Can We Regenerate Neurons?
Cloning of Neuronatin
Rajiv M. Joseph, MD, PhD, FAAN
Attending Neurologist, Presbyterian Hospit...
Overview:
This presentation is divided into two parts.


   Part-1, is a brief overview of normal brain
  development, an ...
SECTIONS x 2

 1: Brain Development

 2: Cloning of Neuronatin
         - a new human gene
Brain Development: Neural Tube
   All vertebrates exhibit a similar pattern and
  framework during their development

   F...
Vertebrates: The Neural Tube

                 “I have two
                 small embryos
                 preserved in
  ...
Neural Proliferation:
   After the formation of the Neural Tube,
  there is a sudden and rapid proliferation of
  the cell...
Neuronal Development

                   Neural Tube

                   Explosive Growth

                   Apoptosis/De...
Growth & Aging:
   We do not fully understand the basis for
  growth and aging

   Fetal brain cells (neurons) grow, but a...
FETAL VS. ADULT NEURONS
                       Fetal Neurons
                       Grow

                       Adult Neu...
Genetic Machinery: 3 Levels
  A cell’s genetic machinery consists of three levels

  Level-1, is the DNA, also called geno...
Genes & Function
                           = Hardware




          /Expression (= Software)


          /Function (= Pro...
Why do Adult Neurons Stop
        Growing?
 An important quest in biology is a better
understanding of why fetal neurons g...
FETAL VS. ADULT NEURONS

                    Both have the
                   same genomic
                             DN...
THE GOAL?




Find Genes Selectively Expressed
  in Growing (Fetal) Neurons
Methods: 3 Steps
A three-step approach was utilized:

  Step-1, find a novel mRNA expressed in the
  fetal brain, but not ...
METHODS
               STEP-2




 /Expression       1
               STEP-


 /Function     STEP-3
Methods: Step-1
  Find fragments of novel genes expressed
 only in fetal brain, not the adult

  Use the novel fragments t...
PROCEDURE:
Step-1




     1. Fragments (Differential Display)
     2. Full length (Screen cDNA Library)
Results: Novel mRNA Fragments
   We studied Neonatal (3 days), Adult (3 months)
  and Aged (33 months) rats

  A technique...
NOVEL
      mRNA FRAGMENTS


                       224 Bases

                       N = Neonatal Rats
                  ...
Results: Full-Length Neuronatin
   Using the novel mRNA (cDNA) fragments, a cDNA library made from
  fetal rat was screene...
NEUROns + NeoNATes + IN = Neuronatin mRNA



Exon-1
                                  1195 Bases
                         ...
Neuronatin: Expressed in Fetal Brain

  Neuronatin mRNA expression was
 studied using a technique called
 Northern Blottin...
Neuronatin mRNA:
           Fetal vs. Adult Human


              Fetal   Adult




Controls      NEURONATIN            Co...
Neuronatin: Expressed in Brain

Neuronatin mRNA expression was
studied using Northern Blotting in
different body organs

N...
Neuronatin mRNA:
   Brain vs. Other Organs


                         Neuronatin




                         G3PDH

     ...
Neuronatin: Expressed in Neurons

 Neuronatin mRNA expression was
further evaluated in neuronal and glial
cell lines

 Neu...
Neuronatin mRNA:
    Neurons vs. Glia




Selective Expression in Neurons
Results of Step-1: Summary
  A novel mRNA sequence is identified
   The mRNA is named,
 NEUROns + neoNATes + IN = NEURONAT...
Methods: Step- 2
 Find the Chromosomal localization for
 Neuronatin Gene

  Sequence Neuronatin DNA (Gene)

  Map Neuronat...
PROCEDURE:
Step-2
                       1. Chromosomal Localization
                       2. Gene Sequence
             ...
Step-2: Chromosomal Localization

   Neuronatin is located on the long arm
  of Chromsome-20 in the segment 11.2-
  12
Neuronatin DNA:
Chromosome-20q11.2-12




    Chromosome: 20

    Long arm: q

    Segment: 11.2-12
Step-2: The Complete Gene
      Sequence of Neuronatin
Neuronatin DNA is 3973 bases long

