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DRAFT

Bi / CNS 150

Lecture 24

Friday November 20, 2015

Two neurodegenerative diseases

Henry Lester

Kandel, Chapters Chapter 43, 44 (p. 1002 - 1012), 59

Alzheimer’s disease

1. Clinical description

2. Genetics

3. Pathophysiology

4. Biomarkers and animal models

5. Heterozygote advantage: none known

6. Therapeutic approaches

We’ll follow this general organization for all neural diseases

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1. Symptoms of Alzheimer’s Disease

1. AD begins with a “pure” impairment of cognitive function. “mild cognitive impairment” does not always lead to dementia.

2. ProgressionA. AD begins slowly. At first, the only symptom may be mild forgetfulness.

In this stage, people may have trouble remembering recent events, activities, or the names of familiar people or things. They may not be able to solve simple math problems. They may begin to repeat themselves every few minutes in conversation.

B. In the middle stages of AD, individuals may forget how to do simple tasks, like

brushing their teeth or combing their hair. They can no longer think clearly. They begin to have problems speaking, understanding, reading, or writing.

C. Late stage: AD patients may become anxious or aggressive, or wander away

from home. Eventually, patients need total care.

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Incidence of Alzheimer’s disease (AD)

AD is the most common degenerative brain disease (est. 5 million USA, 25 million globally) Risk Factors: age

(However, AD is not considered a normal part of aging). The more common form of AD, known as late-onset or sporadic AD, occurs later in life, with no

obvious inheritance pattern. However, several risk factor genes may interact with each other to cause the disease.

Most common risk factor gene identified so far for late-onset AD, is a gene that makes one form of apolipoprotein E (apoE). ApoE4 (prevalence ~ 16%) is the risk factor gene, 3-4 fold dominant increase.

Familial AD, which is rarer, usually starts at age 30 - 60.

65-74 75 80 >85

~5% ~10% ~20% ~50%

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The Anatomical Hallmark of Alzheimer’s Pathology:Amyloid Plaques and Neurofibrillary Tangles in Brain

Amyloid Plaquescontain large amounts of a 42 amino acid peptide termed “-amyloid”, or A42

In the next lecture, we note that -amyloid itself is the initial cause of the pathophysiology that leads to dementia. Amyloid plaques probably contribute to the later stages of pathology

Neurofibrillary tangles: rich in cytoskeletal proteins, especially the microtubule-associated protein, “tau”. In the tangles: heavily phosphorylated proteins, which may cause aggregation and precipitation of the cytoskeleton.

Also generally reduced brain volume, especially in entorhinal cortex and hippocampus

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Red circles: presenilin 1 and APP mutations associated with familial AD

Hardy and Selkoe, 2002, Science

presenilin 1 is part of -secretase, a membrane-associated protease

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There are “tauopathies” as well,

Mutations in tau protein.

These cause frontotemporal dementia with parkinsonism,

linked to Chromosome 17

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3. Pathophysiology. Aβ40 and Aβ42 are proteolytic products formed from APP

Overproduction of A40 and A42 results from altered ratio of proteolytic cleavages at sites termed ,, and.

β-APP = amyloid precursor protein. APP proteins: 110 to 140 kDal.APP expressed by most tissues, especially neurons; reaches axon terminals and dendrites. APP is also found in glial cells.

Kandel et al.,Principles of Neural Science © McGraw-Hill Professional Publishing

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All known genetic risk factors predisposing to Alzheimer’s disease Increase accumulation of Aβ peptides

Chromosome Gene defect Phenotype

21 β-APP mutations ↑All Aβ peptides, or Aβ40 peptidesA673T↓ Aβ peptides, AD, cognitive decline

19 ApoE4 polymorphism(ε4 allele)

↑Density of Aβ plaques & vascular deposits

14 Presenilin 1 mutation ↑Production of Aβ42 peptides

1 Presenilin 2 mutation ↑Production of Aβ42 peptides

6 TREM2 ↑Density of Aβ plaques

“The precise meaning of the amyloid hypothesis changed over the years, and differs among scientists. Originally, it was thought that the actual amyloid is pathogenic—hence the term “amyloid hypothesis”. The more current version of this hypothesis posits that Aβ (especially Aβ42) microaggregates—also termed “soluble Aβ oligomers” or “Aβ-derived diffusible ligands” (ADDLs)—constitute the neurotoxic species that causes AD” – Sheng, 2012

Abnormal states of tau mediate some effects of β-amyloid. This stage may be distal to the more toxic dimers and oligomers.

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Aβ42 peptides form soluble oligomers of ~4 to 40 peptides.

These oligomers interact with other proteins and precipitate to form amyloid plaques.

Soluble oligomers of Aβ42 (containing ~12 to 40 peptides) are toxic to neurons.

