Assessment of lipocalin-1, resistin, cathepsin-D, neurokinin A, agmatine, NGF, and BDNF serum levels in children with Autism Spectrum Disorder

Introduction

Repetitive habits, limited interests, and social communication deficits are characteristics of Autism Spectrum Disorder (ASD), a complex neurological illness. ASD, which begins in early childhood, is a lifelong disease [1]. The World Health Organization (WHO) forecasts that ASD affects one in 160 children globally on average, with males being affected at a rate four to five folds higher than females [1], 2]. ASD’s precise cause is yet unknown. Nonetheless, it has been acknowledged that a complex interplay and combination of immunological dysfunction, genetics, and environmental variables may contribute to the development of ASD [3]. A member of the neurotrophic family, which also contains neurotrophic factors 3 and 4 and nerve growth factor (NGF), is brain-derived neurotrophic factor (BDNF) [4]. Since BDNF’s primary roles include regulating glycogenesis, neurogenesis, and synaptogenesis as well as short- and long-term synaptic interactions that impact neuroprotection, memory processes, and cognition, it has received special attention in the study of ASD [5]. Numerous studies demonstrate that autistic children have higher blood, serum, and brain BDNF levels than typical controls [6]. The characteristic intermediate filament protein of astrocytes and an indicator of astroglial activity is glial fibrillary acidic protein (GFAP). The cerebrospinal fluid of children with autism, Rett syndrome, and several neurological disorders has been shown to have high quantities of this protein [7]. Moreover, recent studies have supported the promising role of GFAP level in blood as a diagnostic and prognostic biomarker of brain and spinal cord disorders [8], 9].

This study aims to examine the levels of important biochemical parameters such as lipocalin-1, resistin, cathepsin-D, neurokinin A, agmatinase, BDNF, NGF and GFAP in children with ASD. The second aim of the study is to evaluate whether some of these parameters can be used as biomarkers in the diagnosis of ASD.

Materials and methods

Results

Of the patients participating in the study, 74.4% were boys and 25.6% were girls. Boys made up 47.5 % and girls made up 52.5 % of the control group. Table 1 shows the comparative biochemical results of the patients and the control groups. Compared to the control group, the patient group exhibited elevated levels of sodium and chloride, whereas magnesium and vitamin B12 levels were reduced. At the p<0.05 level, median comparisons of these values are statistically significant.

Serum levels of lipocalin-1, resistin, cathepsin-D, agmatinase, BDNF, and NGF were high in the patients, and this difference was statistically significant when comparing the median values of serum levels of biomolecules between the patient and control groups in this study. The patient group’s GFAP serum level was found to be statistically substantially decreased (Table 2) (p<0.05).

According to the results of the correlation analysis performed in the control group, strong positive relationships were found between lipocalin-1 and resistin, cathepsin D, agmantinase, GFAP and NGF. The correlations of lipocalin-1 with resistin (r=0.550, p<0.001), cathepsin D (r=0.671, p<0.001), agmantinase (r=0.422, p=0.007), GFAP (r=0.483, p=0.002) and NGF (r=0.415, p=0.008) were found to be statistically significant. In the correlation analysis of the patient group, a significant positive relationship was found between agmantinase and lipocalin-1 (r=0.386, p=0.015). In addition, resistin; correlations with cathepsin D (r=0.555, p<0.001), neurokinin A (r=0.420, p=0.008), GFAP (r=0.643, p<0.001) and NGF (r=0.582, p<0.001) were also found to be statistically significant.

When the disease severity levels of ASD patients participating in the study were evaluated, it was found that most of the patients were at the moderate level. According to clinical data, 74.4 % of ASD patients were mentally retarded. 56.4 % of the patients showed signs of self-mutilation, while 66.7 % of the patients showed signs of coherent sentence formation.

Discussion

The purpose of this study was to characterize the concentrations of certain biomarkers in the serum of both healthy control subjects and those with ASD, as well as to investigate the potential of biomarkers to aid in the diagnosis of ASD. The results of this study showed that serum lipocalin-1, resistin, cathepsin-D, neurokinin A, agmatinase, BDNF, NGF, and GFAP levels were statistically different in healthy controls and children diagnosed with ASD.