There are three Exons and two In...
Neuronatin DNA: The Gene
Full Human Sequence


                                3973 Bases

                               ...
Neuronatin DNA: Gene Map




     PROMOTER   E1   E2   E3
Neuronatin DNA: Promoter




   Basis for Neuronal Specificity
Results of Step-2: Summary
  NEURONATIN DNA is located on Chromosome-
  20q11.2-12

  NEURONATIN DNA has three Exons and t...
Methods: Step- 3

  Predict the protein structure of
 Neuronatin

  Study the role of Neuronatin in cell
 growth
PROCEDURE:
Step-3
                       Neuronatin DNA (Gene)
                       Chromosome 20q11.2-12




    Neuron...
Step-3: Neuronatin Protein

   The predicted Neuronatin protein has
  a hypdrophobic N-terminal and a
  hydrophilic C-term...
Neuronatin Protein
Neuronatin Protein

                  Hydrophobic
                  N-Terminal


                  Hydrophilic
           ...
Step-3: Neuronatin & Cell Growth

   In order to study the role of Neuronatin in cell
  growth, we utilized PC12 cells and...
Neuronatin: Plasmid Constructs


   CONTROL    NNAT       NNAT

              (+)         (-)



      Induces Neuronal Gr...
Neuronatin Function:
PC12 Cells + Plasmid Constructs
             NNAT
              (+)


                               ...
Results of Step-3: Summary
  NEURONATIN Protein has a predicted hydrophobic
  N-terminal and a hydrophilic C-terminal

  N...
The Neuronatin Story
Neuronatin DNA (Gene)
Chromosome 20q11.2-12
Promoter has NRSE

            Neuronatin mRNA
          ...
Neuronatin: Publications
  Joseph, Dou & Tsang, BBRC 1994
  Joseph, Dou & Tsang, Brain Research 1995
  Dou & Joseph, Genom...
The Neuronatin Work Continues
   Since its discovery in 1994, there are
  42 peer-reviewed publications
   (PubMed, August...
Neuronatin:                  June 30, 1994




 42           Peer-Reviewed Publications……….
 (PubMed, 08/18/2008)
Neuronatin & Diabetes
   Neuronatin is expressed in the islet cells of the pancreas, the
   cells that produce insulin

  ...
Neuronatin & Cancer
   Medulloblastoma, Neuroblastoma,
Yokota et al. Oncogene 2004; 23:3444-53 (University of Toronto)
Hig...
Neuronatin & HindBrain Segmentation

   Neuronatin is involved in the
  segmentation of the hindbrain during
  brain devel...
Neuronatin: A Paternally Imprinted Gene

   Imprinted Gene, Paternally Expressed
 Kagitani et al. Nuc Acids Res 1997; 25:3...
Hindbrain
Segmentation                          Imprinted Gene,
Max Planck Institute 1995             Paternally Expressed...
Unsolved…….
              Neuronatin mutations
              Neural Tube Defects?
              Ataxia Syndromes?

       ...
CONCLUSIONS:
          Neuronatin
Is a New Human Gene.

Expressed in Growing
(Fetal) Neurons.
                       Unive...
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Neuronatin presentation

