The gene known as FMR1, Fragile
X Mental Retardation Protein, is a main contender for the spotlight in medical
research of today. There are three specific disorders, called Fragile
X-associated Disorders, that are caused by permutations of this gene and two of
those disorders are studied at the University of California Davis Medical
Center (UCDMC) located in Sacramento, CA. The two kinds of Fragile X-associated
Disorders are discussed as well as final thoughts on the UC Davis internship.
The first of these disorders is
Fragile X Syndrome and is the best known cause of the disorder known as Autism
Spectrum Disorder (ASD) or simply autism. Approximately 1 in 88 children in the
United States are affected by ASD. Those who suffer from ASD show signs such as
delayed speech development, difficulties with social interactions, disrupted
immune responses, and multiple other symptoms that range across the spectrum. This
disorder is studied extensively at the Medical Investigation of
Neurodevelopmental Disorders (MIND) Institute at UC Davis. Stephen Noctor’s lab
focuses on the effect of the mother’s immune response on the neurodevelopment
of the fetus as it is believed this plays a role in the prevalence of autism.
Multiple studies have been conducted that have shown there is some correlation
with the alteration of proteins in the brain of a fetus that was affected by maternal
immune dysregulations during pregnancy. Since proteins play such a large role
in neurodevelopment, it is hypothesized that this is where the problem lies.
In Dr. Noctor’s lab mice, or
rats, are given lipopolysaccharides (LPS). LPS is a sugar/lipid found on
bacteria that triggers an immune response as though the mice had received an
actual bacterium. The immune response triggered mimics that of women during
pregnancy. When someone gets sick, the body releases cytokines that tell the
white blood cells to attack anything possibly perceived as a threat. When this
happens, blood vessels expand and this has direct effect on the placenta and
the blood brain barrier of a developing fetus. The lab continues its study of
the implications this has on autism by studying the structure of brains of
those who had autism.
The fetuses of the mice are
removed one by one from the uterine horns and the fetuses are perfused. This is
done either in the embryonic stage or the very young post birth stage (postnatal)
for the baby mice. The mother is kept alive under anesthesia during this
process until the final fetus is removed after which the animal is killed using
a hand guillotine. The process of perfusion is done under a microscope with
built in lights because the ablation of fetal brains is difficult due to the similarity in coloring
of both the tissue and the layers of protection around the developing brain,
including the still developing skull. The brains are carefully recorded as they
are collected and then stored at four degrees centigrade in a solution made
with sucrose and azide. The purpose of this solution, and occasional washings
with the same solution, is to help avoid contamination. The tissue is later
transferred to tissue collecting solution and placed in the freezer at -20. The
tissue is later frozen in cryomolds in optimal cutting temperature (OCT) gel in
preparation for sectioning on a cryostat. The tissue will then be used for
various studies.
No real conclusions were drawn
during this time but correlation is strongly evident. Should causation be
proved, the implications would be extensive. Women who are instructed not to
take anything when they get sick while pregnant would possibly be advised to
the contrary. If the woman’s family has a history of ASD, it could possibly be
advisable that she take something to stop her immune response should she get
sick while pregnant as this might prevent her fetus from showing with autism. Other
implications include developing methods for earlier detection which would allow
for earlier behavioral therapies.
The second disorder known to
result from a mutation in the FMR1 gene is Fragile X-associated Tremor/Ataxia
Syndrome (FXTAS). FXTAS is a late onset disorder usually seen in men who are
over the age of 50. Recently discovered, FXTAS is sometimes misdiagnosed as
Parkinson’s because symptoms for both include tremors of deliberate motions. It
can also be misdiagnosed as Alzheimer’s because those who have FXTAS suffer
from dementia. Other symptoms include ataxia; short memory loss; disruptions of
mood, including psychiatric problems; and decline of cognitive processes. FXTAS
was discovered in 2001 by a research wife and husband team, the Hagermans, at
the MIND institute. Because they discovered the syndrome, brains are sent to
them of individuals who suffered from this Fragile X-associated Disorder.
Recently, this included the brain of a man only 34 years of age.
The fact that brains are shipped
in for the Hagermans proves beneficial for many labs at the UCDMC. One of the
labs that benefits from this is the lab of Veronica Cerdano-Martinez. Dr.
