IVC / TM / GBA2, Acid ceramidase inhibitor Yuta exec summary, Acid ceramidase / Farber lipogranulomatosis genetics
GBA2
| content | |
|---|---|
| location | At the plasma membrane, cytoplasmic face of the ER and Golgi (ie Non-lysosomal) |
| expression | testis, liver, and brain, in particular in Purkinje cells |
| mutations in the GBA2 gene | the ataxia-spasticity spectrum |
| function | cleaves the same natural and artificial substrates as GBA |
| Inhibition by | N-butyldeoxygalactonojirimycin (NB-DGJ), inhibits GBA2 but does not affect GBA CBE inhibits both. |
| Note | GBA2 activity is regulated by GBA activity, but not vice versa [59]. |
Model system
| Source | Model | Findings |
|---|---|---|
| Cayman | ||
| (Marques, 2016 #603) | COS-7 cell (African green monkey kidney cell), transfected with GBA | Inhibition of GBA with CBE prevented formation of GlcCer + Chol → GlcChol (Fig. 3A). |
| Alfonso, Pilar et al | Miglustat (NB-DNJ) works as a chaperone for mutated acid β-glucosidase in cells transfected with several Gaucher disease mutations | Blood Cells, Molecules, and Diseases 35.2 (2005) 268-276 No info on CBE |
| (Ridley, 2013 #600) | COS-7 cell (not a transfected cell) | Fig 4A, CBE (2.5 mM) completely blocked β-glucosidase activity (ie blocked GBA & GBA2) |
| (Liu, 2015 #601) | Cynomolgus monkeys | Aging: ↑ aSyn oligomer & ↑ p-aSyn, ↓ PPA2A (protein phosphatase 2A), ↓ GBA protein, ↓ GBA activity |
| Primary cultures of rat cortical neurons & MES 23.5 dopaminergic cells | CBE → oligomeric a-syn increased |
Acid Ceramidase inhibitor — Executive summary (Yuta) (PFR-4153-100)
| LGE (AQB1 202303) | PE (FY21 Q4) | CN | |
|---|---|---|---|
| In vitro | Cell free enz assay (IC50 < 100 nM) Cell-based: GBA KO cell (HAP1), ↓ (어느만큼?) GlcSph (IC50 < 100 nM) Effect on GalSph reduction will be confirmed in cellular assay (fibroblast or mouse primary neuron) before in vivo PK/PD study. | GD iPS DA neuron (L444P/L444P) POC for GD: ↓ (어느만큼? Monthly report 보자) GlcSph PD iPS neuron POC for PD: a-syn reduction. 202103 까지 낮췄음, → plan: selective MOA, & Venglu 검사 | |
| In vivo | GalSph reduction in C57/BL mice | Gba D409V mice POC for GD: GlcSph reduction in brain aSyn PFF mice or SNCA-A53T mice (Under discussion) POC for PD: a-syn reduction in brain |
HAP1 cells are a near-haploid cell line derived from the KBM-7 cell line. KBM-7 was found in a patient with chronic myeloid leukemia (CML).
Questions / Research hypothesis
The level of psychosine in GD or GBA-PD?
Lysosome / Research hypothesis?
- Lysosomal dysfunction → ↑ aSyn degradation, then why aSyn?
- aSyn 증가가 positive control 식으로 필요
- Lysosomal dysfunction → global protein degradation → neurodegeneration
- Global proteolysis 감소가 positive control 식으로 필요
- BM discovery effort
- 적어도 aSyn 보이는 model 에서 해야 안하나
- Protein degradation 필요?
- P62 로 되겠나?
- LAMP, p62 등 이미 봤다는 것은 어디?
- iPS에서 comprehensive하게 왔나?
- Work with phReT?
- Human postmortem brain에서 볼 생각 있나?
- Mouse model
- GD CSF (Tottori)
- Interpretation is challenging: eg. trapping of ATG proteins in autophagosomes, the dynamics of ATG proteins, actual change in the autophagy process, reduced autophagosome formation but also to increased autophagosome degradation
Acid ceramidase
| field | content |
|---|---|
| gene name | N-acylsphingosine amidohydrolase 1 (ASAH1) |
| protein name | acid ceramidase (AC, aCDase) |
| tissue distribution | ubiquitous |
| subcellular localization | lysosome |
| function | Hydrolysis of amide on ceramide side-chain by AC generates lyso-sphingolipids in lysosome |
Substrates of AC (ie if AC ↑ → ↓ all below)
- ceramide
- NOTE!) 우리 약으로 AC막으면, ceramide가 늘면 inflammation, insulin signaling inhibition, and Bcl-2 family protein-mediated apoptosis
- glucosylceramide (GlcCer)
- galactosylceramide (GalCer)
Product of AC (ie AC ↑ → ↑ all below)
- sphingoshine (=Sph) (& lipids)
- GlcSph
- galactosylSph (= psychosine, = globoid cell leukodystrophy)
- psychosine induces aggregation of a-syn in NSC34 motor-neuron cells (Abdelkarim et al., 2018, PMID 30127535)
Pathway notes
Ceramide가 다시 lysosome 밖으로 나가서 → GCS에 의해 GlcCer (UGCG 일걸) 가 됨.
GlcCer는 ① lysosome안에서 GBA에 의해 Ceramide만들기도 하지만, ② (Acid ceramidase=ASAH1, 에 의해) GlcSph로 되기도 함. → 이 GlcSph 가 (acid ceramidase 에 의해) Sphingosine 되는 것임 → 이후 다른 sphingolipid 로 진행될걸 (glucosylceramide hydrolysis accounts for 50-90% of sphingolipid production.)
Lysosome cartoons
Healthy people vs Patients with GBA mutations (Golgi → Lysosome):
- Healthy: Cer → GlcCer ----→ GlcCer → Cer (GCS / A C / GlcSph → Sph)
- Patients with GBA mutations: Cer → GlcCer ----→ GlcCer → Cer (GBA↓; GlcSph → Sph)
Specific lysosomes:
- Lysosome of Gaucher disease: GlcCer → AC → GlcSph (GBA ↓)
- Lysosome of Krabbe disease: GlcCer → AC → GalSph (GALC ↓ → Sph)
Genetics ASAH1 / Farber lipogranulomatosis
Variants in the ASAH1 gene lead to severe reduction in acid ceramidase, typically to below 10 percent of normal. As a result, the enzyme cannot break down ceramides properly and they build up in the lysosomes of various cells, including in the lung, liver, colon, muscles used for movement (skeletal muscles), cartilage, and bone. The buildup of ceramides along with the reduction of its fatty breakdown products in cells likely causes the signs and symptoms of Farber lipogranulomatosis. It is unclear whether the level of acid ceramidase activity is related to the severity of the disorder. → Three classic signs occur in Farber lipogranulomatosis: a hoarse voice or a weak cry, small lumps of fat under the skin and in other tissues (lipogranulomas), and swollen and painful joints. Signs and symptoms typically first develop in infancy.
[Robak 2017] In these gene-based analyses, besides the expected result for GBA (P = 0.0001) and confirmation of SMPD1 (P = 0.029), we discover evidence of novel aggregate associations for variants in CTSD (P = 0.002), SLC17A5 (P = 0.005), and ASAH1 (P = 0.031).
Uncertain Spans
| location | transcription | uncertainty |
|---|---|---|
| Healthy people / GBA-mutation cartoons | the diagrams are approximated as text-only abstractions | the source includes paired cell-cartoon diagrams; only the text labels are transcribed. |