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Caron and colleague (2007) conducted a study to determine the similar biological and clinical characteristics that exist in lipodystrophy caused by LMNA mutation and HIV antiretroviral treatments. Observing the cells morphology and function lead them to conclude that prelamin A accumulation, premature ageing of the cells, and oxidative stress is common in both groups. The researches failed to provide a better reasoning for the results they obtained by not analyzing the nuclei of all six mutations and at least three different mitochondrial enzymes. Furthermore, they did not provide adequate amount of background information about the molecular mechanism of this disease. Various minor issues such as the size of the images and the lack of positive and negative control in the methods were perceived. Overall, this article provides essential information that can be used by other researchers to develop HIV treatments that prevent the formation of lipodystrophy.
Lipodystrophy, as its name suggests is a disorder characterized by a loss of fat in different parts of the body. Familial partial lipodystrophy Dunnigan-type (FPLD) is an inherited form of this disease that affects less than 1 out of 15 million people. These individuals exhibit normal phenotype at birth, however, puberty onsets the symptoms of lipodystrophy. They lose subcutaneous fat from the limbs, trunk and other extremities. Approximately forty percent of HIV treated patients that use antiretroviral protease inhibitors (PIs) acquire lipodystrophy and develop similar symptoms (Garg, 2000). Researchers have classified this disorder as a laminopathy since the underlying molecular defect takes place in the nuclear lamina (Caron et al, 2007). These filaments are responsible for maintaining the structure of the nucleus and associate closely with the inner nuclear membrane. They have also been found in nucleoplasm regulating gene expression, DNA replication, and arranging nuclear pores. The gene that encodes this important functional protein is called the LMNA gene, located on chromosome one (q21-22) (Cao, & Hegele, 2000). Alternative splicing of LMNA gene generates different forms of lamina (prelamin A, C, and B) (Wojtanik et al, 2009). Prelamin A and B are the only two filaments that undergo post-transcriptional modification. Initially, the c-terminal region becomes farnesylated by FTase, followed by c-terminal cleavage, methylation and the removal of the downstream farnesylated region by metalloprotease, ZMPSTE24 (Caron et al, 2007). This enzyme is a membrane protein that binds to prelamin A as its substrate and releases the mature form of lamin A after proteolytic cleavage (Boguslavsky, Stewart, and Worman, 2006). Subsequently, mature lamin A/C form heterdimers and bind to emerin, an integral membrane protein (Cao, & Hegele, 2000). To initiate adipose differentiation, sterol response element binding protein 1 (SREBP1) is released from endoplasmic reticulum (ER) where it was originally formed (Szymanski et al, 2007). It enters the nucleus via emerin and interacts with the c-terminal region of mature lamin A (Holt et al, 2001). This filament facilitates the transport of STEBP1 to PPAR-Î³, a transcription factor (TF) which regulates the gene expression of pre-adipocyte differentiation (Capanni et al, 2005). Overall, it appears that the role of lamina in adipose tissue is essential for the formation of fat.