  1. 1. Can We Regenerate Neurons? Cloning of Neuronatin Rajiv M. Joseph, MD, PhD, FAAN Attending Neurologist, Presbyterian Hospital of Plano Clinical Assistant Professor, University of Texas, Southwestern Medical Center Dallas, Texas www.DallasNeurology.com
  2. 2. Overview: This presentation is divided into two parts. Part-1, is a brief overview of normal brain development, an important background for our study Part-2, will address the salient features of work leading to the discovery of Neuronatin
  3. 3. SECTIONS x 2 1: Brain Development 2: Cloning of Neuronatin - a new human gene
  4. 4. Brain Development: Neural Tube All vertebrates exhibit a similar pattern and framework during their development Following fertilization, there is a rapid growth of cells to form a plate like structure, called the Neural Plate. The plate then folds and closes to form a Neural Tube The appearance of the Neural Tube is remarkably similar among the different vertebrate species, including humans
  5. 5. Vertebrates: The Neural Tube “I have two small embryos preserved in alcohol that I forgot to label... Romanes, 1901 They may be lizards, small birds or even mammals”. Karl von Baer 1828
  6. 6. Neural Proliferation: After the formation of the Neural Tube, there is a sudden and rapid proliferation of the cells lining the inner wall of the Neural Tube Thereafter, this increased mass of cells are rearranged and pruned. As part of this remodeling process, there is selective cell death as well
  7. 7. Neuronal Development Neural Tube Explosive Growth Apoptosis/Death E13-15 E16-18
  8. 8. Growth & Aging: We do not fully understand the basis for growth and aging Fetal brain cells (neurons) grow, but adult neurons do not grow We know that both growth and aging are predictable, and would appear to be genetically governed
  9. 9. FETAL VS. ADULT NEURONS Fetal Neurons Grow Adult Neurons Do Not grow Sanjay Gupta at 100! Growth & Aging are Predictable Genetically Determined
  10. 10. Genetic Machinery: 3 Levels A cell’s genetic machinery consists of three levels Level-1, is the DNA, also called genomic DNA. DNA is where the genes reside, and is located in the nucleus of the cell. You could think of DNA as similar to the hardware in a computer Level-2, is the mRNA. mRNA is generated by DNA and helps to transfer genetic information from DNA to the cytoplasm in order to translate proteins that mediate function. Consider mRNA to be similar to a software in a computer Level-3, is the protein. The mRNA that arrives in the cytoplasm from the nucleus is used to generate a protein that effects the specific function of the gene.
  11. 11. Genes & Function = Hardware /Expression (= Software) /Function (= Product)
  12. 12. Why do Adult Neurons Stop Growing? An important quest in biology is a better understanding of why fetal neurons grow, but the adult neuron does not. We know that both fetal and adult neurons must have the same genomic DNA Therefore, it seems reasonable to think that the difference between the fetal and adult neuron may be due to differences in their patterns of mRNA expression
  13. 13. FETAL VS. ADULT NEURONS Both have the same genomic DNA Are Differences in Yakolov mRNA Expression Responsible?
  14. 14. THE GOAL? Find Genes Selectively Expressed in Growing (Fetal) Neurons
  15. 15. Methods: 3 Steps A three-step approach was utilized: Step-1, find a novel mRNA expressed in the fetal brain, but not in the adult Step-2, use the novel mRNA to find and sequence the DNA (Gene) Step-3, study the protein generated by the novel mRNA/Gene
  16. 16. METHODS STEP-2 /Expression 1 STEP- /Function STEP-3
  17. 17. Methods: Step-1 Find fragments of novel genes expressed only in fetal brain, not the adult Use the novel fragments to screen a cDNA (mRNA) library made from fetal brain, and identify the full-length sequence of the novel mRNA
  18. 18. PROCEDURE: Step-1 1. Fragments (Differential Display) 2. Full length (Screen cDNA Library)
  19. 19. Results: Novel mRNA Fragments We studied Neonatal (3 days), Adult (3 months) and Aged (33 months) rats A technique called differential display was used to make the comparisons Five novel mRNA fragments were identified from neonatal rat; one such fragment is shown in the accompanying picture. This first unique sequence was 224 bases long
