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. 2021 Jan 28:15:621173.
doi: 10.3389/fnins.2021.621173. eCollection 2021.

Reduction in Nesfatin-1 Levels in the Cerebrospinal Fluid and Increased Nigrostriatal Degeneration Following Ventricular Administration of Anti-nesfatin-1 Antibody in Mice

Huanhuan Chen  1 , Xuelian Li  1 , Hui Ma  1 , Wei Zheng  2 , Xiaoli Shen  1   2
Affiliations

Reduction in Nesfatin-1 Levels in the Cerebrospinal Fluid and Increased Nigrostriatal Degeneration Following Ventricular Administration of Anti-nesfatin-1 Antibody in Mice

Huanhuan Chen et al. Front Neurosci. .

Abstract

Nesfatin-1 is one of several brain-gut peptides that have a close relationship with the central dopaminergic system. Our previous studies have shown that nesfatin-1 is capable of protecting nigral dopaminergic neurons against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity. A recent study also revealed a reduced blood level of nesfatin-1 in patients with Parkinson's disease (PD). The current study was designed to investigate whether reduced nesfatin-1 in cerebrospinal fluid (CSF) induces nigrostriatal system degeneration. An intra-cerebroventricular (ICV) injection technique was used to administer anti-nesfatin-1 antibody directly into the lateral ventricle of the brain. Enzyme-linked immunosorbent assay (ELISA) results showed that ICV injection of anti-nesfatin-1 antibody into the lateral ventricle of the brain once daily for 2 weeks caused a significant reduction in nesfatin-1 levels in the CSF (93.1%). Treatment with anti-nesfatin-1 antibody resulted in a substantial loss (23%) of TH-positive (TH+) dopaminergic neurons in the substantia nigra pars compacta (SNpc), as shown by immunofluorescence staining, a depletion in dopamine and its metabolites in the striatum detected by high-performance liquid chromatography (HPLC), and obvious nuclear shrinkage and mitochondrial lesions in dopaminergic neurons in the SNpc detected by transmission electron microscopy (TEM). Furthermore, the results from our Western blot and ELISA experiments demonstrated that anti-nesfatin-1 antibody injection induced an upregulation of caspase-3 activation, increased the expression of p-ERK, and elevated brain-derived neurotrophic factor (BDNF) levels in the SNpc. Taken together, these observations suggest that reduced nesfatin-1 in the brain may induce nigrostriatal dopaminergic system degeneration; this effect may be mediated via mitochondrial dysfunction-related apoptosis. Our data support a role of nesfatin-1 in maintaining the normal physiological function of the nigrostriatal dopaminergic system.

