Abstract

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Therapeutic recombinant plant-made copper chaperone for superoxide dismutase (CCS) derived from Ginkgo biloba leaves may establish and maintain physiologic copper levels in neurodegenerative diseases. Through restoration and modulation of biometal metabolism in organ systems of younger Alzheimer patients (> 50 years), medications developed from plant-made copper chaperone proteins may delay progression during early disease stages or even be a basis for a possible causal treatment of preclinical stages of Alzheimer’s disease. Ginkgo biloba leaf extracts seem to be ineligible in the early treatment of dementia syndroms because they may lack specificity as to the binding of metal ions in the cytoplasm.

Introduction

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The dysregulation of biometal (Cu, Zn, Fe) homeostasis and oxidative stress in brain cells are major hallmarks in the pathogenesis of Alzheimer’s disease (AD) [1]. The regulation of metal ion homeostasis in the cytoplasm is strongly influenced by copper chaperone for superoxide dismutase (CCS) and Cu,Zn-superoxide dismutase (SOD-1). The interplay of properly folded CCS and SOD-1 guarantees that free Cu and Zn ions are being complexed by these metal proteins and do not catalyze oxidation processes of proteins, lipids and other molecules in the cells [2]. As soon as these physiological complexation mechanisms do not function properly, oxidative stress and dys-homeostasis of Cu and Zn metabolism give rise to misfolding, accumulation and aggregation of amyloid β (Aβ) peptides [1,2]. The outcome of these pathological processes may lead to incurable progressive neurodegenerative diseases such as Alzheimer’s disease [1,2]. Several therapeutic strategies and nearly all medications used or suggested as Aβ inhibitors, including metal-chelating agents or radical scavengers, at present time, aim at the treatment of AD symptoms only and may either be toxic, reveal a lack of specificity or have unknown mechanisms of action in vivo [2,3].

The aim of this article is to review the interaction of metal ions with novel and early herbal compounds derived from Ginkgo biloba leaves in the treatment of early Alzheimer’s disease. Though Ginkgo biloba leaf extracts are generally administered to treat dementia syndroms in older patients, no preclinical data in cell lines, animal models or patients are available dealing with the possible effects of recombinant plant-made copper chaperone proteins from Ginkgo biloba in younger AD patients (> 50 years old) at present.

Interaction of herbal drugs with metal ions

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The medical community, including the pharmaceutical industry, as well as AD patients, have become aware of the well-known antioxidant effects of ancient herbal medications, namely Ginkgo biloba leaf extracts [4]. A complex mixture of flavonoids and terpenoids represents the main bioactive compounds of these plant extracts [5]. For example, the flavonol quercetin, a major compound of Ginkgo extract, was found to interact with Cu²⁺ and Fe³⁺ [6]. However, an ideal therapeutic drug to dissolve Aβ peptides would involve a compound selective for Cu⁺, Zn²⁺ and Fe³⁺ [7]. A meta-analysis by Birks and Evans revealed that commercially available medicinal plant extracts (EGb 761^® Ginkgo biloba leaf extract) have no consistent pattern of any clinical benefit associated with Ginkgo biloba for people with dementia or cognitive impairment [4]. Furthermore, the results from the study of He and colleagues suggested that high doses of herbal remedies can even be toxic to cells [8]. Thus, Ginkgo biloba extracts may induce unwanted side-effects and may also lack specificity as to the binding of Cu⁺ and Zn²⁺ in the cytoplasm. Likely, these extracts neither reduce metal-based oxidative stress efficiently nor contribute to the homeostatic control of biometals, though in vitro studies have shown that gingkolides may protect against the synapse damage and cognitive loss seen during the early stages of AD [5]. Medicinal plants may contain other more efficient bioactive molecules apart from the well-known flavonoids and terpenoids, namely metal chaperones [9].

Efficacy of plant-made copper chaperones

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As a basis for a new drug development involving metal-chelating agents, the following facts may be important. Endogenous biomolecules such as Cu,Zn-superoxide dismutase, are one of the major means by which cells counteract the deleterious effects of reactive oxygen species (ROS). For proper functioning SOD-1 has to be activated by the metallochaperone protein, copper chaperone for SOD [9-12]. Copper ions are required for enzymatic activity whereas the zinc ion helps to stabilize the enzyme [10]. Studies by Choi and colleagues suggest that recombinant human CCS molecules produced in bacteria provide a potential strategy for therapeutic delivery of these compounds in various human diseases related to ROS and SOD [10]. Drugs derived from recombinant proteins potentially have greater efficacy and fewer side-effects than small organic molecules (e.g., Cu orotate, quercetin), because their action can be more precisely targeted towards the possible cause of Alzheimer’s disease rather than the treatment of AD symptoms [11]. Yet, the pathophysiology of AD is not fully understood saying that CCS-SOD-1 may target one specific but major mechanism in the pathogenesis of Alzheimer’s disease. It is a well-known fact that biometals (Fe, Zn, Cu) are accumulated in the brain with normal ageing [13]. Important factors affecting the balance between metal ion accumulation and deficiency are, for example, genetic dysfunction, environmental exposure, ageing or drug interaction [14]. The inability of the human organism to maintain the metal ion homeostasis due to inactive CCS and SOD molecules in brain cells is suggested as possible cause for preclinical stages, development and progression of AD and other neurodegenerative diseases [9,11,15].

