Journal of Biomolecular Research & Therapeutics

NMDA R: N-Methyl-D-Aspartate Receptor; VDR-Vitamin D Receptor; PD: Parkinson’s Disease

Parkinson’s disease (and other associated movement disorders) is a common condition that is triggered by chemical, environmental, genetic and neurotrophic factors in which dopaminergic neurons are lost and melanin pigment is reduced in the SN [1-3]. If not treated, it can progress rapidly into movement disorders and other cognitive dysfunctions [4]. Since the initial description of this disease and other NDD’s, efforts have been directed towards understanding the molecular mechanism of pigment loss and cell death in the SN [5]. Neurodegenerative Diseases (NDDs) are becoming rampant in sub- Saharan Africa due to food based toxicity and thus, there is an urgent need to conduct cell based research using cheap and appropriate models [6,7]. Previous studies have examined the etiology and cellular mechanisms involved NDDs such as Konzo, tropical ataxic neuropathy and movement disorders often associated with the loss of adrenergic pigmented neurons in the Substantia Nigra (SN) [8,9]. Available cellular models often demonstrate cell death due to aging and have been achieved through manipulation and mutation of the PD genes [10-12]. Other models involves the use of primates; through chemotoxin induced Parkinsonism that selectively targets the dopaminergic cells of the SN [13,14]. Most in vitro models are nonpigmented and thus cannot demonstrate the role of melanosomes in the selective vulnerability of these cells [15]. Also, the in vivo models cannot be observed directly as direct cellular observation is rather invasive [16,17]. Thus, there is a need for the development of in vitro or ex vivo cell models capable of showing synaptic denervation and the roles of pigment vesicles in the cause, progression and therapeutic targeting of PD. An important candidate cell for this purpose is thus the melanocyte in the marine species [18,19]. The melanocytes are adrenergic, pigmented, originates from the neural crest; they can also form extensive cellular processes like neurites. In addition the cells are also concentrated in the stream line of the body of the organism where it detects vibration and maintains the relative position of the organism in water [3,18-20].

It has observed that the fish scale melanocytes can be stimulated by adrenergic effector molecules. Considering the flow from monoamines (dopamine) to catecholamine (epinephrine and norepinephrine) this cell type already has two (2) main features of the SN which is the presence of pigmentation and receptors capable of being stimulated by the adrenergic effector molecules [19,21]. From these, it was inferred that these melanocyte populations in the scale of the fish (Tilapia ) are probably specialized sensors that perform a similar function as those of the SN [3,18]. This function is basically in the determination of the relative position of the organism in space by polymerization and depolymerization of MT-Motor protein assembly to alter the position of melanosomes and thus regulating impulse discharge rate [22-24]. The cause of PD has been broadly described as unclear; in addition loss of adrenergic cells have also been described in the progression of the diseases condition which is characterized by shortening of neuronal projection as a form of synaptic denervation [25]. However, to understand the process involved in this synaptic denervation of adrenergic pigmented neurons, the molecular mechanism needs to be studied at cell and protein level.

Parkinson’s disease has been known to have several causes. An inherited form of the disease has driven the studies to create new in vivo models especially those involving transmission and inheritance of the PD gene [26-28]. Guo described the use of the drosophila as an efficient tool in the study of PD gene inheritance involving pathogenic PD gene mutations [29-32]. This involves the study of the gene products of the two PD genes (PINK1 and Parkin) both involved in a mitochondria fission/fusion pathway [33,34]. These genes have also been observed in both the fly and humans; they also sub serve the same function of recruiting these mitochondria to the site of final removal. The use of drosophila has been linked with in vitro therapeutic targeting [35,36].