Keywords: Parkinson’s disease; apoptosis; degeneration; dopaminergic neuron; mitochondrion; nesfatin-1; nigrostriatal system.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Schematic illustration of the experimental design. (A) Double staining of TH and MC4R in the SNpc to demonstrate the presence of MC4R in the SNpc (n = 6). (B) Anti-nesfatin-1 antibody induces nigrostriatal system degeneration in mice (n = 24, six mice per group). The mice received the substances at the doses indicated by ICV injection for 14 days. The CSF was collected for the determination of the nesfatin-1 concentration. The SNpc and striatum were collected for the determination of TH, caspase-3, and p-ERK1/2 expression, BDNF concentration, mitochondrial morphology, and nuclear morphology. BDNF, brain-derived neurotrophic factor; CSF, cerebrospinal fluid; ELISA, enzyme-linked immunosorbent assay; MAB 201, non-immune anti-mouse IgG antibody; MC4R, melanocortin 4 receptor; p-ERK1/2, phosphorylated ERK1/2; SHU 9119, MC4R receptor inhibitor; SNpc, substantia nigra pars compacta; Str, striatum; TH, tyrosine hydroxylase; ICV, intra-cerebroventricular.
FIGURE 2
FIGURE 2
Double staining of TH and MC4R in the mouse SNpc (n = 6). The images were obtained by immunofluorescence microscopy. TH, tyrosine hydroxylase; MC4R, melanocortin 4 receptor; SNpc, substantia nigra pars compacta.
FIGURE 3
FIGURE 3
Anti-nesfatin-1 treatment induced nigral dopaminergic neuron degeneration. (A) TH(+)-dopaminergic neurons in the SNpc of control mice and mice treated with MAB 201, SHU 9119, or anti-nesfatin-1 antibody are shown. (B) A summary of the data showing the numbers of TH(+)-dopaminergic neurons in the different groups (TH, tyrosine hydroxylase). (C) Nissl-positive neurons in control mice and mice treated with MAB 201, SHU 9119 or anti-nesfatin-1 antibody are shown. The dotted portion indicates the SNpc. (D) A summary of the data showing the numbers of Nissl-positive neurons in the different groups. Each value represents the mean ± SD, n = 6; *p < 0.05. MAB 201, non-immune anti-mouse IgG antibody; SHU 9119, MC4R receptor inhibitor; SNpc, substantia nigra pars compacta; TH(+), TH-positive.
FIGURE 4
FIGURE 4
Anti-nesfatin-1 antibody-induced depletion of DA and HVA in the striatum of the right brain (n = 6). (A) Striatal DA levels in control mice and mice treated with MAB 201, SHU 9119, or anti-nesfatin-1 antibody are shown. (B) Striatal HVA levels in the different groups. (C) Striatal DOPAC levels in the different groups (DA, dopamine; HVA, homovanillic acid; DOPAC, dihydroxyphenylacetic acid). Each value represents the mean ± SD, n = 6; ***p < 0.0001, **p < 0.005, and *p < 0.05. MAB 201, non-immune anti-mouse IgG antibody; SHU 9119, MC4R receptor inhibitor.
FIGURE 5
FIGURE 5
Anti-nesfatin-1 antibody treatment stimulated caspase-3 protein expression in the SNpc (n = 6). (A) An original image of a Western blot showing the caspase-3 protein levels in control mice and mice treated with MAB 201, SHU 9119, or anti-nesfatin-1 antibody. (B) Statistical analysis of caspase-3 protein levels in the different groups. Each value represents the mean ± SD, n = 6; **p < 0.005, and *p < 0.05. MAB 201, non-immune anti-mouse IgG antibody; SHU 9119, MC4R receptor inhibitor; SNpc, substantia nigra pars compacta.
FIGURE 6
FIGURE 6
Anti-nesfatin-1 antibody treatment induced mitochondrial depletion and nuclear shrinkage in dopaminergic neurons (n = 6). (A) Mitochondria in dopaminergic neurons in control and anti-nesfatin-1 antibody-treated mice. (B) A summary of the data showing the numbers of mitochondria in the dopaminergic neurons of control and anti-nesfatin-1 antibody-treated mice. (C) The anti-nesfatin-1 antibody induced nuclear shrinkage. The red arrows indicate the shrunken nuclei. (D) A summary of the data showing the long axis length of mitochondria in the dopaminergic neurons of control and anti-nesfatin-1 antibody-treated mice. Each value represents the mean ± SD, n = 6; ***p < 0.0001, and *p < 0.05. MAB 201, non-immune anti-mouse IgG antibody; SHU 9119, MC4R receptor inhibitor.
FIGURE 7
FIGURE 7
Anti-nesfatin-1 antibody treatment induced an increase in p-ERK protein levels (n = 6). (A) An original image of a Western blot showing the p-ERK protein levels in control mice and mice treated with MAB 201, SHU 9119 or anti-nesfatin-1 antibody. (B) Statistical analysis of p-ERK protein levels in the different groups. Each value represents the mean ± SD, n = 6; *p < 0.05. MAB 201, non-immune anti-mouse IgG antibody; SHU 9119, MC4R receptor inhibitor; p-ERK, phosphorylated ERK.
FIGURE 8
FIGURE 8
Anti-nesfatin-1 antibody treatment elevated BDNF levels in the SNpc (n = 6). Statistical analysis of BDNF protein levels in control mice and mice treated with MAB 201, SHU 9119 or anti-nesfatin-1 antibody. Each value represents the mean ± SD, n = 6; **p < 0.005 and *p < 0.05. BDNF, brain-derived neurotrophic factor; MAB 201, non-immune anti-mouse IgG antibody; SHU 9119, MC4R receptor inhibitor; SNpc, substantia nigra pars compacta.
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