Biology of Neurodegenerative Diseases BIOS E-108

Harvard Extension School Fall 2011

Lucia Pastorino, Ph.D.

Contact:
office 617-735-2234
 Policy of the course
Instructor
Lucia Pastorino, Ph.D.
Instructor in Medicine
Beth Israel Deaconess Medical Center
Harvard Medical School
3 Blackfan Circle CLS #428
Boston, MA 02115
lpastori@bidmc.harvard.edu

Class Location and Time

Tuesday, 7:35-9:35 pm, Science Center, Hall E

Office Hours
Mondays 3.15pm-4.45pm @ 77 Ave. Louis Pasteur, New Research Building NRB, Longwood Medical Area,
Boston. Green Line T stop Longwood. Students who want to meet for office hours have to contact the
instructor with an email ahead of time.

Sections
Required/mandatory for graduate-credit students, optional for undergraduate-credit students Tuesday, 6:30-
7:30 pm. Sections are lead by TAs and are meant to further discuss the topics covered during the previous
class, to stimulate discussion about scientific approaches, techniques and methodologies in the field of the
molecular and cellular biology of neurodegenerative diseases. A research article inherent to the topic covered
in the previous class will be presented by rotating graduate students.
Prerequisites
BIOS E-1A, or the equivalent in cell biology and molecular biology; BIOS E-12 or equivalent in
molecular biology; BIOS E-16/W or equivalent in cell biology are recommended. These prerequisites
indicate the background the students should have to follow this course.

Material for the course:

This course does not use a textbook, but detailed handouts which will be provided by the instructor ahead
of time as powerpoint presentations and pdf files uploaded on the courses website. Students will be
notified with an email when each class is available to be downloaded.

Website
Assignments, research articles, titles of the reviews relevant to the topics covered during the class
(available through Hollis Harvard Library online) and handouts will be posted here ahead of time.
Grading Policy:
Exams
There will be two midterm exams and one final exam.
No make-up or re-take exam will be allowed for any of the exams.

Assignments
Both undergraduate and graduate students will submit one assignment by the date of the final exam. The instructor
will assign it 3 to 4 weeks from the date due to turn it in.

Performance in Section (only for graduate students)

During section, graduate students will present a research article inherent to the topics covered during the previous
class. Graduate students will be evaluated for their overall performance in the section. This will include quality of the
paper presentation, level of participation during the section, attendance and punctuality. Special issues related to
difficulties in attending the section on time should be discussed with the Instructor and with the TA ahead of time.

Final grade components:

Undergraduate: Graduate:
Midterm exam 1: 25% Midterm exam 1: 20%
Midterm exam 2: 25% Midterm exam 2: 20%
Final exam: 30% Final exam: 25%
Assignment: 20% Assignment: 15%
Performance in section: 20%

The score in all the exams and assignment will be assigned to a maximum of 100 points. The final grade will be
calculated as the average between the scores obtained in each exam, assignment and performance in section (this only
for graduate students). The cutoffs for the evaluation of the final grade will be calculated at the end of the course and
will be different for graduates and undergraduates, as undergraduates will have a larger window to discriminate
between the grades.
 Program of the Lectures

PART 1

August 30th /September 6th . Introduction to the course. Neurodegenerative diseases: common features and risk
factors. Mechanisms of protein oxidation, protein aggregation and cell death (apoptosis). Proteostasis and autophagy.

September 13th / September 20th . Parkinsons disease (PD)

Pathological features in PD: Lewy bodies. Molecular mechanisms in PD: role of Parkin1, PINK1, a-synuclein and
other factors that lead to Lewy bodies formation and neuronal death. Mitophagy. Genetics of PD: inherited mutations
on human genes. Diagnosis and treatment.

September 27th. Midterm exam 1

 PART 2

October 4th. Huntington disease (HD)

Genetic of HD: the triplet repeat expansion disease. Huntingtin in protein aggregation and apoptosis.
Animal models. Mechanisms of cell death in HD.

October 11th. Creutzfeldt Jacob disease (CJD)

The prion disease or Transmissible Spongiform Encephalopathy (TSE).
Pathological features: amyloid-like deposit in the brain. Molecular mechanisms in CJD: from cellular
prion protein (Prpc) to the misfolded form scrapie (Prpsc). Prion protein intracellular trafficking.
Diagnosis of CJD: neuro-imaging techniques. Epidemiology of Prion disease: ovine scrapie and
transmission of prions. The vCJD variant. Animal models of CJD.