A number of polyphenols have been reported to play important roles in the inhibition of α-sync which might lead to possible prevention of PD resulting from these mutations. The effect of free radicals has also been implicated in aging related Parkinson’s diseaseoften a product of mitochondria dysfunction. Other studies involved dietary supplements of Nordihydroguaiaretic Acid (NDGA) in drosophila models [35,37]. It was discovered that the loss of movement often observed in this model was delayed when NDGA was included in the diet. In vivo models includes the use of primates and rodents treated with PD causing chemicals agents like MTPT and have been used for studies on how specific drugs affects the progression of PD [38,39]. Novel analogues of MDMA, UWA-101 have been found to improve the therapeutic benefits αin primates being treated with LDopa. It was also observed that this UWA -101 was more effective than MDMA as it lacks psycoactivating and cytotoxic effects [40]. Other laboratory models involves the generation of disease specific stem cell lines from patients with incurable diseases [41]. This is often used for drug screenings and design of systems for understanding disease mechanism. Several nervous system cells have been screened and modified into dopaminergic neurons iPSC. Another cell model mimics the mutation of SNCA gene encoding the pro-oxidant α-sync protein in the budding yeast (S. Cerevisae) ; by studying the increase in cytosolic neutral lipid storage embedded in lipid droplets. The significance of this accumulation was further investigated in a yeast strain which does not possess the machinery to synthesize triglycerides. The outcomes thus show that such strains were more resistant to α-sync toxicity [42].

The major effect of the PD pathogenesis is age related neuronal cell death in the dopaminergic neurons. This has also been found to be the case in mammalian and non-vertebrate models; example is the nematode (C. Elegans ) [43]. Both genetic and drug screens conducted in C. Elegans have aided the identification of small molecules, proteins and discrete biological systems that can impact PD pathology. An example of such system is the identification of the autosomal dominant and idiopathic PD models in C. Elegans due to mutations found within the GTPase and kinase domains, both affecting the molecular motor for vesicular movement [44-46]. Previous studies have shown that such mutations are often linked with kinase hyperactivity. In order to understand this further, transgenic C. Elegans have been developed that can over express LRRK2, GTPase and Kinase similar to those observed in dopaminergic neurons in PD [47,48]. This system also created reduced locomotor activity, memory dysfunction and reduced dopamine production in vivo [47,49].

Although several cellular and in vivo models described above have taken care of most parts of the disease progression in PD, a major point is the selective vulnerability of the pigment cells due to autophagy and induced oxidative stress. Although the cells of the C. Elegans and Drosophila express dopamine, they are however not pigmented as this represents a major limitation of these models. In vivo primate models are however priceless as they represents virtually all the aspects of the PD effects including the role of pigments metabolism and neurotransmission in these cells. It is important to note that study of cellular activities through direct observation are invasive and highly restricted in these rodent models, thus it is important to have an in vitro models where the cells are adrenergic, expresses major process formation and are also pigmented in order to understand the direct cellular effects of therapeutics and disease causing agents. Although the effect of certain drugs have been described as either increasing locomotor activity or improving dopamine level /L-DOPA uptake, direct cellular observation remains a challenge in these models.

It has been observed that dopaminergic neurons of the SN are selectively degenerate during the cause and progression of PD [50-52]. They also represent the most heavily pigmented population of neurons in the brain [50,53,54]. However, the heavy presence of neuromelanin have long being described as an important factor in the susceptibility of these neurons to aging and autophagy - described in the other in vitro models. Previous studies have discussed the unclear nature and role of neuromelanin in this structures, it has been suspected that intra neuronal melanin is neuroprotective through its ability to shield cells from heavy metals and toxins and also excess cathecolamines [2,55]. In contrast melanin released by dying neurons as extraneuronal can trigger inflammation and glia activation [4,50]. Graybiel et al., described the nigrostraital system in induced PD; quantitative analysis however points to selective loss of pigmented cells. Such patterns of loss are important in the study of etiology and clinical symptoms of PD [56].

Glutamate toxicity has been described as a major cause and facilitator of selective vulnerability in dopaminergic neurons [57-59]. The glutamate receptor, NMDA R is important in the astrocyticneuronal glutamate-glucose cycling and glucose metabolism in the brain [60-62]. However during development, pharmacological knock down or genetic deletion of NMDA R have been found to greatly impair neuronal development and circuit formation [63,64]. Depending on the state of development, if the cells do migrate, they will not recognize the final destination in the nervous system [65,66]. Thus NMDA R has been implicated in neurite formation, synapse formation, development and neural circuit maturation [67-69]. Experiments involving the use of excitotoxin such as excess glutamate or glutamate analogues capable of persistent potentiation of the NMDA R have been observed to cause degenerative changes in the adult neurons including autophagy [18,70-72]. Thus the role of the glutamate receptor is switched post maturation [73]. In both development and degeneration, the formation of the cytoskeletal core of the axo-dendritic system is important. Through the work of NGF and other kinase receptors like p75 (LNGFR), the pre and post synaptic systems are established on neurite differentiated through cell elongation and process formation during neuronal cell migration in the developing brain layers [65,66,73]. A major effect of glutamate toxicity in the adult is however linked with autophagy which is much more prominent in pigment cells - the vesicles are observed close and clustered around the nucleus leading to cell death by lysosomal fusion [74,75]. The neuronal cytoskeleton, although forms a cellular track for vesicles and organelle moved to and from the synapses, they also keep organelle in position in these cells [76,77]. Use of depolymerizing agent have shown that loss of the MT assembly will lead to accumulation of these vesicles around the nucleus creating an autophagy scenario in the cell [78,79].

