Parkinsons disease is a prevalent neurodegenerative disease that was associated with Cdk5 and parkin activity. Recent research has shown the mechanism behind the Cdk5 and parkin interaction, but has yet to look at the function of this interaction in relation to Parkinson's disease and whether there is an element of PD causation behind this interaction. A parkin construct with the Cdk5 phosphorylation site residues replaced to render these sites functionless was used in vitro and in vivo parameters. In these experiments, low dopamine levels compared to wildtype were used as a marker for PD. Experiments on mice with these two parkin (parkin construct and wildtype parkin) phenotypes were performed to assess whether there were any behavioral traits similar to mice with PD.
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Parkinson's Disease is a common neurodegenerative disorder that has a lifetime incidence of approximately two percent. The clinical manifestations of it include resting tremor, muscular rigidity, bradykinesia, and postural inability. PD is characterized by the progressive degeneration of dopaminergic neurons in the substantia nigra. A specific pathological feature of PD is the intracytoplasmic inclusion body, known as the Lewy body. The Lewy Body is found in many regions, including the substantia nigra, locus ceruleus, nucleus basalis, hypothalamus, cerebral cortex, cranial motor nerve nuclei, and the central and peripheral divisions of the autonomic nervous system1. One protein that was found to play a key role in both familial and sporadic Parkinson's disease is parkin. Parkin2 belongs to a family of proteins with conserved ubiquitin-like domain (UBL) at the N terminus connected to a C-terminal really interesting new finger (RING) domain by a linker region. The RING finger domain gives parkin the ability to work as an E3 ubiquitin-ligase enzyme. By attaching ubiquitin to a lysine on the target protein through an isopeptide bond, it targets specific protein substrates for degradation by the proteasome. It was found that mutations in parkin are the most common cause of familial PD and also cause a portion of autosomal recessive juvenile parkinsonism3. From the neuropathologic studies4 of patients with parkin mutations, a selective loss of dopaminergic neurons of the substantia nigra and loss of noradrenergic neurons in the locus coeruleus was observed. In a portion of these studies, Lewy bodies were found. Further research, showed that parkin ubiquitylates the ï¡-synclein-interacting protein synphilin-1 and the p38 subunit of aminoacyl-tRNA synthetase/JTV-15. This is important when considering Parkinson's disease because mutations of the ï¡-synclein gene have been found to play a role in some familial forms of PD, and the accumulation of ï¡-synclein protein has been found in the brains of patients with this pathology6. Specifically, parkin protects against the toxicity that may arise from the accumulation of ï¡-synclein. Experiments on Drosophila Parkin null mutants found that they display mitochondria dysfunction7, a sign of PD, and this conveys that Parkin may play a role in the maintenance of mitochondrial function.
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Parkinson's disease, like many other neurological disorders, results in loss of CNS neurons due to apoptotic or necrotic neuronal death. Not only does the apoptotic machinery have crucial functions during development, but it can also be reactivated under pathological conditions in the adult nervous system. One of the components of this apoptotic machinery is Cdk5. Despite being expressed in most tissues, Cdk5 activity is almost exclusively restricted to neurons. Experiments performed by Bibb et al, and White and Cooper, that Cdk5 plays a role in modulating dopamine signaling in dopaminergic. Furthermore, the researchers found that with chronic cocaine administration there would be upregulation of Cdk5. The PD marker mentioned earlier, Lewy bodies, contain Cdk5, further proof that a Cdk5 Parkinson's link does exist(pierre).
In an experiment performed by Avraham et al. (2007), the researchers found parkin was phosphorylated in vitro and in vivo by Cdk5. They deemed that this phosphorylation by Cdk5 regulated the catalytic activity of Parkin, and was responsible for parkin's ability to ubiquitylate its synphilin-1 and p38 substrates. Furthermore, the results they obtained suggested that the phosphorylation of parkin by Cdk5 decreased the E3 ubiquitin-ligase activity. After finding that parkin has four Cdk5 phosphorylation sites, all located in the connecting linker region, in vitro and in vivo phosphorylation experiments were carried out. Previous research had indicated that parkin was phosphorylated at serine residues 101,131, 136, 296, and 378, combining that knowledge with the finding from there research that serine 131 mutants had a significant amount of lower phosphorylation than the other sites. In attempting to find the in vivo specificity of Ser-131 to Cdk5, the researchers generated a parkin construct that had the serine residues at 101, 136, 296, and 278 mutated to alanine and only the serine 131 was left untouched. By using different protein kinase inhibiters that preferentially inhibited different protein kinases, the experimenters found that only the Cdk5 inhibitor, roscovitine, inhibited phosphorylation. This suggests that Cdk5 plays was mainly responsible for the phosphorylation of parkin, and as a result decreased parkin's E3-ubiquitin ligase activity. The decrease in ligase activity may contribute to the accumulation of p38. The toxic protein p38, which accumulates in PD brain tissues and contributes to the death of dopaminergic neurons, is a component of the PD model8 that may be affected by the phosphorylation of parkin by Cdk5.
