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Anonymous, 2013b

Created By: Carolina Jaime

Caregivers for Alzheimer's and Dementia Face Special Challenges 
You are not alone. Whether you need information about early-stage caregiving, middle-stage caregiving, or late-stage caregiving, the Alzheimer's Association is here to help.

Early-stage caregiving
[1] In the early stages of Alzheimer's, a person may function independently. He or she may still drive, work and be part of social activities. Your role as care partner is an important one: to provide support and companionship, and help plan for the future.

What to expect 
"Early stage" refers to people, irrespective of age, who are diagnosed with Alzheimer's disease or related disorders and are in the beginning stages of the disease.[2] A person in the early stages may experience mild changes in the ability to think and learn, but he or she continues to participate in daily activities and give-and-take dialogue. To others, the person may not appear to have dementia. The early stages of Alzheimer's can last for years.

Your role as a care partner
In the early stages, you may act more like a care partner, than a caregiver. [3] Your role is one of support, love and companionship. You are there to help with daily life, as needed, and to help the person with Alzheimer's plan for the future. Since no two people experience Alzheimer's alike, the degree of assistance needed from a care partner in this stage varies.

A person with early-stage Alzheimer's may need cues and reminders to help with memory. For example, he or she may need help with:
Keeping appointments
Remembering words or names
Recalling familiar places or people
Managing money
Keeping track of medications
Doing familiar tasks
Planning or organizing

Tap into the person's strengths and encourage him or her to continue living as independently as possible. You can help the person stay organized with shared calendars, notes, medication schedules and other reminder systems. Establishing a daily routine and maintaining some regularity will be of benefit.

[4] The person also will need emotional support. He or she may feel frustrated, anxious, embarrassed or isolated. You can help by:
Encouraging the person to share his or her feelings, and asking how you can be supportive
Encouraging the person to stay involved in activities he or she enjoys
Helping the person locate a support group for people in the early stages and their care partners

Early-stage issues 
[5] A diagnosis of Alzheimer's is life changing for both the person with the disease and the care partner. Here are some of the issues you may both face

Telling others about the diagnosis.
Telling others about a diagnosis of Alzheimer's or dementia is one of the most difficult steps for people diagnosed in the early stages and their care partners. There may be anxiety surrounding who to tell and worry about social stigma. Be open with friends and family about the changes that are taking place. Educate them on the disease and tell them how they can be supportive. 
Life changes.
Even if changes are small at first, a person with early-stage Alzheimer's will have different needs than he or she did before the diagnosis. Support is critical. As a care partner, you'll need a support system in place, too. You may feel anxiety over how your relationship may change or feel distanced from friends and family. Know that you aren't alone, and that help is available. 

Planning for the future
A Plan in Place: Janet is in the early stages of younger-onset Alzheimer's. She's doing well now, but realizes that eventually she'll have to stop driving. Watch as she talks with her family about a future plan.
It's important to have discussions now about topics that will have to be addressed later. As a care partner, one of the most important things you can do is help the person with early-stage Alzheimer's get legal, financial and care plans in place. Doing so allows the person to share his or her wishes for future decisions, and also allows time to work through the complex issues that are involved in long-term care. This is also the time to talk about future safety topics, such as what to do when driving is no longer an option. 

Staying engaged.
People with early-stage Alzheimer's want to stay as engaged and active as possible for as long as possible. As a care partner, you can help foster this by encouraging involvement in daily life and a healthy lifestyle. Staying engaged and healthy is important for care partners as well. Continue being a part of support systems you have in place. Spend time with friends and family. Be a part of activities you love. And don't forget to eat well, exercise and see the doctor regularly. 

Living alone.
With support and resources, many people in the early stages of Alzheimer's live independently. If you are a family member or caregiver for someone who lives on his or her own, stay involved. Call or visit every day, and make sure the person gets the assistance needed, such as help with housekeeping, meals, transportation, bill paying and other daily chores. Put home safety measures in place, and be aware of any changes that would indicate the need for additional supervision or care.

Middle-Stage Caregiving 
[6] The middle stages of Alzheimer's are typically the longest and can last for many years. As dementia progresses, the person with Alzheimer's will require a greater level of care. During this time, it's important to get the support you need as a caregiver. 
During the middle stages of Alzheimer's, damage to the brain can make it difficult to express thoughts and perform routine tasks. You may notice the person with Alzheimer's jumbling words, having trouble dressing, getting frustrated or angry, or acting in unexpected ways, such as refusing to bathe.
While these changes are difficult for everyone involved, resources are available to help both you and the person with dementia as the disease progresses. There will be challenging days, but there also will be good days. As your relationship with the person with dementia changes, you will find new ways to connect and deepen your bond.

Your role as a caregiver
[7] Being a caregiver for someone in the middle stages of Alzheimer's requires flexibility and patience. As the abilities of the person with Alzheimer's change and functioning independently becomes more difficult, you will have to take on greater responsibility. Daily routines will need to be adapted, and structure will become more important. 
As you gain experience as a middle-stage caregiver, you will develop strategies and ways of coping that work for you and the person with dementia. When abilities diminish further, these will need to be modified. The Alzheimer's Association offers educational workshops and resources that can provide you with the caregiving skills needed to deal with changing needs of someone in this stage of the disease. Sharing information with other Alzheimer's caregivers also can be a great source of information and support. Other caregivers truly understand the complex feelings associated with caring for a person with dementia. 
As caregiving responsibilities become more demanding, it's important take care of yourself. Take breaks, even if it is only for a few moments. Make sure not to isolate yourself. Learn what respite services are available in your community, and take friends and family up on offers to help. Since paying for long-term care can be a big concern and source of stress, research all your options, if plans are not already in place.

Middle-stage concerns

Changes in behavior
Changes in behavior can be some of the most distressing for caregivers and family members. During the middle stages, people may experience depression, anxiety, irritability and repetitive behaviors. As the disease progresses, other changes may occur, including sleep changes, physical and verbal outbursts, and wandering. Understanding what behaviors are common during this stage and how to assist the person with dementia can help. 

As people with Alzheimer's gradually lose their ability to find words, express thoughts and follow conversations, they also have more difficulty understanding others. Communication changes during the middle stages include trouble finding the right word, repeating questions, losing the train of thought, reverting to a native language and relying on non-verbal communication. You can help improve communication by making simple changes, such as speaking slowly and distinctly in a gentle tone. If you notice sudden changes in communication, make sure to contact the doctor, since this could indicate other medical issues or side effects of medication. 

Daily care needs
Eating, dressing and grooming will become more challenging as dementia progresses. This loss of independence and privacy can be a very difficult transition for the person with dementia; your patience and sensitivity will go a long way in helping him or her through it. Once your assistance is needed to complete daily tasks, think about the person's abilities. Encourage the person to do as much as possible, but be ready to help when needed. For example, when dressing, you can give direction indirectly by laying out clothing in the order in which item is put on. 

Activities that provide meaning
In addition to enhancing quality of life, activities can reduce behaviors like wandering and agitation. You don't need to invent new things to do. Think of activities as things we do as part of our daily living. Activities can be making dinner together, gardening, listening to music or going for a walk. 

[8] During the middle stages of the disease, a person with Alzheimer's will need to stop driving. When it is clear that driving is no longer safe, try to involve the person with dementia in the decision to stop. Explain your concerns by giving specific examples. Assure the person you will do everything possible to make rides available. 

Other safety concerns
Early in the middle stages, it will become too difficult or dangerous for a person with Alzheimer's to be left alone. Preventing wandering becomes a crucial part of care, and safety precautions will need to be taken throughout the person's living environment. At this point, if the person is living alone, he or she may need to move in with relatives or to a residential care setting. Go to our free online tool, Alzheimer's Navigator, to receive step-by-step guidance on topics including home safety and driving.

Late-Stage Caregiving 
[9] The late stage of Alzheimer's disease may last from several weeks to several years. As the disease advances, intensive, around-the-clock care is usually required. 

What to expect 
As the disease advances, the needs of the person living with Alzheimer's will change and deepen. A person with late-stage Alzheimer's usually:
Has difficulty eating and swallowing
Needs assistance walking and eventually is unable to walk
Needs full-time help with personal care
Is vulnerable to infections, especially pneumonia
Loses the ability to communicate with words

Your role as a caregiver
[10] During the late stages, your role as a caregiver focuses on preserving quality of life and dignity. Although a person in the late stage of Alzheimer's typically loses the ability to talk and express needs, research tells us that some core of the person's self may remain. This means you may be able to continue to connect throughout the late stage of the disease.

At this point in the disease, the world is primarily experienced through the senses. You can express your caring through touch, sound, sight, taste and smell. For example, try:
Playing his or her favorite music
Reading portions of books that have meaning for the person
Looking at old photos together
Preparing a favorite food
Rubbing lotion with a favorite scent into the skin
Brushing the person's hair
Sitting outside together on a nice day

Late-stage care options 
[11] Since care needs are extensive during the late stage, they may exceed what you can provide at home, even with additional assistance. This may mean moving the person into a facility in order to get the care needed.

Deciding on late-stage care can be one of the most difficult decisions families face. Families that have been through the process tell us that it is best to gather information and move forward, rather than second guessing decisions after the fact. There are many good ways to provide quality care. Remember, regardless of where the care takes place, the decision is about making sure the person receives the care needed.

