Shurong Li

Microglial TIR-domain-containing adapter-inducing interferon-β (TRIF) deficiency promotes retinal ganglion cell survival and axon regeneration via nuclear factor-κB.

BackgroundTIR-domain-containing adapter-inducing interferon-β (TRIF) is the sole downstream adaptor of Toll-like receptor (TLR)3, which is one of the major signaling pathways in immune cells leading to neuroinflammation in the central nervous system. Overexpression of TRIF may lead to activation of inflammatory responses, and contribute to pathophysiological progression in both acute and chronic neurodegenerative retinal diseases. In the present study, was aimed to elucidate the contributions of TRIF to optic nerve (ON) regeneration and retinal ganglion cell (RGC) survival following injury to the ON, a widely studied model of central nervous system injury and of degenerative diseases such as glaucoma.MethodsWe used retrograde labeling with a fluorochrome, hydroxystilbamidine (Fluorogold) to evaluate RGC survival, and immunostaining with growth-associated protein-43 to evaluate axon regeneration in an ON crush model. Changes in microglial cytokines following RGC injury was examined with ELISA and real-time PCR. In vivo studies were carried out in wild-type and trif-/- mice. A Transwell co-culture system and migration test were used to mimic the crosstalk between microglia and RGCs. TRIF-associated downstream adaptors were determined by western blotting.ResultsCompared with wild-type (WT) mice, TRIF knockout (KO) mice displayed a robust ability to regenerate axons 3 or 7 days after nerve injury. In addition, RGC survival was considerably higher in trif-/- than in WT mice. ON lesion induced less microglial activation in trif-/- than in WT mice. and more WT microglia distorted and migrated toward the foramen opticum. In the transwell system, few trif-/- microglia migrated through the membrane when stimulated by the performed lesion on RGC axons in a transwell system. Inactivation of microglial cells in trif-/- mice was associated with reduced production of inflammatory cytokines, as detected with real-time RT-PCR and ELISA. Furthermore western blot analysis showed that activation of known downstream effectors of TRIF, including TBK1, IKKε and NF-κB, were significantly inhibited by TRIF deficiency.ConclusionOur results indicate that TRIF deficiency promotes ON axon regeneration by attenuating microglial activation and consequently reducing the release of harmful cytokines via NF-κB inactivation.

Effects of C3 deficiency on inflammation and regeneration following spinal cord injury in mice

Inflammation can activate the complement system, which in turn enhances inflammation and aggravates secondary injury after spinal cord injury (SCI). As the three complement activation pathways converge at the cleavage of C3, we investigated whether inhibiting complement activation in C3-deficient mice would reduce secondary injury after SCI and improve axon regeneration. Weight-drop contusion injury (5g, 6cm) was created in wild-type or C3-deficient mice. Astrocytes (ASTs) activation, TNF-α expression, and axon regeneration were investigated in vivo. In other studies, dorsal root ganglia (DRGs) were co-cultured with mechanically injured ASTs in vitro to evaluate effects on neurite outgrowth. Our results show that, after injury, C3-deficient mice exhibit higher BBB scores than wild-type mice. In addition, ASTs activation was inhibited, TNF-α expression process was delayed in vivo and inhibited in vitro, and nerve fiber regeneration was improved in C3-deficient mice. DRGs co-cultured with mechanically injured ASTs from C3-deficient mice also showed improved neurite outgrowth. We conclude that C3 deficiency can inhibit inflammation through suppressing ASTs activation and TNF-α expression, thereby reducing secondary injury and improving neural regeneration and functional recovery after SCI. The above results suggest that complement inhibition may be a potential therapy to promote central nervous system regeneration by targeting C3.

