Céline Cluzeau, Ph.D. Postdoctoral Visiting Fellow Biological pathways influencing Purkinje cell...
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![Page 1: Céline Cluzeau, Ph.D. Postdoctoral Visiting Fellow Biological pathways influencing Purkinje cell survival in Niemann- Pick disease type C1.](https://reader030.fdocuments.us/reader030/viewer/2022032414/56649ee55503460f94bf544e/html5/thumbnails/1.jpg)
Céline Cluzeau, Ph.D.
Postdoctoral Visiting Fellow
Biological pathways influencing Purkinje cell survival in Niemann-
Pick disease type C1
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• A fatal, autosomal recessive neurodegenerative disease
• Characterized by the accumulation of unesterified cholesterol and glycosphingolipids in the endosomal/lysosomal system
• Prevalence: 1/100,000-1/150,000 (Western Europe)
• Due to mutations in the NPC1 (95%) or NPC2 genes
• NPC1 encodes a large transmembraneous lysosomal protein• NPC2 encodes a small soluble protein able to bind cholesterol
Niemann-Pick Disease, Type C (NPC)
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• Large variation in age of onset, and severity
• Clinical presentation includes:- Hepatosplenomegaly- Liver disease: cholestasis, elevated plasma enzymes- Neurological symptoms:
Ataxia Progressive dementia
Gelastic cataplexy/SeizuresVertical gaze palsy
• No FDA approved therapy for NPC • Miglustat approved in EU: slow progression• Ongoing clinical trials: 2-hydroxypropyl--cyclodextrin (phase I) and
vorinostat (HDAC inhibitor; in preparation)
NPC Clinical Presentation
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1 wk 3 wks 5 wks 7 wks 9 wks 11 wks
TerminalAtaxia, weight lossTremorsPre-symptomatic
Animal models• Mouse model (BALB/cNctr-Npc1m1N/J)
• Anteroposterior gradient of Purkinje cells degeneration in NPC1 cerebellum (from lobule I-II to X)• Lobule X genuinely resistant to Purkinje cells death even in late stages• Other early pathological signs present in lobule X (axonal degeneration, microgliosis) but progression seems to stop in this posterior lobule
Axonal degenerationMicrogliosis
Astrogliosis Onset of Purkinje cells lossLysosomal accumulation of lipids
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Goals
• Identify the differentially expressed genes between lobules with preserved Purkinje neurons versus lobules with early and late loss of Purkinje cells
• Study the relevance of the genes associated with a different Purkinje cell survival rate to NPC1 pathology
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RNAseq outline
RNA extraction
Enrichment in mRNA
Library building
Preparation of pools of barcoded libraries
Templated bead preparation and sequencing
Bioinformatics analysis
Tissue/sample collection
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Preliminary test
• Lobules very small: how many animals do we need?
• SOLID library building protocol: - 200 to 500ng rRNA-depleted RNA
• rRNA content: 90-95% of total RNA- Need 10g total RNA
• RNA extraction from lobule X of one animal: ~1.7g total RNA- Pool lobules from 6 animals
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Preliminary test: rRNA depletion• Run a Bioanalyzer chip to determine the quality of rRNA-depleted RNA (RNA
pico kit, Agilent)
Bioanalyzer profile after 1st depletion Bioanalyzer profile after 2nd depletion
Total RNA bioanalyzer profile
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Sample collection
Sacrifice of one-month-old Npc1+/+ and Npc1-/- mice (N=24 for each genotype)Cardiac perfusion with cold 1x PBS/0.6% glucose solution with RNase
inhibitors (1/10,000; Protector RNase inhibitor, Roche Diagnostics)
Dissection of cerebellum, and isolation of vermis
Microdissection of each lobule (I-II, III, IV, V-VI, VII, VIII, IX and X)Lobules flash-frozen and kept at -80°C separately
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RNA extraction
• Pool lobules from 6 mice (3 males and 3 females) for RNA extraction:- Lobule III (3 pools; early degeneration)- Lobule V-VI (3 pools; intermediate degeneration) - Lobule X (4 pools; resistant lobule)
• RNA extraction with TRIzol (Life technologies) followed by purification on Qiagen columns (Rneasy Mini kit):
- Good yield and quality
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RNA extraction
• RNA extraction with TRIzol followed by purification on Qiagen columns:
Autoclaved disposable tips
OMNI Inc. Tissue homogenizer
TRIzol
Transfer upper phase in new tubeAdd ethanol
On-column DNase I digestion
2x
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RNA extraction
• RNA concentration measured with Nanodrop (Thermo Sci.)
Sample IDMean RNA
concentration (ng/L)
Total quantity of RNA (g)
WT lobule III 571 17.1
WT lobule V-VI 830 24.9
WT lobule X 374 11.2
Mutant lobule III 529 15.9
Mutant lobule V-VI 563 16.9
Mutant lobule X 334 10
• 10g used for rRNA depletion
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rRNA depletion
1st step with 2x 5g total RNA using RiboMinusTM Eukaryote Kit v2 (Ambion)
2nd step with 1g of rRNA-depleted RNA from 1st step using Low Input RiboMinusTM Eukaryote Kit v2 (Ambion)
• Alternatives: – RiboZero rRNA removal kits (Illumina)– poly(A) selection
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rRNA depletion
Sample IDQuantity of rRNA-depleted RNA (g)
% of starting material
WT lobule III 0.38 3.8 %
WT lobule V-VI 0.39 3.9 %
WT lobule X 0.42 4.2 %
Mutant lobule III 0.46 4.6 %
Mutant lobule V-VI 0.40 4.0 %
Mutant lobule X 0.38 3.8 %
• Slightly less than 5 % of starting material, but within 200-500ng range required for library building
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rRNA depletion
• Run a Bioanalyzer chip to determine the quality of rRNA-depleted RNA (RNA pico kit, Agilent)