New Therapeutic Targets … Ricardo A. Feldman PhD

Stem cell research uncovers disease mechanisms and new therapeutic targets for Gaucher disease.

“Our laboratory is very grateful to the Children’s Gaucher Research Fund (CGRF) for the generous funding that makes it possible for our laboratory to carry out this research.”

Gaucher disease (GD) is a lysosomal storage disorder caused by mutations in GBA1, a gene that encodes the lysosomal enzyme glucocerebrosidase. The disease affects individuals who inherit two copies of the mutated gene, one from each parent.  The GBA1 enzyme 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.

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 higher risk of developing Parkinson’s disease.  Types 2 and 3 GD are caused by severe mutations in GBA1, and in addition to the organs mentioned above there is severe neurological involvement.  Types 2 and 3 GD are known as neuronopathic GD (nGD). 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.

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 the mutant enzyme.  Another approach 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 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 Parkinson’s disease, and for Fabry disease.

A new experimental model for elucidating disease mechanisms and developing new treatments for GD.  A major difficulty to develop new treatments for GD is that the affected tissues, in particular human neurons, are usually not available.  To circumvent this problem, our laboratory has 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.  iPSC are also 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.

Mutant GBA1 interferes with basic cellular functions and normal development.  When we differentiated patient-derived mutant iPSC into blood cells, bone-forming cells, and neurons, we found that all of these cell types had functional abnormalities that reflect clinical manifestations in GD patients.  GD iPSC-derived macrophages had a striking delay in clearance of ingested red blood cells.  This recapitulated a hallmark of GD in which pathological macrophages containing remnants of red blood cells accumulate in bone marrow, liver and spleen, resulting in enlarged organs unable to function properly.  Other types of blood cells were also affected, recapitulating the anemias seen in GD.  iPSC-derived osteoblasts, which are responsible for bone deposition, exhibited developmental and lysosomal defects that interfered with their bone-forming ability.  These osteoblast abnormalities are likely to contribute to the osteoporosis seen in type 1 GD patients.  iPSC-derived neuronal cells also had lysosomal and developmental abnormalities, which are described below.

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 neurodevelopmental network.  We found that neuronopathic GD mutations cause developmental defects 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 rescue neurodevelopmental defects caused by GBA1 mutations.  Consistent with our findings, pharmacological activation of the Wnt pathway reversed the neurodevelopmental defects of nGD progenitors.  Incubation of the mutant cultures with an activator of the Wnt pathway protected b-catenin from degradation.  Importantly, this treatment restored the generation of dopaminergic and other affected neurons in the mutant cultures, showing that pharmacological activators of Wnt/b catenin signaling can bypass the GBA1-induced block in neuronal differentiation.  These studies identify Wnt as a potential therapeutic target to help prevent or ameliorate the neurodevelopmental abnormalities caused by mutant GBA1.  These findings also suggest that treatment of types 2 and type 3 GD 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.  We think that this mature stage is too late for therapeutic intervention, as the neurodevelopmental block caused by mutant GBA1 occurs very early during neuronal development.

mTOR inhibitors restore lysosomal functions affected by nGD.  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.  Because of these clearing functions, autophagy is essential for neuronal survival.  Hyperactivation of mTOR in neuronal cells from patients with nGD 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 Parkinson’s disease, can restore lysosomal functions affected by mutant GBA1.  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 nGD.

Glucosylceramide synthase inhibitors rescue lysosomal function and Wnt/b catenin signaling.  When we tested SRT drugs (glucosylceramide synthase inhibitors) we found that Eliglustat, Gz667161, and Ibiglustat, were able to prevent mTOR hyperactivation and concomitantly, restore lysosomal functions in nGD 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.  As mentioned above, Ibiglustat, a brain-penetrant glucosylceramide inhibitor is in clinical trials for type 3 GD and GBA1-associated Parkinson’s disease.

Acid Ceramidase inhibitors.  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.  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 ameliorate nGD neuronopathy.

Modeling nGD in a 3-D organoid system.  We are now using a self-assembling brain organoid system derived from iPSC to recapitulate the neurodevelopmental defects of nGD in a 3-dimensional system.  Using brain organoids we will precisely map where the developmental abnormalities in nGD occur.  We will also use this system to test candidate drugs for their effectiveness in reversing the neurodevelopmental defects caused by mutant GBA1.

In sum, our disease-in-a-dish model of GD has enabled us to rapidly uncover disease mechanisms and identify new therapeutic targets.  We believe this work will lead to effective therapies for nGD and GBA1-associated Parkinson’s disease.

Ricardo A. Feldman PhD
Associate Professor
Department of Microbiology and Immunology
University of Maryland School of Medicine
Baltimore, Maryland