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 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

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 First – Defining Some Terms

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-Br- cAMP, known outgrowth promoters

Neurite Outgrowth Controls Treated Control

Choosing a Concentration Concentration (μg/ml) F G H I Fraction Values are % lethality

52 pure compounds 27 crude fractions One worth mentioning…..

…Symploca

Symploca 1454 Fractions 1454 A  30 μg/mL:0.53%  10 μg/mL:0.27% 1454 B  30 μg/mL:.32%  10 μg/mL:1.2% 1454 C  30 μg/mL:Toxic  10 μg/mL:.35%

Cont’d 1454 D  1 μg/mL:0.55%  0.3 μg/mL:1.5% 1454 E  10 μg/mL:1.9%  3 μg/mL:.88% 1454 F  3 μg/mL:4.0%  1 μg/mL:.48%

Cont’d 1454 G  1 μg/mL:3.1%  0.3 μg/mL:4.0% 1454 H  0.3 μg/mL:5.2%  0.1 μg/mL:0.9% 1454 I  0.3 μg/mL:3.2%  0.1 μg/mL:1.5%

Sub fractions H-1  μg/mL:4.5%  μg/mL:0.8% H-2  0.1 μg/mL:1.1%  0.03 μg/mL:1.0%

Baseline 1 H-1 Peak Baseline 2

Assay Results H-1 Peak  0.03 μg/mL:1.0%  0.01 μg/mL:1.6% Baseline 1  μg/mL:1.8%  μg/mL:0.5% Baseline 2  μg/mL:2.0%  μg/mL:0.6%

Possible Synergisms? 0.03 μg/mL 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