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This research provided the mechanism behind the phophorylation of parkin by Cdk5, but did not approach the topic of its functional significance with respect to Parkinson's disease. I hope to explore whether the phosphorylation of parkin by Cdk5 leads to Parkinson's disease or other types of neuronal damage. The difference in dopamine release between wildtype neurons and mutants that prevent phosphorylation will be used as a marker for Parkinson's disease. Through the conducting of necessary and sufficient tests, I hope to explore the functionality behind this phosphorylation.
Further elucidation of the Parkinson's disease model needs to occur for there to be effective, targeted drug treatments that help in abating the effects of, or curing PD. One such possible way is to see if Cdk5 inhibitors could be used to treat PD. Before such treatment could be administered, the neurophysiological response of phosphorylation of parkin by Cdk5 must be observed.
To determine if the phosphorylation of parkin by Cdk5 leads to a decrease in dopamine release and if it is either necessary or sufficient for such a phenotype. Also, to observe the behavioral effects this Cdk5 phosphorylation of parkin has on a model mouse organism.
Cell Culture (in vitro)
The in vitro aspect of the experiment will use cells cultured from the striatum of a mutant mouse. The cells are being cultured from the striatum because these cells are dopiminergic, and since the release of dopamine is being studied as the marker for PD it makes sense to use cells from the striatum. The mutations will be made through site-directed mutagenesis. This the same procedure conducted in the Avraham study. Full-length parkin constructs mutated at predicted Cdk5 sites or additional kinase sites will be generated by PCR using primers that contained alanine codons instead of serine ones. The mutations of Cdk5 sites were confirmed by double-strand sequencing. These mutations will inhibit the ability of Cdk5 to phosphorylate parkin, and dopamine levels could then be measured. This test will measure the necessity of the phosphorylation of pakrin by Cdk5. For the in vitro sufficiency test, the wildtype parkin gene should be added back into the mutated culture cells and dopamine levels should be measured. During this step of the procedure the genes responsible for the proteins that have been implicated in Parkinson's disease must all be knocked out, so that only the remaining proteins involved in the Parkinson's disease pathway are parkin and Cdk5. Proteins such as PINK1, HtrA2, and p38 should all be knocked out, or it's expression should be silenced.
The wildtype parkin gene can be added back using a bacterial artificial chromosome (BAC) along with Ampicillin to make sure the gene was taken up. BAC would be used instead of a plasmid vector because the plasmid vector can only contain inserts of about 1-10 kbp while parkin can contain inserts greater than 700 kbp. The parkin wildtype gene is greater than 10 kbp and because of this the plasmid vector cannot appropriately transfer the gene back in. The wildtype parkin gene is inserted into a bacterium and coupled to the gene bla, a gene encoding for ampicillin resistance. After this treatment, the bacteria are then grown in a medium containing ampicillin. The genes that successfully take up the wild-type parkin also possess an ampicllin resistance, thereby, surviving in this ampicillin medium. Striatum cell culture with wild type parkin and wild type Cdk5 should be used as a control and reference for dopamine levels.
The measurement of dopamine in cultured cells can be measured by coulometry9 coupled to a high performance liquid chromatography (HPLC). This approach is better for cell cultures than slices because unlike slices that lack cell bodies or nerve terminals, the cell cultures contain intact neurons.
Model Organism (in vivo)
As in the earlier example, site-directed mutagenesis at the Cdk5 phosphorylation sites on parkin should introduce the parkin construct. Instead of the parkin construct, a Cdk5 knockout could have been used. Like the other Cdks, Cdk5 plays an important role development and to avoid possible confounds a Cdk5 knockout was decided against. Instead of the wildtype parkin gene, the parkin construct should be expressed in my mouse. This serves as an in vivo necessity test. Wildtype parkin and wildtype Cdk5 should be used as a control and serve as a reference for dopamine levels. The level of dopamine can be observed using Positron Emission Tomography (PET).
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Two determine if these two proteins, parkin and Cdk5, interact with one another in both in vivo and in vitro a coimmunoprecipitation assay has to be performed, respectively. The underlying mechanism behind the Co-IP assay is that interaction between two proteins should result in their precipitation together. The assay will be similar to that performed by Avraham et al in that an anti-parkin antibody will be used. This means that Cdk5 and parkin should precipitate together and be captured on an agrose gel. Gel electrophoresis and Western blot analysis will be used to determine if the protein associated with parkin is indeed Cdk5. Another control for both the in vivo and in vitro experiments is testing whether the parkin was actually phosphorylated. This can be done through a radioactive label and autoradiography visualization.
