Expansive Gene Transfer in the Rat CNS Rapidly Produces Amyotrophic Lateral Sclerosis Relevant Sequelae When TDP-43 is Overexpressed

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Expansive Gene Transfer in the Rat CNS Rapidly Produces Amyotrophic Lateral Sclerosis Relevant Sequelae When TDP-43 is Overexpressed David B Wang1, Robert D Dayton1, Phillip P Henning1, Cooper D Cain1, Li Ru Zhao2, Lisa M Schrott1, Elysse A Orchard1,3, David S Knight2 and Ronald L Klein1 Department of Pharmacology, Toxicology, and Neuroscience, Louisiana State University Health Sciences Center, Shreveport, Louisiana, USA; Department of Cellular Biology and Anatomy, Louisiana State University Health Sciences Center, Shreveport, Louisiana, USA; 3Department of Animal Resources, Louisiana State University Health Sciences Center, Shreveport, Louisiana, USA 1 2

Improved spread of transduction in the central nervous system (CNS) was achieved from intravenous administration of adeno-associated virus serotype-9 (AAV9) to neonatal rats. Spinal lower motor neuron transduction efficiency was estimated to be 78% using the highest vector dose tested at a 12-week interval. The widespread expression could aid studying diseases that affect both the spinal cord and brain, such as amyotrophic lateral sclerosis (ALS). The protein most relevant to neuropathology in ALS is transactive response DNA-binding protein 43 (TDP-43). When expressed in rats, human wild-type TDP-43 rapidly produced symptoms ­germane to ALS including paralysis of the hindlimbs and muscle wasting, and mortality over 4 weeks that did not occur in controls. The hindlimb atrophy and weakness was evidenced by assessments of rotarod, rearing, overall locomotion, muscle mass, and histology. The muscle wasting suggested denervation, but there was only 14% loss of motor neurons in the TDP-43 rats. Tissues were negative for ubiquitinated, cytoplasmic TDP-43 pathology, suggesting that altering TDP-43’s nuclear function was sufficient to cause the disease state. Other relevant pathology in the rats included microgliosis and degenerating neuronal perikarya positive for phosphoneurofilament. The expression pattern encompassed the distribution of neuro­pathology of ALS, and could provide a rapid, relevant screening assay for TDP-43 variants and other disease-related proteins. Received 10 June 2010; accepted 16 August 2010; published online 28 September 2010. doi:10.1038/mt.2010.191

Introduction Amyotrophic lateral sclerosis (ALS) involves degeneration of upper and lower motor neurons causing progressive paresis to paralysis, as well as postmortem pathology in the brain and spinal cord.1–3 The main neuropathology is abnormal intracellular aggregates derived from the transactive response DNA-binding

protein 43 kd.1,4,5 This protein has a variety of functions related to DNA binding and RNA processing in the nucleus.6 The ubiquitinated neuropathological lesions in both ALS and forms of frontotemporal lobar degeneration (FTLD) were discovered to be enriched for TDP-43.4 While TDP-43 is normally found in the nucleus, lesions are found in the cytoplasm, so TDP-43 mislocation from the nucleus to the cytoplasm may be a key step in pathogenesis.4 Postmortem TDP-43 pathology is prevalent in ALS and FTLD, occurring in 98% of ALS and 50% of FTLD samples,1,5 as well as in about a third of Alzheimer’s disease and dementia with Lewy ­bodies cases,7 and in certain diseases with Parkinsonism.8 Transgenic mice or rats for TDP-43 result in puissant toxicity when TDP-43 is overexpressed,9–11 as does a TDP-43 viral vector study in rats.12 The vital significance of TDP-43 is underscored by TDP-43 knockdown in mice, zebrafish, or flies;13–17 manipulating TDP-43 levels in either direction produces disease like effects, morbidity, and mortality. Experiments that address loss of or toxic gain of function will be required to determine whether either altered nuclear function, or the build-up of aberrant cytoplasmic TDP-43 causes disease. To mimic the pattern of neuronal loss in ALS and to study effects of TDP-43 in these populations, gene transfer should be directed to upper and lower motor neurons. A number of ­studies have successfully targeted the spinal cord with focal central ­nervous system (CNS), peripheral muscle, or intraventricular adeno-associated virus (AAV) vector injection methods in mice or rats,18–21 but intravenous delivery offers the potential for greater global spread.22–26 Because ALS and FTLD-TDP harbor TDP-43 inclusions in both the spinal cord and brain,3 the capability to simultaneously target both could be of value for study of these diseases. We therefore attempted gene delivery to the spinal cord, brain stem, and cerebral cortex via intravenous administration. The lack of effective therapies for ALS and FTLD may be tied to the lack of appropriate models to study mechanisms and drug development; a more appropriate model could permit development of an efficacious drug. With green fluorescent protein (GFP), we tested the hypothesis that widespread transduction would be achieved in neonatal rats

Correspondence: Ronald L Klein, Department of Pharmacology, Toxicology, and Neuroscience, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130, (318) 675-7830, USA. E-mail: [email protected]

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with AAV9, as previously reported in mice,22,23 in order to expand the spread of gene transfer in the rat CNS and thereby enable the development of new therapeutic strategies in this species. With TDP-43, we tested whether the vector tropism pattern would be well suited to ALS in terms of transduction of target neurons and degeneration of lower motor neurons. The premise of the study is that a vector-based animal model with symptoms of paralysis, muscle wasting, and degeneration of motor neurons would mimic ALS and provide an appropriate assay for mechanistic and therapeutic research. We predicted a specific toxic effect of TDP-43,9–12 and that morbidity would be attributable to effects on spinal cord motor neurons, rather than TDP-43 gene transfer and toxicity in nontarget cells. The excellent germ-line transgenics for TDP-43 provide ­stable platforms for testing hypotheses about this protein’s role in disease.9–11 AAV somatic cell gene transfer also provides ­stable expression levels within subjects, although the technique is undoubtedly more variable across individuals relative to transgenics. We tested whether intravenous AAV TDP-43 delivery would produce a rapid and highly consistent motor phenotype or if the approach would be too variable and unreliable compared to motor characteristics in transgenic lines. A relevant assay with sufficient consistency is important because: (i) embryonic lethality in TDP-43 transgenics could block study;9 (ii) study of variants of TDP-43, such as mutants or truncations could be more rapid and cost-effective; and (iii) greater experimental control of dosing and timing could yield a more gradual and relevant progression.

Results GFP expression The gene transfer strategy was intravenous AAV9 administration to 1-day-old rat pups. AAV was diluted into a volume of 0.1 ml and injected into the temporal vein. The injections were performed by a highly experienced veterinarian (E.A.O.), and success rates were typically noted as 75–100% intravenous, so this was the degree of variability of intravenous dosing, with the remainder being administered extravascularly into subcutaneous tissue. Equal numbers of males and females were distributed in all of the vector groups and all injection conditions (% intravenous) were similar in all groups. Comparison between the TDP-43 and control GFP AAV9 vectors was at a dose of 2 × 1012 vector genomes (vg). A stronger expressing GFP vector that contained the woodchuck hepatitis virus posttranscriptional regulation element (WPRE) was used for tracing purposes and applied at a dose of 3 × 1012 vg. We expected the WPRE to be a reliable means to turn up expression levels of GFP27 to aid in visualizing the transduction pattern. At least two separate virus preps for each of the three AAV9s were used. The spread of GFP expression in the rat CNS was extensive relative to previous focal injections. The rapid assay of biophotonic imaging is useful to view fluorescence from entire tissues at once, like the brain and spinal cord (Figure 1). There was homogeneous GFP expression throughout the spinal cord for up to 12 weeks, with apparently lower relative expression in the brain. In sections, the brain had GFP-expressing neurons and glia, with distribution from the most rostral cerebral cortex and olfactory bulbs to the most caudal coccygeal spinal cord. The advance of widespread CNS expression in rats could benefit gene therapy Molecular Therapy vol. 18 no. 12 dec. 2010