Gonadectomy differentially regulates steroid receptor coactivator‐1 and synaptic proteins in the hippocampus of adult female and male C57BL/6 mice

Hippocampus is one of the most important structures that mediates learning and memory, cognition, and mental behaviors and profoundly regulated by sex hormones in a sex‐specific manner, but the mechanism of underlying sex differences regulation is still unclear. We have previously reported that in the male and female mice, steroid receptor coactivator‐1 (SRC‐1) and some key synaptic proteins share similar developmental profile in the hippocampus, but how circulating sex hormones affect hippocampal SRC‐1 as well as these synaptic proteins remain unclear. In this study, we examined how gonad sex hormones regulate hippocampal SRC‐1, synaptophysin, PSD‐95, and AMPA receptor subtype GluR1 by using immunohistochemistry and Western blot. The results showed that in the female mice, ovariectomy affected hippocampal SRC‐1 and GluR1 were only detected at 2 weeks post operation, then it recovered to sham level; synaptophysin was unaffected at any timepoint examined; significant decrease of PSD‐95 was only detected at 4 weeks post operation. However, in the male hippocampus, SRC‐1 and PSD‐95 were decreased from one week and lasted to 4 weeks after orchidectomy, GluR1 decreased from 2 weeks after orchidectomy, but synaptophysin remained unchanged as in the females. Correlation analysis showed the profiles of SRC‐1 were positively correlated with GluR1 of the females, PSD‐95 and GluR1 of the males, respectively. The above results suggested a distinct regulatory mode between female and male gonad hormones in the regulation of hippocampal SRC‐1 and synaptic proteins, which may be one of the mechanisms contributing to the dimorphism of hippocampus during development and ageing. Synapse, 2012.

Intrastriatal injection of colchicine induces striatonigral degeneration in mice

Recent studies from environmental toxicology and molecular genetics demonstrate that midbrain dopamine (DA) neurons are particularly vulnerable to microtubule depolymerizing agents, indicating the involvement of microtubule dysfunction in the pathogenesis of Parkinson’s disease. Here we show that intrastriatal injection of colchicine (COL), a well‐known microtubule disruptor, induced degeneration of striatonigral pathway. Microtubule disruption caused by unilateral injection of COL blocked the retrograde axonal transport of fluorogold previously injected into striatum and induced substantial death of striatal and DA neurons in substantia nigra pars compacta. Furthermore, COL‐induced pathologic changes were associated with robust glial reaction, which may be conducive to the degeneration of striatonigral pathway. We also found that intrastriatal injection of COL resulted in side bias in spontaneous turning activities and apomorphine‐induced rotational behavior. Together, our results provide in vivo data lending support to the concept that microtubule dysfunction may play a significant role in the death of DA neurons, though glial reaction may be involved and contribute to the degenerative process. Moreover, intrastriatal COL may serve as another experimental model of striatonigral degeneration (Parkinson’s variant of multiple system atrophy), given the concurrent loss of both striatal and DA neurons.

Morphological changes of cholinergic nerve fibers in the urinary bladder after establishment of artificial somatic-autonomic reflex arc in rats

