Academic Appointments

  • Instructor, Ophthalmology

Professional Education

  • PhD, Huazhong Agricultural University, Preventive Veterinary (2017)
  • Master, Shantou University, Biochemistry and molecular biology (2011)
  • Bachelor, Yanshan University, Bioengineering (2008)

All Publications

  • RGC-specific ATF4 and/or CHOP deletion rescues glaucomatous neurodegeneration and visual function. Molecular therapy. Nucleic acids Fang, F., Liu, P., Huang, H., Feng, X., Li, L., Sun, Y., Kaufman, R. J., Hu, Y. 2023; 33: 286-295


    Endoplasmic reticulum (ER) stress has been linked with various acute and chronic neurodegenerative diseases. We previously found that optic nerve (ON) injury and diseases induce neuronal ER stress in retinal ganglion cells (RGCs). We further demonstrated that germline deletion of CHOP preserves the structure and function of both RGC somata and axons in mouse glaucoma models. Here we report that RGC-specific deletion of CHOP and/or its upstream regulator ATF4 synergistically promotes RGC and ON survival and preserves visual function in mouse ON crush and silicone oil-induced ocular hypertension (SOHU) glaucoma models. Consistently, topical application of the ATF4/CHOP chemical inhibitor ISRIB or RGC-specific CRISPR-mediated knockdown of the ATF4 downstream effector Gadd45a also delivers significant neuroprotection in the SOHU glaucoma model. These studies suggest that blocking the neuronal intrinsic ATF4/CHOP axis of ER stress is a promising neuroprotection strategy for neurodegeneration.

    View details for DOI 10.1016/j.omtn.2023.07.015

    View details for PubMedID 37547290

  • Osteopontin drives retinal ganglion cell resiliency in glaucomatous optic neuropathy. Cell reports Zhao, M., Toma, K., Kinde, B., Li, L., Patel, A. K., Wu, K. Y., Lum, M. R., Tan, C., Hooper, J. E., Kriegstein, A. R., La Torre, A., Liao, Y. J., Welsbie, D. S., Hu, Y., Han, Y., Duan, X. 2023; 42 (9): 113038


    Chronic neurodegeneration and acute injuries lead to neuron losses via diverse processes. We compared retinal ganglion cell (RGC) responses between chronic glaucomatous conditions and the acute injury model. Among major RGC subclasses, αRGCs and intrinsically photosensitive RGCs (ipRGCs) preferentially survive glaucomatous conditions, similar to findings in the retina subject to axotomy. Focusing on an αRGC intrinsic factor, Osteopontin (secreted phosphoprotein 1 [Spp1]), we found an ectopic neuronal expression of Osteopontin (Spp1) in other RGCs subject to glaucomatous conditions. This contrasted with the Spp1 downregulation subject to axotomy. αRGC-specific Spp1 elimination led to significant αRGC loss, diminishing their resiliency. Spp1 overexpression led to robust neuroprotection of susceptible RGC subclasses under glaucomatous conditions. In contrast, Spp1 overexpression did not significantly protect RGCs subject to axotomy. Additionally, SPP1 marked adult human RGC subsets with large somata and SPP1 expression in the aqueous humor correlated with glaucoma severity. Our study reveals Spp1's role in mediating neuronal resiliency in glaucoma.

    View details for DOI 10.1016/j.celrep.2023.113038

    View details for PubMedID 37624696

  • Differential effects of SARM1 inhibition in traumatic glaucoma and EAE optic neuropathies. Molecular therapy. Nucleic acids Liu, P., Chen, W., Jiang, H., Huang, H., Liu, L., Fang, F., Li, L., Feng, X., Liu, D., Dalal, R., Sun, Y., Jafar-Nejad, P., Ling, K., Rigo, F., Ye, J., Hu, Y. 2023; 32: 13-27


    Optic neuropathy is a group of optic nerve (ON) diseases withprogressive degeneration of ON and retinal ganglion cells(RGCs). The lack of neuroprotective treatments is a central challenge for this leading cause of irreversible blindness. SARM1 (sterile alpha and TIR motif-containing protein 1) has intrinsic nicotinamide adenine dinucleotide (NAD+) hydrolase activity that causes axon degeneration by degrading axonal NAD+ significantly after activation by axon injury. SARM1 deletion is neuroprotective in many, but not all, neurodegenerative disease models. Here, we compare two therapy strategies for SARM1 inhibition, antisense oligonucleotide (ASO) and CRISPR, with germline SARM1 deletion in the neuroprotection of three optic neuropathy mouse models. This study reveals that, similar to germline SARM1 knockout in every cell, local retinal SARM1 ASO delivery and adeno-associated virus (AAV)-mediated RGC-specific CRISPR knockdown of SARM1 provide comparable neuroprotection to both RGC somata and axons in the silicone oil-induced ocular hypertension (SOHU) glaucoma model but only protect RGC axons, not somata, after traumatic ON injury. Surprisingly, neither of these two therapy strategies of SARM1 inhibition nor SARM1 germline knockout (KO) benefits RGC or ON survival in the experimental autoimmune encephalomyelitis (EAE)/optic neuritis model. Our studies therefore suggest that SARM1 inhibition by local ASO delivery or AAV-mediated CRISPR is a promising neuroprotective gene therapy strategy for traumatic and glaucomatous optic neuropathies but not for demyelinating optic neuritis.

