Daniel K. Borger

A new glucocerebrosidase chaperone reduces α-synuclein and glycolipid levels in iPSC-derived dopaminergic neurons from patients with gaucher disease and parkinsonism

Among the known genetic risk factors for Parkinson disease, mutations in GBA1, the gene responsible for the lysosomal disorder Gaucher disease, are the most common. This genetic link has directed attention to the role of the lysosome in the pathogenesis of parkinsonism. To study how glucocerebrosidase impacts parkinsonism and to evaluate new therapeutics, we generated induced human pluripotent stem cells from four patients with Type 1 (non-neuronopathic) Gaucher disease, two with and two without parkinsonism, and one patient with Type 2 (acute neuronopathic) Gaucher disease, and differentiated them into macrophages and dopaminergic neurons. These cells exhibited decreased glucocerebrosidase activity and stored the glycolipid substrates glucosylceramide and glucosylsphingosine, demonstrating their similarity to patients with Gaucher disease. Dopaminergic neurons from patients with Type 2 and Type 1 Gaucher disease with parkinsonism had reduced dopamine storage and dopamine transporter reuptake. Levels of α-synuclein, a protein present as aggregates in Parkinson disease and related synucleinopathies, were selectively elevated in neurons from the patients with parkinsonism or Type 2 Gaucher disease. The cells were then treated with NCGC607, a small-molecule noninhibitory chaperone of glucocerebrosidase identified by high-throughput screening and medicinal chemistry structure optimization. This compound successfully chaperoned the mutant enzyme, restored glucocerebrosidase activity and protein levels, and reduced glycolipid storage in both iPSC-derived macrophages and dopaminergic neurons, indicating its potential for treating neuronopathic Gaucher disease. In addition, NCGC607 reduced α-synuclein levels in dopaminergic neurons from the patients with parkinsonism, suggesting that noninhibitory small-molecule chaperones of glucocerebrosidase may prove useful for the treatment of Parkinson disease. SIGNIFICANCE STATEMENT Because GBA1 mutations are the most common genetic risk factor for Parkinson disease, dopaminergic neurons were generated from iPSC lines derived from patients with Gaucher disease with and without parkinsonism. These cells exhibit deficient enzymatic activity, reduced lysosomal glucocerebrosidase levels, and storage of glucosylceramide and glucosylsphingosine. Lines generated from the patients with parkinsonism demonstrated elevated levels of α-synuclein. To reverse the observed phenotype, the neurons were treated with a novel noninhibitory glucocerebrosidase chaperone, which successfully restored glucocerebrosidase activity and protein levels and reduced glycolipid storage. In addition, the small-molecule chaperone reduced α-synuclein levels in dopaminergic neurons, indicating that chaperoning glucocerebrosidase to the lysosome may provide a novel therapeutic strategy for both Parkinson disease and neuronopathic forms of Gaucher disease.

Lysosomal storage and impaired autophagy lead to inflammasome activation in Gaucher macrophages.

Gaucher disease, the inherited deficiency of lysosomal glucocerebrosidase, is characterized by the presence of glucosylcer‐amide macrophages, the accumulation of glucosylceramide in lysosomes and the secretion of inflammatory cytokines. However, the connection between this lysosomal storage and inflammation is not clear. Studying macrophages derived from peripheral monocytes from patients with type 1 Gaucher disease with genotype N370S/N370S, we confirmed an increased secretion of interleukins IL‐1β and IL‐6. In addition, we found that activation of the inflammasome, a multiprotein complex that activates caspase‐1, led to the maturation of IL‐1β in Gaucher macrophages. We show that inflammasome activation in these cells is the result of impaired autophagy. Treatment with the small‐molecule glucocerebrosidase chaperone NCGC758 reversed these defects, inducing autophagy and reducing IL‐1β secretion, confirming the role of the deficiency of lysosomal glucocerebrosidase in these processes. We found that in Gaucher macrophages elevated levels of the autophagic adaptor p62 prevented the delivery of inflammasomes to autophagosomes. This increase in p62 led to activation of p65‐NF‐kB in the nucleus, promoting the expression of inflammatory cytokines and the secretion of IL‐1β. This newly elucidated mechanism ties lysosomal dysfunction to inflammasome activation, and may contribute to the massive organomegaly, bone involvement and increased susceptibility to certain malignancies seen in Gaucher disease. Moreover, this link between lysosomal storage, impaired autophagy, and inflammation may have implications relevant to both Parkinson disease and the aging process. Defects in these basic cellular processes may also provide new therapeutic targets.

