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. 2009 Feb 17;17(4):607–613. doi: 10.1038/mt.2009.3

Mimicking Aspects of Frontotemporal Lobar Degeneration and Lou Gehrig's Disease in Rats via TDP-43 Overexpression

Jason B Tatom 1, David B Wang 1, Robert D Dayton 1, Omar Skalli 2, Michael L Hutton 3, Dennis W Dickson 4,5, Ronald L Klein 1,6
PMCID: PMC2835101  PMID: 19223871

Abstract

Since the discovery of neuropathological lesions made of TDP-43 and ubiquitin proteins in cases of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS), there is a burst of effort on finding related familial mutations and developing animal models. We used an adeno-associated virus (AAV) vector for human TDP-43 expression targeted to the substantia nigra (SN) of rats. Though TDP-43 was expressed mainly in neuronal nuclei as expected, it was also expressed in the cytoplasm, and dotted along the plasma membrane of neurons. Cytoplasmic staining was both diffuse and granular, indicative of preinclusion lesions, over 4 weeks. Ubiquitin deposited in the cytoplasm, specifically in the TDP-43 group, and staining for microglia was increased dose-dependently by 1–2 logs in the TDP-43 group, while neurons were selectively obliterated. Neuronal death induced by TDP-43 was pyknotic and apoptotic. TDP-43 gene transfer caused loss of dopaminergic neurons in the SN and their axons in the striatum. Behavioral motor dysfunction resulted after TDP-43 gene transfer that was vector dose-dependent and progressive over time. The cytoplasmic expression, ubiquitination, and neurodegeneration mimicked features of the TDP-43 diseases, and the gliosis, apoptosis, and motor impairment may also be relevant to TDP-43 disease forms involving nigrostriatal degeneration.

Introduction

Nosology of frontotemporal lobar degeneration (FTLD) is a dichotomy of proteinopathies. In one branch, there are microtubule-associated protein tau-positive neurofibrillary tangles that comprise FTLD-tau; in the other branch, there are ubiquitin and TDP- 43-positive inclusions which encompass FTLD-ubiquitin.1 In addition, delving deeper into the nosology, there are FTLD subjects with ubiquitinated lesions negative for tau or TDP-43, and because the tau neurofibrillary tangle lesions are also ubiquitinated, logical future nosology will likely be FTLD-tau, FTLD-TDP-43, and FTLD-protein yet to be identified.2 The clinical symptomologies are shared, for example, changes in behavior and personality and the potential for language and motor deficits including parkinsonism, along with early death.3 However, the neuropathologies are distinct and could require distinct palliatives specific for each type of lesion and sequelae.

TDP-43, the transactive response DNA-binding protein of 43 kd, is a nuclear protein that was first described by Ou et al.4 A number of studies have attributed functions to TDP-43 including modulation of HIV-1 gene expression, exon skipping in cystic fibrosis, transcriptional regulation, exon splicing, and mRNA stability (reviewed in refs. 5 and 6). While roughly 40% of FTLD samples contain tau neurofibrillary pathology, the majority have tau-negative, ubiquitin-positive inclusions.7 Straight filaments in spindle-shaped intranuclear inclusions were viewed in FTLD-ubiquitin cases, although the identity of the filamentous protein was not yet known.7 Mutations in the gene for progranulin were found in familial FTLD-ubiquitin cases,8 and Neumann et al.9 showed that the ubiquitinated lesions in such cases of FTLD-ubiquitin, and in amyotrophic lateral sclerosis (ALS), contained TDP-43 (ref. 9). The pathogenesis of FTLD-U and ALS is thought to involve TDP-43 translocation from the nucleus and aggregation into neuronal cytoplasmic inclusions, an active topic of current research effort considering the emergence of TDP-43 as a hallmark neuropathological marker along with amyloid, tau, synuclein, and polyglutamine. The vast majority of ALS cases harbor TDP-43 neuropathology, except for the small fraction (2%) with superoxide dismutase mutations.10

In an effort to study TDP-43-related diseases, such as FTLD-U and ALS, we generated an animal model with TDP-43 neuropathology. The adeno-associated virus (AAV) somatic cell gene transfer system has been used previously to successfully mimic aspects of Parkinson's disease11 and FTLD-tau,12,13 and we utilized AAV9 in this study for TDP-43 overexpression. The vector-based system affords some unique advantages for modeling neurodegenerative disease relative to germ-line transgenic mice, such as application to adult animals after normal development, avoidance of developmental lethality, and the potential for a shorter and more cost-effective disease progression to study disease mechanisms and therapeutics. Our hypothesis was whether elevated levels of TDP-43 is toxic to brain neurons in vivo in a specific manner relative to glia. Another goal was to test whether the exogenously applied human TDP-43 could precipitate endogenous rat ubiquitin into cytoplasmic neuropathological lesions. For our neurodegeneration model, we chose to inject the substantia nigra (SN), which contains a small and easily quantifiable population of dopamine neurons, and which can also be evaluated with a functional behavioral assay, on the basis of amphetamine-stimulated rotational behavior. Rationale for transducing the nigrostriatal pathway with TDP-43 stems from the nigrostriatal TDP-43 proteinopathy, nigral cell loss, or parkinsonism in cases of FTLD-U,14 ALS,15,16 and other TDP-43 diseases such as Perry syndrome,17 and the parkinsonism–dementia complex and ALS of Guam.18 We studied two doses of AAV9 vectors for either human wild-type TDP-43 or control green-fluorescent protein (GFP) in order to determine a specific neurotoxic effect of TDP-43 on dopamine neurons, and also to evaluate ubiquitination, gliosis, and apoptosis in the induced neurodegenerative disease state.

