Samarjit Patnaik

Noncanonical TGFβ signaling contributes to aortic aneurysm progression in Marfan syndrome mice.

Transforming growth factor–β promotes aortic aneurysm formation through activation of its “noncanonical” signaling pathway. Transforming growth factor–β (TGFβ) signaling drives aneurysm progression in multiple disorders, including Marfan syndrome (MFS), and therapies that inhibit this signaling cascade are in clinical trials. TGFβ can stimulate multiple intracellular signaling pathways, but it is unclear which of these pathways drives aortic disease and, when inhibited, which result in disease amelioration. Here we show that extracellular signal–regulated kinase (ERK) 1 and 2 and Smad2 are activated in a mouse model of MFS, and both are inhibited by therapies directed against TGFβ. Whereas selective inhibition of ERK1/2 activation ameliorated aortic growth, Smad4 deficiency exacerbated aortic disease and caused premature death in MFS mice. Smad4-deficient MFS mice uniquely showed activation of Jun N-terminal kinase–1 (JNK1), and a JNK antagonist ameliorated aortic growth in MFS mice that lacked or retained full Smad4 expression. Thus, noncanonical (Smad-independent) TGFβ signaling is a prominent driver of aortic disease in MFS mice, and inhibition of the ERK1/2 or JNK1 pathways is a potential therapeutic strategy for the disease.

Discovery, structure-activity relationship, and biological evaluation of noninhibitory small molecule chaperones of glucocerebrosidase.

A major challenge in the field of Gaucher disease has been the development of new therapeutic strategies including molecular chaperones. All previously described chaperones of glucocerebrosidase are enzyme inhibitors, which complicates their clinical development because their chaperone activity must be balanced against the functional inhibition of the enzyme. Using a novel high throughput screening methodology, we identified a chemical series that does not inhibit the enzyme but can still facilitate its translocation to the lysosome as measured by immunostaining of glucocerebrosidase in patient fibroblasts. These compounds provide the basis for the development of a novel approach toward small molecule treatment for patients with Gaucher disease.

Induction and reversal of myotonic dystrophy type 1 pre-mRNA splicing defects by small molecules

The ability to control pre-mRNA splicing with small molecules could facilitate the development of therapeutics or cell-based circuits that control gene function. Myotonic dystrophy type 1 (DM1) is caused by the dysregulation of alternative pre-mRNA splicing due to sequestration of muscleblind-like 1 protein (MBNL1) by expanded, non-coding r(CUG) repeats (r(CUG)exp). Here we report two small molecules that induce or ameliorate alternative splicing dysregulation. The thiophene-containing small molecule (1) inhibits the interaction of MBNL1 with its natural pre-mRNA substrates. Compound (2), a substituted naphthyridine, binds r(CUG)exp and displaces MBNL1. Structural models show that 1 binds MBNL1 in the Zn-finger domain and that 2 interacts with UU loops in r(CUG)exp. This study provides a structural framework for small molecules that target MBNL1 by mimicking r(CUG)exp and shows that targeting MBNL1 causes dysregulation of alternative splicing, suggesting that MBNL1 is thus not a suitable therapeutic target for the treatment of DM1.

Macrophage Models of Gaucher Disease for Evaluating Disease Pathogenesis and Candidate Drugs

