The Protein Pallidin and its Prognosis in Schizophrenia

Ece Su Sayin

The mental disorder schizophrenia has been a key research area for the past few decades considering the high number of people that suffer from it. The etiology of schizophrenia is still not known, but there are many theories on it. One of the theories made by Shi et al. (2017) show that Pallidin promotes transcriptional activity of p38. The overexpression of p38 leads to cell differentiation. The problem is that when Pallidin is present, this inhibits growth proteins such as Coronin 1b and Rab13. When this overexpression without the important growth factors takes place, this leads to abnormal neuronal development. The authors hypothesize that Pallidin influence growth of nerve processes via p38 and this causes abnormally developed brains, increasing risk of schizophrenia. What the authors could not fully conclude is when this overexpression happens, does it lead to schizophrenia like they assume.

Key Words: Schizophrenia; Neurodevelopment; Pallidin; P38

mass from the brain was from the lateral cerebral ventricle, hippocampus and prefrontal cortex. The weight increase was due to a decrease in dendritic cells. Researching a protein found throughout the central nervous system and brain, in which it is seen to affect the development of neuronal cells was next. This is why the authors, Shi et al. (2017), wanted to explore the importance of the protein Pallidin in the brain and the central nervous system. The proteins involved Previously, it has been found that the protein Pallidin is highly expressed in the hippocampus of the brain, especially in the glutaminergic neurons. Also, Ach neurons exhibit high levels of Pallidin (Larimore et al., 2014). In the hippocampus of a mouse lidin playing an important role in the regulation of synaptic activity, the protein, p38, has an essential role in apoptosis. In the

Figure 1 Visual summary explaining the paper by Shi et al. Made on BioRender by Ece Su Sayin

Background Information

Schizophrenia is a very different and complex mental disorder that is affecting many people in the world today. Even though one percent of the world population experiences schizophrenialike symptoms (Insel, 2010), the reasons people experience schizophrenia are still unknown. Schizophrenia is defined by the collective onsets of psychosis with paranoid illusions and auditory hallucinations. Schizophrenia only affects a few critical rein the brain together. They hypothesized that Pallidin and p38 are important factors in neurodevelopment, which has to play

gions in the brain and neurotransmitters like dopamine (Javitt, 2010). Since the causes of schizophrenia are not known, there is constant research going on to test various types of proteins, enzymes and complexes in the brain and how changes can lead to schizophrenia.

Direction for Research When it comes to research in this field, there are many aspects one can tackle. In the past, research has been done about the interactions of neurotransmitters in the brain. These studies show that neurotransmitters due play a role in the development of schizophrenia (Carlsson et al., 1999). The interaction between the different kinds of neurotransmitters is essential in the role of neuronal circuits, which ultimately gives insight into antipsychotic activity. The neurotransmitter theory provides validity as there is evidence for genes that play a role in the neurotransmitter system's function. Besides, there is research on the neurotransmitter system abnormality hypothesis (Padeĭskaia et al., 1980). This hypothesis focuses on pieces of evidence found from CAT scans that there is some abnormality in the brain structure from the images. Also, since the structure of the brains looked different from person to person, they weighed them to see if proved that the mass of a brain with schizophrenia versus a healthy brain was, in fact, much lighter. This proves that when people get the onsets of schizophrenia, physical changes due to occur in the brain. With the continuation of this area, Huard et al. (2007) followed up with the research that this decrease in brain, high activity of the Ach neuron suggests that it is involved in the regulation of the synaptic membrane. In addition to Palgenome, research by Shao et al. (2015), it has shown that this protein promotes gene expression and is a crucial protein in neuronal development. Since apoptosis is a crucial and regulatory factor for any development in the body, the protein p38 to have apoptotic abilities ensures successful neuronal development.

Research Question By combining various important key research ideas on the activities that are going on in the brain and the individual function of these proteins, Shi et al. (2017) wanted to test if these two critical proteins are interconnected and play a role in abnormalities there were any noticeable differences. They concluded and

an essential role in the prognosis of schizophrenia. The authors overall wanted to verify if increases in Pallidin and p38 lead to the onsets of schizophrenia and causes behind it.

Major Results

The authors took three different mammalian cells, HEK293A, HCT116 and N2a and treated them with fetal bovine serum, MEM and antibiotic culture medium. Alongside the mammalian cell lines, they took wildtype mice and regular sandy mice in which they extracted the cortical neurons from the cortical tissues. Depending on the study, they performed various experiments on the appropriate cell line or cortical neuron, which is why there was a variety. The authors in this study concluded with three main points in relevance to their hypothesis.

