Promoting Axon Regeneration in Adult CNS by Targeting Liver Kinase B1

Please cite this article in press as: Ohtake et al., Promoting Axon Regeneration in Adult CNS by Targeting Liver Kinase B1, Molecular Therapy (2018), https:// doi.org/10.1016/j.ymthe.2018.10.019

Original Article

Promoting Axon Regeneration in Adult CNS by Targeting Liver Kinase B1 Yosuke Ohtake,1,2,6 Armin Sami,1,2 Xinpei Jiang,1,2 Makoto Horiuchi,1,2 Kieran Slattery,1,2 Lena Ma,1,2 George M. Smith,1,3 Michael E. Selzer,1,4 Shin-ichi Muramatsu,5 and Shuxin Li1,2 1Shriners Hospitals Pediatric

Research Center, Lewis Katz School of Medicine, Temple University, Philadelphia, PA 19140, USA; 2Department of Anatomy and Cell Biology,

Lewis Katz School of Medicine, Temple University, Philadelphia, PA 19140, USA; 3Department of Neuroscience, Lewis Katz School of Medicine, Temple University, Philadelphia, PA 19140, USA; 4Department of Neurology, Lewis Katz School of Medicine, Temple University, Philadelphia, PA 19140, USA; 5Division of Neurology, Jichi Medical University, Shimotsuke, Tochigi 329-0498, Japan

Liver kinase B1 (LKB1), a downstream effector of cyclic AMP (cAMP)/PKA and phosphatidylinositol 3-kinase (PI3K) pathways, is a determinant for migration and differentiation of many cells, but its role in CNS axon regeneration is unknown. Therefore, LKB1 was overexpressed in sensorimotor cortex of adult mice five days after mid-thoracic spinal cord injury, using an AAV2 vector. Regeneration of corticospinal axons was dramatically enhanced. Next, systemic injection of a mutantAAV9 vector was used to upregulate LKB1 specifically in neurons. This promoted long-distance regeneration of injured corticospinal fibers into caudal spinal cord in adult mice and regrowth of descending serotonergic and tyrosine hydroxylase immunoreactive axons. Either intracortical or systemic viral delivery of LKB1 significantly improved recovery of locomotor functions in adult mice with spinal cord injury. Moreover, we demonstrated that LKB1 used AMPKa, NUAK1, and ERK as the downstream effectors in the cortex of adult mice. Thus, LKB1 may be a critical factor for enhancing the growth capacity of mature neurons and may be an important molecular target in the treatment of CNS injuries.

INTRODUCTION There are currently no treatments for functional loss due to axon disconnection in adult CNS. Severed axons within the mammalian CNS do not regenerate after injury, and the lack of rewiring injured fibers usually results in persistent functional deficits in various neurological disorders, including spinal cord injury (SCI). Failure of CNS axons to regenerate is attributed to both the non-permissive environment and reduced intrinsic growth capacity of mature neurons.1 The former includes myelin-associated growth inhibitors,2,3 scar-generating chondroitin sulfate proteoglycans,4,5 repulsive axon guidance cues,6,7 lack of neurotrophic factors,8–10 and physical barrier by reactive glial scar tissues around lesion.11 Among the neuron-intrinsic factors, a number of cell-autonomous molecules have been shown to play significant roles in controlling the growth ability of mature neurons, including cyclic AMP (cAMP),12 RhoA, Krüppel-like factors,13 mammalian target of rapamycin (mTOR),14,15 phosphatase and tensin homolog (PTEN),16 c-myc, and Sox11.17 However, none of these approaches were translated to clinics yet, and thus, there is

a persistent need to identify better targets and improved delivery methods. Liver kinase B1 (LKB1), also known as serine-threonine kinase 11 and Par-4 in C. elegans, is essential for maintaining cell metabolism, energy homeostasis, and cell polarity by activating a number of kinases and regulating vital pathways involved in carbohydrate, lipid, and protein metabolism.18,19 In addition to functioning as a tumor suppressor and associating with development of Peutz-Jeghers syndrome, LKB1 plays critical roles in regulating migration and polarization of several cell types during development by modifying the distribution of the Golgi apparatus in the cytoplasm and through other mechanisms.20–24 In response to stimulation by numerous extracellular factors, including brain-derived neurotrophic factor, neurotrophic factor, Sema3A, netrin-1, reelin, and Wnt, LKB1 is a vital downstream effector of cAMP/protein kinase A (PKA) and phosphatidylinositol 3-kinase (PI3K), the essential signaling pathways for controlling cell survival and elongation.25 LKB1 is required for axon specification during neuronal polarization in the CNS25–30 and for Schwann cell differentiation in the peripheral nervous system (PNS).31,32 During development, LKB1 acts as a major determinant for axon differentiation in some CNS neurons by regulating its initiation and elongation.25,29 LKB1 deletion or downregulation eliminates axon formation in vivo, and overexpression stimulates formation of multiple axons.27,29 As the master upstream kinase of multiple signals for controlling cell growth, LKB1 regulates neuronal polarization largely by activating SAD-A (synapses of amphids defective kinase A), SAD-B, NUAK1 (NUAK family SNF1-like kinase 1), and potentially other kinases.27,28,33,34 Whether LKB1 regulates regrowth of axotomized axons in adult neurons is not known, but if it does, it could be a major molecular target for treating CNS injuries.

Received 23 May 2018; accepted 26 October 2018; https://doi.org/10.1016/j.ymthe.2018.10.019. 6

Present address: Department of Molecular Neuroscience, Graduate School of Medicine, Osaka University, Osaka 565-0871, Japan. Correspondence: Shuxin Li, MD, PhD, Shriners Hospitals Pediatric Research Center, Lewis Katz School of Medicine, Temple University, 3500 N. Broad Street, Philadelphia, PA 19140, USA. E-mail: [email protected]

Molecular Therapy Vol. 27 No 1 January 2019 ª 2018 The American Society of Gene and Cell Therapy.

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Please cite this article in press as: Ohtake et al., Promoting Axon Regeneration in Adult CNS by Targeting Liver Kinase B1, Molecular Therapy (2018), https:// doi.org/10.1016/j.ymthe.2018.10.019

Molecular Therapy

Figure 1. Downregulation of LKB1 Protein in the Cerebral Cortices of C57BL/6 Mice during Development and No Changes of LKB1 Levels in Sensorimotor Cortices of Adult Mice after Mid-thoracic SCI (A and B) Levels of LKB1 protein were measured from the cerebral cortex of mice at postnatal day 3 (P3), 7, and 56 by immunostaining (A) and at P1, P3, P7, P14, P21, P42, and P56 by western blots (B; 4 mice/time point). We detected age-dependent downregulation of LKB1, especially in the cortices of mice >2 weeks old. Scale bars: 25 mm. (C) LKB1 protein levels were determined from the sensorimotor cortices of adult C57BL/6 mice 3 days after dorsal over-transection injury at T7 by western blots. SCI did not significantly alter levels of LKB1 expression in sensorimotor cortex. (D) The selectivity of the LKB1 antibody was confirmed by immunostaining and western blots in MDA-MB231 cell lines infected with AAV2-GFP or AAV2-LKB1. In (A)–(C), n = 4 mice per group. In (D), n = 3 separate experiments.

Here, by using adeno-associated virus (AAV) vectors to increase LKB1 expression, we demonstrate a key role of LKB1 in augmenting the regenerative capacity of CNS neurons in adult mammals using NUAK1 signaling, leading to significantly enhanced recovery of locomotor function. Because AAV vectors have been used safely to treat other neurological disorders,35,36 our new viral constructs have translational potential for treating CNS injuries.

