Direct reprogramming of fibroblasts into spiral ganglion neurons by defined transcription factors

Abstract

Degeneration of the cochlear spiral ganglion neurons (SGNs) is one of the major causes of sensorineural hearing loss and significantly impacts the outcomes of cochlear implantation. Functional regeneration of SGNs holds great promise for treating sensorineural hearing loss. In this study, we systematically screened 33 transcriptional regulators implicated in neuronal and SGN fate. Using gene expression array and principal component analyses, we identified a sequential combination of Ascl1, Pou4f1 and Myt1l (APM) in promoting functional reprogramming of SGNs. The neurons induced by APM expressed mature neuronal and SGN lineage-specific markers, displayed mature SGN-like electrophysiological characteristics and exhibited single-cell transcriptomes resembling the endogenous SGNs. Thus, transcription factors APM may serve as novel candidates for direct reprogramming of SGNs and hearing recovery due to SGN damages.

References

[1] Liu W, Xu X, Fan Z, et al. Wnt signaling activates TP53-induced glycolysis and apoptosis regulator and protects against cisplatin-induced spiral ganglion neuron damage in the mouse cochlea. Antioxid Redox Signal. 2019; 30(11): 1389-1410.

[2] Bao J, Ohlemiller KK. Age-related loss of spiral ganglion neurons. Hear Res. 2010; 264(1-2): 93-97.

[3] Liberman MC. Noise-induced and age-related hearing loss: new perspectives and potential therapies. F1000Research. 2017; 6: 927.

[4] Vlajkovic SM, Ambepitiya K, Barclay M, Boison D, Housley GD, Thorne PR. Adenosine receptors regulate susceptibility to noise-induced neural injury in the mouse cochlea and hearing loss. Hear Res. 2017; 345: 43-51.

[5] Hong G, Fu X, Chen X, et al. Dyslexia-related hearing loss occurs mainly through the abnormal spontaneous electrical activity of spiral ganglion neurons. Adv Sci (Weinh). 2023; 10(16):e2205754.

[6] Wang X, Xu L, Meng Y, et al. FOXO1-NCOA4 Axis contributes to cisplatin-induced cochlea spiral ganglion neuron ferroptosis via ferritinophagy. Adv Sci (Weinh). 2024; 11: e2402671.

[7] Wan G, Gomez-Casati ME, Gigliello AR, Liberman MC, Corfas G. Neurotrophin-3 regulates ribbon synapse density in the cochlea and induces synapse regeneration after acoustic trauma. Elife. 2014; 3: e03564.

[8] Wan G, Corfas G. Transient auditory nerve demyelination as a new mechanism for hidden hearing loss. Nat Commun. 2017; 8: 14487.

[9] Müller U, Barr-Gillespie PG. New treatment options for hearing loss. Nat Rev Drug Discov. 2015; 14(5): 346-365.

[10] Meas SJ, Zhang C-L, Dabdoub A. Reprogramming glia into neurons in the peripheral auditory system as a solution for sensorineural hearing loss: lessons from the central nervous system. Front Mol Neurosc. 2018; 11: 77.

[11] Mertens J, Marchetto MC, Bardy C, Gage FH. Evaluating cell reprogramming, differentiation and conversion technologies in neuroscience. Nat Rev Neurosci. 2016; 17(7): 424-437.

[12] Sancho-Martinez I, Baek SH, Izpisua Belmonte JC. Lineage conversion methodologies meet the reprogramming toolbox. Nat Cell Biol. 2012; 14(9): 892-899.

[13] Bocchi R, Masserdotti G, Gotz M. Direct neuronal reprogramming: fast forward from new concepts toward therapeutic approaches. Neuron. 2022; 110(3): 366-393.

[14] Chen Z, Huang Y, Yu C, Liu Q, Qiu C, Wan G. Cochlear Sox2+ glial cells are potent progenitors for spiral ganglion neuron reprogramming induced by small molecules. Front Cell Dev Biol. 2021; 9: 728352.