At the end of life, another option is hospice. The underlying philosophy of hospice focuses on quality and dignity by providing comfort, care and support services for people with terminal illnesses and their families. To qualify for hospice benefits under Medicare, a physician must diagnosis the person with Alzheimer's disease as having less than six months to live.

Ideally, discussions about end-of-life care wishes should take place while the person with the dementia still has the capacity to make decisions and share wishes about life-sustaining treatment. 

Food and fluids 
One of the most important daily caregiving tasks during late-stage Alzheimer's is monitoring eating. As a person becomes less active, he or she will require less food. But, a person in this stage of the disease also may forget to eat or lose his or her appetite. Adding sugar to food and serving favorite foods may encourage eating; the doctor may even suggest supplements between meals to add calories if weight loss is a problem.

To help the person in late-stage Alzheimer's stay nourished, allow plenty of time for eating and try these tips:
Make sure the person is in a comfortable, upright position.
To aid digestion, keep the person upright for 30 minutes after eating.
Adapt foods if swallowing is a problem.
Choose soft foods that can be chewed and swallowed easily. Make liquids thicker by adding cornstarch, unflavored gelatin or food thickeners (available at pharmacy and health care supply stores) to water, juice, milk, broth and soup. Learn the Heimlich in case of an emergency.
Encourage self-feeding.
Sometimes a person needs cues to get started. Begin by putting food on a spoon, gently putting his or her hand on the spoon, and guiding it to the person's mouth. Serve finger foods if the person has difficulty using utensils.
Assist the person with feeding, if needed.
Alternate small bites with fluids. You may need to remind the person to chew or swallow. Make sure all food and fluid is swallowed before continuing on with the next bite.
Encourage fluids.
Because the sense of thirst diminishes in the late stages of Alzheimer's, the person may not realize that he or she is thirsty. Encourage the person to drink liquids or to eat foods with high liquid content, such as watermelon, peaches, pears or sherbet.
Monitor weight.
While weight loss during the end of life is to be expected, it also may be a sign of inadequate nutrition, another illness or medication side effects. See the doctor to have weight loss evaluated.

Bowel and bladder function
[12] Difficulty with toileting is very common at this stage in the disease. The person may need to be walked to the restroom and guided through the process. Incontinence is also common during late-stage Alzheimer's.

To maintain bowel and bladder function:
Set a toileting schedule.
Keep a written record of when the person goes to the bathroom, and when and how much the person eats and drinks. This will help you track the person's natural routine, and then you can plan a schedule. If the person is not able to get to the toilet, use a bedside commode.

Limit liquids before bedtime.
Limit liquids at least two hours before bedtime, but be sure to provide adequate fluids throughout the day.

Use incontinence products.
Adult briefs and bed pads at night can serve as a backup to the daytime toileting schedule.

Monitor bowel movements.
It is not necessary for the person to have a bowel movement every day, but if there are three consecutive days without a bowel movement, he or she may be constipated. In such instances, it may help to add natural laxatives to the diet, such as prunes or fiber-rich foods (bran or whole-grain bread).

Skin and body health
[13] A person with late-stage Alzheimer’s disease can become bedridden or chair-bound. This inability to move around can cause skin breakdown, pressure sores and "freezing" of joints.

To keep skin and body healthy:

Relieve body pressure and improve circulation.
Change the person’s position at least every two hours to help keep him or her mobile.

Learn how to lift the person.
A care provider, such as a nurse or physical therapist, can provide instructions on how to properly lift the person without causing injury. Make sure not to ever lift by pulling on the person's arms or shoulders.
Keep skin clean and dry.
Since skin can tear or bruise easily, use gentle motions and avoid friction when cleaning. Wash with mild soap and blot dry. Check daily for rashes, sores or breakdowns.
Reduce the risk of bedsores.
Use pillows or pads to protect bony areas such as elbows, heels and hips.
Maintain range of motion in the joints.
"Freezing" of the joints (limb contractures) can occur when a person is confined to a chair or bed. Ask the doctor if range of motion exercises might be beneficial and, if so, how they should be performed.

[14] Infections and pneumonia
The inability to move around during late-stage Alzheimer's disease can make a person more vulnerable to infections.

To help prevent infections:
Keep the teeth and mouth clean.
Good oral hygiene reduces the risk of bacteria in the mouth that can lead to pneumonia. Brush the person's teeth after each meal. If the person wears dentures, remove them and clean them every night. Also, use a soft toothbrush or moistened gauze pad to clean the gums, tongue and other soft mouth tissues.
Treat cuts and scrapes immediately.
Clean cuts with warm soapy water and apply an antibiotic ointment. If the cut is deep, seek professional medical help.
Protect against flu and pneumonia.
The flu (influenza) can lead to pneumonia (infection in the lungs). It's vital for the person with Alzheimer's as well as his or her caregivers to get flu vaccines every year to help reduce the risk. A vaccine to guard against pneumococcal pneumonia is also available. (Usually only one dose is needed, but in certain circumstances, a second dose may be given five or more years after the first dose.)

Pain and illness
Communicating pain becomes difficult in the late stages. If you suspect pain or illness, see a doctor as soon as possible to find the cause. In some cases, pain medication may be prescribed.

To recognize pain and illness:
Look for physical signs .
Signs of pain and illness include pale skin tone; flushed skin tone; dry, pale gums; mouth sores; vomiting; feverish skin; or swelling of any part of the body.
Pay attention to nonverbal signs.
Gestures, spoken sounds and facial expressions (wincing, for example) may signal pain or discomfort.
Watch for changes in behavior.
Anxiety, agitation, shouting and sleeping problems can all be signs of pain.

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Anonymous, 2013

Created By: Carolina Jaime

what is happening to me?
[1] Alzheimer's disease causes gradual,
irreversible changes in the brain. These
changes usually cause problems with
memory, decision making and self care.
The disease also affects the ways we
communicate — both in expressing our
thoughts and in understanding what others
are saying. You may be worried or anxious
about the changes you've noticed so far.

While there is no cure for Alzheimer's,
treatments might help you with some of
your symptoms. And having information
about the disease can help you cope.

It's important to know that:
[2] The changes you are experiencing are
because of the disease. 
You will have good days and bad days.
The disease affects each person
differently, and symptoms will vary. 
Trying different ideas will help you
find comfortable ways to cope. 
Some suggestions may work for you,
and others may not. 
You are not alone
— more than 5
million Americans have Alzheimer's.
There are people who understand what
you're going through and can help you
and your family.

1. what can i do?
Coping with memory loss
While you may clearly remember things
that happened long ago, recent events can
be quickly forgotten. [3] You may have trouble
keeping track of time, people and places.
You may forget appointments or people's
names. It might be very frustrating trying
to remember where you put things.

Suggestions for coping with
memory loss: 
[4] Keep a book with you at all times
that has: 
Important telephone numbers and
addresses, including emergency
numbers and your own contact
People's names and their relationship
to you. 
A to-do list of appointments. 
A map showing where your home is. 
Thoughts or ideas you want to hold
on to. 
Label cupboards and drawers with words
or pictures that describe their contents. 
Get an easy-to-read, digital clock that
displays the time and date, and keep it
in a prominent place.
Use an answering machine or voicemail
to keep track of telephone messages. 
Post phone numbers in large print next
to the telephone; include emergency
numbers along with your address and a
description of where you live. 
Have a dependable friend call to
remind you about meal times,
appointments and medication. 
Keep a set of photos of people you see
regularly; label the photos with names
and who each person is in relation
to you. 
Keep track of the date by marking off
each day on a calendar. 
Use pillboxes to help you organize
your medication; pillboxes with sections
for times of day — like morning and
evening — can help remind you when
you should take your pills.

[5] Finding your way
Sometimes, things that were once familiar
may now seem unfamiliar. A favorite place
may not look the same. Or you might even
get lost.

Suggestions for finding your way: 
Take someone with you when
you go out. 
Don't be afraid to ask for help. 
Explain to others that you have a
memory problem and need assistance. 
Enroll in MedicAlert + Alzheimer's
Association Safe Return , a 24-hour
nationwide emergency response
identification and support program that
will reunite you with your family should
you ever wander. 
Sign up for Alzheimer's Association
Comfort Zone — a Web-based location
management service that ensures you and
your family are always connected.

[6] Doing daily tasks
You may find familiar activities more
For example, you may have
trouble balancing a checkbook, following a
recipe or doing simple household repairs.
Suggestions for doing daily tasks: 
Give yourself a lot of time, and don't
let others hurry you. 
Take a break if something is too difficult. 
Ask for help if you need it. 
Arrange for others to help you with
difficult tasks. 
Maintain a daily routine.
Over time, certain things may become too
difficult for you to do at all. This is because
of the disease. Do the best you can, and
accept help when it's available.

[7] Talking to others
You may have difficulty understanding
what others are saying. You may have
trouble finding the right words to express
your thoughts.

Suggestions for talking to others: 
Take your time. 
Tell people you have difficulty
with thinking, communicating and
Consider with whom you will share
your diagnosis — it's helpful for others
to understand your condition. 
Ask the person to repeat a statement if
you did not understand what was said.
Find a quiet place to converse if loud
noises or crowds are bothering you.

2. is what i'm feeling
[8] Living with the changes caused by
Alzheimer's disease can bring about many
unfamiliar emotions. These feelings are
a natural response to the disease. It is
important to share these reactions with
others. Tell someone with whom you are
comfortable how you feel.

