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Testing five hypotheses for Friedreich s ataxia in human cells Idebenone

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Testing five hypotheses for Friedreich s ataxia in human cells Idebenone Powered By Docstoc
					Testing five hypotheses for Friedreich’s ataxia in human cells

G. Tan, E. Napoli, F. Taroni and Gino Cortopassi
University of California, Davis
Overview:

Friedreich ataxia– a ‘mitochondrial oxidative stress’ disease

Friedreich’s ataxia pathology

Testing 5 hypotheses by microarray

Biochemical confirmations of one model

Causes and effects of oxidative stress

Avenues to therapy

Request for cells
Features of Friedreich’s Ataxia

   1/50,000 incidence
   Commonest inherited ataxia
   Autosomal recessive inheritance
   Triplet repeat disease
   Spinal neuron degeneration
   Phenotype similar to Vitamin E transporter deficiency
Spinal cord dorsal root ganglion cells degenerate
     Control                   Patient
Friedreich’s ataxia pathology--heart
 Hypertrophic cardiomyopathy
 Cardiomyocyte degeneration
 Cardiac accumulation of iron and lipofuscin
What causes Friedriech’s ataxia?

Expansions of GAA reduce frataxin expression
                                          Fxn
         Frataxin                  Fxn
                                               Fxn
normal                                   Fxn



GAAGAAGAAGAAGAAGAAn



           Frataxin                 Fxn

mutant
Larger expansions, less frataxin, more severe disease




                               Campuzano et al. 1997, HMG
What is frataxin?
Frataxin is a nuclear-encoded gene expressed in mitochondria

                   Fxn Fxn
             Fxn
       Fxn
What are Mitochondria?
Mitochondria are plentiful in most human cells
Mitochondria participate in many pathways
   Energy                     Urea
   Generation                 cycle

  Apoptosis                  Fatty acid
                             oxidation
   Ca++                        Heme
   Buffering Nucleotide        Synthesis
             synthesis/salvage
Two points have dominated frataxin research



Question 1: What is frataxin’s correct function in
human cells?

Question 2: How does frataxin loss cause disease?
There are 5 potential functions of frataxin based on work in models:

                                (Mitochondrion)                   fxn
  iron transporter
                                                                  Fe

  iron-binding protein                  fxn
                                              Fe    Fe


                                                                fxn
  Fe-S cluster assembler                                 Fe+S           FeS

  Oxphos stimulator                    fxn         ATP

  Mitochondrial antioxidant
                                                     ROS        fxn
                                                                        ROS
We study the effects of frataxin-deficiency in
human cells
 5 Hypotheses for frataxin’s real function have been generated from
 work in Yeast, Bacteria, and Mice

 However Friedriech’s ataxia is a human disease

 Therefore we wanted to test which function for frataxin is most
 likely in human cells
 Human cell models of Friedreich’s ataxia:
Patient            Patient
                   Lymphoblasts   NT2 frataxin-RNAi neurons
Fibroblasts




Frataxin protein
Affymetrix Microarray format




 Oligonucleotides homologous to 12,599 genes on chip
 Most genes are of known function
Problems with microarray: Signal and Noise

Weak Signal of frataxin-deficiency relative to ‘Noise’.

What makes the ‘Noise’?:

    --inter-patient genetic variability

    --cell type variability

    --biochemical labeling variability
How to filter Signal from Noise:

 Use many chips to determine Mean +/- standard deviation.

 Use cells from many Friedreich’s ataxia patients.

 Compare results from different cell types.

 Use RT-PCR to confirm differential expression.
         Sulfur Amino Acid (SAA) & Iron-Sulfur Cluster (ISC) Synthesis




7 hits
Confirmation of Microarray data by QRT-PCR:

              TNF-microarray                                        TNF-QRTPCR

                                                    200000
600
                                                    180000
500                                                 160000

                                                    140000
400
                                                    120000

300                                                 100000

                                                     80000
200
                                                     60000

                                                     40000
100
                                                     20000

  0                                                      0
      c333   c621   p131   p585   p131-t   p585-t            c333   c621   p131   p585   p131-t   p585-t
        Summary of Microarray Data

The two major frataxin-dependent alterations are:
     A decrease in 7 SAA transcripts
     An increase in apoptosis transcripts

Of the 5 existing hypotheses, the score is 7:0:0:0:0 in favor
of an SAA/ISC hypothesis
Confirmatory tests of an ISC/SAA hypothesis


  Confirmation of decreased SAA/ISC transcripts by RT-PCR

  Measure a decrease in Sulfur Amino Acids concentration.