The 12-mer is the most significant toxic form.

Misfolded Aβ42 may spread from one cell to another, like a prion.

Soluble Oligomers of Aβ42

There are no convincing data suggesting the presence of a single “Aβ receptor”

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Normal function of presenilin’s -secretase

activity:Notch signaling?

APP cleavage would be a

“side effect”

Components of the core γ-secretase proteolytic complex:

Presinilin (either PS1 or PS2),

anterior pharynx-defective 1 (APH-1),

nicastrin (NCT), and

presenilin enhancer 2 (PEN-2)..

Barthet et al, Progress in Neurobiology 201211

Accumulation of erroneous, misfolded, or partially processed proteins continues as a major theme in neurodegenerative disease.

When proteins accumulate in the endoplasmic reticulum, this causes ER stress and the unfolded protein response. An unchecked unfolded protein

response can eventually cause cell death.

General Cellular Processes that Could Account for aβ Pathology

Excitotoxicity, excess Ca2+ influx

Excess excitation overwhelms the cell’s ability to maintain ion gradients via ion-coupled transporters.

Various metabolites accumulate in wrong compartments (extracellular, cytosol, organelle).

Ca2+ also accumulates in the cytosol and organelles, activating transduction systems, overactivating enzymes, and generally leading to cell death.

Protein homeostasis and turnover

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(Injected into ventricles 10 min before high frequency stimulation of the rat Schaffer collateral pathway).

Walsh et al., Nature 2002

CM = “conditioned medium” from a cell line engineered to express Aβ

Wild Type CM

CM + Aβ oligomers

CM + nonspecific“control” antibody

CM + Antibody against Aβ

Specific example of synaptic malfunction: soluble oligomers block induction of long-term potentiation

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Animal models:

mice overexpressing APP, especially with AD-associated mutations.

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4. Biomarkers for AD.

Only an autopsy is conclusive, but progress in two areas:

1. Cerebrospinal fluid analyses of tau, phospho-tau at position 181, and Aβ42.Individual values, or ratios among these.

2. A positron emission tomography (PET) marker, [18F]Florbetapir, binds to plaques containing β-APP.

“A negative scan indicates sparse to no neuritic plaques and is inconsistent with a neuropathological diagnosis of AD at the time of image acquisition; a negative scan result reduces the likelihood that a patient’s cognitive impairment is due to AD. A positive scan indicates moderate to frequent amyloid neuritic plaques; neuropathological examination has shown this amount of amyloid neuritic plaque is present in patients with AD, but may also be present in patients with other types of neurologic conditions as well as older people with normal cognition. [Florbetapir] is an adjunct to other diagnostic evaluations.”

Negative Positive

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5. Heterozygote advantage: none known

Acetylcholinesterase inhibitors (because cholinergic basal forebrain neurons are among the first to die in AD)

donepezil, galantamine, rivastigmine

NMDA inhibitorsmemantine

Still in development β-secretase inhibitors

Failed: latrepirdine = dimebolin (unknown mechanism)

6. Therapeutic approaches

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Two types of candidates target this protease complex:

"Notch-sparing" γ-secretase inhibitors, which block cleavage of APP selectively over that of Notch.

γ-secretase modulators, which shift the proportion of Aβ peptides produced in favor of shorter, less aggregation-prone species.

γ-secretase inhibitors such as semagecestat have failed so far

Gantenerumab (penetrates BBB)

Solanezumab (mildly successfully in mild stages of AD)

Bapineuzumab

Antibodies against β-APP or amyloid-β have given disappointing results

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James Parkinson, apothecary surgeon1817, An Essay on the Shaking Palsy, described "paralysis agitans", from observations of 6 individuals during his daily walks in London

Parkinson’s disease (tremor at rest 3-5 Hz, “pill-rolling”, slow movements, particularly when starting; short, rapid steps)

but most Parkinson patients are either medicated or electrically stimulated

Parkinson’s disease

1. Clinical description

2. Genetics

3. Pathophysiology

4. Biomarkers and non-human models

5. Heterozygote advantage: none known

6. Therapeutic approaches: a, Symptomatic relief; b. Protection

http://www.youtube.com/watch?feature=endscreen&v=j86omOwx0Hk&NR=1x

Excellent dramatization of the large motor problems in a PD patient

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Rodent brain section, stained for tyrosine hydroxylase: coronal view

Dopaminergic neurons in the human brain: Saggital view

Substantia Nigra: Dopaminergic neurons die in PD

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Symptoms appear when “most” (50% -80%)

SNc neurons are lost.

Most people lose dopaminergic neurons throughout life; PD patients have lost more.

Remaining dopaminergic neurons may form additional “sprouts” which partially and temporarily compensate for the loss.

