Mitochondrial dynamics and metabolic remodeling in xenograft of IPSC-derived human neural precursors

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Abstract

It is well recognized that the regulation of mitochondrial functions affects the differentiation and maturation of neurons. The study of these processes is of both fundamental and practical importance for regenerative neurobiology. Aim of the study: to characterize the mitochondrial fission changes and their relation to the activation of oxidative phosphorylation (metabolic shift) during maturation of human IPSC-derived neural precursors grafted into rat striatum. Wistar rats (n = 15) were unilaterally injected into the caudate nucleus with neural precursors derived from human IPSCs. Changes in localization and expression of neuronal differentiation markers: nestin, NeuN, neuronal enolase, as well as mitochondrial outer membrane protein, ATP synthase and mitochondrial fission protein Drp1 were assessed by immunostaining. Measurements were performed on graft cells 2 weeks, 3 and 6 months after surgery. Maturation of grafted neurons was associated with fluctuations morphometric parameters of the mitochondrial fraction and Drp1 levels. Increased mitochondrial fission was detected 3 months after transplantation, before an increase in ATP synthase staining by 6th month and a switch of transplanted cells to oxidative phosphorylation. The conducted experiment demonstrated a link between mitochondrial dynamics and changes in the metabolic profile and maturation of transplanted neurons. The regulation of mitochondrial dynamics may have future implications for developing methods to improve the integration of transplanted neurons into recepient brain structures.

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About the authors

D. N. Voronkov

Research Center of Neurology

Author for correspondence.
Email: voronkov@neurology.ru
Russian Federation, Moscow

A. V. Egorova

Research Center of Neurology; N.I. Pirogov Russian National Research Mediвcal University

Email: voronkov@neurology.ru
Russian Federation, Moscow; Moscow

E. N. Fedorova

Research Center of Neurology; N.I. Pirogov Russian National Research Mediвcal University

Email: voronkov@neurology.ru
Russian Federation, Moscow; Moscow

A. V. Stavrovskaya

Research Center of Neurology

Email: voronkov@neurology.ru
Russian Federation, Moscow

O. S. Lebedeva

Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine

Email: voronkov@neurology.ru
Russian Federation, Moscow

A. S. Olshanskiy

Research Center of Neurology

Email: voronkov@neurology.ru
Russian Federation, Moscow

V. V. Podoprigora

N.I. Pirogov Russian National Research Mediвcal University

Email: voronkov@neurology.ru
Russian Federation, Moscow

V. S. Sukhorukov

Research Center of Neurology; N.I. Pirogov Russian National Research Mediвcal University

Email: voronkov@neurology.ru
Russian Federation, Moscow; Moscow

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Supplementary files

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2. Fig. 1. Detection of tyrosine hydroxylase and mitochondrial markers in the graft: (a) — detection of tyrosine hydroxylase (TH, green) and human nuclear antigen (HNA, red), 6 months after transplantation; (b) — association of the mitochondrial network and Drp1 (SDHB — green, Drp1 — red), 6 months after transplantation, image obtained by deconvolution from 24 focal plans; (c) — detection of the mitochondrial outer membrane (MTC02, green) and the result of the median axis extraction algorithm (white), 2 weeks after transplantation, image obtained using the median filter from 12 focal plans.

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3. Fig. 2. Dynamics of detection of neuronal differentiation marker proteins in the transplant: (a) — fluorescence intensity (8 bits, brightness gradations) for detection of neuronal nuclear protein (NeuN); (b) — fluorescence intensity (8 bits, brightness gradations) for detection of nestin (Nes); (c) — immunoperoxidase staining intensity (8 bits, brightness gradations) for detection of neuronal enolase (NSE). wks — weeks, mth — months * — p < 0.05 compared to 2 weeks after transplantation, ** — p < 0.05 compared to 3 months after transplantation. ANOVA, Tukey's post hoc test.

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4. Fig. 3. Changes in mitochondrial parameters in transplant cells. (a) — fluorescence intensity (8 bits, brightness gradations) when using antibodies to Drp1; (b) — fluorescence intensity (8 bits, brightness gradations) when using antibodies to ATP5A; (c) — changes in the length of MTC02-positive objects (“mitochondria length”) (μm); Designations as in Fig. 1.

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5. Fig. 4. Localization of neuronal and mitochondrial markers in the central region of the transplant at different times after surgery. (a, b, c) – Detection of neuronal enolase (NSE); (d, e, f) – Detection of mitochondrial outer membrane protein (MTC02, green), (e) and (f) additionally show the neuronal marker PGP 9.5 (red); (g, h, i) – Detection of ATP synthase (ATP5A, red) and nestin (Nes, green); (j, k, l) – Detection of Drp1 (red) and neuronal marker NeuN (green). The first column (a, d, g, j) – 2 weeks, the second column (b, e, h, k) – 3 months, the third column (c, f, i, l) – 6 months after transplantation.

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