Honors College Capstone Project
Chen Shi (UIN: 663260971)
Understanding the role of TDP-43 in the Nervous System
Chen Shi
Department of Biological Sciences and Chemistry, The Honors College and College
of Liberal Arts and Sciences, University of Illinois at Chicago, Chicago, IL 60607
E-mail: cshi2@uic.edu
Supervisor: Charles Louis Mix Professor Jane Y Wu
Department of Neurology, Lurie Cancer Center, Center for Genetic Medicine,
Northwestern University School of Medicine, Chicago, IL 60611
E-mail: jane-wu@northwestern.edu
Fellow: Professor Robert Paul Malchow
Department of Biological Sciences, The College of Liberal Arts and Sciences,
University of Illinois at Chicago, Chicago, IL 60607
Email: paulmalc@uic.edu
Keywords: TDP-43 proteinopathy, Drosophila, neurodegeneration, ALS
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Abstract
Background: TAR DNA binding protein-43 (TDP-43) has been identified as an
important player in a wide spectrum of neurodegenerative diseases named TDP-43
proteinopathy. This group of neurodegenerative disorders is heterogeneous both in
neuropathology and clinical manifestations. Molecular mechanisms underlying TDP-43
proteinopathy remain to be elucidated. The specific aim of this study is to examine the
function of human TDP-43 (hTDP-43) protein in vivo using Drosophila as an animal
model. Our working hypothesis is that maintaining appropriate levels of TDP-43
expression in different cell populations is important for the normal development,
maintenance and proper function of the nervous system.
Methods: We generated transgenic flies expressed human TDP-43 (hTDP-43) in
different neuronal subpopulations, including photoreceptor neurons and motor neurons,
while we also knocked down fly endogenous TDP-43 homologue, Tar DNA binding
protein homolog (TBPH) by RNA interference (RNAi). The expression changes have
been confirmed by RT-PCR, western blotting and fluorescent microscopy.
Morphological abnormalities of flies were detected by fluorescent and confocal
microscopy, as well as TUNEL staining. Functional defects of flies were determined by
larval and adult fly motility assay.
Results: Expression of hTDP-43 in fly eyes led to progressive loss of ommatidia and
remarkable signs of neurodegeneration with aging. Overexpressing hTDP-43 in motor
neurons led to axon swelling, aggregate formation in cell bodies and axons, reduction in
axon branches and bouton numbers, neuronal death together with functional deficits.
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TBPH RNAi flies demonstrated the reduced bouton and branch number, as well as the
decreased climbing ability in adult flies.
Conclusions: Flies having either excessive hTDP-43 or insufficient TBPH expression
show deficiency in the nervous system. Overexpression of hTDP-43 in different
subpopulations of neurons led to neurodegeneration with remarkable features of human
TDP-43 proteinopathy. Therefore, proper regulation of TDP-43 expression is critical for
the normal development, maintenance and proper function of the nervous system.
Furthermore, our transgenic flies will be useful for screening genetic modifiers and
chemical compounds that reduce or block the neurodegeneration in TDP-43
proteinopathy patients.