Autophagy itself is described as an intracellular response to stress often characterized by the presence of autophagosomes [80]. Certain studies have demonstrated the autophagy response of cerebellar granule neuron challenged with NMDA (glutamate analogue). It was shown that fluorescently labeled autophagosomes were accumulated in the cell body and neurite at 3 hours post treatment. Lysosomal inhibition studies also reveal that NMDA excitotoxicity diverted the autophagosomes from the usual lysosomal degradation pathway [81]. Another possible path involves the role of cytoskeleton in PD, similar to that of Alzheimer’s disease (AZ). High levels of intracellular calcium can disrupt cytoskeleton and NMDA R stimulation can drive an increase in cellular calcium levels which in turns disrupts cytoskeleton [82,83]. Other studies have shown that neuron that contains calcium binding proteins is less susceptible than the neurons that do not have these proteins. This is an indication that NMDA R stimulation can drive cytoskeletal degradation while calcium binding protein prevents the calcium surge; thus protecting the neurons [84-86]. A link between glutamergic and the nigrostraital system have been described through the glutamergic afferent pathways that projects to the nigrostraital system from the glutamergic tracts of the prefrontal cortex and might play a role in release of glutamate leading to degeneration of the nigrostraital system through excitotoxicity [87-89].

Low serum level of Vitamin D is often associated with PD [90]. The final converting enzyme and the VDR receptor are distributed throughout the brain. Studies have shown that vitamin D is important in neurodevelopment, up-regulation of neurotrophic factors, stabilization of mitochondrial function, and antioxidation [90,91]. The VDR gene codes for the VDR and is responsible for calcium regulation, immune response, neuronal functions [92,93]. VDR polymorphism have been linked with PD aetiology and progression [94,95]. Placebo studies have also employed the use of VDRA in dietary supplements [96]. Improved behaviour and cognitive function have been observed in patients receiving Vitamin D supplements. Nissou et al., 2013 have demonstrated that the role of Vitamin D goes beyond cellular mechanisms, over time it has been found to be associated with up regulation of several genes up to 1.9 folds at transcriptome level [97-100]. The active form of Vitamin D is Vitamin D3 and it acts by binding to the VDR. This in effect regulates several cellular machinery at transcriptome level. Most of it effects have been studies extensively in osteoporosis, cancer, inflammation and immune system. Vitamin D analogues have been employed as therapeutic targets of VDR [101-103]. Vitamin D3 compounds are known to influence melanocyte maturation and differentiation and also to upregulate melanogenesis through pathways activated by specific ligand receptors, such as endothelin receptor and c-kit [104,105]. Studies have shown that although these receptors are highly concentrated in the brain, they are most predominant in the pigmented cell population. Its role in regulating calcium concentration is useful in reduction of calcium ions that might disrupt cytoskeleton, thus helping in the prevention of synaptic denervation [106-109].

Considering the super imposed developmental biology of pigment cells in Humans and Fishes; originating from the neural crest in both organisms, these cell types possess certain features of cells of the nervous system (Figure 1) [110]. The most important candidate considered is the N-Methyl-D-Aspartate Receptor (NMDA R ); that is responsible for neuronal migration, development, process formation and synapse formation at the final site [111]. Our previous findings have shown that these receptors are located on the cell body and cellular projections, similar to what is observable in the mammalian neuronal cells. This was done using a confirmed Human NMDA R1 antagonist (Ketamine) and an agonist (L-Glutamate) to inhibit and potentiate the receptor in live melanocytes using bright field microscopy [3,18].

