Alzheimers Disease

Robert Nagele, Ph.D. Director, Biomarker Discovery Center NJ Institute for Successful Aging and Department of Medicine

Inside the Human Brain

To understand Alzheimers disease, its important to know a bit about the brain
The Brains Vital Statistics
Adult weight: about 3 pounds Adult size: a medium cauliflower Number of neurons: 100,000,000,000 (100 billion) Number of synapses (the gap between neurons): 100,000,000,000,000 (100 trillion)
Slide 8

What is Alzheimers Disease?

Alzheimers disease is an irreversible, progressive brain disease that slowly destroys memory and thinking skills.
*The risk of developing AD increases with age *In most cases, symptoms first appear after age 60 *Familial AD appears early 4% of cases are inherited and nearly all of these are mutations in the presenilin gene *AD is not a part of normal aging it is a fatal brain disease.
Slide 4

Alzheimer Warning Signs

Top Ten
(from the Alzheimers Association)

1. Recent memory loss affecting job 2. Difficulty performing familiar tasks 3. Problems with language 4. Disorientation to time or place 5. Poor or decreased judgment 6. Problems with abstract thinking 7. Misplacing things 8. Changes in mood or behavior 9. Changes in personality 10. Loss of initiative

AD Statistics
AD is the most common cause of dementia among people age 65 and older. Estimate that around 4.5 million people now have AD.

For every 5-year age group beyond 65, the percentage of people with AD doubles.

By 2050, 13.2 million older Americans are expected to have AD - if the current numbers hold and if no preventive treatments become available.
Slide 5

Age vs Percentage of Population with Dementia

Care and its Costs

Where are people with AD cared for? home assisted living facilities (those in the early stages) nursing homes (special care units) The national cost of caring for people with AD is about $150 billion every year.

Slide 6

Preclinical AD and MCI

Signs of AD are first noticed in the entorhinal cortex, then it proceeds to the hippocampus.

Affected brain regions begin to shrink as brain neurons cells die.

Changes can begin 10-20 years before symptoms appear. May notice subtle behavioral and memory changes usually attributed to old age.

Memory loss is the first sign.

Wide variety of symptoms hard to diagnose with certainty
Blue stipple indicates disease spread and extent
Slide 20

Mild to Moderate AD
AD spreads through the brain. The cerebral cortex begins to shrink as more and more neurons stop working, lose their synapses and die. Mild AD signs can include memory loss, confusion, trouble handling money, poor judgment, mood changes, and increased anxiety. Moderate AD signs can include increased memory loss and confusion, problems recognizing people, difficulty with language and thoughts, restlessness, agitation, wandering, and repetitive statements.
Note preferred spread to frontotemporal and more posterior regions
Slide 21

Severe AD
In severe AD, extreme brain shrinkage occurs. Brain retracts from cranial vault brain ventricles expand. Patients are completely dependent on others for care.

Symptoms can include weight loss, seizures, skin infections, groaning, moaning, or grunting, increased sleeping, loss of bladder and bowel control.
Death usually occurs from aspiration pneumonia or other infections. Caregivers can turn to a hospice for help and palliative care.
Note: Pathology is widespread symptoms become similar from one person to the next.
Slide 22

8 years average. Range 2-20 years

CLINICAL MILESTONES IN AD
Emergence of cognitive symptoms Conversion from mild cognitive impairment (MCI) to dementia

Emergence of neuropsychiatric symptoms

Nursing home placement Loss of self-care ADL Death

Typical Clinical Syndrome of AD

1. 2. 3. Amnestic type of memory defect difficulty learning and recalling new information Progressive language disorder beginning with anomia and progressing to fluent aphasia Disturbances of visuospatial skills environmental disorientation difficulty copying figures in the course of mental status examination Deficits in executive function Planning Insight Judgment The patient is typically unaware of memory or cognitive compromise. All cognitive deficits progressively worsen.

4.

5. 6.