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Introduction
TAR DNA binding protein with a molecular weight of 43 KD (TDP-43), a 414-amino
acid nuclear protein, is encoded by a highly conserved gene, TARDBP, on chromosome 1
in the human genome.[1-3] TDP-43 was initially identified as a transcriptional repressor
of HIV-1 gene expression.[4] It is highly conserved and ubiquitously expressed in all
tissues including brain.[2] The normal physiological functions of TDP-43 are diverse and
not fully defined but probably regulate multiple biological processes, such as
transcription, splicing and mRNA stabilization.[5] TDP-43 had been recently identified
as a pathological signature protein in the ubiquitinated inclusions (UBI) from patient
samples of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar dementia
(FTLD).[6, 7] In addition, more recent immunohistochemical studies have identified
TDP-43 positive inclusions in the affected tissues in a range of neurodegenerative
disorders,[8] including Alzheimer’s disease (AD),[9] dementia with Lewy bodies
(DLB)[10] and Guam-parkinsonism-dementia complex.[11] These studies provide the
molecular link among a variety of neurodegenerative diseases characterized by TDP-43
proteinopathy. Therefore, TDP-43 has been added into the growing list of
neurodegenerative proteinopathies, but unique in that it lacks characteristics of brain
amyloidosis.[12]
Accumulating evidences have suggested that abnormally metabolized TDP-43 and the
formation of TDP-43 positive inclusions in affected neurons and glial cells lead to
neurodegenerative diseases characterized by TDP-43 proteinopathy. The molecular and
cellular basis may be related with abnormal cleavage, hyperphosphorylation, cytoplasmic
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mis-localization, decreased protein solubility, and aggregation of TDP-43 protein.[7, 13]
TDP-43 associated cytotoxicity has been observed in cell culture.[14] However, it still
remains elusive that neurodegenerative diseases characterized by TDP-43 proteinopathy
result from a loss of TDP-43 normal function, or a toxic gain of its abnormal function or
both. Therefore, the specific aim of this study is to examine the function of human TDP43 (hTDP-43) protein in vivo using Drosophila as an animal model. We constructed and
characterized transgenic flies overexpressed human TDP-43 (hTDP-43) in the various
neuron populations in flies, while we also generated flies in which the expression of its
endogenous fly homologue, Tar DNA binding protein homolog (TBPH), was knocked
down by RNA interference (RNAi) using two fly lines. When overexpressed in both
photoreceptor neurons and motor neurons, hTDP-43 alone is sufficient to cause neuronal
loss in an age-dependent manner and protein aggregation detectable in neurons and axons.
Such transgenic flies recapitulate a number of critical pathological and clinical features of
ALS patients and provide a useful animal model for TDP-43 proteinopathy, whereas
knocking down endogenous TBPH in flies only demonstrates some critical pathological
and clinical features of ALS patients. Therefore, proper regulation of TDP-43 expression
is important for the normal development, maintenance and proper function of the nervous
system. Furthermore, our transgenic flies will be useful for screening genetic modifiers
and chemical compounds that reduce or block the neurodegeneration in TDP-43
proteinopathy patients.
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Materials and Methods
Constructs and fly stains
The protein sequence alignment of human TDP-43 and its Drosophila homolog TBPH
with predicted alternative splicing isoforms TBPHl and TBPHs (long and short
respectively) was shown (Figure 1A). The open reading frame encoding human TDP-43
was cloned to express a protein fused at its carboxyl terminus to RFP with a hemaglutinin
(HA) tag using the pUAST vector, which contains UAS sequence in the promoter region
(Figure 1C). The expression of the control RFP or hTDP-43-RFP was tested in S2 cell
lines by transient transfection. The control RFP and hTDP-43-RFP constructs were then
sent for customer fly injection service (Rainbow Transgenic Flies, Inc) for generating
transgenic flies in W1118 genetic background. Individual flies with red eyes were
balanced to make stable lines. TBPH-RNAi flies were obtained from Vienna Drosophila
Research Stock Center (VDRC, stock number 38377 and 38379). Different Gal4 lines
were obtained from Bloomington Stock Center and used to drive hTDP-43 or RFP
expression in eye (GMR-Gal4) or subtypes of motor neurons (OK371-Gal4 and RN2Gal4). The expression of hTDP-43 was examined by RT-PCR, western blotting following
standard protocols.
Histology
Fly heads were dissected at day 0, 10, 20, 30 after eclosion and fixed for 48 hours in
2% glutaraldehyde at 4oC. After washed with PBS and post-fixed in 1% OsO4 buffer for
2hrs, samples were washed in distilled water and dehydrated through a series of graded
alcohols. Heads were put through a transition solvent of propylene oxide and a 1:1
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solution of propylene oxide/resin for 30 minutes each, and infiltrated with pure resin
overnight in a 60oC oven. Samples were subjected to semi-thin sectioning. Oriented
vertically, heads were cut for semi-thin sections (1.0μm each for ~20μm per head) and
stained with 0.5% toluidene blue. Light microscope images were obtained with an
Olympus BX60 microscope.