[15] Sun S, Babola T, Pregernig G, et al. Hair cell mechanotransduction regulates spontaneous activity and spiral ganglion subtype specification in the auditory system. Cell. 2018; 174(5): 1247-1263 e1215.

[16] Shrestha BR, Chia C, Wu L, Kujawa SG, Liberman MC, Goodrich LV. Sensory neuron diversity in the inner ear is shaped by activity. Cell. 2018; 174(5): 1229-1246.e1217.

[17] Petitpre C, Wu H, Sharma A, et al. Neuronal heterogeneity and stereotyped connectivity in the auditory afferent system. Nat Commun. 2018; 9(1): 3691.

[18] Li C, Li X, Bi Z, et al. Comprehensive transcriptome analysis of cochlear spiral ganglion neurons at multiple ages. Elife. 2020; 9: e50491.

[19] Zhang B, Hu Y, Du H, et al. Tissue engineering strategies for spiral ganglion neuron protection and regeneration. J Nanobiotechnol. 2024; 22(1): 458.

[20] Daudet N, Ariza-McNaughton L, Lewis J. Notch signalling is needed to maintain, but not to initiate, the formation of prosensory patches in the chick inner ear. Development. 2007; 134(12): 2369-2378.

[21] Aburto MR, Magariños M, Leon Y, Varela-Nieto I, Sanchez-Calderon H. AKT signaling mediates IGF-I survival actions on otic neural progenitors. PLoS One. 2012; 7(1):e30790.

[22] Bok J, Dolson DK, Hill P, Rüther U, Epstein DJ, Wu DK. Opposing gradients of Gli repressor and activators mediate Shh signaling along the dorsoventral axis of the inner ear. Development. 2007; 134(9): 1713-1722.

[23] Shah SM, Kang YJ, Christensen BL, Feng AS, Kollmar R. Expression of Wnt receptors in adult spiral ganglion neurons: frizzled 9 localization at growth cones of regenerating neurites. Neuroscience. 2009; 164(2): 478-487.

[24] Wise AK, Tu T, Atkinson PJ, et al. The effect of deafness duration on neurotrophin gene therapy for spiral ganglion neuron protection. Hear Res. 2011; 278(1-2): 69-76.

[25] Suzuki J, Corfas G, Liberman MC. Round-window delivery of neurotrophin 3 regenerates cochlear synapses after acoustic overexposure. Sci Rep. 2016; 6: 24907.

[26] Akil O, Blits B, Lustig LR, Leake PA. Virally mediated overexpression of glial-derived neurotrophic factor elicits age- and dose-dependent neuronal toxicity and hearing loss. Hum Gene Ther. 2019; 30(1): 88-105.

[27] Raft S, Koundakjian EJ, Quinones H, et al. Cross-regulation of Ngn1 and Math1 coordinates the production of neurons and sensory hair cells during inner ear development. Development. 2007; 134(24): 4405-4415.

[28] Zou D, Silvius D, Fritzsch B, Xu PX. Eya1 and Six1 are essential for early steps of sensory neurogenesis in mammalian cranial placodes. Development. 2004; 131(22): 5561-5572.

[29] Zou D, Erickson C, Kim EH, Jin D, Fritzsch B, Xu PX. Eya1 gene dosage critically affects the development of sensory epithelia in the mammalian inner ear. Hum Mol Genet. 2008; 17(21): 3340-3356.

[30] Ahmed M, Wong EY, Sun J, Xu J, Wang F, Xu PX. Eya1-Six1 interaction is sufficient to induce hair cell fate in the cochlea by activating Atoh1 expression in cooperation with Sox2. Dev Cell. 2012; 22(2): 377-390.

[31] Ahmed M, Xu J, Xu PX. EYA1 and SIX1 drive the neuronal developmental program in cooperation with the SWI/SNF chromatin-remodeling complex and SOX2 in the mammalian inner ear. Development. 2012; 139(11): 1965-1977.

[32] Sherrill HE, Jean P, Driver EC, et al. Pou4f1 defines a subgroup of type I spiral ganglion neurons and is necessary for Normal inner hair cell presynaptic Ca(2+) signaling. J Neurosci. 2019; 39(27): 5284-5298.