October 18th. Multiple Sclerosis (MS)

Pathological features and molecular mechanisms: inflammatory disease, demyelination. Genetic of
MS. Animal models. Diagnosis and treatment: neuro-imaging, biological markers, interferon.

October 25th. Amyotrophic Lateral Sclerosis (ALS)

Motorneuron Disease: pathological features. Molecular mechanisms: apoptosis, oxidative stress, the
protein SOD1 and other factors that induce neuronal death. Genetic of ALS. Animal models.
Therapeutic approaches.

November 1st. Midterm exam 2

 PART 3

November 8th. Alzheimers Disease (AD)

Pathological features: b-amyloid plaques and tangles. APP pathology and tau pathology/taupaties. Diagnosis.
Biological markers. The amyloid precursor protein APP and the generation of b-amyloid peptide.

November 15th /November 22nd. Alzheimers disease (AD)

Molecular basis of APP pathology: the protein APP, role of the secretases (a, b and g) and cholesterol in AD.
b-amyloid peptide: monomers and oligomers function in modulating the neuronal function. Therapeutic
approaches.

November 29th. Alzheimers disease (AD).

Tau pathology in AD. Tau hyperphosphorylation, tangle formation and neurodegeneration.

December 6th. Open discussion. Topics covered: progress in the research in these diseases, progress in early
diagnosis and intervention, ethical issues.

December 13th. Final Exam.

 August 30th 2011

Common features and molecular pathways

in Neurodegenerative diseases
 Characteristics

Irreversible neuronal death

No treatment that can revert the disease

 Degeneration in cortical neurons

Normal Degenerating Dead Neuron

Journal of Cerebral Blood Flow & Metabolism (2011) 31, 328338

 Methods of diagnosis:
advantages and disadvantages

-Identification of biological markers

-Behavioural/Neurological tests (PD, ALS, MS) Cognitive tests (AD)

-PET Scan:
Uses radiolabeled molecules that are able to reach brain areas that are
specific target of the disease. For example dopamine transporter is used for
PET in Parkinsons disease; b-amyloid binding molecules will be used for
PET of Alzheimer diseases, etc.

-MRI
X-rays of the all brain, evaluating changes in brain volume in specific areas
MRI in Alzheimers disease (AD)

Normal Alzheimers diseases

MRI

Computer graphic
PET scan in Parkinsonss disease (PD)
 MRI analysis in Huntingtons disease (HD)

Nuclei Cortex Cerebellum

Huntingtons disease

Normal
 Creutzfeldt-Jacob Disease
(Mad cow disease, BSE or Prion disease)
Progressive cortical degeneration in sporadic CJD

July November
MRI in Amytrophic Lateral Sclerosis (ALS)

Normal ALS
MRI in Multiple Sclerosis (MS)

Image courtesy of Siemens Medical Solutions.

 Common factors in neurodegenerative phenomena
1-Deposition of fibrillar proteinacious material in the intracellular or
extracellular matrix

2- Mitochondrial dysfunction, increased oxidative stress and production

of ROS (many mitochondria, a lot of energy)

3- Increased apoptosis

4- Decreased proteasomal activity (ubiquitin is present in all the lesions,

plaques, Lewy bodies, huntingtin aggregates etc)

5-Decreased autophagy and lysosomal degradation of proteins

6-Excitotoxicity

7-Alterations in the integrity of the cell membrane: implications for

altered levels of intracellular cholesterol
PROTEIN MISFOLDING,
AGGREGATION AND
NEURODEGENERATION
 Deposition of fibrillar proteinacious material
in the intracellular or extracellular matrix

Amyloid: amyloid fibrils are filamentous, hydrophobic structures, width ~10nm, length
between 0.1-10mM. Ribbon-like b-sheets motifs are formed by b-strands and b-turns. These
kind of fibrils are common to different neurodegenerative diseases, from Alzheimers to
Huntingtons disease. Conformation-specific antibodies (raised against specific proteins
which form a specific amyloid formation) can recognize only the b-sheet-polymeric
conformation but not the monomeric-soluble conformation of the protein substrate. Thus, the
b-sheet conformation and amyloid formation may be linked to neurodegenerative diseases.
NH2

COOH

Ross CA, Poirier MA. Nat Med. 2004 Jul;10 Suppl:S10-7. Review.
 How proteins aggregate and form amyloid/insoluble fibrils
Several factors may induce
protein aggregation:

1-Protein oxidation (a-synuclein in PD)

2-Metal chelation (Prion disease and AD) E

a
3-Specific protein cleavage (AD) r
l
y
4-Inefficient protein degradation of
b-sheet proteins/ubiquitin accumulates
within the lesion
(PD, AD, ALS, Prion Disease, HD)

5-Change in intracellular pH (AD)

Late

Ross CA, Poirier MA. Nat Med. 2004 Jul;10 Suppl:S10-7. Review.
 Aggregates

toxic or protective? 100,000,000 $$$$$ question!!!

