Written by: Kaitlyn Sadtler
Written By: Sravya Kotaru
Original article: Yi et al. Human Molecular Genetics 2019
The Gist of It:
Familial or early-onset Parkinson’s disease occurs when individuals inherit harmful changes or mutations in certain genes. A common gene mutated in familial Parkinson’s disease contains the information to produce a protein called Parkin. Parkin gets rid of damaged mitochondria (the cellular compartments that generate energy for a cell to live). If Parkin fails to function properly, a buildup of damaged mitochondria can eventually lead to cell death. When Parkin failure leads to loss of nerve cells controlling skeletal muscles, one loses proper control over body movements and develops Parkinson’s disease, with common symptoms including tremors and stiffness.
Imagine Parkin (or, in fact, any protein) as a tiny machine with different parts that work together to do a job. Mutations can occur in different parts (or domains) of Parkin, each affecting a different coordinated step in Parkin’s function. Some mutations have a minor effect and can be tolerated without complete loss of function – these are called benign (i.e. harmless). Others can be debilitating, causing total failure of Parkin function – these are called pathogenic (i.e. harmful and disease-causing). The best way to ‘fix’ mutant Parkin and treat disease is to identify which pathogenic mutation a patient has and tailor treatment to counteract that specific mutation.
The first step to achieving this kind of customized treatment for Parkin mutations is knowing which are pathogenic and what step of function they interfere with. In this study, researchers classified 215 different Parkin mutations into five categories – pathogenic, likely pathogenic, likely benign, benign, and uncertain significance. This was based on whether each individual mutation causes disease in patients or is tolerated in healthy individuals. They used computer modeling to study the effect of each mutation on Parkin’s structure to determine which step of Parkin’s function was altered, if any. For example, some mutations kept parts (domains) of Parkin from doing their job, some kept Parkin from interacting with partner proteins, whereas some others didn’t have any obvious effect. But the most interesting mutations were those that, when present in combination with a pathogenic mutation, could actually counteract the pathogenic effects of the first mutation and recover normal function. This work opens up the possibility of using drugs that mimic such rescue mutations as a treatment for patients that have the corresponding pathogenic mutation, a.k.a. personalized therapy.
The Nitty Gritty:
Mutations in the E3 ubiquitin ligase Parkin are a leading cause of early-onset or familial Parkinson’s disease, as they interfere with Parkin’s ability to orchestrate the clearance of damaged mitochondria through mitophagy. Most common Parkin mutations are missense and affect function without altering expression, serving as ideal candidates for genotype (mutation)-specific treatment. A prerequisite for such a customized treatment is differentiating pathogenic mutations from non-pathogenic ones. In this study, the authors classified 215 observed Parkin missense mutations into five clinical categories using the Sherloc framework – pathogenic, likely pathogenic, likely benign, benign, unknown significance. This framework uses data from various Parkinson’s disease databases and other exome databases, accounting for segregation within affected families as well as allele frequencies and homozygosity in healthy and diseased individuals. In addition, they also categorized the mutations based on protein stability and mitophagy activity of mutants compared with wild-type Parkin in cell-based assays. They found that clinically pathogenic or likely pathogenic mutations had severely reduced mitophagy and either lower or normal protein levels. Alternatively, clinically benign and likely benign mutations had moderate to no effect on mitophagy with normal protein levels. Moreover, modeling these mutations onto known Parkin structures in different stages of activation indicated that pathogenic mutations often introduce severe steric clashes in various binding and active sites, unlike the benign mutations that only cause minor steric hindrance. An additional group of missense mutations was identified that increased mitophagy compared to wild-type and could rescue some pathogenic Parkin mutations when introduced in cis. Structural modeling showed that these hyperactive mutations relieved the impact of a steric clash from the pathogenic mutations and allowed the necessary protein interactions and structural changes for Parkin activation and function. For example, R234Q and R256C destabilize the RING0:REP interface, whereas M458L destabilizes the RING0:RING2 interface; all of them favoring the Parkin active state.
In conclusion, using a combination of cell-based assays and structural studies predicted the pathogenicity of a mutation. Developing drugs that mimic distinct hyperactive mutations in cis to pathogenic mutations can lead to customized genotype-specific therapies for patients with Parkinson’s disease.