Nestler, Hyman, Malenka, Molecular Neuropharmacology,

© McGraw-Hill Professional Publishing 20

--

-+

+

+

+ +-

-GP, gobus pallidusi, internale, external

STn, subthalamicnucleus

A hallmark of PD pathology:Intracellular “Lewy bodies”, especially in dopaminergic neurons

(Lewy bodies also occur in other diseases, especially some dementias)

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PD patients often have additional symptoms and degeneration

Constipation. Detailed surveys show that most PD patients have constipation long before the clinical symptoms.

Constipation does not predict PD.

Intestinal biopsies show Lewy bodies in the neurons of the intestinal wall.

Braak staging: various neurons with long axons through the brain show Lewy bodies before dopaminergic neurons; but there are few or no symptoms.(Braak & del Tredici, Neurology 2008)

Sleep disorders, especially in rapid eye movement sleep.

Olfactory problems (patients have “anosmia”).

Depression.

Dementia.

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3. Genetics. Familial Parkinson’s disease provides a good review of biochemistry(~ 10% of patients). Onset 30’s to 50’s (rarely earlier or later)

LocusChromosome location

Gene or protein nameInheritance pattern

PARK1 & PARK4

4q21–q23 -synuclein(Membrane fusion in various organelles)

AD

PARK2 6q25.2-q27 ParkinE3 Ubiquitin ligase

AR

PARK3 2p13 Unknown AD, IP

PARK5 4p14 UCH-L1ubiquitin-C-terminal hydrolase-L1

AD

PARK6 1p35-p36 PINK1, PTEN-Induced Putative Kinase 1 AR

PARK7 1p36 DJ-1Uknown function

AR

PARK8 12cen LRRK2leucine-rich repeat kinase 2

AD

PARK9 1p36 ATP13A2 AD

PARK10-PARK16

Various Much less is known various

AD, autosomal dominant; AR, autosomal recessive; IP, incomplete penetranceSee also Table 44-3 23

1. Most cases are unexplained.

2. Dopaminergic neurons may be selectively vulnerable because the cell body must

maintain large amounts of axoplasm and presynaptic proteins.

3. Dopaminergic neurons may be selectively vulnerable because dopamine is highly

reactive.

4. Pesticides (Wang et al, Eur J Epidemiol. 2011).

5. The “frozen addict”. An impurity in synthetic heroin.

Taken up by the dopamine transporter (expressed only in dopaminergic cells). Kills cells.

6. The “encephalitis lethargica” pandemic (worldwide epidemic) of 1918 killed 20

million people. Some people experienced selective damage to dopaminergic

neurons. Presumably an autoimmune reaction was provoked by an still-unknown

infectious agent (virus or bacteria). (“Awakenings”, O. Sacks).

HO

HO

H2C

CH2

NH3+

dopamine

reactive:oxidative damage?

Parkinson’s disease: pathophysiology

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-synuclein has an unknown function; it’s an “intrinsically disordered protein”.

Mutant -synuclein forms fibrils.

Improper mitochondrial fission / fusion may be one of the early events(Prof. D. Chan, Caltech)

Pathogenic hypotheses for toxicity in neurodegenerative disease

Enlarged from Figure 44-6 26

4. Animal Models for Parkinson’s Disease: Drosophila that overexpress synuclein

1. The 4 dopaminergic neurons die preferentially!

We don’t know why.

3. The flies show a “movement disorder”

(2. The cells show dense structures like Lewy bodies)

See also work of Prof. Bruce Hay, Caltech 27

More Models for Parkinson’s Disease

a. Toxin-treated mice, rats, and monkeys

b. Mice with altered PARK genes

c. Yeast that harbor synuclein mutations (Cooper et al, Science 2006)

b. Cultured cells from people carrying PD mutations (“disease in a dish”)

human embryonic stem cells (hESCs) human induced pluripotent stem cells (iPSCs)

But there are still major technical issues in generating dopaminergic neurons that behave like the vulnerable neurons. Distinction between SNc and VTA? Antibody stains for tyrosine hydroxylase and other markers are not yet sufficient to reveal a true phenotype.

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Biomarkers for Parkinson’s Disease

a. Experienced neurologist is the best judge. No effective blood test for PD, Responsiveness to L-dopa is a good criterion.

a. Imaging: [125I]ioflupane, high-affinity dopamine transporter (DAT) ligand.Single-photon emission computed tomography (SPECT)

http://en.wikipedia.org/wiki/File:Datscan.JPG

Normal striatumMay help to differentiate Parkinsonian symptoms from conditions with similar symptoms, such as essential tremor.

Effectiveness as a screening or confirmatory test and for monitoring disease progression or response to therapy has not been established.

