 axis.

Epilepsy is a common and serious neurological disorder characterized by occurrence of recurrent spontaneous seizures, and emerging evidences support the association of mitochondrial dysfunction with epilepsy. Sirtuin 5, localized in mitochondrial matrix, has been considered as an important functional modulator of mitochondria that contributes to ageing and neurological diseases. Our data shows that SIRT5 deficiency strikingly increased mortality rate and severity of response to epileptic seizures, dramatically exacerbated hippocampal neuronal loss and degeneration in mice exposed to kainate, and triggered more severe reactive astrogliosis. We found that the expression of mitochondrial SIRT5 of injured hippocampus was relatively up-regulated, indicating its potential contribution to the comparably increased survival of these cells and its possible neuroprotective role. Unexpectedly, SIRT5 seems not to apparently alter the decline of antioxidant enzymes superoxide dismutase 2 and glutathione peroxidase in hippocampus caused by kainate exposure in our paradigm, which indicates that the protective roles of SIRT5 on seizures and cellular degeneration might through different regulatory mechanisms that would be explored in the future. In the present study, we provided strong evidences for the first time to demonstrate the association between SIRT5 and epilepsy, which offeres a new understanding of the roles of SIRT5 in mitochondrial functional regulation. The neuroprotection of SIRT5 in kainate-induced epileptic seizure and neurodegeneration will improve our current knowledge of the nature of SIRT5 in central nervous system and neurological diseases.