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Acylated but not des ‐ acyl ghrelin is neuroprotective in an MPTP mouse model of Parkinson's disease

View Article: PubMed Central - PubMed

ABSTRACT

The gut hormone ghrelin is widely beneficial in many disease states. However, ghrelin exists in two distinctive isoforms, each with its own metabolic profile. In Parkinson's Disease (PD) acylated ghrelin administration is neuroprotective, however, the role of des‐acylated ghrelin remains unknown. In this study, we wanted to identify the relative contribution each isoform plays using the MPTP model of PD. Chronic administration of acylated ghrelin in mice lacking both isoforms of ghrelin (Ghrelin KO) attenuated the MPTP‐induced loss on tyrosine hydroxylase (TH) neuronal number and volume and TH protein expression in the nigrostriatal pathway. Moreover, acylated ghrelin reduced the increase in glial fibrillary acidic protein and Ionized calcium binding adaptor molecule 1 microglia in the substantia nigra. However, injection of acylated ghrelin also elevated plasma des‐acylated ghrelin, indicating in vivo deacetylation. Next, we chronically administered des‐acylated ghrelin to Ghrelin KO mice and observed no neuroprotective effects in terms of TH cell number, TH protein expression, glial fibrillary acidic protein and ionized calcium binding adaptor molecule 1 cell number. The lack of a protective effect was mirrored in ghrelin‐O‐acyltransferase KO mice, which lack the ability to acylate ghrelin and consequently these mice have chronically increased plasma des‐acyl ghrelin. Plasma corticosterone was elevated in ghrelin‐O‐acyltransferase KO mice and with des‐acylated ghrelin administration. Overall, our studies suggest that acylated ghrelin is the isoform responsible for in vivo neuroprotection and that pharmacological approaches preventing plasma conversion from acyl ghrelin to des‐acyl ghrelin may have clinical efficacy to help slow or prevent the debilitating effects of PD.

Ghrelin exists in the plasma as acyl and des‐acyl ghrelin. We determined the form responsible for in vivo neuroprotection in a mouse model of Parkinson's disease. Although exogenous acyl ghrelin is deacylated in situ to des‐acyl, only acyl ghrelin was neuroprotective by attenuating dopamine cell loss and glial activation. Acyl ghrelin is a therapeutic option to reduce Parkinson's Disease progression.

Cover image: for this issue: doi: 10.1111/jnc.13316.

No MeSH data available.


