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Assignment #4: Review Paper
Mitochondrial pathology and apoptotic muscle degeneration in Drosophila parkin mutants
Jessica C. Greene, Alexander J. Whitworth, Isabella Kuo, Laurie Andrews, Mel B. Feany, and Leo J. Pallanck
PNAS (2003) 100 (7); 4078-4083
Introduction:
Parkinson’s Disease (PD) is a neurodegenerative disorder characterized
by the loss of dopaminergic neurons and the accumulation of intraneural inclusions,
called Lewy bodies. There is no cure for this disease and there is only a
small amount of information known about the disease’s mechanism of action
although it is believed that environmental and genetic factors contribute
to the disease. It has been shown that 1-methyl-4-phenylpyridinium (MPP+)
is a toxin of dopaminergic neurons and that MPP+ induces these cells to die
by inhibiting mitochondrial complex I. This observation led to further speculation
about the correlation between mitochondrial dysfunction and PD and is now
a prominent area of PD research. Genes have been discovered which are known
to cause PD, such as the parkin gene. Mutations of the parkin
gene resulting in a loss of function of the gene results in an early onset
form of PD known as autosomal recessive juvenile parkinsonism (AR-JP). Patients
with AR-JP display most of the same symptoms as patients with idiopathic PD,
but these cases lack Lewy body formation. The parkin gene encodes
a protein with a ubiquitin-like domain, and it has been shown that Parkin
functions as a ubiquitin protein ligase and that when the parkin gene
is mutated, the ubiquitin protein ligase cannot attach to its cellular targets
and this is what causes dopaminergic neuron loss in AR-JP. One potential target
of this ligase is components of Lewy body formation. This model would explain
why patients with AR-JP have no Lewy body formation; if the parkin
gene is mutated, then Parkin cannot attach to Lewy body components and Lewy
body formation does not occur. In this paper, The biological function of parkin
was analyzed by mutating a Drosophila parkin analog and looking for
the subsequent phenotypes in flies and then comparing this phenotype to that
of wt parkin+ flies.
Results:
Figure 1A shows the similarities between the human (H-Park
1) and Drosophila (D-Park 1) Parkin sequence. The human Parkin sequence was
derived from its cDNA sequence and the corresponding Drosophila analog
was searched for using the Berkeley Drosophila Genome Project Database. Both
sequences show an ubiquitin-like domain (boxed), RING finger domains (boxed
and shaded), and an In-Between Ring domain (shaded). The Drosophila sequence
depicted in this figure was the only sequence detected in the fly with these
PD domains, and this fly sequences also exhibits a 59% similarity overall
with the human Parkin sequence. Because of these similarities, it is assumed
that the human Parkin analog in Drosophila has been established and
is as shown in this figure. Figure 1B shows a Northern Blot using poly(A)+
RNA from Drosophila embryos (E), third-instar larvae (L), and adults (A) using
a probe specific to parkin. As is shown by this figure, the parkin
transcript is expressed at each stage of development but is very abundant
in adults. Figure 1C shows the Drosophila parkin gene sequence with
a 71 bp insertion upstream from the parkin start codon (line is called
parkEP(3)LA1, designated parkrvA later in paper) and also
shows three deletion constructs, park13, park45, and park25
that are made from varying pieces of the parkin gene as is shown
by the black bars. The bent arrow shows where the predicted transcription
initiation site of parkin is located. Some deletion constructs contained
no sequence from the parkin coding sequence and represent null alleles
of parkin (park13 and park45). All constructs containing
the parkin null allele are viable through the adult stage, but have
a significantly decreased average lifespan and develop at a slower rate than
their wt countertypes. After observing that all female parkin mutants
were fertile but that all male parkin mutants were sterile, it was
discovered that the Parkin sequence of male parkin mutants contained
two missense mutations, one at amino acid 46 that changes A to T, and another
at amino acid 431 that changes R to a stop codon (Figure 1C).
Figure 2 shows that spermatogenesis is also affected by parkin
mutations. At the individualization stage, a germ-line cyst that normally
separates into mature sperm cells fails to do so in parkin mutants,
as is shown by the arrow in figure 2B. Figure 2A shows wt parkin
flies that show mature sperm cells, as is depicted by the arrowhead, and these
cells are smaller and less clumped together than those cells in figure 2B.
Figures 2E and 2F are higher magnification pictures of figures 2C and 2D respectively
and show other discrepancies between parkin mutants and wt flies. As is shown
in figure 2E, normal mature spermatozoa show the axoneme (A) and the Nebenkern
(N), a mitochondrial derivative. The Nebenkern is surrounded by a uniform
and dense matrix, as is depicted by the black arrow. In figure 2F, the spermatozoa
of parkin mutants show a normal axoneme, but the Nebenkern is practically
absent in some spermatids (arrowhead) and is too abundant in other spermatids
(white arrows). In addition, the matrix surrounding the Nebenkern in parkin
mutants is not nearly as uniform or as dense as that shown in 2E. These results
are consistent with the hypothesis that parkin mutations are due to defective
Nebenkern formation.
