Céline Cluzeau, Ph.D.

Postdoctoral Visiting Fellow

Biological pathways influencing Purkinje cell survival in Niemann-

Pick disease type C1

• 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)

• 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

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

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

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

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

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

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

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

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

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

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

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

rRNA depletion

• Run a Bioanalyzer chip to determine the quality of rRNA-depleted RNA (RNA pico kit, Agilent)