Firstly, the authors concluded that Pallidin does influence transcriptional activity on p38, but when HDAC is bound to p38 and E2F1, it inhibits the transcriptional factor. Following immunocytochemistry, the results showed that in the HCT116 cell line, when the transcriptional activity of HDAC was overexpressed,

this caused an increase in Pallidin, which in return caused an increase in p38. In the western blot analysis that was done, increased levels of Pallidin also caused an increase in p21, and a qPCR showed increased levels of mRNA (Figure 1). This made the search is the general idea is that Pallidin via P38 promotes an increase in transcriptional activity. The enzyme, HDAC, inhibits Pallidin leads to a decrease in the release of growth factors such as Rab1 and coronin 1b. As this cell differentiation occurs in the

authors conclude that Pallidin influences transcriptional activity on p38 even when it is bound to HDAC, an inhibiting protein.

During cell differentiation, if Pallidin levels decrease, this influences the expression of p38. The reduction in p38 has a direct effect on cell differentiation, causing a reduction, which affects cell differentiation. When p38 was knocked down, using realtime PCR, they were able to detect mRNA levels.

Next, using real-time PCR and a western blot, when p38 was knocked down, they measured the levels of mRNA, which showed that the expression of the two proteins, Coronin 1b and Rab13 decreased (Figure 2). The authors noticed that the overexpression of Pallidin caused a decrease in the growth factors, which were downstream genes of p38. For successful neuronal are essential growth factors. When the overexpression of Pallidin inhibits these growth factors, this leads to the abnormal development of the neurons as there are not enough growth factors ensuring the proper development of the neurons in the brain. growth factors. Adapted from “Pallidin protein in neurodevelopment and its relation to the pathogenesis of schizophrenia” by Shi et al. (2017).

Discussion

The primary conclusion that the authors have made in their rethis transcriptional activity by binding to E2F1 and p38. That is why the protein, Pallidin, is required to inhibit HDAC. The overexpression of Pallidin and, in return, continues to the overexpression of p38 when an inhibiting factor is not there, this leads to cell differentiation. The problem is that the overexpression of development, the downstream genes of p38 are required, which

brain without any growth factors, this affects neuronal development. As the neurons in the brain develop irregularly, the authors hypothesize that this abnormal development of the brain and the central nervous system, ultimately leading to schizophrenia.

The authors' conclusion is relevant since they have a good and robust idea about the causes of schizophrenia. In tackling this field with numerous unknowns, it is very safe first to try to figure out what happens in a person's brain, leading to this mental disorder. The authors' theory about the overexpression of Pallidin and p38 and its effects on neurodevelopment are in alignment with other few pieces of research. In an older research paper by Chen et al. (2017), they were able to do a null mutation on Pallidin in Drosophila. Their ideology was that since Pallidin is one of the subunits of the BLOC-1 complex, they wanted to determine if Pallidin played a role in neuronal and synaptic development. They showed that Pallidin promotes fast synaptic vesicle recycling, which is essential to maintain functional vesicle transport during neuronal activity. Both Chen et al. and Shi et al. prove that "Pallidin" plays a vital role in neuronal development. A paper by Ryder & Faundez (2009) showed that DTNBP1, the gene encoding for dysbindin, a Pallidin homolog, is also linked to schizophrenia.

of Pallidin. Adapted from “Pallidin protein in neurodevelopment and its relation to the pathogenesis of schizophrenia” by Shi et al. (2017)

Critical Analysis

Figure 2 (top) shows the western blot indicating increased levels

Every paper that is written and published has both strengths and weaknesses, as no research is considered perfect. When analyzing a paper, the strengths and weaknesses identified by the reader are mostly subjective as there are many ways to conduct research and publish a paper. This paper by Shi et al. was overall well done, and the research they have conducted shows excellent validity. Like all research, this paper also had a few weaknesses, which will also be discussed.

Figure 3 Shows a western blot. Increased levels of Pallidin lead to decreased levels of Coronin 1b and Rab13, which are important

One of the strengths of this paper is that the authors followed a chronological order for the experiments they did. The numerous experiments they have done around the protein Pallidin adds new knowledge to this field of study, building off of previous research. In science, it is essential to follow a chronological order and to have a cohesive strategy as this reduces confusion. The 163

authors did a great job explaining in their introduction, why this protein called “Pallidin” with not much previous research, was essential to study and the mechanisms behind it. Their methodologically, for the most part, followed a precise sequence of events. It made much sense that they first concluded what transcription factor Pallidin inhibited (HDAC), followed by determining the growth factors involved to paint the overall picture. Another strength of this paper is that they tested the same mechanisms on numerous different cell lines and mice cortical neurons from two different mice. Instead of picking just one cell line or a cortical neuron from one mouse and showing results favouring the validity of their hypothesis, they showed a variety. This variety that they showed insured and proved that not all cell lines and even cortical neurons from the same mice produce similar results. Another strength of this paper is the great amount of detail they provided in their methods section. They outlined the ten different steps they took to gather all their data and perform the experiments. In the first part of their methods, they explained where they bought or received all the cell lines and mice that give the places proper credit. Also, the research was approved by the ethics board. This is crucial for the validity of the experiment, as this proves that the steps taken in the experiment follow the ethical guidelines of the institution. The most important strength of this paper is that the hypothesizing mechanism is true, and their results are similar to other papers. Like previously mentioned, the paper by Chen et al. (2017) and Ryder & Faundez (2009) both prove that this protein Pallidin is in the BLOC-1 complex, which is involved in synaptic and neuronal development. The authors of this paper go beyond the research of the others by providing further knowledge on other proteins involved like p38, HDAC, p21, rab13 and coronin 1b, which is an addition to the knowledge in this field.