Neuroprotective effects in Ghrelin KO mice reinstated with acylated ghrelin. (a and b) Plasma analysis of acyl and des‐acyl ghrelin show an elevation in both acylated and des‐acyl ghrelin after acylated ghrelin administration. The red dotted line indicates average circulating levels of acyl and des‐acyl ghrelin in wild‐type mice. (c and d) Plasma non‐esterified fatty acid and Triglyceride levels are elevated post‐MPTP administration. (e) Stereological quantification of tyrosine hydroxylase (TH) neurons in the SN showing chronic acylated ghrelin is protective in Ghrelin KO mice. (f) Overall cell volume showed a significant reduction with MPTP regardless of injection. Stereological quantification of ionized calcium binding adaptor molecule 1 (IBA1) (g) and GFAP (h) shows elevated levels following MPTP, which is attenuated in mice treated with acylated ghrelin. (i  +  j) Representative images showing MPTP induced (i) microglial and (j) astrocyte activation in the SN (green = TH and red = (i) IBA1 or (j) GFAP). (k) When TH cells were separated and plotted based on number distribution, mice treated with MPTP and acylated ghrelin had a significant effect on smaller volume (500–1500 μmᵌ) cells compared to those not treated with acylated ghrelin. (l) Representative western blot images of TH and beta actin in the SN and Striatum. Quantification of TH levels in the SN (m) and Striatum (n) reveals an attenuated loss of TH in MPTP treated with MPTP and acylated ghrelin compared to MPTP alone. (o and p) MPTP significantly reduced both dopamine and DOPAC with no effect of acylated ghrelin. (q) Acylated ghrelin reduced the elevation of the DOPAC:dopamine ratio in MPTP‐treated mice compared saline alone. (r) Plasma corticosterone levels are significantly elevated in response to MPTP regardless of injection. a, significant compared to Saline/saline‐treated mice and b, significant compared to Saline/MPTP‐treated mice. *p < 0.05, **p < 0.01 compared to MPTP/Saline. Data are represented as mean ± SEM (n = 4–8, two‐way anova,). Scale bar = 100 μm.
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jnc13576-fig-0001: Neuroprotective effects in Ghrelin KO mice reinstated with acylated ghrelin. (a and b) Plasma analysis of acyl and des‐acyl ghrelin show an elevation in both acylated and des‐acyl ghrelin after acylated ghrelin administration. The red dotted line indicates average circulating levels of acyl and des‐acyl ghrelin in wild‐type mice. (c and d) Plasma non‐esterified fatty acid and Triglyceride levels are elevated post‐MPTP administration. (e) Stereological quantification of tyrosine hydroxylase (TH) neurons in the SN showing chronic acylated ghrelin is protective in Ghrelin KO mice. (f) Overall cell volume showed a significant reduction with MPTP regardless of injection. Stereological quantification of ionized calcium binding adaptor molecule 1 (IBA1) (g) and GFAP (h) shows elevated levels following MPTP, which is attenuated in mice treated with acylated ghrelin. (i  +  j) Representative images showing MPTP induced (i) microglial and (j) astrocyte activation in the SN (green = TH and red = (i) IBA1 or (j) GFAP). (k) When TH cells were separated and plotted based on number distribution, mice treated with MPTP and acylated ghrelin had a significant effect on smaller volume (500–1500 μmᵌ) cells compared to those not treated with acylated ghrelin. (l) Representative western blot images of TH and beta actin in the SN and Striatum. Quantification of TH levels in the SN (m) and Striatum (n) reveals an attenuated loss of TH in MPTP treated with MPTP and acylated ghrelin compared to MPTP alone. (o and p) MPTP significantly reduced both dopamine and DOPAC with no effect of acylated ghrelin. (q) Acylated ghrelin reduced the elevation of the DOPAC:dopamine ratio in MPTP‐treated mice compared saline alone. (r) Plasma corticosterone levels are significantly elevated in response to MPTP regardless of injection. a, significant compared to Saline/saline‐treated mice and b, significant compared to Saline/MPTP‐treated mice. *p < 0.05, **p < 0.01 compared to MPTP/Saline. Data are represented as mean ± SEM (n = 4–8, two‐way anova,). Scale bar = 100 μm.

Mentions: To examine the neuroprotective properties of des‐acyl ghrelin, we first confirmed that acyl ghrelin attenuated SN dopamine (DA) cell loss in ghrelin KO mice. Administration of acylated ghrelin significantly elevated circulating plasma concentration for acylated (Fig. 1a) and des‐acylated (Fig. 1b) ghrelin, indicating in vivo deacetylation has occurred. There was no significant change in body weight as a result of chronic acylated ghrelin administration (Figure S1a). Administration of MPTP reduced body weight (Figure S1a) and elevated NEFA, Triglycerides and Corticosterone (Fig. 1c, d and r) in the plasma, as well as IBA1 and GFAP in the SN. Microglia (IBA1+ cells) and astrocytes (GFAP+ cells) are activated during cellular damage and are responsible for minimizing overall dopaminergic cell loss (Kohutnicka et al. 1998). A greater number of microglia and astrocytes present indicate a greater amount of cellular damage. Both microglia and astrocytes were significantly elevated post‐MPTP administration with a significant protective effect of acylated ghrelin on microglial numbers only, although there was a trend for acyl ghrelin to reduce GFAP positive astrocytes (Fig. 1g and h). This was concomitant with a significant reduction in the number and size of TH neurons (enzyme marker of dopamine neurons) in the SN post‐MPTP administration, which was significantly attenuated with chronic acylated ghrelin (Fig. 1e, f and k). Acylated ghrelin also attenuated the MPTP‐induced decrease in TH levels in the SN and Striatum (Fig. 1l–n). HPLC analysis of dopamine and DOPAC revealed a significant reduction with MPTP administration (Fig. 1o and p). Acylated ghrelin prevented the increase in the DOPAC:DA ratio observed after MPTP administration (Fig. 1q). Although these results collectively indicate that acylated ghrelin is neuroprotective in PD, data from Fig. 1(b) indicate that injection of acylated ghrelin significantly increases des‐acyl ghrelin. Therefore, it is reasonable to assume that some neuroprotection may have come from elevated des‐acyl ghrelin.