Loss of fat in adipocyte tissues is the result of various mutations in the LMNA gene that encode prelamin A (Garg, 2000). FPLD is caused by a missense mutation (R482Q) that hinders the ability of mature lamin A to bind to SREBP1 (Cao, & Hegele, 2000). It is important to note that this does not inhibit the formation of lamina or its ability to interact with other nuclear membrane proteins such as emerin (Vantyghem et al, 2004). Previous experiments have shown that emerin is still localized in the inner membrane (Holt et al, 2001). Other LMNA mutations (D47Y, L92F, L387V, R399H, L421P and R482W) cause prelamin A accumulation by deleting the cleavage site of ZMPSTE24. This enzyme no longer recognizes prelamin A and the farnesylated region bound to c-terminal domain remains anchored to the protein. The same type of molecular defect has been observed in the adipose tissues of HIV patients that use antiretroviral protease inhibitors (PIs) (Caron et al, 2007). Also, studies in mice have shown that disrupting ZMPSTE24 gene leads to many abnormalities including lipodystrophy (Pendás et al, 2002). As a result of these changes, the polymerization of the filaments in the inner nuclear membrane is disrupted, SREBP1 remains in the cytoplasm, and PPAR-Î³ is down regulated (Capanni et al, 2005). Both acquired and inherited types of LMNA mutation reduce pre-adipocyte differentiation by a variety of different processes (Lloyd, Trembath, and Shackleton, 2002). Caron and colleague (2007) conducted a study to examine the clinical characteristics that are common between LMNA-linked HIV treated patients and familial partial lipodystrophy patients. This comparison was made by extracting skin fibroblasts from female patients that had diabetes, but exhibited abnormal fat distribution. To ensure that lipodystrophy was the cause of this phenotype, LMNA genetic testing was performed and the mutations were determined (D47Y, L92F, L387V, R399H, and L421P). The control skin fibroblasts were taken from non-diabetic women with normal distribution of fat. Abdominal adipose tissues were obtained from four HIV infected patients using protease inhibitors (indinavir and nelfinavir), and HIV infected patients with no protease inhibitors as the control group. To further examine other mutations such as R482W and R439C, they obtained adipose tissue from women suffering from lipodystrophy. Lastly, they compared these tissues to women exhibiting a normal phenotype to obtain more accurate results (Caron et al, 2007).
The results of this experiment provide convincing evidence that HIV treated patients and LMNA mutated genes in lipodystrophy display similar clinical and biological features. Cardiovascular problems, insulin resistance, diabetes, and premature ageing are few clinical characteristics that are common in HIV treated patients and LMNA mutation lipodystrophy (Caron et al, 2007). Previous studies have shown that LMNA-deficient mice show similar nuclear abnormalities and clinical features such as premature ageing (Varela et al, 2005). Caron and colleague (2007) confirmed using western blot analysis that accumulation of farnesylated prelamin A was present in both HIV treated patients and LMNA mutations. To determine the molecular mechanism, the researchers compared HIV fibroblasts treated with protease inhibitors (indinavir and nelfinavir) with fibroblasts treated with atazanavir (nonpeptidodimeric PI). These comparisons lead them to conclude that PI mutates the normal function of ZMPSTE24 and not atazanavir, thereby preventing the cleavage of c-terminal farnesylated region and formation of lamin A. Similarly, mutations in LMNA gene prevent the cleavage site of ZMPSTE24 which result in an increase of prelamin A in the cell. Cultured fibroblasts observed by immunofluorescence microscopy confirmed that nuclear shape abnormalities were common between the two groups. As previously stated, lamin A and B are the only two nuclear laminas that undergo post-transcriptional modification. Approximately sixty percent of the nuclei lamin A/C were dysmorphic while only forty percent of lamin B had an unusual nuclear shape. The cells that contained D47Y mutation showed the highest rate of abnormality (25%) at the initial stage of cellular passage. Also, the shape of their nuclei was composed of many lobules compared to other mutations. Further analysis of immunofluorescence microscopy depicted prelamin A accumulation close to nuclear rim in all six mutations including those treated with PIs (Caron et al, 2007). As a result of this, prelamin A was unable to bind to SREBP1 in all the cultured cells except the control groups (Boguslavsky, Stewart, and Worman, 2006). Improper assembly of lamin A not only prevents the proper formation of nuclei and the expression of adipogenesis, but also leads to premature cell death (Caron et al, 2007).