On the other hand, like most papers, this article had a few weaknesses. Even though it was mentioned earlier that the authors had a clear methodology for their experiment, their explanation of their results section was weak. They did not fully explain their results, and their visuals like graphs and western blots were very hard to understand. The legends of the proposed graphs were not clear, and it was hard to make out the comparison they were trying to make. The authors included four to five big illustrations, with each illustration having six to seven images. This created an overwhelming presentation of data, and they did not clearly explain in detail the relevance of each one. A better way to present their results would have been selecting the most important figures and putting an asterisk on what they wanted their audience to focus on. The balance of the number of visuals and their explanation was low, which left it to the reader to understand and interpret. Another suggestion for the results section would have been to explain the names on the axis, what they stand for, and their relevance. For example, they used short forms like Si-NC, Si-p38 and RA, and not explain what it stands for. The point of publishing literature is to provide a clear explanation of their findings and making sure it is understandable by everyone reading it. cussing the importance of each protein and making connections between the concepts. What makes their discussion/ future directions part weak is that they do not necessarily provide full-on support showing that an abnormally developed brain leads to schizophrenia. They provide great detail about Pallidin via p38 influencing neuronal development, which is in line with previous research, but the connection to schizophrenia is not fully there. The entire point of this paper was to find a mechanism and how overexpression/under expression can lead to the mental disorder of schizophrenia. The authors spend a reasonable amount of time proving the importance of Pallidin but do not necessarily connect it to their main argument. In a way, this is not entirely a weakness as it leaves open for future experiments. However, it would have been a more persuasive argument if they spent more time discussing the connection to schizophrenia.

Future Directions

So far, the research around Pallidin and its involvement in causing schizophrenia has come a long way, showing that when this protein is increased, so does the prognosis of schizophrenia. There are still many missing links in this field of study that limit researchers on making keen connections. The missing links on finding even a simple question like its etiology prevents doctors and health care workers from helping to treat these patients. So far, up to this point in research, scientists have identified the proteins, enzymes and complexes involved in the development of schizophrenia. What would make advancements in this field is by finding a way to make the knockdown of Pallidin in the brains of live mice and test if they develop schizophrenia-like symptoms. This would be very beneficial to research as the authors have left off saying that they predict this abnormal neurodevelopment will lead to schizophrenia. Pallidin is homologous of dysbindin (Shi et al., 2017), which is another subunit Throughout their experiment, the authors do a good job dis

on the BLOC-1 complex. Inhibited levels of Pallidin showed inhibited levels of dysbindin and vice versa (Spiegel et al., 2015), which prove that their functions in the complex are interconnected. In the same paper, it was found that the gene encoding for dysbindin is DTNBP1. This means that if we are successful at knocking down dysbindin, levels of Pallidin will also decrease. The objective of knocking down dysbindin/Pallidin is to verify that experiments in Shi et al. (2017) and to do further testing on the development of schizophrenia. A knockdown in mice can be achieved by RNA interference as this silences the gene of interest for some time. A way to test if this protein has been silenced is by doing an RNA screening similar to DNA screening. This tests to see if the gene of interest has been silenced. The expected results from this experiment would positive, but because it is an unknown field of study, it is hard to come up with definitive answers. If the iRNA does not work, this could mean that there is more to this BLOC-1 complex that researchers do not fully understand. If you cannot induce and cause a mouse to get schizophrenia via the protein Pallidin, there is still another way to test the involvement of Pallidin. What can also be down to test the protein Pallidin and how it is related to schizophrenia would be to use DNA directed RNA

interference. This could be done on mice or humans who already have schizophrenia-like symptoms. This technique of knocking down disease-causing genes has excellent potential in the development of therapeutic drugs (Lisitskaya et al., 2018). This research would be highly translational in humans because it has been found that Pallidin and dysbindin have similar expressions in both mice and human brains (Spiegel et al., 2015). In conclusion, the entire study of neuroscience still has much ground to cover to help the millions of people suffering daily. This protein Pallidin and the entire BLOC-1 complex hold promising results for the etiology of schizophrenia, but there is still much more to uncover and research.

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