    View details for DOI 10.1016/j.omtn.2023.02.029

    View details for PubMedID 36950280

  • Spp1 drives retinal neuron resiliency in glaucomatous neuropathy but not axotomy Zhao, M., Toma, K., Kinde, B., Li, L., Patel, A., Liao, Y., Vila, A., Kriegstein, A., Welsbie, D., Hu, Y., Han, Y., Duan, X. ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2023
  • Neuroprotection of RGC-specific ATF4 Deletion in Mouse Glaucoma Model Li, L., Fang, F., Liu, P., Huang, H., Feng, X., Hu, Y. ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2023
  • Silicone Oil-Induced Glaucomatous Neurodegeneration in Rhesus Macaques. International journal of molecular sciences Moshiri, A., Fang, F., Zhuang, P., Huang, H., Feng, X., Li, L., Dalal, R., Hu, Y. 2022; 23 (24)


    Previously, we developed a simple procedure of intracameral injection of silicone oil (SO) into mouse eyes and established the mouse SOHU (SO-induced ocular hypertension under-detected) glaucoma model with reversible intraocular pressure (IOP) elevation and significant glaucomatous neurodegeneration. Because the anatomy of the non-human primate (NHP) visual system closely resembles that of humans, it is the most likely to predict human responses to diseases and therapies. Here we tried to replicate the mouse SOHU glaucoma model in rhesus macaque monkeys. All six animals that we tested showed significant retinal ganglion cell (RGC) death, optic nerve (ON) degeneration, and visual functional deficits at both 3 and 6 months. In contrast to the mouse SOHU model, however, IOP changed dynamically in these animals, probably due to individual differences in ciliary body tolerance capability. Further optimization of this model is needed to achieve consistent IOP elevation without permanent damage of the ciliary body. The current form of the NHP SOHU model recapitulates the severe degeneration of acute human glaucoma, and is therefore suitable for assessing experimental therapies for neuroprotection and regeneration, and therefore for translating relevant findings into novel and effective treatments for patients with glaucoma and other neurodegenerations.

    View details for DOI 10.3390/ijms232415896

    View details for PubMedID 36555536

  • Longitudinal in vivo Ca2+ imaging reveals dynamic activity changes of diseased retinal ganglion cells at the single-cell level. Proceedings of the National Academy of Sciences of the United States of America Li, L., Feng, X., Fang, F., Miller, D. A., Zhang, S., Zhuang, P., Huang, H., Liu, P., Liu, J., Sredar, N., Liu, L., Sun, Y., Duan, X., Goldberg, J. L., Zhang, H. F., Hu, Y. 2022; 119 (48): e2206829119


    Retinal ganglion cells (RGCs) are heterogeneous projection neurons that convey distinct visual features from the retina to brain. Here, we present a high-throughput in vivo RGC activity assay in response to light stimulation using noninvasive Ca2+ imaging of thousands of RGCs simultaneously in living mice. Population and single-cell analyses of longitudinal RGC Ca2+ imaging reveal distinct functional responses of RGCs and unprecedented individual RGC activity conversions during traumatic and glaucomatous degeneration. This study establishes a foundation for future in vivo RGC function classifications and longitudinal activity evaluations using more advanced imaging techniques and visual stimuli under normal, disease, and neural repair conditions. These analyses can be performed at both the population and single-cell levels using temporal and spatial information, which will be invaluable for understanding RGC pathophysiology and identifying functional biomarkers for diverse optic neuropathies.

    View details for DOI 10.1073/pnas.2206829119

    View details for PubMedID 36409915

  • Maprotiline restores ER homeostasis and rescues neurodegeneration via Histamine Receptor H1 inhibition in retinal ganglion cells. Nature communications Chen, W., Liu, P., Liu, D., Huang, H., Feng, X., Fang, F., Li, L., Wu, J., Liu, L., Solow-Cordero, D. E., Hu, Y. 2022; 13 (1): 6796


    When the protein or calcium homeostasis of the endoplasmic reticulum (ER) is adversely altered, cells experience ER stress that leads to various diseases including neurodegeneration. Genetic deletion of an ER stress downstream effector, CHOP, significantly protects neuron somata and axons. Here we report that three tricyclic compounds identified through a small-scale high throughput screening using a CHOP promoter-driven luciferase cell-based assay, effectively inhibit ER stress by antagonizing their common target, histamine receptor H1 (HRH1). We further demonstrated that systemic administration of one of these compounds, maprotiline, or CRISPR-mediated retinal ganglion cell (RGC)-specific HRH1 inhibition, delivers considerable neuroprotection of both RGC somata and axons and preservation of visual function in two mouse optic neuropathy models. Finally, we determine that maprotiline restores ER homeostasis by inhibiting HRH1-mediated Ca2+ release from ER. In this work we establish maprotiline as a candidate neuroprotectant and HRH1 as a potential therapeutic target for glaucoma.