Staining and microscopy
Fly brains and larval ventral nerve cords were dissected and fixed in 4%
paraformaldehyde. Hoechst was added into the mounting buffer to label nucleus. For
TUNEL staining, samples were fixed in 4% formaldehyde and permeabilized in 50 mM
Sodium Citrate. After blocking with BSA, samples were treated with TUNEL (In Situ
Cell Death Detection Kit) with green fluorescence (Roche). Boutons at the same
anatomic position in dorsal side were examined in the 3rd instar larvae. The nerves and
boutons in live animals were revealed by the expression of mGFP. Fluorescent
microscopy was performed using a Leica DM5500B or a confocal microscope (Zeiss
LSM 510).
Fly motility assays
The larval motility was measured using the late 3rd instar larvae expressed RFP
control or hTDP-43 under the driver of OK371-Gal4, with the number of peristaltic
waves in 2 minutes counted and compared between groups, as previous described.[15]
The adult motility was determined using the climbing assay under the driver of RN2GAL4. Thirty-five virgin female flies were collected and aged in groups, and climbing
ability was examined every 5 days. Flies were transferred into an empty vial and tapped
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to the bottom. After 18s, the number of flies climbing above a 5-cm line was counted,
and the same procedure was repeated 10 times in each test.[16]
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Results
Generation of hTDP-43 overexpressing and TBPH underexpressing flies. To study
hTDP-43 proteinopathy in vivo, we utilized Drosophila, a powerful genetic model widely
used to study neurodegeneration.[17, 18] We generated transgenic flies expressed either
monomeric red fluorescent protein (RFP) as a control or hTDP-43 fused with RFP in the
different neurons using a well-established UAS/Gal4 system (Figure 1C).[19] Both RTPCR and western analysis confirmed the hTDP-43 expression (Figure 1D). RFP signals
could be detected in the eye discs of hTDP-43-RFP larvae when a GMR-Gal4 driver was
used (Figure 1E). In addition, we generated flies in which the expression of its
endogenous fly homologue TBPH was knocked down by RNAi using two fly lines
(number 38377 and 38379). With TBPH-specific primers, we demonstrated that TBPH
was significantly reduced in TBPH-RNAi expressing flies as compared to the control
flies (Figure 1B).
Overexpression of hTDP-43 but not underexpression of TBPH in Drosophila eyes
led to progressive retinal degeneration. To examine effects of hTDP-43 overexpression
on fly eye neurons, we used the GMR-Gal4 driver to express hTDP-43 specifically in
Drosophila eyes beginning at the 3rd instar larval stage. Control flies expressed RFP
showed normal eye morphology (Figure 2A), whereas hTDP43 expressing flies began to
show obvious ommatidia loss at eclosion (Figure 2B). According to the severity of their
eye defects we classified flies into 4 categories: level 1, less than 25% ommatidia loss;
level 2, 25% to 50% ommatidia loss; level 3, 50% to 75% ommatidia loss with small
regions of necrosis (appearing as black dots); level 4, more than 75% ommatidia loss
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with massive regions of necrosis (Figure 2 B1-4). Although there were significant
variations in the eye defects among individual flies with the same age, hTDP-43
overexpressing flies showed progressive degeneration with aging (Figure 2C). In contrast,
we also investigated effect of reduced endogenous fly TBPH on eye morphology using
TBPH-RNAi flies. No obvious eye defect was found in these TBPH RNAi flies,
consistent with the TBPH knock-out flies recently reported by Feiguin, et al.[15]
We further examined eye structures of hTDP-43 transgenic flies in frontal sections of
the retinae following toluidine blue staining. The RFP expressing control flies showed
intact rhabdomere structure throughout their life span (Figure 2D), while the ommatidia
organization was completely lost in hTDP-43 transgenic flies, with large vacuoles
frequently detected in fly eyes (arrow in Figure 2E), although their corn cells remained
largely normal.
