Protein Toxicity in ParkinsonAnd Alzheimer’s DiseaseChuck Sanders, Dept. of BiochemistryRm 5110C MRBIII, chuck.sanders@vanderbilt.edu

Readings on Role of α-Synuclein in Parkinson Disease

100 years of Lewy pathology.Goedert M, Spillantini MG, Del Tredici K, Braak H.Nat Rev Neurol. 2013 Jan;9(1):13-24. 

α-Synuclein: membrane interactions and toxicity in Parkinson's disease.Auluck PK, Caraveo G, Lindquist S.Annu Rev Cell Dev Biol. 2010;26:211-33.

Pathological roles of α-synuclein in neurological disorders.Vekrellis K, Xilouri M, Emmanouilidou E, Rideout HJ, Stefanis L.Lancet Neurol. 2011 Nov;10(11):1015-25

α-Synuclein oligomers and clinical implications for Parkinson disease.Kalia LV, Kalia SK, McLean PJ, Lozano AM, Lang AE.Ann Neurol. 2012 Aug 28. doi: 10.1002/ana.23746.

AD

From: May 2010 US National Vital Statistics Report

PD

Protein Deposition in Neurodegenerative Diseases: A Histological View

A. Abbott Nature Outlook 2011

NEJM 349, 583 (2003)note that list is incomplete and growing

Key Questions About Protein Aggregation in Alzheimer’s and Parkinson Diseases

What triggers protein aggregation? In inherited form of disease?In sporadic (idopathic) form of disease?

What form of the protein is toxic?

Disease promoted by toxic “gain of function”, loss of native function, or both?

What organ, tissue, and cells are affected… as a function of time?

Where in (or out) of cell does protein aggregate, deposit and exert its toxicity?

What is the mechanism of toxicity and is it direct or pathway-based?

Does protein deposition spread from cell to cell and tissue to tissue? If so, how?

Before we explore these issues, we need to review the biophysics and biochemistryof protein folding and misfolding.

Thermodynamic Stability of Proteins

K =eq,fold

[folded]

[unfolded]

Go

Keq,fold

unfolded folded

N

C

Standard Free Energies of Folding for Single Domain Proteins

Protein Gofold (kcal/mol)

lambda repressor -3.0alpha spectrin SH3 domain -2.9arc repressor -6.3cytochrome C (with heme and Fe(II) -15CD2 -8.2procarboxypeptidase -4.1U1A spliceosomal protein -9.3Hpr -4.6

Most folding proteins are only moderately stable:The folded state is usually favored over the unfolded state by only -3 to -10 kcal/mol.

Many Proteins are Natively Unfolded (Natively Disordered)The unfolded state is energetically favored, unless some the protein comes in contactwith something that stabilizes the folded state (e.g., a cognate ligand or a membrane).

Polypeptides <50 Residues Usually Do Not Fold In SolutionUnless They Bind to Another Protein or to A Membrane

Protein Folding Pathways: Kinetics Revisited

Go

Rate depends on the pathway between states and associated G*.

Favorable Go doesn’t mean the process will take place at a significant rate

“Kinetically trapped”

Proteins fold because it is both thermodynamically and kinetically favorable for them to do so.

2 states1 barrier in each direction

Go

G* G*reverseforward

G : free energy of activation*rate is proportional to rate constant, k,which is proportional to: G*

RTeThere is an inverse logrithmic relationshipbetween rate and activation energy.

Kinetics

Examples of Potentially Deadly Mutations

Barrier to folding becomes too high.

Normal barrier to misfolding is reduced.

Folding intermediate is stabilized.

Folded form is destabilized.

Disrupt interactions with the protein folding machinery.

Go

Mutations can perturb the heights of the ratebarriers and/or the energies of the free energystates. Consider a mutation that lowers theenergy of a folding intermediate:

Go

Mutation that lowersenergy of foldingintermediate (only).

Note that this mutation not only makes the intermediatemuch more stable (longer-lived), but that the energybarriers from this state to both initial and final statesare now larger than for wild type.

unfolded

folded

Protein Aggregation

When protein sticks to protein to form very large oligomers

Both folded and unfolded proteins can sometimes aggregate.