Lipocalin-1 has been defined as a molecule that can bind various lipophilic molecules and eliminate harmful lipids and lipophilic molecules from the body [19]. Because of our hypothesis that lipocalin-1 may play a part in immunological processes in autistic individuals, In this investigation, we discovered that the lipocalin-1 levels of the ASD group were statistically substantially greater than those of the control group. Our results are in line with a research published in the literature that found lipocalin-1 abnormalities in saliva samples from autistic people, which may be connected to immune dysregulation processes in autism [20]. Our findings are consistent with a research that measured lipocalin-1 using tear samples from patients with Alzheimer’s disease and another that measured lipocalin-1 using tear samples from patients with diabetic retinopathy, one of the neuroinflammatory illnesses. These studies revealed that the patient group had higher lipocalin-1 levels than the control group, suggesting that tears could be used as a biomarker [21], 22].

Resistin is a proinflammatory cytokine that plays a role in the pathogenesis of various inflammatory central nervous system disorders. Resistin, secreted from adipose tissues, monocytes, and macrophages, is important due to its roles in metabolic functions and immunoinflammatory system [23], 24]. In our study, resistin levels in the ASD group were found to be statistically significantly higher than those in the control group (p=0.008). The results of this study are in line with earlier research that suggests pro-inflammatory cytokine resistin expression may rise in vitro, as well as a study that was published in the literature and suggests that immunological and mitochondrial variables may be involved in the genesis of autism [25], 26]. Another study has shown increases in resistin and visfatin and levels in ASD patients compared to the control group. Therefore, our results indicate that immune dysfunctions may be associated with ASDs [27].

Neurogenic inflammation is mediated by numerous neuropeptides, primarily tachykinins. It has been established that the neuropeptides known as neurokinin A, tachykinins – substance P and neurokinin B are released from the excitatory section of non-adrenergic, non-cholinergic excitatory nervous system nerves in response to allergen exposure [28]. Mammalian neuronal tissue contains the decapeptide neurokinin A, which has the following sequence: His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met-NH2. [29]. Numerous studies have revealed that, in response to inflammatory stimuli, non-neuronal cells, including immune cells, can also produce tachykinins. By changing several facets of immune cell activity, these tachykinins significantly affect inflammatory responses [30]. According to a study, during asthma exacerbations, children’s blood and sputum eosinophil counts significantly correlated with elevated sputum neurokinin A levels [28]. Therefore, the rise in serum neurokinin A levels in autistic children may be due to immune cell activation after exposure to certain environmental antigens (such infectious agents, dietary allergies, and heavy metals) and the ensuing increase in release. The results of this study corroborate previous research by showing that the neurokinin A levels were higher in the autism spectrum group when compared to the control group.

Cathepsin D, the main lysosomal aspartic acid protease, is extensively expressed in the brain and is quite selective in hydrolyzing certain peptide bonds of target proteins [31]. There have been suggestions of multiple functions for cathepsin D both inside and outside of the cell, as well as for its proteolytically inactive precursor, pro cathepsin D. Chronically inflamed organs and malignant tumors have elevated levels of procathepsin D. It has been demonstrated that cathepsin D triggers apoptosis through caspase-8 and is crucial for controlling cellular apoptosis [31], [32], [33]. Furthermore, recent studies have shown that cathepsin D may be released into the cytosol upon inducing apoptosis and cleave the Bcl2 family member Bid, which releases cyt c from mitochondria and activates caspase-9 and -3 [34]. Additionally, a number of studies have shown that procathepsin D causes the production of inflammatory cytokines, including IL-4, IL-8, IL-10, and IL-13 [35]. However, it has been shown that inflammatory cytokines such as TNF-α and IFN-γ increase extracellular pro cathepsin D in primary endothelial cell cultures [36]. In our current findings, it was determined that the cathepsin D level in children diagnosed with autism spectrum was higher than in the control group. Based on these, it could be hypothesized that high cytokine levels may cause increased cathepsin D expression in the autistic brain and that cathepsin D may mediate cytokine-induced apoptosis.

