NDALs Research Activities A. Nazl BAAK - - PowerPoint PPT Presentation

NDAL’s Research Activities

• A. Nazlı BAŞAK

http://www.ndal.boun.edu.tr/posters.php

Neurodegenerative Diseases

• have tremendous adverse effects on the

aging population.....

• people of all nations, races, ethnicity are

affected.....

• 120.000 new cases appear every year...

GREAT BURDEN ! GREAT BURDEN !

AD, PD, HD, ALS, SBMA, Ataxias...

• loss of memory and decrease

in cognitive function: dementia

• deterioration in muscle

coordination: ataxia

• involuntary movements:

chorea

• tremor, rigidity, bradykinesia
• behavioural abnormalities
• paralysis

There are no effective treatments for these disorders yet...

NDAL’s Aims:

Genetic approaches:

• determine mutations and genes leading to

disease

• develop methods for early diagnosis

Molecular and cell biology approaches:

• investigate mechanisms leading to

neurodegeneration

• halt/prevent disease at early stages
• develop molecular treatment modalities

YÜKSEK LİSANS ÖĞRENCİLERİ Suna LAHUT Abdülkadir ÖZKAN Didem ERUSLU Gülçin VARDAR Merve KILINÇ

Why Genetics?

• Genetics plays a major role in any disease;

– cardiovascular disease, cancer, diabetes, infection, neurological/mental disorders

• It is a person’s genetic makeup, which affects

– susceptibility to disease, disease course, response to treatment

• Understanding these genetic contributions is the first

step toward understanding disease mechanisms.

Common Mechanisms: The specific molecular events and selectivity of neuronal death are still unclear, but damage to neurons seems to occur through a combination of several different mechanisms

Selective vulnerability of neurons specific for each disease

Bertram and Tanzi, 2005

HMND

ALS

as

MODEL DISEASE

• Charcot, J.M. & Joffroy, A.

”Deux cas d’atrophie musculaire

progressive avec lesions de la substance grise et des faisceaux antero-lateraux de la moelle epiniere”

• Arch. Physiol. Neurol. Pathol. 2, 744-

754 (1869)

Jean-Martin Charcot (1825-1893)

French Neurobiologist

AL ALS: S: Most Common Motor Neuron Most Common Motor Neuron Disease Disease

• Fatal within 1-5 years

Fatal within 1-5 years

• Incidence: 1-2.5/100 000

Incidence: 1-2.5/100 000

• Prevalence: 4-8 / 100 000

Prevalence: 4-8 / 100 000

• Lou Gehrig

Lou Gehrig: : “ “A bad break’’ A bad break’’

“ “I am so tired, and I simply cannot play well anymore…” I am so tired, and I simply cannot play well anymore…”

AMYOTROPHIC LATERAL SCLEROSIS

• A:

Absence of

• Myo:

Muscle

• Trophic:

Nourishment

• Lateral:

Side (ofspine)

• Sclerosis:

Hardening or scarring Weakness and atrophy of muscles Sclerosis (hardening)

• bserved in the

lateral parts of the spinal cord during autopsy

ALS progressively weakens and paralyses the muscles, controling voluntary movement

HALLMARKS of ALS

• Degeneration and death of

upper upper and lower lower motor neurons in the brain and spinal cord

• Progressive muscle weakness
• Atrophy and spasticity
• Total paralysis
• Denervation of the respiratory

muscles and diaphragm is the fatal event

• “Dying back” of the MN:

– Degeneration begins at the ends of the axon, proceeding back to cell body.

Neighbours matter.....

• A paradigm shift in our thinking about the

nature of ALS has occurred in the last decade.

• ALS is now considered a multisystem

neurodegenerative disease:

– voluntary motor pathways are the first, but not the only (CNS) structures involved – microglia, astrocytes, skeletal muscle are active co-players

The most devastating aspect of ALS...

• Cognitive functions of

the brain remain undamaged

• Sensory nerves are

spared, too...

• Patients are obliged to

watch the demise of their own bodies.