Premature senescence, changes that take place in the cell in response to ageing, was observed during many occasions. For example, population doubling levels (PDL), the number of times the cell doubled since it was isolated, was calculated as a measure of the proliferative rate of fibroblasts. The graph illustrated a gradual decline in the number of cells in both groups of patients. A more pronounced decline was observed in fibroblasts that contained D47Y mutation. On the other hand, the measurements obtained from control cells remained relatively stable. Cell cycle checkpoints inhibitors (p16 and p21) were analysed using western blotting and over expression of these proteins confirmed that the cells of LMNA mutation and PI treated human fibroblasts were undergoing senescence (Caron et al, 2007). The role of cell cycle checkpoints is to prevent cell growth in response to damage, and increase the expression of genes required for repair (Elledge, 1996). Lastly, B-galactosidase activity was observed in each mutation as a measure of cellular senescence. As expected, the control group displayed no B-galactosidase activity, while the expression of this enzyme was increased in mutated fibroblasts and adipose tissues. The cells observed under the microscope appeared enlarged and nuclear abnormalities were still present (Caron et al, 2007). Caron and colleague (2007) established experiments that concluded inhibiting farnesylation will decrease the expression of cell cycle check points such as p16, as well as preventing the formation of reactive oxygen species (ROS). Prelamin A accumulation associated with premature senescence activates various cellular stress pathways to protect the normal function of the cell. As a result, the number of reactive oxygen species increases creating a harmful environment for the cell and other organelles. Also, the free radical theory of aging states that damage to mitochondria generates toxic ROS (Caron et al, 2007). Caron and colleague (2007) examined the mitochondria to look for any changes in the respiratory chain proteins. The normal function of mitochondrial enzymes responsible for peroxidation of lipids became affected which lead to further accumulation of fat. If these harmful substances are not detoxified, substantial changes such as DNA damage will take place (Caron et al, 2007). Overall, it appears that prelamin A accumulation is associated with an indirect increase in ROS; however, the direct step-by-step mechanism has not been determined and requires future studies.
In this article, the researcher's main purpose was to compare the clinical and biological features that exist between HIV treated patients and LMNA mutations. To support their argument, several techniques were used including western blotting, fluorescence microscopy and graphs signifying the statistical changes. Based on the results obtained, they concluded that fibroblast and adipose tissues of the two groups of patients display prelamin A accumulation, oxidative stress caused by the release of ROS and premature cellular ageing (Caron et al, 2007). The major flaw of this study can be attributed to the assumptions the researchers made while explaining some of the molecular mechanisms involved in the study. Originally six mutations were identified from human fibroblasts that had lipodystrophy. However, two out of six mutations (D47Y, R482W) were used to compare the nuclear abnormalities. One mutation in particular showed a different result with a more lobulated membrane. Based on these observations, they stated that LMNA mutations caused by lipodystrophy result in similar cellular abnormalities as those observed in HIV-treated patients. Ultimately, they failed to observe four mutations under immunoflorescence microscopy. In the following section, the same type of procedure was used to analyse the location of prelamin A accumulation. Two new mutations (R399H, L42IP) were compared, while D47Y that showed a unique morphology in the previous section was not tested in this case (Caron et al, 2007). The prelamin A accumulation could have been over expressed in these nuclei or have formed at a different location to give rise to lobulated membranes. As a result of this, the researchers cannot make an assumption that all nuclear abnormalities and prelamin A accumulation are localized at the same place in all six mutations.
Another assumption was made to provide evidence for the malfunction of mitochondria in LMNA mutation and PIs treatments patients. Only the activity of one particular enzyme, cytochrome c-oxidase with different subunits (COXII, COXIV) was studied (Caron et al, 2007). Although COX is an important enzyme in the last step of electron transport chain, the activity of other proteins should be tested to determine whether changes are affecting the entire organelle. For accurate results, the mRNA expression of COX should have been tested to determine the transcription of the mitochondrial genome. If farnesylated prelamin A affected the mitochondrial function, DNA damage could have been observed. Often when researchers study mitochondrial function, citrate synthase is the first enzyme that is studied. This enzyme is responsible for the initial reaction of citric acid cycle and is essential for the breakdown of fatty acids into energy. When fatty acids enter the mitochondria through pyruvate, the carbon atoms form acetyl-coA and enter the citric acid cycle to produce ATP (Lodish et al, 2004). Based on the evidence provided, observing the activity of one enzyme is not sufficient to conclude that all mitochondrial enzymes are affected by the accumulation of prelamin A (Caron et al, 2007).
