亨丁頓舞蹈病症是一種遺傳性神經退行性疾病，由亨丁頓 (Htt) 基因外顯子 1 中有異常 CAG 重複導致細胞內蛋白質聚集。這些有毒的蛋白質聚集會讓HD 患者神經元的形態變差。在先前的研究發現，miR-196a 是 HD 中失調的 miRNA 之一，我們實驗室研究證實miR-196a可改善神經元細胞骨架，尤其是在微管聚合中，並改善 HD 小鼠的運動行為。我們還發現胰島素樣生長因子 2 結合蛋白 3 (IGF2BP3) 是 miR-196a 的靶基因之一，而對胚胎期細胞生長和分化很重要的胰島素樣生長因子 2 (IGF2) 是IGF2BP3下游目標的其中之一。因此，本研究的目標是了解在HD 模型中IGF2在 miR-196a-IGF2BP3 路徑對於軸突生長中的作用。結果我們發現 HD 患者的血液樣本中 IGF2BP3 的 mRNA 水平較高而IGF2較低，在HD 轉基因小鼠的腦樣本中 IGF2BP3 的蛋白質水平較高，而 IGF2 的水平也較低。此外，miR-196a 降低 N2a 細胞中的 IGF2BP3 並增加 IGF2 蛋白質水平。此外，IGF2 的過表達增加了 N2a 細胞和 HD 初級神經元中的總神經突長度，並且 IGF2 的敲低阻止了對 miR-196a 誘導的神經突生長的有益作用。我們進一步發現 IGF2 通過增加 N2a 細胞中 cdc42 的活性來增加絲狀偽足的數量。除了神經突生長之外，IGF2 的下調增加了 N2a 細胞中的突變 HTT 聚集體。總之，我們的研究表明 IGF2 在 HD 中miR-196a 所誘導的軸突生長中起著至關重要的作用。我們證實了在HD中miR-196a神經保護的路徑。我們預計這項研究將為 HD 提供一種新的治療選擇。

Huntington’s disease is an inherited neurodegenerative disease and caused by abnormal CAG repeat expansions at exon 1 of the huntingtin (HTT) gene that results in intracellular aggregate formations in neurons. These toxic aggregates worsen neuronal morphology, such as neurite outgrowth, in HD patients. A previous study in our lab shows that miR-196a, one of dysregulated miRNAs in HD, improves neuronal cytoskeleton, especially in microtubule polymerization, and ameliorates motor behavior in HD mice. We also found Insulin-like growth factor 2 binding protein 3 (IGF2BP3) is one of miR-196a target genes, and Insulin-like growth factor 2 (IGF2), which is important for cell growth and differentiation at embryonic stage, is one of IGF2BP3 downstream targets. Therefore, the goal of this study aims to investigate the role of IGF2 in neurite outgrowth regulated by the miR-196a-IGF2BP3 pathway in HD models. In the results, we found that the mRNA levels of IGF2BP3 was higher and IGF2 was lower in blood samples of HD patients, whereas IGF2BP3 was higher and IGF2 was lower in brain samples in HD transgenic mice as well. In addition, miR-196a decreased IGF2BP3 and increased IGF2 protein levels in N2a cells. Moreover, overexpression of IGF2 increased total neurite length in N2a cells and HD primary neurons and knock down of IGF2 blocked the beneficial effect on neurite outgrowth induced by miR-196a. We further found that IGF2 increased filopodia numbers through increasing active form of cdc42 in N2a cells. In addition to neurite outgrowth, down-regulation of IGF2 increased mutant HTT aggregates in N2a cells. In summary, our studies showed that IGF2 played a crucial role in neurite outgrowth induced by miR-196a in HD. We demonstrate a miR-196a neuroprotective pathway in HD. We anticipate this study will provide a novel therapeutic option for HD.

1. Matlahov, I. and P.C. van der Wel, Conformational studies of pathogenic expanded polyglutamine protein deposits from Huntington's disease. Exp Biol Med (Maywood), 2019. 244(17): p. 1584-1595.
2. Andresen, J.M., et al., The relationship between CAG repeat length and age of onset differs for Huntington's disease patients with juvenile onset or adult onset. Ann Hum Genet, 2007. 71(Pt 3): p. 295-301.
3. Squitieri, F. and J. Jankovic, Huntington's disease: how intermediate are intermediate repeat lengths? Movement Disorders, 2012. 27(14): p. 1714-1717.
4. Fu, H., J. Hardy, and K.E. Duff, Selective vulnerability in neurodegenerative diseases. Nature Neuroscience, 2018. 21(10): p. 1350-1358.
