Identifying iNOS and glycogen as biomarkers for degenerated cerebellar purkinje cells in autism spectrum disorder: Protective effects of erythropoietin and zinc sulfate

Abstract

Autism spectrum disorder (ASD) is a collective neurodevelopmental disorder affecting young children and accounting for 1% of the world's population. The cerebellum is the major part of the human brain affected by ASD and is associated with a substantial reduction in the number of Purkinje cells. An association between ASD and the expression of the nitrosative stress biomarker inducible nitric oxide synthase (iNOS), as well as glycogen deposition in damaged Purkinje cells, has not been previously reported in the medical literature. To explore this correlation, young rats were injected with propionic acid (PPA) (500 mg/kg) for 5 days (model group), while the protection groups were treated with either erythropoietin (EPO, 5,000 U/kg) or 2 mg/kg zinc sulfate immediately after the PPA injections. ASD-like features were developed in the model group, as evidenced by cerebellum damage (degeneration of Purkinje cells) and cerebellar dysfunction (behavioral impairment). This study documented the exclusive expression of iNOS in the degenerated Purkinje cells, along with glycogen deposition in these cells. Additionally, PPA significantly (p < 0.001) modulated cerebellar tissue levels of mammalian target of rapamycin (mTOR), gamma-aminobutyric acid (GABA), GABAA receptor, serotonin, the marker of neuronal loss (calbindin D28K), and social interaction deficit. Some of these parameters were differentially protected by EPO and zinc sulfate, with the former providing greater protection than zinc sulfate. Furthermore, a significant correlation between the iNOS score and these parameters associated with ASD was observed. These findings demonstrate the colocalization of iNOS and glycogen in the damaged Purkinje cells induced by ASD, along with the modulation of ASD parameters, which were protected by EPO and zinc sulfate treatments. Thus, these potential novel biomarkers may offer possible therapeutic targets for the treatment of ASD.

Fig 1. EPO and zinc sulfate protect against PPA-induces ASD in rats.

At the end of the experiment, anxiety-like behavior and cerebellar tissue architecture were assessed. (A-H). H&E images (A,C,E,G 200x; B,D,F,H 400x) of cerebellum tissues obtained from the control (A,B), the model (PPA)(C,D), EPO-treated (PPA+EPO) (E,F), and zinc-treated (PPA + ZnSO₄ (G,H) groups of rats are visualized using light microscopy. The bar graph in (I) represents the quantitative analysis of PC number in cerebellar sections prepared from these rat groups. The bar graphs in (J and K) represent the quantitative analysis of the time spent in open arm (J) and the social interaction time (K) for the groups above. Findings exemplify mean ( ± SD). Presented p values are all significant. * p < 0.05 versus control, #p < 0.05 versus PPA. PPA: propionic acid; ASD: autism spectrum disorder; N: acidophilic neuropil; V: vacuolation in the neuropil of the molecular layer; EPO: erythropoietin; ZnSO₄: zinc sulfate; PC: Purkinje cell; M: molecular layer; P: Purkinje cell layer; G: granular layer.

Fig 2. EPO and ZnSO₄ inhibit PPA-induced iNOS expression in Purkinje cells. iNOS immunohistochemistry of cerebellar tissue sections from rats 17 days after injection with vehicle to the control group (A,200x; B, 400x) and PPA to the model (C,200x; D, 400x) and treated groups; PPA+EPO (E,200x; F, 400x) and PPA + ZnSO₄(G,200x; H, 400x) are illustrated.

Note that arrows in D point to the strong positive iNOS cytoplasmic immunostaining in the degenerated Purkinje cells. Whereas, arrow heads and curved arrows in F and H point to the moderate and mild positive immunostaining, respectively. (I) Degree of iNOS immunostaining in cerebellar sections from groups above is illustrated in a bar chart. Findings exemplify median ±  IQ ±  max/min values. Presented p values are all significant; * p < 0.05 versus control, #p < 0.05 versus PPA. iNOS: inducible nitric oxide synthase; PPA: propionic acid; EPO: erythropoietin; ZnSO₄: zinc sulfate.

Fig 3. EPO and ZnSO₄ protect against the dysregulation of cerebellar tissue levels of ASD biomarkers induced by PPA in rats.

Tissue levels of mTOR gene expression (A), GABAa receptor gene expression (B), GABA (C), serotonin (D), and calbindin-D28K (E) were assessed in all group of rats at the end of the investigation. Findings exemplify mean ( ± SD). Presented p values are all significant. * p < 0.05 versus control, #p < 0.05 versus PPA, $p < 0.05 versus EPO+PPA. ASD: autism spectrum disorder; EPO: erythropoietin; ZnSO₄: zinc sulfate; PPA: propionic acid; mTOR: mammalian target of rapamycin; GABAa: gamma amino-butyric acid receptor type a; GABA: gamma amino-butyric acid.

Fig 4. Induction of glycogen accumulation in Purkinje cells by PPA appears to be ameliorated by EPO and ZnSO₄.

PAS stained pictures (A,C,E,G, 200x; B,D,F,H, 400x) of cerebellar tissue sections from rats 17 days after injection with vehicle to the control group (A,B) and PPA to the model (C,D) and treated groups; PPA+EPO (E,F) and PPA + ZnSO₄ (G,H) are shown. Note that arrows in C and D point to the strong positive PAS stained degenerated Purkinje cells, and arrows in E-H point to few PAS positive cells. (I) Degree of PAS-stained cerebellar tissue sections from groups above are demonstrated in a bar chart. Findings depict median ±  IQ ±  max/min values. Presented p values are all significant; * p < 0.05 versus control, #p < 0.05 versus PPA. PAS: periodic acid Schiff; PPA: propionic acid; EPO: erythropoietin; ZnSO₄: zinc sulfate.

Fig 5. Correlation between the score of Purkinje cells expressing iNOS and cerebellum parameters associated with the induction of autism in rats.

All rats had their iNOS expression in Purkinje cells assessed and the relationship between iNOS and glycogen (A), mTOR (B), GABA_A receptor (C), serotonin (D), calbindin-D28K (E), and interaction time (F) are shown using Spearman correlation coefficient. iNOS: inducible nitric oxide synthase; PAS: periodic acid Schiff; PCs: Purkinje cells; mTOR: mammalian target of rapamycin; GABAa: gamma amino-butyric acid receptor type a; GABA: gamma amino-butyric acid.

Fig 6. Proposed model for PPA induced autism in rats appears protected by EPO and ZnSO₄.

PPA: propionic acid; EPO: erythropoietin; ZnSO₄: zinc sulfate; iNOS: inducible nitric oxide synthase; mTOR: mammalian target of rapamycin; GABAa: gamma amino-butyric acid receptor type a; d1: day one; ASD: autism spectrum disorder.