  Measure a decrease in Iron-Sulfur Cluster dependent
  enzymes.

  Identify a specific defect in ISC-biosynthetic pathway
Rhodanese & Cysteine Desulfurase mRNAs are deficient in mutants:

                Rhodanese           Rhodanese
                 Beta-actin


                                    (Maintains mitochondrial FeS clusters)




             Cysteine desulferase
                                      Cysteine Desulfurase ISC-S
             Beta-actin


                                      (provides Sulfur for FeS clusters)
Sulfur Amino Acid (SAA) & Iron-Sulfur Cluster (ISC) Synthesis
Sulfur Amino acid levels are decreased in FRDA cells:
In whole neural cell extracts inhibited by frataxin RNAi:

 A Acid                Control-NT2        Frataxin-RNAi          P-value
 Homocystine             0.99±0.20         0.29±0.19                0.01*
 Cystathionine         16.16±6.99         10.62±7.23                0.02*
 Serine                 109.7±9.0         88.71±4.91                0.03*


 Ornithine               6.65±2.25         4.80±2.11                0.02*


 Lymphoblast
 mitochondria:
 Cysteine              2.63               0.96                   0.03


 Ornithine             6.0                2.6                    0.03


 Glycine                59                39              0.05
Decrease in ISC-requiring enzyme activity
    Aconitase

    Succinate Dehydrogenase
Membrane potential is generated by TCA/Ox Phos




                Aconitase
Is there a frataxin-specific effect on the Iron-Sulfur Cluster
Biogenesis Machinery?
Sulfur Amino Acid (SAA) & Iron-Sulfur Cluster (ISC) Synthesis
Model of ISC formation from bacteria:
                           ISC-U
Cysteine   Alanine
                           ‘scaffold’

ISC-S              S
                                                              S
                                                              Fe
                                                                   Fe
                                                                   S
                           HscA


           chaperones          HscB



                                                                        Ferredoxin/
                                                                        Glutaredoxin
                                                                   *
                                             Insertion into
                                             Mitochondrial
                        Example:Active SDH   enzymes
ISC-U protein is decreased in patient lymphoblasts
       Lymphoblasts            Fibroblasts
Frataxin may be required for ISC-U stability or expression:

                           ISC-U
Cysteine   Alanine
                           ‘scaffold’

ISC-S              S
                                                                S
                                                                Fe
                                                                     Fe
                                                                     S
                           HscA

                                         fxn
           chaperones          HscB



                                                                          Ferredoxin/
                                                                          Glutaredoxin
                                                                     *
                                               Insertion into
                                               Mitochondrial
                        Example:Active SDH     enzymes
Frataxin may be required for the expression, or stability of ISC-U



                            ISC-U
                                                       Decreased ISCU
                                                       expression
  Cysteine   Alanine
                            ‘scaffold’                      Fe

                                                   S        S


  ISC-S              S
                                                       Fe


                           HscA                                  Decreased ISCU
                                                                 stability
             chaperones         HscB

                          Fxn
Confirmations of an SAA/ISC hypothesis

  7:0:0:0:0 score by Microarray in favor of ISC/SAA hypothesis

  Confirmation of decreased SAA/ISC transcripts by PCR

  Decreases in [SAA]s in cell extracts and mitochondria

  Decreases in Iron-Sulfur Cluster dependent enzymes.

  Identified a specific defect in ISC-U expression in FRDA cells

  Appears to be a direct interaction between ISC-U and frataxin
 How does a defect in FeS clusters cause cell death?


ISC-U             Increased free      Oxidative    Cell
‘scaffold’ Fe     (toxic) iron        Stress       Death
                          (stress pathway)

                              Decreased
               Decreased                          Cell
                              Mitochondrial
Fxn            FeS clusters                       Death
                              Enzyme activity

                  (Cluster-bioenergetic pathway)
 Potential routes to therapy in the stress pathway



                             Oxidative      Cell
Isc-U          Fe            Stress         Death
           Increased free
           (toxic) iron


Possible       Iron           Anti-         Anti-
               chelator       oxidants      apoptotics
Rescue                       Vitamin E      No clinically
                             Trolox         available
Agents        Desferal, L1   (not Vit C.)   anti-
                             Mito-Q         apoptotic..yet
                             Idebenone
 Fenton Chemistry contributes to oxidative stress