Different evidences in the different neurodegenerative
diseases

AD: plaques, soluble Ab, correlate with progression of the disease.

However, Ab oligomers seem to be the more toxic species.

PD: inclusion bodies do not follow the progression of the disease.

HD: aggregates may be present ONLY in surviving neurons.

Winklhofer et al.,
Representation of structural components of protein structure
Protein folding: a folding funnel to change the structure and the
energy of proteins. Only folded, native proteins are functionally
active.

Unfolded states: characterized by higher degree of conformational

entropy and free energy than native states. This leads to unstableness
of proteins when in the unfolded state. As the folding funnel proceeds,
conformational entropy decreases as proteins have lower number of
conformational states, as well as the free energy decreases. The
minimum of the energy level of a protein is reached when its in its
native/folded state.
Thermodynamics of protein folding/misfolding
The energy of the
different conformations
decreases with the
development of organized,
native-like proteins.
Protein misfolding
A common and continuously happening phenomenon in the life of a
protein

Denaturation: the process by which the native structure of proteins is

disrupted. It results in the unfolding of the protein, which then
loses the state of lower energy level.

The protein is then in a highly disorganized structure and tends to

form aggregates to reduce the state of high energy, in a word to
stabilize.
 Steps that lead to formation of aggregates

Unfolding

Nucleation: when proteins attach REVERSIBLY to a growing

core.

Aggregation: when proteins attach IRREVERSIBLY to the core

forming larger aggregates. Aggregation can be triggered by
hydrophobic residues in the sequence of the protein and by b-sheet
structure. Amyloid is one of the forms of protein aggregates that
occurs in nature, is very stable but its formation can be still
reversible. This is not true, unfortunately, for most of the protein
aggregates that are responsible of neurodegenerative diseases.
Factors that might influence protein denaturation and misfolding

-Mutations

-Glucose levels

-Oxidation

-changes in the physiological pH

-Binding to ions

-Levels/concentration of monomers: if low the protein tends not to

aggregate, if high the protein tends to form aggregates
Factors that might affect protein misfolding
Schematic representation of protein misfolding
Summary of protein folding diseases
Deposition of fibrillar proteinacious material in Alzheimers disease

Alzheimers disease: characterized by extracellular depositions, the b-amyloid plaque,

and intracellular depositions, the Neurofibrillary Tangles (NFT) comprised of Paired
Helical Filaments (PHF), aggregates of hyperphosphorylated protein tau.

Bossy-Wetzel E, et al., Nat Med. 2004 Jul;10 Suppl:S2-9. Review.

 Origin of the b-amyloid plaque

1- b-amyloid is DIFFERENT from amyloid. b-amyloid contains specifically the b-

amyloid peptide, an approximately 4kDa peptide (Ab40, Ab42) deriving from the amyloid
precursor protein APP when it undergoes the so called amyloidogenic pathway.
b-amyloid plaque contains also ubiquitin and other proteins coming from degenerative
neurons and glial cells.

2- The b-amyloid peptide is insoluble in water. When released from the precursor protein it
assumes a b-sheet conformation that makes it hydrophobic.

3- b-amyloid peptides forms oligomers that may affect neurotransmitter release and
synaptic plasticity. Oligomers will further aggregates in larger structures called fibrils, that
will form the core of the b-amyloid plaque, depositing in the extracellular matrix.

4- The size of the plaque will increase following the progression of the disease.
Scientific clear evidences are still missing in order to understand whether the plaque is a
consequence or a cause of AD. Indeed, Ab and plaques formation are associated to
neuronal death. Inherited forms of AD lead to substantial increase of Ab production.
 Origin of PHF and NFT

1-NFT are composed of paired helical filaments (PHF), aggregates of phosphorylated

protein tau that form when levels of phosphorylated tau are elevated in the cell.

2-Tau is a microtubule-associated protein that regulates cytoskeleton structure. When

highly phosphorylated, tau is sequestered into PHF, and causes disruption of
microtubules, that ultimately leads to cell death.

3-Phosphorylation of tau by protein kinases such as the neuron-specific cyclin

dependent kinase 5 (cdk5) precedes the formation of PHF that cause
neurodegeneration.

