Biology of Neurodegenerative Diseases BIOS E-108Harvard Extension School Fall 2011

Lucia Pastorino, Ph.D.

Contact:

office 617-735-2234

Policy of the course

InstructorLucia Pastorino, Ph.D.Instructor in MedicineBeth Israel Deaconess Medical CenterHarvard Medical School3 Blackfan Circle CLS #428Boston, MA 02115lpastori@bidmc.harvard.edu Class Location and TimeTuesday, 7:35-9:35 pm, Science Center, Hall E Office HoursMondays 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. SectionsRequired/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.  

PrerequisitesBIOS 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 course’s website. Students will be notified with an email when each class is available to be downloaded. WebsiteAssignments, 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:ExamsThere will be two midterm exams and one final exam. No make-up or re-take exam will be allowed for any of the exams.  AssignmentsBoth 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:Midterm exam 1: 25%Midterm exam 2: 25%Final exam: 30%Assignment: 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.

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

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 . Parkinson’s 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. Alzheimer’s 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. Alzheimer’s 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. Alzheimer’s 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

Irreversible neuronal death

No treatment that can revert the disease

Characteristics

Journal of Cerebral Blood Flow & Metabolism (2011) 31, 328–338

Degeneration in cortical neurons

Normal Degenerating Dead Neuron

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 Parkinson’s disease; -amyloid binding molecules will be used for PET of Alzheimer’ diseases, etc.

-MRIX-rays of the all brain, evaluating changes in brain volume in specific areas

Normal Alzheimer’s diseases

MRI

Computer graphic

MRI in Alzheimer’s disease (AD)

PET scan in Parkinsons’s disease (PD)

MRI analysis in Huntington’s disease (HD)

Huntington’s disease

Normal

Nuclei Cortex Cerebellum

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 0.1-10M. Ribbon-like-sheets motifs are formed by -strands and -turns. These kind of fibrils are common to different neurodegenerative diseases, from Alzheimer’s to Huntington’s disease. Conformation-specific antibodies (raised against specific proteins which form a specific amyloid formation) can recognize only the -sheet-polymeric conformation but not the monomeric-soluble conformation of the protein substrate. Thus, the -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 (-synuclein in PD)

2-Metal chelation (Prion disease and AD)

3-Specific protein cleavage (AD)

4-Inefficient protein degradation of -sheet proteins/ubiquitin accumulates

within the lesion (PD, AD, ALS, Prion Disease, HD)

5-Change in intracellular pH (AD)

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

Early

Late

Aggregates

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

Different evidences in the different neurodegenerative diseases

AD: plaques, soluble A, correlate with progression of the disease. However, A 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 it’s in its native/folded state.

“The energy of the different conformations decreases with the development of organized, native-like proteins”.

Thermodynamics of protein folding/misfolding

Protein misfoldingA 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 -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

Alzheimer’s disease: characterized by extracellular depositions, the -amyloid plaque, and intracellular depositions, the Neurofibrillary Tangles (NFT) comprised of Paired Helical Filaments (PHF), aggregates of hyperphosphorylated protein tau.

Deposition of fibrillar proteinacious material in Alzheimer’s disease

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

Origin of the -amyloid plaque

1- -amyloid is DIFFERENT from amyloid. -amyloid contains specifically the -amyloid peptide, an approximately 4kDa peptide (A40, A42) deriving from the amyloid precursor protein APP when it undergoes the so called amyloidogenic pathway. -amyloid plaque contains also ubiquitin and other proteins coming from degenerative neurons and glial cells.

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

3- -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 -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, A andplaques formation are associated to neuronal death. Inherited forms of AD lead to substantial increase of Aproduction.

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 Parkinson’s disease

Parkinson’s disease: characterized by dopaminergic neuronal loss and by intracellular depositions, the Lewy bodies, comprised of -synuclein and ubiquitin, as the major components. Other components are proteasome and cytoskeletal proteins and other proteins that interact with -synuclein.

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

How -synuclein is involved in Lewy bodies formation

O2

+ H2O2 + O2-

Oxidation of Dopamine and subsequent interaction with -synuclein or with Cys residues on different substrates: first step to the formation of

protofibrils

-synucleinon Tyr, Met or Lys

Long-lived protofibrillar intermediate

Deposition of fibrillar proteinacious material in Huntington’s disease

Huntington’s 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 -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-Jakob’s disease (Prion’s disease)

Prion’s 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.

Alzheimer’s Creutzfeldt-Jakob’s

Pathogenic mechanism of prion protein

Prion: the transmissible principle that causes Transmissible Spongiform Encephalopaties (TSE), also called Mad Cow disease or Prion’s 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 -sheet related proteins lead to hypothesize that these -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

www.alsa.org

Normal

Pharmaceuticals 2010, 3(1), 158-187

HypoxicDamaged

Mitochondria Dysfunction

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

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+

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

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

Heme biliverdin bilirubindegradation reductase

Exogenous scavengers1-Ascorbate or Vitamin C, Vitamin E, Flavonoids (from fruit and vegetable, in particular Ginseng, and Ginko Biloba)

Lipid peroxidation chain reaction can be stopped ONLYby -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.