Memory loss Muscle weakness Loss of voluntary movement Dementia Hallucinations Seizures Jerky body...

Memory loss

Muscle weakness

Loss of voluntary movement

Dementia

HallucinationsSeizures

Jerky body movementsTremors

Rigidity

Confusion

Depression

Language problems

Stiff muscles

Ataxia

Personality changes

Decline in mental abilities

http://en.wikipedia.org/wiki/Neurodegenerative_disease

Assessing -Synuclein Degradation through the

MVB/Endocytosis Pathway

Jaime PérezBIOL 324

Grant Proposal Defense

Common Problem

Parkinson’s Disease

Alzheimer’s Disease

Huntington’s Disease

Prion Disease

ALS

-Synuclein

Amyloid- peptide

Huntingtin

Prion protein

SOD1

Misfolding & Aggregation

Cell Death

Disease Culprit Protein

Common Problem

Parkinson’s Disease

Alzheimer’s Disease

Huntington’s Disease

Prion Disease

ALS

-Synuclein

Amyloid- peptide

Huntingtin

Prion protein

SOD1

Misfolding & Aggregation

Cell Death

Disease Culprit Protein

Road Map

Background: PD & -Synuclein

Major Questions

Why Endocytosis?

Hypothesis

Yeast Model

Specific Aims

Methods

Preliminary Results

Why is this important?

Parkinson’s Disease Facts

• 4 million people affected

• Classic symptoms:Resting tremors Muscular rigidity

• No known cure

Muhammad Ali & Michael J. Fox

Parkinson’s Disease

Substantia Nigra

Basal Ganglia

Motor Cortex Muscle

Less Movement

Giasson, B.I., et al. (1999).Spillantini, M.G., et al. (1998).

Abeliovich, A., et al. (2000).

My Interest in - Synuclein

Lewy Bodies: - Synuclein inclusions

Spillantini, M., et al. (1998).

What is -Synuclein?

• 140 amino acid protein

• Associated with 1. Synaptic membranes

2. Secretory pathway

Jakes, R., et al. (1994).

Davidson, W. S., et al. (1998).

Dixon, C., et al. (2005).

N NAC C

- Synuclein and PD

SPORADIC FAMILIAL

Normal -synuclein

Misfolded

Accumulated

Genes:-synucleinparkinUCH-L1 DJ-1 PINK1

LRRK2

DNA

Point mutations

Polymeropoulos, M.H., et al. (1997), Kitada, T., et al. (1998)

Funayama, M., et al. (2002), Bonifati V., et al. (2003).

Valente, E. M., et al. (2004), Paisan-Ruiz, C., et al. (2004).

ToxinsEnvironmental factors

A30P, A53T, E46K

Wild Type -synuclein

CELL DEATH

Gain of Function Disease

PD-like symptoms

WT & familial mutants (A30P & A53T)

Feany, M., et al. (2000).

Lasko, M., et al. (2003).

Masilah, E., et al. (2000).

Major Questions in Field

Is -synuclein even toxic?

If so, what is causative agent?

Are Lewy bodies involved in onset?

-synuclein accumulation causing PD?

Essential to examine -synuclein degradation routes

Alpha-synuclein

THERAPY!

Proteins1. VERY Important

2. Shape and Function

Normal -synuclein

MisfoldedAccumulated

Degradation Pathways

ProteasomeLysosome

Degrades proteins from cytoplasm or

nucleus

Degrades proteins from plasma membrane or outside cell

-synuclein literature:Rideout, H. J., et al. (2002). Sawada, H., et al. (2003).Bedford, L., et al. (2008). …Evidence

UCHL1parkin

LysosomeOutside Cell

Inside Cell

Lysosome

Autophagy Literature

Lee, H.J., et al. (2004). Ancolio, K., et al. (2000). Cuervo, A. M., et al. (2004). Vogliatzi, T., et al. (2008). Webb, J. L., et al. (2003).

Cell

Cytoplasm

Late Endosome

Lysosome

Early Endosome

MVB

Outside

Endocytosis lit.:Willingham, S., et al. (2003).Kuwahara, T., et al. (2008). Lee, H.J., et al. (2008).…

Why Endocytosis?

ESCRT Machinery

Endosomal Sorting Complexes Required for Transport

Katzmann, D. J., et al. (2002). Katzmann, D. J., et al. (2001).Katzmann, D. J., et al. (2003).Ayala, A. (2009).