5. Soares, T.R., et al., Targeting the proteostasis network in Huntington’s disease. Ageing Research Reviews, 2019. 49: p. 92-103.
6. Carmo, C., et al., Mitochondrial Dysfunction in Huntington's Disease. Adv Exp Med Biol, 2018. 1049: p. 59-83.
7. Mehta, S.R., et al., Human Huntington's Disease iPSC-Derived Cortical Neurons Display Altered Transcriptomics, Morphology, and Maturation. Cell Rep, 2018. 25(4): p. 1081-1096 e6.
8. Oosterloo, M., et al., Clinical and genetic characteristics of late-onset Huntington's disease. Parkinsonism Relat Disord, 2019. 61: p. 101-105.
9. Solberg, O.K., et al., Age at Death and Causes of Death in Patients with Huntington Disease in Norway in 1986-2015. Journal of Huntington's disease, 2018. 7(1): p. 77-86.
10. Wyant, K.J., A.J. Ridder, and P. Dayalu, Huntington's Disease-Update on Treatments. Curr Neurol Neurosci Rep, 2017. 17(4): p. 33.
11. Wild, E.J. and S.J. Tabrizi, Therapies targeting DNA and RNA in Huntington's disease. The Lancet. Neurology, 2017. 16(10): p. 837-847.
12. Lane, R.M., et al., Translating Antisense Technology into a Treatment for Huntington's Disease. Methods Mol Biol, 2018. 1780: p. 497-523.
13. O'Brien, J., et al., Overview of MicroRNA Biogenesis, Mechanisms of Actions, and Circulation. Frontiers in endocrinology, 2018. 9: p. 402-402.
14. Vasudevan, S., Posttranscriptional upregulation by microRNAs. Wiley Interdiscip Rev RNA, 2012. 3(3): p. 311-30.
15. Antoniou, A., et al., The dynamic recruitment of TRBP to neuronal membranes mediates dendritogenesis during development. EMBO reports, 2018. 19(3): p. e44853.
16. Thomas, K.T., C. Gross, and G.J. Bassell, microRNAs Sculpt Neuronal Communication in a Tight Balance That Is Lost in Neurological Disease. Front Mol Neurosci, 2018. 11: p. 455.
17. Mitash, N., J. E Donovan, and A. Swiatecka-Urban, The Role of MicroRNA in the Airway Surface Liquid Homeostasis. International journal of molecular sciences, 2020. 21(11): p. 3848.
18. Magri, F., F. Vanoli, and S. Corti, miRNA in spinal muscular atrophy pathogenesis and therapy. Journal of cellular and molecular medicine, 2018. 22(2): p. 755-767.
19. Chen, F.Z., Y. Zhao, and H.Z. Chen, MicroRNA-98 reduces amyloid β-protein production and improves oxidative stress and mitochondrial dysfunction through the Notch signaling pathway via HEY2 in Alzheimer's disease mice. Int J Mol Med, 2019. 43(1): p. 91-102.
20. Haramati, S., et al., miRNA malfunction causes spinal motor neuron disease. Proceedings of the National Academy of Sciences, 2010. 107(29): p. 13111.
21. Geng, W., et al., Exosomes from miRNA-126-modified ADSCs promotes functional recovery after stroke in rats by improving neurogenesis and suppressing microglia activation. Am J Transl Res, 2019. 11(2): p. 780-792.
22. Kumar, S., et al., MicroRNAs as Peripheral Biomarkers in Aging and Age-Related Diseases. Prog Mol Biol Transl Sci, 2017. 146: p. 47-94.
23. Sinha, M., S. Mukhopadhyay, and N.P. Bhattacharyya, Mechanism(s) of alteration of micro RNA expressions in Huntington's disease and their possible contributions to the observed cellular and molecular dysfunctions in the disease. Neuromolecular Med, 2012. 14(4): p. 221-43.
24. Konovalova, J., et al., Interplay between MicroRNAs and Oxidative Stress in Neurodegenerative Diseases. Int J Mol Sci, 2019. 20(23).
25. Hoss, A.G., et al., MicroRNAs located in the Hox gene clusters are implicated in huntington's disease pathogenesis. PLoS Genet, 2014. 10(2): p. e1004188.
26. Cheng, P.H., et al., miR-196a ameliorates phenotypes of Huntington disease in cell, transgenic mouse, and induced pluripotent stem cell models. Am J Hum Genet, 2013. 93(2): p. 306-12.
