In this cross-sectional study, we demonstrated that serum tau protein levels were elevated in patients with WD, indicating axonal impairment and suggesting that tau protein may be involved in the pathogenesis of tau-mediated neurodegenerative processes in WD.
Tau protein is a highly soluble phosphoprotein and the most important protein of the axonal cytoskeleton [6, 10]. This phosphoprotein mainly presents in the axon microtubules where a high degree of phosphorylation decreases its ability to promote and modulate microtubule stability and axon morphology . Tau regulates microtubule dynamics, axonal transport and neurite outgrowth and might be involved in the establishment and maintenance of neuronal polarity [11, 29].
The axonal-derived phosphor-tau protein is significantly involved in multiple neurotoxic–degenerative mechanisms [12, 13, 30, 31]. In humans, tau hyperphosphorylation results in the formation of neurofibrillary tangles and neurophil threads, which are involved in the pathogenesis of AD and tauopathies. Neurofibrillary tangles associated with AD are composed mainly of paired helical filaments that result from the aggregation of abnormally phosphorylated tau protein. Some researchers have also reported the deposition of aggregates of different tau isoforms in other neurodegenerative diseases[29, 32].
Human studies have demonstrated that high levels of total tau in the cerebrospinal fluid (CSF) are frequently present not only in patients with neurodegenerative diseases, including AD [9, 11, 12, 30] and tauopathies [5, 13], but also in patients with brain trauma , acute ischemic stroke [20, 21], viral encephalitis [4, 17] and severe epileptic seizures , suggesting that tau protein levels in the CSF can reflect axonal brain damage. Other studies have shown high tau levels in patients with cerebral infections . Thus, there is a well-established compilation of tau protein data from numerous clinical and animal studies, but to our knowledge the present study is the first large-scale study to investigate serum tau protein levels in WD patients, with a special focus on the relationship between different clinical forms, age at disease onset and ceruloplasmin and copper levels.
Copper is one of the main biometals, and it plays diverse roles in many vital important processes, including enzyme catalysis, protein stabilization and energy production [1,2,3, 22]. Previous studies have shown that impaired copper homeostasis and transition can enhance amyloid aggregation and neurotoxicity and trigger neurodegeneration [2, 3, 25]. The authors of recent studies [22,23,24] have proposed that copper could affect the accumulation of α-synuclein, a protein involved in the pathogenesis of neuronal death . Mechanisms involving the accumulation of copper to toxic levels may lead to widespread neurodegeneration as free copper is very toxic on its own and can cause irreversible cellular damage. Various mechanisms for impaired copper metabolism, copper intoxication and severe neurodegeneration have been proposed based on experimental findings; these include direct oxidative stress, unregulated apoptosis and loss of caspase-3 inhibitory control [1, 9, 22, 24]. Published studies have shown that toxic copper deposits may induce oxidative stress, modify gene expression and enzyme catalysis and directly inhibit proteins and mitochondria function. In the brain, copper accumulates in astrocytes, leading to impairment of the blood–brain barrier and consequent neuron damage [2, 9, 23, 27, 33].
The results of this study demonstrate that the mean (± SD) level of tau protein increased in patients with WD as compared to the HC (221.7 ± 135.1 vs. 71.14 ± 20.56 pg/mL, respectively), indicating axonal impairment in WD patients and revealing tau-mediated toxicity in WD that might be sufficiently pronounced as to be a potential cause or effect of neurodegeneration. This finding has implications for clinical management as tau level may be a good diagnostic tool to evaluate ongoing axonal damage associated with neurodegenerative processes during life such that tau protein may be an early marker of peripheral axonal damage. Tau protein has become the focus of research in studies on WD diagnostics as serum tau can be easily obtained, thereby providing the means to quantify early axonal injury.
We analyzed the prognostic value of tau protein as a biological marker of axonal damage and tau-mediated neurodegeneration in the 28 patients with the cerebral form and in the 19 patients with the hepatocerebral form who have had this disease for a mean of about 10 years. We report here that the investigated patients with WD had abnormally high levels of tau protein in both the cerebral and hepatocerebral clinical forms.