Overexpression of hTDP-43 in motor neurons caused aggregate formation in cell
bodies and axons. TDP-43-positive pathology has been identified in the human motor
neuron diseases, like ALS. To investigate potential roles of TDP-43 in motor neurons in
vivo, we examined phenotypes of hTDP-43 expressing flies in motor neurons using a
motor neuron-specific driver, OK371-Gal4. We mainly focused on the expression and
localization of hTDP-43 in the transgenic flies by the RFP tag using fluorescent
microscopy. In most motor neurons, hTDP-43 was predominantly localized in the
nucleus. However, hTDP-43 was also detected in the cytoplasm, often accompanied by
signs of neuronal death, including condensation and/or fragmentation of nuclei (as
marked by the arrowhead in Figure 3A). TDP-43 protein aggregation was observed in
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both cell bodies (arrow in Figure 3A) and axons (arrows in Figure 3B and 3C) in these
motor neurons.
Motor neurons in flies overexpressed hTDP-43 showed axon swelling, neuronal
death and functional deficit, distinct from those in flies underexpressed TBPH. We
systematically examined such transgenic flies, focusing on the morphology of motor
neurons in the ventral nerve cord of late 3rd instar larvae. Motor neurons in the control
RFP expressing flies were well organized into clusters in different segments as visualized
by the expression of RFP and mGFP (Figure 4, panels A1 and A2). In contrast, motor
neurons in hTDP-43 expressing larvae were not organized in their normal cluster pattern,
with a wide range of neuronal loss. Such motor neuron loss was more severe in posterior
abdominal segments as compared with the anterior segments. As shown in Figure 4B,
two neurons expressed hTDP-43-RFP marked by arrowheads have lost mGFP signals,
and another neuron marked by the arrow showed swelling of cell body, suggesting
neuronal death. Such neuronal death was also confirmed by TUNEL staining. There was
no TUNEL-positive neuron in the control group; whereas TUNEL-positive cells were
detected in the motor neurons expressed hTDP-43 (Figure 6). Strikingly, axon swelling
was frequently detected in hTDP-43 expressing motor neurons (Figure 3C), similar in
morphology to spheroid structures found in patients with ALS.[20, 21]
We further examined the axonal and synaptic morphology of hTDP-43 expressing
motor neurons, with the focus on neuromuscular junctions (NMJ). Nerves from motor
neurons project to their target muscle, branch a few times, and form a number of synapse
(named bouton) to innervate the muscle. hTDP-43 overexpressing motor neurons showed
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a significant decrease in the number of both big and small boutons, and in the axon
branch number (Figure 4D, Figure 5 panels A and B) as compared to flies in the control
group (Figure 4C, Figure 5 panels A and B). Consistently, the locomotive ability of
hTDP-43 flies was also significantly impaired at both larval and adult stages. The larval
motility was examined from late 3rd instar larvae, and the motility index was quantified as
the number of movement waves in a period of two minutes. As shown in Figure 5C, the
motility index of control flies was 86.1  3.1, whereas it reduced to 46.0  2.3 in hTDP43 expressing flies. Fly mobility defect was also shown as these flies could not hatch and
die at late pupa stage. To examine the fly motility at the adult stage, we used RN2-Gal4
to drive hTDP-43 expression in a small subset of motor neurons in the adult stage.[22]
Fly locomotive ability was examined at different ages using a climbing assay as
previously described.[16] As compared to the control flies, hTDP-43 expressing flies
showed a reduction in their motility that grew progressively worse with aging, with the
significant difference starting from day 10 after eclosion (Figure 5D).
We also examined the morphology and function of motor neurons of TBPH RNAi flies.
Neither axon swelling nor neuron loss was observed in TBPH-RNAi larvae. Nevertheless,
the bouton number and branch number were decreased in both TBPH-RNAi fly lines
(VDRC 38377 and 38379), as compared with the control files (Figure 5, panels A and B).
Additionally, the small bouton and branch number of TBPH RNAi flies was significantly
different from that of flies overexpressed hTDP-43 (Figure 4, panels D-F and Figure 5,
panels A and B). No movement defect was detected in TBPH RNAi larvae (Figure 5C).
