Storyboard Method of Data Management

Rebecca Sappington, Ph.D.Assistant Professor

Ophthalmology and Visual Sciences, Pharmacology and Neuroscience

Responsibilities of a PI Manage progress within projects as well

as for individuals Make strategic decisions regarding project

direction Recognize benchmarks in project Publications/Abstracts/Presentations

Identify new lines of investigation based on outcomes of current project Grant applications

PIs assemble pieces into a cohesive vision.

Need to see the “big picture”

Not so simple…

Multiple projects with intersecting points of interestMultiple individuals contributing to a single projectMultiple individuals contributing to multiple projects with various intersecting points of interest

A successful method of data management is key!

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10011 0010 1010 1101 0001 0100 1011

Needs for Data Management

• Organizing data • Tracking progress• Synthesizing data • Strategizing for future experiments

Goal: Single method of data management that achieves all of the these components of data management.

The Storyboard

David J. Calkins, Ph.D.Director of ResearchAssociate ProfessorVanderbilt Eye Institute

Based on storyboards used to produce movies!

“The Waterhorse” Storyboard: Take 1Craig Woolheater

Application of the Storyboard to Scientific Data

• Identify common threads – can be irrespective of “projects”– For example:

• 3 projects– Neuronal death in disease

» Brain-derived neurotrophic factor inhibits death.– Neuronal-glial interactions in disease

» Brain-derived neurotrophic factor is released by glial cells.

– Neuronal death in development» Deprivation of brain-derived neurotrophic factor is

used to prune neuronal populations.

• Brain-derived neurotrophic factor is a common thread– Data from each “project” has the potential to

contribute to understanding

Structure of the Storyboard Easy-to-use Interface

Microsoft Powerpoint Allows maximum flexibility and easy use

Components of the storyboard evolve as data set evolves Content of slides will be determined by progress of data Content should provide information relevant to current

state of data 100% modifiable

Fits any discipline or data set

Example of a Storyboard at Various Stages of Completion

Storyboard: In the Beginning

Brain-derived neurotrophic factorCollaborative Project:

Calkins Lab and Sappington Lab

Common components for newly initiated storyboards:

•Purpose•Often short, bulletpoint statements

•Experimental Design•Can be a schematic or brief outline

•Data slides •Significant emphasis on method•Questions about data•Follow-up points

Several facts about BDNF:

Brain-derived neurotrophic factor (BDNF) is one of the neurotrophin (NT) family proteins, which includes nerve growth factor (NGF), NT-3, NT-4/5 and NT-6.

All neurotrophins are structurally similar small secreted proteins which play important roles in proliferation, differentiation and survival of neurons in the peripheral and central nervous systems during development.

The gene product of neurotrophins after synthesis are around 35 kDa proteins which are proteolytically cleaved to form biologically active mature molecules (13.5 kD) by removal of the proregions within cells.

The mature molecules form homodimers, are secreted from cells or nerve terminals through constitutive or regulatory pathways and activate their receptors, p75 and trks.

BDNF has particularly strong neuroprotective effect on retinal ganglion cells (RGCs), amacrine cells.

BDNF mRNA and protein are localized in the ganglion cell layer, in the inner nuclear layer and in Muller cells which play important roles in the survival of retinal neurons.

Using antibodies specific for the mature domains of BDNF, immunoreactive proteins with molecular masses of 18 to 32 kD were detected, in addition to the 13.5-kD mature forms of BDNF. This suggested that tissue-specific proteolytic processing of proneurotrphins occurs in vivo.

Select optimal protocol for separation of crude membrane and cytosole fractions of protein extracts from rat adult tissue (Sc, ON, Retina).

Conduct the Western blot analysis of obtained samples with antibody against both forms of BDNF (precursor and mature).

Analyse data of BDNF protein distribution and localization in the adult rat visual system tissue: brain (Superior colliculus), Optic nerve, Retina).

Compare the data obtained from control animal tissue with data from glaucoma model animal tissue.

Our current purpose:

Scheme of cell fractionation of homogenate from Rat tissue (sup. collic, opt. nerve, retina) with HEPES buffer (Vissio P.G., et al., 2008). Homogenation- all-glass, sonication.

HB110mM Hepes, pH 7.81.5mM MgCl210mM KCl1mM DTT0.4mM EDTAPIC

HB220mM Hepes, pH 7.91.5mM MgCl21.2M KCl0.5mM DTT0.4mM EDTA50% glycerolPIC

H (total homogenate) in HB1

P1(nuclei and large debris)

S1(crude cytoplasmicfraction including

subcellular organelles other than nuclei)

Resuspension with HB2,Incubation on ice for 15min,

homogenization

1,000xg

10,000xg

P2(nuclei)

S2(nuclear and

cytoplasmic proteins

S1, S2 → BCA, Coomassie, WB (Bdnf, pro-Bdnf, β-Actin, Histone H3)

Scheme of crude membrane/cytosol preparation from Rat tissue (sup.collic., opt.nerve, retina) with Sucrose-HEPES buffer (Ehlerslab protocol).All-glass homogenization.