Original Research Article: Yi, W., et.al. “The landscape of Parkin variants reveals pathogenic mechanisms and therapeutic targets in Parkinson’s disease.” Hum Mol Genet 28.17 (2019): 2811–2825.
Written By: Abel B. Cortinas
Original Article: Cheng et al. Diabetes 2019
The Gist of It:
Diabetes is a debilitating disease that affects nearly 1 in 10 individuals in the United States (American Diabetes Association, 2015) and nearly 1 in 18 individuals globally (International Diabetes Federation, 2017). Unfortunately, amongst diabetics, one of the most common complications is known as diabetic peripheral neuropathy, sometimes abbreviated as “DPN.” DPN leads to nerve damage that can result in a number of symptoms including numbness, loss of feeling or sensation, and sometimes pain in your feet, legs, or hands. DPN is also associated with an increased susceptibility to foot and ankle fractures and ulcerations, as well as lower-limb amputations. There is currently no disease-modifying treatment or therapy for DPN, which means that the drugs that are available (for example, calcium ion channel inhibitors such as pregabalin, serotonin / norepinephrine reuptake inhibitors such as duloxetine, as well as opioids such as tapentadol) are meant only to help alleviate the many symptoms but don’t help cure the source of DPN. To learn more about the underlying causes of DPN, researchers at the Chinese Academy of Sciences and Shanghai Tech University studied the gene Sarm1, which is known to be responsible for axon degeneration, or put simply the breakdown of nerves that is a key feature of peripheral neuropathies. The researchers wanted to understand the role of Sarm1 in reducing nerve damage, specifically axonal degeneration, as well as the implications on alleviating DPN. Cheng and colleagues uncovered a number of interesting findings. Particularly, diabetic mice that had the Sarm1 gene selectively deleted had alleviated sensitivity to pain, less nerve fiber loss in their feet, and reduced rates of axon degeneration compared with diabetic mice that had the Sarm1 gene intact. Together, these findings support the potential role of the Sarm1 gene and the resulting protein in DPN and highlight a potentially novel strategy for the treatment of DPN either through inhibition of SARM1 or gene silencing / editing.
The Nitty Gritty:
Using a streptozotocin (STZ)-induced diabetic mouse model in animals with a Sarm1-/- gene knockout and with disruptions of exons 3–6, Cheng et al. found that these mice exhibited normal pain sensitivity and intraepidermal nerve fiber density in the footpad comparable to that of wild type mice. Additionally, the Sarm1-deficient diabetic mice were rescued from the modulation of thermal and mechanical pain sensitivity found in STZ-induced diabetic mice with the Sarm1 gene intact when subjected to the hot plate, von Frey filament, as well as tail immersion behavioral tests. Building on this finding, Sarm1 gene deficiency was further studied to determine its protective effects with regard to diabetic peripheral neuropathy (also known as “DPN”). Sarm1 gene deficiency was found to successfully alleviate diabetes-induced intraepidermal nerve fiber loss in diabetic mice. Next, the Sarm1 gene and its protective effects on diabetic axon regeneration were investigated. In an experiment that studied the effects of Sarm1 gene ablation on diabetic neuronal degradation, Sarm1 knockout mice had significantly alleviated diabetes-induced axon degeneration and change of the g-ratio (which is a measurement of myelin sheath thickness and generally considered to be indicative of nerve conduction capacity) as well as a decrease in NAD+ in the sciatic nerve. Lastly, the deletion of Sarm1 also diminished the STZ-induced changes in the gene expression profile of the sciatic nerve. Taken together, Sarm1 gene deficiency provided strong evidence to support the targeting of axon degeneration as a potentially promising strategy to combat DPN.
