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Can We Regenerate Neurons? Cloning of NeuronatinRajiv M. Joseph, MD, PhD, FAANAttending Neurologist, Presbyterian Hospital of PlanoClinical Assistant Professor, University of Texas, Southwestern Medical CenterDallas, Texas
www.DallasNeurology.com
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
Brain Development: Neural TubeAll 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
Vertebrates: The Neural Tube
“I have two small embryos preserved in alcohol that I forgot to label...
They may be lizards, small birds or even mammals”.
Romanes, 1901
Karl von Baer 1828
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
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
FETAL VS. ADULT NEURONS
Fetal Neurons Grow
Adult Neurons Do Not grow
Growth & Aging are Predictable
Genetically Determined
Sanjay Gupta at 100!
Genetic Machinery: 3 LevelsA 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.
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
FETAL VS. ADULT NEURONS
Both have the same genomic
DNA
Are Differences in mRNA Expression
Responsible?Yakolov
Methods: 3 StepsA 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
Methods: Step-1Find 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
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
NOVEL mRNA FRAGMENTS
224 Bases
N = Neonatal RatsY = Young (3 mths)A = Aged (33 mths)
Differential Display
Results: Full-Length NeuronatinUsing 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
NEUROns + NeoNATes + IN = Neuronatin mRNA
1195 Bases EXONS
I: 24 aa
II: 27 aa
III: 30 aa
SPLICED FORMS
Alpha:3 Exons #U08290
Beta: 2 Exons #U09785
RJ et al, BBRC 1994cDNA Library= Full Length
Exon-1
Exon-2
Exon-3
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
Neuronatin mRNA: Fetal vs. Adult Human
Fetal Adult
NEURONATIN ControlsControls
RJ et al, Brain Research, 1995
Expression in Human Fetal Brain
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
Neuronatin mRNA: Brain vs. Other Organs
B L L K
Neuronatin
G3PDH
Selective Expression in Brain Tissue
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
Results of Step-1: SummaryA 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
Methods: Step- 2Find the Chromosomal localization for
Neuronatin Gene
Sequence Neuronatin DNA (Gene)
Map Neuronatin DNA
Step-2: Chromosomal Localization
Neuronatin is located on the long arm of Chromsome-20 in the segment 11.2- 12
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
Neuronatin DNA: The Gene Full Human Sequence
3973 Bases
Exons x 3
Introns x 2
Promoter: NRSE
GenBank #U31767
Dou & RJ, Genomics, 1996
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
Methods: Step- 3
Predict the protein structure of Neuronatin
Study the role of Neuronatin in cell growth
PROCEDURE: Step-3
Neuronatin mRNA
Neuronatin DNA (Gene)
Chromosome 20q11.2-12
1. Protein Structure2. Growth - P12 Cells / Plasmid Constructs
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
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
Neuronatin Function: PC12 Cells + Plasmid Constructs
CTL
NNAT
(+)
NNAT
(-)
RJ et al, Brain Research, 1996
Induces
Neuronal
Growth
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
The Neuronatin Story
Neuronatin ProteinHomology to Ion ChannelsPromotes Neuronal Growth
Neuronatin mRNATwo Spliced FormsNeuron-Specific
Neuronatin DNA (Gene)Chromosome 20q11.2-12Promoter has NRSE
Neuronatin: Publications
Joseph, Dou & Tsang, BBRC 1994Joseph, Dou & Tsang, Brain Research 1995Dou & Joseph, Genomics 1996Dou & Joseph, Brain Research 1996Joseph et al, Brain Research 1996
University of London, PhD Thesis, 1996United States Patent, #5,837,535; 1998
The Neuronatin Work ContinuesSince 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
Neuronatin & DiabetesNeuronatin 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)
Neuronatin & CancerMedulloblastoma, 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 CancerOkubo 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)
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)
Neuronatin: A Paternally Imprinted Gene
Imprinted Gene, Paternally ExpressedKagitani 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)
Protects PC 12 cellsUCLA 2002
Hindbrain SegmentationMax Planck Institute 1995
Imprinted Gene, Paternally ExpressedTokyo Institute of Technology, 1996University of Cambridge, 1997Duke University, 2001
Diabetes & Iset Cell FunctionRoyal Melborne Hospital, 1997Weizman Institute, Israel, 1999Baylor College Medicine, Houston, 2005 Wellcome/CRC, Cambridge, 2001
Medulloblastoma, Neuroblastoma, University of Toronto, 2004Kyushu University, 2007
Neuronatin CONTINUES TO GROW…………..
Pituitary Niigata university, 1997
Lung Cancer University of Tsukuba, 2006 University of Ryukyus, 2007
Unsolved…….Neuronatin mutationsNeural Tube Defects?Ataxia Syndromes?
Genetic EngineeringRekindle Growth & Function
CONCLUSIONS: Neuronatin
Is a New Human Gene.
Expressed in Growing (Fetal) Neurons.
Homology to Ion Channel Proteins.
May Rekindle Growth in Adult Neurons?
University of London, PhD Thesis