Thesis title  

Human induced pluripotent stem cell derived neuronal disease models for mitochondrial neurodegenerationProject Director: Dr. Anu Suomalainen WartiovaaraProject Supervisor: Dr. Christopher JacksonMolecular Neurology research unitBiomedicum, HelsinkiFinland 

Presented by : Shamita MahzabinMaster’s thesis student

Faculty of biochemistry and molecular medicine

Some images of patients having defect in SUCLA2

Congenital muscle dystrophy Hypotonia Dystonia

Different kinds of pathogenic mutations in SUCLA2 Mutations Effect

c.534þ1G>A in intron 4- found in Faroese patients multiple exon skipping resulting in complete absence of functional protein

homozygous change Asp251Asn- 2 Iranian cousins less stable b-subunit

Ala103Asp mutation- Italian patients alter ADP binding resulting in an inactive proteinHeterozygous for Arg284Cys and Gly118Arg8- an Italian/Romanian patient

alters the architecture of the active site and interfere with ADP binding, resulting in a correctly assembled but inactive protein

Missense variant Asp333Gly, homozygous in one patient and compound heterozygous in one

reduce the activity of the otherwise functional enzyme succinyl co-a synthetase.

To be continued… Six carriers of this variant were found among 8078 Finnish control chromosomes, making

the carrier frequency 1/673. The low carrier frequency in the genetically isolated Finland suggests that the variant is recently introduced into the population.

Sequence analysis of SUCLA2 showed that this patient was homozygous for missense variant c. 998A>G (rs140963290) in exon 8, causing an amino-acid change from aspartic acid (Asp) to glycine (Gly)

The amino-acid change, Asp333Gly

Occurred in a highly conserved area Of the protein

Symptoms seen in this patient 5 months: was athetotic and delayed motor development having slightly increased blood

pyruvate and methylmalonic acid (MMA) in urine 2 years: Brain MRI showed atrophy of the caudate and lentiform nuclei. Also, Muscle biopsy

also showed type- II atrophy 4 years: fine motor skills were at the level of a 1.5-year old. 7 years: Hand and feet posturing was dystonic with mild ataxia and sensorineural hearing

deficit. Did not have muscle mtDNA depletion.

Figure: Brain axial T2-weighted magnetic resonance at 7 years showing atrophy of caudate (asterisk) and lentiform (arrow) nuclei.

Scientific questions I am going to addressMy hypothesis: Defect in SUCLA2

- can lead to mitochondrial DNA depletion in the patient neurons.

- Accumulates succinyl co-A, thus profound succinylation can occur in the patient neurons which might have role in the mtDNA depletion

Aim of my study: Analysis of the neuronal cell lines differentiated from induced pluripotent stem cells of this SUCLA2 (A333G) patients to Characterize the mitochondrial dysfunction that occurs in these SUCLA2 defective patients.

Experimentation: IPSc culture & Neuron differentiation

- mtDNA quantification

- Succinylation pattern analysis

- Gene expression analysis

- Single cell level analysis of the neuronal cultures by Immunofluorescence technique

Outline of the works doneand

Result Analysis

mtDNA content in patient fibroblasts & myoblasts- No significant mtDNA depletion, also under starvation conditions

ctrl s

ale 0d

patien

t 0d

ctrl s

ale 2d

patien

t 2d

ctrl s

ale 4d

patien

t 4d

0.0

0.5

1.0

1.5

2.0

2.5

Ctrl & Patient myoblasts (0, 2 & 4 days starved)mtDNA quantification

mtD

NA

Fol

d C

hang

e / B

2M

D-LOOP

ctrl25

24_0

d

ctrl25

24_5

d

patien

t R_0

d

patien

t R_5

d0.0

0.5

1.0

1.5

Ctrl & Patient fibroblasts (0 & 5 days starved)mtDNA quantification

mtD

NA F

old

Chan

ge /

B2M

D-LOOP

mtDNA Quantification in IPSc derived Neurons

0.0

0.5

1.0

1.5

1st week NeuronMitochondrial DNA Quantification

mtDN

A Fo

ld Ch

ange

/ B2M

h D-LOOP h cyt B

0.0

0.5

1.0

1.5

4th week NeuronMitochondrial DNA Quantification

mtDN

A Fo

ld Ch

ange

/ B2M

D-LOOP h ATP8 h Cyt B

Differentiation StatesSamples

Neuronal mtDNA quantity1st week 4th week

H9 (control)

~990 ~680

M108 (control)

~650 ~440

Sucla2 clone 15

~553 ~315

Sucla2 clone 22

~410 ~192

Summary of my findings

Work flow of SUCLA2 project

c Fibroblast

IPSc

Neurosphere

Neuron

Cell culture

(un-starved & starved)

Cell collection (n=2)Freezing (n=2)

Cell collection (n=2) Time Points: Week 2 and 4

Freezing (n=2): week 5 and 7

Cell collection (n=2) Time Points: Week 1, 3 and 4

Freezing (n=3): week 2

DNA ExtractionMitochondrial DNA quantification

Protein ExtractionSuccinylation pattern checking

o RNA extraction & Gene Expression analysiso Immunofluorescence staining

Culture to grow enough amount

Findings until now:

