Research Plan:
Cellular Effects of the PSMC5 P312R Mutation and Search for Potential Therapies
Summary
The research program proposed below is a collaborative effort to understand further the cellular effects of the PSMC5 P312R mutation, and on this basis, to identify genetic manipulations or drugs that may help counter its pathological consequences. Despite many advances in our knowledge about the biochemical mechanisms and physiological importance of the proteasome, we still cannot predict how this change in a single amino acid in one of the proteasome’s six ATPase subunits affects cell function. The proposed research builds on the initial findings already made through studies of fibroblasts from Oliver Myers and his family. An initial goal will be to determine if the cells from Yoni Silverman and family behave similarly. To facilitate further research into the pathogenic mechanisms, we will create new cell lines that differ in only one respect, whether they express the PSMC5 P312R mutant or the normal wild-type proteasome subunit, and this effort will include neuronal cell lines in order to determine if this mutation causes special problems in neurons.
In addition to addressing these fundamental questions, we will use CRISPR technology to screen for proteins or pathways which if inactivated reduce the deleterious effects of this proteasome mutation. Our recent collaborative studies have shown that certain drugs can cause proteasomes to become more active and these agents appear promising treatments for certain models of neurodegerative diseases. We shall test if they also stimulate proteasome function in PSMC5 mutant cells and can protect them against proteotoxic stresses. In addition, a collection of FDA-approved drugs will be screened to learn if any may also have beneficial effects in protecting these cells against toxic conditions.
These studies will build on the biochemical expertise of Drs. Collins and Goldberg in studying proteasome function and intracellular protein degradation and that of Prof Rubinsztein’s lab in genetic analysis of neurological disorders and of autophagy. Also, critical will be the collaboration with Dr. Lior Zangi in using modRNA to analyze the mutant’s cellular consequences and the efforts of the Harvard Cell Biology Core in constructing novel cell lines for these studies. While our immediate goal is to understand the effects of this proteasome mutation and to identify approaches that might benefit Ollie and Yoni, we are convinced that the knowledge gained will also be useful in the study in the growing number of patients with other proteasome mutations and even patients with other proteotoxic diseases affecting brain function.
11 Aims for this Research Project
Because Yoni and Ollie share a common mutation in a proteasomal ATPase subunit, but seem to have had different developmental trajectories, it is important to compare the behavior of their cells, and the biochemical properties of their 26S proteasomes. The Boston Children’s Hospital Human Neuron Core will establish fibroblast lines from Yoni and the Silverman family, while Dr. Siddharth Srivastava will perform an in-depth neurodevelopmental examination. (The Human Neuron Core will also bank the cells for any future studies to generate induced pluripotent stem cells (iPSC) that could be differentiated into neurons, heart, or muscle cells). We will test if Yoni’s fibroblasts differ from those of his immediate family members in exhibiting the key features as Ollie’s cells that we believe are characteristic of cells with defective proteasomes: 1) a greater sensitivity to increased temperatures, which damage cell proteins and thus increase the workload on the proteasome, and 2) increased sensitivity to the proteasome inhibitor, bortezomib, along with 3) a higher cell content of ubiquitin, both of which suggest a reduction in proteasome function. We will also measure the proteasome’s enzymatic activities (rates of peptide hydrolysis), levels of subunit expression, and the cellular content of singly-capped and doubly-capped proteasomes. (See below).
We anticipate carrying out these studies during the first three months of this project and studying further the behavior of the Silverman and Myers family’s cells during months 4 – 6 using other conditions that increase proteasome demand (i.e., other types of proteotoxic stresses – See Aim 6).
For many studies it will be essential to have cells that grow faster than primary fibroblasts and to be able to compare cell lines that differ only in with the PSMC5 P312R mutation. We will use Harvard Medical School’s Cell Biology CRISPR core facility to generate a HCT116 cell line with this mutation. This heterozygous cell line and the wild-type line will allow more rigorous and more efficient biochemical and cell biological studies and will be essential for the screening experiments to be performed by Dr. David Rubinsztein’s laboratory. An important advantage of such cell lines is that they avoid possible complications in interpretation due to other gene differences in the various members of the Silverman and Myers families. Having such cells will also mean that only two lines need to be compared in future studies of the mutation’s effects. In addition to accelerating research, these cell lines will be a valuable resource for future work of other researchers who in the future may build on our studies.