    View details for DOI 10.1038/s41467-022-34682-y

    View details for PubMedID 36357388

  • Single-cell transcriptome analysis of regenerating RGCs reveals potent glaucoma neural repair genes. Neuron Li, L., Fang, F., Feng, X., Zhuang, P., Huang, H., Liu, P., Liu, L., Xu, A. Z., Qi, L. S., Cong, L., Hu, Y. 2022


    Axon regeneration holds great promise for neural repair of CNS axonopathies, including glaucoma. Pten deletion in retinal ganglion cells (RGCs) promotes potent optic nerve regeneration, but only a small population of Pten-null RGCs are actually regenerating RGCs (regRGCs); most surviving RGCs (surRGCs) remain non-regenerative. Here, we developed a strategy to specifically label and purify regRGCs and surRGCs, respectively, from the same Pten-deletion mice after optic nerve crush, in which they differ only in their regeneration capability. Smart-Seq2 single-cell transcriptome analysis revealed novel regeneration-associated genes that significantly promote axon regeneration. The most potent of these, Anxa2, acts synergistically with its ligand tPA in Pten-deletion-induced axon regeneration. Anxa2, its downstream effector ILK, and Mpp1 dramatically protect RGC somata and axons and preserve visual function in a clinically relevant model of glaucoma, demonstrating the exciting potential of this innovative strategy to identify novel effective neural repair candidates.

    View details for DOI 10.1016/j.neuron.2022.06.022

    View details for PubMedID 35952672

  • In Vivo Evaluation of Naive and Diseased RGC Activities at Single-Cell Level Li, L., Fang, F., Feng, X., Zhang, S., Miller, D., Zhuang, P., Huang, H., Liu, P., Liu, J., Sredar, N., Liu, L., Sun, Y., Duan, X., Goldberg, J. L., Zhang, H., Hu, Y. ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2022
  • NMNAT2 and NAD(+) are Downregulated in Glaucomatous RGCs and Overexpression of NMNAT2 Rescues Glaucomatous Neurodegeneration Liu, D., Fang, F., Zhuang, P., Feng, X., Liu, P., Huang, H., Li, L., Chen, W., Liu, L., Sun, Y., Jiang, H., Ye, J., Hu, Y. ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2022
  • Neuroprotection of SARM1 Inhibition in Traumatic and Glaucomatous but not in EAE Optic Neuropathies Liu, P., Huang, H., Chen, W., Fang, F., Li, L., Feng, X., Liu, L., Liu, D., Dalal, R., Sun, Y., Ling, K., Rigo, F., Hu, Y. ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2022
  • NMNAT2 Is Downregulated in Glaucomatous RGCs and RGC-Specific Gene Therapy Rescues Neurodegeneration and Visual Function. Molecular therapy : the journal of the American Society of Gene Therapy Fang, F., Zhuang, P., Feng, X., Liu, P., Liu, D., Huang, H., Li, L., Chen, W., Liu, L., Sun, Y., Jiang, H., Ye, J., Hu, Y. 1800


    The lack of neuroprotective treatments for retinal ganglion cells (RGCs) and optic nerve (ON) is a central challenge for glaucoma management. Emerging evidence suggests that redox factor NAD+ decline is a hallmark of aging and neurodegenerative diseases. Supplementation with NAD+ precursors and overexpression of NMNAT1, the key enzyme in the NAD+ biosynthetic process, have significant neuroprotective effects. We first profile the translatomes of RGCs in naive mice and mice with silicone oil-induced ocular hypertension (SOHU)/glaucoma by RiboTag mRNA sequencing. Intriguingly, only NMNAT2, but not NMNAT1 or NMNAT3, is significantly decreased in SOHU glaucomatous RGCs, which we confirm by in situ hybridization. We next demonstrate that AAV2 intravitreal injection-mediated overexpression of long half-life NMNAT2 mutant driven by RGC-specific mouse gamma-synuclein (mSncg) promoter restores decreased NAD+ levels in glaucomatous RGCs and ONs. Moreover, this RGC-specific gene therapy strategy delivers significant neuroprotection of both RGC soma and axon and preservation of visual function in the traumatic ON crush model and the SOHU glaucoma model. Collectively, our studies suggest that the weakening of NMNAT2 expression in glaucomatous RGCs contributes to a deleterious NAD+ decline and that modulating RGC intrinsic NMNAT2 levels by AAV2-mSncg vector is a promising gene therapy for glaucomatous neurodegeneration.