  20. 20. NOVEL mRNA FRAGMENTS 224 Bases N = Neonatal Rats Y = Young (3 mths) A = Aged (33 mths) Differential Display
  21. 21. Results: Full-Length Neuronatin Using the novel mRNA (cDNA) fragments, a cDNA library made from fetal rat was screened and the full length sequence identified and sequenced. The sequence is 1195 bases long and has three Exons. Exon-1 has 24 amino acids, Exon-2 has 27, and Exon-3 has 30. There are two isoforms of this novel mRNA. The alpha form has all three Exons. The beta form has only two Exons, Exon-1 and Exon-3. The sequence was unique and named Neuronatin, the name derived from finding the gene in Neurons of Neonatal Brain: NEUROns + neoNATes + IN = NEURONATIN GenBank Submissions: Alpha: #U08290 and Beta: #U09785
  22. 22. NEUROns + NeoNATes + IN = Neuronatin mRNA Exon-1 1195 Bases EXONS Exon-2 I: 24 aa II: 27 aa Exon-3 III: 30 aa SPLICED FORMS Alpha:3 Exons #U08290 Beta: 2 Exons #U09785 cDNA Library= Full Length RJ et al, BBRC 1994
  23. 23. Neuronatin: Expressed in Fetal Brain Neuronatin mRNA expression was studied using a technique called Northern Blotting in fetal and adult human brain Neuronatin mRNA was expressed in fetal brain, but not in the adult brain
  24. 24. Neuronatin mRNA: Fetal vs. Adult Human Fetal Adult Controls NEURONATIN Controls Expression in Human Fetal Brain RJ et al, Brain Research, 1995
  25. 25. Neuronatin: Expressed in Brain Neuronatin mRNA expression was studied using Northern Blotting in different body organs Neuronatin mRNA was expressed in brain, but not in lungs, liver and kidney, the other organs we studied
  26. 26. Neuronatin mRNA: Brain vs. Other Organs Neuronatin G3PDH B L L K Selective Expression in Brain Tissue
  27. 27. Neuronatin: Expressed in Neurons Neuronatin mRNA expression was further evaluated in neuronal and glial cell lines Neuronatin mRNA was expressed in neuronal cell lines, but not in glial or supporting cells
  28. 28. Neuronatin mRNA: Neurons vs. Glia Selective Expression in Neurons
  29. 29. Results of Step-1: Summary A novel mRNA sequence is identified The mRNA is named, NEUROns + neoNATes + IN = NEURONATIN NEURONATIN mRNA is expressed in Fetal Humans, but not in Adults NEURONATIN mRNA is expressed in Brain, but not in several other organs studied NEURONATIN mRNA is expressed in Neurons but not in glia and supporting brain cells
  30. 30. Methods: Step- 2 Find the Chromosomal localization for Neuronatin Gene Sequence Neuronatin DNA (Gene) Map Neuronatin DNA
  31. 31. PROCEDURE: Step-2 1. Chromosomal Localization 2. Gene Sequence 3. Gene Map Neuronatin mRNA
  32. 32. Step-2: Chromosomal Localization Neuronatin is located on the long arm of Chromsome-20 in the segment 11.2- 12
  33. 33. Neuronatin DNA: Chromosome-20q11.2-12 Chromosome: 20 Long arm: q Segment: 11.2-12
  34. 34. Step-2: The Complete Gene Sequence of Neuronatin Neuronatin DNA is 3973 bases long There are three Exons and two Introns The promoter has a unique Neural-Restrictive- Silencer-Element (NRSE) that governs neuron-specific expression of the gene GenBank Submission #U31767
  35. 35. Neuronatin DNA: The Gene Full Human Sequence 3973 Bases Exons x 3 Introns x 2 Promoter: NRSE GenBank #U31767 Dou & RJ, Genomics, 1996
  36. 36. Neuronatin DNA: Gene Map PROMOTER E1 E2 E3
  37. 37. Neuronatin DNA: Promoter Basis for Neuronal Specificity
  38. 38. Results of Step-2: Summary NEURONATIN DNA is located on Chromosome- 20q11.2-12 NEURONATIN DNA has three Exons and two Introns NEURONATIN DNA has a promoter that has a unique Neural-Restrictive Silencer Element (NRSE) that governs neuron-specific expression
  39. 39. Methods: Step- 3 Predict the protein structure of Neuronatin Study the role of Neuronatin in cell growth
  40. 40. PROCEDURE: Step-3 Neuronatin DNA (Gene) Chromosome 20q11.2-12 Neuronatin mRNA 1. Protein Structure 2. Growth - P12 Cells / Plasmid Constructs
  41. 41. Step-3: Neuronatin Protein The predicted Neuronatin protein has a hypdrophobic N-terminal and a hydrophilic C-terminal The protein structure is similar to a class of proteins called proteolipids, including PMP1 and phospholamban, that may function as ion-channels
  42. 42. Neuronatin Protein