Results

TDP-43 gene transfer

The initial test of the human wild-type TDP-43 plasmid was transient transfection of 293-T human embryonic kidney cells. TDP-43 expression was clearly elevated on western blots (not shown) or by immunofluorescence, 2 days after transfection of the plasmid (4 µg per 2 × 106 cells on a 6 cm dish), compared to the same amount of the GFP plasmid (Figure 1a,b). The TDP-43 mainly colocalized with nuclei marked with DAPI (not shown). Next, we injected the AAV9 vectors for either TDP-43 or GFP into the SN of rats. The AAV9 TDP-43 vector prep was high titer, and in initial tests with undiluted vector (dose of 5.9 × 1010 vg in 1 µl) at a 4-week interval, there was complete obliteration of the nigrostriatal pathway. For expression analysis, we, therefore, chose a 2-week time point and a lower dose to avoid such loss of transduced cells. With 3 rats per vector group equally dosed at 1 × 1010 vg, we estimated a threefold increase in TDP-43 levels in the TDP-43 group relative to their GFP counterparts (Figure 1c). The control GFP vector group appeared to have similar TDP-43 levels as nontransduced or vehicle-injected tissue, using an antibody for TDP-43 that recognizes both rat and human TDP-43, and normalizing to the housekeeping band, glyceraldehyde-3-phosphate dehydrogenase (Figure 1c). This small degree of upregulation indicated high levels of endogenous TDP-43. Further upregulation of transgene expression with AAV9 with increasing time and dose is generally expected, but not in the case of a toxic gene product. The vector-derived TDP-43 expression was well targeted to the SN, as demonstrated by the human specific TDP-43 antibody (Figure 1d,e). To evaluate the transduction pattern of the vector, we used the control AAV9 GFP and tracked colocalization with cellular markers. As expected,13,19 there was apparently 100% overlap with the neuronal marker NeuN, but no overlap with the microglial marker CD11b, or the astroglial marker glial fibrillary acidic protein (GFAP; Supplementary Figure S1). In viewing the fluorescently labeled sections with the fluorescent counterstain 4′-6-diamidino-2-phenylindole (DAPI), we observed a striking spike in cell densities in the areas of TDP-43, but not GFP vector injections (Supplementary Figure S2).

Figure 1.

Figure 1

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Human wild-type TDP-43 overexpression. (a) TDP-43 immunofluorescence in vitro. Transfection of 293-T cells with the TDP-43 plasmid produced a mainly nuclear-staining pattern. (b) Control DNA transfection (blank). (c) Western blot of TDP-43 overexpression in the substantia nigra (SN) at 2 weeks postinjection. The TDP-43 adeno-associated virus (AAV) consistently upregulated the 43 kd band relative to green-fluorescent protein (GFP) AAV9 control vector, uninjected (U) or vehicle (V)-injected tissues. This antibody recognizes both rat and human TDP-43. There was a threefold increase in the expression in the TDP-43-treated animals compared to GFP controls after normalization to the band for the housekeeping gene product glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 38 kd band) from the same blot. Three subjects for either vector group are shown, which all received an equivalent dose of 1 × 1010 vector genomes. (d) Rat SN, 2 weeks after a TDP-43 AAV9 injection, stained with an antibody specific for human TDP-43. The staining pattern was mainly nuclear. (e) Control vector (blank). SNC, SN pars compacta; SNR, SN pars reticulata. On the order of 1% of the TDP-43-positive nuclei were associated with cytoplasmic TDP-43 labeling, examples shown in Figure 2. a,b, bar = 84 µm; d,e, bar = 134 µm.

TDP-43 and ubiquitin pathology

A small fraction of the TDP-43 transduced cells displayed cytoplasmic TDP-43, on the order of 1%, examples in Figure 2a–c. The staining spread through the cytoplasm diffusely (Figure 2c) and in some cells, appeared granular (Figure 2a,b), which we interpreted to be prefilamentous rather than mature inclusions. The staining method in Figure 2a–d used the nonspecies-specific antibody, and yielded typical nuclear staining (Figure 2d), although pathological, cytoplasmic localization was associated only with TDP-43 AAV injections. Counterstaining for hematoxylin and eosin revealed pyknosis, or abiotrophic type neuronal death after TDP-43 gene transfer, while cells on the control side remained viable (Figure 2e,f). Remarkably, ubiquitin deposited into neuronal cytoplasm in the areas of the pyknotic cells (Figure 2g,h). Cytoplasmic ubiquitin labeling, though not found in controls, was found in the TDP-43 samples, however. This finding validated that TDP-43 vector gene transfer could precipitate endogenous rat ubiquitin and model a specific aspect of FTLD–ubiquitin pathology, which encompasses ubiquitinated neuronal cytoplasmic inclusions.20 Confocal micrographs colocalized the human TDP-43 expressed from the vector with the neuronal marker, β III tubulin, and showed expression in nuclei as well as dotted along the plasma membrane of transduced neurons (Figure 2i–k).

Figure 2.