    View details for DOI 10.1016/j.ymthe.2022.01.035

    View details for PubMedID 35114390

  • Osteopontin drives retinal ganglion cell resiliency in glaucomatous neuropathy Kinde, B., Zhao, M., Toma, K., Li, L., Hu, Y., Han, Y., Duan, X. ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2021
  • OCRL regulates lysosome positioning and mTORC1 activity through SSX2IP-mediated microtubule anchoring. EMBO reports Wang, B., He, W., Prosseda, P. P., Li, L., Kowal, T. J., Alvarado, J. A., Wang, Q., Hu, Y., Sun, Y. 2021: e52173


    Lysosomal positioning and mTOR (mammalian target of rapamycin) signaling coordinate cellular responses to nutrient levels. Inadequate nutrient sensing can result in growth delays, a hallmark of Lowe syndrome. OCRL mutations cause Lowe syndrome, but the role of OCRL in nutrient sensing is unknown. Here, we show that OCRL is localized to the centrosome by its ASH domain and that it recruits microtubule-anchoring factor SSX2IP to the centrosome, which is important in the formation of the microtubule-organizing center. Deficiency of OCRL in human and mouse cells results in loss of microtubule-organizing centers and impaired microtubule-based lysosome movement, which in turn leads to mTORC1 inactivation and abnormal nutrient sensing. Centrosome-targeted PACT-SSX2IP can restore microtubule anchoring and mTOR activity. Importantly, boosting the activity of mTORC1 restores the nutrient sensing ability of Lowe patients' cells. Our findings highlight mTORC1 as a novel therapeutic target for Lowe syndrome.

    View details for DOI 10.15252/embr.202052173

    View details for PubMedID 33987909

  • Chronic mild and acute severe glaucomatous neurodegeneration derived from silicone oil-induced ocular hypertension. Scientific reports Fang, F., Zhang, J., Zhuang, P., Liu, P., Li, L., Huang, H., Webber, H. C., Xu, Y., Liu, L., Dalal, R., Sun, Y., Hu, Y. 2021; 11 (1): 9052


    Recently, we established silicone oil-induced ocular hypertension (SOHU) mouse model with significant glaucomatous neurodegeneration. Here we characterize two additional variations of this model that simulate two distinct glaucoma types. The first is a chronic model produced by high frequency (HF) pupillary dilation after SO-induced pupillary block, which shows sustained moderate IOP elevation and corresponding slow, mild glaucomatous neurodegeneration. We also demonstrate that although SO removal quickly returns IOP to normal, the glaucomatous neurodegeneration continues to advance to a similar degree as in the HF group without SO removal. The second, an acute model created by no pupillary dilation (ND), shows a greatly elevated IOP and severe inner retina degeneration at an early time point. Therefore, by a straightforward dilation scheme, we extend our original SOHU model to recapitulate phenotypes of two major glaucoma forms, which will be invaluable for selecting neuroprotectants and elucidating their molecular mechanisms.

    View details for DOI 10.1038/s41598-021-88690-x

    View details for PubMedID 33907301

  • Delineating the organization of projection neuron subsets in primary visual cortex with multiple fluorescent rabies virus tracing. Brain structure & function Li, L., Tang, Y., Sun, L., Yu, J., Zhang, S., Gong, H., Webber, H. C., Zhang, X., Hu, Z., Li, X., Rahman, K., Shi, X., Fu, Z., Dai, J., Cao, G. 2021


    The impressive functions of the brain rely on an extensive connectivity matrix between specific neurons, the architecture of which is frequently characterized by one brain nucleus/region connecting to multiple targets, either via collaterals of the same projection neuron or several, differentially specified neurons. Delineating the fine architecture of projection neuron subsets in a specific brain region could greatly facilitate its circuit, computational, and functional resolution. Here, we developed multiple fluorescent rabies viruses (RV) to delineate the fine organization of corticothalamic projection neuron subsets in the primary visual cortex (V1). By simultaneously retrograde labeling multiple distinct subsets of corticothalamic projection neurons in V1 from their target nuclei in thalamus (dLGN, LP, LD), we observed that V1-dLGN corticothalamic projection neurons were densely concentrated in layer VI, except for several sparsely scattered neurons in layer V, while V1-LP and V1-LD corticothalamic projection neurons were localized to both layers V and VI. Meanwhile, we observed a fraction of V1 corticothalamic projection neurons targeting two thalamic nuclei, which was further confirmed by fMOST whole-brain imaging. The multiple fluorescent RV tracing tools can be extensively applied to resolve the architecture of projection neuron subsets in certain brain regions, with a strong potential to delineate the computational and functional organization of these brain regions.

    View details for DOI 10.1007/s00429-021-02250-7

    View details for PubMedID 33710409

  • Post-translational modification of Sox11 regulates RGC survival and axon regeneration. eNeuro Chang, K., Bian, M., Xia, X., Madaan, A., Sun, C., Wang, Q., Li, L., Nahmou, M., Noro, T., Yokota, S., Galvao, J., Kreymerman, A., Tanasa, B., Hu, Y., Goldberg, J. L. 2021