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6. Therapeutic approaches to Parkinson’s disease

a. Symptomatic relief: L-dopa

5. Heterozygote advantage: none known

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levodopa, “L-dopa”zwitterion

permeates into brainvia a transporter

dopamine

does not enter brain

enzyme:decarboxylase

HO

HO

H2C NH3

+

CO2-

HO

HO

H2C

CH2

NH3+

Used with carbidopa, which inhibits decarboxylase.

Prevents hydrolysis in the blood and in the peripheral nervous system. HO

HOH2C

C

HN

NH3+

CO2-H3C

D2 receptor agonist is often added. This seems to reduce dyskinesias (next slide).

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L-dopa remains effective only as long as sufficient dopaminergic neurons remain to take up and secrete dopamine.

Dyskinesias (“bad movements”, very common in people who have used L-dopa for many years, often seen in TV appearances of medicated PD patients).

Mechanism is unknown. “Outside-in” continued activation of Gi-coupled pathways?

Visual hallucinations, other psychotic symptoms, sleep disturbances, and confusion.

“On-off” phenomenon: abrupt and transient fluctuations in the severity of parkinsonism at intervals during the day; such fluctuations are unrelated to ongoing drug dosage.

Side effects of L-dopa

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Monoamine oxidase (MAO type B) inhibitors

Muscarinic antagonists may prolong the action of certain key interneurons in the striatum

Amantadine (blocks NMDA Receptors, may decrease excitotoxicity)

Mitochondrial stabilizers, such as coenzyme Q10 and creatine

Adenosine receptor antagonists (Gs coupled, A1 or A2A), mechanism uncertain

Other drugs for relief of PD symptoms.

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Activate neurons?

Silence neurons?

Axons passing through

Deep brain stimulation for Parkinson’s Disease

Developed from ablation

Nestler, Hyman, Malenka, Molecular Neuropharmacology,

© McGraw-Hill Professional Publishing 34

6. Therapeutic approaches to Parkinson’s disease

b. Arrest the degeneration Goal: intervene in early-stage PD with drug taken from that point.

Some degeneration has already occurred.

Good example of the interplay between diagnosis & therapy.

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Does deep brain stimulation for

Parkinson’s disease delay degeneration?

Data are unclear, both in humans

and animal models.

Nestler, Hyman, Malenka, Molecular Neuropharmacology,

© McGraw-Hill Professional Publishing

See also work of Prof. Gradinaru, Caltech

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Smokers get less Parkinson’s disease.Inverse correlation: between a person’s history of smoking, and the risk of PD

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B. Nicotine itself is probably a part of smoking’s apparent neuroprotective action

1. In rodents and monkeys, nicotine protects dopaminergic neurons against toxins

2. α4 nicotine receptor knockout mice lack this protection

A. Tobacco use protects against PD, vs PD patients use less tobacco

1. Clinical trial under way for early-stage PD patients given nicotine patches

2. Nicotine itself is a perfect addictive drug but a sub-optimal therapeutic drug.

3. Nicotine itself activates several nicotinic receptors; many people cannot tolerate patches.

D. Simply use nicotine patches?

C. We need additional human data. Will vapers get less PD?

Enlarged from Figure 44-6 38

Smoking-relevant nicotine concentration attenuates the unfolded protein response in dopaminergic neurons

 R. Srinivasan, BM Henley, BJ. Henderson, T Indersmitten, BN Cohen, C Kim, S McKinney, P Deshpande, C Xiao, HA LesterJ. Neurosci, in press

Usually, more neurons initially appear in a ganglion or nucleus than are required to innervate the target cells.

The excess neurons die

This occurs because the target secretes a limiting amount of a trophic factor

The “limiting neurotrophic factor” hypothesis for neuronal survival

Figure 53-14

PD Therapeutic approaches: protection by proteins or gene therapy

See lectures on development

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GDNF, a neurotrophic factor

Since the early 1990’s, several pharmaceutical companies have experimented with pumps that infuse glial cell derived neurotrophic factor (GDNF) in or near the substantia nigra or its target, the striatum.

Several companies abandoned, despite anecdotal stories of success; others continue.

PDB: 2GHO

artemin ( a GDNF homolog)

Part of a growth factor receptor

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Attempts at Gene therapy for PD

Using adeno-associated viruses modified to express the protein of interest. “AAV2” is a viral “vector”. Lentivirus vectora also show promise.

Further attempts with GDNF

Attempts with neurturin. Insufficient encouraging results

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Gene therapy withglutamate decarboxylase

in subthalamic nucleus

“AAV2-GAD”

Nestler, Hyman, Malenka, Molecular Neuropharmacology,

© McGraw-Hill Professional Publishing

Safety established; effectiveness unknown.Phase II shows initialEncouraging results(LeWitt et al., Lancet Neurol, 2011)

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End of lecture

Henry Lester cannot attend “office” hours today (Friday)