Figure 2

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TDP-43 pathology. (a–c) TDP-43 labeling was found in the cytoplasm of cells after TDP-43 AAV9 injections. The cytoplasmic staining (arrows in a–c) was spread diffusely and, in some cases, was granular as in b. (d) Only nuclear staining was found in control vector tissues using the nonspecies-specific antibody as in a–d. Hematoxylin and eosin staining of a (e) control vector or (f) TDP-43 vector injection site. The cells on the control side are viable, while neurons on TDP-43 side show pyknotic- or abiotrophic-type neuronal death (arrows in e,f). Ubiquitin labeling of (g) control or (h) TDP-43 injection sites. Ubiquitin deposited into neuronal cytoplasm in the areas of the pyknotic cells only in TDP-43 cases, two cells shown. (i–k) Confocal double immunofluorescence for human-specific TDP-43 (red) and a neuronal perikaryal marker, β III tubulin (green). TDP-43 outlined the plasma membrane in transduced neurons in a patchy manner. a–h, interval 3 weeks, dose for either AAV9 TDP-43 or control vector was matched at 3 × 1010 vector genomes. i–k, 2 weeks, 1 × 1010 vector genomes TDP-43 vector. a–h, bar = 3 µm; i–k, bar = 8.5 µm.

Gliosis

The TDP-43 gene transfer caused striking gliosis both for microglia (Figure 3, Supplementary Figures S3 and S4) and astrocytes (Figure 3). We have previously observed a slight astrogliosis associated with robust GFP expression,19 and slight increases in microglial and astroglial staining with GFP relative to uninjected tissue in this study, although the TDP-43 effect was exponentially greater, and consistent to the observations with DAPI, which showed increased cell densities (Supplementary Figure S2). Matching images for the GFP and TDP-43 vectors in Figure 3 demonstrate elevated numbers in glia as well as enlarged glia, specifically after TDP-43 gene transfer. To track the TDP-43-induced gliosis, we viewed a 1-, 3-, 7-, 14-, and 28-day time course of microglial staining after AAV9 GFP or AAV9 TDP-43 injections (Supplementary Figure S3). The autofluorescence from the needle scars generally attenuated by 28 days, with little indication of any gliosis away from the needle scar in the GFP vector group. However, beginning at 7 days, distinct gliosis, away from the needle track developed in the TDP-43 group, was increased further at both 14 and 28 days (Supplementary Figure S3h–j). The widespread and prodigious microgliosis caused by TDP-43 gene transfer at 4 weeks appeared to be dose dependent (Supplementary Figure S4). We quantified the microgliosis at 4 weeks by an optical density method. Each section in the analysis came from unilateral treatment groups, and, for each section, the density was normalized to the uninjected side with the same imaging conditions; therefore, every measurement was appropriately controlled, with five evenly spaced sections through the SN per subject. The ratios for the injected/uninjected side were averaged as follows: GFP, dose 3.0 × 1010 vg, 1.40 ± 0.22, N = 5; TDP, dose 1.0 × 1010 vg, 20.55 ± 9.07, N = 4; and TDP, dose 3.0 × 1010 vg, 110.6 ± 27.16, N = 6. There was a 40% elevation in the GFP group relative to the uninjected side, and a 20- to 110-fold induction by TDP-43 depending on the dose. We compared high-dose TDP-43 vector to high-dose GFP vector and low-dose TDP-43 vector. Furthermore, the ANOVA/Bonferroni analyses revealed that high-dose TDP-43 was different from high-dose GFP (P < 0.001) and that there was a significant dose difference between the two TDP-43 groups (P < 0.01). The gliosis at 1–4 weeks after TDP-43 vector injections matched well with the onset of transgene expression from single-stranded AAV vectors,13 although the TDP-43-induced cell loss could also have contributed to the observed gliosis.

Figure 3.

Figure 3

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Micro- and astrogliosis induced by TDP-43. (a–d) Microglial labeling with the antibody IBA1 in (a,c) control vector or (b,d) TDP-43 vector tissues. There is a massive induction in the number of (a,b) stained microglia and also an (c,d) increase in their size. (e,f) Labeling of glial fibrillary acidic protein for astroglia in control (e) vector or (f) TDP-43 vector tissues. There are more and larger astroglia after TDP-43 gene transfer. Interval of 3 weeks and equal vector doses of 3 × 1010 vector genomes. a,b, bar = 14 µm; c–f, bar = 7 µm.

Apoptotic nuclei

End labeling of exposed 3′-OH groups on nuclear DNA revealed many apoptotic nuclei, with the high-dose TDP-43 vector at 2 weeks, but not in controls (Figure 4). Of note, there was an indication of stages of apoptosis, ranging from examples of lightly stained large nuclei to intensely stained condensed nuclei. There was faint evidence for end labeling in low-dose TDP-43 subjects at 2 weeks, with fewer cells than with the high dose, and with light labeling spread over large nuclei (not shown) rather than in condensed nuclei as in Figure 4.

Figure 4.

Figure 4

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Labeling for apoptotic nuclei. End labeling with biotinylated nucleotides was visualized with a diaminobenzidine chromagen with a methylene green counterstain. End labeling occurred after (a) TDP-43 injections, but not after (b) control injections (c) higher magnification of end labeling as in a. Interval of 2 weeks and equal vector doses of 3 × 1010 vector genomes. a,b, bar = 34 µm; c, bar = 21 µm.