4-Importantly, the formation of PHF and NFT is a hallmark in AD and many different
neurodegenerative diseases, which together are called tauopathies.
Deposition of fibrillar proteinacious material in Parkinsons disease

Parkinsons disease: characterized by dopaminergic neuronal loss and by

intracellular depositions, the Lewy bodies, comprised of a-synuclein and ubiquitin,
as the major components. Other components are proteasome and cytoskeletal proteins
and other proteins that interact with a-synuclein.

Bossy-Wetzel E, et al., Nat Med. 2004 Jul;10 Suppl:S2-9. Review.

How a-synuclein is involved in Lewy bodies formation
Oxidation of Dopamine and subsequent interaction with a-synuclein or with
Cys residues on different substrates: first step to the formation of
protofibrils

O2

a-synuclein
on Tyr, Met or Lys
Long-lived protofibrillar
+ H 2 O 2 + O2 - intermediate
 Deposition of fibrillar proteinacious material
in Huntingtons disease

Huntingtons disease: a progressive neurodegenerative disease characterized by CAG repeats

causing glutamine expansion motifs (polyQ) in the N-terminal region of the protein huntingtin.
Onset of the disease inversely correlates with the number of CAG repeats. Huntingtin can
aggregate forming intracellular inclusion bodies that contain also proteasome proteins and
ubiquitin. Huntingtin aggregates contain b-sheet structures similar to amyloid.

Ross CA, Poirier MA. Nat Med. 2004 Jul;10 Suppl:S10-7. Review.
 Deposition of fibrillar proteinacious material
in Creutzfeldt-Jakobs disease (Prions disease)

Prions disease: neurodegenerative disorder caused by prions, via environmental stimuli or

genetic mutations. Alteration in the prion protein lead to both intracellular and extracellular
accumulation of amyloid aggregates, plaques, similar to those characteristic of AD, and
positive to prion protein staining. Probably, replication and accumulation of the protease
insensitive PrPsc results in fibril formation and plaque deposition.

Alzheimers Creutzfeldt-Jakobs
 Pathogenic mechanism of prion protein
Prion: the transmissible principle that causes Transmissible Spongiform Encephalopaties
(TSE), also called Mad Cow disease or Prions disease or Creutzfeldt-Jakob disease

It is caused by the replication of a protease-resistant modified form of the cellular prion protein.
Cellular prion protein PrPc is converted to Scrapie prion protein PrPsc. The infectious principle
may consist of i) PrPsc subspecies, ii) unstable intermediate of PrPc, iii) or a complex with PrPsc
and other host-derived proteins
 Deposition of fibrillar proteinacious material
in Amyotrophic Lateral Sclerosis (ALS)
ALS: a progressive fatal disease caused by the degeneration of lower motor neurons in the
lateral horn of the spinal cord and the upper motor neurons of the cortex. Insoluble
cytoplasmic inclusions are observed in the brain of ALS patients. These inclusions are
composed of SOD1 protein and ubiquitin. However, SOD1 does not form aggregate in vitro
and is not usually observed in sporadic cases.

Ross CA, Poirier MA. Nat Med. 2004 Jul;10 Suppl:S10-7. Review.
How the cell tries to cope with the presence of aggregates
In conclusions:
Most of the neurodegenerative diseases are characterized by intracellular or
extracellular deposition of insoluble material.

Whether this is a cause or a consequence of the diseases is

not known yet.

It is speculated that the early species in this process might be most toxic,
by being involved in abnormal interactions with other cellular proteins.

However, the fact that all these diseases are characterized by the same
common factors, and the observation that inherited forms of these diseases
cause a massive increase in the production of b-sheet related proteins lead
to hypothesize that these b-sheet proteins and the subsequent formation of
the insoluble lesions may be upstream the cascade of events that lead to
neurodegeneration.
 Common cellular pathways that lead to increased levels of
proteins or peptides that form insoluble aggregates

Many of them are related to aging, or can be triggered by particular

toxins or by loss-of-function or gain-of-function protein mutants.

1- Mitochondrial dysfunction and increased oxidative stress, increased

production of ROS

2-Increased apoptosis

3-Decreased chaperones and proteasomal activity

4-Alterations in the integrity of the cell membrane: implications for altered

levels of intracellular cholesterol
Mitochondrial dysfunction and oxidative stress
The neuron
Neuronal synapse
The mitochondrion
 Mitochondria Dysfunction

Damaged Hypoxic

Normal

www.alsa.org

Pharmaceuticals 2010, 3(1), 158-187

 Mitochondrial activity

The role of the mitochondria is to produce ATP through a process called Oxidative
Phosphorylation. This process occurs thanks to a complex system of redox reactions
that moves H+ and e- between the inner membrane and the inter space of the
mitochondrion, generating H+ gradient that moves the reactions.