However, adult flies of TBPH RNAi lines exhibited a reduction in their climbing ability
(Figure 5D).
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Discussion
TDP-43 is a novel neurodegenerative proteinopathy in a wide range of
neurodegenerative diseases, such as ALS.[12] TDP-43 proteinopathy is characterized by
the presence of TDP-43 positive neuronal and glial inclusions, progressive and agedependent neuronal loss, and consequently muscle atrophy and weakness. We
constructed and characterized transgenic flies overexpressed human TDP-43. Such
hTDP-43 transgenic flies demonstrated abnormal morphologic features such as the
aggregate formation in motor neurons and axons, and progressive and age-dependent
neuronal loss found in photoreceptor neurons (Figure 2) and motor neurons (Figure 3, 4,
and 6). These features are strikingly similar to the pathologic characteristics of human
TDP-43 proteinopathy, and functional defects in flies mimic those found in patients
affected by TDP-43 proteinopathy. It strongly suggested that simply increasing TDP-43
protein expression is sufficient to trigger progressive neuronal death. In contrast, TBPH
underexpressing flies only showed partial critical pathological features found in patients
with ALS. Therefore, proper regulation of TDP-43 expression is important for the normal
development, maintenance and proper function of the nervous system. Furthermore, our
transgenic flies will be useful for screening genetic modifiers and chemical compounds
that reduce or block the neurodegeneration in TDP-43 proteinopathy patients.
ALS is a progressive neurologic disorder affecting motor neurons that innervate
skeletal muscle involved in voluntary muscle movements and in respiration, usually
leading to age-dependent impairment of locomotive function and respiration, with
resultant devastating disability and death.[23] The neuropathology of ALS is
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characterized by progressive degeneration of motor neurons, with the presence of
chromatolytic neurons (“balloon-like cells”), axonal spheroids (swellings), and synapse
loss.[24, 25] Often, the reduction of synapses in anterior horns of the spinal cord in
patients with ALS is closely associated with the degree of neuronal loss.[26] In some
cases, axonopathy with giant axon swellings in the corticospinal tracts has been found,
suggesting possible distal axonal degeneration during the early stage of the disease.[25]
When clinical signs of muscle weakness and wasting are apparent, there has already been
a loss of as many as 80% of the anterior horn motor neurons.[23, 24, 27] In our
transgenic flies, hTDP-43 expressing motor neurons showed abnormal axon swelling and
synapse reduction, mimicking the spheroids/axon swellings found in ALS patients. In
addition, these flies exhibited age-dependent deficits in locomotion. Therefore,
overexpression of hTDP-43 is sufficient to induce the disease found in patients with ALS.
In both FTLD and ALS, there are significant variations in clinical manifestations and
neuropathologic features. In familial cases, this occurs even among patients from the
same family. The duration of these diseases can vary significantly, ranging from months
to decades. For example, the age of onset for sporadic ALS (sALS) varies significantly,
generally between 55–65 years with a median age of onset of 64 years and about 5% of
cases having onset before the age of 30.[27, 28] Muscle weakness and atrophy can occur
either symmetrically or asymmetrically. Likewise, our hTDP-43 transgenic flies
exhibited a range of variations in neuronal death, axonal changes and functional deficits,
recapitulating the phenotypic variation found in patients affected by TDP-43
proteinopathy. Such pathologic and clinical variations suggest the possible involvement
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of environmental and epigenetic factors as well as genetic modifiers in influencing
phenotypic manifestations and clinical outcomes of TDP-43 proteinopathy.