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Untreated

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Figure 1  Biophotonic imaging of GFP 12 weeks after intravenous AAV9 delivery to a neonatal rat. The rat receiving the AAV9 GFP-WPRE vector displays intense and uniform GFP expression levels in the spinal cord, with apparently less GFP in the brain, whereas the untreated, age-matched control rat is blank for GFP. AAV9, adeno-associated virus ­serotype-9; GFP, green fluorescent protein.

modalities as well as study of disease-related proteins such as TDP-43. Unequivocally stronger GFP detection derived from the AAV9 GFP-WPRE vector than the AAV9 GFP control vector that did not have the WPRE, although the pattern of cells transduced did not seem to be affected by the WPRE. Results from 4 to 12 weeks after gene transfer show similar widespread and uniform density of GFP-expressing cells across the motor cortex at the two time points (Figure 2a–d). Upper and lower motor neurons were of greatest interest for the study of TDP-43 and ALS. Upper motor neurons in layer V of motor cortex (Figure 2e) and lower motor neurons in the ventral horn of the spinal cord (Figures 2f,g, and 3a–f), as well as ALS relevant motor neurons in the brainstem (Figure 2r), were clearly transduced, although a selectivity for the vector to transduce only neurons that degenerate in ALS was not evident. Estimation of lower motor neuron transduction derived from colabeling GFP with nonphosphorylated neurofilament (SMI 311), an efficient marker for spinal motor neurons (Figure 3d–f). The AAV9 GFP-WPRE vector yielded 77.9 ± 5.5% transduction at 12 weeks (N = 3) and the AAV9 GFP (without the WPRE) yielded 31.6 ± 4.9% at 4 weeks (N = 4), though comparison of transduction levels was not the main goal. Neurons throughout the cerebral cortex were transduced, without specificity for one cortical layer or region. The transduction was largely neuronal, although a minor fraction of the GFP-expressing cells was non-neuronal, demonstrated by double-labeling studies for the neuronal marker, NeuN (Figure  2k). Viewing layer V neurons in the motor cortex colabeled with either NeuN or SMI 311 suggested that the transduction rate of upper motor neurons was not as robust as for spinal lower motor neurons. Most of the non-neuronal cells in cortex, hippocampus, and thalamus had consistent morphology (smaller and more uniform than neurons) and were determined to be astroglia using an antibody for the marker glial acidic fibrillary protein (data not shown). The nuclei or regions with the highest densities of GFP-labeled neuronal perikarya were lateral septum (Figure  2c,d), striatum (Figure 2c,d,i), dorsal thalamic nuclei (Figure 2j), pontine nuclei (Figure  2q), cerebellar cortex, especially ventral cerebellar cortex (Figure 2o), and the circumventricular organs (median 2065

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Figure 2  Widespread CNS gene transfer from intravenous AAV9 GFP gene delivery. (a) Frontal cortex and olfactory nuclei 4 weeks after gene transfer; lo, lateral olfactory tract. (b) A similar area as in a, though more caudal, at 12 weeks. (c) Cingulate cortex (cg), lateral septum (ls), and striatum (st) at 4 weeks. (d) A similar area to c, at 12 weeks. The transduction patterns at the two time points were similar comparing a,b or c,d. (e) Motor cortex; neurons in layer V were transduced. (f) Lumbar spinal cord; the dorsal white matter and dorsal horn (dh) had intense labeling, whereas the ventral horn (vh) had relatively lower GFP levels. (g) Higher magnification of GFP-expressing neurons in the ventral horn of lumbar spinal cord. (h) Fibers in cross-section in the gracile and cuneate (cu) fasciculi and the dorsal corticospinal tract (cs) in lumbar spinal cord. (i) Somatosensory cortex (cx) of the upper lip and jaw and striatum (st). (j) Hippocampus (hc) and thalamus (th). (k) Higher magnification of hippocampal pyramidal neurons near the midline, counterstained with the neuronal marker NeuN; arrows, non-neuronal cells (NeuN negative) expressing GFP. (l) Thalamus; GFP expression in optic tract (ot). (m) Midbrain; GFP expression in superior colliculus (sc) and oculomotor nucleus (3). (n) Inferior colliculus (ic). (o) Cerebellum; high fraction of Purkinje neurons were transduced, particularly in the ventral lobules. (p) Higher magnification of Purkinje neurons; NeuN counterstain delineates gray matter. (q) Pontine nucleus. (r) Brainstem; expression in neurons of the facial nucleus (fn) and fibers of the spinal trigeminal nucleus (spt). (s) Brainstem; expression in area postrema (ap) and gracile and cuneate nuclei. (t) Dorsal root ganglion. All images are GFP immunofluorescence except for t, which is native fluorescence. a,c,h,t, 4 weeks; b,d,e–g,i–s, 12 weeks. Bar, a–d,j,l,m–o,r,s = 536 µm, f,i = 268 µm, e,h,q = 134 µm, g,t = 84 µm, k = 67 µm, p = 42 µm. AAV9, adeno-associated virus serotype-9; CNS, central nervous system; GFP, green fluorescent protein.

eminence, subfornical organ, area postrema, Figure 2s), and other regions along the midline, which is consistent with vector flow to the ventricles. Cerebellar Purkinje neurons (Figure 2o,p) and primary sensory neurons of the dorsal root ganglia (Figure  2t) were transduced at considerable efficiency accounting for many GFP-expressing axons in the dorsal columns and dorsal horn of the spinal cord. Other brain regions with relatively strong densities of GFP-expressing neurons were all areas of cerebral cortex (Figure 2a–e,i) and hippocampus (Figure 2j), with the strongest 2066

cortical regions being cingulate (Figure  2c,d), retrosplenial and piriform cortices and somatosensory cortical areas relating to the jaw and upper lip (Figure  2i). The midline of both cortex (cingulate and retrosplenial cortex) and hippocampus were strongly transduced. Subcortical nuclei having the highest densities of transduced neurons were the olfactory nuclei (Figure 2a,b), septofimbrial nuclei, anterior hypothalamic area, supraoptic nuclei, bed nuclei of the accessory olfactory tract, antero- and laterodorsal thalamic nuclei, ventral posteromedial and medial thalamic www.moleculartherapy.org vol. 18 no. 12 dec. 2010

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nuclei, posterolateral and posteromedial cortical amygdaloid nuclei, zona incerta, para- and presubiculum, superficial gray and optic nerve layer of superior colliculi (Figure  2m), oculomotor nuclei (Figure  2m), external cortex of inferior colliculi (Figure  2n), superior olivary nuclei, dorsal periolivary region, deep cerebellar nuclei (Figure  2r), cochlear nuclei (Figure  2r),

Figure 3 Transgene expression in large ventral horn neurons. (a–c) GFP expression (immunofluorescence), the neuronal marker NeuN in red, and merger. Arrows indicate nontransduced neurons, while most of the neurons are transduced. (d) Staining for the neuronal marker nonphosphorylated neurofilament (SMI 311) which we used to estimate percent transduction of the large ventral horn neurons. (e,f) Mergers of SMI 311 and GFP labeling at two magnifications, visualizing transduced and nontransduced neurons, the latter indicated with arrows. (g–i) Human TDP-43 immunofluorescence in red, the neuronal marker β III tubulin in green, and merger. A major fraction of the lower motor neurons were transduced by either the AAV9 GFP or the AAV9 TDP-43. (j) Higher magnification of TDP-43 expression in the large neurons of the ventral horn, indicating clear nuclear, but not cytoplasmic localization. (k) A control sample from the GFP group is blank when stained for the human TDP-43 antibody. (l) Merger of k with β III tubulin, the blank control relative to i. Bar, a–c,f,g–i,k,l = 67 µm, d,e = 134 µm, j = 42 µm. AAV9, adeno-associated virus serotype-9; GFP, green fluorescent protein; TDP-43, transactive response DNA-binding protein 43.