The Alzheimer's Association can refer you
to a support group where you can meet
others who are living with Alzheimer's. You
can also connect with people who relate to
your experiences through ALZConnected
(alzconnected.org), an online social
networking community powered by the
Alzheimer's Association.
You may find yourself saying:
“I worry more than usual.”
It's important to talk to your family and
friends about your concerns. You may worry
about what's going to happen to you in the
future. Or you may wonder how quickly
the disease will progress.
While there are no definite answers to
these questions, most people find that doing
something they enjoy — like walking or
gardening — helps them take their mind
off their worries.
“I sometimes think I'm
going crazy.”
[9] The disease can make you feel as if you
are losing control. Telling those around
you how you feel may give you comfort.
Sharing your feelings with others who are
living with Alzheimer's may also help.

“I sometimes get into a
bad mood.”
It's normal to experience mood changes.
On these days, it is important to remember
that tomorrow could be a better day. Try to
do things that will lift your spirits.
“Sometimes I feel angry.”
Feeling angry is natural. Sometimes being
part of a support group or talking to a
counselor who knows about Alzheimer's
can help. Your doctor or the Alzheimer's
Association can refer you.
It’s normal to go through a range of
emotions. You’re facing many challenges
and adjustments. It's important to find
ways to cope with these feelings.

“I sometimes feel sad.”
You may feel sadness when faced with the
changes that the disease brings to your life.
It may help to spend time with friends or
family, or to do something you enjoy. You
might also consider consulting your doctor
about medications that may help ease
feelings of sadness.
“When things go wrong, I feel
really embarrassed.”
[10] Getting lost, forgetting a once-familiar face
or not being able to find the right word
can feel embarrassing. But this is a part
of the disease. Explain to people that you
have memory problems to help ease any
awkward feelings. Keeping
a sense of humor,
whenever possible, can also be very helpful.

why you are feeling this way. See if there
is anything that you, or those around you,
can do to make things easier.

“Sometimes I feel very lonely.”
You may think that the people around
you do not understand what you're going
through. It can be comforting to talk to
others who are living with Alzheimer's
disease. The Alzheimer's Association can
refer you to a support group. You can
also connect with others online through
ALZConnected (alzconnected.org).

“I feel guilty asking for help.”
Few of us like to ask for help. We often
resist relying on others. [11] Over time, you
will find it necessary to ask for help more
often. Try to accept the assistance you
need. Chances are that others will be
pleased to provide it. 

how else can i take
care of myself?
Two of the most important ways to maintain
your well-being are to stay healthy and safe.

Take good care of your body.
Suggestions for your health: 
Rest when you are tired. 
Exercise regularly, with your doctor's
Eat properly. 
Cut down on alcohol — it can make
your symptoms worse. 
Take your medications as prescribed, and
ask for help if it is difficult to remember
when they should be taken. 
Reduce stress in your daily life. 

Memory problems, difficulties with decision
making, and communication changes can all
create new safety concerns.
Suggestions for your safety: 
Consider a companion
The person you live with may worry
about leaving you alone for long
periods of time. While you may feel you
will be fine alone, having a companion
can help the time pass more pleasantly.
It can also lessen worry for those close
to you. 
Stop driving when it's no longer safe
Memory loss can hinder your ability
to drive safely. You may also become
less able to make decisions and react
quickly. While it is not easy to give
up your license, at some point it will
no longer be safe for you to drive.
Look into other ways to get around,
like friends, family, taxi cabs or public
Just as people can wander while
walking, they can also become lost
when driving or taking a bus, train or
airplane. Some wander hundreds of
miles away from home.
Visit alz.org/safety for information,
tips, and resources to assist you with
safety inside and outside of the home,
wandering and getting lost, and driving
and dementia. 
Be mindful of electrical appliances
Leave written reminders to yourself
like, “turn off the stove” or “unplug the
iron.” Be sure you have an automatic
shut-off feature on the appliances you
use most often — especially the ones
that can cause harm if left unattended. 
Use smoke detectors
Make sure your home has working
smoke detectors, which could save your
life in a fire. Put a reminder in your
calendar to change the batteries. 
Be careful of people you don't recognize
If someone you don't recognize comes
to your door, don't let them in. Instead,
write down the person's name and
telephone number. Later, you or a
family member can call the person.

what if i live on my own?
Many people with Alzheimer's continue to
live successfully on their own during the
early stages of the disease. Making simple
adjustments, taking safety precautions and
having the support of others can make
things easier.

Suggestions for living on your own: 
Get advice from the Alzheimer's
Association or your doctor about
where to get help for things like
housekeeping, meals and transportation. 
Inform your bank if you have difficulty
with record-keeping and keeping track of
your accounts; they may provide special
services for people with Alzheimer's. 
Arrange for direct deposit of checks,
such as your retirement pension or
Social Security benefits. 
Plan for home-delivered meals, if
available in your community. 
Have a family member regularly sort
your closet and dresser drawers to
make it easier for you to get dressed.
Leave a set of house keys with a
neighbor you trust. 
Schedule family, friends or a community
service to make a daily call or visit; keep
a list of things to discuss.
At some point, living alone will become
too difficult or dangerous. Make plans
now for where you will live as the disease
progresses. You may want to get a helpful
roommate, live with relatives or move to a
residential care setting. 

what about the future?
Alzheimer's disease is a progressive illness,
and the symptoms you're experiencing will
gradually worsen. You will need more help.
There is no way to predict how or when
this will happen. It's a good idea for you to
make decisions about your future as early in
the course of the disease as possible.
Suggestions for future plans:
Make arrangements at work 
Talk to your employer about
Alzheimer's. Take someone with you to
help explain and clarify your symptoms
and particular situation. 
Cut down on your hours or
responsibilities, if possible. 
If you own your own business, put
plans in place for its future operations.
Consider future living arrangements 
Talk to your family or friends about
where you want to live, and with
whom, to prepare for the time when
you will need more care. 
Consider all of the options available,
including adult day programs, in-home
care and hospice services. 

Settle your money and legal matters 
Consider naming a person to make
health care decisions for you when you
are unable to do so. This person should
know your wishes about your health care
and future living arrangements. 
Make sure your money matters are in
the hands of someone you trust, like
your spouse or domestic partner, your
child or a close friend. 
See a lawyer about naming a person to
legally take care of your money matters
when you can no longer do it. 
Take someone with you to the lawyer to
help explain your situation and to help
interpret what the lawyer says. 
Find out about any available options for
long-term care insurance.
Planning ahead ensures that your future will be
in good hands. It also helps those close to you
make the right decisions for you in the future. 

Map out your plan to approach Alzheimer’s
The new Alzheimer’s Association Alzheimer’s
Navigator™ (alzheimersnavigator.org) online
assessment program can help you create a
customized action plan to proactively face
this disease.
Implementing your action plan is easy
with help from local resources located
one click away via the Alzheimer’s
Association Community Resource Finder
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Anonymous, 2011

Created By: Carolina Jaime

A Nasal Spray Vaccine For Alzheimer's Disease

By News Staff | February 28th 2011 11:24 AM

Up to 12 percent of Americans may get Alzheimer's disease, current statistics say. [1] In the quest to prevent Alzheimer's, or at least make it manageable like diabetes, a group of researchers are working on a nasally-delivered vaccine that promises to protect against Alzheimer's. Bonus: It may help prevent strokes also. The new vaccine repairs vascular damage in the brain by using the body's own immune system and, in addition to its prophylactic effect, it can work even when Alzheimer's symptoms are already present, according to the paper in Neurobiology of Aging.

[2] "Using part of a drug that was previously tested as an influenza drug, we've managed to successfully induce an immune response against amyloid proteins in the blood vessels," says Dr. Dan Frenkel of Tel Aviv University's Department of Neurobiology, who collaborated on the project with Prof. Howard L. Weiner of Brigham and Women's Hospital, Harvard Medical School. "In early pre-clinical studies, we've found it can prevent both brain tissue damage and restore cognitive impairment," he adds.

Modifying a vaccine technology owned by Glaxo Smith Kline, their approach activates a natural mechanism in our bodies that fights against vascular damage in the brain. The vaccine activates macrophages, large proteins in the body that swallow foreign antigens, and they clear away the damaging build-up of waxy amyloid proteins in our brain's vascular system. Animal models showed that once these proteins are cleared from the brain, further damage can be prevented, and existing damage due to a previous stroke can be repaired.

An Alzheimer's cure?

[3] Ignoring the media hype that results around a lot of experiments done in animal models, could it be a vaccine and a cure for Alzheimer's disease? "It appears that this could be the case," says Frenkel. "We've found a way to use the immune response stimulated by this drug to prevent hemorrhagic strokes which lead to permanent brain damage."

[4] In the animal models in mice, they did MRIs and the used "object recognition" experiments, testing the cognitive functions both before and after administration of the vaccine. MRI screenings confirmed that, after the vaccine was administered, further vascular damage was prevented, and the object recognition experiments indicated that those animals treated with the new vaccine returned to normal behavior.

Frenkel believes that this approach, when applied to a human test population, will be able to prevent the downward health spiral of Alzheimer's and dementia. The vaccine could be given to people who are at risk, those who show very early symptoms of these diseases, and those who have already suffered strokes to repair any vascular damage.
So far the vaccine has shown no signs of toxicity in animal models and Frenkel is hopeful that this new approach could lead to a cure, or at least an effective treatment, for the vascular dementia found in 80% of all people with Alzheimer's.
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Burica, 2010

Created By: Carolina Jaime

Discovery may lead to Alzheimer’s cure
Written by Cassie Burica, Daily Vidette Reporter
Tuesday, 23 March 2010 20:44

[1] A new study suggests that a protein known for causing Alzheimer’s may actually be a natural defense against invading microbes and bacteria in the brain.