The formation of TDP-43 inclusions is a prominent histopathologic feature of TDP-43
proteinopathy. It remains controversial whether the protein aggregate is the cause or the
consequence of neuronal death, or whether it accelerates or prevents neurotoxicity. Axon
swelling and movement defects were found in all hTDP-43expressing flies, while only
about 10-20% of these flies showed cytoplasmic aggregates containing TDP-43 in the
cell bodies and axons of the motor neurons, although larvae with aggregates showed
more severe locomotive defects than their siblings without detectable aggregates. It is
possible that early neurotoxic species of TDP-43 protein exist in forms that are below
microscopic detection level. It seems to be a case with Abeta oligomers in Alzheimer
disease.[29] The formation of large TDP-43 protein aggregates detectable by fluorescent
microscopy represents the late-stage byproducts. Alternatively, as in Huntington disease
and other trinucleotide repeat disorders,[30] such large TDP-43 protein aggregates may
represent reactive or even protective responses by neurons. In any case, generation of
such transgenic flies provides a power system for us to address these issues using in vivo
imaging when more sophisticated imaging techniques are developed in future studies.
To investigate whether the pathogenic mechanism underlying TDP-43 proteinopathy is
a toxic gain of abnormal TDP-43 function or a loss of normal TDP-43 function or both,
we also examined the effects of down-regulation of fly TDP-43 homologue TBPH on the
formation and function of the nervous system. As a result of alternative splicing,
Drosophila TBPH gene is predicted to produce four RNA transcripts encoding two
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protein isoforms. The short protein isoform (TBPHs) shares 49.1% identity and 68.4%
similarity with hTDP-43; and the long protein isoform (TBPHl) has 31.1% identity and
42.7% similarity (Figure 1A). With TBPH-specific primers, the long isoform (TBPHl)
was detected as the predominant isoform in fly heads, and it was significantly reduced in
TBPH-RNAi expressing flies as compared to that in the control flies (Figure 1B). These
RNAi flies showed axonal and synapse losses as well as impaired locomotion in the adult
stage, suggesting that TBPH is required for the proper function and maintenance of the
nervous system in Drosophila, and implying that TDP-43 proteinopathy may be partially
due to a loss of normal TDP-43 function, although the precise biological function of fly
TBPH gene remains to be elucidated.
On the other hand, the knock-down TBPH model and the hTDP-43 transgenic flies
showed a number of distinct pathologic and functional features. First, in TBPH-RNAi
flies, no TDP-43 protein aggregate was detected. Second, some neuron populations, such
as photoreceptors, were not affected by decreased endogenous TBPH expression,
suggesting that endogenous expression of TBPH is low in the eye, and/or that TDP-43 is
not essential for eye development. Consistent with these results, there is a recent report
that a TBPH knock-out fly had normal eye morphology.[15] Overexpression of hTDP-43,
however, induced severe degeneration of photoreceptors, but left the external corn
structure largely unaffected, indicating the relative specificity of the effects of TDP-43.
Third, although the bouton number and axon branching of motor neurons were decreased
in both cases, hTDP-43 overexpressing flies had significantly fewer small boutons and
axon branches than TBPH knock-down flies. Fourth, the neuron loss and axon swelling
in hTDP-43 expressing larvae were not detected in TBPH knock-down larvae.
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Correlating with these morphologic findings, no locomotive defect was found at the
larval stage in TBPH RNAi flies, and they hatched normally when OK371-Gal4 was used
as the driver. Larvae of hTDP-43 transgenic flies, on the other hand, could not hatch and
exhibited severely impaired motility when TDP-43 expression was driven by the OK371Gal4 driver. Interestingly, adult flies of either TBPH RNAi or TDP-43 transgenic lines
exhibited progressive reduction in locomotion, suggesting that although the endogenous
TBPH in motor neurons may not be essential for motor neuron function at larval stage, it
becomes critical in adult flies. This may also provide an explanation for the observation
that TBPH knock-out flies began to die at the age of 3 days after hatching.[15] Thus, it is
most likely that both TDP-43 over- and under-expression contribute to neuronal
dysfunction in TDP-43 proteinopathy, while overexpression of hTDP-43 has much more
neuronal toxicity. This might be the reason that recent reported mutations in TDP-43 are
predominantly point mutations rather than functional loss mutation.[31]
Taken together, Flies having either excessive hTDP-43 or insufficient TBPH
expression could generate critical features of neurodegenerative diseases with human
TDP-43 proteinopathy. However, the disease severity between two models is quite
different. Overexpression of hTDP-43 in different subpopulations of neurons led to
neurodegeneration with remarkable features of human TDP-43 proteinopathy. It is likely
that these two models mimic the different stages of neurodegenerative diseases with
TDP-43 proteinopathy, and consequently useful animal models for our future
investigations into the role of TDP-43 protein in the pathogenesis of TDP-43
proteinopathy, and the underlying molecular mechanisms of neurodegenerative diseases.