RESULTS LKB1 Protein Is Downregulated in the Cortices of Developing Mice

To assess expression changes of LKB1 signal in developing mammalian brain, we measured the expression levels of this protein in cerebral cortex of mice at different ages, postnatal day 1 (P1), P3, P7, P14, P21, P42, and P56, by western blotting and

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immunohistochemistry. LKB1 was upregulated at P3 compared with P1 but was downregulated during development, reaching its lowest level in adult mice (Figures 1A and 1B). Immunostaining indicated that LKB1 signals are mainly co-localized with neuronal marker NeuN, although they also are present in non-neuronal cells. Previous studies showed age-dependent expression changes of other genes regulating axon growth, including progranulin and phosphorylated mTOR in mouse brain.37,38 We also examined alterations in LKB1 expression in the sensorimotor cortex of adult mice (8 weeks old) 3 days after a dorsal over-hemisection at T7. LKB1 levels were very low in the cortices of both uninjured and injured mice (Figure 1C), and SCI did not alter its expression levels. Thus, reduced LKB1 levels in adult CNS may contribute to reduction in growth ability of mature neurons during development or after injury.

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Figure 2. Infection with AAV2-LKB1 Vector Increased Neurite Outgrowth of DRG Neurons Cultured on Coverslips Spotted with Purified Aggrecan or CNS Myelin (A) Schematic drawing shows generation and package of AAV2-LKB1 viral vector using HEK293 cells. Three prepared AAV2 plasmids that carry the ITR-containing GFPLKB1, Rep-Cap, and helper genes were co-transfected into HEK293 cells for viral packaging. The AAV2-LKB1 viral vector was then harvested and purified. (B) Schematic drawing indicates protocols for neurite growth assay of cultured DRGs shown in (C) and (D). (C) Adult DRG neurons were transfected with AAV2-GFP (Ctrl) or AAV2-LKB1 and cultured in dishes. Two days after growth, DRGs were replated onto coverslips spotted with purified aggrecan. Five days after culture on coverslips, DRG neurites (stained with Tuj1) that crossed the aggrecan rim were evaluated from each spot. The representative examples indicate that a number of neurites in DRGs transfected with AAV2-LKB1 crossed aggrecan rim, in contrast to no neurites crossing in control. (D) Two days after growth in culture dishes, transfected adult DRGs were replated and cultured for 5 days on coverslips spotted with purified CNS myelin and stained for Tuj1. Very few neurites were detected on myelin spots in DRGs infected with AAV2-GFP, but many neurites were detected on myelin spots in DRGs infected with AAV2-LKB1. The numbers indicate means ± SEM from 11–24 aggrecan or myelin spots per group (from 3 separate experiments). Scale bar: 75 mm.

To verify the specificity of the LKB1 antibody used for immunostaining in this study, we transfected AAV2-GFP or AAV2-LKB1 to MDA-MB-231 cultures. MDA-MB-231cell lines have previously been shown to be LKB1 negative.39 In contrast to lack of LKB1 signals in two groups of controls infected with AAV2-GFP or treated with PBS (not shown), immunostaining with the LKB1 antibody revealed obvious expression of LKB1 protein in the cytoplasm of cells infected with AAV2-LKB1 seven days after transduction (Figure 1D). Western blot assays using the same antibody selectively detected a band of predicated size for the fused GFP-LKB1 protein at 97 kDa only in AAV2-LKB1-infected cells (Figure 1E). With the same GFP-LKB1 plasmid, another group reported a protein of identical size in SKMEL-28 melanoma cell lines.40 Therefore, the signals detected by the chosen LKB1 antibody are highly selective to the target gene. LKB1 Overexpression by AAV2 Viral Vector Infection Enhances Neurite Outgrowth on Inhibitory Substrates In Vitro

To evaluate the role of LKB1 in regulating growth of mature neurons, we tested whether its overexpression with an AAV2 vector would enhance neuronal elongation in vitro by analyzing neurite outgrowth

in adult mouse dorsal root ganglion (DRG) cultures seven days after cell plating and viral infection. Because the optimal time to express the targeted genes is 5 days after AAV2 transduction (not shown), we measured neurite growth in adult DRG cultures 7 days after addition of AAV2: the 7-day period included 2 days of growth in 60-mm dishes and 5 days of growth on aggrecan (one of the lectican family of CSPGs; 600 mg/mL) or CNS myelin-spotted coverslips. Aggrecan and CNS myelin spot assays are frequently used to study axon growth on inhibitory substrates.41,42 Compared with AAV2-GFP controls, AAV2-LKB1 dramatically increased the numbers of axons that crossed an inhibitory gradient of aggrecan and that grew on myelin spots (Figure 2), which contain a very high concentration of purified CNS myelin (200 mg/mL).42 To confirm the AAV2-transduction efficiency, we examined the number of GFP+ cells co-localized with the neuronal marker Tuj1. Most neuronal cell bodies in DRG cultures exhibited GFP signals, but only some neurites crossing the aggrecan rim or grown on myelin spots displayed clear GFP signals (Figures S1A and S1B). We also attempted to evaluate the effects of LKB1 on DRG growth without inhibitory substrates, but the extremely high density of neurites in most areas of the coverslips at more than 4 days after plating precluded reliable measurements (Figure S1C). Similarly, although 80% of DRG neuronal cell bodies are GFP+ when cultured without

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Molecular Therapy

axon growth inhibitors, only a portion of neurites showed various levels of GFP signals (Figures S1C and S1D). The lengths of GFP+ neurites showed a trend toward enhanced growth in the AAV2LKB1 group (Figure S1E). Thus, overexpression of LKB1 by AAV2 viral transduction increased neuronal growth on axon growth inhibitors in vitro, suggesting that LKB1 signaling contributes to the growth failure of adult mammalian neurons. Treatment with AAV2-LKB1 Viral Vector Delivered Five Days after Injury Promotes Regrowth of Corticospinal Tract Axons in Adult Mice

To determine whether our AAV2 vectors are effective for promoting axon regeneration in vivo when delivered at a therapeutically realistic time, we performed dorsal over-hemisection at T7 in 8-week-old C57BL/6 mice and 5 days later injected AAV2 vectors (2  1012 genomic copy/mL) for GFP (Ctrl) or LKB1 into the left sensorimotor cortex. Because signals of expressed GFP were not strong enough to visualize axonal structures following AAV2 infection, anterograde tracing with (biotinylated dextran amine) BDA was used to examine regrowth of the corticospinal tract (CST) 8 weeks after SCI (4 months old). Mice treated with the LKB1 vector showed higher densities of CST sprouts out of the dorsal CSTs rostral to the lesion (not shown). In contrast to SCI controls, animals treated with the LKB1 vector exhibited remarkable CST axon regeneration into the lesion area and distal (caudal) to it (Figure 3). Many of the regenerated CST axons typically paralleled the GFAP+ reactive astrocytic processes surrounding the dorsal lesion epicenter and grew into the deeply transected areas close to ventral spinal cord. CST axons regrew approximately 1 mm into the caudal spinal cord in most mice but in others reached more than 4 mm caudal to the lesion. CST axons in the caudal spinal cord exhibited unusual meandering and branching patterns, the features of regenerated CSTs.43 We did not detect BDA-traced axons in the original locations of dorsal and lateral CSTs in transverse sections at lumbar spinal cord levels (not shown), indicating regenerative CSTs in AAV2-LKB1 group. Immunostaining for GFAP indicated that the sizes of the injury and reactive scar tissue areas were similar in LKB1 and control animals, although it is difficult to measure accurate lesion depth based on GFAP staining because it mainly outlined scar tissues and cavities around the lesion. Therefore, our AAV2LKB1 viral vector, delivered 5 days after SCI, promoted dramatic regrowth of CSTs in adult rodents. In control SCI mice treated with AAV2-GFP, all the dorsal, dorsolateral, and lateral CST fibers labeled by BDA terminated at the lesion site (Figure 3), as we reported previously.42,44 We also confirmed the upregulation of LKB1 protein in the sensorimotor cortex following local injections of AAV2-LKB1 vector (Figure S2). Quantification of LKB1+ cells co-localized with the neuronal marker NeuN in the motor cortex indicated that 81% of neurons exhibited apparent LKB1 signals. Although LKB1 signals were present in approximately 50% of cortical neurons in the AAV2-GFP group, they were generally weaker than those in the AAV2-LKB1 animals. Moreover, we did not detect GFP-LKB1 signals in the regenerative CST axons caudal to the lesion. Further sections through the region