The protein, which is known as beta amyloid, or A-beta, forms into plaque that destroys signals between nerves. The buildup causes people to lose their memory and suffer from a personality change.

“It was thought that when these fragments accumulated and formed plaques, inflammation and damage killed off the neurons, resulting in the symptoms of Alzheimer’s disease,” Laura Vogel, professor of immunology, said.

In a recent study, researchers at Harvard have suggested that the protein may actually have a useful function in defending the brain.

Upon looking at a list of genes that were associated with Alzheimer’s disease, Rudolph E. Tanzi, the lead researcher of the study, discovered that many resembled those associated with the innate immune system.

“Many cells and molecules in your body are part of the innate immune system, including neutrophils and macrophages that engulf and digest germs. Another important part of the innate immune system are small protein fragments called anti-microbial peptides.

“These are small molecules that exist all the time in your body so they are ready to attack pathogens the minute you get infected,” Vogel said.

[2] Tanzi and a colleague also discovered that A-beta closely resembled the protein LL-37, which has been known to protect against infections in rodents.

A-beta killed eight out of 12 of the microbes in an experiment, which is just as efficient, and in some cases more efficient at killing the microbes than LL-37.

Now that scientists understand that A-beta may actually be a necessary protein, the treatments may take a different route.

[3] Instead of Alzheimer’s being caused by A-beta, some scientists believe the disease is only caused by an excess of it.

“There could be significant clinical applications since many of the approaches to treat Alzheimer’s are trying to eliminate A-beta; if it turns out that you need A-beta to protect yourself from infection, getting rid of the protein may be a bad move,” Vogel explained.

H. Tak Cheung, professor of biology, explained that if the trigger of A-beta is a microorganism- based infection, controlling it may result in a cure.

“The extensive production of A-beta perhaps is the brain’s attempt to destroy a microorganism. So far we have not been able to find microorganisms in the brain using Alzheimer’s disease,” he said.

[4] “Maybe remnants of microorganisms that enter into the brain trick the brain to think it is infected by microorganisms and it responds in the production of A–beta.”

Cheung said that the discovery was similar to other medical breakthroughs in the past, such as the treatment of ulcers that were once thought to be only caused by stress but can now be treated with antibiotics.

“If this turns out to be correct, it could provide major importance to the treatment and cure of Alzheimer’s,” he said. 

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Kim, 2009

Created By: Carolina Jaime


ADAM10, a member of a disintegrin and metalloprotease family, is an α-secretase capable of anti-amyloidogenic proteolysis of the amyloid precursor protein. [1] Here, we present evidence for genetic association of ADAM10 with Alzheimer's disease (AD) as well as two rare potentially disease-associated non-synonymous mutations, Q170H and R181G, in the ADAM10 prodomain. These mutations were found in 11 of 16 affected individuals (average onset age 69.5 years) from seven late-onset AD families. Each mutation was also found in one unaffected subject implying incomplete penetrance. Functionally, both mutations significantly attenuated α-secretase activity of ADAM10 (>70% decrease), and elevated Aβ levels (1.5–3.5-fold) in cell-based studies. In summary, we provide the first evidence of ADAM10 as a candidate AD susceptibility gene, and report two potentially pathogenic mutations with incomplete penetrance for late-onset familial AD.

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[2] Alzheimer's disease (AD) is a progressive neurodegenerative disorder and the leading cause of dementia in the elderly. AD is pathologically characterized by abundant amyloid plaques and neurofibrillary tangles. The amyloid β-protein (Aβ), the primary component of the amyloid plaques, is generated via serial proteolytic cleavage of the amyloid precursor protein (APP) by β-secretase followed by γ-secretase, which employs the presenilins as the catalytic subunits. Over 200 missense mutations in the APP and the presenilin 1 and 2 (PSEN1; PSEN2) genes cause early-onset, autosomal dominant, familial AD. APP is usually cleaved toward the middle of the Aβ sequence by α-secretase, which precludes Aβ production. This anti-amyloidogenic pathway also generates a soluble N-terminal fragment (sAPPα), for which neurotrophic and neuroprotective mechanisms have been proposed (1–4). α-Secretase activity can be regulated by several signaling pathways involving protein kinase C (PKC), tyrosine kinases, the mitogen-activated protein kinases and extracellular signal-regulated kinases (5–7). Activation of the PKC signaling cascade by phorbol esters has been shown to increase sAPPα secretion and to significantly alleviate Aβ formation (8–13). [3] Three members of the ADAM (a disintegrin and metalloprotease) family, ADAM9, ADAM10 and ADAM17, have been shown to possess α-secretase activity (14). Single knockout mutants of the ADAM proteases do not completely abolish α-secretase activity. For example, ADAM10-deficient mice are embryonic lethal due to defective Notch signaling; however, embryonic fibroblasts from these mice maintain α-secretase activity (15). In ADAM17 (tumor necrosis factor-α convertase, TACE)-knockout mice, phorbol ester-induced secretion of sAPPα was abolished while the constitutive release of sAPPα was preserved (16,17). Finally, in vivo deletion of ADAM9 did not lead to changes in α-secretase activity (18). Hence, these three ADAM proteases may exhibit considerable functional redundancy (14,15). [4] Each ADAM member has unique features, i.e. ADAM10 has been shown to be responsible for both constitutive and regulated α-secretase activities (19,20), while TACE appears to be mainly involved in regulated activity (16,17,21,22). Although all three proposed α-secretase candidates contribute to APP cleavage, ADAM10 is unique due to its combined constitutive and regulated activity, high enzymatic stability under conditions for α-secretase cleavage (23) and coordinated mRNA expression with APP expression in mouse and human cortical neurons (24). Reduced levels of ADAM10 have been reported in sporadic AD patients along with lower sAPPα levels (25). Additionally, moderate neuronal overexpression of ADAM10 in mice has been shown to lead to elevated sAPPα release, reduction in Aβ formation and plaque deposition and improved cognition (26).

The domain structure of ADAMs consists of a prodomain, a metalloprotease domain, a disintegrin domain, a cystein-rich domain, an EGF-like domain, a transmembrane domain and a cytoplasmic tail (27). The primary function of the prodomain is to maintain the metalloprotease site in an inactive state via a cysteine switch (28). A zinc atom in the catalytic site is coordinated by a conserved cysteine residue in the prodomain and the metalloprotease domain remains in a latent conformation. Inactivation prevents ADAMs from auto-catalysis during biosynthesis. To generate the active protease, the prodomain must be cleaved from the rest of the protein by proprotein convertases in trans-Golgi network (TGN) (20,29). In ADAM10, proprotein convertase recognition sequence (RKKR) is essential for activation of the zymogen, and both furin and PC7 can act as proprotein convertases (29). Following maturation, the majority of the mature ADAM10 is transported to the plasma membrane, where it functions as an ectodomain sheddase for several cell surface proteins. In addition, the prodomain is suggested to act as an intramolecular chaperone during biosynthesis and secretion of the catalytic domain, facilitating proper folding and structure of the catalytic active site, as well as assisting transit throughout the secretory pathway of the protease (27).

Late-onset AD is a genetically complex and heterogeneous disease. The ε-4 allele of apolipoprotein E (APOE) is the only established genetic risk factor for late-onset AD. However, it has been suggested that APOE and the three early-onset familial AD genes, APP, PSEN1 and PSEN2 account for less than half of the genetic variance of AD (30). [5] With the aim of assessing the potential role of ADAM10 as an AD susceptibility gene, we initially tested nine single nucleotide polymorphisms (SNPs) in this gene for genetic association with AD in over 400 AD families comprising the National Institute of Mental Health (NIMH) AD Genetics Initiative family sample. These association findings were later confirmed by data generated in a recently completed genome-wide association study (GWAS) in the same sample (31). Ultimately, these data led to the identification of two rare, non-synonymous mutations in the region of ADAM10 encoding the prodomain. We assessed the functional consequences of these two prodomain mutations by examining their effects on α-secretase activity, Aβ levels, as well as ADAM10 maturation and biogenesis in a series of Chinese hamster ovary (CHO) cell lines stably overexpressing APP and ADAM10 constructs.

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Family-based association of ADAM10 SNPs in the NIMH AD family sample

To test the potential role of ADAM10 as an AD susceptibility gene, we initially tested a total of nine SNPs in the ADAM10 gene for genetic association with AD in the NIMH AD Genetics Initiative family sample (1439 DNAs from 436 multiplex AD families (32)). The nine SNPs were chosen from publicly available databases as proxies to tag the most common variants in the ADAM10 gene. One of these SNPs (rs2305421) showed evidence of genetic association in the NIMH families (P-value = 0.003; Table 1). Upon stratification of families by APOE-ε4, the association with rs2305421 became more pronounced (P-value = 0.0005 in the APOE4+ families), and two other SNPs of the nine tested (rs605928 and rs4775083) now showed marginal association (P-values = 0.02 and 0.06, respectively; Table 1). These associations were later confirmed on a larger number of SNPs genotyped as part of a GWAS on the same families from the NIMH collection (31). On the GWAS array, there were a total of 53 SNPs ± 100 kb from the ADAM10, and 11 of these showed nominal association (P-value ≤ 0.05) with AD risk, in good agreement with the SNPs genotyped in the initial phase of this study (Supplementary Material, Table S1). Despite the consistent evidence for association between AD risk and genetic variants in ADAM10 in the NIMH sample, none of the initially genotyped SNPs showed evidence of association in the independent Consortium on Alzheimer's Genetics (CAG) sample consisting of primarily of discordant sibpairs (489 DNAs, 217 families (33)), most likely due to lower power of this sample (see Supplementary Material, Table S2). Nonetheless, the combined NIMH and CAG samples still yielded overall significant evidence for association of AD with ADAM10 SNP rs2305421 (P-value = 0.008 in the unstratified NIMH and CAG samples and P-value = 0.0007 in the APOE ε4-pos families only).