Stephen Hawking

Most ALS Cases Are Isolated Incidences  

% 90 % 90

 

% 10 % 10

sALS

fALS

 Familial ALS : fALS

(Mendel gen.: AD/AR inh.)

 Sporadic ALS : sALS

(no documented family history, genetic contribution?)

fALS and sALS are clinically similar!!

Aging

Environment

Genes

SOD1 Risk

Low High

? retrovirus ? pesticides smoking high glut diet Risk Age Youth Risk

The development of ALS The development of ALS probably probably reflects an interplay reflects an interplay between environmental and genetic factors, between environmental and genetic factors, influenced by aging influenced by aging. .

Seven genes involved in fALS

 SOD1  Alsin  Senataxin  VAPB  Dynactin  TDP-43  FUS

fALS genes encode proteins

• Involved in a diverse range of cellular processes:

– dismutation – RNA processing – axonal transport – angiogenesis – vesicle transport – endosomal trafficking – DNA/RNA binding

• This diversity indicates that the pathogenesis of ALS is

probably associated with several different processes – leading to a cascade of neurodegeneration.

SOD1: the first and major gene

1993(Rosen et al.)

chromosome 21 All the other genes account for 11% (blue), leaving 69% (green) undefined. SOD1 accounts for 20% of all familial forms (and for 2 % of all ALS cases).

SOD1 catalyzes the conversion of superoxide to SOD1 catalyzes the conversion of superoxide to

• xygen and hydrogen peroxide.
• xygen and hydrogen peroxide.
• extremely stable dimer:

153 aa/monomer

• Cu catalytic,

Zn structural

• ubiquitously expressed
• present in all organisms

above bacteria

• aminoacid sequence is

highly conserved

– crucial funtion in cellular homeostasis

All 133 SOD1 mutations result in typical ALS!

• Alsin, SETX, DCTN1, VAPB

Atypical !

Mutations confer a destructive property to SOD1:

• Gain of a novel toxic function!
• Autosomal dominant inheritance!
• How such a diverse range of mutations can result in a

novel function

– producing the same relatively homogeneous phenotype is

still a mystery!

Passinelli & Brown, 2006

Mt SOD1 impairs multiple cellular functions, leading to death of MNs

ALS in Turkey ????

192 ALS patients from different regions of Turkey

• FALS: 32 patients *

– 6 families/ 15 members – 17 single cases

• Juvenile: 11 patients

– 1 family/ 2 children – 9 single cases

• SALS: 149 apparently sporadic cases

*ALS was classified as familial, when at least two members were clinically affected in the same pedigree.

Clinical Characteristics of Patients

FALS SALS

# of patients* 32 (15 fam.) 149 Gender (Male/Female) 13 / 19 93 / 56 Ratio 1 : 1. 5 1.6 : 1

Age of onset (years) Mean

39.3 47.5

Range 16-68 18-79

* Juvenile cases not included

SOD1 Results

• FALS: 5mut/32 patients: 15.6%

– H71Y – N86S* * recessive inheritance – D90A* – L144F – A4S – IVS-III-34 (A:C)

• SALS:

– 6 cases: IVS-III-34 (A:C)

NORTH-SOUTH gradient in the frequency of SOD1 mutations across Europe.

23% 23% 50% 50% 16% 16% 20% 20% 64% 64% 13% 13% 15% 15%

EUROPE: SOD1 mts in FALS

15.6% 15.6%

D90A, N86S autosomal recessive behaviour

• Evidence for a cis-acting regulatory

polymorphism as disease modifier?

– decreases ALS susceptibility in heterozygotes – slows disease progression in homozygotes

• Comparative haplotype analysis

Genome-wide Association Studies in ALS (Collaboration Brown Lab)

• Aim: to identify genetic variants associated with

– Susceptibility for sALS – phenotypes in SALS

• site of onset
• age of onset
• risk of disease
• duration of disease
• Materials: 1821 SALS DNA + 2258 Control DNA

– 288,357 SNPs

Reduced Expression of the Kinesin-Associated Protein 3 (KIFAP3) Gene Increases Survival in SALS Landers et al, 2009, PNAS in print

• A single SNP (rs1541160) in the noncoding

region of KIFAP3 showed significant value, regarding the disease duration in ALS.