Original Research Article: Cheng, Y., et al. “Sarm1 gene deficiency attenuates diabetic peripheral neuropathy in mice.” Diabetes (2019). doi: 10.2337/db18-1233
Written by: Abby Stahl
Original Article: Sohn et al. Neuron 2019
The Gist of It
Frontotemporal dementia (FTD), also known as Pick’s disease, occurs when cells in the front and sides of the brain die or become dysfunctional. The disease can cause changes in behavior and personality as well as difficulties with understanding or producing language. FTD is typically diagnosed in individuals between 40–60 years of age and is inherited in a third of all cases. One specific change, or mutation, in a protein called Tau has been identified as a major cause of FTD. Scientists wanted to know how this mutated Tau protein could affect the electrical signals that brain cells use to communicate. To answer this, they looked at areas called axon initial segments (AIS), a part of the exterior barrier of the cell that is essential in controlling the sending of these electrical signals. They compared the AIS in brain cells taken from healthy patients with those taken from patients with FTD. The researchers found that brain cells from patients with FTD have a shortened AIS and send electrical signals erratically. How does this happen? The team discovered that mutant Tau binds to another protein called EB3 more strongly than expected and brings it to the AIS; having the extra EB3 at the AIS causes the cell to become more rigid and less flexible, preventing the brain cell from sending electrical signals properly. When the amount of EB3 or mutant Tau was decreased in the cell, the AIS length was extended, and electrical signals returned to normal. This study nicely demonstrated how mutant Tau protein can stop the brain from signaling and identified potential new targets for FTD treatment.
The Nitty Gritty
As signals enter neurons, they are integrated at the axon initial segment (AIS). If the strength of the signal surpasses a given threshold, the cell undergoes depolarization and emits an electrical signal down the axon. Interestingly, the AIS becomes physically shorter during depolarization, which can be measured by tracking the length of an associated protein called Ankyrin G (AnkG). In patients with FTD, mutant Tau is thought to impact the stability of AnkG at the AIS; however, the mechanism is unknown.
To study brain signaling ex vivo, researchers collected skin cells from patients with Tau mutation V337M and transformed them into induced pluripotent stem cells, then differentiated them into cortical neurons. Furthermore, the scientists made “isogenic control cells” from the same patient cells, in which they corrected the Tau mutation with CRISPR/Cas9 gene editing, so they could compare wild type and mutant Tau phenotypes within the same genetic background. Interestingly, the authors noticed that AnkG was shorter in cells with mutant Tau compared with healthy and isogenic control cells. When they added potassium chloride (KCl) to induce depolarization, AnkG could not get any shorter in the mutant cells. To test whether the contraction of the AIS would influence electrical signaling, they used multi-electrode arrays and found “firing rates” were similar, but cells with mutant Tau had more spikes in electrical signal within a given burst of activity and fewer network bursts per minute, meaning they were not functioning normally. To understand exactly how mutant Tau was causing these changes in AnkG length and electrical signaling, the authors studied EB3, which is known to be regulated by Tau and stabilize AnkG in the AIS. Using nuclear magnetic resonance (NMR) spectroscopy, the group found three potential binding sites where Tau could interact with EB3. They found that mutant Tau can bind to EB3 four times more strongly than the wild type Tau! Furthermore, EB3 was dispersed nicely throughout healthy cells but accumulated at the AIS in cells with mutant Tau, suggesting the interactions were playing a role in electrical signaling. Next, they treated cells with a small interfering RNA (siRNA) to block EB3 protein from being made. When EB3 was not available to interact with mutant Tau, AnkG was elongated, and normal electrical signaling was restored. Conversely, when EB3 was overexpressed within healthy cells, AnkG became shorter and did not decrease in size during depolarization, mimicking the diseased state. When siRNA was directed against Tau in mutant cells, EB3 levels decreased in the AIS, AnkG was elongated, and normal electrical signaling was restored. Finally, when Tau was suppressed but EB3 was overexpressed, there was no benefit in the disease cells, definitively showing that the improvements in electrical signaling were made through blocking the interactions of mutant Tau and EB3.
In summary, the authors showed that mutant Tau binds strongly to EB3, causing shortening of AnkG at the AIS. Shortened AnkG prevents normal electrical signals from being fired and likely contributes to the dysfunction of brain cells observed in FTD. Importantly, EB3 may be a new therapeutic target for treating FTD and restoring neuronal plasticity.
Original Research Article: Sohn, P.D., et al. “Pathogenic Tau impairs axon initial segment plasticity and excitability homeostasis.” Neuron (2019). doi: 10.1016/j.neuron.2019.08.008
Written by: Kaitlyn Sadtler