Dopaminergic nigrostriatal system

High-dose TDP-43 vector erased tyrosine hydroxylase (TH) immunoreactivity in the SN at 4 weeks. The loss of neurons in the SN was confirmed with the neuronal marker NeuN (Supplementary Figure S5). Despite the large increase in glial cell densities after TDP-43 injections (Figure 3, Supplementary Figures S3 and S4), the neurons in the SN were clearly obliterated.

We attempted to titrate the dopaminergic neuronal loss with two diluted vector doses along with equal-dose comparisons to GFP and were able to observe reductions in TH immunoreactivity in the SN from partial to more complete lesions (Figure 5). TDP-43 was compared to GFP at two vector doses to quantify its specific, dose-dependent neurotoxicity. Both the 1 and 3 × 1010 vg doses of AAV9 GFP vector preserved the full complement of TH immunoreactive cells in the SN relative to untreated samples (Table 1). The four vector groups in Table 1 were compared by ANOVA that showed differences overall, F(3, 27) = 40.89, P < 0.0001, and in Bonferroni posttests, which showed less cells in the low-dose TDP-43 group relative to low-dose GFP (P < 0.001), less cells in the high-dose TDP-43 group relative to the high-dose GFP (P < 0.001), and less cells in the high-dose TDP-43 group relative to the low-dose TDP-43 (P < 0.01). The dopamine neuron population of the rat SN demonstrated TDP-43-induced neurodegeneration in a dose-dependent manner, specifically in relation to dose-matched controls. In perfect agreement with the results in the SN, density of TH-immunoreactive nigrostriatal axons in the striatum was affected by TDP-43 gene transfer (Figure 6). Using the contralateral uninjected side as an internal control, the unilateral GFP gene transfer did not alter TH fiber density. In contrast, the TDP-43 lowered fiber density, and in a dose-dependent manner. When the four vector groups in Table 1 were compared for striatal TH staining, there was an overall effect, F(3, 27) = 41.07, P < 0.0001, and in the Bonferroni posttests, lower density in the low-dose TDP-43 group relative to the low-dose GFP, lower density in the high-dose TDP-43 group relative to the high-dose GFP, and lower density in the high-dose TDP-43 group relative to the low-dose TDP-43 (P < 0.001 for each).

Figure 5.

Figure 5

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TDP-43 gene transfer reduces tyrosine hydroxylase (TH) immunoreactivity in the substantia nigra (SN). (a) TH labeling in the SN pars compacta in uninjected tissue. (b) 4 weeks after a dose of 3 × 1010 vector genomes (vg) of the green-fluorescent protein (GFP) AAV9 vector, there was no change in TH staining. Section in a is from the contralateral side of b. (c,d) Contralateral, uninjected, and TDP-43 AAV9-transduced tissue, injected at a dose of 1 × 1010 vg. (e,f) Contralateral, uninjected, and TDP-43 AAV9-transduced tissue, injected at a dose of 3 × 1010 vg. There was partial and more complete lesioning of the SN with TDP-43 gene transfer at 4 weeks, depending on the dose. a–f, bar = 145 µm.

Table 1.

TDP-43 gene transfer causes dopaminergic neurodegeneration

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Figure 6.

Figure 6

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TDP-43 gene transfer reduces tyrosine hydroxylase (TH) immunoreactivity in the striatum (4 weeks). For each subject, the right side is the uninjected side and the left side received either green-fluorescent protein (GFP) or TDP-43 vector at two doses (low dose 1 × 1010 vector genomes; high dose 3 × 1010 vector genomes). While GFP vector injections did not produce side-to-side differences in the density of TH axons in the (a,b) striatum, the TDP-43 injections appeared to reduce fiber density relative to the contralateral side, and in a (c,d) dose-dependent manner. a–d, bar = 610 µm.

The rat nigrostriatal dopamine system provides a functional index as long as large losses in dopamine and its markers occur.12 Rats were tested for amphetamine-stimulated rotations to assess whether the TDP-43-induced cell loss was behaviorally significant. The TDP-43 vector gene transfer drove circling toward the injected side, consistent with a loss of dopamine, in the high-dose group at 4 weeks (more ipsilateral turns compared to contralateral turns, P < 0.05, t test), but no directional bias was observed at the earlier 2-week interval or in the low-dose TDP-43 or GFP vector groups (Figure 7). A behavioral phenotype occurred which was progressive between 2 and 4 weeks, in only the high-dose TDP-43 group, which underscored the requirement of large lesions for amphetamine-stimulated rotations.

Figure 7.

Figure 7

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Progressive development of amphetamine-stimulated rotational behavior after high-dose TDP-43 gene transfer. (a) Green-fluorescent protein (GFP) AAV9 low-dose (1 × 1010 vector genomes) group, tested at 2 and 4 weeks. (b) GFP AAV9 high dose (3 × 1010 vector genomes). (c) TDP-43 AAV9 low dose as in a. (d) TDP-43 AAV9 high dose as in b. There was a significant ipsilateral turning bias only in the high-dose TDP-43 group, and at 4, but not 2 weeks. *P < 0.05, t test.