Figure 3 shows that warped wing posture is also a characteristic
of parkin mutants. Figures 3A and 3A’ both show 1-day old wt
flies with normal wing posture. Figures 3B and 3B’ show age-matched
parkin mutant flies (parkin25) and from these figures it
is easily seen that the mutants have a warped wing posture. Because of this
abnormality, the flight and climbing ability of parkin mutants is
compromised as is shown by Figures 3C-3F. Figure 3C shows that for the control
parkin allele, parkrvA, the flight index is much higher
than for the parkin mutants. Figure 3D shows that as the age of the
fly increases, the climbing index decreases. This is true for the control
allele (parkrvA) and for the mutated allele (park13), although
the climbing index is larger at each point for the control allele than for
the mutated allele. Figures 3E and 3F show that when the UAS/GAL4 system is
used to express parkin in defined tissues, the mutated alleles show
a rescue effect in that the flight ability and climbing index of the mutants
increases. Rescue 1 and 2 are different in that each rescue uses a different
enhancer sequence upstream of the GAL4 sequence. Rescue 1 uses a 24B-GAL4
sequence and rescue 2 uses a Dmef2-GAL4 sequence. The controls depict parkin
mutant flies in which the rescue effect is not allowed to take place
because of the lack of either the UAS or the GAL4 sequence (control 1- no
UAS, control 2 and 3- no GAL4). This clarifies that Parkin is indeed necessary
for wing posture and flight and climbing capabilities and for the musculature
in general.
Figure 4 looks at the musculature defects in parkin
mutants in more detail. Figures 4A-4C show longitudal sections of the indirect
flight muscles (IFMs) from wt parkin flies, parkin mutants,
and mutants that have undergone transgenic rescue. As is shown, parkin
mutants show decreased muscle integrity as is depicted by the vacuole
formation (arrow) and the accumulation of cellular debris (arrowhead). Rescued
alleles show muscle integrity between that of the wt flies in 4A and the mutant
flies in 4B. Figures 4D-4L show that parkin mutants show decreased
myofibril density, decreased sarcomere length, and swollen and degenerating
mitochondria as compared to the wt parkin flies and the parkin-rescued
flies. These figures basically show that parkin mutants generally
show mitochondrial pathology that most likely leads to muscle dysfunction
and degeneration and that differs from normal myofibril and mitochondrial
integrity.
Figure 5 shows that muscle dysfunction and degeneration proceeds
through an apoptotic mechanism. The IFM in age-matched wt parkin
flies and mutant flies underwent terminal deoxynucleotidyltransferase-mediated
dUTP end labeling (TUNEL) staining, and it was found at 96 and 120 hours after
puparium formation that no TUNEL staining was observed in parkin wt
and mutant flies (Figures 5A and 5B) but that there was a significant amount
of staining in 1-day old mutants (5D) as compared to 1-day old control flies
(5C). The increased staining in parkin mutants suggests that the
IFM is indeed formed but that it disintegrates later on in development. This
ultimately means that muscle mitochondrial defects result in cell death by
an apoptotic mechanism.
The goal of figure 6 is to determine the role of Parkin in the brain. Histologic analysis of brain sections from 30-day old parkin wt and mutant flies is shown in figures 6A and 6B respectively. As is shown in these figures, there are no structural abnormalities in the parkin mutant, and also that no increase in neurodegeneration is evident in the mutants as compared to the wt flies. Figures 6A and 6B look practically identical. Figures 6C and 6D show that there is no difference in the number of neurons in the dorsomedial cluster between parkin wt (6C) and parkin mutant (6D) flies. The neurons were stained with tyrosine hydroxylase. In figure 6D the cell body is smaller for the parkin mutant than for the wt fly (shown by arrow), and that the proximal dendrite shows less staining for the mutant than the wt fly (shown by arrowhead), but these defects do not effect on the number of neurons present in the parkin mutant fly.
Discussion:
The authors restate the findings that loss of parkin function in flies results in male sterility, deformed wing posture, impaired flight and climbing abilities, muscle degeneration, mitochondrial pathology, and structural changes in dopaminergic neurons. One difference between the Drosophila parkin mutants and AR-JP individuals is that in the fly mutants, the most striking phenotype is the muscle degeneration and germ-line pathology, but in humans, the most striking phenotype is the loss of dopaminergic neurons. They proceed to say that despite these phenotypic differences, the affected tissues undergo alterations and deformations by the same mechanism. The differences that are observed in parkin mutant flies and humans as compared to their wt counterparts arises through mitochondrial dysfunction. They conclude that further work will need to be done to elucidate the mechanism by which mitochondrial dysfunction affects cell death and the mechanism by which loss of parkin affects the mitochondrial system.
