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Caroline Bennett's Review Paper
paper being reviewed:
Mitochondrial pathology and apoptotic muscle degeneration in Drosophila parkin mutants
By Jessica C. Greene, Alexander J. Whitworth, Isabella Kuo, Jaurie A. Andrews, Mel B. Feany, and Leo J. Pallanck
From PNAS, Volume 100, Number 7, 1 April 2003: 4078-4083
Edited by Kathryn V. Anderson
Summary / Critique
Abstract and Introduction
This April 1, 2003 article from PNAS focuses on Parkinson’s
Disease (PD), particularly an early-onset familial form of the disease called
autosomal recessive juvenile parkinsonism (AR-JP). The Abstract and Introduction
provide adequate background regarding previous research done to understand the
function and mechanism of AR-JP. PD in general is a neurodegenerative disorder
that causes loss of dopaminergic neurons in the substantia nigra pars compacta
and the accumulation of proteinaceous intraneuronal inclusions called Lewy bodies.
The molecular mechanism for these defects thus far remains unclear, but scientific
evidence points to mitochondrial dysfunction as the major causative factor.
In the Introduction, Greene et al. describe the previous identification of loci
responsible for rare monogenic forms of PD as insightful to mechanistic understanding
of the disorder; this approach identified the gene parkin. A paper
published in 1998 illustrated that loss-of-function mutations in parkin
result in early onset of AR-JP and lack of Lewy body formation. The parkin
gene encodes a polypeptide with an N-terminus ubiquitin-domain, two C-terminus
ring-finger domains, and an in-between ring-finger domain. In accordance with
this structure, parkin has previously been found to act as an ubiquitin protein
ligase; in other words, dopaminergic neuron loss in AR-JP results from the failure
to label specific cellular targets with ubiquitin.
Greene et al. recognized the need for an animal model of this disorder in order
to investigate the relevance of these Parkin substrates to dopaminergic neuron
loss. They applied a functional approach to further the scientific understanding
of the molecular mechanism responsible for selective cell death in AR-JP by
creating a Drosophila model for the disease. This Abstract and Introduction
give the reader a clear general understanding of Parkinson’s Disease and
the research that has been done on AR-JP thus far. It also explains that the
null mutants for parkin generated in this study exhibit reduced life
span, locomotion defects, and male sterility, indicating that the defects result
from mitochondrial dysfunction. In the Abstract, the authors write that these
results “raise the possibility that similar mitochondrial impairment triggers
the selective cell loss in AR-JP” (4078). Greene et al. chose these words
carefully, explaining what the data “indicate” and “suggest”
rather than “prove.”
Methods
Initially, the researchers probed the Berkeley Drosophila Genome Project Database
using a human Parkin polypeptide query sequence. They found the DNA sequence
encoding Drosophila parkin ortholog and fully sequenced it. Parkin
mutants were generated by inducing transposition of a P element insertion, and
a single mutant line, parkEP(3)LA1, was recovered via PCR analysis
of genomic DNA from the offspring of approximately 5,500 flies. ParkEP(3)LA1
has an insertion 71 bp upstream of the parkin start codon. They introduced
modified cDNA (parkin cDNA clone SD01679) into the Drosophila
germ line after being ligated into the P{UAST} vector. Northern blot analysis
followed.
Greene et al. then conducted three behavioral assays on the mutant flies to analyze the functional differences between flies with mutant parkin and wild-type flies. The first assay tested the longevity of flies. It was conducted in triplicate for each genotype, and the researchers recorded the number of dead flies when 0-24 hr old flies were transferred to new vials every 2-3 days. The test for flight was a bit more complicated, and used the Benzer apparatus. At least 100 flies of each type (1-2 days old) were tested for flight. In general, flies were released into a graduated cylinder and then stuck to an acetate sheet coated with vacuum grease; with this method, flies stick wherever they alight and flight efficiency can be calculated based on where they stick. To test climbing efficiency in mutant vs. wild type flies, at least 60 flies of each genotype were involved in climbing assays using a multi-chambered apparatus. By counting the number of flies able to climb to various chambers in the apparatus during timed trials, a climbing index was calculated in the same manner as the flight index. It is important to note that these behavioral results are based on averages of many flies; the researchers were conscious of the fact that large sample sizes provide more accurate results. However, the statistical values (indexes) calculated seem to have significant ranges of error, based on the graphs in Figure 3. This issue will be discussed again in the analysis of the results, but it illustrates the difficulty of behavioral tests.
Frontal brain and muscle tissue were sectioned and stained with hematoxylin and eosin or immunostained with a polyclonal antibody to tyrosine hydroxylase. Also, tissues for electron microscopic analysis were prepared by dissecting testes from 6-hr old males, thoraces from 1- to 2-day old adults, and aged pupae.