Martinez has been studying neurodegenerative diseases as they are associated
with stem and progenitor cells at the Shriner’s Children’s Hospital of Northern
California. Her lab most recently has been studying the levels of iron in the
brain of those who suffered from FXTAS. Many neurodegenerative diseases are
associated with levels of iron in the brain, including Parkinson’s and
Alzheimer’s. An incorrect amount of iron in the developing fetus has
detrimental effects on the structure of the brain later in life because iron
plays a role in the neurogenesis of cells in the brain. Iron deficiency can be
seen affecting the amount of dendrites, the function and location of
oligodendrocytes, and to some extent myelin sheathing. Because of this, large
perforations in the white matter can be seen in those who suffered from iron
deficiencies – like those who suffered from FXTAS. In order to understand this
more fully, the lab strives to analyze the amount of iron in the tissues
available.
Brains are brought to the lab
and are sectioned small enough to keep and later section on a cryostat or
occasionally a vibratome. The tissues are sectioned and placed on slides.
Slides are stored at -20 until they are used for immunohistochemistry (IHC).
Slides are placed in a pressure cooker/decloaker to break the connections
between proteins in the tissue allowing for IHC. The tissue is then hydrated to
allow the rest of the IHC to take place. Hydration includes moving the slides through
xylene substitute, isopropanol, and various percentages of ethanol. To see the
levels of iron in the tissue, a four day IHC is used for both ceruloplasmin and
transferrin. Ceruloplasmin and transferrin are both iron transport
proteins.
Each case was stained for iron
found in the neurons (NeuN), microglia (Iba), the protein Sox10, and the
protein S100. The tissue is treated with primary antibodies of the four
mentioned and then secondary antibodies of donkey anti goat or donkey anti
mouse depending on which primaries are used. The tissue is stained with AB blue
kit as well as NOVA red and DAB. The four day process takes several hours each
day. At several places, where the staining is placed on the tissue, knowing
when to stop the development is determined by careful watch with the use of a
microscope.
After the IHC is complete, the
slides are dehydrated and coverslipped using permount and stored in a slide
case until they can be counted. After the slides have dried, they are counted
using either ImageJ software or a microscope. An overall knowledge of how much
transferrin or ceruloplasmin can be seen on each slide is determined by using
ImageJ. In order to get a more precise idea of the signal available per slide,
the counting is done under a microscope. Each slide is stained for either NeuN
with S100 or they are stained for both Sox10 and Iba. Differentiating between
the two on each slide is difficult with a two dimensional picture on the
computer and therefore needs the microscopes ability to move through the tissue
by altering the focus.
The iron amounts associated with
neurodegenerative disorders has been studied extensively but there is still
room for further understanding. Should causation be more firmly established it
would be possible for the creation or improvement of therapies or interventions
that could help those who might suffer later in life with neurodegenerative
diseases due to their birth with iron deficiencies.
Martinez’s lab was also
searching to establish new recipe/protocols for golgi staining as current
protocols are not sufficient for the lab’s needs. Golgi staining is a precious
tool as it allows for the staining of complete structure including neurons. Current
theories of the problem with golgi staining are mostly biology based and the
lab proposes to establish that the problem lies in the chemistry rather than
the biology of the stain. Brain tissue are used at both 100 and 200 um and
placed in solutions made with Potassium Dichromate and Potassium Chromate at
various molarities, and tested for both one week and two weeks to discover if
any improvements could be established in golgi protocols. At the time of this
paper no final conclusions have been drawn.
Other techniques learned and
utilized in the lab settings include the use of chemistry and math in order to
create solutions including TBS, PBS, various ethanol percentages, sucrose
mixtures, DIVA for the decloaking machine, and others. The proper process for
sterilizing dishes was learned. The collection of brain tissue was inventoried
and photographed. Ablations and perfusions at both labs was observed and
performed at the first. A protocol for IHC with hematoxylin was also performed.
The use of high functioning microscopes was learned including Olympus and
Keyence microscopes.
