Xiangmei Kong

Chronic Hypoxia Impairs Murine Hippocampal Development and Depletes the Postnatal Progenitor Pool by Attenuating Mammalian Target of Rapamycin Signaling

Chronic hypoxia (CH) is a major risk factor for impaired cognitive function in various disease states, particularly in the context of cyanotic congenital heart disease. Although most brain development occurs prenatally, the dentate gyrus (DG) of the hippocampus harbors progenitor stem cells that contribute to its ongoing development postnatally. It is unclear how exposure to CH might affect postnatal hippocampal development, so we used a transgenic mouse that expresses enhanced green fluorescent protein (eGFP) within this progenitor population to determine the effect of CH on the DG. We find that exposure to 10% oxygen from postnatal d 3 to 28 results in a smaller DG with long-term impairment of hippocampal neurogenesis. Because the mammalian target of rapamycin (mTOR) pathway is a well-known regulator of cell proliferation and growth and is sensitive to hypoxia, we investigated its activation on exposure to CH and find it to be attenuated specifically in neural progenitor cells. Systemic inhibition of the mTOR pathway using rapamycin also caused impairment of hippocampal neurogenesis that mimics exposure to CH. Our findings demonstrate that CH results in long-term impairment of hippocampal neurogenesis and is mediated, in part, by attenuation of the mTOR pathway.

Single Dose of Glycoengineered Anti-CD19 Antibody (MEDI551) Disrupts Experimental Autoimmune Encephalomyelitis by Inhibiting Pathogenic Adaptive Immune Responses in the Bone Marrow and Spinal Cord while Preserving Peripheral Regulatory Mechanisms

Plasma cells and the autoreactive Abs they produce are suspected to contribute to the pathogenesis of multiple sclerosis, but recent attempts to target these components of humoral immunity have failed. MEDI551, an anti-CD19 Ab that depletes mature B cells including plasma cells may offer a compelling alternative that reduces pathogenic adaptive immune responses while sparing regulatory mechanisms. Indeed, our data demonstrate that a single dose of MEDI551, given before or during ongoing experimental autoimmune encephalomyelitis, disrupts development of the disease. Leukocyte infiltration into the spinal cord is significantly reduced, as well as short-lived and long-lived autoreactive CD138+ plasma cells in the spleen and bone marrow, respectively. In addition, potentially protective CD1dhiCD5+ regulatory B cells show resistance to depletion, and myelin-specific Foxp3+ regulatory T cells are expanded. Taken together, these results demonstrate that MEDI551 disrupts experimental autoimmune encephalomyelitis by inhibiting multiple proinflammatory components whereas preserving regulatory populations.

Pharmacological inhibition of the mTOR pathway impairs hippocampal development in mice

Brain injury is an important cause of morbidity in infants at risk for exposure to chronic hypoxia. Using a transgenic mouse that expresses green fluorescent protein (GFP) within this progenitor population we have previously shown that exposure to chronic hypoxia significantly decreases the progenitor stem cell pool in the dentate gyrus of the hippocampus that is in part mediated by inhibition of the mammalian target of rapamycin (mTOR) pathway. Hence we hypothesized that pharmacological inhibition of the mTOR pathway using rapamycin will alter the progenitor stem cell pool and impair the development of the dentate gyrus. We find that prolonged inhibition of the mTOR pathway causes a decrease in the early progenitor stem cell pool, demonstrated by decreased GFP-expressing progenitors, which persists long term. However there is a significant increase in proliferating progenitor cell pool, as seen by increased BrdU that is coupled with increased apoptosis thereby leading to fewer Neu N-expressing mature neurons. Further inhibition of the mTOR pathway leads to depletion of the astrocyte and microglial pool in the dentate gyrus as well. Overall our findings demonstrate that pharmacological inhibition of the mTOR pathway leads to impaired development of the DG, raising the concern that in young children could impair cognitive development.