This will enable us to carry out systematic studies on the in vivo pathologic effects of
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hTDP-43 and the resulting alterations in neuronal morphology and function in the distinct
neuronal populations affected in human TDP-43 proteinopathy. Application of this TDP43 proteinopathy fly models in the future may also allow us to establish the temporal
sequence and possible causal relationships between events occurring during
neurodegeneration in TDP-43 proteinopathies, such as axonal swelling, synapse loss, and
neuronal death. This will also facilitate future development of effective targeted therapy
for the TDP-43 proteinopathies.
Acknowledgement
I wish to express my gratitude to my supervisor, Charles Louis Mix Professor Jane Y
Wu, for her extraordinary guidance through this scientific journey, for her never-ending
encouragement and enthusiasm, and for providing the facilities for conducting the study.
I am grateful to my fellow, Professor Robert Paul Malchow, for his everlasting
encouragement, constant help, enthusiasm, and optimism. I am sincerely thankful to Dr
Yan Li for teaching me the experiential technology, for sharing her invaluable ideas and
time with me whenever I needed. I am grateful to Drs. E. Bigio, A. Kolodkin, L. Li, L.
Luo, M. Mesulam, T. Siddique for generously providing invaluable suggestions, reagents
and support and critical reading of the manuscript, to Joy Wu for kind help, and to A
Sibtain for technical assistance. I thank the members of Wu laboratory for helpful
discussion and critical reading of the manuscript. I thank NIH (GM070967 and
EY014576 to JYW) and Searle Foundation (to JYW) for grant support.
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Figure legends:
Figure 1. Generation of transgenic flies expressed hTDP-43. (A) The protein sequence
alignment of human TDP-43 and its Drosophila homolog TBPH with predicted
alternative splicing isoforms TBPHl and TBPHs (long and short respectively). (B) The
endogenous TBPH RNAs were detected by radioactive RT-PCR in adult fly heads. ActGal4 was used to drive gene expression in all cells: the vector control (lane 1), TBPHRNAi (VDRC lines 38377 and 38379, lanes 2 and 3 respectively). Another nuclear RNA
binding protein Prp31 was administrated as a loading control. (C) A diagram illustrating
the constructs used to generate transgenic flies expressed hTDP-43-RFP or control RFP
proteins under the UAS sequence. (D) The expression of hTDP-43 was detected in
transgenic flies by RT-PCR and Western Blotting. Panel D1 showed RT-PCR results
using specific primers to detect hTDP-43 expression in GMR-Gal4/UAS-hTDP-43 flies
with Drosophila Prp31 as a control. (E) RFP signals revealed hTDP-43 expression in the
eye discs of a GMR-Gal4/UAS-TDP43-RFP fly.
Figure 2. Overexpression of human TDP-43 in fly eyes led to progressive eye defects
in an age-dependent manner. Panels A and B showed the morphology of fly eyes, (A)
GMR-Gal4/UAS-RFP fly eye as control; (B) GMR-Gal4/UAS-hTDP-43-RFP flies
exhibited obvious ommatidia loss and necrosis formation in their eyes. Panels B1-B4
demonstrated representative images of hTDP-43 expressing flies with ommatidia loss at
different levels as described. (C) Quantification of the eye defects in hTDP-43 expressing
flies with aging, showing significant difference from that in the control RFP expressing
flies (p<0.0001 in CHI square test). The eye defects were classified into 4 levels: 1, less
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than 25% ommatidia loss; 2, 25% to 50% ommatidia loss; 3, 50% to 75% ommatidia loss
with small regions of necrosis (appearing as black dots); and 4, more than 75%
ommatidia loss with massive regions of necrosis. The percentages of flies with eye
defects at each level were shown in different colors (1: blue; 2: red; 3: yellow; and 4:
cyan). By day 6, a majority of flies expressed hTDP-43 showed moderate to severe eye
defects. (D and E) Retinal sections stained with toluidine blue showed intact ommatidial
and rhabdomere structure in GMR-Gal4/UAS-RFP control fly eye (D, enlarged in the
insert). The rhabdomere structures were disintegrated and ommatidia arrangement was
disrupted in GMR-Gal4/UAS-hTDP-43-RFP fly eyes (E). Arrow: a big vacuole in the
eye.