    The failure of adult CNS neurons to survive and regenerate their axons after injury or in neurodegenerative disease remains a major target for basic and clinical neuroscience. Recent data demonstrated in the adult mouse that exogenous expression of Sry-related high-mobility-box 11 (Sox11) promotes optic nerve regeneration after optic nerve injury, but exacerbates the death of a subset of retinal ganglion cells, alpha-RGCs. During development, Sox11 is required for RGC differentiation from retinal progenitor cells (RPCs), and we found that mutation of a single residue to prevent sumoylation at lysine 91 (K91) increased nuclear localization and RGC differentiation in vitro Here we explored whether this Sox11 manipulation similarly has stronger effects on RGC survival and optic nerve regeneration. In vitro, we found that non-SUMOylatable Sox11K91A leads to RGC death and suppresses axon outgrowth in primary neurons. We furthermore found that Sox11K91A more strongly promotes axon regeneration but also increases RGC death after optic nerve injury in vivo in adult mouse. RNA sequence data showed that Sox11 and Sox11K91A increase the expression of key signaling pathway genes associated with axon growth and regeneration but downregulated Spp1 and Opn4 expression in RGC cultures, consistent with negatively regulating the survival of alpha-RGCs and ipRGCs. Thus Sox11 and its sumoylation site at K91 regulate gene expression, survival and axon growth in RGCs and may be explored further as potential regenerative therapies for optic neuropathy.Significance Statement Sox11 expression promotes optic nerve regeneration but also increases RGC death after optic nerve injury. Here we demonstrate that mutation of a single SUMOylation site on Sox11 (Sox11K91A) leads to stronger effects in vivo RNA sequencing analysis reveals that Sox11 and Sox11K91A differentially regulate downstream gene expression related to axon growth and guidance. Understanding these effects of post-translational modification of Sox11 in regulating regeneration in vivo suggests a potent therapeutic strategy for vision restoration in optic neuropathies.

    View details for DOI 10.1523/ENEURO.0358-20.2020

    View details for PubMedID 33441400

  • Neuronal NMNAT2 Overexpression Does Not Achieve Significant Neuroprotection in Experimental Autoimmune Encephalomyelitis/Optic Neuritis. Frontiers in cellular neuroscience Liu, P., Huang, H., Fang, F., Liu, L., Li, L., Feng, X., Chen, W., Dalal, R., Sun, Y., Hu, Y. 2021; 15: 754651


    Optic neuritis, inflammation, and demyelination of the optic nerve (ON), is one of the most common clinical manifestations of multiple sclerosis; affected patients suffer persistent visual symptoms due to ON degeneration and secondary retinal ganglion cell (RGC) death. The mouse experimental autoimmune encephalomyelitis (EAE) model replicates optic neuritis and significant RGC soma and axon loss. Nicotinamide mononucleotide adenylyltransferases (NMNATs) are NAD+-synthetic enzymes that have been shown to be essential for axon integrity, activation of which significantly delays axonal Wallerian degeneration. NMNAT2, which is enriched in axons, has been proposed as a promising therapeutic target for axon injury-induced neurodegeneration. We therefore investigated whether activation of NMNAT2 can be used as a gene therapy strategy for neuroprotection in EAE/optic neuritis. To avoid the confounding effects in inflammatory cells, which play important roles in EAE initiation and progression, we used an RGC-specific promoter to drive the expression of the long half-life NMNAT2 mutant in mouse RGCs in vivo. However, optical coherence tomography in vivo retina imaging did not reveal significant protection of the ganglion cell complex, and visual function assays, pattern electroretinography, and optokinetic response also showed no improvement in mice with NMNAT2 overexpression. Postmortem histological analysis of retina wholemounts and semithin sections of ON confirmed the in vivo results: NMNAT2 activation in RGCs does not provide significant neuroprotection of RGCs in EAE/optic neuritis. Our studies suggest that a different degenerative mechanism than Wallerian degeneration is involved in autoimmune inflammatory axonopathy and that NMNAT2 may not be a major contributor to this mechanism.

    View details for DOI 10.3389/fncel.2021.754651

    View details for PubMedID 34707482

  • Longitudinal Morphological and Functional Assessment of RGC Neurodegeneration After Optic Nerve Crush in Mouse. Frontiers in cellular neuroscience Li, L. n., Huang, H. n., Fang, F. n., Liu, L. n., Sun, Y. n., Hu, Y. n. 2020; 14: 109


    The mouse optic nerve crush (ONC) model has been widely used to study optic neuropathies and central nervous system (CNS) axon injury and repair. Previous histological studies of retinal ganglion cell (RGC) somata in retina and axons in ON demonstrate significant neurodegeneration after ONC, but longitudinal morphological and functional assessment of RGCs in living animals is lacking. It is essential to establish these assays to provide more clinically relevant information for early detection and monitoring the progression of CNS neurodegeneration. Here, we present in vivo data gathered by scanning laser ophthalmoscopy (SLO), optical coherence tomography (OCT), and pattern electroretinogram (PERG) at different time points after ONC in mouse eyes and corresponding histological quantification of the RGC somata and axons. Not surprisingly, direct visualization of RGCs by SLO fundus imaging correlated best with histological quantification of RGC somata and axons. Unexpectedly, OCT did not detect obvious retinal thinning until late time points (14 and 28-days post ONC) and instead detected significant retinal swelling at early time points (1-5 days post-ONC), indicating a characteristic initial retinal response to ON injury. PERG also demonstrated an early RGC functional deficit in response to ONC, before significant RGC death, suggesting that it is highly sensitive to ONC. However, the limited progression of PERG deficits diminished its usefulness as a reliable indicator of RGC degeneration.