Induced pluripotent stem cell models of lysosomal storage disorders

ABSTRACT Induced pluripotent stem cells (iPSCs) have provided new opportunities to explore the cell biology and pathophysiology of human diseases, and the lysosomal storage disorder research community has been quick to adopt this technology. Patient-derived iPSC models have been generated for a number of lysosomal storage disorders, including Gaucher disease, Pompe disease, Fabry disease, metachromatic leukodystrophy, the neuronal ceroid lipofuscinoses, Niemann-Pick types A and C1, and several of the mucopolysaccharidoses. Here, we review the strategies employed for reprogramming and differentiation, as well as insights into disease etiology gleaned from the currently available models. Examples are provided to illustrate how iPSC-derived models can be employed to develop new therapeutic strategies for these disorders. We also discuss how models of these rare diseases could contribute to an enhanced understanding of more common neurodegenerative disorders such as Parkinson’s disease, and discuss key challenges and opportunities in this area of research. Summary: This Review discusses how induced pluripotent stem cells (iPSCs) provide new opportunities to explore the biology and pathophysiology of lysosomal storage diseases, and how iPSCs have illuminated the role of lysosomes in more common disorders.

Varied autopsy findings in five treated patients with Gaucher disease and parkinsonism include the absence of Gaucher cells

Enzyme replacement therapy is standard of care for patients with Gaucher disease, as it significantly improves skeletal, visceral, and hematological symptoms. Few pathological studies have documented the extent of pathological findings in treated patients. Autopsy findings in five treated patients, who ultimately developed parkinsonism, ranged from the complete absence of Gaucher pathology to extensive involvement of multiple tissues, without correlation to age, genotype, spleen status, or dose/duration of therapy. Additional autopsies may elucidate modifiers and biomarkers contributing to disease burden and response to therapy.

Efferocytosis is impaired in Gaucher macrophages

Gaucher disease, the inherited deficiency of lysosomal glucocerebrosidase, is characterized by the presence of glucosylceramide-laden macrophages resulting from impaired digestion of aged erythrocytes or apoptotic leukocytes. Studies of macrophages from patients with type 1 Gaucher disease with genotypes N370S/N370S, N370S/L444P or N370S/c.84dupG revealed that Gaucher macrophages have impaired efferocytosis resulting from reduced levels of p67phox and Rab7. The decreased Rab7 expression leads to impaired fusion of phagosomes with lysosomes. Moreover, there is defective translocation of p67phox to phagosomes, resulting in reduced intracellular production of reactive oxygen species. These factors contribute to defective deposition and clearance of apoptotic cells in phagolysosomes, which may have an impact on the inflammatory response and contribute to the organomegaly and inflammation seen in patients with Gaucher disease.

Applications of iPSC-derived models of Gaucher disease

Gaucher disease (GD) is an autosomal recessive disorder caused by loss-of-function mutations in the GBA1 gene, which codes for the lysosomal hydrolase glucocerebrosidase (GCase). GCase deficiency leads to accumulation of un-metabolized glycolipid substrates, primarily in cells of the macrophage lineage. GD usually manifests with visceral, hematological, and skeletal involvement, and common symptoms include hepatosplenomegaly, anemia, thrombocytopenia, and osteopenia. More severe enzyme deficiency can also lead to neuronal glycolipid accumulation and central nervous system symptoms.