(Inline Takeda slide titled “High-Level Guideline for Activities/Deliverables in Pre-Clinical Stages”; preserved as body_r01 evidence.)
| Project Start | Lead Generation | Lead Optimization | Candidate Selection | Candidate Nomination | |
|---|---|---|---|---|---|
| Target | Likely target indication and high level description of unmet medical need | Preliminary TPP, Showed scientific rationale toward target indication, Competitive status | (refined TPP) | (translational evidence) | (IND-supportive) |
| Chemotype/Molecule | Obtained at least one lead chemotype or able to design one based on disclosed | ||||
| Screening & assays | Confidence in hit ID or already obtained tractable molecules to be modified/optimized | Showed screening flow including proper 2nd in vitro/vivo screening systems | |||
| Pharmacology | Demonstrated the concept by use of tool compounds | In house native cell pharmacological/physiological data to support progression of hit series, Indicated PD marker or its candidate | |||
| DMPK/Safety | No severe class effect reported/expected or proposed its countermeasure | Clarified risks of target and compound(s) by Early Safety Review and related mitigation plan | |||
| Translational/early clinical | Possible path forward to demonstrate hypothesis in the clinic | Showed potential translational plan (e.g. clinical biomarker candidate) |
Mitochondria
Astrocyte
- {Tsukada, 2019 #903} The brain is very metabolically active, accounting for around 20% of total oxygen and 25% of total glucose, and each cell relies on mitochondria to produce energy as adenosine triphosphate (ATP). The brain consists of neuronal and glial cells, each of which uses the different metabolic pathways to produce ATP: astrocytes are highly glycolytic, while neurons depend on oxidative phosphorylation (OXPHOS) in the mitochondria.
What is the structural basis of glycolysis-based ATP generation in glial mitochondria?
| Neurons | Astrocyte | |
|---|---|---|
| Generate ATP by use of O2 | ATP by use of glucose | |
| Oxidative phospohorylation | glycolysis | |
| neuronal function is highly dependent on mitochondrial metabolism | Fiebig 2019) astrocytes require a functional ETC and oxPhos machinery for proliferation and neuroprotection under injury conditions | |
| Survival 에 든다. (deletion → 사멸) | Fiebig 2019) stroke model mouse, Mitochondrial dysfunction did not impair survival of astrocytes, but caused a reactive gliosis in the cuolere under physiological conditions {S. astrocyte Mc1을 ko 후 본것일 듯} Function: ATP {S 마니기능?? cf MC1MICE 에 서는, 26개월에 LFP 보임} Js: astrocyte 가 mt impairment 에서 죽지않으니까 chronic neuroinflammation 가능?(MC1을 ko 한 mouse가 26 m 에 LPS 발현) | |
| {Lopez-Fabuel, 2016 #906} 중요! | neurons show complex I to be mostly embedded into supercomplexes, thus resulting in high mitochondrial respiration and low ROS production. | most complex I is free, but high ROS production. (cf Mitochondrial respiratory chain (MRC) complexes can be organized in higher structures called supercomplexes.) (Inline figure thumbnail "A · Complex I" with bar chart Neurons vs Astrocytes (n.s.); preserved as body_r03 evidence.) |
| mitophagy | (2019 Jackson 2nd) Genetic ablation of Pink1 decreases mitochondrial function, Mc1 (decreased) mitochondrial mass, ATP production, and increased ROS generation) and attenuates astrocyte proliferation in primary cultures of astrocytes (즉, Parkin-PD 인데도 Asto에 ascis. ?), mitophagy → MC1, Mass (↑ MC1) MC1 imaging, so will not mass reduced MC1 imaging in neurons), {ATP하라} 그리고 not death. Review도 빠르게 나옴 ? | |
| S/X) 2019 Jackson | ||
| (lopez-fabuel 2016) complex I activity was very similar in both cell types. (fig3a) MC1 complex I (rotenone-sensitive NADH-ubiquinone (oxidoreductase) activity, as assessed spectrophotometrically in cell homogenates, in astrocytes and neurons. | ||
asyn and mitochondria
| a-syn can localize to the intermembrane space, to the matrix [3-6] or to mitochondria-associated ER membranes | |
| Physical interaction | {Devi, 2008 #1382} that the N-terminal 32 amino acids of human α-syn contains a cryptic mitochondrial targeting signal, which is important for mitochondrial targeting of α-syn |
| {Devi, 2008 #1382} postmortem: associateion of aSyn with MC1 | |