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facial nuclei (Figure 2r), lateral paragigantocellular nuclei, principal and spinal trigeminal nuclei (Figure 2r), ­cuneate and gracile nuclei (Figure 2s), and hypoglossal nuclei. The red nucleus, substantia nigra, globus pallidus, and other parts of the amygdala had relatively fewer GFP-expressing neurons, than the nuclei above. The GFP expression in fibers of the cells above was widespread and robust. The strong GFP signal in the spinal cord on biophotonic imaging arose from strong expression in the dorsal roots, dorsal white matter, the cuneate and gracile fasciculi, and dorsal horn (Figure 2f,h), and likely originated in large part from dorsal root ganglion cells (Figure  2t). The spinal trigeminal nuclei (Figure 2r), which are continuous with the dorsal horns of the spinal cord, had the greatest density of GFP fibers in the brainstem, consistent with the GFP fibers in the dorsal horn. Other sensory tracts such as the olfactory (Figure 2a,b), optic (Figure 2l), auditory, gustatory (solitary) tract, and vestibular tracts (Figure  2r) were also densely transduced. Motor tracts were positive for GFP in the internal capsule, pyramidal tract, corticospinal tract (Figure  2h), and the ventral roots of the spinal cord, although the sensory areas had relatively more GFP expression. The areas that could be visualized with strong GFP expression in fibers and neuropil were: lateral olfactory tract (Figure 2a,b), olfactory tubercle, optic chiasm, optic tract (Figure  2l), internal capsule, globus pallidus, substantia nigra pars reticulata, lateral geniculate nuclei (Figure 2l), medial terminal nuclei of the accessory optic tract, lateral posterior thalamic nuclei, ventral posteromedial thalamic nuclei, medial lemniscus, paralemniscal nuclei, ventral and intermediate nuclei of lateral lemniscus, cerebellar peduncles (Figure  2r), oculomotor nuclei (Figure  2m), superior colliculi (Figure 2m), inferior colliculi (Figure 2n), vestibular nuclei and nerve (Figure 2r), cochlear nuclei (Figure 2r), nuclei of solitary tract, pyramidal tract, sensory root of trigeminal nerve, principal and spinal trigeminal nuclei (Figure 2r), cuneate and gracile nuclei (Figure 2s), cuneate and gracile fasciculi (Figure 2f,h), and corticospinal tract (Figure 2h). The intravenous AAV9 injections also produced GFP expression in other organs such as liver and heart, although there was little to no expression found in spleen or kidney (Figure 4). The widespread GFP expression pattern in the CNS of rats is similar to previous intravenous AAV gene transfer in mice with respect to relatively strong transduction of cerebellar Purkinje neurons and dorsal root ganglia and circumventricular areas,23 transduction in lower motor neurons,22–24 as well as transduction of non-neuronal tissues.25,28 Upper and lower motor neurons were unequivocally transduced, although relatively more GFP expression appeared to

Figure 4  GFP expression in liver and heart at 4 weeks. (a) Liver sample stained for GFP immunofluorescence from a non-GFP transduced control rat. (b) GFP expression in liver cells in the AAV9 GFP group. (c) GFP expression in heart. (d) Kidney; little or no expression. (e) Spleen; few GFP cells found. Bar, a,b,d,e = 67 µm, c = 134 µm. AAV9, adeno-associated virus serotype-9; GFP, green fluorescent protein.

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derive from sensory pathways, and there was strong expression outside the CNS.

TDP-43 expression The vector for human wild-type TDP-43 expression did not contain the WPRE expression enhancer, and the detection of human TDP-43 was less sensitive than for GFP, even when the WPRE was not used with the GFP. Toxicity of the TDP-43 (i.e., loss of TDP-43-expressing cells), or clearance of excess TDP-43 could have contributed to the low expression levels. However, expression of human TDP-43 in large ventral horn neurons of the spinal cord, only in the TDP-43 group, was unequivocal (Figures 3g–i and 5a). The human TDP-43 detected was found only in nuclei (Figure  3g,j), and there was no evidence for cytoplasmic deposition of human TDP-43, or for ubiquitin positive aggregates at 4 weeks. Colabeling with the neuronal marker β III tubulin demonstrated TDP-43 expression in large ventral horn neurons (Figure 3g–i), whereas samples from the GFP group were blank for the vector-derived human TDP-43 (Figure 3k,l). As with the GFP transduction, in the cerebral cortex, the retrosplenial cortex appeared to have a relatively high density of human TDP-43expressing cells (Figure 5b), as did the dorsal thalamus and hippo­ campus (Figure  5c), posterolateral cortical amygdaloid nucleus (Figure  5d), cerebellum (Figure  5e), and dorsal root ­ganglia (Figure  5f). Overall the percent of lower motor neurons in the spinal cord that could be detected expressing TDP-43 was minor, although an efficient example of transduction of lower motor ­neurons was obtained in Figure 3g–i. The consistent pathophysiological effects described below were highly specific to the TDP-43 group, and reliably occurred even when expression levels were barely detectable for our human TDP-43 staining assay. Pronounced morbidity, mortality, and motor phenotype in TDP-43 rats The TDP-43 gene transfer was highly toxic to rats, in a specific manner relative to the control gene transfer group. For the first week, the weight gain of GFP and TDP-43 rats was similar, but diverged thereafter, whereas the weight gain curve for GFP superimposed untreated rats (Figure  5g). Repeated measures analysis of variance comparing the weights of GFP and TDP-43 rats resulted in an effect of vector group (F1,60 = 10.92, P < 0.005), age 2068

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Long-term expression and lack of morbidity in GFP rats The high-level expression was maintained for at least 12 weeks (Figures 1 and 2), so the promoter system provided stable expression over time after intravenous vector administration, as expected, based on long-term data from focal injections.27 Despite the high levels of GFP, AAV9 GFP-WPRE rats showed no signs of morbidity in terms of grooming, weight gain, or motor behaviors. To determine whether the GFP expression yielded an unwanted behavioral side effect, a small group of AAV9 GFP-WPRE rats (N = 4; two male and two female) was compared to a group of agematched, uninjected rats (N = 6; all male). There were no statistical differences between the two groups for weight or any locomotor behaviors, and most importantly, for rotarod performance and rearing, which were affected with great sensitivity by TDP-43.

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Figure 5 TDP-43 expression in spinal cord and brain 4 weeks after intravenous gene delivery. The detection of TDP-43-expressing cells was most sensitive in (a) spinal cord lower motor neurons, (b) retrosplenial cortex, (c) hippocampus and dorsolateral thalamus, (d) posterolateral cortical amygdaloid nucleus, (e) cerebellum, and (f) dorsal root ganglia, all areas that were also transduced by the control GFP vector. A dotted line outlines the gray matter of the spinal cord in a. CA4 pyramidal neurons and thalamus (th) are indicated in c. Images are immunofluorescence (a,c,f) or immunoperoxidase staining that was inverted to a negative image (b,d,e). (g,h) Weight gain and survival changes in rats overexpressing TDP-43. (g) The weights of TDP-43 rats diverged from control GFP rats over time, whereas the GFP gene transfer did not alter weight relative to untreated rats. The TDP-43 and GFP groups were compared by repeated measures ANOVA, yielding an effect of treatment group, time, and an interaction (P < 0.005, N = 8–9/group). *P < 0.05, Bonferroni post-test at 4 weeks. (h) A group of seven untreated rats had 100% survival, which is the top curve. In the GFP group, 95% survived to 4 weeks. There was a difference in survival rate for TDP-43 and GFP rats over 4 weeks (P < 0.002, Kaplan–Meyer/ log rank analysis, N = 21–22/group). Bar in a = 168 µm, b–e = 134 µm, c = 42 µm. AAV9, adeno-associated virus serotype-9; ANOVA, analysis of variance; GFP, green fluorescent protein; TDP-43, transactive response DNA-binding protein 43.