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of the motor cortex doubly stained for LKB1 and BDA tracer revealed dramatically increased LKB1 signals in the AAV2-LKB1 animals (Figure S3A). Most LKB1+ cells were co-labeled with BDA tracer, but LKB1 signals were largely restricted to cell bodies and were rarely detected in individual axon cylinders. We then examined LKB1 immunostaining in transverse sections of spinal cord 5–7 mm rostral to the lesion and observed enhanced levels in the dorsal CST area of AAV2LKB1 mice but did not detect them in individual CST sprouts (Figure S3B). Systemic mAAV9 Viral Vector for LKB1 Delivered before SCI Promotes Robust CST Regeneration in Adult Mice

We then generated a neuron-specific LKB1 viral vector that can efficiently penetrate into the CNS following a systemic application by inserting GFP (control) or GFP-LKB1 plasmid into tyrosine mutant pseudotype AAV9. In this construct, two surface-exposed tyrosine residues in AAV9 capsid protein are removed and replaced by phenylalanine residues (Figure 4A), which increase the blood-brain barrier transference and infectivity of viral vectors.45 The synapsin I promoter was employed for selective expression in neurons after systemic application.46 We injected mutant AAV9 (mAAV9)-GFP or mAAV9-LKB1 intravenously into 6-week-old C57BL/6 mice (50 mL/mouse, 2  1012 genomic copy/mL) and performed T7 dorsal over-hemisection 2 weeks after vector application (8 weeks old). Eight weeks after SCI (4 months old; Figure 4B), we examined regrowth of CST axons by anterograde labeling with BDA and detected increased densities of CST sprouts rostral to the lesion in the mAAV9-LKB1-treated group (Figure 4C). In dramatic contrast to controls, animals treated with mAAV9-LKB1 exhibited remarkable regeneration of CST axons into the lesion area and caudal spinal cord (Figure 4D). Similar to results with AAV2-LKB1 treatment, most regenerated CST axons paralleled GFAP+ reactive astrocytic processes surrounding the dorsal lesion and some labeled axons were present ventral to the deeply transected area close to the ventral margin of the spinal cord. Most mice showed long-distance regeneration of CST axons, extending more than 2 mm caudal to the lesion, and some CSTs reached more than 4 mm caudal to the center of the lesion. Immunostaining for GFAP indicated comparable extents of injury and reactive scar tissues in animals treated with either mAAV9-GFP or mAAV9-LKB1. Camera lucida drawings of CST fibers from parasagittal sections around the lesion were made to quantify the dramatic regeneration into the caudal spinal cord observed in all 6 mice treated with mAAV9-LKB1 (Figure 4E). Measurements of traced CST fibers in the caudal spinal cord showed much greater CST regeneration in the LKB1 vector group than in GFP controls (Figure 4F). Moreover, some mAAV9-LKB1treated mice had regenerated CST axons into the upper lumbar levels of the spinal cord, 5–7 mm caudal to the lesion (Figure 4G), indicating long-distance CST regeneration into the lumbar enlargement, which contains motor neurons that supply the lower limbs. Double labeling for CSTs and vGlut1 (a marker for glutamatergic axon terminals) suggested the presence of synaptic terminals of regenerated CST axons at the lumbar spinal cord levels

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Figure 3. Local Injections of AAV2-LKB1 into the Sensorimotor Cortex Initiated 5 Days after SCI Stimulate Robust Regeneration of Injured CST Axons into the Caudal Spinal Cord of Adult Mice (A) Schematic drawing indicates the experimental procedures and time protocols in adult mice intracortically treated with AAV2 vectors. (B) Neuronal tracer BDA was injected into the motor cortex 6 weeks after SCI, and BDAlabeled CST axons were evaluated 8 weeks after SCI. Parasagittal sections around the lesion in SCI controls indicated termination of injured dorsal CST axons, and there are no regenerated CST axons in the caudal spinal cord. In contrast, similar sections in two representative AAV2-LKB1-treated mice indicated regeneration of a great number of CST axons into the lesion area and caudal spinal cord. Scale bars: 250 (left and middle) and 100 (right) mm. (C) Camera lucida drawings indicate BDAlabeled CST axons from all the parasagittal sections of 4 representative mice, 1 from AAV2-GFP group and 3 from AAV2-LKB1 group. CST axons had short sprouting around lesion but terminated without apparent regeneration in SCI control. In contrast, the animals treated AAV2LKB1 display robust regrowth of CST axons around the lesion and in the caudal spinal cord. (D) BDA-labeled CST fibers were traced from all parasagittal sections of the spinal cord 0–3.2 mm caudal to the lesion, and the total length of CST axons was quantified from each bin box of 0.8-mm spinal cord caudal to the lesion center. Most regenerated CST axons reached the spinal cord approximately 2 mm caudal to the lesion, and some of them regrew longer than 3.2 mm caudal to the lesion center, a level close to the lumbar enlargement in this model. Dorsal is up in all sections. The numbers indicate means ± SEM from 4 or 5 mice per group. Scale bars: 250 mm.

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Figure 4. Systemic mAAV9-LKB1 Vector Delivered 14 Days before SCI Promotes CST Sprouting and Robust Regeneration of Injured CST Axons into the Caudal Spinal Cord in Adult Mice (A) Schematic drawings show the maps for mutant AAV9 (Y446F and Y731F) and for the plasmid that carries the ITR-containing neuronal promoter of synapsin-I and transgene of GFP-LKB1. (B) Schematic drawing indicates the time protocols for in vivo experiments with adult mice intravenously treated with mAAV9 vectors. (C) Representative images of transverse spinal cord sections indicate increased sprouting of BDA-traced dorsal CST axons in the spinal cord 5 mm rostral to the lesion in mAAV9-LKB1-treated mice. Graph shows quantification of the ectopic dorsal CST sprouts in two groups of SCI mice. Scale bar: 100 mm. (D) Parasagittal sections around the lesion in SCI control indicated termination of injured dorsal CST axons, and there are no regenerated CSTs in the caudal spinal cord. In contrast, similar sections in two mAAV9-LKB1-treated mice indicated regeneration of a great number of CST axons into the lesion area and caudal spinal cord. Scale bar: 250 mm. (E) Camera lucida drawings indicate BDA-labeled CST axons from all the parasagittal sections of 8 representative mice, 2 from the mAAV9-GFP group and 6 from the mAAV9-LKB1 group. CST axons sent out short sprouts near the lesion but terminated proximal to the lesion, without obvious regeneration in SCI controls. In contrast, the animals treated with mAAV9LKB1 displayed robust regrowth of CST axons around the lesion and in the caudal spinal cord. Scale bar: 250 mm. (F) BDA-labeled CST axons were traced from all parasagittal sections of the spinal cord 0–3.2 mm caudal to the lesion. The total length of CST axons was quantified from each bin box of 0.8-mm spinal cord caudal to the lesion center. Most regenerated CST axons reached the spinal cord 1 mm caudal to the lesion, but some of them regrew >3.2 mm in the caudal spinal cord, a level close to the lumbar enlargement in this model. (G) Transverse sections of the spinal cord 5–7 mm caudal to the lesion indicate regenerated CST axons in mice treated mAAV9-LKB1, in contrast to no CST axons in mAAV9-GFP controls. The glutamatergic axon terminal marker vGlut is co-localized to some of CST axons presented in the caudal spinal cord. Scale bar: 25 mm. Dorsal is up in all these sections. The numbers indicate means ± SEM from 6–8 mice per group.