Discussion

The rat/AAV system was successful in reproducing some key aspects of the TDP-43 diseases, FTLD-ubiquitin, and ALS, particularly those involving signs of nigrostriatal degeneration.14,15,16,17,18 Human wild-type TDP-43 was expressed in both the nucleus and the cytoplasm of rat midbrain neurons, causing a ubiquitination response in the host rat, thus validating the model for studying the TDP-43/ubiquitin cytoplasmic lesions of FTLD-ubiquitin and ALS. TDP-43, in a specific fashion relative to dose-matched control vector, induced gliosis, neurodegeneration, and progressive motor dysfunction. It remains to be proven whether the induced disease state via overexpression in rats truly mimics a TDP-43 disease as there are currently no known forms associated with elevated expression. While no animal system can recapitulate human diseases, the rat model provides a starting point to improve the mimicry, such as disease-related mutations, targeting the upper and lower motor neurons of the brain and spinal cord relevant to ALS, and coexpression of enzyme factors and inhibiting RNAs that modulate TDP-43, which will hopefully tell us about the TDP-43 disease mechanism and provide a therapeutic lead. Wild-type TDP-43 was expressed in the cytoplasm of midbrain neurons and the cytoplasmic pattern was diffuse and granular, consistent with preinclusions. We expect that more mature TDP-43 inclusions would form given time past 4 weeks. Truncating TDP-43's nuclear localization signal would increase cytoplasmic expression,21 as could potentially other pathogenic familial forms of TDP-43 (ref. 22), or blocking progranulin.23 We would be most interested in pursuing wild-type TDP-43 for its broad relevance to sporadic disease forms and to study modulating cofactors that induce its cytoplasmic localization. Alternatively, from a neuroprotective angle, we would be interested in coexpressing a protein factor that could potentially lead to TDP-43 degradation such as CHMP2B,24 or parkin to increase TDP-43 ubiquitination,25 or an antiapoptotic factor,26 considering the apoptotic nuclei observed in the high-dose TDP-43 group.

The microgliosis induced by TDP-43 was robust compared to observations during this study and previously with GFP, and tau gene transfer. We have observed modest upregulation of astroglia with AAV9 GFP gene transfer compared to vehicle injections,19 and consistently robust microgliosis induced by AAV9 tau compared to GFP,27 though the gliosis caused by TDP-43 in this study was even more pronounced, up to 100-fold. It remains difficult to attribute the observed gliosis to either TDP-43 transgene expression or the robust neurodegeneration induced by TDP-43. However, we successfully titrated the gliosis, the dopamine neuron loss, and the rotational behavior. The shared dose dependence suggests the readouts are causally linked.

The nigrostriatal dopamine system served as a quantifable index of neuronal cell loss and motor behavior function, while there is rationale for studying TDP-43 in this system.14,15,16,17,18 We wish to study TDP-43 pathophysiological processes relevant to FTLD-ubiquitin and Lou Gehrig's disease to pursue a new small molecule therapy. In light of TDP-43's functions outside of the nucleus, its localization to dendritic RNA granules, and activity-dependent restructuring,6,28 not to mention viewing membrane TDP-43 expression here, the overexpression might also be used for studying synaptic plasticity.

Materials and Methods

TDP-43 DNAs and AAVs. The human wild-type TDP-43 was purchased from Invitrogen (Carlsbad, CA). The TDP-43 cDNA was moved into an expression cassette flanked with AAV2 terminal repeats. The hybrid cytomegalovirus/chicken β-actin promoter was used to drive expression. We packaged either of the TDP-43 plasmids or a similar GFP plasmid into AAV9 as described.13 Human embryonic kidney 293-T cells were cotransfected with either TDP-43 or GFP transgene plasmid along with two packaging plasmids needed to make AAV9 by the calcium-phosphate method. The cell lysate was applied to a discontinuous gradient of iodixanol (OptiPrep; Greiner Bio-One, Longwood, FL) 3 days after transfection and centrifuged (350,000g for 1 hour). The AAV was then removed, diluted twofold with lactated Ringer's solution (Baxter, Deerfield, IL), and then washed and concentrated by Millipore (Billerica, MA) Biomax 100 Ultrafree-15 units. The final stocks were sterilized by Millipore Millex-GV syringe filters into low adhesion tubes (USA Scientific, Ocala, FL). Vectors were aliquoted and stored frozen. Encapsidated genome copies were titered by dot blot. The titer of the AAV9 TDP-43 was 5.9 × 1013 vector genomes (vg)/ml and the AAV9 GFP was 1.0 × 1013 vg/ml. Equal dose comparisons were made by normalizing titers with the diluent, lactated Ringer's solution.

Animals and stereotaxic injections. Male Sprague–Dawley rats, 3 months old from Harlan (Indianapolis, IN), were anesthetized with a cocktail of 3 ml xylazine (20 mg/ml; Butler, Columbus, OH), 3 ml ketamine (100 mg/ml; Fort Dodge Animal Health, Fort Dodge, IA), and 1 ml acepromazine (10 mg/ml; Boerhinger Ingelheim, St Joseph, MO) administered intramuscularly at a dose of 1 ml/kg. Viral stocks were injected through a 27-gauge cannula connected via 26-gauge internal diameter polyethylene tubing to a 10 µl Hamilton syringe mounted to a microinjection pump (CMA/Microdialysis, North Chelmsford, MA) at a rate of 0.2 µl/min with 3 µl of vector/diluent injected. The stereotaxic injection coordinates for the SN were 5.3 P, 2.1 L, and 7.6 V.29 The needle remained in place at the injection site for one additional minute before the cannula was removed slowly (over 2 minutes). The skin was sutured, and the animal was placed on a heating pad until it began to recover from the surgery, before being returned to its individual cage. All animal procedures followed protocols approved by our institutional Animal Care and Use Committee as well as the National Institutes of Health Guide for Care and Use of Laboratory Animals.