Aggregates can be disordered (amorphous) or can be ordered.

Amyloid fibrils are a common type of ordered aggregate.

The rate of formation of ordered aggregates is usually limited bythe rate of a requisite nucleation step.

Figure: Aguzzi and Polymenidou, Cell, 2004

The Formation of Ordered Aggregates Is Rate-Limited By A Nucleation EventThis could involve the rare collision of minor conformations of the protein or could depend on the appearance of post-translationally modified form of a protein that can serve as an aggregation seed. The rareness of the seeding species and events can make it very hard topin down the exact nature of the nucleation event, especially in living tissue.

Selkoe, Nature, 2003

cross-β fibril formation on the road to mature amyloid; initial self-association might be staged from native or denatured states in someinstances.

Protein Folding in the Cell

The main locations of protein folding in the cell are the cytosol and the lumen of endoplasmic

reticulum.

ER: Membrane proteins, secreted proteins, andorganellar proteins.

Protein folding in the cell…

3-D structure is determined by sequence.

Folding pathways differ from test tube:

Folding is often co-translational.

Chaperone proteins play roles.

Other folding accessory proteins.

Folding is monitored by quality control.

Folding-defective protein is degraded.

There are two major degradation protein pathways in mammalian cells.

The ubiquitination/proteasome pathway.

The autophagy/lysosomal pathway(s).

Members of Alois Alzheimer’s research group at the Royal Psychiatric Clinic of the University of Munich, Germany in 1910

Goedert, M. et alNat Rev Neurol 9, 13-24, 2013

Alois Alzheimer

Fritz Jakob Heinrich Lewey

http://coloradodementia.orgMarwan Sabbagh

Extracellular Amyloid Plaques Are One of the Hallmarks of AD; Central Component: The Amyloid-β Polypeptide

A Second Hallmark of Alzheimer’s Disease Are Intracellular Deposits Called “Neurofibrillary Tangles”.

The Central Component of NFTs is the Hyperphosphorylated Tau Protiein

Intracellular Lewey Bodies Are the Hallmark Parkinson DiseaseCentral Component: α-Synuclein

The superstructure of amyloid-β in

(i) amyloid fibrils in amyloid plaques

(ii) hyperphosphorylated tau in neurofibrillar tangles,

(iii) α-synuclein in Lewey bodies

is thought to be organized around

“amyloid” cross-beta fibrils formed

by each of these three proteins.

The Amyloidogenic Pathway and Alzheimer’s Disease

secretasecleavage

Amyloid PrecursorProtein

cytosol

secretasecleavage

C99

cytosolAmyloid-Beta

Oligomers

NeuronCell Death

The Amyloid Precursor Proteins and The Amyloid-β Polypeptides

Long forms Aβ42 and Aβ43 thought to be more toxic than short forms Aβ38 and Aβ40

Retaining half of the TM segment of the APP, the Aβ polypeptides (especially) the long forms are only marginally water-soluble. They have a high propensity to aggregate and may also retain significant affinity for membrane surfaces.

The function of APP and of its fragments (extracellular domain: β-APP,Aβ, and APP intracellular domain:AICD) are not well understood. Lots of bitsand pieces of evidence in support of various roles. However, APP-knockout miceare a little slow and a little stupid, but live. Lethality occurs only when two homologs of APP– the APP-like proteins (ALP1 and ALP2) are also knocked out.This implies that AD is not strongly related to the loss of native function of APP orof its derived fragments.

Schematic of the protein sequence of human α-synuclein.

Bosco D A et al. Cold Spring Harb Perspect Biol 2011;3:a007500

©2011 by Cold Spring Harbor Laboratory Press

Natively disordered in solution.Binds to membrane surface via N-terminaldomain, which is thought to form helices. C-terminal domain remains disordered, butengages in complexes with other proteins.

Functions of α-Synuclein

α-synuclein stabilizes synaptic vesicles (and other vesicles such as the ER-to-Golgitransport vesicles) to prevent premature membrane fusion and loss of vesiclecontents. However, knockout of α-synclein is not fatal and does not lead to neurodegeneration. Parkinson’s does not seem to be primarily loss of function in nature.

Annual Reviews