To establish an artificial somatic-autonomic reflex arc in rats and observe the following distributive changes of neural fibers in the bladder. Adult Sprague-Dawley rats were randomly divided into three groups: control group, spinal cord injury (SCI) group, and reinnervation group. DiI retrograde tracing was used to verify establishment of the model and to investigate the transport function of the regenerated efferent axons in the new reflex arc. Choline acetyltransferase (ChAT) in the DiI-labeled neurons was detected by immunohistochemistry. Distribution of neural fibers in the bladder was observed by acetylcholine esterase staining. DiI-labeled neurons distributed mainly in the left ventral horn from L3 to L5, and some of them were also ChAT-positive. The neural fibers in the bladder detrusor reduced remarkably in the SCI group compared with the control (P < 0.05). After establishment of the somatic-autonomic reflex arc in the reinnervation group, the number of ipsilateral fibers in the bladder increased markedly compared with the SCI group (P < 0.05), though still much less than that in the control (P < 0.05). The efferent branches of the somatic nerves may grow and replace the parasympathetic preganglionic axons through axonal regeneration. Acetylcholine is still the major neurotransmitter of the new reflex arc. The controllability of detrusor may be promoted when it is reinnervated by the pelvic ganglia efferent somatic motor fibers from the postganglionic axons. 观察体神经, 内脏神经人工反射弧建立后?大鼠膀胱肌间神经丛分布的改变以及神经肌肉接头处的变化。 Sprague-Dawley 大鼠随机分为三组: 对照组、 脊髓横断组和手术重建组。 手术重建组大鼠术后饲养 3 个月, 与脊髓横断组大鼠一起进行脊髓横断, 再继续饲养3 个月, 对照组不做任何处理。 DiI 进行逆行神经追踪; 免疫荧光的方法显示 DiI 阳性标记细胞中的胆碱乙酰转移酶(choline acetyltransferase, ChAT); 改良的 Karnovsky-Roots法 检测膀胱铺片中神经纤维的分布。 DiI 阳性标记细胞主要分布于脊髓 L3 尾部至 L5 头侧前角, ChAT 阳性细胞和 DiI 阳性标记细胞部分重叠。 手术重建组和对照组相比, 膀胱肌间神经纤维的数量较少, 染色浓度也较浅(P < 0.05); 而手术重建组神经纤维密度较脊髓横断组增大, 染色浓度增强(P < 0.05); 且出现明显的神经再分布。 人工体内脏神经反射弧建立后, 新的传出支为体神经, 可以长入副交感神经纤维, 传出神经元的递质仍为乙酰胆碱, 膀胱内胆碱能神经纤维再生和乙酰胆碱活性增强且出现神经再分布, 这可能在膀胱的控制性排尿中起作用。

Complement 3-deficient mice are not protected against MPTP-induced dopaminergic neurotoxicity.

Recent studies have invoked inflammation as a major contributor to the pathogenesis of Parkinsons disease (PD). Emerging evidence indicated that components of complement system may be involved in such disorder and contribute to its development. We thus observed the influence of deficiency of complement 3 (C3), the key component of complement system, on the death of dopaminergic neurons in substantia nigra pars compacta (SNpc) and the loss of dopaminergic fibers in striatum induced by acute or chronic administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Immunohistochemical staining of dopaminergic neurons in SNpc and neurochemical analysis of dopamine and its metabolites in striata revealed that there was no significant difference between the two genotypes. Longer survival time also indicated that C3 might not mediate the spontaneous recovery of dopaminergic fibers in mouse striatum acutely challenged by MPTP. We conclude that, despite growing evidence indicating the involvement of complement system in the pathogenesis of PD, our data do not support a role for C3 in this established model of PD, as indicated by results from HPLC analysis and immunohistochemical staining.

Spatial and Temporal Distribution of Dopaminergic Neurons during Development in Zebrafish

As one of the model organisms of Parkinson’s disease (PD) research, the zebrafish has its advantages, such as the 87% homology with human genome and transparent embryos which make it possible to observe the development of dopaminergic neurons in real time. However, there is no midbrain dopaminergic system in zebrafish when compared with mammals, and the location and projection of the dopaminergic neurons are seldom reported. In this study, Vmat2:GFP transgenic zebrafish was used to observe the development and distribution of dopaminergic neurons in real time. We found that diencephalons (DC) 2 and DC4 neuronal populations were detected at 24 h post fertilization (hpf). All DC neuronal populations as well as those in locus coeruleus (LC), raphe nuclei (Ra) and telencephalon were detected at 48 hpf. Axons were detected at 72 hpf. At 96 hpf, all the neuronal populations were detected. For the first time we reported axons from the posterior tubercle (PT) of ventral DC projected to subpallium in vivo. However, when compared with results from whole mount tyrosine hydroxylase (TH) immunofluorescence staining in wild type (WT) zebrafish, we found that DC2 and DC4 neuronal populations were mainly dopaminergic, while DC1, DC3, DC5 and DC6 might not. Neurons in pretectum (Pr) and telencephalon were mainly dopaminergic, while neurons in LC and Ra might be noradrenergic. Our study makes some corrections and modifications on the development, localization and distribution of zebrafish dopaminergic neurons, and provides some experimental evidences for the construction of the zebrafish PD model.

