Stem cell research uncovers disease mechanisms and new therapeutic targets for GBA1-associated Parkinson’s disease.
“Our laboratory is very grateful to the Parkinson’s and Brain Research Foundation for the generous funding that makes it possible for our laboratory to carry out this research.”
Nearly two decades ago, Ellen Sidransky’s group at the NIH made the observation of an increased incidence of Parkinson’s disease (PD) in families of individuals with Gaucher disease (GD). The genetic link between GBA1 and PD has been confirmed by analysis of thousands of patients in multiple international studies, and it is now well established that mutations in GBA1 are the highest risk factor for developing PD. It is estimated that mutations in GBA1 increase the risk of developing PD by 5- to 20-fold, and that 7% of all PD patients have mutations in GBA1.
The GBA1 gene encodes the enzyme glucocerebrosidase, a lysosomal enzyme that breaks down lipids called glucocerebrosides. When this enzyme is mutated it does not function properly, and toxic levels of these otherwise normal lipids accumulate in lysosomes, causing damage to the cells. Lysosomes are intracellular organelles involved in the recycle of cellular materials and the disposal of harmful protein aggregates. Individuals who inherit two copies of a mutant GBA1 gene, one from each parent, develop GD. GD has a wide spectrum of clinical manifestations from mild to fatal, depending on the severity of the GBA1 mutation, genetic background, and environmental factors. Mild GBA1 mutations (type 1 GD) cause damage in liver, spleen, bone marrow and bone, but overt neurological involvement is not evident. However, type 1 GD patients are at increased risk of developing Parkinson’s disease. Even healthy carriers of GD, who inherit only one mutated copy of GBA1, are at increased risk of developing Parkinson’s disease. Types 2 and 3 GD, known as neuronopathic GD (nGD), are caused by severe mutations in GBA1. In addition to damage to the organs mentioned above, nGD is characterized by severe neurological involvement. Type 2 GD is a devastating disorder that is fatal within 2 years of life, whereas type 3 GD is a more chronic form of nGD.
An experimental model for elucidating disease mechanisms and developing new treatments for GD and GBA1-associated PD. A major difficulty to develop new treatments for GD and GBA1-associated PD is that the affected tissues, in particular human neurons, are usually not available. To circumvent this problem, we have used a novel reprogramming technology that enabled us to generate induced pluripotent stem cells (iPSC) from skin biopsies of affected patients. iPSC are self-renewing, providing unlimited numbers of patient-derived stem cells for study. Another major advantage of iPSC is that they are capable of differentiating into virtually any cell type, including neurons, in a process akin to biological alchemy. Using this experimental system we identified a number of cellular abnormalities that recapitulate clinical manifestations, and have developed cell-based assays to identify drugs that can reverse the abnormalities caused by GBA1 deficiency.
Identification of new pharmacological targets to treat GBA1-associated neurodegeneration. Work in our laboratory has identified two important effects of GBA1 deficiency that shed light on disease mechanisms: 1) GBA1 deficiency interferes with the Wnt/b-catenin pathway, which is involved in neuronal development, and 2) GBA1 deficiency disrupts the normal functioning of lysosomes through hyperactivation of the mTOR pathway. As described below, these findings have important implications for therapy.
Mutant GBA1 disrupts a major developmental network, uncovering a link between Gaucher and Parkinson’s disease. We found that GBA1 mutations cause the loss of dopaminergic neurons due to interference with the Wnt/b-catenin pathway. This is a signaling network that plays a central role in brain development. GBA1 mutations caused a depletion of neuronal progenitors that are dependent on Wnt activity, including midbrain dopaminergic progenitors. As Wnt signaling is required for the survival of dopaminergic progenitors and mature dopaminergic neurons, the cells that are lost in Parkinson’s disease, these findings provide a mechanistic link between GBA1 mutations and Parkinson’s disease. Our studies showed that mutant GBA1 inhibits Wnt signaling by inducing the degradation of b-catenin, a key component of the Wnt pathway.
Pharmacological Wnt activators restore the generation of midbrain dopaminergic neurons. Consistent with our findings, incubation of the mutant cultures with an activator of the Wnt pathway protected b-catenin from degradation. Importantly, pharmacological Wnt activation prevented the depletion of dopaminergic progenitors, enabling the generation of mature midbrain dopaminergic neurons. These findings identify Wnt as a potential therapeutic target and suggest that Wnt activators may help prevent or delay the onset of PD. In fact, a number of clinical investigators are taking advantage of the efficacy of Wnt activators to increase the yield of clinical-grade dopaminergic neurons derived from pluripotent stem cells. In upcoming clinical trials, these stem cell-derived neurons will be injected into the brains of patients afflicted by Parkinson’s disease. Our studies also suggest that treatment for GBA1-associated neuropathies should start early, before the damage to the nervous system becomes irreversible. This is a novel concept, as much of the work on GBA1-associated neurodegeneration is focused on analyzing mature neurons.