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Table 1.
Association results for nine SNPs in ADAM10 gene region and AD

We next sequenced 32 families of the NIMH sample that showed particularly strong evidence of association, i.e. those in which affected individuals were homozygous for the risk allele of the best-associated SNP (rs2305421). This led to the identification of two rare, non-synonymous changes in exon 5 of ADAM10, Q170H and R181G, located near the consensus sequence for the cysteine switch in the prodomain of ADAM10 in two families out of 32. Screening of the remaining NIMH samples for the Q170H and R181G mutations revealed further three families, resulting in a total of five families (three for Q170H and two for R181G) carrying these potential ADAM10 mutations (Table 2). Combining both mutations to one aggregate genotype revealed significant evidence for association with AD risk across the NIMH sample [P-value = 0.0043 (including all individuals), and a P-value = 0.06 (after exclusion of probands initially selected for sequencing)]. While none of the unaffected individuals of these families were found to be mutation carriers, for each mutation there was one family in which one of the affected siblings was not a carrier. The affected R181G non-carrier also carried an APOE-ε4 allele, while the affected Q170H non-carrier did not. Neither mutation was found in the total set of 678 unaffected subjects in the combined NIMH and CAG samples.

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Table 2.
Seven AD families harboring either Q170H or R181G mutation in ADAM10 gene

To search for additional AD families carrying these mutations, we further screened 1111 individuals from 351 AD pedigrees from the National Institute on Aging (NIA) Study Sample (Table 2). We found one additional AD family for each of the mutations. In both of these families, the mutations did not perfectly segregate with disease status (Table 2). For the Q170H mutation, one AD patient, with onset age of 63 years, carried the mutation while the other two (onset age of 65 and 71 years) did not. In addition, a 63-year-old unaffected family member carried the mutation. These results suggest that other genetic factors contribute to AD risk in this family and, potentially, incomplete penetrance of the Q170H mutation. It is worth noting that the patient with the earliest onset carries the Q170H mutation as well as an APOE-ε4 allele. For the R181G mutation, one of two affected individuals carried the mutant allele (onset 80 years) while the other (onset 74 years) did not. In addition, an unaffected family member (age 83 years) carried the mutant allele. All three family members were heterozygous for APOE-ε4. As was observed with Q170H, these findings suggest incomplete penetrance for the R181G mutation, and the probable involvement of additional genetic factors. Combining both mutations to one aggregate genotype revealed significant evidence for association with AD risk across the NIMH and NIA samples [P-value = 0.0186 (including all individuals), and a P-value = 0.212 (after exclusion of probands initially selected for sequencing)].

Novel prodomain mutations in ADAM10 attenuate constitutive α-secretase activity

To investigate the potential functional effects of Q170H and R181G mutations in the ADAM10 prodomain, we generated CHO cell lines stably expressing wild-type or mutant ADAM10 together with APP. For this purpose, CHO-APP751 cells were transfected with a HA-tagged ADAM10 cDNA, with or without the prodomain mutations. CHO-APP751 cells transfected with empty vector or a dominant negative (E384A) mutant form of ADAM10 were also generated to serve as negative and positive controls, respectively. Among several ADAM10-expressing, single cell-originated clones, we selected multiple clones for the mutant constructs, two for Q170H, three for R181G and two for E384A, and then measured sAPPα levels. As shown in Figure 1A and B, relative levels of sAPPα normalized to full-length APP (FL-APP) levels were >4-fold higher in wild-type ADAM10 cells (W) when compared with empty vector-transfected cells (EV), indicating increased α-secretase activity resulting from overexpression of ADAM10. However, overexpression of ADAM10-Q170H and ADAM10-R181G mutant forms did not elevate sAPPα levels to the same extent as wild-type ADAM10. sAPPα levels, normalized to either FL-APP (sAPPα/FL-APP) or FL-APP and ADAM10 (sAPPα/FL-APP/ADAM10), were significantly decreased (by >72%) in cells expressing ADAM10 harboring the Q170H (M11, M12) and R181G mutations (M21, M22, M23) versus those in wild-type cells. sAPPα levels in the Q170H and R181G cells were comparable to those in cells expressing a known dominant negative (E384A) ADAM10 mutant (DN1 and DN2). The inhibitory effects of the dominant negative (E384A) ADAM10 mutation on the constitutive and regulated α-secretase activities of ADAM10 have been previously defined (20). Decreased sAPPα levels were observed in the multiple independent mutant clones and could be restored by transient overexpression of the wild-type ADAM10 (Fig. 1C), suggesting that the observed results were not a clone-specific effect of stable cells but due to genuine changes in ADAM10 catalytic activity.

Figure 1.
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Figure 1.
Decrease in constitutive α-secretase activity in CHO cells stably expressing APP and wild-type or prodomain mutant forms of ADAM10. CHO-APP751 cells were used to generate cell lines stably overexpressing different ADAM10 constructs, wild-type (W), Q170H mutant (M11, M12), R181G mutant (M21–M23), E384A dominant negative mutant (DN1, DN2) and empty vector (EV). Each stable cell line was grown to exponential phase and conditioned media and cell lysates were collected to measure levels of sAPPα, ADAM10 and APP. (A) Representative western blots. FL-APP, full length APP; imm-ADAM10, immature ADAM10; mat-ADAM10, mature ADAM10. (B) Graphs for relative sAPPα levels normalized with full-length APP levels (sAPPα/FL-APP) and further with ADAM10 levels (sAPPα/FL-APP/ADAM10). n = 4–8, mean ± SEM. (C) Restoration of the reduced sAPPα levels in ADAM10 mutant cells by overexpression of wild-type ADAM10. Each ADAM10 stable cell line was transiently transfected with wild-type ADAM10 construct and levels of sAPPα and ADAM10 were measured. (D) Ratios of mature and immature ADAM10. Mature and immature ADAM10 bands in the western blot in (A) were quantified and their ratio in each cell was calculated. n = 3–6, mean ± SEM. Significance of changes in the mutant cells was calculated when compared with wild-type. *P < 0.05, **P < 0.005 (two-tailed Student's t-test).

To confirm these results, levels of APP C-terminal fragment (CTF) of α-secretase cleavage were examined in the ADAM10 stable cells (Fig. 2). In order to accurately measure the production of CTFs, the γ-secretase inhibitor (DAPT) was used to inhibit cleavage of APP-CTFs. Consistent with the observed changes in sAPPα levels, the relative CTFα (C83) levels were significantly lower in the Q170H and R181G mutants when compared with cells expressing wild-type ADAM10. These data further support defective α-secretase activity in the cells expressing ADAM10 harboring the Q170H and R181G mutations. In contrast, we observed no detectable increase in levels of APP-CTFβs [C89 and C99; confirmed by treatment with a BACE1 inhibitor and by western blotting with Aβ1–16 specific antibody (data not shown)] in the mutant versus wild-type ADAM10 cells.

Figure 2.
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Figure 2.
Decrease in APP-CTFα levels in CHO cell lines stably expressing prodomain mutant forms of ADAM10. CHO-APP-ADAM10 stable cell lines were treated with 250 nM DAPT for 24 h to inhibit γ-secretase and APP-CTFs (C83, C89, C99) levels were examined in total cell lysates. (A) Representative western blots. (B) Graphs for relative CTFα levels normalized with full-length APP levels (C83/FL-APP) and further with ADAM10 levels (C83/FL-APP/ADAM10). n = 7, mean ± SEM. Significance of changes in the mutant cells was calculated when compared with wild-type. *P < 0.05, **P < 0.005 (two-tailed Student's t-test). EV, empty vector-transfected cells; W, wild-type ADAM10 cell; M11, M12, Q170H mutant cells; M21–M23, R181G mutant cells; DN1, DN2, E384A dominant negative mutant cells.

Since the Q170H and R181G mutations are located close to the potential consensus sequence for the cysteine switch in the ADAM10 prodomain and proprotein convertase recognition sequence (RKKR) in the metalloprotease domain, we next assessed the synthesis and maturation of ADAM10 in the mutant versus wild-type ADAM10 cells. Protein levels of total ADAM10, mature ADAM10 (64 kDa) and immature ADAM10 (90 kDa), were detectable but variable across the mutant clone cell lines (Fig. 1A). Variable levels were not likely to result from an enhanced protein degradation or auto-catalysis during ADAM10 biosynthesis because the ADAM10 levels were not consistent among cells expressing the same mutations nor did they correlate with α-secretase activity. The ratio of steady-state levels of mature to immature ADAM10 did not exhibit any significant or consistent changes in the mutant versus wild-type ADAM10 cell lines (Fig. 1D). Collectively, these data suggest that these two mutations do not alter ADAM10 maturation.