Macrophages differentiated from monocytes or induced pluripotent stem cells derived from patients with Gaucher disease facilitate investigation of disease pathogenesis and validation of new candidate drugs. Emptying the Trash Studies of Gaucher disease, caused by a deficiency of the enzyme glucocerebrosidase, have been hindered by the lack of cellular models that show glycolipid accumulation in the lysosomes of macrophages, a hallmark characteristic of the disease. Using blood and skin samples from patients with Gaucher disease, Aflaki et al. now have developed macrophage models that recapitulate characteristics of the disease, including loss of glucocerebrosidase activity, glycolipid accumulation in lysosomes, and impaired macrophage function. Treatment of patient-derived macrophages with a new small-molecule drug corrected the enzyme deficiency, reduced lysosomal storage of lipids, and restored macrophage function. These new cellular models of Gaucher disease should facilitate our understanding of this disorder and the development of new drugs. Gaucher disease is caused by an inherited deficiency of glucocerebrosidase that manifests with storage of glycolipids in lysosomes, particularly in macrophages. Available cell lines modeling Gaucher disease do not demonstrate lysosomal storage of glycolipids; therefore, we set out to develop two macrophage models of Gaucher disease that exhibit appropriate substrate accumulation. We used these cellular models both to investigate altered macrophage biology in Gaucher disease and to evaluate candidate drugs for its treatment. We generated and characterized monocyte-derived macrophages from 20 patients carrying different Gaucher disease mutations. In addition, we created induced pluripotent stem cell (iPSC)–derived macrophages from five fibroblast lines taken from patients with type 1 or type 2 Gaucher disease. Macrophages derived from patient monocytes or iPSCs showed reduced glucocerebrosidase activity and increased storage of glucocerebroside and glucosylsphingosine in lysosomes. These macrophages showed efficient phagocytosis of bacteria but reduced production of intracellular reactive oxygen species and impaired chemotaxis. The disease phenotype was reversed with a noninhibitory small-molecule chaperone drug that enhanced glucocerebrosidase activity in the macrophages, reduced glycolipid storage, and normalized chemotaxis and production of reactive oxygen species. Macrophages differentiated from patient monocytes or patient-derived iPSCs provide cellular models that can be used to investigate disease pathogenesis and facilitate drug development.

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.

Activation of β-Glucocerebrosidase Reduces Pathological α-Synuclein and Restores Lysosomal Function in Parkinson's Patient Midbrain Neurons

Parkinsons disease (PD) is characterized by the accumulation of α-synuclein (α-syn) within Lewy body inclusions in the nervous system. There are currently no disease-modifying therapies capable of reducing α-syn inclusions in PD. Recent data has indicated that loss-of-function mutations in the GBA1 gene that encodes lysosomal β-glucocerebrosidase (GCase) represent an important risk factor for PD, and can lead to α-syn accumulation. Here we use a small-molecule modulator of GCase to determine whether GCase activation within lysosomes can reduce α-syn levels and ameliorate downstream toxicity. Using induced pluripotent stem cell (iPSC)-derived human midbrain dopamine (DA) neurons from synucleinopathy patients with different PD-linked mutations, we find that a non-inhibitory small molecule modulator of GCase specifically enhanced activity within lysosomal compartments. This resulted in reduction of GCase substrates and clearance of pathological α-syn, regardless of the disease causing mutations. Importantly, the reduction of α-syn was sufficient to reverse downstream cellular pathologies induced by α-syn, including perturbations in hydrolase maturation and lysosomal dysfunction. These results indicate that enhancement of a single lysosomal hydrolase, GCase, can effectively reduce α-syn and provide therapeutic benefit in human midbrain neurons. This suggests that GCase activators may prove beneficial as treatments for PD and related synucleinopathies. SIGNIFICANCE STATEMENT The presence of Lewy body inclusions comprised of fibrillar α-syn within affected regions of PD brain has been firmly documented, however no treatments exist that are capable of clearing Lewy bodies. Here, we used a mechanistic-based approach to examine the effect of GCase activation on α-syn clearance in human midbrain DA models that naturally accumulate α-syn through genetic mutations. Small molecule-mediated activation of GCase was effective at reducing α-syn inclusions in neurons, as well as associated downstream toxicity, demonstrating a therapeutic effect. Our work provides an example of how human iPSC-derived midbrain models could be used for testing potential treatments for neurodegenerative disorders, and identifies GCase as a critical therapeutic convergence point for a wide range of synucleinopathies.

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.

