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

SECTIONS x 2

1: Brain Development

2: Cloning of Neuronatin- a new human gene

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

Neuronal Development

Neural Tube

Explosive Growth

Apoptosis/Death

E13-15 E16-18

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.

Genes & Function= Hardware

/Expression (= Software)

/Function (= Product)

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

THE GOAL?

Find Genes Selectively Expressed in Growing (Fetal) Neurons

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-2

/Expression STEP- 1

/Function STEP-3

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

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 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

Neuronatin mRNA: Neurons vs. Glia

Selective Expression in Neurons

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

PROCEDURE: Step-2

1. Chromosomal Localization2. Gene Sequence3. Gene Map

Neuronatin mRNA

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 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

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 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

Neuronatin Protein

Neuronatin Protein

Hydrophobic

N-Terminal

Hydrophilic

C-Terminal

Homology to 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: Plasmid Constructs

CONTROL NNAT

(+)

NNAT

(-)

Induces Neuronal Growth

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: June 30, 1994

42 Peer-Reviewed Publications………. (PubMed, 08/18/2008)

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