[33] Deng M, Yang H, Xie X, Liang G, Gan L. Comparative expression analysis of POU4F1, POU4F2 and ISL1 in developing mouse cochleovestibular ganglion neurons. Gene Expr Patterns. 2014; 15(1): 31-37.

[34] Huang M, Sage C, Li H, Xiang M, Heller S, Chen ZY. Diverse expression patterns of LIM-homeodomain transcription factors (LIM-HDs) in mammalian inner ear development. Dev Dyn. 2008; 237(11): 3305-3312.

[35] Thaler JP, Lee S-K, Jurata LW, Gill GN, Pfaff SL. LIM factor Lhx3 contributes to the specification of motor neuron and interneuron identity through cell-type-specific protein-protein interactions. Cell. 2002; 110(2): 237-249.

[36] Appler JM, Lu CC, Druckenbrod NR, Yu WM, Koundakjian EJ, Goodrich LV. Gata3 is a critical regulator of cochlear wiring. J Neurosci. 2013; 33(8): 3679-3691.

[37] Yu WM, Appler JM, Kim YH, Nishitani AM, Holt JR, Goodrich LV. A Gata3-Mafb transcriptional network directs post-synaptic differentiation in synapses specialized for hearing. Elife. 2013; 2: e01341.

[38] Nishimura K, Weichert RM, Liu W, Davis RL, Dabdoub A. Generation of induced neurons by direct reprogramming in the mammalian cochlea. Neuroscience. 2014; 275: 125-135.

[39] Noda T, Meas SJ, Nogami J, et al. Direct reprogramming of spiral ganglion non-neuronal cells into neurons: toward ameliorating sensorineural hearing loss by gene therapy. Front Cell Dev Biol. 2018; 6: 16.

[40] Kempfle JS, Luu NC, Petrillo M, Al-Asad R, Zhang A, Edge ASB. Lin28 reprograms inner ear glia to a neuronal fate. Stem Cells. 2020; 38: 890-903.

[41] Li X, Bi Z, Sun Y, Li C, Li Y, Liu Z. In vivo ectopic Ngn1 and Neurod1 convert neonatal cochlear glial cells into spiral ganglion neurons. FASEB J. 2020; 34(3): 4764-4782.

[42] Hu W, Qiu B, Guan W, et al. Direct conversion of normal and Alzheimer's disease human fibroblasts into neuronal cells by small molecules. Cell Stem Cell. 2015; 17(2): 204-212.

[43] He Z, Guo L, Shu Y, et al. Autophagy protects auditory hair cells against neomycin-induced damage. Autophagy. 2017; 13(11): 1884-1904.

[44] Cai T, Jen HI, Kang H, Klisch TJ, Zoghbi HY, Groves AK. Characterization of the transcriptome of nascent hair cells and identification of direct targets of the Atoh1 transcription factor. J Neurosci. 2015; 35(14): 5870-5883.

[45] Scheffer DI, Shen J, Corey DP, Chen ZY. Gene expression by mouse inner ear hair cells during development. J Neurosci. 2015; 35(16): 6366-6380.

[46] Shen J, Scheffer DI, Kwan KY, Corey DP. SHIELD: an integrative gene expression database for inner ear research. Database. 2015; 2015: bav071.

[47] Ringnér M. What is principal component analysis? Nat Biotechnol. 2008; 26(3): 303-304.

[48] Jolliffe IT, Cadima J. Principal component analysis: a review and recent developments. Philos Trans A Math Phys Eng Sci. 2016; 374(2065):20150202.

[49] Takahashi K, Tanabe K, Ohnuki M, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007; 131(5): 861-872.

[50] Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006; 126(4): 663-676.

[51] Xie H, Ye M, Feng R, Graf T. Stepwise reprogramming of B cells into macrophages. Cell. 2004; 117(5): 663-676.

