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Neuronatin web3
1.
2. Can We Regenerate Neurons?
Cloning of Neuronatin
Rajiv M. Joseph, MD, PhD, FAAN
Attending Neurologist, Presbyterian Hospital of Plano
Clinical Associate Professor, University of Texas,
Southwestern Medical Center
Dallas, Texas
www.DallasNeurology.com
3. 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
4. SECTIONS x 2
1: Brain Development
2: Cloning of Neuronatin
- a new human gene
5. 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
6. 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
7. 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
9. 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
10. FETAL VS. ADULT NEURONS
Fetal Neurons
Grow
Adult Neurons
Do Not grow
Sanjay Gupta at 100!
Growth & Aging
are Predictable
Genetically
Determined
11. 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.
13. 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
14. FETAL VS. ADULT NEURONS
Both have the
same genomic
DNA
Are Differences in
Yakolov mRNA Expression
Responsible?
16. 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
18. 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
20. 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
21. NOVEL
mRNA FRAGMENTS
224 Bases
N = Neonatal Rats
Y = Young (3 mths)
A = Aged (33 mths)
Differential Display
22. 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
23. 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
24. 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
25. Neuronatin mRNA:
Fetal vs. Adult Human
Fetal Adult
Controls NEURONATIN Controls
Expression in Human Fetal Brain
RJ et al, Brain Research, 1995
26. 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
27. Neuronatin mRNA:
Brain vs. Other Organs
Neuronatin
G3PDH
B L L K
Selective Expression in Brain Tissue
28. 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
29. Neuronatin mRNA:
Neurons vs. Glia
Selective Expression in Neurons
30. 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
31. Methods: Step- 2
Find the Chromosomal localization for
Neuronatin Gene
Sequence Neuronatin DNA (Gene)
Map Neuronatin DNA
35. 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
36. Neuronatin DNA: The Gene
Full Human Sequence
3973 Bases
Exons x 3
Introns x 2
Promoter: NRSE
GenBank #U31767
Dou & RJ, Genomics, 1996
39. 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
40. Methods: Step- 3
Predict the protein structure of
Neuronatin
Study the role of Neuronatin in cell
growth
42. 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
44. Neuronatin Protein
Hydrophobic
N-Terminal
Hydrophilic
C-Terminal
Homology to Ion Channels
45. 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
48. 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
49. 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
50. 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
51. 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
52. Neuronatin: June 30, 1994
54 Peer-Reviewed Publications……….
(PubMed, 08/18/2008)
53. 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)
54. 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)
55. 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)
56. 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)
57. 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…………..
59. 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?