(F4,60 = 654.8, P < 0.0001), and an interaction of the two (F4,60 = 13.32, P < 0.0001), whereas the Bonferroni post-test only resulted in a vector group difference at 4 weeks (P < 0.05). There was no difference in the weight of the TDP-43 group at 1, 2, and 3 weeks in the post-tests, so there was a time-dependent and progressive effect, underscored by the interaction. Males and females were affected similarly. Survival up to 4 weeks in the TDP-43 group was 52% (11/21) and 95% (21/22) for the GFP control group, with males and females affected similarly. A group of seven untreated rats had 100% survival. Kaplan–Meyer/log rank analysis resulted in a survival difference between the two vector groups (χ2 = 10.72, P < 0.002; Figure  5h). Individual tissues weighed at necropsy were: brain, spinal cord, hindlimb muscles, liver, heart, kidney, and spleen (Supplementary Table S1). The body mass and organ weights were lower in the TDP-43 group compared to control GFP by 26–36% (P < 0.005, t-test), except for the brain which only trended lighter by 5% (P = 0.06, t-test). That the brain developed www.moleculartherapy.org vol. 18 no. 12 dec. 2010

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glucose (malnutrition). Total protein was 8% lower in the TDP-43 rats compared to GFP control rats (P < 0.05, t-test, N = 5/group), although glucose or albumin levels were not different between the groups (Supplementary Table S2). Blood urea nitrogen was lower in the TDP-43 group by 19% (P < 0.05, t-test, N = 8–13/group), although an increase, rather than a decrease, in blood urea nitrogen would indicate kidney dysfunction. Based on the GFP expression found in liver and heart, we cannot rule out that some of the morbidity and mortality in the TDP-43 rats was due to expression outside of the CNS, although the metabolic profile did not indicate impairment in liver or kidney function. By 2 weeks, a clear phenotype manifested in the TDP-43 rats in terms of a hindlimb escape reflex. GFP rats extended their hindlimbs outward when picked up by their tail (like untreated rats; Figure 6a). However, all of the TDP-43 rats clasped their hindlimbs

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to its normal weight in the TDP-43 rats, supports that the lower weights of the other tissues could involve atrophy rather than developmental retardation. Due to the GFP expression observed in liver and heart, we studied a metabolic panel in order to determine whether TDP-43 expression affected the function of non-CNS organs. The assay panel included the following: ions calcium, sodium, potassium, chloride, and the anion gap (an index of acidosis); CO2 (indicator of acidosis/alkalinosis and lung function); blood urea nitrogen and creatinine levels (kidney function); alanine transaminase and aspartate transaminase (released into the blood stream when the liver is damaged); alkaline phosphatase (marker for bile duct obstruction or liver disease); total bilirubin (marker for hemolysis or jaundice); albumin (related to liver function, malnutrition, and protein deficiencies); total protein (malnutrition); and

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Figure 6  Motor phenotype in TDP-43 rats at 4 and 15 weeks of age. (a) A control GFP rat displays the normal hindlimb extension escape response when raised by its tail. (b) In contrast, a TDP-43 rat crosses its hindlimbs to the midline (center) when raised and also shows loss of muscle tone in the hindlimbs (right). (c,d) A GFP rat in an activity-monitoring chamber shows normal walking stance up on its haunches and normal rearing. (e,f) A TDP-43 rat has deficient motor control of its hindlimbs and therefore cannot mount full rears. (g,h) A similar phenotype with dysfunctional hindlimbs and rearing persisted at 15 weeks. Measurements demonstrated consistent motor phenotype in TDP-43 rats at 4 weeks of age. (i) Behavioral measures. Rotarod: TDP-43 rats could not control their hindlimbs for wheel walking. P < 0.0001, t-test. Rearing: the deficient hindlimbs impaired number of rears (P < 0.05), rearing distance (P < 0.005), and time spent rearing (P < 0.002) during 30 minute activity-monitoring sessions; t-tests; GFP, N = 16; TDP-43, N = 11. GFP, green fluorescent protein; TDP-43, transactive response DNA-binding protein 43.

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inward, and also showed loss of muscle tone, consistent with late stage denervation (Figure  6a,b; Supplementary Videos S1 and S2). In accordance with the loss of the hindlimb reflex, all of the TDP-43 rats were impaired on the rotarod (Figure 6i), displaying

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a striking phenotype relative to control (P < 0.0001, t-test). The rotarod involves coordination of all four limbs and every TDP-43 rat was affected because their hindlimbs were severely paralyzed. In activity-monitoring sessions, the TDP-43 rats had lost control of their hindlimbs, which would drag along the floor, demonstrating paresis and paralysis (Figure 6c,e; Supplementary Video S3). Despite the fact that the TDP-43 rats propelled themselves mainly by their forelimbs with their abdomen touching the floor, the distance traveled over 30 minutes did not reach ­statistical ­difference relative to controls (P = 0.06, t-test; Supplementary Table S3). Loss of control of the forelimbs was less likely but variable, with some of the TDP-43 rats displaying paretic forelimbs as well. The rats that showed noticeable forelimb impairment were overall more severely affected by TDP-43 and the ones that were likely to die prematurely (usually by 10–14 days). Such severely affected rats had lower weights and became so paralyzed days before dying that it was difficult to detect any limb movements and even their breathing movements. The situation was reminiscent of a transgenic rat line for familial mutant superoxide dismutase where the hindlimbs were typically more sensitive to the disease state than the forelimbs, but subjects with forelimb impairment had the most severe and rapidly progressing disease state.29 The ability to rear was greatly lost due to the paralyzed hindlimbs at 4 weeks (Figure 6d,f), and the hindlimb paralysis was stable for up to 16 weeks, the longest time point studied (15 weeks shown in Figure 6g,h). The loss of hindlimb function at 4 weeks was evident in terms of the number of rears (P < 0.05, t-test; Figure 6i), the rearing distance (P < 0.005, t-test), and the rearing time (P < 0.002, t-test), during the 30-minute test period. Some of the animals were analyzed for gait by dipping their paws in ink and having them walk through a corridor with recording paper on the floor. The GFP rats produced uniform tracings with each forepaw and hindpaw on one side landing exactly on the same points along the path, whereas TDP-43 rats showed mismatch of forepaw and hindpaw placement, and the paretic hindlimbs caused shuffled, brushstroke like traces versus the precise paw prints in controls (Supplementary Figure S1).

