1. Introduction
Alzheimer’s disease (AD) is a progressive, incurable neurodegenerative disease that is the most common form of dementia in the elderly. The causes of this disease are still not fully understood, and there are no effective methods of its treatment. The main pathogenesis of AD is the amyloid theory, according to which the main phenomenon is the deposition of Aβ42 peptide (42 amino acid amyloid protein) in the brain, which causes the death of neurons and the development of clinical symptoms of dementia. Secondary to amyloidosis are increased levels of oxidative stress, chronic inflammatory response, disturbances in mineral components metabolism homeostasis, formation of pathological ion channels, or changes in the transmission of neurotransmitters [
1]. The second disease the process behind the development of AD, in addition to beta-amyloid (β-amyloid) deposition, is the formation of the so-called neurofibrillary ganglia, which contain double-helical filaments composed of the abnormally phosphorylated tau protein. In the process of abnormal transformations involving β- and γ-secretases, it is fragmented into insoluble forms of β-amyloid, which is deposited inside and then extracellularly in the form of senile plaques. β-amyloid is also deposited in the walls of small cerebral vessels in the cortical layer, and this process may lead to the formation of microbleeding foci located in the cortical structures. The number and distribution of senile plaques and neurons with signs of neurofibrillary degeneration are the basis for the neuropathological classification of AD diagnosis. The above pathological processes result in a decrease in the level of messenger substances, of which the reduction of acetylcholine content is the most important for the memory system [
2].
Among the pathomechanisms involved in the process of neuronal death, there are also disorders in mitochondrial function, an abnormal reaction to oxidative stress, activation of cytokines and other pro-inflammatory factors, disorders in the systems of neurotrophins and their receptors [
3,
4].
So far, no effective treatment for AD has been developed. Only symptomatic treatment is used, which includes drugs affecting cognitive functions, including acetylcholinesterase inhibitors (donepezil and rivastigmine registered in Poland) and memantine, which is an antagonist of the NMDA receptor (N-methyl-D-aspartic acid), as well as antidepressant, antipsychotics, and anti-anxiety drugs. The causal treatment of AD is still in clinical trials. It should be used in the period when the loss of neurons in strategic areas for the disease does not exceed 50%. That is why it is so important to identify patients in the initial period of the disease in order to inhibit the neurodegenerative process and the full symptoms of the disease. Targeted treatments are available to prevent the toxic effects of two key proteins in AD: β-amyloid and tau. These are either anti-toxic Aβ antibodies or β-secretase and γ-secretase inhibitors, which are pathological enzymes involved in the amyloid cascade [
5].
Enzymes such as E1, E2, and E3, deubiquitinating enzymes (DUBs) involved in UPS regulate disease-causing proteins by controlling the degree of ubiquitination. An interesting issue is the development of therapies that target enzymes in degenerative diseases such as AD [
6,
7,
8]. Studies have shown that the ubiquitin-proteasome system (UPS), the major intracellular system for protein quality control in eukaryotic cells, is associated with the pathogenesis of AD. Growing evidence points to a close relationship between β-amyloid and UPS. Impairment of the UPS system in AD can degrade β-amyloid and lead to abnormal accumulation. At the same time, β-amyloid inhibits proteasome activity and disrupts the multi-vesicular body (MVB) sorting pathway, creating an interaction between β-amyloid and UPS. Mutant ubiquitin (Ub) and ubiquitin-1 like ubiquitin (UBL) are associated with β-amyloid accumulation. Meanwhile, E2 coupling enzymes, E3 ligases, and deubiquitinating enzymes play a key role in the proteasomal degradation of β-amyloid. The ubiquitin-proteasome system has a profound effect on the amyloidogenic pathway of processing the amyloid precursor protein (APP) that generates β-amyloid. Upregulation of proteasomal degradation of BACE1 and gamma-secretase components leads to reduced accumulation of β-amyloid. An in-depth look at the mechanism underlying the interaction between β-amyloid and UPS could provide alternative therapeutic targets and lead to the development of new drugs and therapies [
9].
Ubiquitin carboxy-terminal hydrolase L1 (UCHL1) is a deubiquitinating enzyme (223 amino acid protein encoded by 9 exons) [
10]. This enzyme is found in all nerve cells in the brain (accounts for 1–2% of total brain protein) as well as ovary and testis [
11,
12]. AD and Parkinson’s disease are related to dysregulation of UCHL1 [
13,
14]. Loss of the amount of UCHL1 is occurred in amyotrophic lateral sclerosis and AD patients [
14,
15]. A decrease in the amount of UCHL1 increases the tendency of pancreatic beta-cells to programmed cell death. This protein protective function in neuroendocrine cells and explain the connection between diabetes and neurodegenerative diseases [
16]. UCHL1 products hydrolyze small C-terminal adducts of ubiquitin to produce the ubiquitin monomer. This enzyme has both hydrolase and ligase activities [
17]. Hydrolase activity deletes and converts ubiquitin molecules from degraded proteins. Moreover, it is also associated with proteasomal activity [
18]. Ligase activity combines ubiquitin molecules for use in tagging proteins to delete [
12]. Inactivation of the deubiquitinating enzyme inhibits ubiquitin-mediated proteolysis when the free ubiquitin will be spent or and saturation of the proteasome with polyubiquitin chains. Moreover, deubiquitination also plays a specific regulatory role. UCHL1 is relevant for maintaining free ubiquitin pool and for the proper function of the ubiquitin-proteasome system since inhibition of UCHL1 causes a 50% reduction of free ubiquitin in vitro [
17]. In AD, the process of ubiquitination is crucial due to the fact that neurofibrillary tangles are positive for immunostaining to ubiquitin [
19,
20].