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1.0

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mtDNA quantification of IPSc's

mtDNA

Fold C

hange

/ hAPP

hCYTb

0.0

0.5

1.0

1.5

mtDNA quantification of6 week Neurospheres

mtDNA

Fold C

hange

/ B2M

h D-loop hCYTb

SamplesmtDNA Amount

Control Patient

Myoblasts

Un-starved

~640 ~900

starved ~620 ~1000 Fibroblasts

Un-starved

~1300 ~1000

starved ~750 ~650 Neurospheres

Age: 5 week ~400 ~400Age: 6 week ~300 ~200

2. Gene Expression analysis

oWhile generation of iPSCs is fairly robust, differentiation of the iPSCs and achieving pure target cell populations is really difficult.

oNow it is necessary to analyze the gene expression of the iPSCs derived neurons

In order to see do the mutant and wild type neurons differentiated equally

Do they produce similar neuron / glia proportionsAlso to assess purity of neuronal culturesTo see is the mitochondrial maturation going similarly or not

Neuronal markers Corresponding gene expression

MAP2 (Microtubule-associated protein 2) 

Early neuronal markers, weak expression in neuronal precursors but it increases during neuron development process.

MSI 1 (Musashi RNA-binding protein 1) Proliferating neuronal markers

GAD2 (GABAenergic neurons) Mature neuronal marker expressed in GABAergic interneurons.

VGLUT (vesicular glutamate transporter 1 encoded by solute carrier genes Slc17a7)

Glutamatergic neuronal markers (later staged neurons, in the developmental process)

0.0

0.5

1.0

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1st week Neuronal Gene Expression

mRNA

Fold C

hange

/ Actin

MAP2 MSI1

0

1

2

3

3rd week Neuronal Gene Expression

mRNA

Fold C

hange

/ Actin

MAP2 MSI1

0.0

0.5

1.0

1.5

2.0

2.5

4th week Neuronal Gene Expression

mRNA

Fold C

hange

/ Actin

MAP2 MSI1

Neuronal Gene Expression analysis- Similar expression observed in all cell lines & timepoints

Other markers tested Corresponding gene expression

Cytb (Cytochrome b) Mitochondrial marker

SUCLA2 (Succinyl CoA ligase β subunit encoded by the SUCLA2 gene)

Neuronal marker

SUCLG2 (Succinyl CoA ligase β subunit encoded by the SUCLG2 gene)

Expressed ubiqutously

- Mitochondrial and SUCLA2 Gene expression is reduced in the SUCLA2 clones- Corresponding to the findings with mtDNA depletion - SuclG2 expression increased in Sucla2 clones0.0

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1.0

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1st week Gene Expression

mRNA

Fold

Chan

ge / A

ctin

cytB SUCLA2 SUCLG2

0.0

0.5

1.0

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4th week Gene Expression

mRNA

Fold

Chan

ge / A

ctin

cytB SUCLA2 SUCLG2

3. Immunofluorescence Staining- characterization of the neuronal population in our culture

Map2 + GFAP Map2 + Anti-DNA Map2 + Tom20 Map2 + SuclA2 (did not work) Map2 + SuclG2 Map2 + Pan-anti succinyllysine (too much background)(too much background

Beta Tubulin + Tom20

Map2 + Complex 2 (Did not work)

Tuj1(Did not work) + Sucla2 NeuN (Did not work) + Sucla2

Beta tubulin+Tom20; 1st week NeuronsControl H9 SUCLA2 22Control H9

20µM 20µM

MAP2+SUCLG2Immunostaining4th week Neurons

4. Succinylation pattern check

5. Succinate treatment :

Further necessary findings

• Justifying the previous findings

•Checking succinylation in Neurons

•Basic functional characterization of the Neurons- MEA experimentation (mean firing rate)

•Live mitochondrial staining to analyze their structures in the patient mitochondria compared to the healthy individuals (Imaging approaches)

Conclusion Characterization of this particular SUCLA2 mutation (A333G)

Any novel finding- Characterization of other sucla2 defects

Find out precise reasoning of these SUCLA2 defects at a molecular level to shed some light towards a possible therapeutic solution.

Acknowledgements Most special Thanks

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0.5

1.0

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2.0

1st week Astrocytic Gene Expression

mRNA

Fold

Chan

ge / A

ctin

GFAP S100B

0

1

2

3

3rd week Astrocytic Gene Expression

mRNA

Fold

Chan

ge / A

ctin

GFAP S100B

0

1

2

3

4th week Astrocytic Gene Expression

mRNA

Fold

Chan

ge / A

ctin

GFAP S100B

Astrocytic markers Corresponding gene expression

GFAP (Glial fibrillary acidic protein) Astrocytic and ependymal marker

S100b (S100b calcium-binding protein B)

Constitutive astrocytic (glial) marker