Mounting Evidence that Endocytosis Regulates -Synuclein

Degradation

Jaime Pérez Pineda

Advanced Cell Biology

Fall 2009

Memory loss

Muscle weakness

Loss of voluntary movement

DementiaHallucinations

Seizures

Jerky body movements

Tremors

RigidityConfusion

DepressionLanguage problems

Stiff muscles

Ataxia

Personality changes

Decline in mental abilities

Symptoms ofNeurodegenerative

Diseases

Road Map

INTRODUCTION:•Neurodegenerative Diseases•Culprit Protein in Parkinson’s•Protein Degradation Pathways•Endocytosis and ESCRT•Hypothesis and Model Organism

RESULTS:•Three Aims & Goals

DISCUSSION:•Major Findings

Introduction to Parkinson’s Disease

Common Problem in NDD

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

Parkinson’s Disease

• 4 million people affected

• Classic symptoms:Resting tremors Muscular rigidity

• No known cure Muhammad Ali & Michael J. Fox

SPORADIC FAMILIALNormal -synuclein

Misfolded

Accumulated

Genes:-synucleinparkinUCH-L1 DJ-1 PINK1

LRRK2

DNA

Point mutationsToxinsEnvironmental factors

A30P, A53T, E46K

Normal (Wild Type) -synuclein

CELL DEATH

Sporadic vs. Familial PD

>90% <10%

What Happens in the PD Brain?

My Interest in -Syn:

Lewy Bodies: - Synuclein

inclusions

Basal Ganglia

Substantia Nigra

Motor Cortex

Less movement!

Muscle

Culprit Protein: -Synuclein • 140 amino acid protein

• Associated with:

1. Cytoplasm2. Synaptic Machinery3. Extra cellular matrix4. Secretory pathway

• Loves to aggregate and bind to membrane

Proof of -Synuclein Involvement:

PD-like symptoms

WT & familial mutants (A30P & A53T)

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

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

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

What causes -synuclein toxicity?

Essential to examine -synuclein degradation routes

Alpha-synuclein

THERAPY!

Is -synuclein accumulation causing PD?

Normal -synuclein MisfoldedAggregated

-Synuclein accumulates

Proteins1. VERY Important

2. Shape and Function

Normal Protein

MisfoldedOld

Degradation Pathways

ProteasomeLysosome

Degrades proteins from cytoplasm or

nucleus

Degrades proteins from plasma

membrane, organelles or outside cell

-synuclein literature:

EvidenceUCHL1parkin

Knowing where -synuclein is found, which mechanism will degrade it?

Lysosome

Outside Cell

Inside Cell

Why Endocytosis?

Dixon et al. (2005)

Lee et al. (2008)

Sharma et al. (2006)

Lysosome

Cell

Cytoplasm

Late Endosome

Lysosome

Early Endosome

MVB

Outside

Endocytosis

Golgi

ESCRT Pathway

Endosomal Sorting Complexes Required for Transport

MVB

Cytoplasm

? ?

Gap in Knowledge and Hypothesis

My hypothesis is that -synuclein interacts with the pre-ESCRT proteins while using the MVB/endosome pathway as a route to the lysosome.

Model OrganismBudding Yeast (S. cerevisiae)

• Easy to grow• Cost effective• Genome sequence

available• ESCRT genes

conserved in humans• MVB/endosome

pathway best studied in yeast

Experimental Design

EndocytosisIntact

No vps34 No vps27

Budding yeast with…

vs. and

CONTROL

Does -synuclein…1. aggregate more 2. accumulate more3. cause cell toxicity

…in endocytosis compromised yeasts

Results: What I Found

What I found:Strain:

Localization

Toxicity

Accumulation

Pre-ESCRT

vps34 vps27

Weak Weak

Non Toxic Non Toxic

Strong (WT)

None

ESCRT-I: mvb12 vps23 vps28 vps37

None Weak Weak Strong

Non Toxic Non Toxic

Weak Non Toxic

Weak Weak Weak Strong

ESCRT-II: vps25 vps36

Weak Weak

Non Toxic Non Toxic

Weak Strong

ESCRT-III: vps20 vps24

Weak Strong

Non Toxic

Toxic (A30P)

Strong None

Localization:

GFP

Method:WT A30P E46KGFP

24 hrs

48 hrs

EndocytosisIntact

WT A30P E46KGFP

24 hrs

48 hrs

No vps34

Accumulation:

Method:

V5

PGK

24 hrGAL

HeatSDS

Class BeadsProtein Gel

Membrane

YY

Anti-PGK

Anti-V5

BY4741vps34

WT

A3

0P

E4

6K

GF

P

anti-PGK

MW

anti-V5

WT

A3

0P

E4

6K

GF

P

Method:

V5

PGK

24 hrGAL

HeatSDS

Class Beads

Protein GelMembrane

YYAnti-V5

24 hrGLU

GLU

0 hrGLU

6 hrGLU

12 hrGLU

BY4741vps34

Ga

l

Ga

l

MW061224 0 61224

GluGlu

WT Loss of Induction

anti-PGK

anti-V5

Cell Toxicity:

Method:

Spectrophotometer CultureGAL

Time0 3 6 12 18 24 36 48

Ab

sorb

an

ce

Abs

orba

nce

Time (hrs)

No vps34

Mounting Evidence:Strain:

Localization

Toxicity

Accumulation

Pre-ESCRT

vps34 vps27

Weak Weak

Non Toxic Non Toxic

Strong (WT)

None

ESCRT-I: mvb12 vps23 vps28 vps37

None Weak Weak Strong

Non Toxic Non Toxic

Weak Non Toxic

Weak Weak Weak Strong

ESCRT-II: vps25 vps36

Weak Weak

Non Toxic Non Toxic

Weak Strong

ESCRT-III: vps20 vps24

Weak Strong

Non Toxic

Toxic (A30P)

Strong None

vps22

vps32vps2

Hse1

Final Thoughts-synuclein

Hypothesis supported!

Hardships, inconvenience and costs

Mounting Evidence that Endocytosis Regulates -Synuclein

Degradation

Jaime Pérez Pineda

Advanced Cell Biology

Fall 2009

Acknowledgements

BIOL 324: Keith, Carlos, Will, Rebecca, Alina and DannySTOCKROOM: Beth, Tasheka, Kaylyn, Jordanah, and Madhavi

Lastly: Mike and Dr. DebBurman!

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Two Very Important Proteinsvps27:forms complex with Hse1p;Required for recycling Golgiproteins, forminglumenal membranesand sorting ubiquitinatedproteins

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-I

Endosome Membrane