    View details for DOI 10.3389/fncel.2020.00109

    View details for PubMedID 32410964

    View details for PubMedCentralID PMC7200994

  • Mouse gamma-Synuclein Promoter-Mediated Gene Expression and Editing in Mammalian Retinal Ganglion Cells. The Journal of neuroscience : the official journal of the Society for Neuroscience Wang, Q. n., Zhuang, P. S., Huang, H. n., Li, L. n., Liu, L. n., Webber, H. C., Dalal, R. n., Siew, L. n., Fligor, C. M., Chang, K. C., Nahmou, M. n., Kreymerman, A. n., Sun, Y. n., Meyer, J. S., Goldberg, J. L., Hu, Y. n. 2020


    Optic neuropathies are a group of optic nerve (ON) diseases caused by various insults including glaucoma, inflammation, ischemia, trauma and genetic deficits, which are characterized by retinal ganglion cell (RGC) death and ON degeneration. An increasing number of genes involved in RGC intrinsic signaling have been found to be promising neural repair targets that can potentially be modulated directly by gene therapy, if we can achieve RGC specific gene targeting. To address this challenge, we first used adeno associated virus (AAV)-mediated gene transfer to perform a low throughput in vivo screening in both male and female mouse eyes and identified the mouse γ-synuclein (mSncg) promoter, which specifically and potently sustained transgene expression in mouse RGCs and also works in human RGCs. We further demonstrated that gene therapy that combines AAV-mSncg promoter with CRISPR/Cas9 gene editing can knockdown pro-degenerative genes in RGCs and provide effective neuroprotection in optic neuropathies.Significance Statement:Here we present an RGC-specific promoter, mouse γ-synuclein (mSncg) promoter, and perform extensive characterization and proof-of-concept studies of mSncg promoter-mediated gene expression and CRISPR/Cas9 gene editing in RGCs in vivo To our knowledge, this is the first report demonstrating in vivo neuroprotection of injured RGCs and optic nerve by AAV-mediated CRISPR/Cas9 inhibition of genes that are critical for neurodegeneration. It represents a powerful tool to achieve RGC-specific gene modulation, and also opens up a promising gene therapy strategy for optic neuropathies, the most common form of eye diseases that cause irreversible blindness.

    View details for DOI 10.1523/JNEUROSCI.0102-20.2020

    View details for PubMedID 32300046

  • In Vivo Two-photon Calcium Imaging in Dendrites of Rabies Virus-labeled V1 Corticothalamic Neurons NEUROSCIENCE BULLETIN Tang, Y., Li, L., Sun, L., Yu, J., Hu, Z., Lian, K., Cao, G., Dai, J. 2019


    Monitoring neuronal activity in vivo is critical to understanding the physiological or pathological functions of the brain. Two-photon Ca2+ imaging in vivo using a cranial window and specific neuronal labeling enables real-time, in situ, and long-term imaging of the living brain. Here, we constructed a recombinant rabies virus containing the Ca2+ indicator GCaMP6s along with the fluorescent protein DsRed2 as a baseline reference to ensure GCaMP6s signal reliability. This functional tracer was applied to retrogradely label specific V1-thalamus circuits and detect spontaneous Ca2+ activity in the dendrites of V1 corticothalamic neurons by in vivo two-photon Ca2+ imaging. Notably, we were able to record single-spine spontaneous Ca2+ activity in specific circuits. Distinct spontaneous Ca2+ dynamics in dendrites of V1 corticothalamic neurons were found for different V1-thalamus circuits. Our method can be applied to monitor Ca2+ dynamics in specific input circuits in vivo, and contribute to functional studies of defined neural circuits and the dissection of functional circuit connections.

    View details for DOI 10.1007/s12264-019-00452-y

    View details for Web of Science ID 000500858000002

    View details for PubMedID 31808041

  • A Reversible Silicon Oil-Induced Ocular Hypertension Model in Mice. Journal of visualized experiments : JoVE Zhang, J., Fang, F., Li, L., Huang, H., Webber, H. C., Sun, Y., Mahajan, V. B., Hu, Y. 2019


    Elevated intraocular pressure (IOP) is a well-documented risk factor for glaucoma. Here we describe a novel, effective method for consistently inducing stable IOP elevation in mice that mimics the post-operative complication of using silicone oil (SO) as a tamponade agent in human vitreoretinal surgery. In this protocol, SO is injected into the anterior chamber of the mouse eye to block the pupil and prevent inflow of aqueous humor. The posterior chamber accumulates aqueous humor and this in turn increases the IOP of the posterior segment. A single SO injection produces reliable, sufficient, and stable IOP elevation, which induces significant glaucomatous neurodegeneration. This model is a true replicate of secondary glaucoma in the eye clinic. To further mimic the clinical setting, SO can be removed from the anterior chamber to reopen the drainage pathway and allow inflow of aqueous humor, which is drained through the trabecular meshwork (TM) at the angle of the anterior chamber. Because IOP quickly returns to normal, the model can be used to test the effect of lowering IOP on glaucomatous retinal ganglion cells. This method is straightforward, does not require special equipment or repeat procedures, closely simulates clinical situations, and may be applicable to diverse animal species. However, minor modifications may be required.