As shown above this internship
has proved to be beneficial in that many techniques were learned as well as an
increased understanding of neurodevelopmental disorders (NDs). To see two labs
in two different locations was incredibly interesting, as it allowed for the
comparison of techniques used and
increased the knowledge gained of NDs. Dr. Noctor’s lab is a friendly
atmosphere where graduate students are very dedicated in their research and the
intern is expected to help them where needed. In Dr. Martinez’s lab the lab
itself is more open and placed in a large room in close proximity to other
labs. It is also on the sixth floor of a hospital and has a different feel to
the atmosphere. While still employing grad students, those students are not
quite so open or friendly but it is guessed that this is due in part to the
intern’s lack of being a part of their work. In Martinez’s lab interns are more
or less given their own work to add to the overall arching research project
while also performing the tasks of an intern, mostly shared in the previous
paragraph.
While intrigued with the
research of Fragile X-associated Disorders, the hands on work of being an
intern was less than appealing. While enjoying the everyday tasks of bench
work, the perfusions and ablations were distasteful. A grad student in Dr.
Martinez’s lab is also studying the spinal cord of mice and the image of the
spinal cord being grotesquely hacked from the body of a mouse is something not
easily forgotten.
This internship is an invaluable
experience for students seeking future education or careers in science, whether
research is a desired part or not. To learn hands on how research is done and
the process in making research articles happen is a valuable lesson. The two
labs collaborate every other week to study together a research article of a grad
student’s choosing. This article club increases the knowledge of current
research and allows for better understanding of the papers chosen as questions
are freely asked and answered. A club such as this could be extremely useful
for those students at Brigham Young University-Idaho who desire to enter this
internship as it will better prepare them to understand the work they will be a
part of. Much of the research done today is not new but built on the back bone
of other previous research. It is suggested that future interns come with the
ability to read papers, pull needed information out, and formulate questions to
ask. Researchers enjoy talking about their work and questions are always
welcomed
Outside the lab, the ability to
spend a few months in California was also an interesting experience. For those
born inside Utah or Idaho, it is necessary to live for a time somewhere not
filled with members of the Church of Jesus Christ of Latter-day Saints. The
interactions and friendships made were beneficial and the many chances to share
parts of the gospel outside of the lab, on the bus etc., are valuable life
lessons for every member of the Church.
Being a part of the research
done by faculty at BYUI would be good preparation. Because the research done under
the advisement of Clair Eckersell includes perfusions, IHCs, and cryostat work
it is good preparation for the labs of Noctor or Martinez. It would also be
suggested that future interns be aware that it is their job to say when they do
not know how to do something and to make sure what they are doing is in correct
sequence. Careful keeping of a lab journal proved extremely beneficial in both
labs. Future interns should be aware that a lot of research is waiting. Actions
are performed followed by a lot of five minute, one hour, two hour waits and
that can be frustrating.
Fragile
X-associated Disorders are intensely interesting. The ability to study both ASD
and FXTAS is a memorable experience that will prove useful for future
endeavors, both in career settings and education. The process of research,
starting from hypothesizing, researching and then writing articles for
submission is neat to watch and in part experience. Future interns should feel
lucky to be a part of a program like this. A final set of suggestions for those
who will be joining this internship: bring a bathing suit and a lot of sunscreen.
Future interns should be reminded to not forget to visit the beach while
remembering the words of Dwayne “The Rock” Johnson that formed the mantra for the
last three months, “Be humble, hungry, and the hardest worker in the room.”
Pictures
These are bat brains that Dr. Martinez travelled to collect for us. Well, obviously they are bat heads. The brains aren’t out yet. No project has been started with the bat brains yet but I had to transfer them into sucrose for storing.
References
http://www.fxtas.org
Crichton, R.,
Dexter, D., Ward, R. (2011). Brain iron metabolism and its perturbation in neurological
diseases. Journal of Neural Transmission.
118, 301-314.
Luo, Y., Shan,
G., Guo, W., Smrt, E., Johnson, E., Li, X., … Zhao, X. (2010). Fragile X mental
retardation protein regulates proliferation and differentiation of adult neural
stem/progenitor cells. PLOS Genetics.
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Saffary, R.,
Xie, Z. (2010). FMRP regulates the transition from radial glial cells to
intermediate progenitor cells during neocortical development. The Journal of Neuroscience. 31, 1427-1439
Braunshweig,
S., Krakowiak, P., Duncanson, P., Boyce, R., Hansen, R., Ashwood, P., … Van de
Water, J. (2013). Autism-specific maternal autoantibodies recognize critical
proteins in developing brain. Translational
Psychiatry. 3
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