Western blots. Transfected 293-T cells were washed and collected in homogenization buffer [1% Nonidet-P40/0.5% sodium deoxycholate/0.1% sodium dodecyl sulfate/phosphate-buffered saline (PBS)] with protease inhibitors (Halt protease inhibitor cocktail kit; Pierce, Rockford, IL), then sonicated and centrifuged to make a soluble fraction. The SN was dissected and frozen on dry ice. The samples were put in homogenization buffer with protease inhibitors, and the soluble fraction was prepared by Dounce homogenization and centrifugation. Protein content was determined by Bradford assay reagents (Bio-Rad, Hercules, CA) and subjected to 12% sodium dodecyl sulfate/polyacrylamide gels (Bio-Rad), with the gel loaded with equal protein in each lane. The primary antibodies for the immunoblot were TDP-43 (1:1,000; Proteintech Group, Chicago, IL) or glyceraldehyde-3-phosphate dehydrogenase (1:1,000; Ambion, Austin, TX). The secondary antibody was from Santa Cruz (1:10,000; Santa Cruz, CA) and the ECL reagent from Amersham/GE Healthcare (Buckinghamshire, UK).

Immunostaining. Primary antibodies for immunostaining included the following: nonspecies-specific TDP-43 (1:5,000; Proteintech Group, Chicago, IL), human-specific TDP-43 antibody (1:1,000; Abnova, Taipei City, Taiwan); TH (1:2,000; Pel-Freez, Rogers, AR) for marking dopamine neurons, glial fibrillary acidic protein (1:400; Chemicon) for astroglia, IBA1 (1:500; Abcam, Cambridge, MA) or CDllb (1:400; Chemicon, Millipore, Billerica) for microglia, NeuN for neuronal nuclei (1:500; Chemicon), neuron-specific β III tubulin (1:500; Abcam), and antiubiquitin (1:2,000). Anesthetized animals were perfused with PBS, followed by cold 4% paraformaldehyde in PBS. The brain was removed and immersed in fixative overnight at 4 °C. Brains were equilibrated in a cryoprotectant solution of 30% sucrose/PBS at 4 °C. Coronal sections (50 µm thick) were cut on a sliding microtome with a freezing stage. Alternatively, some brains were paraffin embedded for thin sections (5 µm; Figures 2 and 3). Primary antibody incubations on free-floating sections were kept overnight at 4 °C on a shaking platform. For immunoperoxidase staining, endogenous peroxidase activity was quenched with 0.1% H2O2/PBS for 10 minutes. The sections were washed in PBS and incubated for 5 minutes in 0.3% Triton X-100/PBS, and washed before applying primary antibody. Biotinylated secondary antibodies for peroxidase staining were from DAKO Cytomation (1:2,000; Carpinteria, CA), incubated for 1 hour at room temperature. The sections were washed with PBS and labeled with horseradish peroxidase-conjugated Extravidin (1:2,000; Sigma, St Louis, MO) for 30 minutes at room temperature. The chromogen was diaminobenzidine (0.67 mg; Sigma) in 0.3% H202, 80 mmol/l sodium acetate buffer containing 8 mmol/l imidazole and 2% NiSO4. After mounting on slides, the sections were dehydrated in a series of alcohol and xylene and coverslipped with Eukitt (Electron Microscopy Sciences, Hatfield, PA). For immunofluorescence, sections were incubated in primary antibody overnight, washed and incubated with Cy3-conjugated secondary antibodies (1:300; Jackson ImmunoResearch, West Grove, PA) for 2 hour, followed by DAPI counterstaining (1 µg/ml), washing, and coverslipping with glycerol/gelatin (Sigma).

Apoptotic nuclei. TdT-FragEL DNA Fragmentation Detection Kit from Calbiochem (a brand of EMD, an affiliate of Merck, Damstadt, Germany) was used as per the instructions. The kit uses a combination of biotinylated nucleotides, horseradish peroxidase, and diaminobenzidine for visualization of exposed 3′-OH ends of DNA fragments generated during apoptosis, and includes methylene green counterstain.

Stereological estimates. The number of SN pars compacta neurons expressing TH immunoreactivity was estimated by unbiased stereology using the MicroBrightfield system. Eight sections evenly spaced throughout the SN pars compacta structure were analyzed for each probe. Optical dissectors were 50 × 50 × 16 µm cubes spaced in a systematic random manner 150 × 150 µm apart and offset 2 µm from the section surface. The fractionator sampling was optimized to yield about 150 counted cells per animal, for Gundersen error coefficients <0.10.13

Optical-density measurements for striatal TH analysis and microgliosis. Five sections evenly spaced through the striatum were processed for TH immunohistochemistry. The specific TH staining in the striatum was quantified using the Scion (Frederick, MD) imaging program. The striata were traced and then measured for optical density of staining (pixels). The ratio of the injected side relative to the contralateral uninjected side was calculated for each section. The value for each animal was an average from five sections.