Animal Behavioral Studies
To get a holistic understanding of the effects Cdk5 has on parkin and its role in the development of Parkinson's disease, it may be beneficial to perform behavioral studies that test for PD. Such experiments have been performed before, and they mainly comprise of looking at behavioral symptoms that are associated with Parkinson's disease. Tests for akinesia (immobility) will be performed by placing the different variants of mice described above on a flat surface, and latency is will be measured until the mouse moves all four of its limbs. Catalepsy, or the inability of the animal to correct an externally imposed posture, can be quantified by putting the animal's fore-limbs and hind-limbs on a small bar that is a few centimeters above the ground, and determining the time it takes for the animal to correct its position (this behavior is normally observed in normal mice). There is also a swim test that can be performed on the animals to assess the level of motor impairment. In this experiment mice were put in a water basin and there swimming techniques were dichotomized for active vs. passive floating. Lastly, the pole test introduced by Ogawa et al (1985) to measure bradykinesia in mice will be used. The test uses a 50cm high, gauze-taped pole that is 1cm in diameter with a small ball of cork at the top and places mice with their head upwards right below the top. The time until the animals have turned by 180 degrees, and the time they have come to the ground is measured. In such experiments, control animals have been show to turn around and descend in about 20 seconds.12
Expected Outcomes and Interpretations
The in vitro assay with the parkin construct should exhibit phosphorylation when examined by autoradiography, this result is predicted because there are no sites for Cdk5 phosphorylation and only served as a control. Since there are Cdk5 phosphorylation sites on the wildtype parkin, phosphorylation should be observed at these sites, specifically at the serine residue 131, a major site of Cdk5 phosphorylation. The coimmunoprecipitation assay should reveal that only in the case of wildtype parkin did Cdk5 also precipitate along with it. Since there is phosophorylation, and thus no interaction, between the mutated parkin and Cdk5 the two proteins should not precipitate together. The dopamine readings from the cell culture that contains the parkin construct should have more elevated levels than the ones with knockin wildtype parkin because the wildtype parkin has unblocked Cdk5 phosphorylation sites, phosphorylation that has been linked to apoptosis and is a component of the apoptotic machinery. As a result, this experiment should give both necessary and sufficient results for the elevation of dopamine levels in non-Cdk5 phosphorylated parkin. The direct link between PD and the phosphorylation of parkin cannot be found in these experiments. Further research may aim to find this direct link, and it could be guided by the Brion et al. (1995) experiment that found cortical and brainstem-type Lewy bodies that were immunoreactive for Cdk5. This research could aim to find whether parkin was also present in these cells, and illustrate a physiological link between the interaction of Cdk5 and parkin to Parkinson's pathology.
The in vivo experiment with mice possessing mutated parkin should show greater dopamine activity, this is the same result as those expected in culture. Without a key component in apoptotic function (Cdk5 activity), the dopaminergic neurons should continue persisting and as a result keep producing dopamine. The mice with wildtype parkin should show less dopamine activity because this apoptotic mechanism is still conserved. Coimmunopercipitation and autoradiography for phosphorylation should illustrate the same results it did in vitro, this is because the interplay of the proteins and the parameters are essentially the same. One aspect that is different between the two experiments (in vitro vs. in vivo) is that there were both necessary and sufficiency tests performed in the in vitro parameter, but only necessary tests were performed in the in vivo case. This was done because knocking out all the genes that have been previously implicated in Parkinson's disease would have had tremendous developmental consequences, and the mice probably would not have survived to have any substantial tests run on them.
All four behavioral tests would not have provided any link between Cdk5 phosphorylation of parkin and symptoms of Parkinson's disease, meaning all the mice variants would exhibit similar behavior. There would be no significant data found from such experiments unless behavioral manifestations of the interaction between Cdk5 and parkin were found. This is expected because there are a vast number of proteins and mechanisms such as mitochondria dysfunction and deregulation of the apoptotic cycle that has been associated with PD.
Possible Pitfalls and Solutions
One of the main pitfalls of the experiment is that there is no measurable way to link this interaction to the topic of concern, Parkinson's disease. A more direct marker for the disease would be to observe the formation of Lewy bodies. The problem with that approach is that Lewy bodies may be formed through means unrelated to the dopaminergic neurons observed, whereas, the survivability of dopaminergic neurons and therefore the production of dopamine can be directly observed with the striatum neurons being tested upon.
Another main pitfall of the experiment is that the Parkinson's disease model associates many different proteins and pathways with the causation of the disease. Examples of proteins implicated in this model are: PINK-1 that recruits parkin to mitochondria during mitophagy, and p38 that forms a complex with PINK-1 to activate HtrA2 activity to induce the degradation of misfolded proteins. Nonetheless, this experiment may further clarify the role of Cdk5 and parkin in the loss of dopaminergic neurons, an important aspect of PD.