1. Céline Cluzeau, Ph.D. Postdoctoral Visiting Fellow Biological pathways influencing Purkinje cell survival in Niemann- Pick disease type C1

2. 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)

3. 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/Seizures Vertical 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

4. 1 wk3 wks5 wks7 wks9 wks11 wks TerminalAtaxia, weight lossTremorsPre-symptomatic Animal models Mouse model (BALB/cNctr-Npc1 m1N /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 degeneration Microgliosis AstrogliosisOnset of Purkinje cells loss Lysosomal accumulation of lipids

5. 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

6. 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

7. 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 10  g total RNA RNA extraction from lobule X of one animal: ~1.7  g total RNA - Pool lobules from 6 animals

8. Preliminary test: rRNA depletion Run a Bioanalyzer chip to determine the quality of rRNA-depleted RNA (RNA pico kit, Agilent) Bioanalyzer profile after 1 st depletion Bioanalyzer profile after 2nd depletion Total RNA bioanalyzer profile

9. 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

10. 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

11. 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 tube Add ethanol On-column DNase I digestion 2x

12. RNA extraction RNA concentration measured with Nanodrop (Thermo Sci.) Sample ID Mean RNA concentration (ng/  L) Total quantity of RNA (  g) WT lobule III57117.1 WT lobule V-VI83024.9 WT lobule X37411.2 Mutant lobule III52915.9 Mutant lobule V-VI56316.9 Mutant lobule X33410 10  g used for rRNA depletion

13. rRNA depletion 1 st step with 2x 5  g total RNA using RiboMinus TM Eukaryote Kit v2 (Ambion) 2 nd step with 1  g of rRNA-depleted RNA from 1 st step using Low Input RiboMinus TM Eukaryote Kit v2 (Ambion) Alternatives: –RiboZero rRNA removal kits (Illumina) –poly(A) selection

14. rRNA depletion Sample ID Quantity of rRNA- depleted RNA (  g) % of starting material WT lobule III0.383.8 % WT lobule V-VI0.393.9 % WT lobule X0.424.2 % Mutant lobule III0.464.6 % Mutant lobule V-VI0.404.0 % Mutant lobule X0.383.8 % Slightly less than 5 % of starting material, but within 200-500ng range required for library building

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