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Figure 5. Systemic mAAV9-LKB1 Vector Delivered 14 Days Stimulates Significant Regrowth of Injured Descending 5-HT and TH Axons into the Caudal Spinal Cord of Adult Mice (A) Cartoon shows locations of spinal cord tissue processing around the lesion for in vivo mouse experiments. (B) The representative images (one example per group) and camera lucida drawings (1–3 examples per group) of parasagittal sections show increased density of 5-HT axons in the caudal spinal cord of SCI mice treated with mAAV9-LKB1. Graph shows the length of 5-HT axons measured from all parasagittal sections 0–3.2 mm caudal to the lesion. Treatment with mAAV9-LKB1 resulted in greater 5-HT axons in the caudal spinal cord. (C) Transverse sections of the spinal cord 5–7 mm caudal to lesion at upper lumbar levels displayed reduced 5-HT fibers 8 weeks after SCI, but treatment with mAAV9-LKB1 increased density of 5-HT fibers in the caudal spinal cord. Graph shows length of 5-HT axons in two groups. (D) Transverse sections of the spinal cord 5–7 mm caudal to the lesion indicate that treatment with mAAV9-LKB1 enhanced density of TH axons in the caudal spinal cord. Consistently, graph shows increased length of TH axons in mAAV9-LKB1 group. Dorsal is up in all these sections. Means ± SEM were shown from 6–8 mice per group. Scale bars: 200 mm (B) and 50 mm (C and D).

(Figure 4G). Similarly, we did not detect clear GFP-LKB1 signals in the regenerative CST axons caudal to the lesion. We confirmed the high-transduction efficiency of our mAAV9 vectors in the brain (Figure S4) and enhanced LKB1 signals in the motor cortex (Figure S5A) and in the dorsal CST areas of spinal cord rostral to the lesion (Figure S5B), but no clear GFP-LKB1 signals were seen in individual axon cylinders in either brain or spinal cord. Systemic mAAV9-LKB1 Delivered before SCI Stimulates Regrowth of Descending Serotonergic and TH Axons in Adult Mice

Because our mAAV9 vectors include the synapsin I promotor, systemic mAAV9-LKB1 application should selectively upregulate LKB1 in neurons. Therefore, we used immunohistochemistry to determine whether mAAV9-LKB1 treatment stimulates regrowth of other descending fiber tracts, including 5-hydroxytryptamine (5-HT+) serotonergic axons and tyrosine hydroxylase (TH)+ dopa-

minergic and noradrenergic axons. Both spinal 5-HT and TH tracts contribute to behavioral recovery after SCI.47–51 Because our dorsal over-hemisection at T7 typically transects approximately 70% of the spinal cord area, this lesion spares some ventral spinal cord,44 including a small portion of 5-HT and TH tracts. We characterized the descending serotonergic fibers from parasagittal and transverse sections of the spinal cord by immunostaining for 5-HT. Eight weeks after dorsal over-hemisection, the density of 5-HT fibers was reduced in the spinal cord caudal to the lesion, compared to rostral sections.52 However, in parasagittal sections containing the lesion site, the number of 5-HT axons projecting into the caudal spinal cord was greater in mAAV9-LKB1-treated mice than in controls (Figures 5A and 5B). Camera lucida drawings of 5-HT axons from parasagittal sections showed enhanced density of these fibers at different levels of spinal cord caudal to the lesion (not shown). Measurements of fiber length from parasagittal sections showed an almost 2-fold increase in 5-HT axons in the caudal spinal cord in mAAV9-LKB1 group. Moreover, mAAV9-LKB1 treatment significantly increased the fiber length of both 5-HT and TH tracts in the transverse dorsal and ventral spinal cord 5–7 mm caudal to the lesion at the upper lumbar level (Figures 5C and 5D). Staining intensity for LKB1 protein was consistently greater in multiple regions of the brain in mice systemically treated with mAAV9-LKB1 vector than in controls (Figure S4), including the motor cortical area (Figure S5A).

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Figure 6. Treatments with Intracortical AAV2-LKB1 or Systemic mAAV9-GFP-LKB1 Vector Enhanced Locomotor Recovery Several Weeks after SCI (A) Graph indicates the locomotor BMS scores in SCI mice treated with AAV2-GFP or AAV2-LKB1. AAV2LKB1-treated mice exhibit increased BMS scores several weeks after SCI. (B) Graph indicates grid walk errors in 2 groups of mice 4–8 weeks after SCI. AAV2-LKB1-treated mice had reduced grid walk errors compared with SCI controls. n = 5 mice per group; *p < 0.05; **p < 0.01. (C) Graph indicates BMS locomotor scores in two groups of SCI mice during an 8-week period of observation. mAAV9 LKB1 group has increased BMS locomotor scores several weeks after SCI. (D) Graph indicates grid walk errors in two groups of mice 4 and 8 weeks after SCI. (E) Graph shows grasping rates evaluated at several time points after SCI. mAAV9-LKB1 group also had better performance for this test 4 and 8 weeks after SCI. n = 6–8 mice per group; *p < 0.05; **p < 0.01.

Upregulation of LKB1 by Intracortical AAV2 or Systemic mAAV9 Vector Treatment Promotes Significant Recovery of Locomotor Function in SCI Mice

We evaluated functional recovery in SCI mice treated with intracortical AAV2 vectors or systemic mAAV9 vectors during an 8-week period of observation by performing several behavioral tests in a blinded manner. Two days after SCI, all groups of mice had similar severities of injury, with locomotor BMS scores of approximately 3.5 (Figures 6A and 6C). Several weeks after injury, AAV2GFP- or mAAV9-GFP-treated controls showed partial recovery, but their recovery reached a stable level by 2 or 3 weeks after SCI. However, the BMS scores in either AAV2-LKB1 or mAAV9-LKB1-treated mice continued to increase 3–8 weeks after SCI, and most of these mice had better coordination than controls (Figures 6A and 6C). Evaluation of hindlimb grid walk indicated that the SCI mice treated with intracortical AAV2-LKB1 or systemic mAAV9-LKB1 made fewer errors, more correctly placing their hindpaws on the grid at 4 or 8 weeks after SCI than did

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the corresponding injury controls (Figures 6B and 6D). Moreover, mAAV9-LKB1-treated mice performed better in the touch-grasping test, increasing the hindlimb grasping rate at 4 and 8 weeks after SCI (Figure 6E). Therefore, upregulation of LKB1 by either intracortical AAV2 or systemic mAAV9-LKB1 treatment significantly improved regrowth of descending fiber tracts and recovery of locomotor functions in adult mammals with SCI. AAV2LKB1 mice showed further reduction in grid walk errors compared with mAAV9-LKB1 treatment, although the molecular and cellular basis for this difference remains unknown. It will be interesting to dissect whether upregulation of LKB1 in cortical glia by local AAV2 vector application also contributes to functional recovery after CNS injury. Systemic mAAV9-LKB1 Delivered 3 Days after SCI Stimulates Vigorous CST Regeneration in Adult Mice

