Sovateltide (IRL-1620) activates neuronal differentiation and prevents mitochondrial dysfunction in adult mammalian brains following stroke

Amaresh K Ranjan, Seema Briyal, Anil Gulati, Amaresh K Ranjan, Seema Briyal, Anil Gulati

Abstract

The development of effective drugs for stroke is urgently required as it is the 2nd largest killer in the world and its incidence is likely to increase in the future. We have demonstrated cerebral endothelin B receptors (ETBR) as a potential target to treat acute cerebral ischemic stroke. However, the mechanism of ETBR mediated neural regeneration and repair remains elusive. In this study, a permanent middle cerebral artery occluded (MCAO) rat model was used. Sovateltide (an ETBR agonist) injected intravenously showed better survival and neurological and motor function improvement than control. Higher neuronal progenitor cells (NPCs) differentiation along with better mitochondrial morphology and biogenesis in the brain of sovateltide rats were noted. Exposure of cultured NPCs to hypoxia and sovateltide also showed higher NPC differentiation and maturation. This study shows a novel role of ETBR in NPCs and mitochondrial fate determination in cerebral ischemia, and in improving neurological deficit after stroke.

Conflict of interest statement

Dr. Anil Gulati (A.G.) is an employee of Pharmazz, Inc, and has issued and pending patents related to this study. All other authors declare no competing interests.

Figures

Figure 1
Figure 1
Effect of sovateltide on neurological and motor performance in the MCAO rats. Data with Mean ± SEM were plotted (supporting data tables are provided in Table S1).
Figure 2
Figure 2
Expression of neural progenitor marker Doublecortin (DCX) in stroked brain and after sovateltide treatment. (A) Diagrammatic representation of adult rat brain isolation and tissue collection for western blots. (B,C) Blots and densitometry graphs of sham, vehicle and sovateltide (Sova) treated rat right hemisphere (RH) and left hemisphere (LH) brain tissues at 24 h post MCAO. All blots are representative of four different experiments with similar results in rat brains. Values are expressed as mean ± SEM. β-Actin was developed after re-probing of Doublecortin blots with anti-β-Actin and used as a loading control and normalization (full blots in Fig. S1A).
Figure 3
Figure 3
Expression of NPCs differentiation marker HuC/HuD in stroked brain and after sovateltide treatment. (A,B) western blots and densitometry graphs of sham, vehicle and sovateltide (Sova) treated rat right hemisphere (RH) and left hemisphere (LH) brain tissues at 24 h post MCAO. All blots are representative of four different experiments with similar results in rat brain. Values are expressed as mean ± SEM. β-Actin was developed after re-probing of HuC + HuD blots with anti-β-Actin and used as a loading control and normalization (full blots in Fig. S1B).
Figure 4
Figure 4
Expression of NPCs differentiation marker NeuroD1 in stroked brain and after sovateltide treatment. (A,B) western blots and densitometry graphs of sham, vehicle and sovateltide (Sova) treated rat right hemisphere (RH) and left hemisphere (LH) brain tissues at 24 h post MCAO. All blots are representative of four different experiments with similar results in rat brain. Values are expressed as mean ± SEM. β-Actin was developed after re-probing of NeuroD1 blots with anti-β -Actin and used as a loading control and normalization (full blots in Fig. S1C). (C) Expression of neuronal differentiation marker, NeuroD1 in cultured adult rat neural progenitor cells in hypoxia. Representative immunofluorescence microscopy images of sovateltide (1 ng/ml) treated cultured neural progenitor cells after 24 h of hypoxia exposure (supporting images are provided in Fig. S2). Higher expression of NeuroD1 (green) and mature neuronal marker, NeuN (red) was observed in sovateltide than vehicle treated samples. Nuclei were stained with DAPI (blue). Bar scale = 75 µm. (D,E) Fluorescence intensity graphs of NeuroD1 (D) and NeuN (E). Values are expressed as mean ± SEM. (F) Diagrammatic representation of dissection of adult rat brain and tissue collection for cell culture.
Figure 5
Figure 5
Expression of mitochondrial fission and fusion markers in stroked brain tissues. (A,B) western blots and densitometry graphs, (C,D) immunofluorescence images of fission marker (Drp1) and fusion marker (Mfn 2) in sham, vehicle and sovateltide (Sova) treated rat right hemisphere (RH) and left hemisphere (LH) brain tissues at 24 h and day 7 post MCAO. (A,B) densitometry values are expressed as mean ± SEM and β-Actin was developed after re-probing of protein blots with anti-β-Actin and used as a loading control and normalization (full blots in Fig. S3). *p < 0.001 compared to sham, #p < 0.0001 compared vehicle. (C,E) representative immunofluorescence images showing expression of Drp 1 (green, C) and Mfn 2 (green, E). Nuclei were stained with DAPI (blue), Scale bar = 20 µm (C,E). n = 4. (D,F) fluorescence intensity graphs of Drp 1 (D) and Mfn 2 (F) at 24 h and day 7 post MCAO. Values are expressed as mean ± SEM. (G) Diagrammatic representation of dissection of adult rat brain and tissue collection for tissue sectioning and immunostaining.
Figure 6
Figure 6
Transmission electron microscopic (TEM) analysis of mitochondria in brain tissues. (A) Diagrammatic representation of dissection of adult rat brain and tissue sectioning for TEM. (B) representative TEM images of sham, vehicle and sovateltide (Sova) treated rat right hemisphere (RH) brain tissues at 24 h and day 7 post MCAO (supporting images are provided in Fig. S4). Black arrows indicate representative mitochondria. Image magnification 3,000×, scale bar = 0.2 µm. (B) Measurement of mitochondrial cross-sectional area × number at 24 h and day 7 post MCAO, respectively. (C) Measurement of percent mitochondrial to tissue area ratio at 24 h and day 7 post MCAO, respectively. Values are expressed as mean ± SEM (B,C).
Figure 7
Figure 7
In situ PCR analysis of mitochondrial biogenesis in brain tissues. (A) Diagrammatic representation of dissection of adult rat brain and tissue sectioning for in situ PCR. (B) representative in situ PCR images of sham, vehicle and sovateltide (Sova) treated rat right hemisphere brain tissues at day 7 post MCAO (supporting images are provided in Fig S5). Red fluorescence indicates amplified MT-ATP8 DNA in mitochondria. Image magnification 200×. (B) fluorescence intensity graph of MT-ATP8 DNA. Values are expressed as mean ± SEM.
Figure 8
Figure 8
Plausible mechanism of action of ETBR signaling involved in neuronal differentiation of NPCs. Activation of ETBR signaling after sovateltide treatment increases HuC/HuD expression in NPCs probably (indicated with ‘??’ in the diagram) through increased transcription or stability of HuC/HuD RNAs. Increased expression of HuC/HuD would be pushing the NPCs towards neuronal differentiation with increasing the stability of RNAs involved mainly in NPCs differentiation, neuronal maturation and cell cycle exit (A). SATB 1 RNA is one of them, which is known to be stabilized after binding of HuD on its 3′ UTR. SATB 1 is a transcription factor and positively regulates the expression of NeuroD1, a master regulator of neuronal differentiation and also of HuD (positive feedback loop, shown in broken red circle) (B).

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