    View details for DOI 10.3791/60409

    View details for PubMedID 31789319

  • A novel inducible and reversible mouse glaucoma model: Silicone Oil-Induced Ocular Hypertension Under-detected (SOHU) Zhang, J., Li, L., Huang, H., Webber, H., Li, S., Tang, P., Mahajan, V. B., Sun, Y., Zhang, M., Hu, Y. ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2019
  • Neuroprotection of Retinal Ganglion Cells by AAV2-gamma-Synuclein Promoter-Mediated CRISPR/Cas9 Gene Editing Li, L., Wang, Q., Huang, H., Sun, Y., Goldberg, J. L., Hu, Y. ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2019
  • Stem cell-derived retinal ganglion cell differentiation and its transplantation Chang, K., Wu, S., Li, L., Sun, C., Xia, X., Knasel, C., Nahmou, M., Wernig, M., Goldberg, J. L. ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2019
  • Loss of optineurin C-terminus causes significant retinal ganglion cell degeneration Webber, H., Huang, H., Li, L., Zhang, J., Zhuang, P., Wang, Q., Hu, Y. ASSOC RESEARCH VISION OPHTHALMOLOGY INC. 2019
  • Memantine can relieve the neuronal impairment caused by neurotropic virus infection JOURNAL OF MEDICAL VIROLOGY Sun, L., Zhou, M., Liu, C., Tang, Y., Xiao, K., Dai, J., Gao, Z., Siew, L., Cao, G., Wu, X., Li, L., Zhang, R. 2019; 91 (6): 935–40

    View details for DOI 10.1002/jmv.25396

    View details for Web of Science ID 000465087100005

  • Silicone oil-induced ocular hypertension and glaucomatous neurodegeneration in mouse ELIFE Zhang, J., Li, L., Huang, H., Fang, F., Webber, H. C., Zhuang, P., Liu, L., Dalal, R., Tang, P. H., Mahajan, V. B., Sun, Y., Li, S., Zhang, M., Goldberg, J. L., Hu, Y. 2019; 8
  • The anatomy and metabolome of the lymphatic system in the brain in health and disease. Brain pathology (Zurich, Switzerland) He, W. n., Jing, Y. n., Wan, Q. n., Xiao, K. n., Chen, K. n., Lu, Y. n., Li, L. n., Tang, Y. n., Deng, Y. n., Yao, Z. n., Yue, J. n., Cao, G. n. 2019


    Recent studies have demonstrated that the brain is equipped with a lymphatic drainage system that is actively involved in parenchymal waste clearance, brain homeostasis and immune regulation. However, the exact anatomic drainage routes of brain lymph fluid (BLF) remain elusive, hampering the physiological study and clinical application of this system. In this study, we systematically dissected the anatomy of the BLF pathways in a rat model. Moreover, we developed a protocol to collect BLF from the afferent lymphatic vessels of deep cervical lymph nodes (dcLNs) and cerebrospinal fluid (CSF) from the fourth ventricle. Nuclear magnetic resonance spectroscopy showed that BLF contains more metabolites than CSF, suggesting that BLF might be a more sensitive indicator of brain dynamics under physiological and pathological conditions. Finally, we identified several metabolites as potential diagnostic biomarkers for glioma, Parkinson's disease and CNS infectious diseases. Together, these data may provide insight into the physiology of the lymphatic system in the brain and into the clinical diagnosis of CNS disorders.

    View details for DOI 10.1111/bpa.12805

    View details for PubMedID 31747475

  • Digestion-ligation-only Hi-C is an efficient and cost-effective method for chromosome conformation capture NATURE GENETICS Lin, D., Hong, P., Zhang, S., Xu, W., Jamal, M., Yan, K., Lei, Y., Li, L., Ruan, Y., Fu, Z. F., Li, G., Cao, G. 2018; 50 (5): 754-+


    Chromosome conformation capture (3C) technologies can be used to investigate 3D genomic structures. However, high background noise, high costs, and a lack of straightforward noise evaluation in current methods impede the advancement of 3D genomic research. Here we developed a simple digestion-ligation-only Hi-C (DLO Hi-C) technology to explore the 3D landscape of the genome. This method requires only two rounds of digestion and ligation, without the need for biotin labeling and pulldown. Non-ligated DNA was efficiently removed in a cost-effective step by purifying specific linker-ligated DNA fragments. Notably, random ligation could be quickly evaluated in an early quality-control step before sequencing. Moreover, an in situ version of DLO Hi-C using a four-cutter restriction enzyme has been developed. We applied DLO Hi-C to delineate the genomic architecture of THP-1 and K562 cells and uncovered chromosomal translocations. This technology may facilitate investigation of genomic organization, gene regulation, and (meta)genome assembly.