Alzheimers Disease (Az Staging)

Stage 1: No Impairment (Subclinical) Stage 2: Very Mild Decline (Subclinical) Stage 3: Mild Decline Stage 4: Moderate Decline (Early Stage) Stage 5: Moderately Severe Decline (Mid Stage) Stage 6: Severe Decline (Mid Stage) Stage 7: Very Severely Declined (Late Stage)

Note: Because disease is progressive, the borders between each stage are fuzzy

Dementia, Depression, Delirium: Case (AZ Staging)

Stage 3: Mild cognitive decline Early-stage Alzheimer's can be diagnosed in some, but not all, individuals with these symptoms Friends, family or co-workers begin to notice deficiencies. Problems with memory or concentration may be measurable in clinical testing or discernible during a detailed medical interview. Common difficulties include: -Word- or name-finding problems noticeable to family or close associates -Decreased ability to remember names when introduced to new people -Performance issues in social or work settings noticeable to family, friends or coworkers -Reading a passage and retaining little material -Losing or misplacing a valuable object -Decline in ability to plan or organize

HPI: 84 year old presents to the clinic for an evaluation of memory loss. She has lost her keys and finds them in unusual places (i.e. recently found them in the mailbox) PMHx: Osteoporosis FMHx: Mother w/ Dementia MMSE 29/30 GDS: 3/15

Dementia, Depression,Delirium: Case (AZ Staging)

HPI: 84 year old with AZ dementia and resident of a LTC facility notes a 8lb weight loss during the past two months and has recently required hand-feeding assistance. During this time she spent most of her time in her wheelchair although the nursing staff assists her with ambulation using a wheeled walker daily for exercise. She does brighten w/ pet therapy PMHx: AZ Dementia, COPD, HTN, OA/DJD, Cholelithiasis SOC: resident of LTC Stage 7: Very severe cognitive decline (Severe or latestage Alzheimer's disease) -This is the final stage of the disease when individuals lose the ability to respond to their environment, the ability to speak and, ultimately, the ability to control movement. -Frequently individuals lose their capacity for recognizable speech, although words or phrases may occasionally be uttered -Individuals need help with eating and toileting and there is general incontinence of urine -Individuals lose the ability to walk without assistance, then the ability to sit without support, the ability to smile, and the ability to hold their head up. Reflexes become abnormal and muscles grow rigid. Swallowing is impaired.

RISK FACTORS FOR AD

Age Gender female has more risk Low education and low IQ (e.g., the Nun Study) Low income Positive family history of AD or dementia Apolipoprotein alleles (esp. Apo E4 genotype ApoE4 has been shown to be a carrier of the amyloid peptide across the blood-brain barrier) Systolic arterial hypertension relate to blood-brain barrier leakage High cholesterol Mutations in Amyloid Precursor Protein (APP) generate more amyloid Mutations in genes processing APP (presenilin 1 & 2) generate more amyloid Downs syndrome (chr. 21) contains APP gene - 100% incidence of Alzheimers disease generate more amyloid Head injury (Note: Recent studies of sports-related head injuries) Low serum levels of folate and vitamin B12 evidence weak Elevated plasma and total homocysteine levels evidence weak

PROTECTIVE FACTORS FOR AD

High education mental activity Overall cardiovascular health - Exercise NSAIDS? Statins? Red wine?, Beer? Curcumin? - curry Blueberries? its the color - antioxidant Intellectual leisure activities, socialization

Diagnosing AD
Tools used to diagnose AD: a detailed patient history

information from family and friends

physical and neurological exams and lab tests

Neuropsychological/cognitive tests
imaging tools such as CT scan, or magnetic resonance imaging (MRI).

Very Soon: A simple blood test

AD vs Other Dementias
Alzheimers Type Dementia (50-75 % of cases)
*Vascular cognitive impairment (formerly vascular dementia (10 to 20 %) *Dementia with Lewy bodies (10 to 15 %)

Frontotemporal dementia (5 to 15 %)
*Difficult to distinguish from AD on clinical grounds alone

Atypical Features that Suggest a Diagnosis Other than Alzheimer's Disease

Feature
Abrupt onset

Diagnostic consideration
Vascular dementia (multi-infarct)

Stepwise deterioration

Vascular dementia: involves brain quadrants fed by major vessel that becomes blocked or has blood-brain barrier breakdown
Frontotemporal dementia Frontotemporal dementia Frontotemporal dementia, vascular dementia Vascular dementia, hydrocephalus Delirium due to infection, alcohol, medications, or other causes; dementia with Lewy bodies; seizures Delirium due to infection, medications, or other causes; dementia with Lewy bodies Parkinsonian syndromes, vascular dementia

Prominent behavior changes Profound apathy Prominent aphasia Progressive gait disorder Prominent fluctuations in levels of consciousness or cognitive abilities Hallucinations or delusions Extrapyramidal signs or gait

Why Diagnose AD Early?