Because our vectors are thought to promote axon regrowth mainly by enhancing intrinsic regenerative programs, delivery initiated several days after injury may be as effective as pre-injury injection. To develop a practical strategy for treating patients with acute CNS injuries, we then delivered mAAV9 viral vectors to C57BL/6 mice 3 days after SCI. This time frame applies to most acute patients with axonal injuries, including SCI. We examined regeneration of BDA-traced CST axons 8 weeks after SCI as above. Consistently, we detected termination of severed CST axons in controls treated with mAAV9-GFP but significant regeneration of CST axons into the caudal spinal cord of SCI mice treated with mAAV9-LKB1 (Figure 7). Some regenerated CST axons in mAAV9-LKB1 mice directly crossed the transected spinal cord surrounding the central canal area,

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although others bypassed the lesion in the ventral spinal cord. Notably, mAAV9-LKB1 mice displayed long-distance regeneration of CST axons >2–4 mm caudal to the lesion center (see 3 examples in Figure 7C). Moreover, evaluation of transverse sections 5–7 mm caudal to the lesion at the upper lumbar levels indicated a number of regenerated CST axons (not shown). Therefore, post-injury mAAV9-LKB1 treatment is also highly effective for stimulating regeneration of injured CNS axons in adult mammals. LKB1 Used AMPK1, NUAK1, and ERK as Downstream Signals in Adult CNS

The signaling pathways by which LKB1 controls neuronal growth in adult CNS have not been studied previously. As the master upstream kinase for multiple signals controlling cell growth, LKB1 regulates axon development largely by activating NUAK1, SAD-A, and SAD-B kinases.27,28,33,34 To provide insight into LKB1-mediated regeneration in adult CNS, we investigated the potential downstream effectors of LKB1 in the motor cortices of adult mice 14 days after local intracerebral injections of AAV2-GFP or AAV2-LKB1. After confirming upregulation of the fused GFP-LKB1 protein (at 97 kDa) in the viral-injected cortex by western blots (Figure 8A),40 we measured a number of signaling proteins associated with LKB1 function and also known to regulate neuronal growth. Because LKB1 can phosphorylate and activate several serine-threonine kinases, including AMPK, we examined whether upregulating LKB1 would alter levels of phosphorylated AMPKa (p-AMPKa) at Thr172. AMPK becomes activated when phosphorylation occurs at Thr172 by an upstream kinase.53 We detected the increased levels of p-AMPKa in the cortex of mice infected with AAV2-LKB1 (Figure 8B). Previously, AMPK has been shown to regulate several neuronal functions, including synaptic remodeling in retinas of aged mice54 and axogenesis and axon growth during metabolic stress.55

Figure 7. Systemic mAAV9-LKB1 Vector Delivered 3 Days after SCI Stimulates Vigorous Regeneration of Injured CST Axons into the Caudal Spinal Cord in Adult Mice (A) Schematic drawing indicates the time protocols in adult mice intravenously treated with mAAV9 vectors 3 days after SCI. (B) Representative images of parasagittal sections around the lesion indicated regeneration of a great number of CST axons into the lesion area and caudal spinal cord in mAAV9-LKB1-treated mice, in contrast to no CST regeneration in the caudal spinal cord of mAAV9-GFP controls. Scale bars: 200 (low magnification) and 100 mm (high magnification). (C) Repre-

We next evaluated whether the AMPK-related kinases, NUAK1 and NUAK2, would serve as the effectors of LKB1 in adult CNS. NUAK1 is not detectable in the cortex of control mice treated with AAV2GFP, but AAV2-LKB1 infection significantly upregulated NUAK1 levels (Figure 8C). In contrast, NUAK2 is expressed in the cortex of adult control mice and upregulation of LKB1 by AAV2-LKB1 did not alter its levels. Consistently, the LKB1-NUAK1 pathway has been reported to regulate axon branching by promoting immobilization of mitochondria during development.28 Phosphorylation of LKB1 on S431 phosphorylates and activates PAR-1-related kinases, especially SAD-A and SAD-B at the T-loop residue Thr-175 and Thr-187, respectively. SAD-A and -B are principally expressed in the brain and spinal cord of embryonic and postnatal mice, and their activation by LKB1 is essential for controlling axon differentiation.25 We thus determined the levels of SAD-A and -B in the cortex of adult

sentative examples of camera lucida drawing from all parasagittal sections of the spinal cord indicated termination of severed CST axons in SCI control but regeneration of CSTs into the lesion area and caudal spinal cord in three mAAV9-LKB1treated mice. Scale bar: 250 mm.

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Molecular Therapy

Figure 8. Upregulation of GFP-LKB1 Altered the Levels of Several Signaling Proteins Related to Neuronal Growth in the Motor Cortex of Adult Mice 14 Days after Intracortical Injections of AAV2-LKB1 (A) Levels of fused GFP-LKB1 and endogenous LKB1 proteins were determined from the motor cortex of adult mice 14 days after intracortical injections of AAV2-GFP or AAV2-LKB1. AAV2-LKB1 induced the expression of fused GFP-LKB1 protein at 97 kDa and also slightly downregulated endogenous LKB1 at 54 kDa. (B) Levels of phosphorylated AMPKa (p-AMPKa) were measured from the motor cortex of adult mice 14 days after intracortical injections of AAV2-GFP or AAV2-LKB1. AAV2-LKB1 significantly increased the levels of p-AMPKa at 62 kDa. (C) Levels of NUAK1 and NUAK2 proteins were measured from the motor cortex of adult mice 14 days after intracortical injections of AAV2-GFP or AAV2-LKB1. AAV2-LKB1 significantly increased the levels of NUAK1, but not NUAK2. (D) Levels of SAD-A and -B kinases were measured from the motor cortex of adult mice 14 days after intracortical injections of AAV2-GFP or AAV2-LKB1. AAV2-LKB1 significantly reduced the levels of both kinases. (E) Levels of phosphorylated Akt, S6, and 4E-BP1 (p-Akt, p-S6, and p-4E-BP1) were measured from the motor cortex of adult mice 14 days after intracortical injections of AAV2-GFP or AAV2-LKB1. AAV2-LKB1 significantly decreased the levels of p-S6 but did not alter the levels of p-Akt and p-4E-BP1. (F) Levels of phosphorylated ERK (p-ERK) were measured from the motor cortex of adult mice 14 days after intracortical injections of AAV2-GFP or AAV2-LKB1. AAV2-LKB1 significantly increased the levels of p-ERK. (G) Table summarizing the results of signaling proteins evaluated in this study. n = 4 mice per group.

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mice following LKB1 upregulation and detected basic levels of both kinases in control mice. However, AAV2-LKB1 treatment reduced their expression levels (Figure 8D). We attempted to measure the levels of phosphorylated SADs with the antibodies used previously27 but failed to obtain these antibodies. Because AMPK activation by LKB1 could activate tuberous sclerosis 1 and 2 (TSC1/TSC2), which consequently inhibit activities of Rheb and mTOR,25,56 we next measured the activities of several signaling proteins along the PI3K/mTOR pathway, a known regulator for CNS regeneration failure.57,58 Upregulation of LKB1 did not alter the levels of phosphorylated Akt and 4-E-BP1 but significantly attenuated levels of phosphorylated S6 kinase (Figure 8E). Because previous studies indicated the interactions between ERK and LKB1 pathways,59,60 we also examined ERK activity by measuring the levels of phosphorylated p44/42 MAPK (Erk1/2) at Thr202/ Tyr204 and found that LKB1 upregulation increased its levels (Figure 8F). Constantly, neuronal ERKs appear critical for regulating protein synthesis and maintaining levels of proteins required for growth in response to various extracellular cues, especially neurotrophins.61,62 Thus, upregulation of GFP-LKB1 in the motor cortex of adult mice activated AMPKa and ERK signals and upregulated NUAK1 protein but suppressed S6 kinase activity and downregulated SAD-A and B (see summary in Figure 8G). It did not alter the activities of Akt and 4E-BP1 and the expression levels of NUAK2. Notably, AAV2LKB1 application slightly reduced the levels of endogenous LKB1 at 54 kDa (Figure 8A), but the overall levels of LKB1 (i.e., GFPLKB1 + endogenous LKB1) were significantly greater in the AAV2LKB1 group than in AAV2-GFP controls (not shown).

