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3.1 Overview


image_3.png
Selegiline inhibits Monoamine Oxidase B (MAO-B) leads to increase in dopamine. (Adapted from Rang, H. P., et al. (2001))

MAO inhibitors are prescribed to alleviate the symptoms of a multitude of disorders such as schizophrenia, anti-social behavior, & depression/bipolar. MAO-B Inhibitors (MAO-BI) are most commonly used to treat the symptoms of Parkinson’s disease (PD).

The most common movement disorder in the world today is Parkinson’s disease (PD). The symptoms of PD are tremors in the limbs, bradykinesia, & impaired movement coordination[1] . Currently there is no cure for PD but there are treatments such as Selegiline and Rasagiline that alleviate or improve motor function[2] . The symptoms of PD are due to the apoptosis of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNc)[3] [4] .

Mitochondrial dysfunction, accumulation of anomalous proteins, and oxidative stress are some of the processes believed to lead to the death of DA neurons in the SNc. Currently it is believed that these processes trigger the apoptotic mechanism[5] .Despite whatever mechanism might underlies the death of DA neurons in the SNc the neurodegeneration process can’t be stopped or slowed down.

3.2 Apoptosis- ER stress induced


3.21 Overview
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Substantia nigra and Parkinson’s disease. (Adapted from PubMed Health)

A lot of different mechanisms have been proposed to lead to the death of the dopaminergic neurons in the SNc such as oxidative stress, mitochondrial dysfunction, and the accumulation of anomalous proteins[6] [7] . Currently, most of the evidence points toward apoptosis underlying the neurodegeneration that occurs in the brain of PD patients[8] [9] .
Apoptosis is one of several mechanisms in which programmed cell death can occur. Apoptosis can occur via three distinct pathways. An intrinsic pathway, extrinsic pathway, & Endoplasmic reticulum (ER) stress induced pathway. ER stress pathway is triggered by a buildup of misfolded or unfolded proteins in the ER lumen [10] [11] (10), (11).

3.22 UPR

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Schematic overview of the UPR Pathway. http://www.bijvoet-center.nl/cpc/research/ERstress/UPR
The Unfolded Protein Response (UPR) occurs before the ER
stress induces the apoptotic pathway. UPR is a cellular stress response involving the ER. This cellular response is conserved from yeast to mammals. In the mammalian response the buildup of misfolded or unfolded proteins in the lumen of the ER activates the UPR. Initially this response will try to correctly fold the misfolded or unfolded proteins by arresting translation and increasing the production of molecular chaperones. If the misfolded proteins are not correctly folded in a certain time frame then UPR leads to apoptosis[12] [13] .

3.23 ER stress – Pathway


The complete cellular mechanism of ER stress-induced apoptotic pathway is not known. It is known that this pathway involves proteins of the B-cell lymphoma-2 (BCL-2) family and caspase proteins[14] . BAX, BAK and BCL-XL are three proteins in the BCL-2 family and have been implicated in this pathway. BAX & BAK are pro-apoptotic while BCL-XL is anti-apoptotic. One study showed that even though these two types of proteins have opposing effects they both mediate cell death. It turns out the BH3- only proteins interact with some of the anti-apoptotic proteins in the BCL-2 family, such as BCL-XL, leading to an overall signaling pathway towards apoptosis.The BH3-only proteins are in a distinct family from the BCL-2 but they share a common motif; the BH3 domains through which the interaction occurs. Depending on the molecule that activates BCL-XL it can be either pro-apoptotic or anti-apoptotic[15] . BAX & BAK are normally found in the cytosol but are activated via ER stress signaling. ER stress signaling pathway activates BH3-only protein, BIM. BIM can interact with BAK and BAX activating them towards the apoptotic pathway[16] . Once activated BAK can insert into the outer mitochondrial membrane (OMO) and increase permeability of the membrane to certain apoptogenic factors such as cytochrome c and second mitochondria-derived activator of caspases (SMAC)[17] . These apoptogenic factors lead to the activation of caspases and subsequently apoptosis[18] .

3.24 ER stressER permeability


Most studies focus on BCL-2 proteins and their effects on the permeability of the Outer mitochondrial membrane (OMO). The study by Wang, Olberding, White, & Li (2010) looked at the effects of BCL-2 proteins on ER membrane permeability. They showed that there was an increase in ER membrane permeability that was dependent on BCL-2 family proteins, BAX and BAK. A plasmid encoding a fluorescent protein was used to transfect the ER lumen of the wild type mouse embryonic fibroblasts (MEF). At first the fluorescence was only localized to the ER lumen. Next the cells were treated with ER stress inducers, thapsigargin or tunicamycin. Within minutes of adding the ER stress inducers the fluorescence was no longer localized exclusively to the ER lumen but also to the cytoplasm and nucleus. They added the ER stress inducers to BAX-/-BAK-/- MEF cells and the fluorescence stayed, mostly, within the ER lumen. Further studies are required to elucidate which molecules translocate between the cytosol and ER and the cellular pathway induced[19] .