Safety (driving, cooking, etc.) Family stress and misunderstanding (blame, denial) Early education of caregivers on how to handle patient Advance planning while patient is competent (will, proxy, power of attorney, advance directives) Patients and Familys right to know Make use of specific treatments now available
May help delay symptoms May delay nursing home placement

Assessment
History Of The Development Of The Dementia
Ask the Patient What Problem Has Brought Him to See You Ask the Family, Companion about the Problem Specifically Ask about Memory Problems Ask about the First Symptoms Inquire about Time of Onset Ask about Any Unusual Events Around the Time of Onset, e.g., stress, trauma, surgery Ask about Nature and Rate of Progression

Physical Examination Neurological Examination Laboratory Tests Neuropsychological / Cognitive Assessment

CLINICAL TOOLS FOR COGNITIVE ASSESSMENT

FOLSTEIN MINI-MENTAL STATE EXAM (MMSE) CLOCK DRAWING ANIMAL NAMING (1 minute) SHORT BLESSED MATTIS DEMENTIA RATING SCALE ALZHEIMERS DISEASE ASSESSMENT SCALE (ADAS) ACTIVITIES OF DAILY LIVING GLOBAL CLINICAL SCALE CLINICAL DEMENTIA RATING SCALE GLOBAL DETERIORATION SCALE / FAST

Mini-Mental State Exam (MMSE)

Orientation to time Orientation to place Registration Attention & Calculation Recall Language 5 points 5 points 3 points 5 points 3 points 9 points 30 points

MMSE Considerations
Specificity is good (96%) But the sensitivity is poor (63%) Using a standard cutoff score of 24 will leave a substantial proportion of cases of early dementia undetected. Asking patients and knowledgeable informants about deficits may enhance detection of early stages of AD
abnormalities in learning and retaining new information difficulty handling complex tasks impaired reasoning ability changes in language or behavioral alterations

ALZHEIMERS DISEASE ASSESSMENT - MMSE

Estimate MMSE as a function of time
30
MMSE score

25 20 15 10 5 0 -10 -8 -6 -4

24

-2

10

Estimated years into illness

Limitations: False-positives (usually depression with pseudodementia) False negatives (early dementia in high functioning patients) Lack of comprehensiveness

Important Note: AD begins many years before symptoms are detectable

General Atrophy in Alzheimers Disease

Brain shrinks due to widespread neuron cell death cerebral cortex and hippocampus. Note larger space between cerebral cortex and cranial vault Ventricles filled with cerebrospinal fluid (CSF) get larger to compensate for cell loss

PET scans showing much reduced glucose utilization in the AD brain compared to controls

Reason: Cell death means less cells capable of metabolizing glucose

Course of Brain Aging, MCI and AD

Brain Aging Cognitive Decline Brain Aging Brain AD
AAMI / ARCD

MCI

Mild

Moderate

Clinical AD

Moderately Severe Severe

Time (Years)

(Ferris, 4/03)

Potential Treatment Outcomes in Alzheimers Disease

Cure
Functional ability

Maintenance of function Slowing of disease progression Symptomatic benefit Natural Progression of AD

Time
(Ferris, 8/03)

Treatment

Other Treatments of Alzheimers Disease? Big Pharma Pipeline

Slowing Progression and Prevention
Antioxidants (dietary supplements) Anti-inflammatory drugs (ADAPT) Amyloid antagonists
secretase inhibitors amyloid vaccines

Anti-neurofibrillar drugs Genetic engineering Stem Cells Reducing vascular risk

statins, homocysteine reduction Blocking blood-brain barrier breakdown* * In my opinion, the only one with promise

Problems in Demonstrating a Real Effect on Disease Progression

Modest drug effects are difficult to detect and measure requires long term studies and very large sample sizes in clinical trials Cannot clearly differentiate symptomatic effects versus slowing of the disease process Biological markers are not well validated No established criteria for proving that the disease is slowing

Current therapies for AD are limited

*Current therapies treat the symptoms and improve performance. But - disease progression continues unchecked

*NO THERAPIES TREAT THE PATHOLOGICAL PROGRESSION OF AD. Why? Because we dont understand the pathology of AD. The field is in a state of confusion.