DISCUSSION CNS neurons can regenerate their axons robustly during development but lose their growth capability during maturation. Thus, axons cannot regenerate past an injury site to form functional synaptic reconnections in the injured mature mammalian CNS. Expression changes of a few cell-autonomous molecules, including PTEN, SOCS3, cAMP, Krüppel-like factors, and c-myc can regulate neuronal growth in mammals.12,13,15,16,63,64 PTEN deletion in knockout mice produced remarkable CST regrowth, but the regrown CSTs typically reach less than 2 mm caudal to the injury.16,65 PTEN short hairpin RNAs (shRNAs)66,67 and antagonist peptides42 administered before or after SCI induced only limited CST regrowth, and developing a successful strategy for regenerating injured CST axons in adult mammals has proved to be very challenging. Using novel AAV2 and mAAV9 viral vectors, we have demonstrated that LKB1 plays an important function in regulating the growth capacity of mature neurons in the mammalian CNS. This study expands our understanding of the factors that boost regenerative capacity of mature neurons after CNS injury. Of particular interest, systemic application of our mAAV9 vector to enhance neuronal LKB1 activity potentially could lead to development of a non-invasive therapy to recover lost functions in humans with CNS injuries.

We demonstrated robust regeneration of injured CST fibers in adult mice treated either intracortically or systemically with viral vectors to overexpress LKB1 protein in neurons, supporting the critical role of this kinase in controlling neuronal growth capacity in adult mammals. We identified that most CSTs bypassed or passed the injury epicenter and projected into spinal gray matter. CSTs in the caudal spinal cord morphologically meet the criteria of regenerative axons defined previously,43 including locations close to axotomy, unusual course through CNS tissue environment of gray matter, and highly branching patterns. Because we did not detect ventral CSTs in C56BL/6 mice and transected dorsal spinal cord at a depth of 1 mm (confirmed by passing the sharp part of a 30G needle across the dorsal spinal cord 5 times), our lesions should have severed all dorsal and lateral CST axons. We also confirmed the absence of BDA-labeled axons in original locations of CSTs from transverse sections of caudal spinal cord. Transgenic PTEN deletion showed similar patterns of CST regeneration after dorsal hemisection (0.8 mm lesion depth) in mice.16 With single-cell intracellular electrophysiology, one group demonstrated formation of novel polysynaptic relay contacts of injured CSTs to interneurons around contusion lesion at T10 in rats.68 LKB1 is a serine-threonine kinase required for maintaining cell metabolism, energy homeostasis, and polarity by activating AMPactivated protein kinase (AMPK) (on Thr-172) and at least 12 other kinases. It is widely expressed in many embryonic and adult tissues, including the developing brain, with its highest levels in forebrain.27,69 LKB1 is subcellularly concentrated in the nucleus, cytosol, and mitochondria and is also present in the axons and extracellular space. LKB1 controls epithelial cell and neuronal polarization during development by regulating Golgi apparatus distribution in the cytoplasm. As the convergent downstream effector of cAMP/PKA and PI3K signaling pathways in response to stimulation by various extracellular factors, including neurotrophins, guidance cues, and ECM molecules, LKB1 regulates neuronal migration, axon initiation, axon extension, and synapse formation.25,29,54 Thus, LKB1 is a major determinant of axon differentiation. LKB1 overexpression stimulates formation of multiple axons, whereas deletion or downregulation of LKB1 in mice blocks axon formation in vivo.27,29 Moreover, LKB1 is required for maintaining stability and myelination of peripheral axons by regulating the function of Schwann cells.31,32,70 The present report shows that LKB1 plays an important role in controlling the regenerative capacity of injured adult mammalian CNS neurons in vivo. Previously, LKB1 was recognized as a tumor suppressor because its mutations were present in cancers of several tissues, including cervix, breast, intestine, testicle, pancreas, and skin. Germline inactivating mutations of LKB1 also are associated with Peutz-Jeghers syndrome, an autosomal dominant genetic disease characterized by multiple benign polyps in the gastrointestinal system.71 During development, cAMP/PKA and PI3K signals are critical for controlling cell growth in response to stimulation by many extracellular factors. These pathways are activated in parallel and converge on common downstream effectors that regulate axon differentiation.

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Molecular Therapy

Among the downstream effectors, LKB1 phosphorylation represents an early essential signal for axon formation during neuronal polarization.25 A number of highly conserved residues on LKB1 are phosphorylated either by auto-phosphorylation (Thr-185, Thr-189, Thr-336, and Ser-404) or by upstream kinases (Ser-31, Ser-325, Thr-366, and Ser-431). LKB1 has a kinase domain at its N terminus and regulatory domains at both N and C termini. Activation of LKB1 usually requires formation of a complex with its cofactors STE20-related kinase adaptor alpha (STRAD) and calcium binding protein 39 (also known as MO25)72 in the nucleus, and its expression pattern is usually similar to that of STRAD and MO25. In fact, accumulation of LKB1 and STRAD correlates with axon differentiation, and their overexpression stimulates the neuron to give rise to multiple axons.29 After activation and translocation to the cytoplasm, LKB1 is phosphorylated at Ser-431 by active cytoplasmic PKA and then activates downstream signaling pathways, including AMPK, SAD, and other kinases.25 Therefore, somatic expression of LKB1 appears critical for controlling the growth of cells, including neurons. Accordingly, we detected clear and strong LKB1 protein signals in the somata of AAV2-LKB1-infected neurons in vitro and in vivo. By contrast, only a small portion of axons exhibited weak to moderate LKB1 signals in adult neurons. Therefore, we did not visualize LKB1 signals in regenerated CST axons in the caudal spinal cord of adult mice. We demonstrated that LKB1 upregulation activated AMPKa and ERK signals and increased expression of NUAK1 in the cortical neurons of adult mice. In adult rodents, ERK activation by viral infection promoted CST regeneration following subcortical axotomy.62 During neural development, NUAK1 controlled axon branching by regulating axonal mitochondria immobilization.28 In contrast, we found reduced levels of SAD-A and -B following LKB1 overexpression in adult cortex, suggesting that SADs are not the major regulators downstream of LKB1 in mature CNS neurons. During neural development, in addition to LKB1,27 the signals downstream of PI3K, TSC1, and TSC2 could activate SADs by locally restricting their translation.73 SADs represent a point of convergence whereby PKA/LKB1 and PI3K/Akt pathways regulate axon formation by phosphorylating the axon protein Tau on Ser-262. This controls Tau binding to microtubules and subsequent microtubule organization.27,33,34 Accordingly, deletion of both SAD-A and -B causes a striking deficiency of cortical axon formation.33 Because AAV2-LKB1 suppressed the activity of S6 kinase, regeneration of certain populations of CNS neurons appears to be independent of mTOR/S6 kinase. Further studies are required to clarify the detailed signaling pathway of LKB1 in mature neurons. Because AAV vectors have favorable safety profiles, efficiently transduce a wide range of cell types, and are frequently used to treat neurological disorders,35,36 this study may help us identify innovative, practical strategies to treat CNS disorders by enhancing regeneration and reconnection between neurons. Of particular interest for the development of a practical and non-invasive treatment strategy for CNS injury, we systemically applied a mAAV9 vector with the neuronal promotor synapsin I to upregulate LKB1 in multiple neuronal popu-

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lations. This vector promoted robust axon regeneration and significant functional recovery in adult rodents. Because almost all currently available small- and large-molecule drugs fail to cross the blood-brain barrier, including most serotypes of AAV vectors, our systemically deliverable vector could have therapeutic potential for treating CNS disorders, including SCI.