Figure 3. Overexpression of hTDP-43 in motor neurons resulted in the protein
aggregation in the cytoplasm and axons, together with axon swelling. (A and B)
Overexpression of hTDP-43-RFP resulted in protein aggregates detectable in the
cytoplasm (A, arrow) and axons of motor neurons (B). The expression of hTDP-43 was
targeted to motor neurons using a motor neuron-specific driver, OK371-Gal4. (C) All
hTDP-43 overexpressing flies exhibited axon swelling, which was absent in the control
flies. Red: hTDP-43-RFP; green: mGFP; blue: Hoechst staining.
Figure 4. Overexpression of hTDP-43 caused morphological defects of motor
neurons. (A) Motor neurons in the dorsal clusters were well organized in the ventral
nerve cord (VNC) in the control fly larvae. (B) hTDP-43 expressing motor neurons
showed a significant neuronal loss and morphological defects in dorsal clusters,
especially in the last 3 segments of VNC. The arrow marked a representative neuron
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showing neuronal swelling (with an enlarged mGFP area); and arrowheads marked
neurons with fragmented or condensed nuclei and their membrane GFP signals lost. (D)
Overexpression of hTDP-43 in motor neurons led to a significant reduction in the number
of axon branches and boutons, as compared with the control (C) with the quantification
data from abdominal segments 2-4 shown in Figure 5 panels A and B. Genotype of
control flies used in A-D: OK371-Gal4/UAS-mGFP/UAS-RFP. Experimental flies in AD: OK371-Gal4/UAS-mGFP/UAS-hTDP-43-RFP.
Figure 5. Both overexpression of hTDP-43 and underexpression of endogenous
TBPH led to a significant reduction in the number of axon branches and boutons of
motor neurons as compared to control flies (p<0.0001), and functional defects of
motor neurons. (A and B) The number of small boutons and axon branches was
significantly less in the larvae expressed hTDP-43 than that in the larvae expressed
TBPH-RNAi (*, p<0.05). More than 56 larvae were collected in each group from neural
muscle junctions at the same position in abdominal segments 2-4. (C) The larval motility
was impaired in fly motor neurons expressed hTDP-43 as compared to the control group,
but not TBPH knock-down flies. More than 30 larvae were tested in each group. ***,
p<0.0001. (D) Both hTDP-43 expressing flies and TBPH knock-down flies exhibited
progressive decrease with aging in the climbing ability at the adult stage, while the
control group remained stable. The difference between two groups became significant
beginning from day 15. Genotype of control flies used in A and B: OK371-Gal4/UASmGFP/UAS-RFP; C: OK371-Gal4/UAS-RFP; D: RN2-Gal4/UAS-RFP. Experimental
flies in A and B: OK371-Gal4/UAS-mGFP/UAS-hTDP-43-RFP; C: OK371-Gal4/UAShTDP-43-RFP; D: RN2-Gal4/UAS-hTDP-43-RFP.
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Figure 6. TUNEL staining. Apoptosis was revealed in hTDP-43 expressing motor
neurons (panel B2). Such TUNEL positive cell was not detectable in the control flies
expressed RFP (panel A). OK371-Gal4 was used to drive hTDP-43-RFP or RFP
expression in motor neurons. Scale bar: 20m.
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Figure 1
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Figure 2
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Figure 3
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Figure 4
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Figure 5
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Figure 6
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