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-Br cAMP, known outgrowth promoters

Neurite Outgrowth Controls

25 20 15 10 5 0 Treated RPMI w/ 30μL DMSO 5 μM L a c t a c y s t i n 24 hrs 48 hrs 72 hrs Control

I F G H Choosing a Concentration 30 91 95 97 97 Concentration (μg/ml) 10 74 90 96 96 3 19 64 95 96 1 66 61 Values are % lethality 0.3

21 21 0.1

11

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

…Symploca

www.sitesatlas.com

Symploca 1454 Fractions   

1454 A

  30 μg/mL: 10 μg/mL:

1454 B

 30 μg/mL:  10 μg/mL:

1454 C

  30 μg/mL: 10 μg/mL: 0.53% 0.27% .32% 1.2% Toxic .35%

Cont’d   

1454 D

  1 μg/mL: 0.3 μg/mL:

1454 E

 10 μg/mL:  3 μg/mL:

1454 F

  3 μg/mL: 1 μg/mL: 0.55% 1.5% 1.9% .88% 4.0% .48%

Cont’d   

1454 G

  1 μg/mL: 0.3 μg/mL:

1454 H

 0.3 μg/mL:  0.1 μg/mL:

1454 I

  0.3 μg/mL: 0.1 μg/mL: 3.1% 4.0% 5.2% 0.9% 3.2% 1.5%

Sub fractions  

H-1

  0.003 μg/mL: 0.001 μg/mL:

H-2

  0.1 μg/mL: 0.03 μg/mL: 4.5% 0.8% 1.1% 1.0%

3.00

2.50

2.00

1.50

1.00

Baseline 1 0.50

0.00

H-1 Peak Baseline 2 996 at 254.00

996 at 230.00

996 at 216.00

996 at 340.00

10.00

20.00

30.00

40.00

Minutes 50.00

60.00

70.00

Assay Results 

H-1 Peak

  0.03 μg/mL: 0.01 μg/mL: 1.0% 1.6% 

Baseline 1

  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?

0.003 μg/mL 0.03 μg/mL 0.03 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

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2001

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25

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1998

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273

, 8545  Aoki et al., Biochem. Biophys. Res. Com.

2001

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