Elevated Aβ levels in the presence of the novel prodomain mutations in ADAM10

Next, we investigated potential effects of the ADAM10 prodomain mutations on Aβ levels in the CHO stable cell lines as measured by ELISA using Aβx-40-specific antibodies. The Aβ40 levels were significantly (P < 0.0005) decreased upon overexpression of wild-type ADAM10 (Fig. 3). In contrast, overexpression of the Q170H and R181G mutants did not lead to reduced Aβ40 levels, consistently showing Aβ levels equivalent to or higher than those of EV control. Aβ40 levels were significantly increased about 1.5-fold and 2∼3.5-fold in the Q170H mutants (P < 0.0005) and the R181G mutants (P < 0.05), respectively, when compared with those in wild-type cells. Collectively, these results suggest that the ADAM10 Q170H and R181G mutations attenuate constitutive α-secretase activity of ADAM10, concurrently increasing Aβ levels.

Figure 3.
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Figure 3.
Increase of Aβ40 levels in the prodomain mutant ADAM10 stable cell lines. CHO-APP-ADAM10 stable cell lines were grown to exponential phase. Twenty four hour after changing media, the conditioned media were collected and levels of Aβx-40 were measured by ELISA. Aβ40 levels were normalized with amount of total protein in each corresponding cell lysate. n = 6–12, mean ± SEM. Significance of differences of the mutant and empty-vector transfected cells versus wild-type was calculated. *P < 0.05, ***P < 0.0005 (two-tailed Student's t-test). EV, empty vector-transfected cells; W, wild-type ADAM10 cell; M11, M12, Q170H mutant cells; M21–M23, R181G mutant cells.

Attenuation of PKC-inducible α-secretase activity of ADAM10 by two novel prodomain mutations

We next investigated whether phorbol ester-inducible α-secretase activity of ADAM10 is affected by the Q170H and R181G mutations. For this purpose, CHO-APP-ADAM10 stable cell lines were treated with PMA (Fig. 4). In the wild-type ADAM10 cells, sAPPα levels (normalized to FL-APP and ADAM10) were significantly increased (>2.5-fold) following PMA-treatment when compared with DMSO-treated controls. However, none of the mutant ADAM10 cells (Q170H, R181G and E384A) showed significant increases in sAPPα levels upon PMA treatment, leading to further decrease in sAPPα levels (>85%) in the mutant versus the wild-type ADAM10 cells under PKC-activation. These data indicate that the two novel prodomain mutations not only reduce constitutive activity of ADAM10, but also impair PKC-inducible α-secretase activity of ADAM10.

Figure 4.
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Figure 4.
Attenuated PMA-inducible α-secretase activity in CHO cells stably expressing the prodomain mutant forms of ADAM10. Each CHO-APP-ADAM10 stable cell line was treated with 1 µM PMA or equivalent amount of DMSO for 6 h and harvested to monitor sAPPα levels. (A) Representative western blots. (B) Graphs for relative sAPPα levels. n = 4–6, mean ± SEM. **P < 0.005 (two-tailed Student's t-test).

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We have observed genetic association of ADAM10 with AD risk and identified two novel non-synonymous mutations in the ADAM10 prodomain in seven AD families; we also assessed their effects on ADAM10 function and APP processing. Using CHO-stable cell lines expressing ADAM10 and APP, we demonstrated that Q170H and R181G mutations significantly attenuate α-secretase activity of ADAM10, leading to decreased levels of sAPPα and C83, and increased levels of Aβ. Although the two novel mutations are located in the proximity of the conserved sequences for cysteine switch and proprotein convertase recognition, they did not affect either the maturation or biosynthesis of ADAM10.

The two novel ADAM10 prodomain mutations are rare, segregating in only seven AD families (three for one mutation and four for the other) out of 1004 AD families screened. For each mutation, there were two families in which one (or two) affected individuals were found to be non-carriers, suggesting that in these individuals disease onset was influenced by other genetic factors. Because multiple genetic and environmental factors likely contribute to risk for late-onset of the disorder, onset in one (or two) affected individuals in the absence of each ADAM10 mutation is not entirely unexpected. A similar situation has been observed for AD mutations observed in presenilin 2 (N141I (34)) and presenilin 1 (A79V (35)), most likely owing to high prevalence of AD in the elderly population. In addition, for each mutation, there was one family in which one unaffected subject carried each mutation, suggesting incomplete penetrance for late-onset AD.

Support for the potential pathogenicity of the ADAM10 Q170H and R181G mutations was derived from experiments assessing the functional impact of these missense mutations on ADAM10 activity. For this purpose, we tried to generate cell lines stably overexpressing both APP and ADAM10 using H4 neuroglioma cells, HEK293 cells and CHO cells. We were only able to select wild-type and mutant ADAM10-expressing clones in CHO cells, most likely due to cytotoxicity caused by overexpression of both ADAM10 (36,37) and APP in the two human cell lines. In the CHO-APP-ADAM10 stable cells, the Q170H and R181G mutants caused a dramatic reduction in the generation of sAPPα and APP-C83 when compared with wild-type ADAM10 (Figs 1 and 2). However, western blot analysis did not reveal increases in sAPPβ and APP-CTFβ (C99 and C89) levels in the mutant cell lines (data not shown and Fig. 2). This may be due to the relatively low sensitivity of our western blot analyses for sAPPβ and APP-CTFβ. Moreover, regulated α-secretase activity by TACE has previously been shown to directly compete with β-secretase activity for APP cleavage in TGN (38). However, catalytically active ADAM10 is mainly localized in plasma membrane (20); thus, concurrent changes in sAPPβ and APP-CTFβ levels may be difficult to detect upon ADAM10 overexpression using western blot analysis. Aβ levels, determined by a sensitive ELISA, were consistently increased in the presence of the two ADAM10 prodomain mutations. Collectively, these data indicate that both ADAM10 mutations lead to defective α-secretase activities and subsequently elevation in Aβ levels in vitro, suggesting potentially pathogenic roles for these mutations, pending confirmation in vivo.

The molecular mechanism underlying the effects of the two novel ADAM10 mutations likely involves the prodomain function. Both mutations are located near the proprotein convertase recognition sequence (RKKR), which is required for proteolytic activation of the zymogen (29). However, since steady-state levels of mature and immature ADAM10s were not altered significantly (Fig. 1D), the mutations most likely do not affect ADAM10 maturation. The mutations may instead affect the intramolecular chaperone function of the ADAM10 prodomain. The role of prodomain in proper folding of ADAM proteins, particularly the metalloprotease domain, is also supported by studies of other members of ADAM protein family. For example, the secreted soluble form of ADAM 12 (ADAM12-S) lacking the prodomain remains in the early endomembrane system and is not secreted from cells, suggesting that the prodomain might be required in folding of the metalloprotease domain to a secretion-competent conformation (39). Truncated forms of ADAM10 and TACE lacking the prodomain have been shown to be catalytically inactive (29,40). Intracellular degradation of TACE lacking the prodomain and its rescue by prodomain expression in trans strongly suggests a chaperone role for the prodomain. Moreover, the ability of the prodomain to hold the catalytic domain of TACE in a relatively open conformation that is inactive has also been shown (40,41). In the case of ADAM10, the absence of the prodomain does not lead to defective biogenesis and secretion/trafficking of the protease. ADAM10 lacking the prodomain and expressed at high levels has been detected on the plasma membrane; however, it was proteolytically inactive (29). Interestingly, as with TACE, the ADAM10 prodomain expressed in trans was able to restore the catalytic activity of ADAM10 lacking the prodomain. Prodomain expression has also been shown to inhibit proteolytic activity of endogenous and overexpressed wild-type ADAM10, implying its direct interaction with mature ADAM10 (29). Future studies will be necessary to test whether the Q170H and R181G mutations can affect the chaperone function of the ADAM 10 prodomain.

[6] In summary, we have discovered two rare, partially penetrant, familial late-onset AD mutations in the ADAM10 gene that lead to defective α-secretase activity. The fact that these two mutations were both found in late- versus early-onset familial AD may reflect the relatively modest effects on Aβ accumulation relative to those of the early-onset familial mutations in APP, PSEN1 and PSEN2. Furthermore, since α-secretase activity is also exerted by TACE and ADAM9, one might expect a defect in ADAM10 activity to be compensated by molecular redundancy, possibly explaining the relatively late onset of AD in carriers of these two mutations and incomplete penetrance. The novel findings presented here provide the first genetic evidence in support of a possible role for the ADAM10 gene in the etiology and pathogenesis of late-onset AD. Moreover, given the location of these two mutations in ADAM 10, these data suggest that modulation of ADAM10 activity via the prodomain could represent a novel therapeutic target for the treatment and prevention of AD.

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AD family samples

The National Institute of Mental Health (NIMH) AD Genetics Initiative Study Sample. Subjects were collected from January 1991 until September 1997 following a standardized protocol applying NINCDS/ADRDA criteria for the diagnosis of AD (42,43). Over the first 17 years that the participating families had been followed, a clinical diagnosis of AD was confirmed at autopsy in 94% of the cases (44). The full NIMH sample includes 1439 individuals (68.9% female) from 436 families with at least two affecteds, including 995 affected individuals [n = 995 affecteds (mean age of onset 72.4 ± 7.7 years, range 50–97 years), n = 411 unaffecteds, n = 34 with phenotype unknown; see Schjeide et al. for details (45)].