Spinal cord atrophy, lower motor neuron degeneration, and muscle wasting in TDP-43 rats Viewing the spinal cord sections by various methods including Nissl (Figure  7a,b), human TDP-43, neuronal markers NeuN Figure 7 Spinal cord neuropathology induced by TDP-43. (a–d) Nissl staining of control GFP (left column) or TDP-43 (right column) spinal cords. The TDP-43 samples were consistently smaller than controls, with the most noticeable atrophy in the dorsal white matter: the dorsal roots and the gracile and cuneate fasciculi, arrows. (c,d) Nissl staining of ventral horn of lumbar spinal cord. Lumbar segments 2–5 were quantified for number of large motor neurons. There was a 14% loss of motor neurons in L2–5 in the TDP-43 group (P < 0.01, t-test, N = 8–9/group). (e,f) Phospho-neurofilament (p-NF) labeling of cuneate fasciculus fibers in cross-section shows a severe loss of immunoreactivity with TDP-43. (g,h) When comparing the ventral roots labeled with β-tubulin, specific shrinkage and loss of tissue in the TDP-43 group was not as noticeable as it was for the dorsal spinal cord in e,f. (i,j) The aberrant presence of neuronal perikarya labeled for phospho-neurofilament was prevalent in the TDP-43 samples, consistent with neurodegeneration. (k,l) Trend of more pronounced microglial staining (CD11b antibody) in the spinal cord in the TDP-43 group. Bar, a,b = 335 µm, c,d,i,j = 67 µm, e–h = 42 µm, k,l = 134 µm. GFP, green fluorescent protein; TDP-43, transactive response DNA-binding protein 43.

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a

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b GFP

GFP, H&E

c

TDP-43, H&E

TDP-43

a.t. m.g. sol.

Figure 8  Hindlimb muscle wasting in TDP-43 rats. (a) Atrophy of anterior tibialis (a.t.), medial gastrocnemius (m.g.), and soleus (sol.) in a TDP-43 rat relative to a control GFP rat at 32 days old. (b) Hematoxylin and eosin (H&E) stain of medial gastrocnemius from a GFP rat. (c) The myofibers are severely atrophied in a TDP-43 rat (same magnification as b). The smaller and angulated myofibers are consistent with denervation. Bar = 67 µm. GFP, green fluorescent protein; TDP-43, transactive response DNA-binding protein 43.

and  β III tubulin, phospho-neurofilament, and the microglial marker CD11b, there was obvious shrinkage in the TDP-43 group relative to control. In particular the TDP-43 spinal cords had shrunken dorsal roots and cuneate and gracile fasciculi (arrows in Figure 7a,b). Nissl analysis focused on the L2–5 segments, which contain the motor neurons controlling the principal hindlimb muscles.30 The shrinkage of the gray and white matter of the spinal cord in the TDP-43 group was quantified. The cross-­sectional area of the cord in this region was shrunken by 17% in the TDP-43 group relative to control (GFP, 4.90 ± 0.20 versus TDP-43, 4.07 ± 0.15 mm2; P < 0.01, t-test, N = 8–9/group; Figure  7a,b), the area of the gray matter by 12% (GFP, 2.50 ± 0.08 versus TDP-43, 2.20 ± 0.06 mm2; P < 0.01, t-test, N = 8–9/group), and the area of the white matter by 22% (GFP, 2.40 ± 0.13 versus TDP-43, 1.88 ± 0.10 mm2; P < 0.01, t-test, N = 8–9/group), consistent with the noticeable loss of the dorsal white matter. We had the most success visualizing the large motor neurons of the ventral horn with the Nissl staining compared to choline acetyltransferase, NeuN, or β III tubulin staining. The object size limits on the imaging program were set so only large ventral horn neurons (cross-sectional area >500 µm2), which we assumed to be motor neurons, were counted. The results for counts of the number of lower motor neurons in L2–5 were similar to those for the loss of gray matter with 14% fewer cells in the TDP-43 group relative to control (GFP, 591.9 ± 19.5 versus TDP-43, 508.1 ± 19.2 cells counted; P < 0.01, t-test, N = 8–9/group). The neuronal soma size was not different between the groups (GFP, 935.0 ± 20.1 versus TDP-43, 923.8 ± 27.0 µm2, N = 8–9/group). The region of the spinal cord that was most affected was the dorsal roots and dorsal white matter (Figure 7e,f), consistent with effects of TDP-43 expression in dorsal root ganglia neurons (Figure 5f). By comparison, the ventral roots were not as affected by the TDP-43 (Figure  7g,h) as much as the dorsal roots and white matter were (Figure 7e,f). However, the phosphoneurofilament marker provided evidence of neurodegeneration in ventral horn neurons (Figure 7i,j). Under normal conditions, neurofilaments are highly phosphorylated in axons (Figure  7e), but not in neuronal perikarya, thus phospho-neurofilament is an axonal marker.31 During ALS and other neurodegenerative diseases, abnormal phospho-neurofilament accumulates in neuronal perikarya along with other signs of neurodegeneration, such as loss of Nissl substance, displacement of the nucleus, and Molecular Therapy vol. 18 no. 12 dec. 2010

ballooning of the cell body.32–34 There was both a striking loss of axonal phospho-neurofilament in the TDP-43 group (Figure 7e,f), and a consistent expression in neuronal cell bodies in the ventral horn, that was not found in controls (Figure 7i,j). There was also a qualitative, but consistent trend for elevated microglial staining in TDP-43 spinal cords (Figure 7k,l), that could either be a response to, or could underlie the neurodegeneration. We probed for astrogliosis in the spinal cord and brain and microgliosis in the brain. There was a weak indication of elevated glial fibrillary acidic protein immunoreactivity in the spinal cords and brains, and elevated CD11b in the brains, of TDP-43 rats (data not shown). Hindlimb muscles were grossly shrunken in TDP-43 rats relative to control (Figure 8a), as were myofibers of the gastrocnemius. The uniform shrinkage of the myofibers (Figure  8b,c) indicates widespread denervation, although we cannot rule out that low-level TDP-43 expression in muscle could have caused the myopathy.

Discussion TDP-43 had a powerful impact when overexpressed in rats, in clear contrast to the efficient control gene transfer, which was well tolerated. Both in our study with focal expression in the substantia nigra12 and here with much more widespread expression, augmenting TDP-43 levels penetrantly manifested a motoric phenotype. In terms of the spread of the control GFP transgene expression and number of cells transduced in the rat CNS, such returns have never been attained before. Biophotonic data visualized complete spread of GFP throughout the spinal cord. With this degree of expression, it is not surprising that TDP-43 had such a functional impact. Relative to intravenous AAV9 vector delivery to neonatal mice,23 the CNS transduction in neonatal rats produced a similar pattern in general, which is an advance because: (i) it helps confirm that global gene transfer is not species-specific and may therefore have relevance to humans;22,26 (ii) the larger rat is advantageous for anatomical manipulations; and (iii) there are well-characterized pharmacological and behavioral paradigms that are facilitated with rats, which could enable drug development. The motor symptoms of paresis/paralysis of the hindlimbs evidenced by hindlimb reflex, rotarod, rearing, and gait, as well as the profound loss of muscle tone and mass, and rapidly progressing morbidity and mortality are germane to the pathogenesis and symptomatology of ALS. We predicted that changes in 2071