Homogenization buffer (HB)0.32M Sucrose10mM Hepes, pH7.42mM EDTA0.1mM PMSF1mM Na3VO450mM NaF2mM EGTAPIC

HEPES-Lysis buffer (HLB)50mM Hepes, pH7.42mM EDTA0.1mM PMSF1mM Na3VO450mM NaF2mM EGTAPIC

H (total homogenate) in HB

1,000xg

P1(nuclear fraction)

S1

200,000xgS2P2

(crude membrane pellet)(crude cylosol)

resuspendpellet in HB

200,000xgP2’

(washed crude membrane pellet)

Resuspend pellet in HLB

BCA, Coomassie, WB (Bdnf, pro-BDNF, β-Actin, Histone H3)

Bdnf

Protein extraction using Buffered sucrose

with the following subcellular fractionation

using ultracentrifugation. (Fawcett JP. at all, 1997)

ON Retina Super. Collic. Cortex

Syn

40 40 40 40 50 50 50 50 50 50 µg/lane

?

Syn

Bdnf

Retina SC Cortex

70 100 96 81 100 100 100 96 100 100 µg/lane

?

Storyboard: Intermediate (Abstract level)

54 slides

Common components for intermediate storyboards:

•Purpose•More detailed background information •Layout of hypothesis

•Experimental Design•Geared more towards future experiments needed - identifying holes in current data set

•Data slides •Highly variable in content at this stage

•More advanced components will look more figure-like •More recent experiments will look more like the initial storyboard slides

•Notation of needs for publication, grant, etc.•For example, need to re-photograph sample to better highlight conclusion

•Start putting slides in order that follows a data-driven line of reasoning for publication/grant application!

The Hook• Widely accepted that BDNF produced in the lateral

geniculate nucleus (primates) or superior colliculus(rodents) is the main source of trophic support for RGCs.

• Also, widely accepted that BDNF must be retrogradelytransported from target to RGC soma to provide support.

• Early work in primate and rodent models of ocular hypertension suggested that transport of BDNF was blocked at the optic nerve head, the proposed site of nerve compression. This hypothesis, which is now dogma, is supported by experiments with poor design and little data.

• We found that a reduction in BDNF labeling at the optic nerve head is present in naïve rodents. Furthermore, the perceived magnitude of this reduction is dependent on the site of antibody recognition. To delineate the anatomical properties of BDNF trophic support for RGCs, we examined cell type-specific expression and subcellular localization of BDNF in the early visual pathway.

C57

Same pattern of labeling in a C57 mouse and rat without IOP elevation. Retinal labeling in RGC soma also present.

Need re-image of C57 whole eye and SD whole eye BDNF alone!

Can use SMAC material!

ADD BDNFi and BNDFc picture for mouse and rat optic tract and superior colliculus!!!!

Again, could this be BDNF or pro-BDNF associated with vesicles? Appears to be mostly in rat.

Could be pro-BDNF (mature BDNF + prodomain), which has an expected molecular weight of 32kDa, 28kDa and 24kDa. Pig shows only smallest pro-BDNF isoform and only in nerve.

BDNF is present in retina, ONH and ONm

REMOVE PORCINE!!!

Perform western blot for superior colliculus in rat?

Potential Caveats

• BDNF c-terminal antibody also recognizes, to a lesser extent, NT-3 (M.W. 35kDa)

• BDNF internal antibody also recognizes, to a lesser extent, NGF (M.W. mature = 13kDa; M.W. precursor = 27kDa)

Storyboard: Paper in Progress

Common components for final storyboards:

•Purpose•More detailed background information •Layout of hypothesis

•Data slides •Publication quality figures with figure legends•Notation of needs for publication, grant, etc.

•For example, need to re-photograph sample to better highlight conclusion•By the end, the storyboard should be a complete outline of your manuscript for publication!

Figure 1

Figure 1. Expression profile of BDNF varies across the early visual pathway. A. Quanitative PCR reveals the presence of BDNF mRNA in retina, optic nerve and superior colliculus of mice. B. Subcellular fraction and immunoblotting for BDNF in rat superior colliculus , myelinated optic nerve , optic nerve head and retina. BDNF protein is present in multiple conformations across all sites and in multiple fractions including, the crude cystosolic fraction (S2), the crude membrane and synaptosomal fraction (P3) and the synaptic vesicle-enriched fraction (P4).

Figure 2

Figure 2. BDNF localizes mainly to RGCs in mouse retina. A. Confocal micrographs of co-immunohistochemical localization of BDNF (green) and the RGC marker Brn3a (red) in longitudinal sections of mouse retina counterstained with DAPI (blue). BDNF localizes mostly to the GCL, where it co-localizes with Brn3a (arrows). Scale is consistent for all. Abbreviations: outer nuclear layer (ONL); outer plexiform layer (OPL); inner nuclear layer (INL); inner plexiform layer (IPL); ganglion cell layer (GCL).

Common Elements of the Storyboard Slides Annotation of data

Short method Gel lanes Immunohistochemical labels (antibodies including company)

Interpretation of data Range from one or two sentences to full figure legends

Location of data Electronic location (filename and directory) Physical location

Source of data Personnel Date (for lab notebook references)

Advantages: PIProject Management– Assess progress with a glance at any time– Increase productivity of meetings with laboratory personnel

Publications– Reduces time to publication – Helps avoid data backlog

Grants and Presentations– Spend less time looking for data– Data already compiled into an intellectually cohesive unit– For presentations, simply cut and paste!

Advantages: Lab PersonnelIncreases intellectual investment for personnel at all levels.Particularly useful for students and postdocsregarding experimental design and preparation for publication.Reduces time spent preparing presentations and poster for lab meetings, scientific meetings, graduate programs, etc.Teaches organizational skills!

Disadvantages

Requires diligent participation by all members of the laboratory.– Lag time between data generation and

incorporation into the storyboard greatly impacts the success of this system!

– Lack of participation can also negate many of the advantages!

Thank You!!!

Acknowledgements: Dr. David Calkins