• Genetic factors modify phenotypes in sALS
• Cellular motor proteins are determinants of

motor neuron viability.

From SOD1 to GWAS in ALS

• Aslıhan Özoğuz, PhD Thesis

– Altar Sorkaç

• Collaboration with Brown’s Lab

– John Landers

• Collaboration with Andersen’s Lab
• Tubitak SBAG 1001

Alsin in the pathology of ALS

 Alsin mutations lead to related, but clinically distinct motor neuron degenerative diseases:

 ALS ALS  PLS  HSP  SMA

May be an important molecule in the elucidation of a common May be an important molecule in the elucidation of a common mechanism for MN degeneration! mechanism for MN degeneration!

Alsin

 Ubiquitously expressed and abundant in neurons

 Function not fully understood  Many interacting domains  Investigations on Alsin may be crucial for the elucidation of a common mechanism for MND

Alsin Protein (G nt exchange factor)

Yeast-2-Hybrid screen

• Alsin is known to inhibit the neurotoxicity of mt SOD1; its

SOD1 interacting region is DH/PH.

• the DH/PH region of Alsin revealed 6 novel i.a,

– UXT Xp11 – PMM1 22q13 – PCMB4 1q21 – SRPK2 7q22-q31.1 – NDUFV1 11q13 – VARSL 6p21

• These proteins are expected to give clues about the

function of Alsin.

2 New Candidate Proteins for ALS

• UXT (Ubiqutiously Expressed Transcript)

– Coactivator of Androgen Receptor – Essential cofactor in NF-kB transcriptional enhanceosome

• SRPK2 (Serine Rich Protein Kinase 2)

– Phosphorylates many key proteins involved in cell survival

• Serine rich splicing factors
• Apoptosis related protein (Acinus)
• Cell cycle related protein (Cyclin A1)

Candidate proteins: Functions?

• The interaction of UXT and SRPK2 with Alsin will be

investigated: – In homogeneous/heterogeneous cell culture models

• to characterize their functions in vivo
• to characterize their interactions with other proteins
• to see if they are involved in protein complexes

– in transgenic and knock-out ALS Drosophila models

• Characterization of Drosophila homologs of the

human proteins

• In long term: drug development studies

Strategy Strategy

a. Identify interacting partners of Alsin d. Determine their relation with Alsin h. Investigate their relation with ALS

• Yeast Two Hybrid

Screens

• Cell culture /Animal

Models

• Mutation/polymorphism

screen in patients

Animal Models Animal Models

LONG TERM GOAL

• Drosophila M. Cell Cultures

– Characterise insect homologues

• f mammalian proteins under

investigation

• Transgenic Drosophila Models

– Further approach to real life processes of the disease

 Neuronal N2a Cells: Mouse Albino Neuroblastoma.  SH-SY5Y Cells: Human Neuroblastoma.  U-138 MG Cells: Human Glioblastoma.  U-87 MG Cells: Human Glioblastoma Astrocytoma.  C2C12 Cells: Mouse myoblast cells.  NSC-34 Cells: Hybrid cell line derived from the fusion of N18TG2 (mouse aminopterin-sensitive neuroblastoma) and  Mouse embryonic day 12-14 spinal cord cells.

* All cells are commercially available except NSC-34.