Agmatine is a naturally occurring polyamine metabolite that is produced by arginine decarboxylase from the amino acid l-arginine. When agmatine is metabolized by the agmatinase enzyme, it is converted into putrescine, spermidine and spermine [37]. Agmatine has been shown in numerous prior studies to have neuromodulatory, antiapoptotic, neurogenic, antioxidant, and anti-inflammatory activities [38], 39]. Also, agmatine has demonstrated beneficial effects on many preclinical studies assessing its therapeutic effects, including hypoxic ischemia, epileptic seizures, morphine tolerance, anxiety disorders and memory, depression, and neuropsychiatric conditions like Parkinson’s illness [40]. Numerous animal experiments have demonstrated that treating mice with agmatine reduces the symptoms of obsessive-compulsive disorder [41], 42]. In an experimental animal study, it was suggested that the increase in agmatinase caused by the upregulation of the agmatinase gene and the resulting agmatine deficit led to a depressive phenotype [43]. According to another study, it was determined that agmatinase expression increased significantly in postmortem measurements in individuals with bipolar and unipolar disorders, and it was stated that agmatine degradation may be responsible for the pathogenesis [44]. According to the results of the current study, children with ASD had greater levels of agmatinase than the control group.

BDNF belongs to the neurotrophic family, which also includes NGF and neurotrophic factors 3 and 4 [45]. BDNF has drawn particular focus in the research of ASD because to its significant functions in regulating glycogenesis, neurogenesis, and synaptogenesis as well as short- and long-term synaptic contacts that affect neuroprotection, memory functions, and cognition [46]. Serum levels of BDNF were found to be greater in children with ASD than in the control group. A statistically significant positive change was observed. In this study, there was a noteworthy positive association between the patients’ blood levels of BDNF and agmatinase. Another study found that the average BDNF levels in the sick group were considerably greater than those in the control group, which supports our findings [47]. According to the findings of a meta-analysis research that measured five neurotrophic factors in 2,486 children with ASD and 4,303 healthy controls, elevated peripheral blood concentrations of BDNF, NGF, and VEGF were identified as an indication of ASD in children. This result confirms clinical data showing children with ASD have aberrant neurotrophic factor profiles [48]. According to a different study, autistic children had higher levels of BDNF in their brain, blood, and serum when compared to normal controls [49].

The activation of astrocytes is indicated by GFAP, the distinctive intermediate filament protein of astrocytes. It has been discovered that children with Rett syndrome, autism, and other neurological disorders have elevated amounts of this protein in their cerebral fluid [50]. Furthermore, recent research has confirmed the blood level of GFAP as a promising biomarker for brain and spinal cord disorders, both in terms of diagnosis and prognosis [51], 52]. In a study, it was found that autistic people’s brains had noticeably greater amounts of GFAP. This data suggests that autism is associated with microglial and astrocyte activation. and is associated with astrocyte activation [53]. The buildup of GFAP molecules in the intracellular space and decreased microglial and astrocyte activation may be indicated by low blood GFAP levels in children with autism. Our study’s comparison of the GFAP levels of the ASD and control groups revealed that the ASD group’s GFAP levels were statistically substantially lower than the control group’s. Nerve growth factor is a multifunctional molecule that influences energy homeostasis, synaptic plasticity, and neuro-immune processes. It is currently unclear how NGF-induced neuronal differentiation impacts ASD, despite the fact that its processes are widely characterized [54]. NGF levels were examined in a 2013 research including 49 children with ASD and 49 healthy children; the findings indicated that the ASD group’s NGF levels were noticeably greater than the control group’s [55]. It was consistent with our results in another study that plasma NGF levels of the ASD group were significantly higher than those of the control group [56].

Conclusions and recommendations

This study investigated the relationships between agmatinase, BDNF, neurokinin A, cathepsin D, lipocalin-1, resistin, GFAP, and NGF levels, as well as some essential routine biochemical parameters of ASD patients. While several studies have looked into biomarkers in the etiology of ASD, none have evaluated all eight of these biomarkers together. This is significant because this study is the first in the literature. Understanding the pathophysiology of autism can be greatly aided by identifying molecular alterations in the disorder through experimental research.