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motor function could be attributed to effects on motor neurons and that TDP-43 expression in motor neurons would be necessary to mimic ALS. With the dose-matched control vector at 4 weeks, we predicted 32% transduction efficiency of lumbar motor neurons, whereas there was a 14% loss of these cells in the TDP-43 group. The detection of human TDP-43 was less sensitive than for the GFP transgene product, so the degree of cell loss could have reflected the transduction rate. However, the effect on lower motor neurons seems too small to account for the pronounced motor phenotype, not to mention the weight loss and early death. The data therefore disfavor the hypothesis that the disease state can be fully explained by effects on TDP-43 expression in lower motor neurons. There could be a greater functional impact of TDP-43 on the motor neurons than indicated by the cell loss, or effects of TDP-43 on other cells contributed to the disease state. In any case, major loss of motor neurons is not required for pronounced behavioral outcomes after manipulating TDP-43 levels in transgenic mice and rats, where 15–25% losses of spinal motor neurons were found.9–11 While the neuropathological relevance of expressing TDP-43 in the nucleus remains in question, the aberrant phospho-neurofilament expressing neuronal perikarya32–34 and microgliosis35,36 are relevant to ALS pathology. The motor phenotype was highly consistent for the functional readouts of hindlimb reflex, gait, rotarod, and rearing, and similar to the effects reported in transgenic TDP-43 lines9–11 and earlier models of ALS.10,29 Though there is inherently greater interindividual variability in a vector gene transfer strategy relative to transgenic mice, transgene expression was stable for as long as 16 weeks. We conclude that the vector/rat method is a rapid and reliable assay for studying some aspects of ALS such as functional paralysis, muscle wasting, and loss of motor neurons. The smaller TDP-43 rats could be explained by less food intake, which was likely, although this too could be relevant to bulbar onset ALS and dysarthria and dysphagia2,37 if TDP-43 was expressed in the parts of the brainstem that control mastication and swallowing. Locomotor deficits could also have potentially contributed to less feeding as well. The metabolic panel served its purpose as a control by demonstrating that functions of the liver, kidney, and lungs were not deficient in TDP-43 rats, supporting the specificity of a neurological effect. While the upper and lower target motor neurons for ALS were transduced, so too were many other neuronal populations as well as non-neuronal tissues, and despite the ALS-like disease sequelae, the high amplitude transduction of primary sensory neurons was unwanted. More work is required to improve the selectivity for neurons most relevant to ALS pathophysiology, potentially by manipulating the promoter or the AAV capsid. Considering the transduction pattern in dorsal root ganglia and the dorsal fasciculi, we can infer that sensory impairment had an impact. While most areas of the brain had a more diffuse transduction pattern than the high density that can be achieved with focal injections over limited spread, dorsal root ganglia and Purkinje neurons were densely transduced, and the results from dorsal root ganglia did appear similar to those from direct injections to this tissue.38 Proprioception is important for coordination as is the cerebellum, so dysfunction of dorsal root ganglion proprioceptor neurons and Purkinje neurons likely contributed to the complete and drastic 2072

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effect on the rotarod, although in TDP-43 rats, the hindlimbs appeared paralyzed and completely disabled for the rotarod task. The strong impact of the nuclear TDP-43 expression was underscored by the rapid disease progression over a short period of a few weeks, even in TDP-43 animals with barely detectable levels of the vector-derived human TDP-43 protein. The longest time point studied for a TDP-43 rat was 16 weeks, so an adult model is possible, which is significant because ALS is a disease of older adults.2 ALS pathogenesis begins before symptoms are present,2 so it could be possible to study presymptomatic changes in a more slowly progressing model. Control strategies to slow the disease state progression in rats, and therefore improve the relevance could include: dose titration, waiting longer after birth for gene transfer, expression in adults using mannitol,24,25 or regulated expression. The human wild-type TDP-43 that was expressed is most relevant to sporadic forms of disease with TDP-43 proteinopathy, and therefore the largest portion of the TDP-43 disease population. A model using wild-type TDP-43 for testing therapeutics would be appropriate for sporadic forms of ALS, FTLD, cases of AD, and Parkinsonism with TDP-43 pathology.1,5,7,8 However, comparing familial mutant forms of TDP-43 to wild type in the vector/rat model could be of interest based on the toxicity­enhancing effects of mutant TDP-43 in various models,11,39,40 and the propensity for mislocalization to the cytoplasm,40,41 stabilization of mutant TDP-43,42 and disease-relevant molecular interactions.41,42 The vector/rat method could be a cost-effective route to compare different forms of TDP-43 such as mutants and truncations found in disease.43 Coexpression of TDP-43 disease modifying ­factors is of interest, for example a disease-related mutant form of valosin-containing protein that promotes cytoplasmic deposition of TDP-43.41 The expanse of gene transfer in the rat CNS has been extended, although a more relevant vector/ rat assay for ALS will have improved selectivity for expression in motor neurons, cytoplasmic TDP-43 pathology, and slower progression in older adults.

Materials and Methods DNAs and AAVs. Expression of the human wild-type TDP-43 was driven by a hybrid cytomegalovirus/chicken β-actin promoter.27 A similar GFP plasmid was used as the control for TDP-43. In addition, another GFP plasmid containing the WPRE, which confers stronger expression, was used for tracing purposes.27 The constructs have AAV2 terminal repeats and were packaged into AAV9 capsids as previously described.12 The final stocks were sterilized by Millipore (Billerica, MA) Millex-GV syringe ­filters, aliquoted, and stored frozen. Encapsidated genome copies were titered by dot-blot, with all preps containing over 4 × 1013 vg/ml. Equal dose comparisons were made by normalizing titers with the diluent, lactated Ringer’s solution (Baxter Healthcare, Deerfield, IL). Over the course of the study, at least two different batches of the GFP control, GFP-WPRE, and TDP-43 AAV9 ­vectors were tested, and yielded consistent results across batches. Animals, timeline, dosing, vector injections. Experiments derived from

eight litters of Sprague–Dawley rats (Harlan, Indianapolis, IN). The groups included untreated rats, GFP AAV9 at a dose of 2 × 1012 vg, TDP-43 AAV9 at the same dose, or in some cases the stronger GFP-WPRE AAV9 vector at a higher dose of 3 × 1012 vg for tracing the transduction pattern. Gene transfer occurred at postnatal day 1, and the animals were studied for either 4, 12, or 16 weeks, as indicated. Both males and females were represented www.moleculartherapy.org vol. 18 no. 12 dec. 2010

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equally in all groups. Rats were wrapped in a latex glove and submerged up to their neck in an ice bath for 45 seconds for anesthesia/immobilization. The rat was then manually injected with 100 µl of diluted AAV9 into the temporal vein using a 30-gauge needle. An estimate of efficiency for direct intravenous delivery was recorded for each injection. Tattoo ink (~10 µl; Spaulding Color, Voorheesville, NY) was gently injected into the paws and/or tail for identification. The pups were placed on a heating pad and under a lamp to recover before being returned to the mother cage. Weaning was at 3 weeks. A total of 60 rats were injected, and 6 rats died within the first 48 hours postinjection (three GFP and three TDP). These deaths were attributed to the injections and not included in the ­mortality data presented. 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. Biophotonic imaging. Rats were anesthetized with a cocktail of 3 ml xyla-

zine (20 mg/ml, from Butler, Columbus, OH), 3 ml ketamine (100 mg/­ml, from Fort Dodge Animal Health, Fort Dodge, IA), and 1 ml acepromazine (10 mg/ml, from Boerhinger Ingelheim, St Joseph, MO) administered intramuscularly at a dose of 1 ml/kg, and then perfused with 100 ml ­phosphate-buffered saline (PBS). Tissues were then extracted and stored in PBS. Within 30 minutes, the brains were placed in the Xenogen (Alameda, CA) IVIS 100/XFO-12 apparatus and imaged with the GFP filter set.