mTOR inhibitors restore lysosomal functions affected by mutant GBA1. A second major effect of GBA1 mutations uncovered in our studies is the hyperactivation of mTOR, an enzyme that regulates metabolism and lysosomal functions including autophagy. Autophagy is a lysosome-dependent process for recycling cellular materials and the removal of harmful protein aggregates of a-synuclein. Because of these clearing functions, autophagy is essential for neuronal survival. Hyperactivation of mTOR in neuronal cells harboring GBA1 mutations caused lysosomal depletion and disruption of autophagy. Using sensitive assays developed in our laboratory we found that mTOR inhibitors, which are already in use to treat epilepsy and certain cancers, and in clinical trials for PD, can restore lysosomal functions in the mutant neurons. In preliminary experiments another drug, Metformin, also prevented mTOR hyperactivation and improved lysosomal function. Metformin is a drug used by millions of people to treat type II diabetes. Further analysis will determine whether mTOR inhibitors and Metformin can help prevent or ameliorate GBA1-associated PD.
Glucosylceramide synthase inhibitors rescue lysosomal function and Wnt/b catenin signaling. Type 1 GD can be successfully treated by periodic infusions of recombinant glucocerebrosidase, but as this enzyme cannot cross the blood-brain-barrier, it cannot prevent the neurological damage caused by GBA1 deficiency. Another approach used to reduce the accumulation of toxic lipids is Substrate Reduction Therapy (SRT). SRT is based on preventing the synthesis of the harmful lipids in the first place. This is accomplished using pharmacological inhibitors of glucosylceramide synthase, the enzyme that is responsible for the synthesis of these lipids. One of these SRT drugs, Eliglustat, has been approved for the treatment of type 1 GD. Ibiglustat, an SRT drug that is accessible to the brain is in Phase II clinical trials for type 3 GD, for GBA1-associated PD, and for Fabry disease. When we tested Eliglustat, and the brain-penetrant Gz667161 and Ibiglustat, we found that these drugs were able to prevent mTOR hyperactivation and restored lysosomal functions in the mutant neurons. SRT compounds were also able to restore Wnt/b-catenin signaling, highlighting the broad effect of SRT in reversing the deleterious effects of mutant GBA1.
Acid Ceramidase inhibitors also restore lysosomal function. More recently, we found that inhibitors of Acid ceramidase, another enzyme involved in the accumulation of neurotoxic lipids, can also protect the mutant neurons. Acid ceramidase is a lysosomal enzyme that converts glucosylceramide, the primary substrate of GBA1 enzyme into glucosylsphingosine. Glucosylsphingosine is a highly neurotoxic lipid that is elevated several hundred-fold in nGD brains, and also builds up during aging and in PD. Treatment of nGD neurons with Carmofur, an inhibitor of Acid ceramidase, reduced mTOR hyperactivation and prevented lysosomal loss. In future work we will explore the therapeutic potential of Acid ceramidase inhibitors to help prevent or treat GBA1-associated PD.
Modeling GBA1-associated PD in a 3-D organoid system. We are now setting up a self-assembling brain organoid system derived from iPSC to recapitulate midbrain development in a 3-dimensional system. Using midbrain organoids we will study neurodevelopmental abnormalities that may contribute to the dopaminergic loss that leads to PD. We will also use this system to test candidate drugs for their effectiveness in preventing the loss of midbrain dopaminergic neurons in GBA1-associated PD.
In perspective, the disease-in-a-dish model of GBA1-associated neuronopathy has enabled us to rapidly uncover disease mechanisms and identify new therapeutic targets. The extent of the neuronal abnormalities observed was dependent on severity of the GBA1 mutation. This may explain why in individuals with type 1 GD and in GD carriers, where the GBA1 mutations they harbor have low penetration, it may take decades for dopaminergic loss to take hold. It should be noted that although GBA1 mutations are a risk factor for PD, only a small percentage of these individuals will develop the disease. This is because with type 1 GD and GD carriers, progression to PD is also dependent on other unknown genetic and environmental factors. As GBA1 mutations are the highest risk factor for PD, the study of GD is helping to understand the mechanisms leading to the far more common Parkinson’s disease. Studies on GD are also guiding drug discovery for PD. This is best illustrated by the ongoing clinical trials for PD using drugs developed for treating GD. Our laboratory is studying both disorders, in the knowledge that what we learn from one disease with help develop effective treatments for the other. As mentioned earlier, another important concept emerging from our studies is that treatment of GBA1-associated PD should start early, before dopaminergic neurons have been significantly depleted. This will require identification of the earliest possible markers of the disease, an endeavor that we are actively pursuing.
Ricardo A. Feldman PhD
Associate Professor
Department of Microbiology and Immunology
University of Maryland School of Medicine
Baltimore, Maryland