H2O is a product of this reaction

oxidase
O2 + e - O2-. Superoxide ion
dismutase
O2-. + e- + 2H+ H2O2 Hydrogen peroxide
catalase
H2O2 + e- + H+ OH. + H2O Hydroxyl radical

OH. + e- + H+ H2O

This is the last step, last reaction of the cellular respiration upon the action of
cytochrome c oxidase, aka Complex IV.
 Cellular Respiration

1- This process occurs thank to a H+ gradient generated through the transport of electrons
between the two sides of the inner membrane. Many proteins are involved in this process,
which is based on a chain of redox reactions. Among these, the NADH dehydrogenase,
also called Complex I, is a proton pump, crucially involved in the transport of 4 H+,
creating a strong H+ gradient. Lack or loss of function of Complex I leads to e- leakage in
the intermembrane space, and in the cytosol, initiating the process that leads to the formation
of Reactive Oxygen Species, ROS.

2- e- are transported to proteins thank to e- transporters like Fe-S and cytochrome c,

involved in different moments of the mitochondrial respiration. The succinate
dehydrogenase, also called Complex II, moves e- from intermediate species, and
cytochrome bc complex, also called Complex III, move e- to the cytochrome c and pumps
back 4 H+ from the inter-space to the inner-space, creating a strong H+ gradient.

3- Cytochrome c oxidase, also called Complex IV, removes the e- from cytochrome c
molecules and transfer them to molecular oxygen O2, creating H2O. It also moves 4 H+ back
out of the inner space of the mitochondrion, re-initiating the H+ gradient.
During synthesis of molecules of water, numerous Reactive Oxygen
Species (ROS) are generated

mitochondrial deficit related to either aging or protein dysfunction

may lead to leakage of reactive species out of the mitochondrion into
the cytosol through Voltage Dependent Anion Channel (VDAC), in
particular

O2-. Superoxide ion and e-

This phenomenon increases oxidative stress and promotes the

generation of other ROS within the cell, being toxic for the cell.
 How oxidative stress is toxic?

By promoting oxidation of

1- proteins

2-lipids

3-cathecolamine (adrenaline, noradrenaline, dopamine)

4-DNA
 Transition Metal Ions
All transition metal ions (many are co-enzymes) are toxic

1-by participating to the Haber Weiss reaction, that leads to production

of ROS, and subsequent disruption of the FeS cluster in the
mitochondria by O2-.

H2O2 + Me+ OH. + OH- + Me2+

Me2+ + O2-. Me+ + O2

2- by initiating in vivo a chain reaction of lipid peroxidation, with a
mechanism that is still unknown, but that involves both forms of the
metal ion

LOOH + Fe2+ LO. +OH-+ Fe3+

LOOH + Fe3+ LOO. +H+ + Fe2+
 However, the cell has a fine mechanism
to control the amount of ROS

Endogenous scavengers, low-molecular weight proteins that work

as reducing substrates of peroxidases
1- Glutathion 2GSH GSSG (GSH levels are higher in astrocytes
than in neurons)

2- Bilirubin, than can be oxidized to biliverdin again

degradation reductase
Heme biliverdin bilirubin

Exogenous scavengers
1-Ascorbate or Vitamin C, Vitamin E, Flavonoids (from fruit and
vegetable, in particular Ginseng, and Ginko Biloba)
Lipid peroxidation chain reaction can be stopped ONLY
by a-tocopherol or Vitamin E

LOO. +Toc-OH LOOH + Toc-O.

Toc-O. is a non-reactive species

 Antioxidant proteins and enzymes
Superoxide Dismutase SOD: dismutes ion supeeroxide O2-. to
hydrogen peroxide H2O2

- 3 different types, SOD1, SOD2, SOD3 localized in different

intracellular compartments

-SOD1 (CuZnSOD): localized mainly in cytosol and nucleus and in

the intermembrane space of the mitochondria. Abundantly expressed
ubiquitously, in the brain, and high levels of expression in the spinal
cord. More than 100 mutations are found in genetic ALS, motor neuron
disease in which SOD1 shows a gain of toxic function (SOD1 KO
mice have no motorneuron degeneration)

-SOD2 (MnSOD): essential for mitochondrial integrity. When

nitrosylated by peroxinitrate, SOD2 loses its function.

-SOD3: low expression profile in brain.