An article requires adequate amount of background information to provide the reader with a general understanding of the subject being studied. Unfortunately, the researchers of this article failed to provide simple and clear understanding of molecular mechanisms that cause lipodystrophy. Also, they did not mention the basic symptoms associated with the disease (loss of fat from extremities). The most important step in developing lipodystrophy is the down regulation of PPAR-Î³ due to the accumulation of prelamin A (Capanni et al, 2005). Changing the conformation of lamin A prevents the binding of its c-terminal region to the N-terminal region of SREBP-1 (Lloyd, Trembath, and Shackleton, 2002). Binding of these two proteins causes the localization of SREPB-1 via emerin to the nucleoplasm. The expression of PPAR-Î³ TF is required for adipose differentiation in the tissues (Capanni et al, 2005). This basic molecular mechanism was not provided, therefore, plenty of prior knowledge was required in order to comprehend the article. SREBP1 was mentioned once in the discussion section with no clear understanding of its function. The authors assumed that their intended audience are those with strong background knowledge about this topic. The only molecular mechanism provided was regarding post-transcriptional modification where prelamin A forms into the mature form after a membrane protein, ZMPSTE24 cleaves the farnesylated region (Caron et al, 2007). The last major problem that may have hindered the results of this experiment involves the subjects of the study. The methodology section states that only female patient's adipose tissues and fibroblast were used (Caron et al, 2007). The results obtained here might not apply to male individuals because of hormonal changes especially in FPLD when lipodystrophy starts with the onset of puberty. Proper research is done when male to female ratio is one, otherwise, the results do not explain the molecular mechanism of all lipodystrophy patients.
Although the previously described problems had a major effect on the outcome of results, the following are only minor issues. The images provided by micrographs to view the cells are extremely small and comparing the mutations between different cell types are difficult to grasp. Perhaps larger images with higher resolutions could have been used and labelled along with arrows to indicate where the mutations were mostly frequent. Another minor issue was found while analyzing the results. Western blotting analysis in some cases included positive and negative controls whereas majority of the time only one control was used. For example, prelamin A expression display on gel electrophoresis was shown as a single dark band in all six mutations, while there was no band present in one control group. More accurate results may be obtained by manipulating prelamin A farnesylation in vitro to form a positive and negative control. Afterwards, the formation of bands on the gel would be compared to cultured fibroblasts created in vivo. Lastly, some terms that were critical for understanding the results were not defined. Throughout this article, the PI treated fibroblasts were grouped into indinavir and nelfinavir, yet no explanation was given to compare and contrast the chemical properties associated with these two treatments (Caron et al, 2007). For example, researchers could provide information about a common chemical found in both of these antiretroviral therapies that lead to the mutation of ZMPSTE24's normal function. By obtaining this information and the research done on this paper, they can prevent the addition of that particular chemical in antiretroviral therapies and other drugs used to treat HIV.
Despite the issues found, this article provides excellent information regarding the molecular mechanism of lipodystrophy created by HIV treatments. Other researchers can use the information provided here to discover other antiretroviral therapies that do not cause any side effects. Caron and colleague (2007) showed that these treatments mutated ZMPSTE24 enzyme that would normally recognize the cleavage site of farnesylated prelamin A. The current treatments used lead to the development of lipodystrophy because adipogenesis did not take place. LMNA mutations are not found in these individuals, thus they are not predisposed to this disease. By switching to treatments that prevent the mutation of ZMPSTE24, the number of HIV patients developing lipodystrophy will substantially decrease (Caron et al, 2007). In addition, the researchers provided detailed methodologies that were designed to allow other researchers to replicate the experiment. Future research can look specifically at ZMPSTE24 and the amino acid change that occurs when it becomes mutated.