3.3 Treatment for Parkinson’s disease


3.31 Current MAO-B Inhibitor overview

MAO-BIs have been used to slow down the degradation of dopamine. This allows dopamine to persist longer in the synapse, which prolongs the effects of endogenous or exogenous dopamine[20] .Currently there is no cure for PD but there are treatments that improve the motor deficits[21] . All the drugs currently used to treat PD including the most common drugs, Levodopa, Rasagiline, and Selegiline are symptomatic. These drugs work to decrease the motor deficits of PD but none slow down the neurodegenerative process (7).
In addition, some studies have shown that MAO-BIs such as Selegiline, & Rasagiline have some neuroprotective effects in different experimental models of PD such as the
MPTP Parkinson's disease mouse model
[22] [23] . Presently none of the drugs, including Selegiline and Rasagiline, with a potential neuroprotective effect slows down the neurodegeneration process in clinical trials[24] .

3.32 PF9601N (Potential MAO-B Inhibitor)


3.321 Overview

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Chemical structure of PF9601N. (Adapted from Mercedes, U., et al. (2011)).

PF9601N is considered a second generation MAO-BI that was identified through a series of screening processes by many groups[25] [26] [27] . PF9601N inhibits MAO hence preventing MAO from degrading dopamine and leading to an increase of dopamine in the synapse. This drug is more potent & selective than Selegiline and Rasagiline in inhibiting MAOB. In addition, PF9601N does not generate amphetamine as a byproduct when metabolized unlike selegiline (23). Amphetamine could potentially contribute to the degeneration process but there is no evidence of that as of now.Not only does PF9601N prevent the metabolism of dopamine but it prevents the reuptake of dopamine in clinical trials as well as in rat striatal synapses[28] [29] . It also decreases the loss of dopaminergic neurons after selective injection of 6-Hydroxydopamine in the nigrostriatal pathway of rat brains[30] . In addition, PFN601N has been tested in cell culture models of PD and shown to protect DA neurons of a cell line SH-SY5 from degeneration[31] .

3.322 Anti-apoptotic mechanism of PF9601N

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Semi-quantitative RT-PCR analysis of GADD153/CHOP expression. β-Actin is the control. (Adapted from Sanz, E., et al. (2009)).

In Sanz et al., (2009) described how ER stress induced cell death was halted by PF9601N. No other MAO-BI’s have been shown to prevent cell death induced via ER stress. PF9601N is able to prevent ER induced apoptosis by blocking Unfolded Protein Response (UPR) & as a result prevented the downstream apoptosis signaling pathway (31).
Brefeldin A (BFA) is a protein that disrupts the trafficking from the ER to the Golgi which leads to the buildup of proteins in the ER. The buildup of proteins activates UPR and subsequently the apoptotic pathway. They pretreated the SH-SY5Y cells with the same concentration of PF9601N and increased the concentration of BFA each trial. The results indicated that PFN601N prevented the BFA from inducing an ER stress response compared to the control. They also pretreated the SH-SY5Y cells with Selegiline before adding BFA. Selegiline could not block the ER stress pathway by blocking UPR. Next they checked for the levels of CHOP via semi-quantitative RT-PCR after the addition of BFA to SH-SY5Y cells that were either pretreated with PF9601N or Selegiline. CHOP is activated by BFA. CHOP is the protein that begins the apoptotic signaling pathway[32] . Only PF9601N treated SH-SY5Y cells managed to keep the mRNA level of CHOP low enough to prevent the start of the apoptotic signaling cascade.

3.4 Clinical Potential


PF9601N

PF9601N is more selective than Selegiline and Rasagiline. PF9601N does not inhibit MAO-A, unlike Selegiline. When MAO-A is inhibited it leads to an increase in tyramine, a vasopressor, derived from diet which can cause hypertension. Therefore due to the selective nature of PF9601N it is less likely to lead to unfavorable side effects.
Both PF9601N & Seligiline undergo metabolism in the liver by microsomal enzymes via different cytochrome P450 (CYP) enzymes[33] . Most drugs and many organic substances are catalyzed by CYP enzymes found in the liver[34] . Selegiline is metabolized by CYP2B6 & CYP2C19. PF9601N is metabolized by CYP3A. Selegiline inhibits CYP2B6 & CYP2C19, hence effecting its own metabolism. In addition, Selegiline lowers the CYP microsomal content thereby delaying the clearance of PF9601N. The prolonged presence of seligiline or PF9601N can lead to unwanted side effects or interactions[35] . PF9601N has neuroprotective effectives, antiapoptotic effects, & positive metabolic profile which encourages potential clinical use but PFN601N still requires further investigation.

See also


References



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