Aims of future AD therapies

*Treat the pathology directly. *But, we must first understand the pathology and identify pathological targets before we can propose effective treatment strategies.

Research

Whats happening in our brains?

Why do our brain cells die?

Brain Regions and Vulnerable Neurons in AD

Basal forebrain cholinergic system (nucleus basalis) Monoaminergic system Hippocampus (CA1 and CA2 pyramidal cells) Amygdala Frontal cortex Entorhinal cortex Neocortex

PATHOPHYSIOLOGY OF AD
Deposition of insoluble amyloid peptide both in and around neurons primarily involves pyramidal neurons amyloid may or may not be directly toxic (e.g., excessive accumulation of anything can be bad) Formation of millions of amyloid (neuritic) plaques small spherical accumulations of amyloid each may be the end result of a single neuron cell death event Formation of neurofibrillary tangles containing primarily the hyperphosphorylated tau protein the tau protein associates with microtubules in axons and dendrites of neurons Inflammatory response -astrocytosis (activation of astrocytes an early event involved in clearing of synaptic debris) -microgliosis (activation of microglia a later event involved in clearing of dead cell debris and initiating a more global inflammation) Cholinergic deficit loss of acetylcholine receptors in cholinergic neurons each neuron less able to perform acetylcholinesterase inhibitors like Aricept help resolve this

NEUROPATHOLOGICAL HALLMARKS of AD
Causes Amyloid plaques
beta-amyloid protein (Primary problem occur early) Neurofibrillary tangles (Mostly a secondary problem occur later) hyperphosphorylated tau Cerebrovascular amyloidosis (Primary problem) (Clifford et al. 2007)

Blood-brain barrier breakdown (Primary problem) (Clifford et al., 2007). Brain-reactive autoantibodies in blood (Primary problem) (Nagele et al.,
2011)

Effects Neurotransmitter losses

Acetylcholine (Ach) major loss of nicotinic receptors Norepinephrine, serotonin, glutamate, GABA

Inflammatory responses Loss of synapses Death of neurons Appearance and progression of symptoms

Morphology and Distribution of Neuritic Plaques and Neurofibrillary Tangles

-Pathologic diagnosis of AD require the presence of both neuritic plaques and neurofibrillary tangles in excess of the abundance anticipated for age-matched healthy controls. -Neuritic plaques consist of a central core of amyloid protein surrounded by astrocytes, microglia, and dystrophic neurites often containing paired helical filaments. -Neurofibrillary tangles are the second major histopathological feature of AD. They contain paired helical filaments of abnormally phosphorylated tau protein that occupy the cell body and extend into the dendrites
Copyright restrictions may apply.

Cummings, J. L. et al. JAMA 2002;287:2335-2338.

Amyloid (Abeta42) deposition makes neurons sick. Sick neurons retract their axons and dendrites and lose synaptic connections with each other Loss of memory and cognition
Sick neuron

AP

Amyloid plaque
Retracted corkscrew dendrites

Amyloid plaques contain Abeta42

Abeta42 Arises From Sequential Cleavage of the Amyloid Precursor Protein

Dense-core plaque
APP

AD brain

Alzheimers Disease
How Amyloid (Abeta42) Deposits in the Brain
Old Model Our New Model
Amyloid plaques

Intracellular amyloid

Normal healthy brain

AD brain Extracellular amyloid

AD brain Intracellular amyloid

New model shows that amyloid deposits within neurons first; amyloid plaques are the residue left by dead neuron New Model Means New Potential Drug Targets

Neurons accumulate large amounts of Abeta42 (amyloid peptide)

Pyramidal Neurons Nucleus

Sequestered AB42

Brain Regions with Abundant Plaques Are Generally Devoid of Pyramidal Cells

Brain Regions Exhibiting Few Plaques Contain Numerous Abeta42-burdened Pyramidal Neurons

Amyloid Plaque

Pyramidal Neurons

AD and the Brain

Neurofibrillary Tangles

Neurons have an internal support structure partly made up of microtubules. A protein called tau helps stabilize microtubules. In AD, tau changes, causing microtubules to collapse, and tau proteins clump together to form neurofibrillary tangles. Slide 18