[52] Feng R, Desbordes SC, Xie H, et al. PU.1 and C/EBPalpha/beta convert fibroblasts into macrophage-like cells. Proc Natl Acad Sci U S A. 2008; 105(16): 6057-6062.

[53] Ieda M, Fu JD, Delgado-Olguin P, et al. Direct reprogramming of fibroblasts into functional cardiomyocytes by defined factors. Cell. 2010; 142(3): 375-386.

[54] Marro S, Pang ZP, Yang N, et al. Direct lineage conversion of terminally differentiated hepatocytes to functional neurons. Cell Stem Cell. 2011; 9(4): 374-382.

[55] Davis RL, Weintraub H, Lassar AB. Expression of a single transfected cDNA converts fibroblasts to myoblasts. Cell. 1987; 51(6): 987-1000.

[56] Kim J, Efe JA, Zhu S, et al. Direct reprogramming of mouse fibroblasts to neural progenitors. Proc Natl Acad Sci U S A. 2011; 108(19): 7838-7843.

[57] Pfisterer U, Kirkeby A, Torper O, et al. Direct conversion of human fibroblasts to dopaminergic neurons. Proc Natl Acad Sci U S A. 2011; 108(25): 10343-10348.

[58] Caiazzo M, Dell'Anno MT, Dvoretskova E, et al. Direct generation of functional dopaminergic neurons from mouse and human fibroblasts. Nature. 2011; 476(7359): 224-227.

[59] Lee H, Lee HY, Lee BE, et al. Sequentially induced motor neurons from human fibroblasts facilitate locomotor recovery in a rodent spinal cord injury model. Elife. 2020; 9: 9.

[60] Son EY, Ichida JK, Wainger BJ, et al. Conversion of mouse and human fibroblasts into functional spinal motor neurons. Cell Stem Cell. 2011; 9(3): 205-218.

[61] Colasante G, Lignani G, Rubio A, et al. Rapid conversion of fibroblasts into functional forebrain GABAergic interneurons by direct genetic reprogramming. Cell Stem Cell. 2015; 17(6): 719-734.

[62] Blanchard JW, Eade KT, Szűcs A, et al. Selective conversion of fibroblasts into peripheral sensory neurons. Nat Neurosci. 2015; 18(1): 25-35.

[63] Xiao D, Deng Q, Guo Y, et al. Generation of self-organized sensory ganglion organoids and retinal ganglion cells from fibroblasts. Sci Adv. 2020; 6(22):eaaz5858.

[64] Chen J, Yen A, Florian CP, Dougherty JD. MYT1L in the making: emerging insights on functions of a neurodevelopmental disorder gene. Transl Psychiatry. 2022; 12(1): 292.

[65] Matsushita F, Kameyama T, Kadokawa Y, Marunouchi T. Spatiotemporal expression pattern of Myt/NZF family zinc finger transcription factors during mouse nervous system development. Dev Dyn. 2013; 243(4): 588-600.

[66] Mall M, Kareta MS, Chanda S, et al. Myt1l safeguards neuronal identity by actively repressing many non-neuronal fates. Nature. 2017; 544(7649): 245-249.

[67] Vierbuchen T, Ostermeier A, Pang ZP, Kokubu Y, Südhof TC, Wernig M. Direct conversion of fibroblasts to functional neurons by defined factors. Nature. 2010; 463(7284): 1035-1041.

[68] Guo Z, Zhang L, Wu Z, Chen Y, Wang F, Chen G. In vivo direct reprogramming of reactive glial cells into functional neurons after brain injury and in an Alzheimer's disease model. Cell Stem Cell. 2014; 14(2): 188-202.

[69] Matsuda T, Irie T, Katsurabayashi S, et al. Pioneer factor NeuroD1 rearranges transcriptional and epigenetic profiles to execute microglia-neuron conversion. Neuron. 2019; 101(3): 472-485.e477.

[70] Mattugini N, Bocchi R, Scheuss V, et al. Inducing different neuronal subtypes from astrocytes in the injured mouse cerebral cortex. Neuron. 2019; 103(6): 1086-1095.e1085.