Immunostaining. Animals were anesthetized as above and perfused with PBS, followed by cold 4% paraformaldehyde in PBS. Tissues were removed, weighed, and immersed in fixative overnight at 4 °C. Brains and spinal cords 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. Primary antibody incubations on freefloating sections were overnight at 4 °C on a shaking platform. Primary antibodies included: human-specific TDP-43 antibody (Abnova, Taipei City, Taiwan; 1:100); GFP (Invitrogen, Carlsbad, CA; 1:500); glial fibrillary acidic protein (Millipore; 1:400) for astroglia, CD11b (Millipore; 1:400) for microglia, NeuN for neuronal nuclei (Millipore; 1:500), neuron-specific β III tubulin (Abcam, Cambridge, MA; 1:100), phosphorylated high and middle rat neuro­filament subunits (p-NF, Millipore; 1:500), nonphosphorylated neuro­filament SMI 311 (Covance, Emeryville, CA). 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 (Carpinteria, CA; 1:2,000), incubated for 1  hour at room temperature. The sections were washed with PBS and labeled with horseradish peroxidase–conjugated Extravidin (Sigma, St Louis, MO; 1:1,000) for 30 minutes at room temperature. The chromogen was diaminobenzidine (0.67 mg; Sigma) in 0.3% H2O2, 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 either Alexa Fluor 488 or Cy3 conjugated secondary antibodies (Invitrogen or Jackson ImmunoResearch, West Grove, PA, respectively, 1:300) for 2 hours, followed by DAPI (Sigma) counterstaining (1 µg/ml), washes and coverslipping with glycerol/gelatin (Sigma). Some tissues were stored in 50% ethanol/PBS after fixation and then paraffin embedded and sectioned at 5-µm thickness onto slides, and stained using an automated system (Biogenex I6000, San Ramon, CA). Nissl and hematoxylin and eosin staining followed standard methods.12,44 Blood chemistry. Blood was collected from rats by cardiac puncture before being perfused. Plasma separator tubes with lithium heparin (BectonDickinson, Franklin Lakes, NJ) were used and the samples were frozen and Molecular Therapy vol. 18 no. 12 dec. 2010

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then submitted to the diagnostic laboratory at our hospital for a standard metabolic profile. Behaviors. Animals were assessed for performance on several motor

related behaviors: hindlimb escape reflex, rotarod, and open field. For the hindlimb escape reflex, rats were gently raised by their tail to observe the normal outward extension of the hindlimbs for 10 seconds, and this was viewed at 2, 3, and 4 weeks after gene transfer. Rotarod (Rota-rod/RS; Letica Scientific Instruments, Barcelona, Spain) testing was studied at 4 weeks and in some cases at 12 weeks as indicated. The rats were trained on the rotarod for 1 minute at 4 r.p.m. Sessions involved an accelerating rotarod from 4 to 40 r.p.m. over 2 minutes. The length of time spent on the wheel before falling was averaged from three trials. Rats were tested in a photobeam activity-monitoring system (Truscan 2.0; Coulbourn Instruments, Whitehall, PA) for locomotor and rearing behaviors in a 30 minute trial in a dark room, at 4 or 12 weeks after gene transfer.

Analysis of lower motor neurons. The fraction of large motor neurons in the ventral horn that was transduced by the GFP vectors was estimated using the marker SMI 311 for nonphosphorylated neurofilament. Twelve sections evenly spaced throughout lumbar spinal cord segments 2–4 were colabeled with SMI 311 and GFP antibodies. At least two observers scored the samples for the fraction of colabeled neurons in the ventral horn, counting over 150 cells, and the observers’ scores were averaged for the percentage of SMI 311 positive neurons expressing GFP. Nissl staining was used to measure the area of the spinal cord and quantify the size and number of lower motor neurons. Cross-sections from L2–5 were imaged with the Scion Program (Frederick, MD). Discriminating for objects with crosssectional area >500 µm2, only the motor neurons of the ventral horn were analyzed. Fifteen evenly spaced sections through L2–5 hindlimb enlargement were analyzed. The number of motor neurons was summed, and the cell area was averaged for each rat. Statistics. Data are expressed as mean ± SEM. Statistical tests included

repeated measures analysis of variance and Bonferroni’s post-tests, Kaplan–Meier/log rank test, or t-tests as indicated.

SUPPLEMENTARY MATERIAL Figure S1.  Gait prints of control GFP and TDP-43 rats. Table S1.  Body and tissue weights (g) at 31 ± 1 days old. Table S2.  Metabolic panel from blood samples collected at 32 days of age. Table S3.  Activity monitoring at 4 weeks old, in 30 minute sessions. Video S1.  A control GFP rat displays the normal hindlimb extension escape response, and muscle tone, when raised by its tail. Video S2.  In contrast to Video S1, the hindlimbs cross to the midline and there is loss of muscle tone in a TDP-43 rat. Video S3.  Motor impairment caused by TDP-43 gene transfer.

ACKNOWLEDGMENTS This work was supported by National Institute of Neurological Disorders and Stroke R01 NS048450. We thank Jean-Charles Paterna (Eidgenössische Technische Hochschule, Zürich, Switzerland), David Finkelstein (Mental Health Research Institute, Parkville, Australia), Robert Schwendimann (LSUHSC-Shreveport), Ikuo Tsunoda (LSUHSCShreveport), Omar Skalli (LSUHSC-Shreveport), and Dennis Dickson (Mayo Clinic, Jacksonville, FL) for advisement, and Jacqueline Moran and Jessica Cruzan for technical assistance.

References

1. Mackenzie, IR, Bigio, EH, Ince, PG, Geser, F, Neumann, M, Cairns, NJ et al. (2007). Pathological TDP-43 distinguishes sporadic amyotrophic lateral sclerosis from amyotrophic lateral sclerosis with SOD1 mutations. Ann Neurol 61: 427–434. 2. Eisen, A (2009). Amyotrophic lateral sclerosis: A 40-year personal perspective. J Clin Neurosci 16: 505–512. 3. Geser, F, Martinez-Lage, M, Kwong, LK, Lee, VM and Trojanowski, JQ (2009). Amyotrophic lateral sclerosis, frontotemporal dementia and beyond: the TDP-43 diseases. J Neurol 256: 1205–1214.