Potential mechanisms of neuroprotection induced by low dose total-body γ-irradiation in C57 mice administered with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)

Low dose total-body gamma-irradiation (TBI) was reported to confer neuroprotection against MPTP-induced dopaminergic neurotoxicity. After being pretreated with a single low dose (0.5Gy, 2.0Gy or 3.5Gy) TBI, C57BL/6 mice were administered with MPTP (15mg/kg, four times, 2h apart) intraperitoneally (i.p.). In the group pretreated with 2.0Gy TBI, with lower lymphocytes number, neuroprotection was found by High Performance Liquid Chromatography (HPLC) determination of the striatal dopamine. Contrarily, in the group pretreated with 0.5Gy TBI, with higher lymphocytes number, dopaminergic neuron toxicity was enhanced. So it was probably the decrease of lymphocytes, not the radiation hormesis that rendered the potential neuroprotection. And it was the balance between radiation injury and lymphocytopenia neuroprotection that decided the effect of low dose gamma-irradiation on MPTP-induced dopaminergic neurotoxicity.

Identification of a NEP1-35 recognizing peptide that neutralizes CNS myelin inhibition using phage display library.

Nogo-A has been identified as an inhibitory molecule to neurite outgrowth after injury in adult mammalian central nervous system (CNS). The C-terminal fragment of Nogo-A, Nogo-66, inhibits axonal regrowth through NgR1 signaling. Residues 1-32 of Nogo-66 cover two regions that contribute most affinity of Nogo-66 to NgR1. It is unclear whether blocking the two regions with specific small ligands could neutralize the inhibition of Nogo-66. Therefore in this study we explored two phage display peptide libraries to screen small peptides that might bind Nogo-66. NEP1-35 containing 1-33 residues of Nogo-66 was taken as the target for panning. We found that phage-borne peptides with stronger affinity to NEP1-35 contained a relatively conserved motif, RRXXXXXXXRRX. Afterwards one identified peptide, NH(2)-RRQTLSHQMRRP-COOH was synthesized and tested in neurite outgrowth assay, in which this small molecule showed moderate ability to neutralize CNS myelin inhibition in vitro. Our results demonstrated that short peptides could act as adaptors to Nogo-66 and neutralize CNS myelin inhibition in vitro. Additionally, the results also suggested that phage display could help to discover novel small molecules with high affinity to CNS regrowth inhibitors, which might be able to promote CNS regeneration with fewer side effects since they could block only the corresponding regions of inhibitors.

Small Nogo-66-binding peptide promotes neurite outgrowth through RhoA inhibition after spinal cord injury

Abortive regeneration in the adult mammalian central nervous system (CNS) is partially mediated through CNS myelin proteins, among which Nogo-A plays an important role. Nogo-66, which is located at the C-terminus of Nogo-A, inhibits axonal regrowth through the Nogo-66/NgR signalling pathway. In this study, two small peptides were tested in a neurite outgrowth assay and spinal cord injury (SCI) model to examine the effects of these molecules on the inhibition of Nogo-66/NgR signalling. PepIV was selected from a phage display peptide library as a Nogo-66 binding molecule. And PepII was synthesized as a potential NgR antagonist. The results indicated that PepIV and PepII decrease the mRNA levels of the small GTPase RhoA and partially neutralize CNS myelin inhibition to cultured cerebellar granule cells (CGCs). Moreover, treatment with both peptides was propitious to maintaining residual axons after SCI, thereby promoting regeneration and locomotion recovery. Because RhoA plays a role in stabilizing the cytoskeleton in growth cones and axons, enhanced neurite outgrowth might reflect a decrease in RhoA expression through PepIV and PepII treatment. Moreover, PepIV induced lower RhoA mRNA expression compared with PepII. Therefore, PepIV could block Nogo-66/NgR signalling and reduce RhoA mRNA level, and then contribute to neuronal survival and axonal regrowth after SCI, showing its ability to reverse CNS myelin inhibition to regeneration. Furthermore, selected small peptide might cover some unknown active sites on CNS myelin proteins, which could be potential targets for improving neurite outgrowth after injury.