Critique:
I believe that all of the experiments comparing the phenotypes of parkin mutant flies to their counterpart wt flies are very well done and well controlled. For every figure of a parkin- fly, there is a corresponding figure of a parkin+ fly. I was forced sometimes to trust the author’s conclusions about figures and trust that the differences between the wt and mutant flies corresponds to what they say it does, which is frustrating, but is necessary since I am not an expert in Drosophila eye, muscle, and brain structure and pathology. For instance, in Figures 2A and 2B, I see a difference between these figures, but I must trust the authors’ claim that this difference is attributed to the presence or lack of mature sperm cells. Despite having to blindly believe that their interpretation of the data is accurate, I was satisfied with the results and presentation of the results and thought that the data were well-controlled and explained well.
My only actual criticism of this paper is that they fail to go into detail
about something seen in figure 6 that is particularly striking to me. The
authors state in the introduction that AR-JP is characterized primarily by
a loss of dopaminergic neurons, but in figure 6, the parkin- flies
show absolutely no dopaminergic neuron loss. The authors briefly mention this
observation as is seen in figure 6, but do not focus much on it. I just have
a hard time believing that the mechanisms by which the parkin mutation
acts on the fly and human system are that similar when the outcomes seem drastically
different. Yes, the parkin mutant fly exhibits traits that derive
from mitochondrial defects as is shown in this paper (figure 4), and yes,
the parkin mutant humans exhibit traits that derive from the inhibition
of mitochondrial complex I, but since the phenotypic expression of the loss
of parkin seems so different between the two systems, I am finding
it difficult to assume so many similarities between the mechanisms underlying
these very different phenotypic outcomes. If it had been found that at least
some dopaminergic neuron loss had been found in parkin mutant flies
but that the muscle degeneration and mitochondrial pathology was just more
prominent in mutant flies, I would be more satisfied with the conclusion that
the phenotypic expression of parkin mutations in these systems result
from similar mechanisms because then there would be dopaminergic neuron loss
in both systems. After all, PD is primarily characterized by neuron loss.
I would also have liked to see some other data from AR-JP individuals, such as spermatogenesis development and muscle and mitochondrial pathology to determine if these characteristics are common to both flies and humans. I do not know if any of the phenotypic expression that results from this mutation is common to both species, but it would have been nice to see if there was any similarities so that the argument that the phenotypic mutations result from the same mechanism would have been stronger.
Future Work:
As I just mentioned, I would like to see some data comparing the phenotypic expressions of parkin mutants in humans and flies to see if there are any similarities that exist between these species. I would like to see a figure such as figure 1, but using AR-JP individuals as well, and I also think it would be nice to see a figure like figure 6 using AR-JP individuals (along with the appropriate controls like in this paper). I do not know if human biological structure and function is similar to those in flies, but I think it would be interesting to see if the extent of sperm maturation, muscle degeneration, mitochondrial pathology, and brain development in AR-JP individuals. Then, with this data from AR-JP individuals, it would be possible to compare the phenotypic expression of the parkin mutant flies to that of AR-JP humans to see if any other phenotypic similarities exist besides the proposed mitochondrial mechanism.
As the authors suggest, I think the next big step in this research is to determine
the specific mechanism by which the loss of the parkin gene affects
mitochondrial structure and function. I am having a hard time with the authors
assumption that the loss of parkin function in the fly and human
is extremely similar, and so I would feel more confident about this conclusion
if data were presented that elucidated the mechanism by which loss of parkin
affects sperm maturation, muscle degeneration, mitochondrial pathology,
and brain structure data for both flies and humans. In order to accomplish
this goal, it seems necessary to look for transcription factors or other proteins
that are affected by the parkin mutation in humans and see if these equivalent
proteins are affected in flies. This isolation of transcription factors or
other proteins involved in this mechanism might be accomplished by producing
a radioactive or labeled Parkin antibody and inserting it into various tissues
(muscle, mitochondria, brain) and seeing where it stuck. Once the Parkin receptor
was identified and sequenced for both humans and flies, it could be determined
if the receptor protein is similar for both species. Then the same process
can be repeated by making a labeled antibody for the receptor protein and
see what protein the first protein stuck to. In this way, the mechanism for
how Parkin affects proteins in the body can be elucidated. In addition, at
the end of every step and once the affected protein is sequenced, the amino
acid sequence can be compared between flies and humans to see if the Parkin
protein affects the same sorts of proteins and if the process is similar.
In this way, the mechanism for how the expression or nonexpression of the
Parkin protein affects bodily processes can be elucidated for humans and for
flies and the mechanisms can be compared to each other to determine if, in
fact, they both involve a mitochondrial pathway.
Chemistry Individual Research Project, May 2002
Send comments, questions, and suggestions to cawilliford@davidson.edu