Normal versus degenerating neuron

Cell death

Effects of Intraneuronal Amyloid Accumulation on Neurons

The neuron cell body maintains the dendrite tree and axon. Abeta42 accumulates within the cell body Cell body gets sick can no longer support extensive dendrite tree and long axon dendrites and axons collapse loss of synapses cell dies plaque is cell grave marker

Loss of synapses precedes neuron death and the appearance of amyloid plaques in AD brains
Synaptophysin Synaptophysin

ML

ML

Synaptic loss

Age-matched control

AD brain

What is the source of the Abeta42 that accumulates in the Alzheimers brain?
Two possibilities
1. Cells generate their own Abeta42 internally.
2. Abeta42 leaks into the brain from the blood, binds to neurons and is internalized in neurons.

What has changed in the elderly to allow Abeta42 to leak into the brain from the blood? Answer: Aging-associated changes in blood vessels leads to breakdown of the blood-brain barrier this allows things in the blood (like soluble Abeta42 peptide and immunoglobulin) to leak into the brain tissue

Artery Capillary

Medium-sized arteriole in the brain

Atherosclerosis and diabetes damage the endothelial lining of the vessel

In all AD brains, the BBB is defective.

Plasma components (including Abeta42) can now leak into the brain. A42
capillaries

C1q

Abeta42 leak cloud

Art

C1q leak cloud

C5b-9
Plaque

Ig

Ig leak cloud

Neuron

Art

Proof that the blood can serve as the main source of the Abeta42 that deposits in the brain?
Experiment: We directly demonstrated that Abeta42 can leak from the blood into the brain?

Abeta42 accumulates in the brain

Inject fluorescent Abeta42 into tail vein of mouse with defective BBB

Clifford et al., 2007 Brain Research

Abeta42 accumulates in specific types of neurons. The same neurons are affected in human AD brains

Neurons with Abeta42

Fluorescent image

DAPI nuclear stain Mouse brain

Neurons accumulate fluorescent Abeta42 (green) that originated in the blood

cortex
dendrite

cortex
pyramidal cell

N pyramidal cell

dendrite

intracellular accumulated AB42

neg neurons

pos neurons

neurons

What we know so far

1. The blood is the main source of Abeta42. 2. Abeta42 leaks through a defective BBB and enters the brain. 3. Once in the brain, Abeta42 can bind to and accumulate within certain neurons.

Question?
What drives Abeta42 inside the neuron? Answer: Neuron-binding autoantibodies

We all have autoantibodies in our blood that can bind to brain neurons. A defective BBB allows these autoantibodies to leak into the brain and bind to neurons.

The neurons clean their surfaces and internalize the antibodies

Autoantibodies from the blood bind to the same neurons that accumulate Abeta42 in AD brains
AD brain AD brain

neurons

Conclusion: Antibody binding to neurons may play a role in the accumulation of Abeta42 in these cells.

How?
What does the nerve cell do with the antibody stuck to its surface?

Answer
It cleans its surface, brings it all inside via endocytosis and tries to digest it.

A New Problem
Abeta42, also present on the cell surface, is brought inside the cell with the antibody Abeta42 cannot be degraded
Accumulation of Abeta42 within neurons begins.

Test for the role of neuron-binding autoantibodies in intraneuronal amyloid (Abeta42) accumulation in vivo

Kopf Stereotaxic Device Direct injection of Abeta42 and human Ig

into mouse brain to bypass the BBB

Abeta42 accumulates in brain

Micro-injector with Abeta42

mouse

Mouse Brain

Testing for the role of autoantibodies in amyloid (Abeta42) deposition in neurons in vitro
Mouse Hippocampal Slice Cultures

neurons

Note: The hippocampus is an important memory center that is highly vulnerable to AD pathology in humans
Nagele et al., 2011 J. Alzheimers Disease

Autoantibodies Accelerate Neuronal Amyloid Accumulation in vitro

Adult mouse brain slice (organotypic) cultures

Enhancement of intraneuronal A42 accumulation by human sera

0.14

0.12

Relative Abeta42 content per neuron

0.1

0.08

0.06

0.04

0.02

0 No Tx HS1 only HS2 only HS3 only Abeta42 only HS1 + Abeta42 HS2 + Abeta42 HS3 + Abeta42

What can happen when the BBB leaks and autoantibodies enter the brain tissue?
Brain-reactive autoantibodies prevalent in human sera increase amyloid beta 1-42 (AB42) deposition in neurons
RG Nagele1,PM Clifford1, G Siu1, EC Levin1 , NK Acharya1, M Han1, MC Kosciuk1, V Venkataraman2, S Zavareh1, S Zarrabi1, K Kinsler1, N Thaker1, EP Nagele3, J Dash1, H-Y Wang4 and A Levitas5
1New