The Consortium on Alzheimer's Genetics (CAG) Study Sample. Subjects in this second, independently ascertained AD family sample were collected under the auspices of the ‘Consortium on Alzheimer's Genetics.’ NINCDS/ADRDA criteria were used for a clinical diagnosis of AD (42), and probands were included only if they had at least one unaffected living sibling willing to participate in this study. Unlike the NIMH sample, no affected individual beyond the proband was required, thus the majority of families include only one sample affected. Most sibships were consisted of just one discordant sibpair, while in 41 families, there were more than two siblings available. Data and specimen collection began in October 1999 (still ongoing), and is currently completed for 489 individuals (62.6% female) from 217 sibships in which all affected individuals displayed an onset age ≥50 years [n = 222 affecteds (mean age of onset 69.2 ± 9.0 years, range 50–89 years), n = 267 unaffecteds; see Schjeide et al. for details (45)].

The National Institute on Aging (NIA) Study Sample. Subjects of this AD family sample were obtained from the National Repository of Research on Alzheimer's Disease (NCRAD), and ascertainment and collection details can be found at the NCRAD website (http://www.ncrad.org). For this study, we used families of self-reported European ancestry with DNA available from at least two first-degree relatives (concordant or discordant) and in which all individuals affected with AD showed onset ages ≥50 years. It comprises 1111 subjects (62.1% female) from 351 pedigrees, including 803 affected individuals [n = 803 affecteds (mean age of onset 74.1 ± 7.1 years, range 52–98 years), n = 290 unaffecteds; see Schjeide et al. for details (45)].

Genotyping and association analyses

A total of nine SNPs (rs605928, rs593742, rs514049, rs442495, rs7161889, rs714696, rs2305421, rs4775083 and rs1869135) located in ADAM10 were initially identified from publicly available databases. SNP genotypes were generated by fluorescent polarization detected single-base extension (FP-SBE, on a ‘Criterion Analyst AD’, Molecular Devices, Inc.), using individually optimized PCR and single-base extension protocols as described previously (33). Approximately 10% of samples were run in duplicate to assess the genotyping efficiency (average across all nine SNPs >96.5%) and error rate (average across all nine SNPs <0.5%). Primer sequencing and genotyping conditions are available from the authors upon request. All nine SNPs were tested in the NIMH sample, and four of these (rs605928, rs714696, rs2305421 and rs4775083) were tested in the full CAG sample. Subsequently, NIMH families in which two risk alleles of the best-associated SNP (rs2305421) were transmitted to affected individuals were chosen for re-sequencing of the ADAM10 coding regions from genomic DNA, overall a total of 32 families.

GWAS SNPs were generated in a separate project on the Affymetrix' GeneChip Human Mapping 500K Array Set, using individually optimized genotyping and allele-calling procedures (31). SNPs selected for this project needed to be located within 100 kb of the start-/stop-codon of ADAM10, and show no deviation from Hardy–Weinberg equilibrium (P-value ≥ 0.01), and have an allele frequency of ≥0.001. Overall, this yielded 53 SNPs spanning a chromosomal interval of ∼340 kb that could be used in the statistical analyses (Supplementary Material, Table S1).

Statistical analyses

To test for association between SNPs in ADAM10 and AD risk, we used PBAT (v3.6) with an additive model and the same parameters as used in our GWAS (31). All analyses were first restricted to families of self-reported ‘Caucasian’ ancestry, and then repeated using families of all ancestries (with no change in results; data not shown). Hardy–Weinberg equilibrium was determined using ‘Haploview’ (v4.1; (46)). Association results were combined across the NIMH and CAG samples for overlapping SNPs P-values using the method described by Fisher (47) taking into account the direction of the transmissions in each individual sample.

DNA sequencing

We re-sequenced all exons and adjacent introns (∼200 bp) of ADAM10 in 96 individuals from 32 ADAM10-associated AD families (66 affected with AD and 30 unaffected) using standard capillary electrophoresis. In addition, we also re-sequenced ∼4 kb of the adjacent 5′ and ∼3 kb of the 3′ regions of ADAM10. The DNA sequence of exons 5 and 6 of ADAM10, encoding the cysteine switch and proprotein cleavage site, were further determined in all of the individuals making up the NIMH AD sample. Exons 9 and 10, encoding the active site of ADAM10, were sequenced in all 994 affected individuals from the NIMH AD sample. We used standard protocols for DNA sequencing, with minor modifications. PCR primers were designed to yield amplified DNA fragments between 500 and 900 bp in length. PCR products were amplified from ∼30 ng genomic DNA using HotStarTaq® (Invitrogen, Carlsbad, CA, USA) and individually optimized PCR conditions. The amplified DNA fragments were purified on QIAquick 96 PCR purification plates (QIAGEN, Valencia, CA, USA), and the yields were determined using PicoGreen (Molecular Probes, Eugene, OR, USA) according to the manufacturer's protocol. Sequencing reactions were performed using the BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA), according to the manufacturer's instructions, except that the volume of BigDye mix was reduced to 1 µl and supplemented with 2 µl 5x BigDye v3.1 sequencing buffer in each 20 µl reaction. The reactions were subjected to analysis on an ABI 3700 DNA sequencer (Applied Biosystems), and the data were evaluated using SequencherTM (Gene Codes Corporation, Ann Arbor, MI, USA) analysis software.

Plasmid construction

Mammalian expression peak12 plasmid containing human ADAM10 wild-type cDNA sequence with a C-terminal HA-tag and the empty vector were kindly provided by Dr Stefan Lichtenthaler at Ludwig-Maximilians-University, Germany (48). Q170H, R181G and E384A mutations were introduced to the wild-type ADAM10 sequence by site-directed mutagenesis using PCR with oligomeric primers containing the mutations and insert swapping with restriction enzyme digestion. The entire cDNA sequences of all ADAM10 constructs were verified.

Cell culture, transfection and generation of stable cells

The CHO cell line was grown in Dulbecco's modified Eagle's medium (BioWhittaker) supplemented with 10% fetal bovine serum (Sigma), 2 mm l-glutamine (Sigma), 100 units/ml penicillin (Sigma) and 100 µg/ml streptomycin (Sigma). CHO cells overexpressing APP751 were grown in the same medium with 200 µg/ml G418 (Sigma). To generate stable CHO cell lines expressing ADAM10, each cDNA construct harboring either wild-type, Q170H, R181G or E384A ADAM10 was transfected into the CHO-APP751 cells using the Amaxa nucleofector system (Lonza) as described in the manufacturer's instructions. Clonal ADAM10-expressing CHO-APP751 cells were selected in 250 µg/ml puromycin and 200 µg/ml G418. ADAM10 expression was confirmed by western blot analysis. Among several single cell-originated ADAM10-expressing clones, cell lines with similar levels of APP were identified by western blot analysis. The selected CHO-APP-ADAM10 stable cells were plated at the constant number and grown to exponential phase, followed by replacing the medium with the constant volume of fresh medium. After 24 h of incubation, the conditioned medium was collected and the cell lysate was prepared in M-PER mammalian protein extraction reagent (Pierce) for measuring sAPPα and ADAM10 levels, respectively. To monitor levels of APP-CTFs, the γ-secretase specific inhibitor DAPT (N-[N-(3,5-Difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester, Sigma) was added to the cells (250 nM) and 24 h later cell lysate was collected and analyzed by western blot analysis. The phorbol ester effect was characterized 6 h after 1 µM of PMA (phorbol 12-myristate 13-acetate, Sigma), which was added to the cells.

For rescue of decreased sAPPα levels in ADAM10 mutant cells by wild-type ADAM10, the wild-type ADAM10 cDNA construct was transiently transfected to the CHO-APP-ADAM10 stable cells containing the mutants. Transfection efficiency was measured to be ∼90% based on counting of enhanced green fluorescent protein-positive cells. The medium was replaced with a constant volume of fresh medium 24 h after transfection. After 24 h of incubation, the conditioned medium and the cell lysate were collected.

Western blot analysis

Total protein lysates or conditioned media (the equal volume) were separated on 4–12% or 12% Bis-Tris polyacrylamide gel (Invitrogen), and transferred to immunoblot polyvinylidene difluoride membrane (Bio-Rad). For primary antibodies, anti-Aβ monoclonal antibody (6E10, Covance) was used to detect sAPPα levels in conditioned medium. Anti-APP C-terminal antibody (A8717, Sigma) was utilized for full-length APP and CTFs in cell lysates and anti-HA antibody (6E2, Cell Signaling) for HA-tagged ADAM10. Anti-actin monoclonal antibody (pan Ab-5, Neo-Markers) was used as control signal for equal loading. Protein signal was visualized using SuperSignal West Femto Maximum Sensitivity Substrate (Pierce) and the chemiluminescence signal was quantified by VersaDoc imaging system and Quantity One quantification software (Bio-Rad).


Levels of Aβx-40 in cell culture media were determined using a human/rat Aβ-specific chemiluminescence ELISA kit (Wako Chemicals USA, Inc., VA, USA). Briefly, samples and Aβ standards were aliquoted to 96 well plates coated with monoclonal antibody against Aβ11–28 region. After overnight incubation, anti-Aβ40 specific antibody conjugated with horseradish peroxidase (HRP) was added to the captured Aβ in each well. Plates were then developed with a chemluminescent HRP substrate and the absorbance at 450 nm was read with microplate reader.
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Murray, et. al 2013

Created By: Carolina Jaime

Chapter 57. Biochemical Case Histories

Robert K. Murray, MD, PhD & Peter L. Gross, MD, MSc, FRCP(C)

Case 2: Alzheimer Disease (AD)

Before studying this case, the reader is advised to consult the material in Chapter 5 on AD.