  43. 43. Neuronatin Protein Hydrophobic N-Terminal Hydrophilic C-Terminal Homology to Ion Channels
  44. 44. Step-3: Neuronatin & Cell Growth In order to study the role of Neuronatin in cell growth, we utilized PC12 cells and plasmids with and without a functional Neuronatin gene. Two different clones of PC12 cells were generated, one expressing Neuronatin and the other not expressing Neuronatin. In preliminary experiments, the PC12 cell clone expressing Neuronatin grew faster than the clone not expressing Neuronatin
  45. 45. Neuronatin: Plasmid Constructs CONTROL NNAT NNAT (+) (-) Induces Neuronal Growth
  46. 46. Neuronatin Function: PC12 Cells + Plasmid Constructs NNAT (+) Induces CTL NNAT Neuronal (-) Growth RJ et al, Brain Research, 1996
  47. 47. Results of Step-3: Summary NEURONATIN Protein has a predicted hydrophobic N-terminal and a hydrophilic C-terminal NEURONATIN Protein has homology to a group of proteins called proteolipids that may function as ion channels NEURONATIN, in preliminary experiments, may promote cell growth
  48. 48. The Neuronatin Story Neuronatin DNA (Gene) Chromosome 20q11.2-12 Promoter has NRSE Neuronatin mRNA Two Spliced Forms Neuron-Specific Neuronatin Protein Homology to Ion Channels Promotes Neuronal Growth
  49. 49. Neuronatin: Publications Joseph, Dou & Tsang, BBRC 1994 Joseph, Dou & Tsang, Brain Research 1995 Dou & Joseph, Genomics 1996 Dou & Joseph, Brain Research 1996 Joseph et al, Brain Research 1996 University of London, PhD Thesis, 1996 United States Patent, #5,837,535; 1998
  50. 50. The Neuronatin Work Continues Since its discovery in 1994, there are 42 peer-reviewed publications (PubMed, August 18, 2008) Neuronatin has been shown to be an imprinted gene involved in Diabetes, Cancer and Hindbrain Segmentation
  51. 51. Neuronatin: June 30, 1994 42 Peer-Reviewed Publications………. (PubMed, 08/18/2008)
  52. 52. Neuronatin & Diabetes Neuronatin is expressed in the islet cells of the pancreas, the cells that produce insulin The Neuronatin Promoter has a binding site for Beta-2/Neuro- D1, a transcription factor that is known to stimulate glucose- mediated insulin secretion Niwa et al. Endocrinology; 1997 138:1419-26 (Royal Melbourne Hospital, Australia) Arava et al. Diabetes 1999; 48:552-6 (Weizman Institute, Israel) Chu & Tsai. Diabetes 2005; 54:1064-73 (Baylor College Medicine)
  53. 53. Neuronatin & Cancer Medulloblastoma, Neuroblastoma, Yokota et al. Oncogene 2004; 23:3444-53 (University of Toronto) Higashi et al. J Pediatr Hematol Oncol 2007; 29:551-6 (Kyushu University) Lung Cancer Okubo et al. J. Thorac Oncol 2006; 1:780-6 (University of Tsukuba) Uchihara et al. J. Thorac Oncol 2007; 2:796-801 (University of Ryukyus) Pituitary Usui et al. J. Mol Neurosci 1997; 9:55-60 (Niigata university)
  54. 54. Neuronatin & HindBrain Segmentation Neuronatin is involved in the segmentation of the hindbrain during brain development Wijnholds et al. Dev Biol 1995; 17:73-84 (Max Planck Institute, Germany)
  55. 55. Neuronatin: A Paternally Imprinted Gene Imprinted Gene, Paternally Expressed Kagitani et al. Nuc Acids Res 1997; 25:3428-32 (Tokyo Institute of Technology) Kikyo et al. Dev Biol 1997; 190:66-77 (University of Cambridge) Evans et al. Genomics 2001; 77:99-104 (Duke University)
  56. 56. Hindbrain Segmentation Imprinted Gene, Max Planck Institute 1995 Paternally Expressed Tokyo Institute of Technology, 1996 University of Cambridge, 1997 Duke University, 2001 Diabetes & Pituitary Niigata university, 1997 Iset Cell Medulloblastoma, Function Royal Melborne Hospital, 1997 Neuroblastoma, Weizman Institute, Israel, 1999 University of Toronto, 2004 Baylor College Medicine, Houston, 2005 Kyushu University, 2007 Wellcome/CRC, Cambridge, 2001 Lung Cancer Protects PC 12 cells University of Tsukuba, 2006 UCLA 2002 University of Ryukyus, 2007 Neuronatin CONTI NUES TO GR OW…………..
  57. 57. Unsolved……. Neuronatin mutations Neural Tube Defects? Ataxia Syndromes? Genetic Engineering Rekindle Growth & Function
  58. 58. CONCLUSIONS: Neuronatin Is a New Human Gene. Expressed in Growing (Fetal) Neurons. University of London, Homology to Ion PhD Thesis Channel Proteins. May Rekindle Growth in Adult Neurons?

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