Mammalian Cell Lines Stock in Our Lab:

ALSIN-RELATED PROTEIN DYNAMICS: Strategy

• Differentiation N2a, C2C12 and NSC-34 cells
• Determine gene expression by qRT-PCR
• Knock down alsin by RNAi
• Determine gene expression by qRT-PCR
• Investigate possible protein interactions

– Immnunoprecipitation

Gene/protein set:

 Dctn1 (p150Glued)  Dctn3 (p22)  Kif3b  UXT*  PKN1  SRPK2* Retrograde Transport Anterograde Transport Ser/Thre kinase (neurofilaments and intermediate filaments) Coactivator of Androgen Receptor cofactor in NF-kB transcriptional enhanceosome Phosphorylates many key cell survival proteins Serine rich splicing factors Apoptosis related protein (Acinus) Cell cycle related protein (Cyclin A1)

Ongoing Y-2-H data will continuously supply new candidate genes...

• Izzet Enünlü TBAG-1001

– Alpay Burak Seven – Arman Aksoy – Rana Özdeşlik

• Mehmet Ozansoy SBAG-1001

– Aslı Şahin BAP-08HB-102 – Suna Lahut, MSc Thesis (İ.Kıraç fellow)

PARKINSON’S DISEASE

• Bradykinesia
• Rigidity
• Rest tremor
• Good initial response to

levodopa

FAMILIAL FORMS <10% SPORADIC CASES 90%

Environmental toxins Proteasome dysfunction Genetic susceptibility factors Inflammation Monogenic forms Oxidative Stress & Mitochondrial dysfunction

ETIOLOGY OF PARKINSON’S DISEASE

From a Sporadic to a Genetic Disease

 α

α-synuclein

• synuclein

 Parkin

Parkin

 PINK1

PINK1

 DJ1

DJ1

 LRRK2

LRRK2

Genetic Analyses Results of the 78 Turkish PD Patients

The Lady of PD...

• Caroline Pirkevi, PhD. Thesis (completed)

– Xalid Bayramlı – Gülçin Vardar – Onur Birol

• BAP 07HB105D; BAP 08HB101D
• TÜBITAK-PIA-12 105S163

MYOTONIC DYSTROPHY

• Defined by Hans Steinert in 1909.
• Neuromuscular disease with multisystemic effects

– Skeletal and cardiac muscles – Endocrine system – Gastrointestinal system – Central nervous sytem

• Typical myotonic face
• CTG repeat in the 3’ UTR, but

– autosomal dominant inheritance!!!

• Toxic RNA hypothesis for the first time

– Alters splicing pattern of other proteins

METHODOLOGY

DNA isolation from peripheral blood Triplet-repeat-primed-PCR & Flanking PCR Fragment analysis with ABI 310 or ABI 3130XL Peak scanner or Chromas analysis of the results

P4 primers randomly bind to CTG repeats on the DMPK region Resulting products are amplified with the P1 and P3 primers

TP-PCR: Possible adaptation to other Triplet Repeat Disorders

RESULTS

117 affected or at risk patients: 21 families with 69 members 48 isolated cases  67 DM1 positive (57%)  50 DM1 negative (43%)

DM: A Triplet Repeat Disorder, not Obeying Rules...

• Didem ERUSLU, MSc thesis (İ.Kıraç fellow)

– Merve Kılınç

• Boğaziçi University, Research Funds

Temporal Analysis of Gene Expression in Brain Vascular Development

• Angiogenesis, the growth of new blood vessels,

is a crucial force

– for shaping the nervous system – protecting it from disease

• Angiogenesis factors, especially VEGF, are

known to have roles in

– birth of new neurons (neurogenesis), – prevention or mitigation of neuronal injury (neuroprotection), – pathogenesis of stroke, AD and ALS

Venous Angioma Arteriovenous Malformations Aneurysms Cavernous Malformations

Brain vascular malformations give not only rise to hemorrhage, stroke, paralysis...