We anticipate establishing this line during the first four months of this study, and during months 5 and 6, examining the behavior of these cells to confirm that they exhibit the same defects as Yoni’s and Ollie’s fibroblast lines (as measured in Aim 1).
For many studies it will be essential to have cells that grow faster than primary fibroblasts and to be able to compare cell lines that differ only in with the PSMC5 P312R mutation. We will use Harvard Medical School’s Cell Biology CRISPR core facility to generate a HCT116 cell line with this mutation. This heterozygous cell line and the wild-type line will allow more rigorous and more efficient biochemical and cell biological studies and will be essential for the screening experiments to be performed by Dr. David Rubinsztein’s laboratory. An important advantage of such cell lines is that they avoid possible complications in interpretation due to other gene differences in the various members of the Silverman and Myers families. Having such cells will also mean that only two lines need to be compared in future studies of the mutation’s effects. In addition to accelerating research, these cell lines will be a valuable resource for future work of other researchers who in the future may build on our studies.
We anticipate establishing this line during the first four months of this study, and during months 5 and 6, examining the behavior of these cells to confirm that they exhibit the same defects as Yoni’s and Ollie’s fibroblast lines (as measured in Aim 1).
For many studies it will be essential to have cells that grow faster than primary fibroblasts and to be able to compare cell lines that differ only in with the PSMC5 P312R mutation. We will use Harvard Medical School’s Cell Biology CRISPR core facility to generate a HCT116 cell line with this mutation. This heterozygous cell line and the wild-type line will allow more rigorous and more efficient biochemical and cell biological studies and will be essential for the screening experiments to be performed by Dr. David Rubinsztein’s laboratory. An important advantage of such cell lines is that they avoid possible complications in interpretation due to other gene differences in the various members of the Silverman and Myers families. Having such cells will also mean that only two lines need to be compared in future studies of the mutation’s effects. In addition to accelerating research, these cell lines will be a valuable resource for future work of other researchers who in the future may build on our studies.
We anticipate establishing this line during the first four months of this study, and during months 5 and 6, examining the behavior of these cells to confirm that they exhibit the same defects as Yoni’s and Ollie’s fibroblast lines (as measured in Aim 1).
In addition to the HCT116 cells (Aim 2), we will also collaborate with Harvard Medical School’s Cell Biology CRISPR core facility to generate SH-SY5Y lines bearing the PSMC5 P312R mutation. SH-SY5Y are a human neuroblastoma cell line that grows quickly but can be stimulated to stop dividing and to differentiate into neurons (Ross et al., 1983). If such a cell line can be established, it will offer important advantages for our studies. It will enable us to determine whether neurons for some reason are especially sensitive to the PSMC5 P312R mutation. Possibly non-proliferating cells (e.g., neurons or cardiac muscle cells) are especially sensitive to impaired proteasomes because they cannot dilute out misfolded proteins, which cannot be degraded due to the PSMC5 mutation, by repeated cell divisions (Erjavec et al., 2008; Lindner et al., 2008; Shcheprova et al., 2008). Also, although the 26S proteasomes are identical in all tissues, other components of the ubiquitin-proteasome system differ in specific ways between cell types and may influence their resistance to proteotoxic stresses; (for example, the proteasome activating protein, ZFAND5, studied below is normally present in high amounts, only in brain and heart (Lee et al., 2018). It is noteworthy in this regard that the most common diseases caused by accumulation of misfolded proteins are neurodegenerative diseases of the elderly (e.g. Alzheimer’s, Parkinson’s diseases, and ALS (Cohen and Kelly, 2003; Selkoe, 2003)), but these pathways are also affected in neurodevelopmental disorders (e.g. certain forms of autism and intellectual disability (Louros and Osterweil, 2016)). We shall study therefore the susceptibility of growing and neuronal SH-SY5Y mutant and control SH-SY5Y cells to the same stressful conditions as in Aim 1 and 2. If the neuronal cells prove to be more sensitive, this cell line will be the focus in subsequent studies in Dr. Rubinsztein’s and our labs.
Because the Harvard Medical School Cell Biology’s CRISPR core has not previously worked with the SH-SY5Y cells, it may take six months to establish lines carrying the mutation. We therefore assume that characterization of the proliferating neuronal cell lines will then be carried out during months 7 – 10, but perhaps earlier.