Functionsvps27:Protein that forms a complexwith Hse1p; requiredfor recycling Golgiproteins, forminglumenal membranesand sorting ubiquitinatedproteins destined for degradation

vps34:Kinase responsible for the

synthesis of one

phospholipid; forms

membrane complex with

Vps15p to regulate protein

sorting

Yeast Genome Database

vps34 vps27MVB Cargo

Endosome Lumen

Off to ESCRT-1

Endosome Membrane

Gap in Knowledge

The MVB/endosome pathway is a route through which -synuclein is targeted to the lysosome

Is alpha-synuclein being degraded through the MVB/endosome pathway?

Hypothesis

Why Budding Yeast?

• Easy to grow• Cost effective• Genome sequence

available• ESCRT genes conserved

in humans

• MVB/endosome pathway

best studied in yeast

Outeiro, T.F., et al. (2003).

Willingham, S., et al. (2003).

Specific Aims

Assess -synuclein…1. Accumulation

2. Localization

3. Toxicity

in cells compromised for endocytosis at the pre-ESCRT step

PYES.2 Plasmid

GAL

-Syn GFP

V5

URA-3

Transformation

Yeast

LiAc Heat ShockssDNA

Vector

YPD -URA GLU

Yeast Constructs

PP

GFP

WT

A30P

E46K

URA-3

GAL

GFPV5

URA-3

GAL

GFP

V5

URA-3

-Syn

GAL

GFP

V5

URA-3

-Syn

GAL

GFP

V5

URA-3

-Syn

vps27

vps34

BY4741Controls

Rationales

• Accumulation– Western Blot Ayala, A. (2009) & Sharma, N., et al (2006).

– Loss of Induction

• Localization– GFP Microscopy Outeiro, T.F., et al. (2003), Nobel Prize 2008

• Toxicity– Growth Curves Ayala, A. (2009) & Sharma, N., et al (2006).

– Spotting

Aim One

Assess a-synuclein accumulation in cells compromised for

endocytosis at pre-ESCRT step.

Accumulation:Western Blot

Method:

V5

PGK

24 hrGAL

HeatSDS

Class BeadsProtein Gel

Membrane

YY

Anti-PGK

Anti-V5

Accumulation:Loss of Induction

Method:

V5

PGK

24 hrGAL

HeatSDS

Class Beads

Protein GelMembrane

YY

Anti-PGK

Anti-V5

24 hrGLU

GLU

0 hrGLU

6 hrGLU

12 hrGLU

Predictions

Anti-PGK

Anti-V5

BY4741 vps

Gal

GalMW 0 6 12 24 0 6 12 24

GluGlu

Loss of Induction

BY4741 vps

WT

WT

A30

P

A30

P

E46

K

E46

K

GF

P

GF

P

MW

Aim Two

Assess a-synuclein localization in cells compromised for

endocytosis at pre-ESCRT step.

Localization:GFP Microscopy

Method:

Predictions

24 hr

GFP WT

48 hr

24 hr

WT E46K

48 hr

BY4741

vps

A30P E46K

Aim Three

Assess a-synuclein toxicity in cells compromised for

endocytosis at pre-ESCRT step.

Toxicity:Growth Curves

Method:

Spectrophotometer CultureGAL

Time0 3 6 12 18 24 36 48

Ab

sorb

an

ce

Toxicity:Spotting

GLU GAL

Predictions

Time

0 3 6 12 18 24 36 48

Ab

sorb

an

ce

4741, PP, GFP

-Syn

GLU4741, PP, GFP

-Syn

GAL

4741, PP, GFP

-Syn

Repeats and Analysis

Strain OD SP WB LOI MC

vps27 _ _ _ _ _ _ _

vps34 _ _ _

Analysis Cumulative & with t-test

N/A N/A N/A Graph

Preliminary Data: vps34Cumulative

0.0

0.5

1.0

1.5

2.0

2.5

3.0

0 6 12 18 24 30 36 42 48

Time (hrs)

Absorbance

PPGFPWTA30PE46K

PPGFP

WTA30P

E46K

GalactoseGlucose

BY4741 vps34

WT

WT

A30

P

A30

P

E46

K

E46

K

GF

P

GF

P

MW

Anti-V5

Anti-PGK

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24 hr

48 hr

GFP WT A30P E46K

Lysates 34MC 27 MC 27 Western 34

Lysates 27 Western 27

OD 27SP 27

Western 27

REPEATS and LOI if necessary

Why Important to Study?

• Hardships, inconvenience, and cost:– NEED to understand mechanism that will lead to cure!

• We are training well-prepared undergraduates for diverse biological careers in research, medicine and allied health.