Acylated but not des ‐ acyl ghrelin is neuroprotective in an MPTP mouse model of Parkinson's disease
Neuroprotective effects in Ghrelin KO mice reinstated with acylated ghrelin. (a and b) Plasma analysis of acyl and des‐acyl ghrelin show an elevation in both acylated and des‐acyl ghrelin after acylated ghrelin administration. The red dotted line indicates average circulating levels of acyl and des‐acyl ghrelin in wild‐type mice. (c and d) Plasma non‐esterified fatty acid and Triglyceride levels are elevated post‐MPTP administration. (e) Stereological quantification of tyrosine hydroxylase (TH) neurons in the SN showing chronic acylated ghrelin is protective in Ghrelin KO mice. (f) Overall cell volume showed a significant reduction with MPTP regardless of injection. Stereological quantification of ionized calcium binding adaptor molecule 1 (IBA1) (g) and GFAP (h) shows elevated levels following MPTP, which is attenuated in mice treated with acylated ghrelin. (i  +  j) Representative images showing MPTP induced (i) microglial and (j) astrocyte activation in the SN (green = TH and red = (i) IBA1 or (j) GFAP). (k) When TH cells were separated and plotted based on number distribution, mice treated with MPTP and acylated ghrelin had a significant effect on smaller volume (500–1500 μmᵌ) cells compared to those not treated with acylated ghrelin. (l) Representative western blot images of TH and beta actin in the SN and Striatum. Quantification of TH levels in the SN (m) and Striatum (n) reveals an attenuated loss of TH in MPTP treated with MPTP and acylated ghrelin compared to MPTP alone. (o and p) MPTP significantly reduced both dopamine and DOPAC with no effect of acylated ghrelin. (q) Acylated ghrelin reduced the elevation of the DOPAC:dopamine ratio in MPTP‐treated mice compared saline alone. (r) Plasma corticosterone levels are significantly elevated in response to MPTP regardless of injection. a, significant compared to Saline/saline‐treated mice and b, significant compared to Saline/MPTP‐treated mice. *p < 0.05, **p < 0.01 compared to MPTP/Saline. Data are represented as mean ± SEM (n = 4–8, two‐way anova,). Scale bar = 100 μm.
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jnc13576-fig-0001: Neuroprotective effects in Ghrelin KO mice reinstated with acylated ghrelin. (a and b) Plasma analysis of acyl and des‐acyl ghrelin show an elevation in both acylated and des‐acyl ghrelin after acylated ghrelin administration. The red dotted line indicates average circulating levels of acyl and des‐acyl ghrelin in wild‐type mice. (c and d) Plasma non‐esterified fatty acid and Triglyceride levels are elevated post‐MPTP administration. (e) Stereological quantification of tyrosine hydroxylase (TH) neurons in the SN showing chronic acylated ghrelin is protective in Ghrelin KO mice. (f) Overall cell volume showed a significant reduction with MPTP regardless of injection. Stereological quantification of ionized calcium binding adaptor molecule 1 (IBA1) (g) and GFAP (h) shows elevated levels following MPTP, which is attenuated in mice treated with acylated ghrelin. (i  +  j) Representative images showing MPTP induced (i) microglial and (j) astrocyte activation in the SN (green = TH and red = (i) IBA1 or (j) GFAP). (k) When TH cells were separated and plotted based on number distribution, mice treated with MPTP and acylated ghrelin had a significant effect on smaller volume (500–1500 μmᵌ) cells compared to those not treated with acylated ghrelin. (l) Representative western blot images of TH and beta actin in the SN and Striatum. Quantification of TH levels in the SN (m) and Striatum (n) reveals an attenuated loss of TH in MPTP treated with MPTP and acylated ghrelin compared to MPTP alone. (o and p) MPTP significantly reduced both dopamine and DOPAC with no effect of acylated ghrelin. (q) Acylated ghrelin reduced the elevation of the DOPAC:dopamine ratio in MPTP‐treated mice compared saline alone. (r) Plasma corticosterone levels are significantly elevated in response to MPTP regardless of injection. a, significant compared to Saline/saline‐treated mice and b, significant compared to Saline/MPTP‐treated mice. *p < 0.05, **p < 0.01 compared to MPTP/Saline. Data are represented as mean ± SEM (n = 4–8, two‐way anova,). Scale bar = 100 μm.
Mentions: To examine the neuroprotective properties of des‐acyl ghrelin, we first confirmed that acyl ghrelin attenuated SN dopamine (DA) cell loss in ghrelin KO mice. Administration of acylated ghrelin significantly elevated circulating plasma concentration for acylated (Fig. 1a) and des‐acylated (Fig. 1b) ghrelin, indicating in vivo deacetylation has occurred. There was no significant change in body weight as a result of chronic acylated ghrelin administration (Figure S1a). Administration of MPTP reduced body weight (Figure S1a) and elevated NEFA, Triglycerides and Corticosterone (Fig. 1c, d and r) in the plasma, as well as IBA1 and GFAP in the SN. Microglia (IBA1+ cells) and astrocytes (GFAP+ cells) are activated during cellular damage and are responsible for minimizing overall dopaminergic cell loss (Kohutnicka et al. 1998). A greater number of microglia and astrocytes present indicate a greater amount of cellular damage. Both microglia and astrocytes were significantly elevated post‐MPTP administration with a significant protective effect of acylated ghrelin on microglial numbers only, although there was a trend for acyl ghrelin to reduce GFAP positive astrocytes (Fig. 1g and h). This was concomitant with a significant reduction in the number and size of TH neurons (enzyme marker of dopamine neurons) in the SN post‐MPTP administration, which was significantly attenuated with chronic acylated ghrelin (Fig. 1e, f and k). Acylated ghrelin also attenuated the MPTP‐induced decrease in TH levels in the SN and Striatum (Fig. 1l–n). HPLC analysis of dopamine and DOPAC revealed a significant reduction with MPTP administration (Fig. 1o and p). Acylated ghrelin prevented the increase in the DOPAC:DA ratio observed after MPTP administration (Fig. 1q). Although these results collectively indicate that acylated ghrelin is neuroprotective in PD, data from Fig. 1(b) indicate that injection of acylated ghrelin significantly increases des‐acyl ghrelin. Therefore, it is reasonable to assume that some neuroprotection may have come from elevated des‐acyl ghrelin.