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4. Neumann, M, Sampathu, DM, Kwong, LK, Truax, AC, Micsenyi, MC, Chou, TT et al. (2006). Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Science 314: 130–133. 5. Neumann, M (2009). Molecular neuropathology of TDP-43 proteinopathies. Int J Mol Sci 10: 232–246. 6. Buratti, E and Baralle, FE (2008). Multiple roles of TDP-43 in gene expression, splicing regulation, and human disease. Front Biosci 13: 867–878. 7. Nakashima-Yasuda, H, Uryu, K, Robinson, J, Xie, SX, Hurtig, H, Duda, JE et al. (2007). Co-morbidity of TDP-43 proteinopathy in Lewy body related diseases. Acta Neuropathol 114: 221–229. 8. Wider, C, Dickson, DW, Stoessl, AJ, Tsuboi, Y, Chapon, F, Gutmann, L et al. (2009). Pallidonigral TDP-43 pathology in Perry syndrome. Parkinsonism Relat Disord 15: 281–286. 9. Wegorzewska, I, Bell, S, Cairns, NJ, Miller, TM and Baloh, RH (2009). TDP-43 mutant transgenic mice develop features of ALS and frontotemporal lobar degeneration. Proc Natl Acad Sci USA 106: 18809–18814. 10. Wils, H, Kleinberger, G, Janssens, J, Pereson, S, Joris, G, Cuijt, I et al. (2010). TDP-43 transgenic mice develop spastic paralysis and neuronal inclusions characteristic of ALS and frontotemporal lobar degeneration. Proc Natl Acad Sci USA 107: 3858–3863. 11. Zhou, H, Huang, C, Chen, H, Wang, D, Landel, CP, Xia, PY et al. (2010). Transgenic rat model of neurodegeneration caused by mutation in the TDP gene. PLoS Genet 6: e1000887. 12. Tatom, JB, Wang, DB, Dayton, RD, Skalli, O, Hutton, ML, Dickson, DW et al. (2009). Mimicking aspects of frontotemporal lobar degeneration and Lou Gehrig’s disease in rats via TDP-43 overexpression. Mol Ther 17: 607–613. 13. Feiguin, F, Godena, VK, Romano, G, D’Ambrogio, A, Klima, R and Baralle, FE (2009). Depletion of TDP-43 affects Drosophila motoneurons terminal synapsis and locomotive behavior. FEBS Lett 583: 1586–1592. 14. Kabashi, E, Lin, L, Tradewell, ML, Dion, PA, Bercier, V, Bourgouin, P et al. (2010). Gain and loss of function of ALS-related mutations of TARDBP (TDP-43) cause motor deficits in vivo. Hum Mol Genet 19: 671–683. 15. Kraemer, BC, Schuck, T, Wheeler, JM, Robinson, LC, Trojanowski, JQ, Lee, VM et al. (2010). Loss of murine TDP-43 disrupts motor function and plays an essential role in embryogenesis. Acta Neuropathol 119: 409–419. 16. Sephton, CF, Good, SK, Atkin, S, Dewey, CM, Mayer, P 3rd, Herz, J et al. (2010). TDP43 is a developmentally regulated protein essential for early embryonic development. J Biol Chem 285: 6826–6834. 17. Wu, LS, Cheng, WC, Hou, SC, Yan, YT, Jiang, ST and Shen, CK (2010). TDP-43, a neuropathosignature factor, is essential for early mouse embryogenesis. Genesis 48: 56–62. 18. Peel, AL, Zolotukhin, S, Schrimsher, GW, Muzyczka, N and Reier, PJ (1997). Efficient transduction of green fluorescent protein in spinal cord neurons using adenoassociated virus vectors containing cell type-specific promoters. Gene Ther 4: 16–24. 19. Kaspar, BK, Lladó, J, Sherkat, N, Rothstein, JD and Gage, FH (2003). Retrograde viral delivery of IGF-1 prolongs survival in a mouse ALS model. Science 301: 839–842. 20. Towne, C, Schneider, BL, Kieran, D, Redmond, DE Jr and Aebischer, P (2010). Efficient transduction of non-human primate motor neurons after intramuscular delivery of recombinant AAV serotype 6. Gene Ther 17: 141–146. 21. Passini, MA, Bu, J, Roskelley, EM, Richards, AM, Sardi, SP, O’Riordan, CR et al. (2010). CNS-targeted gene therapy improves survival and motor function in a mouse model of spinal muscular atrophy. J Clin Invest 120: 1253–1264. 22. Duque, S, Joussemet, B, Riviere, C, Marais, T, Dubreil, L, Douar, AM et al. (2009). Intravenous administration of self-complementary AAV9 enables transgene delivery to adult motor neurons. Mol Ther 17: 1187–1196. 23. Foust, KD, Nurre, E, Montgomery, CL, Hernandez, A, Chan, CM and Kaspar, BK (2009). Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes. Nat Biotechnol 27: 59–65. 24. Gray, SJ, McCown, TJ and Samulski, RJ (2009). Global gene delivery to the central nervous system via adeno-associated virus vectors administered intravenously. Program No. 643.6. Neuroscience Meeting Planner. Society for Neuroscience: Chicago, IL.

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25. McCarty, DM, DiRosario, J, Gulaid, K, Muenzer, J and Fu, H (2009). Mannitolfacilitated CNS entry of rAAV2 vector significantly delayed the neurological disease progression in MPS IIIB mice. Gene Ther 16: 1340–1352. 26. Foust, KD, Wang, X, McGovern, VL, Braun, L, Bevan, AK, Haidet, AM et al. (2010). Rescue of the spinal muscular atrophy phenotype in a mouse model by early postnatal delivery of SMN. Nat Biotechnol 28: 271–274. 27. Klein, RL, Hamby, ME, Gong, Y, Hirko, AC, Wang, S, Hughes, JA et al. (2002). Dose and promoter effects of adeno-associated viral vector for green fluorescent protein expression in the rat brain. Exp Neurol 176: 66–74. 28. Inagaki, K, Fuess, S, Storm, TA, Gibson, GA, Mctiernan, CF, Kay, MA et al. (2006). Robust systemic transduction with AAV9 vectors in mice: efficient global cardiac gene transfer superior to that of AAV8. Mol Ther 14: 45–53. 29. Matsumoto, A, Okada, Y, Nakamichi, M, Nakamura, M, Toyama, Y, Sobue, G et al. (2006). Disease progression of human SOD1 (G93A) transgenic ALS model rats. J Neurosci Res 83: 119–133. 30. McHanwell, S and Watson, C. (2008). Location of motoneurons in the spinal cord. In: Watson, C, Paxinos, G and Kayalioglu, G (eds). The Spinal Cord: A Christopher and Dana Reeve Text and Atlas. Academic Press: Sydney, pp. 94–114. 31. Sternberger, LA and Sternberger, NH (1983). Monoclonal antibodies distinguish phosphorylated and nonphosphorylated forms of neurofilaments in situ. Proc Natl Acad Sci USA 80: 6126–6130. 32. Manetto, V, Sternberger, NH, Perry, G, Sternberger, LA and Gambetti, P (1988). Phosphorylation of neurofilaments is altered in amyotrophic lateral sclerosis. J Neuropathol Exp Neurol 47: 642–653. 33. Munoz, DG, Greene, C, Perl, DP and Selkoe, DJ (1988). Accumulation of phosphorylated neurofilaments in anterior horn motoneurons of amyotrophic lateral sclerosis patients. J Neuropathol Exp Neurol 47: 9–18. 34. Sobue, G, Hashizume, Y, Yasuda, T, Mukai, E, Kumagai, T, Mitsuma, T et al. (1990). Phosphorylated high molecular weight neurofilament protein in lower motor neurons in amyotrophic lateral sclerosis and other neurodegenerative diseases involving ventral horn cells. Acta Neuropathol 79: 402–408. 35. Engelhardt, JI and Appel, SH (1990). IgG reactivity in the spinal cord and motor cortex in amyotrophic lateral sclerosis. Arch Neurol 47: 1210–1216. 36. Kawamata, T, Akiyama, H, Yamada, T and McGeer, PL (1992). Immunologic reactions in amyotrophic lateral sclerosis brain and spinal cord tissue. Am J Pathol 140:691–707. 37. Tomik, B and Guiloff, RJ (2008). Dysarthria in amyotrophic lateral sclerosis: A review. Amyotroph Lateral Scler (epub ahead of print). 38. Mason, MR, Ehlert, EM, Eggers, R, Pool, CW, Hermening, S, Huseinovic, A et al. (2010). Comparison of AAV serotypes for gene delivery to dorsal root ganglion neurons. Mol Ther 18: 715–724. 39. Sreedharan, J, Blair, IP, Tripathi, VB, Hu, X, Vance, C, Rogelj, B et al. (2008). TDP43 mutations in familial and sporadic amyotrophic lateral sclerosis. Science 319: 1668–1672. 40. Barmada, SJ, Skibinski, G, Korb, E, Rao, EJ, Wu, JY and Finkbeiner, S (2010). Cytoplasmic mislocalization of TDP-43 is toxic to neurons and enhanced by a mutation associated with familial amyotrophic lateral sclerosis. J Neurosci 30: 639–649. 41. Ritson, GP, Custer, SK, Freibaum, BD, Guinto, JB, Geffel, D, Moore, J et al. (2010). TDP-43 mediates degeneration in a novel Drosophila model of disease caused by mutations in VCP/p97. J Neurosci 30: 7729–7739. 42. Ling, SC, Albuquerque, CP, Han, JS, Lagier-Tourenne, C, Tokunaga, S, Zhou, H et al. (2010). ALS-associated mutations in TDP-43 increase its stability and promote TDP-43 complexes with FUS/TLS. Proc Natl Acad Sci USA 107: 13318–13323. 43. Zhang, YJ, Xu, YF, Cook, C, Gendron, TF, Roettges, P, Link, CD et al. (2009). Aberrant cleavage of TDP-43 enhances aggregation and cellular toxicity. Proc Natl Acad Sci USA 106: 7607–7612. 44. Paxinos, G and Watson, C (1998). The Rat Brain in Stereotaxic Coordinates, 4th edn. Academic Press: San Diego, CA.

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