Tau is a new biomarker of interest in WD. Elevated serum tau protein levels have been associated with the presence of the late-onset cerebral clinical WD form, with median baseline serum tau levels tending to be higher in patients with the cerebral form of WD (p = 0.5611). This result suggests that tau protein may serve as a prognostic biomarker for cerebral forms of WD. Our two study groups (patients and HC) did not appreciably differ in terms of disease duration, age of disease onset and serum ceruloplasmin levels; also, men and women showed no differences in tau concentrations. The elevated serum levels of tau-phosphor protein seems to reflect the destruction of neurons in the brain associated with baseline copper dyshomeostasis that was observed in all WD cases. Very high tau concentrations were found in 29.78% patients with WD, with all patients with WD being on a treatment course consisting of a stable dose of penicillamine.
WD is a disease leading to the progressive cell death of neurons and reactive astrogliosis where tau partly reflects WD neurodegeneration pathology [1,2,3]. Tau protein can be secreted across a damaged blood–brain barrier (BBB) and released into systemic circulation after the disintegration of neuron cells . If the axonal integrity is interrupted for any reason, tau protein passes into the extracellular space and then to the CSF. Two pathophysiological mechanisms that cause this axonal loss have been proposed in the literature: inflammation and degeneration. According to this hypothesis, the source of tau protein in the CSF and blood is the transected axons [4, 12]. The increase in CSF tau concentration, found not only in numerous neurodegenerative diseases but also in patients with MS [18,19,20] and brain trauma , support this hypothesis. One recent study, conducted on patients with AD, suggests that tau protein may be released extracellularly by an exosome-based mechanism .
We can conclude that tau itself is the prominent agent implicated in the pathogenesis of many neurodegenerative diseases with axonal degeneration, but the key question on whether the elevation of serum tau protein level could reflect the primary disease pathogenesis processes or whether it occurs secondary to neurodegeneration in WD remains to be assessed in future studies. Tau-related neurodegeneration plays a specific role in our current understanding of the neurochemistry and pathogenesis of WD, and the mechanisms underlying these interactions are largely unknown.
In the brain, copper accumulates predominantly in astrocytes, leading to BBB impairment with consequent damage to neurons and oligodendrocytes; the basal ganglia and brainstem are the regions with the highest susceptibility to copper toxicity. The increased level of free copper in the brain may also originate from the copper mobilized from brain parenchyma cells [35, 36], and not only from the serum. The copper-related toxic effects on the brain and BBB will lead to a worsening of the neurological conditions, which may be reflected in secondary neurodegeneration, which was measured here for the first time using the parameter of tau protein level. On the other hand, we cannot deny that tau protein level could reflect the pathological processes of the primary disease. However, the exact molecular mechanisms underlying abnormal copper metabolism, neurodegeneration, and tau protein levels are still insufficiently understood. The clinical presentation of WD can vary widely; consequently, the diagnosis of WD is not always straightforward . Key WD features are liver disease and neuropsychiatric disturbances, and the diagnosis is especially difficult when WD presents with liver pathology, as none of the available laboratory tests is perfect. Gaining a better understanding of the molecular mechanism underlying the development of WD is imperative. Increased tissue copper levels may induce a series of harmful biochemical reactions, particularly oxidative stress, which can damage the structure and integrity of mitochondria, leading to neuron injury [3, 22, 25]. A better understanding of the exact molecular mechanisms causing aberrant copper deposition and of the mechanisms of tau-induced brain pathology are keys to developing effective management approaches for neurodegeneration where early detection of axonal impairment and early intervention are important steps in the prevention of disease progression.
Based on our results, we suggest that while elevated tau protein levels could be one of the causes of tau-mediated neurodegeneration processes in WD, they are most likely a result of neurodegenerative processes related to disease outcome. However, all patients in our study were on pharmacotherapy and it is more likely that copper can cause the release of tau into the circulation where the elevated tau level should be a mirror of neuronal damage. Our results also support the role of tau level as a prognostic factor in patients with the cerebral form of WD. We found that serum tau protein levels were increased in patients with WD and that tau levels tended to be higher in patients with the cerebral form. Taken together, our findings increase our knowledge of WD pathogenesis in humans.
There are a number of inherent limitations to this study. The main limitation is the lack of objective measures of cognitive functions and neuroimaging abnormalities, such as the extent of atrophy rates (either hippocampal or whole brain) and contrast enhancing pattern to determine BBB breakage. However, we believe that it is extremely difficult to obtain these measures when examining patients with severe WD. The exact serum tau profile  in relation to cognitive dysfunction remains to be further assessed; such results will provide simple non-invasive tests to be useful in the stratification of the disease clinical form. The relatively small number of patients in this study limits us to conclude that tau protein may have a useful role in the monitoring of disease progression. Prospective investigations in this area should be performed.