Figure 1 and 2a, 2b: (1) The control melanocyte (2a) the treatment group involving pharmacological knockdown of NMDA R by ketamine, a non-competitive antagonist. The cells showed elongation of cellular processes far higher than those recorded in the control. The cellular process elongation reveals the reverse role of the NMDA R in the embryonic system versus the adult system as these cells are also derived from the neural crest. (2b) Rectangles represent sites of structural intercellular connection.

Potentiating the NMDA R with glutamate caused process formation on the cell body, while inhibiting the receptor in vitro facilitated formation of processes having an appearance similar to axo-dendritic process formation pattern in developing neurons (Figure 2A and 2B). Bright field imaging techniques were also used to capture process formation and intercellular structural interactions. At this point, formation of cellular processes does not represents axons but provides an appropriate premise for studying pigmented cellular processes similar to those of axons of pigmented neurons. Extensive process formation and cellular connections were also observed post inhibition of NMDA R using a non-competitive open channel blocker, ketamine (Figure 2A and 2B). It can also be used to combine the study of microtubule-motor protein assembly and autophagy in pigmented adrenergic cells [3,18,63].

Other studies examined impact of Vitamin D receptor (VDR) and Vitamin D receptor agonists (VDRA) interaction on process formation in this model [112,113]. It was observed that both inhibition of NMDA R and VDR stimulation by VDRA facilitated process formation (Figure 3). Certain differences were noted some of which includes; short processes were created by VDR stimulation [114] as compared to longer processes seen in NMDA R inhibition [115], the blobbed ends of processes are well seen following VDRA stimulation [3,18]. Extent of branching in VDR shows short projections originating directly from the cell body while in NMDA R inhibition, larger processes were observed before rapidly branching to give smaller processes. The cellular process involved in the VDR stimulation suggests a rapidly branching dendritic network facilitating polymerization of the MT system and creating more branches similar to the dendritic nucleation assembly (Figure 3).

Figure 3: Inverted grey scale cell process measurement for Control cells, Ketamine treatment, VDRA+Ketamine and VDRA Only. Increase in process length was observed in all the treatment categories. Ketamine treatment gave the longest cell process while VDRA treatment induced shorter process formation (Scale bar: 10 μm) (From Ogundele et al., [18])

In this study, we have first described the mechanism involved in neuronal loss in the SN and the general structure of the fish scale melanocyte. Using bright field imaging techniques, live cell imaging was conducted in vitro to show the various changes observed in the melanocytes upon manipulation of the NMDA R and the VDR. The outcome shows that the fish scale melanocyte contains NMDA R on its membrane just like the human neuronal cells, although it is much more localized on the axon-like processes; while VDR is localized on the cell body and short dendrite-like processes. Upon inhibition of this receptor (using ketamine; a human NMDA R antagonist), the cell projections forms wide array networks of cellular processes following a similar pattern to what is observed in neuronal axon-dendrite formation. Glutamate treatment (NMDA R potentiation) also caused formation of cellular projections but not as extensive as that seen in NMDA R inhibition. These findings therefore creates a premise for the study of pigmented neuronal cells in vitro as this cell type (fish scale melanocyte) expresses NMDA R and the role of this receptor in cellular process formation has also been seen to be similar to the pattern observed in neuronal axon-dendrite formation. Inverted gray scale image analysis shows that this cell upon inhibition of NMDA R shows temporary connections between the formed processes, an association similar to the synapse that has been observed in the human neurons. VDR stimulation facilitated more of short process formation radiating directly from the cell body suggesting its role in a cellular process similar to that of the dendritic nucleation assembly in neurons.

We acknowledge the Directorate of Research, Afe Babalola University and the Laboratory Staff of the Department of Biological Sciences, Anatomy and Physiology. Also we acknowledge the contributions of Elizabeth Peters of the College of Medicine for assisting in the revision of this work. All other members of my research team are acknowledged for their support and contribution. The Afe Babalola University Sponsored Our Research paper Presentations on “NMDA R1 as a tool in Neuroprotection and Nerve cell reprogramming” made at the SONA conference in Morocco (June 13-18, 2013).