27. Mancarella, C. and K. Scotlandi, IGF2BP3 From Physiology to Cancer: Novel Discoveries, Unsolved Issues, and Future Perspectives. Frontiers in cell and developmental biology, 2020. 7: p. 363-363.
28. Jia, M., H. Gut, and J.A. Chao, Structural basis of IMP3 RRM12 recognition of RNA. Rna, 2018. 24(12): p. 1659-1666.
29. Wächter, K., et al., Subcellular localization and RNP formation of IGF2BPs (IGF2 mRNA-binding proteins) is modulated by distinct RNA-binding domains. Biol Chem, 2013. 394(8): p. 1077-90.
30. Degrauwe, N., et al., IMPs: an RNA-binding protein family that provides a link between stem cell maintenance in normal development and cancer. Genes Dev, 2016. 30(22): p. 2459-2474.
31. Bell, J.L., et al., Insulin-like growth factor 2 mRNA-binding proteins (IGF2BPs): post-transcriptional drivers of cancer progression? Cellular and molecular life sciences, 2013. 70(15): p. 2657-2675.
32. Mori, H., et al., Expression of mouse igf2 mRNA-binding protein 3 and its implications for the developing central nervous system. J Neurosci Res, 2001. 64(2): p. 132-43.
33. Nielsen, J., et al., A Family of Insulin-Like Growth Factor II mRNA-Binding Proteins Represses Translation in Late Development. Molecular and Cellular Biology, 1999. 19(2): p. 1262-1270.
34. Yin, H., et al., miR-9-5p Inhibits Skeletal Muscle Satellite Cell Proliferation and Differentiation by Targeting IGF2BP3 through the IGF2-PI3K/Akt Signaling Pathway. Int J Mol Sci, 2020. 21(5).
35. Chen, P., et al., The distribution of IGF2 and IMP3 in osteosarcoma and its relationship with angiogenesis. J Mol Histol, 2012. 43(1): p. 63-70.
36. Bryan, M.R. and A.B. Bowman, Manganese and the Insulin-IGF Signaling Network in Huntington's Disease and Other Neurodegenerative Disorders. Advances in neurobiology, 2017. 18: p. 113-142.
37. Allodi, I., et al., Differential neuronal vulnerability identifies IGF-2 as a protective factor in ALS. Sci Rep, 2016. 6: p. 25960.
38. Garcia-Huerta, P., et al., Insulin-like growth factor 2 (IGF2) protects against Huntington's disease through the extracellular disposal of protein aggregates. Acta Neuropathol, 2020. 140(5): p. 737-764.
39. Hawkes, C. and S. Kar, The insulin-like growth factor-II/mannose-6-phosphate receptor: structure, distribution and function in the central nervous system. Brain Res Brain Res Rev, 2004. 44(2-3): p. 117-40.
40. Selfe, J. and J.M. Shipley, IGF signalling in germ cells and testicular germ cell tumours: roles and therapeutic approaches. Andrology, 2019. 7(4): p. 536-544.
41. DeChiara, T.M., E.J. Robertson, and A. Efstratiadis, Parental imprinting of the mouse insulin-like growth factor II gene. Cell, 1991. 64(4): p. 849-59.
42. Mellott, T.J., et al., IGF2 ameliorates amyloidosis, increases cholinergic marker expression and raises BMP9 and neurotrophin levels in the hippocampus of the APPswePS1dE9 Alzheimer's disease model mice. PLoS One, 2014. 9(4): p. e94287.
43. Cheng, B. and M.P. Mattson, IGF-I and IGF-II protect cultured hippocampal and septal neurons against calcium-mediated hypoglycemic damage. J Neurosci, 1992. 12(4): p. 1558-66.
44. Bracko, O., et al., Gene expression profiling of neural stem cells and their neuronal progeny reveals IGF2 as a regulator of adult hippocampal neurogenesis. J Neurosci, 2012. 32(10): p. 3376-87.
45. Cianfarani, S., Insulin-like growth factor-II: new roles for an old actor. Frontiers in Endocrinology, 2012. 3(118).
46. Li, C.Q., et al., Development of anxiety-like behavior via hippocampal IGF-2 signaling in the offspring of parental morphine exposure: effect of enriched environment. Neuropsychopharmacology, 2014. 39(12): p. 2777-87.
47. Liu, J., P. Speder, and A.H. Brand, Control of brain development and homeostasis by local and systemic insulin signalling. Diabetes Obes Metab, 2014. 16 Suppl 1: p. 16-20.