| {Guardia-Laguarta, 2014 #1383} wild-type α-syn from cell lines, and brain tissue from humans and mice, is present not in mitochondria but rather in MAM | |
| {Vicario, 2018 #1384, review} a fraction of cellular a-syn can selectively localize to mitochondrial sub-compartments upon specific stimuli → | |
| {Krzystek, 2021 #1662} i) excess full-lenth a-syn causes mitochondrial problem in motility and fragmentation and then oxidation ii) but excess C-term- (1/120) truncated aSyn causes fragmentation but still healthy mitochondria | |
| {Devi, 2008 #1382} Accumulation of wild-type α-syn in the mitochondria of human dopaminergic neurons caused reduced MC1 activity | |
| {Guardia-Laguarta, 2014 #1383} that α-syn operates downstream of the mitochondrial fusion/fission machinery, which is impaired by pathogenic mutations in α-syn. | |
| {Vicario, 2018 #1384, review} plethora of mitochondrial processes such as mitochondrial respiration, complex I activity, mitochondrial proteostasis, cytochrome c release, calcium homeostasis, control of mitochondrial membrane potential and ATP production, is directly influenced by a-syn. | |
| {Vicario, 2018 #1384, review} a-syn localization within mitochondria was also account for its aggregation state, |
Evidence in sPD
| Postmortem | (Schapira, 1990 #877) n=9, SN, |
↓ decreased complex I activity in SN of PD patients (reviewed in reference [4]). Parker et al. [5] found decreased complex I activity in frontal cortex = mito mass, ↓ rotenone-sensitive NADH cytochrome c reduction (a complete mitochondrial complex I & III, was normal) (cf. antimycin A sensitive- succinate cytochrome c reductase activity, which assessed Complex II and III, was also a little overlap, although statistically significant; reductase) Rotenone-sensitive NADH cyto c reductase (MC1-specific) ↓ (by 30%. 2.34±0.76 vs 3.36±0.44, so there is a 30% reduction in cytochrome c reductase (MC1-specific) xxxi) = MC1 mass (staining intensities of subunits coded by mitochondrial and nuclear genomes) Conclusion: selective ↓ complex I activity | ||||||||||||||||||
| (Gatt, 2016 #1387) PD N=41, PDD n=40, control 33, prefrontal cortex |
In PD] = MC1 activity, = mt DNA (PCR), = porin (VDAC1), - TFAM, - NDUFB8, MC3 (30 Xa subunit), MC3 (CORE 2), - mc4 (COX II), - ATP synthase subunit alpha In PDD] ↓ (27%) MC1 activity, ↓ (18%) mtDNA, but - porin (VDAC1), - TFAM, - NDUFB8, MC3 (30 Xa subunit), MC3 (CORE 2), - mc4 (COX II), - ATP synthase subunit alpha | |||||||||||||||||||
| (Garcia-Esparcia, 2018 #1388) control n=34, iPD n=15, PD n=20, PDD n=4 |
• MC1,2,3,4, 5 mRNA in frontal cortex: table 2) ↑ NDUFA7, ↑ NDUFA10, ↑ NDUFB10, NDUFA13 in iPD (PD에선 X) • MC1,2,3,4, 5 mRNA in angular gyrus: table 4) - 다 - • Fig1) NDUFA7, NDUFA10, NDUFB10, NDUFA13 in angular gyrus의 mRNA에서 - 일반 PD에선 X, 단 PDD에선 ↑ • MC1,2,3,4, 5 activity 들이 대개 PDD에선 -, 단 일반 PD에선 ↑ | |||||||||||||||||||
| (Grünewald, 2016 #935) |
[single SN neurons from 10 sPD patients isolated with laser-capture microdissection] = mito mass (porin and GRP75) , ↓ MC 1 mRNA (postmortem in SN) (figure NDUFA13), ↓ transcription/replication-associated mtDNA mutations factors TFAM & TFB2M, ↑ mtDNA heteroplasmy | |||||||||||||||||||
| (Keeney, 2006 #944) N=10 sPD, frontal cortex |
complex I shows: 11% increase in ND6 (20 kDa), 34% decrease in 8 kDa subunit (NDUFA13). 47% more protein carbonyls localized to catalytic subunits coded for by mitochondrial and nuclear genomes. , By 32% MC1 (rotenone sensitive, fig.표 없는듯) | |||||||||||||||||||
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Uncertain Spans
| location | transcription | uncertainty |
|---|---|---|
| Astrocyte vs Neurons table | Korean fragment about Mt-impairment chronic neuroinflammation | The mixed Korean-English notes contain abbreviations whose endings (e.g. “이 LPS 발현”) are partially clipped. |
| Lopez-Fabuel row | most complex I is free, but high ROS production | Continuation of the sentence on the right is partly cut by the embedded thumbnail. |
| Schapira row | ↓ (by 30%. 2.34±0.76 vs 3.36±0.44, so there is a 30% reduction in cytochrome c reductase (MC1-specific) | The trailing parenthetical structure is unbalanced and may be cropped. |
| Keeney row | 8 kDa subunit (NDUFA13) | Small subunit number; could read NDUFA12 or NDUFA13. |