MATERIALS AND METHODS Characterization of LKB1 Protein Expression in the Nervous System

All the experimental procedures with animals were approved by the Institutional Animal Care and Use Committee at Temple University. We evaluated the expression level of LKB1 protein in the cortices of developmental C57BL/6 mice by western blots. For sample preparation, mice were perfused with ice-cold PBS for 5 min and fresh tissue blocks of both sides of cortices were collected immediately and then prepared in lysis buffer (50 mM Tris-HCl [pH 7.2], 10 mM MgCl2, 300 mM NaCl, and 1.5% IGEPAL) containing various protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 2 mM orthovanadate, 10 mg/mL leupeptin, and 10 mg/mL aprotinin). The supernatants of tissue lysates containing equal amounts of total protein were loaded onto Tris-glycine gels and transferred to nitrocellulose membranes. Membranes were blocked with 5% blotting grade milk (Bio-Rad), blotted with a rabbit anti-LKB1/STK11 antibody from Novus Biologicals (for Figures 1B and 1C), and mouse anti-actin clone C4 antibody (MP Biomedicals; for loading control) and then incubated with secondary antibodies conjugated to IRDye 800CW goat anti-mouse immunoglobulin G (IgG) (LI-COR Biosciences) or IRDye 680RD goat anti-rabbit IgG (LI-COR Biosciences). The western blot bands were visualized and quantified with the LI-COR Biosciences Odyssey scanner and Odyssey ImagingStudio software. To examine expression alterations of the LKB1 protein in the sensorimotor cortex of adult mice after SCI, we performed dorsal over-transection injury at T7 in C57BL/6 mice (8 weeks old) and collected fresh tissues of sensorimotor cortices 3 days after injury. We then measured levels of LKB1 from these samples by western blotting. At least 3 or 4 separate experiments were performed, and the representative blots are shown in Figure 1. To verify neuronal expression of LKB1 in the cortex, coronal brain sections from C57BL/6 mice of various ages were immunostained for LBK1 (antibody cat no.: 3050S; Cell Signaling Technology) and for the neuronal marker NeuN. MDA-MB-231 Cell Cultures and Verification of Selectivity of LKB1 Antibody

MDA-MB-231 cell lines were purchased from the American Type Culture Collection and maintained in DMEM with high glucose (Life Technologies) and 10% fetal bovine serum (FBS) on coverslips inside of 12-well plates (for immunocytochemistry) or on 60-mm culture dishes (for western blots) at 37 C with 5% CO2. Cells were infected with AAV2-GFP and AAV2-LKB1 in the presence of etoposide (250 nM) or treated with saline as additional controls. Seven days after infection, cells were fixed for immunostaining or collected fresh for western blot assay. For the former, cell cultures were fixed in 4% paraformaldehyde in PBS for 20 min. After several rinses with PBS,

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coverslips were incubated in a blocking solution with 0.1% Triton X-100 for 1 hr at room temperature and then incubated with LKB1 antibody (1:1,000) overnight at 4 C. After rinses several times with PBS, cell cultures were incubated in appropriate secondary antibody for 1 hr. Multiple coverslips were employed for each group of individual experiments. For western blots, cells were prepared in 300 mL cold lysis buffer supplemented with protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 2 mM orthovanadate, 10 mg/mL leupeptin, and 10 mg/mL aprotinin). Samples were clarified by centrifugation at 15,000 g for 20 min at 4 C, and total protein was determined with Bio-Rad DC protein assay reagents. Samples containing the same amount of total protein were aliquoted into multiple tubes and used for western blotting assay as described above. The primary LKB1 antibody was used at a dilution of 1:1,000, and the secondary antibody was used at 1:10,000 dilution.

DRG cultures were fixed in 4% paraformaldehyde in PBS for 30 min. After several rinses with PBS, coverslips were incubated in blocking solution (5% normal goat serum + 0.2% BSA in 0.1% Triton X-100 PBS) for 1 hr at room temperature and then incubated with antibIII-tubulin (1:400; Covance) overnight at 4 C. After rinses several times with PBS, DRG cultures were incubated in appropriate secondary antibody (Thermo Fisher Scientific) for 1 hr at room temperature (RT). The number of bIII-tubulin-positive axons crossing spot rim were counted from each aggrecan spot, and the total length of neurites on each CNS myelin spot was measured with Photoshop and NIH image software.75 Multiple coverslips were employed for each group of individual experiments.

Production of AAV2-GFP or AAV2-LKB1 Vectors and Evaluation of Their Function with Neurite Outgrowth Assay in Primary Neuronal Cultures

To lesion the spinal cord of C57BL/6 mice (8–10 weeks old), we exposed the dorsal spinal cord by T6-7 laminectomy. A dorsal over-hemisection (1 mm in depth and approximately 1.5 mm in dorsoventral diameter) was performed at T7 with a 30G needle and microscissors to completely sever the dorsal CST. The lesion depth of 1 mm was ensured by passing a marked 30G needle at least 5 times across the dorsal spinal cord. To upregulate LKB1 in cortical motor neurons of adult C57BL/6 mice, 5 days after SCI, we used a Nanoinjector to inject AAV2-GFP or AAV2-LKB1 (2  1012 genomic copy/mL) into 5 sites within the left sensorimotor cortex (anterior-posterior coordinates from Bregma in mm: 1.0, 0.5, 0, 0.5, 1.0; all at 1.0 mm lateral and at a depth of 1.0 mm). Six weeks after SCI, the mice received BDA (10,000 molecular weight [MW]) tracer injections into 5 sites of the sensorimotor cortex at the same coordinates as for AAV2 vector injections. Mice were perfused 2 weeks after BDA injection, and fixed spinal cords were dissected for histology as below.

To generate AAV2-based vectors, we inserted GFP (Ctrl) or GFPLKB1 into the pAM/chicken b-actin/woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) vector and packaged viral vectors in HEK293 cells using helper and packaging vectors. The AAV2 vector plasmids contain the expression cassettes, including chicken b-actin promoter, cDNA encoding GFP (Ctrl) or GFPLKB1, WPRE, and SV40 polyA between inverted terminal repeats (ITRs). The recombinant GFP-LKB1 plasmid was cloned and generated by PCR in our lab. The vector, pAAV-RC2 (AAV2 rep and cap expression plasmid) and helper plasmid pHelper were co-transfected into HEK293 cells by polyethyleneimine.74 The prepared AAV2 vectors were purified by double centrifugation with cesium chloride and then dialyzed overnight. The vector titers used in this study were initially determined by infecting fibroblast cells and confirmed in transfected adult DRG cultures by immunostaining for GFP or LKB1. To evaluate the role of LKB1 in regulating axon growth in vitro, we performed neurite outgrowth in DRG cultures 7 days after growth, including 2 days in 60-mm dish with AAV2 viral infection and 5 days on aggrecan or CNS myelin spotted coverslips after replating. DRGs were harvested from adult C57BL/6 mice (7 or 8 weeks) and incubated in collagenase (100 U/mL; Worthington Biochemical, Lakewood, NJ) and then with collagenase plus 0.25% trypsin/ EDTA. After wash with culture medium, dissociated DRG neurons were plated onto 60-mm dish and transfected with AAV2-GFP or AAV2-LKB1 vector. Two days after growth in the dish, DRGs were collected and dissociated gently and then replated onto plastic coverslips and grown in culture medium (DMEM/F12 mixture plus 10% fetal bovine serum, 2 mM glutamine, 100 mg/mL penicillin, and 100 mg/mL streptomycin) for 5 days at 37 C.75,76 Before cell replating, coverslips (15 mm) coated with poly-L-lysine were spotted with a 2-mL solution of aggrecan (600 mg/mL) and laminin (10 mg/mL) in Hank’s balanced salt solution (HBSS)-calcium/magnesium free (CMF) (2 spots/coverslip) or with 2 mL of purified CNS myelin (200 mg/mL) plus 10 mg/mL of laminin. Five days after the replating,