- B) Studies to Understand the Cellular Mechanisms Causing Pathology
It will be very important to determine how a mutation in just one copy of a proteasome subunit gene leads to these major cellular problems. Two general mechanisms exist. (1) Having a defective proteasome subunit could mean that Ollie’s and Yoni’s cells are functioning with only half the normal proteasomes. Geneticists call this a loss-of-function mutation, and it means that half the proteasomes in their cell lines (i.e., ones bearing the mutation) are inactive. (2) Or, these defective proteasome subunits may compromise the function of proteasomes which have normal subunits. If so, one may be able to improve overall cellular proteasome function by removing the PSMC5 P312R from cells. Geneticists call this a dominant-negative mutation.
If the P312R mutation causes a loss-of-function, then adding back an extra copy of the wild-type proteasome gene should rescue this genetic defect, and the cells would not be as sensitive to heat shock or to proteasome inhibitors and would have normal levels of ubiquitin. In collaboration with Dr. Lior Zangi, we shall attempt to reverse the effects of the PSMC5 P312R mutation by adding back the wild-type (normal) gene using mod-RNAs. In such studies, we would need to check whether these newly added proteasome subunits are being incorporated into the fully assembled proteasome particles
(which will require a chemical tag on the PSMC5 gene encoded in the mod-RNA) and to check if they are working properly (by measuring 26S peptidase activity in the lysate). Expected time frame: months 1 – 4.
Alternatively, if the P312R mutation acts by a dominant-negative mechanism. We shall attempt to mimic the deleterious effects of the PSMC5 P312R mutation by expressing the PSMC5 P312R mutation in wild-type cells with Dr. Zangi’s mod-RNAs. As above, in such studies, it will also be necessary to confirm that these mod-RNA-encoded proteasome subunits are being incorporated into the proteasomes and are enabling the proteasomes to function. (Expected time frame: 1 – 4 months).
If the mod-RNAs indicate that PSMC5 P312R is a loss-of-function mutation or if the mod-RNA subunits are not incorporated into the proteasomes, we shall analyze the loss of function by using siRNAs directed against only the wild-type mutant PSMC5. On-the-other hand, if the PSMC5 P312R mutation is a dominant-negative mutation, we will try to specifically inhibit the expression of the altered allele using RNA inhibition. If we can successfully target the mutated gene, we will test if the sensitivity of these cells to heat shock and proteasome with PSMC5 P312R knocked down (Expected time frame: months 4 – 6).
It is well established that yeast cells bearing mutations in different proteasome subunits can survive and proliferate by producing increased amounts of proteasomes, even if they are defective in certain respects. Similarly, human cells respond to drugs that block proteasome function by making more proteasomes. This compensatory synthesis of new proteasomes involves the transcription factor, Nrf1, whose activation we have methods to monitor easily (Sha and Goldberg, 2014). (In fact, we have found this Nrf1-dependent production of new proteasomes in a mouse model of a neurological disease where proteasomes have reduced activity (VerPlank et al., 2018)). So, we shall test if Ollie’s and Yoni’s cells may be surviving through such a compensatory mechanism. It is also possible that Yoni’s cells may have compensated in this way although Ollie’s cells may have not done so, which if true could perhaps account for their different developmental histories.
In addition, if their mutant cell lines have not adapted by increasing their proteasome content, we shall test if increasing by genetic manipulation the amount of proteasome in the cell can restore the cellular phenotype to normal. The over-expression of Nrf1 or also of two specific proteasome subunits, PSMB5 and PSMD11 have been shown to increase the total number of proteasomes in cells (Lu et al., 2014; Vilchez et al., 2012). We will test if mod-RNAs of PSMB5 or PSMD11 increase the number of proteasomes in the fibroblast, HCT116, and SH-SY5Y cell lines, and if by doing so, it may be possible to help protect Ollie’s and Yoni’s cells against heat shock, proteasome inhibitors, or other proteotoxic stresses. (Expected time frame: months 7 – 10).