Autoreactive CD19 + CD20 − Plasma Cells Contribute to Disease Severity of Experimental Autoimmune Encephalomyelitis

The contribution of autoantibody-producing plasma cells in multiple sclerosis (MS) remains unclear. Anti-CD20 B cell depletion effectively reduces disease activity in MS patients, but it has a minimal effect on circulating autoantibodies and oligoclonal bands in the cerebrospinal fluid. Recently we reported that MEDI551, an anti-CD19 mAb, therapeutically ameliorates experimental autoimmune encephalomyelitis (EAE), the mouse model of MS. MEDI551 potently inhibits pathogenic adaptive immune responses, including depleting autoantibody-producing plasma cells. In the present study, we demonstrated that CD19 mAb treatment ameliorates EAE more effectively than does CD20 mAb. Myelin oligodendrocyte glycoprotein–specific Abs and short-lived and long-lived autoantibody-secreting cells were nearly undetectable in the CD19 mAb–treated mice, but they remained detectable in the CD20 mAb–treated mice. Interestingly, residual disease severity in the CD20 mAb–treated animals positively correlated with the frequency of treatment-resistant plasma cells in the bone marrow. Of note, treatment-resistant plasma cells contained a substantial proportion of CD19+CD20− plasma cells, which would have otherwise been targeted by CD19 mAb. These data suggested that CD19+CD20− plasma cells spared by anti-CD20 therapy likely contribute to residual EAE severity by producing autoreactive Abs. In patients with MS, we also identified a population of CD19+CD20− B cells in the cerebrospinal fluid that would be resistant to CD20 mAb treatment.

Preconditioning-induced CXCL12 upregulation minimizes leukocyte infiltration after stroke in ischemia-tolerant mice.

Repetitive hypoxic preconditioning creates long-lasting, endogenous protection in a mouse model of stroke, characterized by reductions in leukocyte–endothelial adherence, inflammation, and infarct volumes. The constitutively expressed chemokine CXCL12 can be upregulated by hypoxia and limits leukocyte entry into brain parenchyma during central nervous system inflammatory autoimmune disease. We therefore hypothesized that the sustained tolerance to stroke induced by repetitive hypoxic preconditioning is mediated, in part, by long-term CXCL12 upregulation at the blood–brain barrier (BBB). In male Swiss Webster mice, repetitive hypoxic preconditioning elevated cortical CXCL12 protein levels, and the number of cortical CXCL12+ microvessels, for at least two weeks after the last hypoxic exposure. Repetitive hypoxic preconditioning-treated mice maintained more CXCL12-positive vessels than untreated controls following transient focal stroke, despite cortical decreases in CXCL12 mRNA and protein. Continuous administration of the CXCL12 receptor (CXCR4) antagonist AMD3100 for two weeks following repetitive hypoxic preconditioning countered the increase in CXCL12-positive microvessels, both prior to and following stroke. AMD3100 blocked the protective post-stroke reductions in leukocyte diapedesis, including macrophages and NK cells, and blocked the protective effect of repetitive hypoxic preconditioning on lesion volume, but had no effect on blood–brain barrier dysfunction. These data suggest that CXCL12 upregulation prior to stroke onset, and its actions following stroke, contribute to the endogenous, anti-inflammatory phenotype induced by repetitive hypoxic preconditioning.

Quantification of Neurovascular Protection Following Repetitive Hypoxic Preconditioning and Transient Middle Cerebral Artery Occlusion in Mice

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week protocol for repetitive hypoxic preconditioning (RHP) induces long-term protection against central nervous system (CNS) injury in a mouse model of focal ischemic stroke. RHP consists of 9 stochastic exposures to hypoxia that vary in both duration (2 or 4 hr) and intensity (8% and 11% O2). RHP reduces infarct volumes, blood-brain barrier (BBB) disruption, and the post-stroke inflammatory response for weeks following the last exposure to hypoxia, suggesting a long-term induction of an endogenous CNS-protective phenotype. The methodology for the dual quantification of infarct volume and BBB disruption is effective in assessing neurovascular protection in mice with RHP or other putative neuroprotectants. Adult male Swiss Webster mice were preconditioned by RHP or duration-equivalent exposures to 21% O2 (i.e. room air). A 60 min transient middle cerebral artery occlusion (tMCAo) was induced 2 weeks following the last hypoxic exposure. Both the occlusion and reperfusion were confirmed by transcranial laser Doppler flowmetry. Twenty-two hr after reperfusion, Evans Blue (EB) was intravenously administered through a tail vein injection. 2 hr later, animals were sacrificed by isoflurane overdose and brain sections were stained with 2,3,5- triphenyltetrazolium chloride (TTC). Infarcts volumes were then quantified. Next, EB was extracted from the tissue over 48 hr to determine BBB disruption after tMCAo. In summary, RHP is a simple protocol that can be replicated, with minimal cost, to induce long-term endogenous neurovascular protection from stroke injury in mice, with the translational potential for other CNS-based and systemic pro-inflammatory disease states.