Jersey Institute for Successful Aging, University of Medicine and Dentistry of New Jersey, Stratford, New Jersey 08084, USA 2Department of Cell Biology, University of Medicine and Dentistry of New Jersey, Stratford, New Jersey 08084, USA 3Durin Technologies, Inc., 120 Albany Street, New Brunswick, NJ 08901, USA 4Departments of Physiology and Pharmacology, Sophie Davis School of Biomedical Education, The City University of New York Medical School, New York, NY, 10031, U.S.A. 5Department of Psychiatry, University of Medicine and Dentistry of New Jersey, Stratford, New Jersey 08084, USA

Conclusion: Brain-reactive autoantibodies contribute to the disease process

Proposed Mechanism and Therapeutic Implications BBB breakdown, Ig leak, dendrite collapse, amyloid accumulation
Atherosclerosis Cerebrovascular amyloidosis Microinfarct
Dendrite collapse

Receptor stripping

Cell death Inflammation

Nagele et al., 2011 J. Alzheimers Disease

Blood-brain barrier integrity protects the brain from brain-reactive autoantibodies in the blood. BBB breakdown plays a role in neurodegenerative diseases.
Intact BBB + Few Reactive Auto-Ig Intact BBB + Many Reactive Auto-Ig Disrupted BBB + Few Reactive Auto-Ig Disrupted Tight Junctions Disrupted BBB + Many Reactive Auto-Ig Disrupted Tight Junctions

Endothelial Cell Tight Junctions

Endothelial Cell Tight Junctions

No Damage Healthy Neurons

No Damage Healthy Neurons

Minor Damage Most Neurons Recover

Extensive Damage Neuronal Cell Death

Disease severity depends on extent of BBB breakdown and autoantibody profile. Symptoms depend on the specific brain region affected.
Levin et al., 2010

Hypothesis
Aging Diabetes High Cholesterol
Traumatic Brain Injury High Blood Pressure

Disrupt Blood brain barrier (BBB)

Influx of Plasma Components into Brain Loss of Brain Homeostasis Disruption of Neuronal Function

Triggers aging-associated neurodegenerative diseases Triggers neurodegeneration resulting from traumatic brain injury

A global therapy for neurodegenerative diseases Goal: Blocking blood-brain barrier breakdown

Darapladib maintains/repairs blood-brain barrier integrity

Block initiation and progression of all neurodegenerative diseases (Nagele-GlaxoSmithKline patent)
Atherosclerosis Cerebrovascular amyloidosis Local chronic inflammation Traumatic brain injury
Dendrite collapse

X
Receptor stripping

amyloid plaque

Potential treatment to block neurodegeneration resulting from traumatic brain injury?

Darapladib blocks BBB breakdown

DMHC pigs showed the greatest extent of BBB breach

Amount of material leaking from arterioles, venules and capillaries

Extent of vessel leak per sq. mm cortex
0.08 0.07 0.06 0.05

n=3
0.04 0.03 0.02 0.01 0

n = 13 n = 12

Control

DMHC Treatment groups

Treatment groups DMHC/C DMHC/Rx Rx/C P-value 0.033088 0.097645 0.298917

Rx

Acharya et al., 2013 J. of Alzheimers Disease

Darapladib blocks intraneuronal amyloid deposition

DMHC pig brains show the greatest extent of amyloid (A42) accumulation
Acharya et al., 2013

