Discovery of Neuro-regenerative
Natural Products from Marine Algae
and Cyanobacteria
Amanda Cordes, Dr. Doug
Goeger, Dr. William Gerwick
Background


At present, there are an estimated
243,000 people living with spinal cord
injuries in the United States
53% of new injuries occur within the
16-30 age group
Goals

Identify a small molecule from
algae/cyanobacteria which exhibits
activity in stimulating nerve
regeneration



Many algae compounds are similar to
retinoic acid and eicosanoids
A marine sponge compound,
Lembehyne A, promotes neurite
outgrowth
Known outgrowth promoters stop cell
cycle progression, as do many of the
lab’s characterized compounds
First – Defining Some Terms

Neuro 2a Neuroblastoma Cells: A mouse
cancer cell line deriving from neurons

Neuron: Cell with capability of transmitting
electric signals, found in nervous system

Neurite: Long, branching outgrowth from a
neuron

Differentiate: Cells mature, adopt distinctive
functions, less likely to divide
http://cancerweb.ncl.ac.uk
Neuro 2a Cells with Neurites
Experimental Protocol



Neuro 2a cells are cultured in serum
supplemented medium
Cells then exposed to novel marine
extracts, observed in 24 hr increments
Neurite outgrowth compared against
untreated control cells and ones
treated with Lactacystin and with 8-BrcAMP, known outgrowth promoters
% of cells showing outgrowths
Neurite Outgrowth Controls
25
Treated
20
RPMI w/
30μL DMSO
5 μM
15
10
L a c ta c y s tin
5
0
24 48 72
hrs hrs hrs
Control
Choosing a Concentration
1454 Fraction
Concentration (μg/ml)
30
10
3
1
0.3
0.1
F
91
74
19
G
95
90
64
H
97
96
95
66
21
11
I
97
96
96
61
21
Values are % lethality

52 pure compounds

27 crude fractions

One worth mentioning…..
…Symploca
www.sitesatlas.com
Symploca 1454 Fractions

1454 A



0.53%
0.27%
1454 B



30 μg/mL:
10 μg/mL:
30 μg/mL:
10 μg/mL:
.32%
1.2%
1454 C


30 μg/mL:
10 μg/mL:
Toxic
.35%
Cont’d

1454 D



0.55%
1.5%
1454 E



1 μg/mL:
0.3 μg/mL:
10 μg/mL:
3 μg/mL:
1.9%
.88%
1454 F


3 μg/mL:
1 μg/mL:
4.0%
.48%
Cont’d

1454 G



3.1%
4.0%
1454 H



1 μg/mL:
0.3 μg/mL:
0.3 μg/mL:
0.1 μg/mL:
5.2%
0.9%
1454 I


0.3 μg/mL:
0.1 μg/mL:
3.2%
1.5%
Sub fractions

H-1



0.003 μg/mL:
0.001 μg/mL:
4.5%
0.8%
H-2


0.1 μg/mL:
0.03 μg/mL:
1.1%
1.0%
3.00
996
996
996
996
at
at
at
at
254.00
230.00
216.00
340.00
H-1 Peak
2.50
2.00
AU
1.50
1.00
Baseline 1
Baseline 2
0.50
0.00
10.00
20.00
30.00
40.00
Minutes
50.00
60.00
70.00
Assay Results

H-1 Peak



1.0%
1.6%
Baseline 1



0.03 μg/mL:
0.01 μg/mL:
0.003 μg/mL:
0.001 μg/mL:
1.8%
0.5%
Baseline 2


0.003 μg/mL:
0.001 μg/mL:
2.0%
0.6%
Possible Synergisms?
0.03 μg/mL
0.03 μg/mL
0.003 μg/mL
0.003 μg/mL
…..perhaps….

H-1 Peak and Baseline 1


2.3%
H-1 Peak and Baseline 2



4.3%
6.5%
4.3%
…..results, conclusions, and
future plans…

Further investigation of Symploca
compounds

Continued screening of crude fractions
and newly purified compounds
Acknowledgements

Rebecca Medina

Gerwick Lab

Kevin Ahern & HHMI
References

Blanco et al., Cell Biol. Int. 2001, 25,
909

Fenteany et al., J. Biol. Chem.1998,
273, 8545

Aoki et al., Biochem. Biophys. Res.
Com. 2001, 289, 558