These studies will attempt to define further the cellular consequences caused by the PSMC5 mutation. Our prior studies identified two conditions that increase the workload on the proteasome and reveal the defects caused by the PSMC5 P312R mutation: heat shock and proteasome inhibitors. Building on these findings, we shall test if the PSMC5 P312R mutation also makes Yoni’s and Ollie’s cells more sensitive to misfolded or damaged proteins than those of their parents and siblings. (Once they are available, such comparisons of the HCT116 or the SH-SY5Y wild-type and mutant lines, will be more informative and easier to study). In order to develop better screening techniques to be used by Dr. Rubinsztein’s laboratory in their efforts to find suppressor mutations or drug candidates. We shall investigate the
sensitivity of the cells bearing the mutation to other proteotoxic conditions. We will compare the viability of the fibroblast and CRISPR mutation lines with arsenite (which causes free radical damage to proteins), with the amino acid analog canavanine (which replaces arginine in proteins and interferes with normal protein folding), and upon inhibition of the protein-folding chaperone Hsp70. In each case, we shall test if these treatments selectively impact growth and viability of cells expressing the PSMC5 mutation. (Expected time frame: months 4 – 7).
Many neurodegenerative diseases (e.g., Alzheimer’s and Parkinson’s Diseases and ALS, and certain cardiac and muscular diseases) are due to the accumulation of aggregation-prone proteins (Cohen and Kelly, 2003; Selkoe, 2003), which impair proteasomes (Deriziotis et al., 2011; Thibaudeau et al., 2018). We will test the viability of CRISPR mutation lines after transfection of mutant Tau, SOD1, FUS, and crystallin and measure whether over expression of these disease-associated proteins leads to decreased growth of the mutant cells and a greater build-up of these toxic proteins in the PSMC5 mutant lines than in wild-type cells. Aside from their mechanistic interest, such studies may suggest potential disease-susceptibilities for the families to be aware of. (Expected time frame: months 8 – 10).
Much of the research proposed here assumes that the PSMC5 mutation reduces protein degradation rates. Yet, it will be valuable to define how much the PSMC5 P312R mutation disrupts protein. We will measure rates of degradation of cell proteins in the HCT116 CRISPR line (or SH-SY5Y if it displays more exaggerated effects) using radioactive amino acids to label cell proteins and following their degradation back to the constituent amino acids. We will initially measure the degradation of long-lived proteins, which comprise the bulk of cell proteins, both at normal temperatures (37°C) and increased temperatures (e.g., during heat shock) where defects are more pronounced. These are methods our lab uses routinely. Such quantitative information will also provide a basis for the studies below on whether different drugs or suppressor mutations can enhance the capacity of the mutant cells to degrade proteins. (Expected time frame: months 7 – 8).
We shall first measure the degradation of the long-lived cell proteins which comprise the bulk of the cell’s protein content. However, many of the proteins that biologists consider “most interesting and more important” are rapidly degraded regulatory proteins. We will also measure the ability of these cells to degrade short-lived proteins and degradation of several specific proteins that have important regulatory functions (e.g., HIF1α or p53) and reporter substrates of the proteasome. (Expected time frame: months 9 – 12).
To really understand how the PSMC5 P312R mutation impairs protein degradation, it will be necessary to study further how this mutation changes the enzymatic activities of purified proteasomes. Because this mutation is in one of the proteasomes’ six ATP-consuming subunits (Collins and Goldberg, 2017), we shall compare how rapidly ATP is hydrolyzed by the mutant and wild-type proteasomes both at rest, and when proteasome function is stimulated by binding of a ubiquitinated protein substrate. In addition, we have identified two types of proteins that can stimulate 26S proteasome activities: proteins containing a UBL-domain (Collins and Goldberg, 2020; Kim and Goldberg, 2018) and ZFAND5 (which binds to proteasomes and activates their function in heart, brain, and atrophying muscles (Lee et al., 2018)). We will also measure the rates of degradation of pure ubiquitinated protein substrates (e.g., Sic1, ODC-RFP,
and Cyclin B) by the purified proteins. Our lab has developed methods for affinity purification of 26S proteasomes and for assaying these enzymatic reactions. Such studies will rigorously define the relative efficiencies of mutant and wild-type. They will also test if proteins that stimulate proteasomes’ function can enhance the ATPase activity and may even have therapeutic potential. Aside from further defining the molecular basis for Ollie’s and Yoni’s developmental problems, these experiments may also clarify further the 26S proteasomes biochemical mechanisms. (Estimated time frame: 11 – 16)
- C) Testing Possible Pharmacological Treatments to Enhance Proteasome Function