48. García-Huerta, P., et al., Insulin-like growth factor 2 (IGF2) protects against Huntington's disease through the extracellular disposal of protein aggregates. Acta Neuropathol, 2020. 140(5): p. 737-764.
49. Huang, S., et al., Increased miR-124-3p in microglial exosomes following traumatic brain injury inhibits neuronal inflammation and contributes to neurite outgrowth via their transfer into neurons. Faseb j, 2018. 32(1): p. 512-528.
50. Xin, H., et al., Exosome-mediated transfer of miR-133b from multipotent mesenchymal stromal cells to neural cells contributes to neurite outgrowth. Stem Cells, 2012. 30(7): p. 1556-64.
51. Shen, L.M., et al., miR-181d-5p promotes neurite outgrowth in PC12 Cells via PI3K/Akt pathway. CNS Neurosci Ther, 2017. 23(11): p. 894-906.
52. Hogel, M., R.B. Laprairie, and E.M. Denovan-Wright, Promoters are differentially sensitive to N-terminal mutant huntingtin-mediated transcriptional repression. PLoS One, 2012. 7(7): p. e41152.
53. Her, L.S., et al., miR-196a Enhances Neuronal Morphology through Suppressing RANBP10 to Provide Neuroprotection in Huntington's Disease. Theranostics, 2017. 7(9): p. 2452-2462.
54. Pascual-Lucas, M., et al., Insulin-like growth factor 2 reverses memory and synaptic deficits in APP transgenic mice. EMBO Mol Med, 2014. 6(10): p. 1246-62.
55. Kocerha, J., et al., microRNA-128a dysregulation in transgenic Huntington's disease monkeys. Mol Brain, 2014. 7: p. 46.
56. Muller, S., In silico analysis of regulatory networks underlines the role of miR-10b-5p and its target BDNF in huntington's disease. Transl Neurodegener, 2014. 3: p. 17.
57. Beletskiy, A., E. Chesnokova, and N. Bal, Insulin-Like Growth Factor 2 As a Possible Neuroprotective Agent and Memory Enhancer-Its Comparative Expression, Processing and Signaling in Mammalian CNS. Int J Mol Sci, 2021. 22(4).
58. Marchenko, O.O., et al., A minimal actomyosin-based model predicts the dynamics of filopodia on neuronal dendrites. Mol Biol Cell, 2017. 28(8): p. 1021-1033.
59. Arstikaitis, P., et al., Proteins that promote filopodia stability, but not number, lead to more axonal-dendritic contacts. PLoS One, 2011. 6(3): p. e16998.
60. Wang, C., et al., Insulin-like growth factor 2 regulates the proliferation and differentiation of rat adipose-derived stromal cells via IGF-1R and IR. Cytotherapy, 2019. 21(6): p. 619-630.
61. Agrogiannis, G.D., et al., Insulin-like growth factors in embryonic and fetal growth and skeletal development (Review). Mol Med Rep, 2014. 10(2): p. 579-84.
62. Dominguez, R. and K.C. Holmes, Actin structure and function. Annu Rev Biophys, 2011. 40: p. 169-86.
63. Zhao, T., et al., Compartment-Dependent Degradation of Mutant Huntingtin Accounts for Its Preferential Accumulation in Neuronal Processes. J Neurosci, 2016. 36(32): p. 8317-28.
64. Chen, D.Y., et al., A critical role for IGF-II in memory consolidation and enhancement. Nature, 2011. 469(7331): p. 491-7.
65. Lehtinen, M.K., et al., The cerebrospinal fluid provides a proliferative niche for neural progenitor cells. Neuron, 2011. 69(5): p. 893-905.
66. Suvasini, R., et al., Insulin growth factor-2 binding protein 3 (IGF2BP3) is a glioblastoma-specific marker that activates phosphatidylinositol 3-kinase/mitogen-activated protein kinase (PI3K/MAPK) pathways by modulating IGF-2. J Biol Chem, 2011. 286(29): p. 25882-90.
67. Sainath, R. and G. Gallo, Cytoskeletal and signaling mechanisms of neurite formation. Cell Tissue Res, 2015. 359(1): p. 267-78.
68. Dugina, V., et al., Interaction of microtubules with the actin cytoskeleton via cross-talk of EB1-containing +TIPs and gamma-actin in epithelial cells. Oncotarget, 2016. 7(45): p. 72699-72715.
69. Papandreou, M.J. and C. Leterrier, The functional architecture of axonal actin. Mol Cell Neurosci, 2018. 91: p. 151-159.