Results
When probing the Drosophila genome for parkin, a single was gene identified. The fact that the human homolog recognized this gene and that this gene is the only one in the Drosphila genome encoding a polypeptide bearing a ubiquitin-like domain, ring-finger domains, and IBR domain indicate that the correct gene was isolated in this process. This 468 aa gene shares 59% overall similarity with human Parkin, based on sequence analysis. Description of Figure 1B explains that “embryos, larvae, and adults detected transcripts at all developmental stages with particularly high abundance in adults” (4079). While the figure does illustrate that the parkin transcript is detected in each of the three stages of development shown, I am skeptical whether this data can actually be quantified in this way. The blot shows no loading controls to indicate that the same amount of RNA was loaded in each lane. I would have appreciated seeing mw markers as well.
After identifying and isolating the fly Parkin ortholog, Greene et al. generated
a disruption of the parkin gene. They did this using a transposon mutagenesis
screen using a P element mapping close to parkin. This process yielded
the single mutated line ParkEP(3)LA1 with an insertion 71 bp unstream
from the parkin start codon. They further generated more severe alleles
via the screening process. Of the collection of deletion alleles via the transposon
mutagenesis screen method, some were null alleles lacking all of the parkin
coding sequence. Figure 1C accurately illustrates various mutants and the portions
of the parkin coding sequence deleted with each. A vital component
of their work is the control chromosome generated as a standard of comparison
(parkrvA).
Greene et al. then studied the physical effects of replacing the wild type
parkin allele with the null allele (the Df(3L)Pc-MK deletion chromosome
was responsible for removing the wt parkin gene).
Observed physical effects in flies with the null allele include:
1) Flies live to adulthood, but average lifespan is significantly
shorter (27 days with a max. of 50 days vs. 39 days with a max. of
75 days).
2) Complete male sterility was observed in flies tested. With
this knowledge, the sterile lines were screened for additional parkin mutations.
Sequencing two of the mutants recovered from this screen revealed missense and
premature stop codon mutations; this illustrates that the observed phenotype
does in fact result from loss of parkin function. Analysis of testes
of parkin mutants compared to wt testes (shown in Figure 2) illustrates
that male sterility is caused from a late defect in spermatogenesis. The defect
occurs at the level of individualization; germ-line cysts that typically separate
into individual sperm cells fail to do so. Figure 2 clearly shows this structural
defect. The inset in Panel 2B shows that spermatids do form in the mutant cells,
but they fail to individualize like the sperm shown in panel 2A (wt). Panels
2E and 2F illustrate conservation of axoneme structure, while the Nebenkern
structure is disrupted. Some spermatids have multiple Nebenkern (which are specialized
mitochondrial derivitives), and others have only a reduced Nebenkern component.
Since this specific structural change is observed and the axoneme structure
is not disrupted, it is logical for Greene et al. to suggest that defective
Nebenkern formation and/or function may underlie the failure of spermatid individualization.
3) All flies with null parkin alleles also exhibit abnormal
wing posture. The images in Figure 3 illustrate this “partially
penetrant downturn wing phenotype,” a condition that progresses significantly
with age (4080). Locomotor ability and climbing ability was prompted as a result
of this finding; panels 3C and 3D show the significant defects in both of these
functions. Both of these two graphs base their analysis on the control phenotype,
and the climbing index is significantly lower for the mutant flies compared
to wt; also, the proportional difference between wt and mutant is consistent
at each varying age level. In panel 3C, the mutants are again significantly
lower in flight index, and one mutant flight index is lower than the other two.
The authors do not address this slightly lower average index. I am concerned
the significant error bars for each bar graph in Figure 3. This error range
could alter the difference in index values between wt and mutant flies, and
therefore cause the data to be less consistent that it appears in this graph.
Panels 3E and 3F show an important technique used to support the role of parkin.
A GAL4-UAS system was used to as a “rescue” mechanism to return
parkin expression to defined tissues. The bar graphs in 3E and 3F provide
compelling evidence that Parkin function is required in the musculature. Two
GAL4 lines were found to rescue wing posture, flight, and climbing phenotypes
of mutants to the level of wt or near-wt levels. The fact that expressing parkin
in mesoderm cells can fully restore gene function is positive, convincing evidence
for gene function.
Figure 4 further demonstrates the physical consequences of the null allele through histological analysis of the major flight muscles in Drosophila. As expected, the parkin mutants have severe disruption of muscle integrity; acute degeneration is characterized by vacuole formation and accumulation of cellular debris (Figure 4B). Amazingly, transgenic expression of parkin again rescues this phenotype (although occasional vacuoles are still seen). This raises one question, however. Why isn’t recovery always 100% complete if you are restoring the exact gene that encoded the muscle structure seen in the control (4A)? This observation suggests that perhaps the recovery process used is not a completely efficient method. The occasional occurrence of vacuoles in the transgenic tissue could also be a result of experimental error. I think it is unlikely that this observation undermines the role of the parkin gene. It is curious that the authors fail to include data to fully support their claim that “only a subset of muscles are affected by loss of parkin function” (4080). The paper is not very clear about exactly which muscles are and are not affected by parkin, and I don’t know whether they know this information and simply did not include it or whether the specific muscles are still being investigated.
