Academic Appointments


  • Basic Life Science Research Associate, Bioengineering

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

    Abstract

    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

  • The UPR Maintains Proteostasis and the Viability and Function of Hippocampal Neurons in Adult Mice INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES Liu, P., Karim, M., Covelo, A., Yue, Y., Lee, M. K., Lin, W. 2023; 24 (14)

    Abstract

    The unfolded protein response (UPR), which comprises three branches: PERK, ATF6α, and IRE1, is a major mechanism for maintaining cellular proteostasis. Many studies show that the UPR is a major player in regulating neuron viability and function in various neurodegenerative diseases; however, its role in neurodegeneration is highly controversial. Moreover, while evidence suggests activation of the UPR in neurons under normal conditions, deficiency of individual branches of the UPR has no major effect on brain neurons in animals. It remains unclear whether or how the UPR participates in regulating neuronal proteostasis under normal and disease conditions. To determine the physiological role of the UPR in neurons, we generated mice with double deletion of PERK and ATF6α in neurons. We found that inactivation of PERK and ATF6α in neurons caused lysosomal dysfunction (as evidenced by decreased expression of the V0a1 subunit of v-ATPase and decreased activation of cathepsin D), impairment of autophagic flux (as evidenced by increased ratio of LC3-II/LC3-I and increased p62 level), and accumulation of p-tau and Aβ42 in the hippocampus, and led to impairment of spatial memory, impairment of hippocampal LTP, and hippocampal degeneration in adult mice. These results suggest that the UPR is required for maintaining neuronal proteostasis (particularly tau and Aβ homeostasis) and the viability and function of neurons in the hippocampus of adult mice.

    View details for DOI 10.3390/ijms241411542

    View details for Web of Science ID 001038454800001

    View details for PubMedID 37511300

    View details for PubMedCentralID PMC10380539

  • 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

    Abstract

    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

  • 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

    Abstract

    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

    Abstract

    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

    Abstract

    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

  • Multiplex CRISPR genome regulation in the retina Guo, L., Bian, J., Davis, A. E., Liu, P., Kempton, H., Zhang, X., Chemparathy, A., Gu, B., Lin, X., Rane, D., Jamiolkowski, R. M., Hu, Y., Wang, S., Qi, L. 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
  • 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
  • Multiplexed genome regulation in vivo with hyper-efficient Cas12a. Nature cell biology Guo, L. Y., Bian, J., Davis, A. E., Liu, P., Kempton, H. R., Zhang, X., Chemparathy, A., Gu, B., Lin, X., Rane, D. A., Xu, X., Jamiolkowski, R. M., Hu, Y., Wang, S., Qi, L. S. 2022

    Abstract

    Multiplexed modulation of endogenous genes is crucial for sophisticated gene therapy and cell engineering. CRISPR-Cas12a systems enable versatile multiple-genomic-loci targeting by processing numerous CRISPR RNAs (crRNAs) from a single transcript; however, their low efficiency has hindered in vivo applications. Through structure-guided protein engineering, we developed a hyper-efficient Lachnospiraceae bacterium Cas12a variant, termed hyperCas12a, with its catalytically dead version hyperdCas12a showing significantly enhanced efficacy for gene activation, particularly at low concentrations of crRNA. We demonstrate that hyperdCas12a has comparable off-target effects compared with the wild-type system and exhibits enhanced activity for gene editing and repression. Delivery of the hyperdCas12a activator and a single crRNA array simultaneously activating the endogenous Oct4, Sox2 and Klf4 genes in the retina of post-natal mice alters the differentiation of retinal progenitor cells. The hyperCas12a system offers a versatile in vivo tool for a broad range of gene-modulation and gene-therapy applications.

    View details for DOI 10.1038/s41556-022-00870-7

    View details for PubMedID 35414015

  • Multiplexed Genome Regulation In Vivo with Hyper-Efficient Cas12a Guo, L., Bian, J., Davis, A. E., Liu, P., Kempton, H. R., Zhang, X., Chemparathy, A., Gu, B., Lin, X., Rane, D. A., Jamiolkowski, R. M., Hu, Y., Wang, S., Qi, L. S. CELL PRESS. 2022: 103
  • 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

    Abstract

    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

  • 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

    Abstract

    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

  • 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

    Abstract

    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