 .O2- + Fe3+ => Fe2+ + O2
Superoxide
(produced by mitochondria)



Fe2+ + H2O2 => .OH +                    HO- + Fe3+
             Hydrogen        Hydroxyl
             Peroxide        Radical
             (toxic)         (toxic)
Frataxin levels affect free mitochondrial iron
                                 120


                                 100
          Filtrable Mito. iron
          (pmol/mg prote in)



                                  80

  Bound
                                  60


                                  40
  Free

                                  20                     Free Mito. Iron

                                   0
                                       Ctl   CH   CH-t
Peroxides are increased in FRDA mitochondria
                                                  A

                                  350
       (pmol/mg protein/60 min)
                                  300

                                  250
             DCF content




                                  200

                                  150

                                  100

                                  50

                                   0
                                        Control       FRDA   FRDA-t
  Potential routes to therapy in the stress pathway



                              Oxidative      Cell
Isc-U          Fe             Stress         Death
           Increased free
           (toxic) iron


Possible       Iron            Anti-         Anti-
               chelator        oxidants      apoptotics
Rescue                        Vitamin E      No clinically
                              Trolox         available
Agents        Desferal, L1    (not Vit C.)   anti-
                              Mito-Q         apoptotic..yet
                              Idebenone
 Potential routes to therapy in the Cluster-bioenergetic pathway


              (Cluster-bioenergetic pathway)
                                 Decreased
ISC-U         Decreased
                                 Mitochondrial
                                                       Cell
              FeS clusters                             Death
                                 Enzyme activity


              Supplement          Supplement        Anti-apoptotics
Potential     Non-enzymatic       Mitochondrial
Rescue        FeS Cluster         Activity
Agents        Synthesis

              Thiosulfate?         Idebenone,         Not yet
Examples      Sulfur               Creatine           available
              compounds?
Sulfide rescues Friedreich’s ataxia cells sensitivity to Oxidative Stress
                             120


                             100
  (% of untreated control)



                                                                 Untreated
                             80
                                                                 t-BOOH
          Viability




                                                                 Vitamin B12
                             60
                                                                 Sulfate
                                                                 Sulfite
                             40
                                                                 Sulfide

                             20


                              0
                                   control    FRDA
                              1                             Control
ZVAD protects FRDA cells                       *
From apoptosis              0.75
                                                   - z-VAD.fmk


                             0.5



  1.5                       0.25


                                    + z-VAD.fmk
                              0
                                   0               200        400        600         800
                                                            H2 O2 (uM)
                                                            FRDA
   1                                1         *
                                                   **
                               0.75
                                                           **
  0.5                              0.5
                                                                + z-VAD.fmk


                               0.25
                                             - z-VAD.fmk
   0
                                    0
                                         0          200       400    600       800
           control   FRDA                                   H2O2(uM)
  Potential routes to therapy in the Cluster-bioenergetic pathway


               (Cluster-bioenergetic pathway)
                                  Decreased
ISC-U          Decreased                                Cell
                                  Mitochondrial
               FeS clusters                             Death
                                  Enzyme activity


               Supplement          Supplement        Anti-apoptotics
Potential      Non-enzymatic       Mitochondrial
Rescue         FeS Cluster         Activity
Agents         Synthesis

               Thiosulfate?         Idebenone,         Not yet
Examples       Sulfur               Creatine           clinically
               compounds?                              available
Summary
1. Microarray only supports one of 5 hypotheses for frataxin.
2. There are multiple confirmations of FeS in human cells:
      Decreased ISC/SAA transcripts by QRT-PCR
      Decreased SAA levels
      Defects in ISC-dependent enzymes
      Defects in expression of ISC-U
3. There is evidence for increased apoptosis in human cells
4. Some therapy is available, and some others may soon
     be possible, but there is a very long way to go to
     specific, curative FRDA therapy
  (We have a need for more cells from individuals with FRDA,
  and hope you could help)
Guolin Tan        UC Davis- VM:Molecular Biosciences
Eleonora Napoli   UC Davis & Universita di Padova


Gino Cortopassi   UC Davis

Franco Taroni     Istituto Neurologico ‘Carlo Besta’, Milan, Italy




                                                PHS/NIH
                                                FARA Foundation

				
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Description: Testing five hypotheses for Friedreich s ataxia in human cells Idebenone