As far as their analysis of the indirect flight muscles (IFMs), Figure 4 shows
grossly swollen mitochondria with “severe disruption and disintegration
of the cristae” in parkin mutants (4080). Also, overall density
of myofibrils decreases, the myofibril Z-line broadens, and the sarcomere shortens
in mutants. As expected, transgenic parkin expression results in recovery
of myofibril integrity and mitochondrial morphology.
The next question Greene et al. asked was: does muscle degeneration proceed through an apoptotic mechanism?
To investigate this issue, IFM in parkin mutants and controls were subjected to TUNEL staining. A significant increase in TUNEL-positive nuclei appeared in the IFM of 1-day-old adult parkin mutants (Figure 5), suggesting that the muscle mitochondrial defects do indeed ultimately result in cell death through an apoptotic mechanism.
Greene et al. further investigated the role of parkin in the brain by histologic analysis. No clear neuronal loss was observed when brain sectioned were immunostained with tyrosine hydroxylase. However, as illustrated in Figure 6, cells of the dorsomedial dopaminergic cell cluster reliably showed shrinkage of the cell body and decreased immunostaining in proximal dendrites in aged mutants relative to controls. Why is this observed? The authors mention the fact that this specific cluster of neurons has enhanced toxicity to a protein implicated in familial PD as an interesting connection. I did not find this connection very striking, but that may be because I am not familiar with the toxin.
Discussion and Future Experiments
Greene et at. successfully create a Drosophila model for AR-JP by disrupting a highly conserved Drosophila parkin ortholog. Their extensive analysis of null mutant flies illustrates the phenotypical effects of loss of parkin function. They also resaonably hypothesize the mechanisms through which these physical effects occur. Based on histological analysis, they propose that male sterility results from a spermatid individualization defect in the male germ line, and that locomotion defects arise from apoptotic muscle degeneration.
Although loss of parkin function in Drosophila mutants and in AR-JP targets differnt tissues, it is likely that AR-JP operates via a similar molecular mechanism as the Drosophila model studied. Mitochondrial defects are a common characteristic of the pathology of male germ lines and IFM in parkin mutants (under ultrastructural examination). But the possible mechanistic relationship between mitochondrial pathology and lack of spermatid individualization is still unknown. Greene et al. target initial mitochondrial disfunction as the common origin of the Drosophila parkin mutant phenotypes. The logical next step for understanding this mechanism is to investigate exactly how loss of parkin can trigger mitochondrial pathology and ultimately cell death at the molecular level.
More in depth structural analysis of the protein that parkin encodes is a good start. We know that Parkin functions as a ubiquitin protein ligase, tagging cellular targets with ubiqutin. In Parkin mutants, failure to tag the targets causes dopaminergic neuron loss in AR-JP. The Introduction explains that it was recently found that Parkin functions this way in a pathway with ubiquitin-activating (E1) and ubiquitin-conjugating (E2) enzymes. What exactly does the ubiquitin protein ligase do in order to cause neuron loss and what significance is there that it acts in a pathway with two other enzymes? And does the Drosophila parkin operate in a pathway as well? How conserved is the exact mechanism through which Parkin operates? It might be helpful to stain the protein in wt fly cells from various tissues and follow its path and mechanism over a time period. You could use Green Flourescence Protein labeling in order to stain the protein while the cell remains alive and functional. You could use GFP in human wt cells and conpare the observations.
Another logical next step following this paper is to create a mammalian model for the disease in mice. You could test mice in a similar manner as Greene et al. tested flies. You would isolate the parkin ortholog in the mouse genome by probing it with the human sequence and then sequence the mouse version of parkin. You could also analyze phenotypical defects in mutant mice, although this procedure would be more difficult in mammals with longer gestation periods. If mutant mice show muscle degeneration and locomotive defects, this is strong evidence that the gene is highly conserved in mice as well. You would analyze muscle tissue for degeneration and the mitochondrial pathology within muscle tissue, looking out for swollen mitochondria. You would also investigate the sterility of male mice and then analyze the testes on a cellular level for the mechansim for spermatid individualization. You would expect to see Nebenkern abnormalities. A mouse model for AR-JP would be incredibly imformative and a fascinating. It would be another key to understanding evolutionary mechanistic conservation in relation to PD.
Althoght this field still requires extensive research in order to gain the
ability to more effectively treat and/or cure the disease, this study represents
a valuable step toward understanding the mechanism of AR-JP and PD in general.
The findings presented by Greene et al. certainly have greater implications
for a potential mechanism for idiopathic PD. In the Discussion, the authors
point out that "a substantial body of evidence suggests that mitochondrial
disfunction and apoptosis are important functions underlying neurodegeneration
inidiopathic PD" (4083). If we can uncover the complete mechanism for Parkinson's
at the level of Drosophila (or mice), we will have reason to believe that the
mechanism of the disease in humans is highly conserved. Undertanding the mechanism
of Parkinson's Disease will inform drug research in the direction of what to
target in order to treat the diesease effectively.
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Please direct questions or comments to Caroline Bennett at cabennett@davidson.edu.