Erythropoietin-mediated neuroprotection in a pediatric mouse model of chronic hypoxia

Chronic hypoxia (CH), a disease state that accounts for significant morbidity and mortality in pediatrics, occurs in many children during critical periods of hippocampal development and cortical myelination. Hippocampal neurogenesis occurs throughout postnatal life and is important for normal development, thus impairment results in long-term cognitive deficits. Erythropoietin (EPO), a drug commonly known for its role in erythrogenesis, has recently been evaluated in neuroprotection in neonatal injury models and preterm brain injury. However, the effects of EPO therapy on hippocampal neurogenesis and myelination in pediatric CH are unknown. We show that CH decreases hippocampal neurogenesis in a pediatric mouse model. This decrease in early and late progenitors, and actively dividing cells is rescued with EPO treatment. Furthermore, we show that CH during this critical time decreases oligodendrocyte progenitor (OPC) populations in the cortex, leading to defective myelination. However, EPO therapy is only able to rescue the OPC but not the loss of mature myelin. Overall, our findings demonstrate that CH in developing mice has significant effects on hippocampal neurogenesis and OPCs, which can be rescued with EPO treatment. Future studies should confirm the role of this FDA-approved therapy in neuroprotection in at-risk pediatric populations.

Leucine Zipper-Bearing Kinase Is a Critical Regulator of Astrocyte Reactivity in the Adult Mammalian CNS

SUMMARY Reactive astrocytes influence post-injury recovery, repair, and pathogenesis of the mammalian CNS. Much of the regulation of astrocyte reactivity, however, remains to be understood. Using genetic loss and gain-of-function analyses in vivo, we show that the conserved MAP3K13 (also known as leucine zipper-bearing kinase [LZK]) promotes astrocyte reactivity and glial scar formation after CNS injury. Inducible LZK gene deletion in astrocytes of adult mice reduced astrogliosis and impaired glial scar formation, resulting in increased lesion size after spinal cord injury. Conversely, LZK overexpression in astrocytes enhanced astrogliosis and reduced lesion size. Remarkably, in the absence of injury, LZK overexpression alone induced widespread astrogliosis in the CNS and upregulated astrogliosis activators pSTAT3 and SOX9. The identification of LZK as a critical cell-intrinsic regulator of astrocyte reactivity expands our understanding of the multicellular response to CNS injury and disease, with broad translational implications for neural repair.

Selective Non-nuclear Estrogen Receptor Activation Decreases Stroke Severity and Promotes Functional Recovery in Female Mice

Estrogens provide neuroprotection in animal models of stroke, but uterotrophic effects and cancer risk limit translation. Classic estrogen receptors (ERs) serve as transcription factors, whereas nonnuclear ERs govern numerous cell processes and exert beneficial cardiometabolic effects without uterine or breast cancer growth in mice. Here, we determined how nonnuclear ER stimulation with pathway-preferential estrogen (PaPE)-1 affects stroke outcome in mice. Ovariectomized female mice received vehicle, estradiol (E2), or PaPE-1 before and after transient middle cerebral artery occlusion (tMCAo). Lesion severity was assessed with MRI, and poststroke motor function was evaluated through 2 weeks after tMCAo. Circulating, spleen, and brain leukocyte subpopulations were quantified 3 days after tMCAo by flow cytometry, and neurogenesis and angiogenesis were evaluated histologically 2 weeks after tMCAo. Compared with vehicle, E2 and PaPE-1 reduced infarct volumes at 3 days after tMCAo, though only PaPE-1 reduced leukocyte infiltration into the ischemic brain. Unlike E2, PaPE-1 had no uterotrophic effect. Both interventions had negligible effect on long-term poststroke neuronal or vascular plasticity. All mice displayed a decline in motor performance at 2 days after tMCAo, and vehicle-treated mice did not improve thereafter. In contrast, E2 and PaPE-1 treatment afforded functional recovery at 6 days after tMCAo and beyond. Thus, the selective activation of nonnuclear ER by PaPE-1 decreased stroke severity and improved functional recovery in mice without undesirable uterotrophic effects. The beneficial effects of PaPE-1 are also associated with attenuated neuroinflammation in the brain. PaPE-1 and similar molecules may warrant consideration as efficacious ER modulators providing neuroprotection without detrimental effects on the uterus or cancer risk.