Orphan GPR110 (ADGRF1) targeted by N -docosahexaenoylethanolamine in development of neurons and cognitive function

Docosahexaenoic acid (DHA, 22:6n-3) is an omega-3 fatty acid essential for proper brain development. N-docosahexaenoylethanolamine (synaptamide), an endogenous metabolite of DHA, potently promotes neurogenesis, neuritogenesis and synaptogenesis; however, the underlying molecular mechanism is not known. Here, we demonstrate orphan G-protein coupled receptor 110 (GPR110, ADGRF1) as the synaptamide receptor, mediating synaptamide-induced bioactivity in a cAMP-dependent manner. Mass spectrometry-based proteomic characterization and cellular fluorescence tracing with chemical analogues of synaptamide reveal specific binding of GPR110 to synaptamide, which triggers cAMP production with low nM potency. Disruption of this binding or GPR110 gene knockout abolishes while GPR110 overexpression enhances synaptamide-induced bioactivity. GPR110 is highly expressed in fetal brains but rapidly decreases after birth. GPR110 knockout mice show significant deficits in object recognition and spatial memory. GPR110 deorphanized as a functional synaptamide receptor provides a novel target for neurodevelopmental control and new insight into mechanisms by which DHA promotes brain development and function.

A Novel Brain Penetrant NPS Receptor Antagonist, NCGC00185684, Blocks Alcohol- Induced ERK-Phosphorylation in the Central Amygdala and Decreases Operant Alcohol Self- Administration in Rats

The Neuropeptide S receptor, a Gs/Gq-coupled GPCR expressed in brain regions involved in mediating drug reward, has recently emerged as a candidate therapeutic target in addictive disorders. Here, we describe the in vitro and in vivo pharmacology of a novel, selective and brain penetrant NPSR antagonist with nanomolar affinity for the NPSR, NCGC00185684. In vitro, NCGC00185684 shows biased antagonist properties, and preferentially blocks ERK-phosphorylation over intracellular cAMP or calcium responses to NPS. In vivo, systemic NCGC00185684 blocks alcohol-induced ERK-phosphorylation in the rat central amygdala, a region involved in regulation of alcohol intake. NCGC00185684 also decreases operant alcohol self-administration, and lowers motivation for alcohol reward as measured using progressive ratio responding. These effects are behaviorally specific, in that they are observed at doses that do not influence locomotor activity or reinstatement responding following extinction. Together, these data provide an initial validation of the NPSR as a therapeutic target in alcoholism.

Allosteric Inhibitors of the Eya2 Phosphatase Are Selective and Inhibit Eya2-mediated Cell Migration

Background: The phosphatase activity of Eya is important for transformation, invasion, migration, and metastasis of breast cancer cells. Results: A class of N-arylidenebenzohydrazide compounds specifically inhibits the phosphatase activity of Eya2 but not Eya3. Conclusion: This class of compounds likely acts through an allosteric mechanism. Significance: These inhibitors may be developed into chemical probes or anti-cancer drugs. Eya proteins are essential co-activators of the Six family of transcription factors and contain a unique tyrosine phosphatase domain belonging to the haloacid dehalogenase family of phosphatases. The phosphatase activity of Eya is important for the transcription of a subset of Six1-target genes, and also directs cells to the repair rather than apoptosis pathway upon DNA damage. Furthermore, Eya phosphatase activity has been shown to mediate transformation, invasion, migration, and metastasis of breast cancer cells, making it a potential new drug target for breast cancer. We have previously identified a class of N-arylidenebenzohydrazide compounds that specifically inhibit the Eya2 phosphatase. Herein, we demonstrate that these compounds are reversible inhibitors that selectively inhibit the phosphatase activity of Eya2, but not Eya3. Our mutagenesis results suggest that this class of compounds does not bind to the active site and the binding does not require the coordination with Mg2+. Moreover, these compounds likely bind within a site on the opposite face of the active site, and function as allosteric inhibitors. We also demonstrate that this class of compounds inhibits Eya2 phosphatase-mediated cell migration, setting the foundation for these molecules to be developed into chemical probes for understanding the specific function of the Eya2 phosphatase and to serve as a prototype for the development of Eya2 phosphatase specific anti-cancer drugs.