For the restoration and modulation of metal ion homeostasis in the treatment of AD, we have proposed another class of pharmacologically active plant ingredients as antioxidants: copper chaperone for superoxide dismutase derived from medicinal plants (e.g., Ginkgo biloba) [9,11]. In molecular farming approaches transgenic plants (e.g., tobacco) may serve as an efficient production platform for medications in regard to protein yield, quality and stability [16]. Recombinant CCS proteins produced in transgenic plants may cross the blood-brain barrier and are relatively free from side-effects [9,11]. Being properly-folded, plant-made CCS may have the ability to bind and deliver Cu⁺/Cu²⁺, Zn²⁺ and Fe³⁺ ions and to normalize the SOD-1 activity via specific protein-protein interactions in the nervous system and peripherally [9,11].

In contrast to plant-made pharmaceuticals (PMPs), organisms like yeast, mammals or bacteria used for molecular farming approaches, express properly folded as well as improperly folded recombinant therapeutic proteins. These medications may lack stability and pharmacological efficiency in protein-misfolding diseases, e.g., Alzheimer’s disease [16]. Furthermore, PMPs may have several advantages in terms of cost, scalability or safety issues compared to the other genetically modified organisms [16].

Because of their specific biochemical behavior, plant-made CCS from medicinal plants may be efficient in the treatment of patients with preclinical stages of AD. The endogenous levels of this essential copper protein may be important, since a mild copper deficiency has been described in AD patients [17]. Furthermore, the expression level of CCS has been found to reflect the Cu status of patients and thus, may serve as a marker for in vivo copper levels [18]. The metal levels in patients with Alzheimer’s disease compared with healthy individuals are important parameters for developing drugs that restore the intracellular metal ion metabolism [14]. It is anticipated that plant-made CCS may establish physiologic copper levels through restoration and modulation of biometal metabolism in diseased organ systems of AD patients [9,11]. However, no experimental data are available to prove these theoretical considerations.

The effects of recombinant CCS in the treatment of AD can be characterized as follows: unfolded SOD-1 in diseased blood involving denatured metalloproteins and peptides, may be activated by copper ion incorporation via specific CCS-SOD interaction. Copper-demetallated CCS and Cu cofactor-containing CCS complexes may pass through the blood-brain barrier and activate unfolded SOD-1 by Cu ion transfer or bind free Cu, Zn and Fe ions in brain cells, respectively [9,11]. Copper homeostasis is an important factor in the complex pathogenesis of AD [19]. Thus, restoring of the homeostatic control of Cu metabolism may positively affect early disease stages of AD patients because oxidative and anti-oxidative processes in the brain cells are being balanced and protein-misfolding processes implying Aβ peptides, CCS and SOD-1 molecules are prevented or minimized [9, 11, 19]. The interactions of plant-derived CCS medications with unfolded human SOD-1 and free metal ions may trigger a cascade of other biochemical reactions, such as the degradation of misfolded proteins and Aβ deposits by molecular chaperones and the ubiquitin proteasome system [20].

To test this hypothesis it will be an important step to evaluate the relative biochemical impact of therapeutic metalloproteins by using a multifaceted approach for the determination of dose, bioavailability and mechanisms of action of recombinant plant-made CCS in younger AD patients (> 50 years) and control populations as previously proposed [9, 11].

Conclusion

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For the restoration and modulation of metal ion homeostasis and for balancing intracellular pro-oxidative and antioxidative processes in the treatment of Alzheimer’s disease, plant-made copper chaperone for superoxide dismutase (CCS) proteins potentially have greater efficacy and fewer side-effects compared to small organic molecules (e.g., quercetin) from medicinal plant extracts or therapeutic recombinant proteins produced in bacteria, fungi or mammals. Possibly suitable for use in younger AD patients (> 50 years), CCS proteins derived from medicinal plants may be targeted more towards possible causes of AD rather than the treatment of AD symptoms. I suggest that recombinant plant-made CCS derived from Ginkgo biloba leaves might be promising in the treatment of patients suffering from preclinical symptoms of Alzheimer’s disease.

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Acknowledgement

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The author is grateful to Univ.-Prof. em. Dr. med. Jürgen Horst and Ass. Prof. Dr. med. Basil Horst for revising this article. Dr. Kerstin A. Nagel and Dr. David E. Garfin are greatly acknowledged for their advice.

Source(s) of Funding

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None

Competing Interests

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The author declares that he has no competing interests

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