Insufficient vessel growth and abnormal vessel regression cause neurodegenerative diseases

Zacchigna, Nature 2008

Understanding the molecular pathologies of diseases Designing possible therapies. Analyzing the temporal gene expression in brain vascular development using mouse models pathway-specific arrays

Investigating Brain Vascular Development is important for

q-RT-PCR Western blot

Immunohistochemistry

Angiogenesis-specific membrane-array: 113

angiogenesis-related genes

• With different

functions

– Growth Factors – Receptors – Adhesion Molecules – Proteases – Inhibitors – Matrix Proteins – Transcription Factors – Cytokines & Chemokines – other related genes

Oligo-probes specific for angiogenic genes

Three angiogenesis-related genes

Previously not shown in vascular/brain development: – BAI-1 (Brain-specific Angiogenesis Inhibitor-1) – NPR-1 (Natri-uretic Peptide Receptor-1) – NUDT-6 (Nudix Type motif 6) – Further analysis

– RNA expression, localization and protein expression

insight into the angiogenic developmental stages of the brain at molecular level

From Brain Vascularization to Neuron Degeneration...

• Kadir Özkan- MSc thesis

– İbrahim Taştekin – Atilla Biçer

• In collaboration with Marmara University,

Neurosurgery Dept.

– Prof. Türker Kılıç

• Tübitak-SBAG 108S080
• Boğaziçi University BAP 07HB104
• Marmara University SAG-B-030108-0008

Molecular Diagnoses at NDAL

Aslı Gündoğdu, MSc. Irmak Şahbaz

Neurological 1997- Hematological 1987-

• >2300
• Triplet repeats
• HD
• SCA 1, 2, 3, 6, 7,

8, 17

• FRDA
• SBMA
• DRPLA
• DM
• PD (parkin,

α- synuclein)

• AD (ApoE)
• ALS (SOD1)
• >2500
• Alpha-thal

(>50patients)

• Beta-thal

(>2000patients)

• Abnormal Hbs

(>500patients)

• Hb S
• Hb E
• Hb E Saskatoon
• Hb D Ankara
• Hb D Iran
• Many more...

Large collection of patient material: huge potential for research

Molecular Analyses are important…

• To help patients/families
• To assist physicians

– differential diagnosis

• For research

– novel mutations… – novel genes… – novel disease mechanisms…

Ethical Issues

• Consultance by physicians and geneticists
• informed consent to be signed
• Approval obtained from the local

ethical committees for medical research

• humans
• animals

Dissemination of information materials

No lab has access to all the elements required to conquer a disease...

– Bob Brown, UMASS Med School, Worcester – Jeffrey Macklis, HMS, Harvard Med School, Boston – Alexis Brice, INSERM, Salpêtrière Hospital, Paris – P. Hande Özdinler, Les Turner ALS Lab, Chicago – Hilmi Özçelik, Mount Sinai Hospital, Toronto – Justin Fallon, Brown Univ, Neuroscience Dept. – Jon Warner, Western General Hospital, Edinburgh – Peter Andersen, Umea Med. School/ Neurology, Sweden – Türker Kılıç, Marmara Univ., Neurosurgery Dept. – Clinicians in Neurology and Hematology Departments of several hospitals throughout Turkey

International Projects

• EU-Ithanet Project, 2007-2009

“Electronic Infrastructure for Thalassemia Research Network”

• EU-Cost Project, 2008-2010

“Brain Plasticity and Neural Regeneration”

• EU- e-rare Project, 2009-2011

“Ribosomal Translation Mechanisms and Lithium Treatment of SCA2 in European Cohorts”

Published Papers NDAL 2005-2008

In the Pipeline... 2009-

Conferences/Exhibitions

BAP - DSS

Acknowledgement: former students

• Hilmi Özçelik
• Ayşe Özer
• Şükrü Tüzmen
• Ghazi Tadmouri
• Edward Senga
• Shahid Baig
• Özlem Taştan
• Onur Bilenoğlu
• Ayşe Latif
• Baki Yokeş
• Nagehan Ersoy
• Nazan Saner
• Mine Güzel
• Sinem Hocaoğlu
• Begüm Erdoğan
• Pınar Gencer
• Charlotte
• Esra Soydan

Thank you !

You are all welcome to the

• II. Suna Kıraç Conference
• n Neurodegeneration

“Stem cells, RNAi and Neurodegeneration”

20-22 July, 2009 Pera Museum, Istanbul