[71] Niu W, Zang T, Zou Y, et al. In vivo reprogramming of astrocytes to neuroblasts in the adult brain. Nat Cell Biol. 2013; 15(10): 1164-1175.

[72] Tai W, Wu W, Wang LL, et al. In vivo reprogramming of NG2 glia enables adult neurogenesis and functional recovery following spinal cord injury. Cell Stem Cell. 2021; 28(5): 923-937.e924.

[73] Torper O, Ottosson DR, Pereira M, et al. In vivo reprogramming of striatal NG2 glia into functional neurons that integrate into local host circuitry. Cell Rep. 2015; 12(3): 474-481.

[74] Chen YC, Ma NX, Pei ZF, et al. A NeuroD1 AAV-based gene therapy for functional brain repair after ischemic injury through in vivo astrocyte-to-neuron conversion. Mol Ther. 2020; 28(1): 217-234.

[75] Qian H, Kang X, Hu J, et al. Reversing a model of Parkinson's disease with in situ converted Nigral neurons. Nature. 2020; 582(7813): 550-556.

[76] Zhou H, Su J, Hu X, et al. Glia-to-neuron conversion by CRISPR-CasRx alleviates symptoms of neurological disease in mice. Cell. 2020; 181(3): 590-603.e516.

[77] Wu Z, Parry M, Hou XY, et al. Gene therapy conversion of striatal astrocytes into GABAergic neurons in mouse models of Huntington's disease. Nat Commun. 2020; 11(1): 1105.

[78] Wang H, Chen Y, Lv J, et al. Bilateral gene therapy in children with autosomal recessive deafness 9: single-arm trial results. Nat Med. 2024; 30(7): 1898-1904.

[79] Lv J, Wang H, Cheng X, et al. AAV1-hOTOF gene therapy for autosomal recessive deafness 9: a single-arm trial. Lancet. 2024; 403(10441): 2317-2325.

[80] Qi J, Zhang L, Tan F, et al. Preclinical efficacy and safety evaluation of AAV-OTOF in DFNB9 mouse model and nonhuman primate. Adv Sci (Weinh). 2024; 11(3):e2306201.

[81] Qi J, Tan F, Zhang L, et al. AAV-mediated gene therapy restores hearing in patients with DFNB9 deafness. Adv Sci (Weinh). 2024; 11(11):e2306788.

[82] Duarte MJ, Kanumuri VV, Landegger LD, et al. Ancestral adeno-associated virus vector delivery of opsins to spiral ganglion neurons: implications for optogenetic cochlear implants. Mol Ther. 2018; 26(8): 1931-1939.

[83] Zhang L, Chen X, Wang X, et al. AAV-mediated gene cocktails enhance supporting cell reprogramming and hair cell regeneration. Adv Sci (Weinh). 2024; 11(29):e2304551.

[84] Liao M, Hu Y, Zhang Y, et al. 3D Ti(3)C(2)T(x) MXene-Matrigel with electroacoustic stimulation to promote the growth of spiral ganglion neurons. ACS Nano. 2022; 16(10): 16744-16756.

[85] Wei H, Chen Z, Hu Y, et al. Topographically conductive butterfly wing substrates for directed spiral ganglion neuron growth. Small. 2021; 17(38):e2102062.

[86] Truong K, Leigh B, Vecchi JT, et al. Interaction of micropatterned topographical and biochemical cues to direct neurite growth from spiral ganglion neurons. Hear Res. 2021; 409: 108315.

[87] Tao T, Chen C, Sun J, Peng Y, Zhu M. A bacterial artificial chromosome transgenic mouse model for visualization of neurite growth. Sci China Life Sci. 2015; 58(4): 373-378.

[88] Qiu LQ, Lai WS, Stumpo DJ, Blackshear PJ. Mouse embryonic fibroblast cell culture and stimulation. Bio Protoc. 2016; 6(13):e1859.

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2024 The Author(s). Cell Proliferation published by Beijing Institute for Stem Cell and Regenerative Medicine and John Wiley & Sons Ltd.