[1] Deposition of amyloid peptide (A42) in certain parts of the brain is believed by many neuroscientists to be one major cause of AD. It is thought that this 42-amino-acid peptide, occurring as beta sheets, oligomerizes and is deposited around neurons; the oligomers may be toxic to neurones. Deposition of A42 may be due to excessive formation or decreased removal of the peptide. In certain cases of familial AD, specific genes have been identified (eg, these encoding amyloid precursor protein [APP], presenilins 1 and 2, and apolipoprotein E4), which affect the production or removal of A42.

History and Physical Examination

A 72-year-old woman who lived on her own was found wandering around her neighborhood at 2 A.M. Her husband had died 3 years previously and her one son lived some distance away. The lady was confused and taken to the hospital. The son was notified and came immediately to see his mother. She was not able at the time of admission to give a clear history. The son volunteered that she had been diagnosed by a neurologist as having early AD, but had refused to go into a nursing home. She had home help during the day, and had not previously wandered out of her home. Sometimes a lady friend visited and spent the night at her house. In fact, she had appeared relatively normal prior to the present situation, as her son spoke to her on the phone every day of the week. However, her short-term memory had become worse in recent months, and he had become concerned about her. She was on a medication (donepezil) for AD. Otherwise she had no significant medical history. She was kept in the hospital for a couple of days, during which time her family doctor and the neurologist were consulted.


There is no specific treatment for AD at the present time. [2] Donepezil and several other drugs that are used in the management of AD inhibit the activity of cholinesterase, an enzyme that hydrolyzes acetylcholine (ACh) to acetate and choline. They are used because some studies had shown lower than normal levels of ACh in specimens of brains from subjects who had died of AD. They appear to produce a modest improvement of brain function and memory in some patients. Memantine, a drug that antagonizes N-methyl-D-aspartate receptors, may cause some slowing of the progression of AD. Symptoms such as depression, agitation, anxiety, and insomnia can be treated by appropriate drugs and antipsychotics may be required if psychosis intervenes. A possible preventive role of omega-3 fatty acids is under study. However, overall, there is as yet no effective therapy for AD. This patient was kept on donepezil and admitted to a nursing home that specialized in caring for patients with AD. Apart from high-quality basic care, the nursing home offered a variety of programs, including music therapy and exercise programs.


[2] AD is a progressive neurodegenerative condition in which decline of general cognitive function occurs, usually accompanied by affective and behavioral disturbances. At least 2 million people in the United States suffer from AD, and its prevalence is likely to increase as more people live longer. Some cases have a familial (genetic) basis, but the great majority (~90%) appears to be sporadic. AD is the most common cause of dementia, which can be defined as a progressive decline in intellectual functions, due to an organic cause, that interferes substantially with an individual's activities. AD places a tremendous burden on families and on the health care system, as, sooner or later, most patients cannot look after themselves. The usual age at onset is over 65 years, but the disease can have an early onset (eg, in the 40s), particularly in cases where there is a genetic predisposition (see below). Survival ranges from 2 to 20 years. It is estimated that about 40% of people over 85 years of age have variable degrees of AD. Loss of short-term memory is often the first sign. AD usually progresses inexorably, and many patients are eventually completely incapacitated.

The diagnosis is usually one of exclusion. A complete neurologic exam is necessary and also a recognized mental status exam. Other forms of dementia (Lewy body, vascular, etc) must be excluded, as must other organic and psychiatric problems; various lab tests may thus be indicated to do this. [3] In certain cases an MRI or CT scan may be indicated; these will usually reveal variable degrees of cortical atrophy and enlargement of ventricles if AD is present. Considerable research is underway to develop laboratory tests (eg, on blood or cerebrospinal fluid) that will assist in making the unequivocal diagnosis of AD.

The basic pathologic picture is of a degenerative process characterized by the death and consequent loss of cells in certain areas of the brain (eg, the cortex, hippocampus and certain other sites). Apoptosis (a programmed type of cell death in which various mechanisms, particularly the activities of proteolytic enzyme known as caspases, are activated within a cell leading to rapid cell death, may be involved in the cell death occurring in AD. At the microscopic level, neuritic plaques containing aggregated amyloid peptide (A, a peptide of 42 amino acids, occurring in beta sheets) surrounded by nerve cells containing neurofibrillary tangles (paired helical filaments formed from a hyperphosphorylated form of the microtubule associated protein, tau) are hallmarks. Deposits of A2are frequent in small blood vessels.

Intensive research is under way to determine the cause of AD. [4] Particular interest has focused on the presence of A42, the major constituent of the plaques found in AD. The term "amyloid" refers to a group of diverse extracellular protein deposits found in many different diseases. Amyloid proteins usually stain blue with iodine, like starch, which accounts for the name (amylo denotes starch). The amyloid cascade hypothesis proposes that deposition of A42 is the cause of the pathologic changes observed in the brains of the victims of AD and that other changes, such as neurofibrillary tangles and vascular alterations, are secondary. A42 is derived from a larger precursor protein named amyloid precursor protein (APP), whose gene is located on chromosome 21 close to the area affected in Down syndrome (trisomy 21). Individuals with Down syndrome who survive to age 50 often suffer from AD.

As shown in Figure 57–2, APP is a transmembrane protein that can be cleaved by proteases known as secretases. In step 1, APP is cleaved by either -secretase or -secretase to produce small nontoxic products. Then in step 2, cleavage of the -secretase product by -secretase results in either the toxic A42 (containing 42 amino acids) or the nontoxic A40 peptide. Cleavage of the -secretase product by -secretase produces the nontoxic P3 peptide. When split off from its parent protein, A42 forms an insoluble extracellular deposit. Aggregation of A42, produced by its oligomerization and formation of beta sheets, is thought by some to be a key event in causing AD. Recent studies have suggested that impairment of clearance of A42 may be an important part of the problem in AD.

Figure 57–2 Add to 'My Saved Images'

Simplified scheme of the formation of A42. Amyloid precursor protein (APP) is digested by -, -, and -secretases. A key initial step (Step 1) is the digestion by either -secretase or -secretase, producing smaller nontoxic products. Cleavage of the -secretase product by -secretase (Step 2) results in either the toxic A42 (containing 42 amino acids) or the nontoxic A40 peptide. Cleavage of the -secretase product by -secretase produces the nontoxic P3 peptide. Excess production of A42 is a key initiator of cellular damage in Alzheimer disease (AD). Among research efforts on AD have been attempts to develop therapies to reduce accumulation of A42 by inhibiting - or -secretases, promoting -secretase activity or clearing A42 by use specific antibodies. (Reproduced, with permission, from Fauci AS et al [editors,] Harrison's Principles of Internal Medicine, 17th ed, McGraw-Hill, 2008, p. 2542.)

Mutations in certain genes have been found in some patients with AD (familial AD). These mutations often predispose to early onset AD. One of these genes is that encoding APP. Table 57–1 summarizes some aspects of the principal genes so far discovered. In general, the effects of the products of these genes are to enhance deposition of amyloid or to diminish its removal. Precise dissection of their mechanisms of action is underway.

Table 57–1 Some Genes Involved in Familial Types of Alzheimer Disease (AD)1

A second part of the amyloid cascade hypothesis is that A or A-containing fragments are directly or indirectly neurotoxic. There is evidence that exposure of neurons to A can increase their intracellular concentrations of Ca2+. Some protein kinases, including that involved in phosphorylation of tau, are regulated by levels of Ca2+. Thus, the increase of Ca2+ may lead to hyperphosphorylation of tau and formation of the paired helical filaments present in the neurofibrillary tangles. Interference with synaptic function is also probable, perhaps secondary to neuronal damage.

Further research may reveal unexpected developments that alter the validity of the amyloid cascade theory as presented above.

Work on AD has shown the probable importance of an abnormally folded peptide in the causation of this important brain disease. It is hoped that further research on AD may result in drugs that will prevent, arrest, or even reverse AD. For example, it may be possible to develop small molecules that prevent formation or deposition of A42, prevent its aggregation, or accelerate its clearance. In addition, it is possible that specific antibodies to A42 or tau could prevent their putative toxic actions.

[5] AD is one of the so-called conformational diseases in which abnormally folded proteins play a central role in the causation of a disease. Other examples of these diseases are cystic fibrosis, alpha1-antitrypsin disease, and the prion diseases.

The study of various neurodegenerative diseases is providing dramatic evidence of the importance of protein structure and function in their causation. For example, abnormal forms of the protein huntingtin play an important role in Huntington disease, abnormalities of -synuclein play a role in some cases of Parkinson disease, and prions have been found to play key roles in the causation of bovine spongiform encephalopathy (BSE) and Creutzfeldt–Jacob disease. The application of genomic and proteomic techniques is also beginning to throw light on the causation of major psychiatric disorders, such as bipolar disease and schizophrenia. The importance of genetic and biochemical approaches in understanding disease processes has never been more clear. A simplified scheme of the causation of AD is shown in Figure 57–3. 
A tentative scheme of the possible sequence of events in at least certain cases of AD. 

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