Density of A42 positive neurons per pig

No. of A42 pos neurons per 20x viewing field
12

10

Treatment groups and individual pigs

control Diabetes/hypercholesterolemia (DMHC) 93 DMHC/Darapladib

R55 R54 R47 R37 R35 R34 R30 R29 R27 R24 R21 R19 R14

C50 C49 C41

D46 D45 D43 D40 D38 D37 D28 D26 D22 D20 D17 D16 D15

Goal: Do what you can to prevent breakdown of the blood-brain barrier

Cardiovascular exercise

Bob

Minimize the risk factors that we have some control over

Cardiovascular disease; diabetes; hyperlipidemia

Mild Resistance

aerobic

Fun

Early Treatment is Essential for Drug Success

Alzheimers pathology is underway 8-10 years before symptoms appear

Minimental State Exam Results

Estimate MMSE as a function of time
30 25 20 15 10 5 0 -10

MMSE score

Disease Starts
-8 -6 -4 -2 0 2 4 6 8 10

Early Detection Allows Early Treatment (prior to appearance of symptoms)

Estimated years into illness

Autoantibodies (IgG) are surprisingly numerous in human serum

Results using human protein microarrays Protein Microarray Nearly 2,000 autoantibodies are detected using 9,486 human protein targets representing roughly 1/3 of the total human proteome) Effects of Age and Gender on Total Autoantibody Count
Age
< 45 45 - 65 > 65

N
10 32 15

% Female
33.3 18.2 60

Antibody Count P value

1498.2 545.7 2335.6 1009.5 2647.8 1139.2 <45 vs. 45-65: 0.0021 45-65 vs. >65: 0.37 <45 vs. >65: 0.0028

Sex
Female Male Total

N
18 39 166

Age
57.6 18.7 53.1 15.1 62.4 16.3

Antibody Count P value

2772.5 714.8 2039.3 1092.7 1996.9 1051.9 0.004

E. Nagele et al., 2012

Kits for the Detection and Diagnosis of Neurodegenerative Diseases Alzheimers Disease Parkinsons Disease Multiple Sclerosis
Technology based upon the identification of disease specific autoantibody profiles in blood samples employing proprietary patterns of protein targets.

Alzheimers disease
Slide with human protein antigen targets

Preparation and Processing

Slide with fluorescent targets

Autoantibody production
1. Neurons die which type of neurons depends on brain region affected 2. Dead neurons release their debris into the surrounding cerebrospinal fluid (CSF) 3. Debris enters bloodstream and activates the immune system 4. Immune system generates many autoantibodies to clear debris 5. Durin detects disease-specific autoantibodies as biomarkers
Incubation with human serum Incubation with fluorescentlabeled secondary antibodies Fluorescentlabeled secondary antibodies bind to autoantibodies

Autoantibody biomarkers bind to targets

Analysis of fluorescent signal patterns and detection of disease-specific autoantibody biomarkers

Alzheimers disease results

Sensitivity 96.0% Specificity 92.5%

Parkinsons disease results

Sensitivity 93.1% Specificity 100%

1 drop of blood

Durins Alzheimers Diagnostic Test Published August 3, 2011

A Multi-Disease Diagnostic Strategy

Applying the same strategy identifies autoantibodies as highly accurate diagnostic indicators for Parkinsons disease (PD)

Results: PD sera were differentiated from control sera with a 97.1% overall accuracy; sensitivity of 93.1% and specificity of 100%

MJFF is currently funding a validation study with two goals:

1. To confirm that our panel of 10 autoantibody biomarkers can accurately diagnose PD in a larger population. 2. To determine if our diagnostic strategy can be used for early detection of PD. We have thus far achieved early detection of PD with 94.5% accuracy!

Progress and Plans

Durin Technologies

*Alzheimers disease Completed Alzheimers early detection in progress Parkinsons disease Completed

*Parkinsons early detection in verification

Multiple Sclerosis (MS) in progress Traumatic brain injury - planned Autism in progress Early stage breast cancer in verification

Our Dream
To Construct a Multi-Disease Diagnostic Blood Test
Only 10-30 autoantibodies are needed for each diagnostic. Each array can currently hold 23,000 protein targets. There is plenty of room for many diagnostic tests on a single microarray. Neurodegenerative diseases Various cancers

Alz

Park

MS

Breast Lung Pancreatic

NJISAs Biomarker Discovery Center

NIH Biomarker Consortium NIH National Institute of Aging National Cancer Institute

Venture Funds

Private Foundations Alzheimers Association Michael J Fox Foundation Multiple Sclerosis Society Autism Speaks Breast Cancer Society American Cancer Society Osteopathic Heritage Foundation

Collaborations and consultations with individual investigators In house investigators Outside investigators Collaborations and consultations with Pharma and Biotechs Johnson & Johnson General Electric