    View details for DOI 10.1038/s41588-018-0111-2

    View details for Web of Science ID 000431394900019

    View details for PubMedID 29700467

  • In vivo fiber photometry of neural activity in response to optogenetically manipulated inputs in freely moving mice JOURNAL OF INNOVATIVE OPTICAL HEALTH SCIENCES Li, L., Tang, Y., Sun, L., Rahman, K., Huang, K., Xu, W., Yu, J., Dai, J., Cao, G. 2017; 10 (5)
  • Rabies viruses leader RNA interacts with host Hsc70 and inhibits virus replication ONCOTARGET Zhang, R., Liu, C., Cao, Y., Jamal, M., Chen, X., Zheng, J., Li, L., You, J., Zhu, Q., Liu, S., Dai, J., Cui, M., Fu, Z. F., Cao, G. 2017; 8 (27): 43822–37


    Viruses have been shown to be equipped with regulatory RNAs to evade host defense system. It has long been known that rabies virus (RABV) transcribes a small regulatory RNA, leader RNA (leRNA), which mediates the transition from viral RNA transcription to replication. However, the detailed molecular mechanism remains enigmatic. In the present study, we determined the genetic architecture of RABV leRNA and demonstrated its inhibitory effect on replication of wild-type rabies, DRV-AH08. The RNA immunoprecipitation results suggest that leRNA inhibits RABV replication via interfering the binding of RABV nucleoprotein with genomic RNA. Furthermore, we identified heat shock cognate 70 kDa protein (Hsc70) as a leRNA host cellular interacting protein, of which the expression level was dynamically regulated by RABV infection. Notably, our data suggest that Hsc70 was involved in suppressing RABV replication by leader RNA. Finally, our experiments imply that leRNA might be potentially useful as a novel drug in rabies post-exposure prophylaxis. Together, this study suggested leRNA in concert with its host interacting protein Hsc70, dynamically down-regulate RABV replication.

    View details for DOI 10.18632/oncotarget.16517

    View details for Web of Science ID 000405498000019

    View details for PubMedID 28388579

    View details for PubMedCentralID PMC5546443

  • Induction of HepG2 cell apoptosis by Irgarol 1051 through mitochondrial dysfunction and oxidative stresses TOXICOLOGY IN VITRO Wang, L., Liang, B., Li, L., Liu, W. 2013; 27 (6): 1771–79


    In this study, HepG2 cells were exposed to 0.04-40 mg/L Irgarol 1051. Results show that Irgarol 1051 can damage cell morphology and cause a significant decrease in cell viability. Positive staining by Annexin V, caspase-3 activity enhancement, and the damage in cell ultrastructure indicated an apoptotic mode of cell death for 4.0mg/L Irgarol 1051 treatment. At the same time, caspase-9 was also significantly induced by 0.4 and 4.0mg/L Irgarol 1051 at 72 h, which suggests that the intrinsic mitochondria pathway was involved in the apoptosis. The mitochondrial membrane potential decreased significantly after the HepG2 cells were exposed to Irgarol 1051 for 6 and 72 h. Especially, the translocation of cytochrome c from mitochondria to cytosol was recorded, supporting the idea that the mitochondrial pathway was involved in the apoptosis signal pathways induced by Irgarol 1051. The significantly increased levels of intracellular reactive oxygen species (ROS) and an immediate ROS burst were also recorded. The results here may imply that Irgarol 1051 induces HepG2 cell apoptosis through mitochondrial dysfunction and oxidative stresses. Although it is possible that this chemical has no detrimental effects on human health at the environmentally relevant concentration, it may cause problems to top coastal predators due to bio-accumulation through the food chain.

    View details for DOI 10.1016/j.tiv.2013.05.006

    View details for Web of Science ID 000324847800021

    View details for PubMedID 23722069

  • Enhanced proliferation and differentiation of neural stem cells grown on PHA films coated with recombinant fusion proteins ACTA BIOMATERIALIA Xie, H., Li, J., Li, L., Dong, Y., Chen, G., Chen, K. C. 2013; 9 (8): 7845–54


    Polyhydroxyalkanoates (PHAs) belong to a family of copolyesters with demonstrated biocompatibility. We hypothesize that genetically fusing evolutionarily preserved cell binding motifs, such as RGD or IKVAV, to the PHA-binding protein phasin (PhaP) for surface functionalization of PHA materials could better support the growth and differentiation of neural stem cells (NSCs). This hypothesis is tested on three polyester materials of the same aliphatic family: poly(L-lactic acid) (PLA) and two PHB copolymers, poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate) (PHBVHHx). Experimental results indicate that surface coating of the two fusion proteins, PhaP-RGD and PhaP-IKVAV, provides short-term advantages in promoting the adhesion, proliferation and neural differentiation of rat NSCs compared to the PhaP-coated or uncoated material. Among the tested samples, the combination of coating PhaP-IKVAV on an PHBVHHx surface yields the highest levels in cell adhesion and proliferation, while the PLA film coated with PhaP-IKVAV promotes better neural differentiation and neurite outgrowth in the early stage. Because both PhaP-RGD and PhaP-IKVAV could be produced in an inexpensive manner, our data suggest that PhaP-IKVAV is an ideal nonspecific coating agent to functionalize hydrophobic biomaterials in the application of neural tissue engineering.

    View details for DOI 10.1016/j.actbio.2013.04.038

    View details for Web of Science ID 000322207700025

    View details for PubMedID 23639778