Building upon insights from the studies described above, the genetic screens and the screens of drug libraries in Dr. Rubinsztein’s lab will be aimed at finding agents that can suppress the deleterious effects of the PSMC5 mutation. In this regard, our laboratory has recently described several mechanisms that organisms have evolved to stimulate proteasome activity and enhance the cell’s capacity for protein degradation under specific conditions (Lokireddy et al., 2015; VerPlank et al., 2019; VerPlank et al., 2020). These pathways can be activated by drugs now widely used to treat other diseases. Building on these findings, we shall test if these drugs have the capacity to enhance proteasome function in Yoni and Ollie’s fibroblasts, cells as well as CRISPR-derived neural cells. We have found that agents that raise the cellular content of cAMP (i.e., activators of adenylate cyclase and inhibitors of PDE4) through protein kinase A modify the 26S proteasome and increase its activity. In addition, we recently showed that agents that raise levels of cGMP (stimulators of guanylate cyclase and inhibitors of PDE5) through protein kinase G activate the proteasome, stimulate ubiquitination, and promote cellular protein breakdown. A number of FDA-approved drugs can raise cAMP or cGMP, and we shall test if they also activate the mutant proteasomes’ activity and can enhance overall protein degradation by the ubiquitin-proteasome pathway in cells bearing the PSMC5 P312R mutation as they do in wild-type cells. If so, it will be of particular interest then to test if these treatments can also help protect the cells from the lethal consequences of increased temperatures and other proteotoxic stressors. (Expected time frame: months 11 – 17).
Our cells contain several mechanisms by which they adapt to the presence of misfolded proteins and thus can withstand their toxic effects (Balch et al., 2008). Three mechanisms, which involve expression of a set of stress-related genes, may be allowing Ollie’s and Yoni’s cells to function relatively normally despite their mutation. Alternatively, the induction of these responses might help increase their cells’ resistance to the proteotoxic stress. Therefore, we shall analyze if Ollie’s and Yoni’s cells may have induced these protective mechanisms to adapt to their PSMC5 mutation. For example, our cells express a specific set of genes in response to an accumulation of misfolded proteins in the cytosol (the “Heat Shock Response”) or to misfolded membrane or secreted proteins (the “Unfolded Protein Response”). Drugs that block proteasomal function can elicit both these protective responses (Bush et al., 1997). We shall assay the expression of genes characteristic of these responses under basal growth conditions, and under the proteotoxic stress conditions identified earlier. (Expected time frame: months 12 – 17). We also know that if such stressful conditions are prolonged the cells can trigger a programmed cell death response (i.e., apoptosis (Walter and Ron, 2011)). Therefore, it will be important to learn if the characteristic signs of apoptosis are elicited more easily in cells bearing the PSMC5 mutation.
- D) Future Studies – (To Be Undertaken After Obtaining Outside Grant Support)
In the future we hope to build upon insights gained in these studies, to determine, through collaborations with European investigators, whether the cells from patients with mutations in other proteasome subunits show similar defects in vitro in dealing with proteotoxic stresses (e.g., heat shock and proteasome inhibitors). Also, if we are able to identify treatments that increase proteasome function in the PSMC5 P312R mutant cells, we would hope to learn if these agents may also help protect cells of patients bearing other proteasome mutations from proteotoxic stress. At least 70 other patients have now been reported, in the published literature or in the GeneMatcher database, to have mutations in 15 of the 18 subunits in the 19S proteasome regulatory complex (like PSMC5). Thus, Yoni and Ollie are the tip of the iceberg of individuals with proteasome mutations, who vary in their degree of intellectual disability, and may differ in the capacity of their proteasomes to degrade proteins. The cell lines of these patients should also be analyzed using the selective conditions we have found, and the possible beneficial effects of genetic or pharmacological treatments described above should be examined.
Furthermore, we believe that insights gained in studies of Yoni and Ollie’s cells may also help us understand other diseases that may involve a problem in protein homeostasis or in which the cells are producing large amounts of misfolded proteins (such as Alzheimer’s Disease due to misfolded tau) or even Down’s Syndrome due to the trisomy of chromosome twenty-one. It is quite possible that the assays and screens developed here may also be useful in clarifying the mechanisms of these other proteotoxic diseases and will be used by others to accelerate progress in those areas.
Our Research Timeline
Funds Needed for this Research
The funds required for this research will be put forth directly towards the Harvard and University of Cambridge labs. The main purpose of these funds will be to positively treat not just Ollie and Yoni, but also to those other children who have PSMC5 gene mutation challenges.