View Article: PubMed Central - PubMed

ABSTRACT

The gut hormone ghrelin is widely beneficial in many disease states. However, ghrelin exists in two distinctive isoforms, each with its own metabolic profile. In Parkinson's Disease (PD) acylated ghrelin administration is neuroprotective, however, the role of des&#8208;acylated ghrelin remains unknown. In this study, we wanted to identify the relative contribution each isoform plays using the MPTP model of PD. Chronic administration of acylated ghrelin in mice lacking both isoforms of ghrelin (Ghrelin KO) attenuated the MPTP&#8208;induced loss on tyrosine hydroxylase (TH) neuronal number and volume and TH protein expression in the nigrostriatal pathway. Moreover, acylated ghrelin reduced the increase in glial fibrillary acidic protein and Ionized calcium binding adaptor molecule 1 microglia in the substantia nigra. However, injection of acylated ghrelin also elevated plasma des&#8208;acylated ghrelin, indicating in&nbsp;vivo deacetylation. Next, we chronically administered des&#8208;acylated ghrelin to Ghrelin KO mice and observed no neuroprotective effects in terms of TH cell number, TH protein expression, glial fibrillary acidic protein and ionized calcium binding adaptor molecule 1 cell number. The lack of a protective effect was mirrored in ghrelin&#8208;O&#8208;acyltransferase KO mice, which lack the ability to acylate ghrelin and consequently these mice have chronically increased plasma des&#8208;acyl ghrelin. Plasma corticosterone was elevated in ghrelin&#8208;O&#8208;acyltransferase KO mice and with des&#8208;acylated ghrelin administration. Overall, our studies suggest that acylated ghrelin is the isoform responsible for in&nbsp;vivo neuroprotection and that pharmacological approaches preventing plasma conversion from acyl ghrelin to des&#8208;acyl ghrelin may have clinical efficacy to help slow or prevent the debilitating effects of PD.

Ghrelin exists in the plasma as acyl and des&#8208;acyl ghrelin. We determined the form responsible for in&nbsp;vivo neuroprotection in a mouse model of Parkinson's disease. Although exogenous acyl ghrelin is deacylated in&nbsp;situ to des&#8208;acyl, only acyl ghrelin was neuroprotective by attenuating dopamine cell loss and glial activation. Acyl ghrelin is a therapeutic option to reduce Parkinson's Disease progression.

Cover image: for this issue: doi: 10.1111/jnc.13316.

No MeSH data available.