The proteasome is a protein complex that plays the essential role of deleting damaged or unnecessary proteins by proteolysis (chemical reaction is the breakdown of proteins into smaller polypeptides or amino acids). It is believed that the proteasome is the main component in the protein degradation pathway [
21]. Proteasomes are found in the plasma of patients suffering from autoimmune and inflammatory diseases [
22,
23,
24]. Interestingly in inflammatory conditions, the concentration of proteasomes in the blood correlates with the activity of the disease [
25]. It has been observed that proteasome inhibition is a mediator of increased concentrations of aggregated protein, oxidized protein, and neuronal death in the brains of AD patients [
26]. Studies show that proteasome activity is decreased in the brains of these patients [
19,
27,
28,
29,
30].
The objective of the study was to estimate the concentration of UCHL1 and proteasome in patients with AD in terms of their clinical condition, dietary habits, smoking cigarettes, and selected indicators of oxidative stress.
4. Discussion
Contemporary literature data describe the ubiquitin-proteasome system as a potential therapeutic target in AD. This system is crucial for protein degradation in eukaryotes. Ubiquitin hydrolase (UCHL1) has been shown to increase the cellular levels of monoubiquitin and thus increase the protein turnover rate of the above system. Low levels of UCHL1 are associated with the accumulation of Aβ in AD [
38].
UCHL1 is a deubiquitinating enzyme that is involved in the pathogenesis of neurodegenerative diseases, including AD. The main enzyme role is the elimination of misfolded proteins [
39]. Our research allowed us to assess that AD patients are characterized by a higher median UCHL1 than healthy individuals (56.05 vs. 7.98 ng/mL,
p < 0.000001). However, the decrease of this enzyme in brain tissue was observed in both ischemic injury and AD [
40]. It is possible that the increased UCHL1 level in serum should compensate for the decrease in the enzyme concentration in brain tissue. A higher concentration of UCHL1 may also reflect the body’s metabolic response to acute inflammation; it is considered a biomarker for various forms of acute CNS damage [
40].
Our research showed a tendency to have a negative link between the concentration of UCHL1 and Zn and between UCHL1 and TAS. Zinc deficiency has been shown to reduce UCHL1 expression in the rat hippocampus [
41]. In contrast, studies on cultured hippocampal neurons showed that zinc modulated UCHL1 expression, suggesting that UCHL1 downregulation may be involved in memory dysfunction caused by a deficiency of this element [
42].
Regression analysis showed that frequent consumption of honey, cooked vegetables, and milk may be negatively correlated with UCHL1 concentration, while consumption of fruit, other cold cuts (sirloin, ham), yellow and processed cheese, legumes, jams, and sausages may be positively correlated with this parameter. This group of products includes highly processed foods that are not recommended in the diet of AD patients. In 2017, a systematic review was published on the link between nutrition and AD. The authors found such a link in 50 of the 64 studies [
43]. For example, a study of 5386 participants found a link between the consumption of trans fat, saturated fat, cholesterol, and total fat and the development of AD [
44]. An excess of simple sugars and saturated fatty acids in the diet is a significant risk factor for AD. The so-called Western diet may result in, inter alia, the occurrence of obesity, dysbiosis of the intestinal microflora, and acceleration of low-grade systemic inflammation. These changes can lead to impairment of the blood-brain barrier and the development of neuroinflammation in addition to amyloid accumulation. The consequence may be dysfunction of synaptic transmission, neurodegeneration, and impairment of cognitive functions and memory [
45].
In turn, the concentration of the proteasome may be positively affected by fruit consumption, and disadvantageously by the consumption of canned fish. Fish is rich in omega-3 fatty acids, including docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). Shin et al. (2017) showed that DHA mediates intermolecular bonds of proteins through oxidation, and the resulting protein aggregates strongly reduce the activity of proteasomes—which has been proven both in vitro and in cultured cells. In cell models characterized by the overexpression of aggregation-prone proteins, such as for example tau, significantly increased levels of tau aggregates and total ubiquitin conjugates were found in the presence of DHA. In summary, DHA is described as a potent inducer of cellular protein aggregates that inhibit proteasome activity and retard systemic protein degradation in pathological conditions [
46].
Other nutritional factors that are relevant to dementia and cognitive disorders include extra-virgin olive oil, nuts, berries, coffee, tea, cocoa, garlic, curcumin, omega-3 fatty acids, ginkgo biloba, resveratrol, phytoestrogens, and alcohol [
47].
Donepezil and rivastigmine are cholinesterase inhibitors, while memantine is the N-methyl-d-aspartate receptor blocker [
48]. In our study, we found no difference in UCHL1 levels in the rivastigmine and memantine groups compared to the memantine and donepezil groups. In the future, it is possible to conduct gene therapies in patients with impaired UCHL1 function.
The research by Öhrfelt et al. (2016) indicates that UCHL1 levels can be used as a cerebrospinal AD biomarker. The pilot study showed that the median UCHL1 in cerebrospinal fluid in the control group (
n = 31) was 4.5 µg/L, while in the AD group (
n = 10) it was significantly higher: 12 µg/L. This trend has been confirmed in the larger population. Finally, the control group was characterized by UCHL1 at the level of 7.2 µg/L, and the AD group at a significantly higher concentration—at the level of 11 µg/L [
49]. Our research also showed a significant increase in UCHL1 in patients with AD.
Our results show that the SPRI technique with specific biosensors for the determination of UCHL1 and proteasome may be a very useful tool in the investigation of AD including effectiveness therapy, the recommended diet, etc.