A similar method was adopted for quantifying microglial staining. Immunofluorescent images for CD11b in the SN were captured under equal conditions for the injected side and contralateral uninjected side with a ×10 lens. Grayscaled images were analyzed with the Scion program, with specific staining density measured over the entire ×10 field under the same settings for the two sides. The ratio of injected/uninjected side for each section was averaged for five evenly spaced sections through the SN.

Rotational behavior. Animals were challenged with d-amphetamine (free base, 2 mg/kg in saline, intramuscular; Sigma). The amphetamine was injected 20 minutes before placing the animals in an automated rotometer system from San Diego Instruments (San Diego, CA) for 10 minutes. Pilot studies determined that locomotor activity peaks by 20 minutes after injection and that a 10-minute measurement is sufficient to determine whether a side-to-side rotational bias is present.

Statistics. Data are expressed as mean ± SEM. Statistical tests included ANOVAs and Bonferroni's multiple comparisons, or t tests as indicated.

Supplementary MaterialFigure S1. Co-localization with GFP (4 weeks, 3 × 1010 vector genomes). A) Merged image of GFP and the microglial marker CD11b in red; no overlap. B) Merged image of GFP and the astroglial marker glial fibrillary acidic protein in red; no overlap. C) Merged image of GFP and neuronal nuclei, NeuN, in red; every GFP positive cell was associated with a NeuN positive nucleus. D-F) Same section shown for GFP, NeuN, and merger. SNC, substantia nigra pars compacta; SNR, substantia nigra pars reticulata. A-C, bar = 134 μm. D-F, bar = 54 μm.Figure S2. TDP-43 gene transfer induces a noticeable increase in cell numbers, labeled by 4′-6-diamidino-2-phenylindole (DAPI; 4 weeks; equal vector doses of 3 × 1010 vector genomes). A, B) Uninjected tissue. C, D) GFP e×pression from AAV9 vector. E, F) DAPI from same section as C. G, H) TDP-43 vector, increased density of DAPI staining. SN, substantia nigra. A, C, E, G, bar = 268 μm. B, D, F, H, bar = 134 μm.Figure S3. Microglial staining with antibody CD11b in animals treated with a fixed dose of 1 × 1010 vector genomes of either AAV9 GFP (A–E) or AAV9 TDP-43 (F–J). Microglial staining with antibody CD11b in animals treated with a fi×ed dose of 1 × 1010 vector genomes of either AAV9 GFP (A-E) or AAV9 TDP-43 (F-J). TDP-43 induced microgliosis relative to GFP away from the needle track in the substantia nigra in H-J, with apparent increases in staining at intervals 3-7 days, 7-14 days, and 14-28 days, the longest time point studied. CD11b was labeled with a red fluorescent antibody. A-J, bar = 134 μm.Figure S4. Dose-dependent microgliosis induced by TDP-43 vector (4 weeks). The low dose was 1.0 × 1010 vector genomes and the high dose was 3.0 × 1010 vector genomes. At both doses, microglial staining was elevated in the TDP-43 groups (B, C), relative to either low (not shown) or high dose GFP (A), and there was elevated staining in the high dose TDP-43 group (C) relative to the low dose group (B). CD11b was labeled with a red fluorescent antibody. A-C, bar = 67 μm.Figure S5. TDP-43 obliterates neurons, stained with a red-fluorescent antibody for neuronal nuclei, NeuN (2 weeks, equal vector doses of 3 × 1010 vector genomes). TDP-43 obliterates neurons, stained with a red fluorescent antibody for neuronal nuclei, NeuN (2 weeks, equal vector doses of 3 × 1010 vector genomes). A) Uninjected substantia nigra (SN). B) GFP e×pression from AAV9 GFP. C) NeuN from the same section as B. D) TDP-43 AAV9 injected into the SN at this dose destroys the vast majority of neurons in the region. SNC, substantia nigra pars compacta; SNR, substantia nigra pars reticulata. A-D, bar = 134 μm.

Supplementary Material

Figure S1.

Co-localization with GFP (4 weeks, 3 × 1010 vector genomes). A) Merged image of GFP and the microglial marker CD11b in red; no overlap. B) Merged image of GFP and the astroglial marker glial fibrillary acidic protein in red; no overlap. C) Merged image of GFP and neuronal nuclei, NeuN, in red; every GFP positive cell was associated with a NeuN positive nucleus. D-F) Same section shown for GFP, NeuN, and merger. SNC, substantia nigra pars compacta; SNR, substantia nigra pars reticulata. A-C, bar = 134 μm. D-F, bar = 54 μm.

Click here for supplemental data (44.3KB, pdf)
Figure S2.

TDP-43 gene transfer induces a noticeable increase in cell numbers, labeled by 4′-6-diamidino-2-phenylindole (DAPI; 4 weeks; equal vector doses of 3 × 1010 vector genomes). A, B) Uninjected tissue. C, D) GFP e×pression from AAV9 vector. E, F) DAPI from same section as C. G, H) TDP-43 vector, increased density of DAPI staining. SN, substantia nigra. A, C, E, G, bar = 268 μm. B, D, F, H, bar = 134 μm.

Click here for supplemental data (38.9KB, pdf)
Figure S3.

Microglial staining with antibody CD11b in animals treated with a fixed dose of 1 × 1010 vector genomes of either AAV9 GFP (A–E) or AAV9 TDP-43 (F–J). Microglial staining with antibody CD11b in animals treated with a fi×ed dose of 1 × 1010 vector genomes of either AAV9 GFP (A-E) or AAV9 TDP-43 (F-J). TDP-43 induced microgliosis relative to GFP away from the needle track in the substantia nigra in H-J, with apparent increases in staining at intervals 3-7 days, 7-14 days, and 14-28 days, the longest time point studied. CD11b was labeled with a red fluorescent antibody. A-J, bar = 134 μm.

Click here for supplemental data (29.5KB, pdf)
Figure S4.

Dose-dependent microgliosis induced by TDP-43 vector (4 weeks). The low dose was 1.0 × 1010 vector genomes and the high dose was 3.0 × 1010 vector genomes. At both doses, microglial staining was elevated in the TDP-43 groups (B, C), relative to either low (not shown) or high dose GFP (A), and there was elevated staining in the high dose TDP-43 group (C) relative to the low dose group (B). CD11b was labeled with a red fluorescent antibody. A-C, bar = 67 μm.

Click here for supplemental data (24.8KB, pdf)
Figure S5.

TDP-43 obliterates neurons, stained with a red-fluorescent antibody for neuronal nuclei, NeuN (2 weeks, equal vector doses of 3 × 1010 vector genomes). TDP-43 obliterates neurons, stained with a red fluorescent antibody for neuronal nuclei, NeuN (2 weeks, equal vector doses of 3 × 1010 vector genomes). A) Uninjected substantia nigra (SN). B) GFP e×pression from AAV9 GFP. C) NeuN from the same section as B. D) TDP-43 AAV9 injected into the SN at this dose destroys the vast majority of neurons in the region. SNC, substantia nigra pars compacta; SNR, substantia nigra pars reticulata. A-D, bar = 134 μm.

Click here for supplemental data (21.2KB, pdf)

Acknowledgments

National Institute of Neurological Disorders and Stroke R01 NS048450 and the Society for Progressive Supranuclear Palsy supported the work.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1.

Co-localization with GFP (4 weeks, 3 × 1010 vector genomes). A) Merged image of GFP and the microglial marker CD11b in red; no overlap. B) Merged image of GFP and the astroglial marker glial fibrillary acidic protein in red; no overlap. C) Merged image of GFP and neuronal nuclei, NeuN, in red; every GFP positive cell was associated with a NeuN positive nucleus. D-F) Same section shown for GFP, NeuN, and merger. SNC, substantia nigra pars compacta; SNR, substantia nigra pars reticulata. A-C, bar = 134 μm. D-F, bar = 54 μm.

Click here for supplemental data (44.3KB, pdf)
Figure S2.

TDP-43 gene transfer induces a noticeable increase in cell numbers, labeled by 4′-6-diamidino-2-phenylindole (DAPI; 4 weeks; equal vector doses of 3 × 1010 vector genomes). A, B) Uninjected tissue. C, D) GFP e×pression from AAV9 vector. E, F) DAPI from same section as C. G, H) TDP-43 vector, increased density of DAPI staining. SN, substantia nigra. A, C, E, G, bar = 268 μm. B, D, F, H, bar = 134 μm.

Click here for supplemental data (38.9KB, pdf)
Figure S3.

Microglial staining with antibody CD11b in animals treated with a fixed dose of 1 × 1010 vector genomes of either AAV9 GFP (A–E) or AAV9 TDP-43 (F–J). Microglial staining with antibody CD11b in animals treated with a fi×ed dose of 1 × 1010 vector genomes of either AAV9 GFP (A-E) or AAV9 TDP-43 (F-J). TDP-43 induced microgliosis relative to GFP away from the needle track in the substantia nigra in H-J, with apparent increases in staining at intervals 3-7 days, 7-14 days, and 14-28 days, the longest time point studied. CD11b was labeled with a red fluorescent antibody. A-J, bar = 134 μm.

Click here for supplemental data (29.5KB, pdf)
Figure S4.

Dose-dependent microgliosis induced by TDP-43 vector (4 weeks). The low dose was 1.0 × 1010 vector genomes and the high dose was 3.0 × 1010 vector genomes. At both doses, microglial staining was elevated in the TDP-43 groups (B, C), relative to either low (not shown) or high dose GFP (A), and there was elevated staining in the high dose TDP-43 group (C) relative to the low dose group (B). CD11b was labeled with a red fluorescent antibody. A-C, bar = 67 μm.

Click here for supplemental data (24.8KB, pdf)
Figure S5.

TDP-43 obliterates neurons, stained with a red-fluorescent antibody for neuronal nuclei, NeuN (2 weeks, equal vector doses of 3 × 1010 vector genomes). TDP-43 obliterates neurons, stained with a red fluorescent antibody for neuronal nuclei, NeuN (2 weeks, equal vector doses of 3 × 1010 vector genomes). A) Uninjected substantia nigra (SN). B) GFP e×pression from AAV9 GFP. C) NeuN from the same section as B. D) TDP-43 AAV9 injected into the SN at this dose destroys the vast majority of neurons in the region. SNC, substantia nigra pars compacta; SNR, substantia nigra pars reticulata. A-D, bar = 134 μm.

Click here for supplemental data (21.2KB, pdf)

Articles from Molecular Therapy: the Journal of the American Society of Gene Therapy are provided here courtesy of The American Society of Gene & Cell Therapy

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