Spinal Cord Injury, Intracortical Treatments with AAV2-GFP or AAV2-LKB1 Vectors, and Tracer Injections to Label CST Axons in Adult Mice

Production of Recombinant mAAV9-GFP or AAV9-GFP-LKB1 Vectors and Systemic Treatment with These Vectors in Mice

mAAV9-LKB1 and AAV9-GFP vector plasmids consist of neuronspecific synapsin I promoter followed by cDNA encoding GFPLKB1 or GFP (Ctrl), WPRE, and a simian virus 40 polyadenylation signal sequence (SV40 poly[A]) between ITRs of AAV3. mAAV9 vp was produced by substitutions of thymidine for adenine at positions 1,337 and 2,192, which results in amino acid changes from tyrosine to phenylalanine at positions 446 and 731.45,46 These vectors were produced by transfection of HEK293 cells with vector plasmids, AAV3 rep and mAAV9 vp expression plasmid, and adenoviral helper plasmid pHelper.77 Recombinant viruses were then purified in CsCl gradients. To infect neurons by systemic treatment, in the first set of study, we slowly injected a solution of these viral vectors (titer: 1.5  1012 genomic copies/mL) into the tail vein of C57BL/6 wildtype (WT) mice (6 weeks old; 50 mL per mouse). Two weeks after the viral injection, we performed dorsal over-hemisection of the spinal cord as described above. To evaluate the efficacy of mAAV9 vectors delivered after injury, in the second batch of experiments, we made dorsal over-hemisection at T7 in adult mice (8–10 weeks

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Molecular Therapy

old) and injected mAAV9-GFP or mAAV9-LKB1 (same titer as above) into the tail vein 3 days after the injury. Histology, Axon Quantification, and Behavioral Evaluations

The spinal cord extending from 0 to 4 mm rostral to and caudal to the lesion (8 mm long; containing the injury site) was cut parasagittally (30 mm; see drawing in Figure 5A). The spinal cord 5–7 mm rostral to and caudal to injury was transversely sectioned (30 mm). All the parasagittal sections were processed for BDA tracer with tyramide signal amplification (TSA) systems (PerkinElmer). For raphe axon labeling (for the experiments with mAAV9 vector treatment), all the parasagittal sections were immunostained with a rabbit anti-5HT serotonergic antibody (ImmunoStar) and an Alexa-488-conjugated secondary antibody. To visualize the lesion area, all parasagittal sections were also stained for GFAP (Sigma) with an anti-mouse Alexa-Fluor-647-conjugated secondary antibody. Some transverse sections rostral to and caudal to the lesion were used for processing BDA tracer and for immunostaining (for mAAV9 experiments) with a 5-HT serotonergic antibody or with a rabbit anti-TH antibody (Millipore). To confirm the expression levels of LKB1 in SCI mice, some brain sections in the region of the motor cortex and transverse sections of spinal cord 5–7 mm rostral or caudal to the lesions were doubly stained for LKB1 (cat no. 3050S; Cell Signaling Technology) and NeuN or for LKB1 and BDA tracer. To compare axon numbers in the caudal spinal cord between different groups, we determined the length of BDA-labeled CST(in AAV2 and mAAV9 experiments) and 5-HT (in mAAV9 study) axons in all parasagittal sections of spinal cord from 0 to 3.2 mm caudal to the lesion epicenter in each animal. The injury center was determined as the midpoint of histological abnormalities produced by lesion cavitation, reactive astrocytes, and morphological changes of injured axons. The 5-HT and CST axons caudal to the lesion were traced manually in each of the parasagittal sections, and their total length inside of several bin boxes at 0.8, 1.6, 2.4, and 3.2 mm caudal to lesion center was measured using Photoshop and ImageJ software. To determine ectopic sprouting of CSTs in the transverse spinal cord sections 5–7 mm rostral to lesion (around T2 or 3 levels), we manually traced individual BDA-labeled CST fibers outside of dorsal CSTs in two-side spinal cord from images captured with a 10 objective. The density of traced CST fibers was measured from 4 randomly selected sections in each mouse using Photoshop and ImageJ software. The 5-HT and TH fibers were measured in the ventral and dorsal half of transverse sections from 5 to 7 mm caudal to the lesion (at the upper lumbar levels) in the mAAV9 study. To determine functional recovery, we evaluated locomotion alterations during 8 weeks of survival by measuring locomotor Basso mouse scale (BMS) scores at two days after SCI and weekly thereafter and grid walk performances at 4 and 8 weeks after SCI. We also measured grasping rate in mice treated with viral vectors at 2 days and then at 2, 4, and 8 weeks after SCI. The BMS scores were evaluated while mouse was walking in an open field and confirmed from digital video records. The grid walk errors were counted from video-

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tapes played at a slow speed (4 separate trials per test) and averaged from different trials. For grasping analysis, the mouse was pulled steadily until it lost forelimb grip and then the peak force was measured by the force meter. Contact-evoked grasping rate was measured by lowering the hindpaws toward a wire cage lid and determining the percent of times that it was grasped successfully. All the behavioral tests were performed by two persons who were unaware of animal identifications. Characterization of Downstream Signals of LKB1 by Western Blots in Adult Mice Intracortically Treated with AAV2-GFP or AAV2-LKB1

To study the downstream signals of LKB1 in cortical neurons of adult C57BL/6 mice, we injected AAV2-GFP or AAV2-LKB1 (2  1012 genomic copy/mL) into 5 sites of the sensorimotor cortex with the coordinates described above. Two weeks after the AAV2 vector injections, mice were perfused with cold PBS, and fresh tissues in the region of AAV2-injected cortex were collected and prepared for western blot assays as above. The following primary antibodies were used for western blot assays shown in Figure 8: rabbit LBK1 (cat no.: 3050S); rabbit phospho-AMPKa (Thr172; 40H9); rabbit NUAK1 (ARK5); NUAK2 (SNARK; cat no.: 4100S); rabbit monoclonal antibody (mAb) BRSK2 (D29B6; SAD-A); rabbit BRSK1 (D10F2; SAD-B); mouse mAb phospho-Akt (Ser473; 587F11); rabbit mAb phospho-S6 ribosomal protein (Ser235/236); rabbit mAb phospho-4E-BP1 (eukaryotic initiation factor 4E-binding protein 1; Thr37/46; 236B4); rabbit mAb phospho-Erk1/2 (p44/42); and rabbit or mouse beta actin. All of these antibodies were purchased from Cell Signaling Technology. Statistical Analysis

GraphPad Prism software was used for statistical analysis. Data in graphs are shown as means ± SEM. Comparisons between two groups at multiple time points were analyzed with a repeated-measures ANOVA. The experiments comparing a single determination of means between two independent groups were analyzed with Student’s t test. Differences between groups with p < 0.05 were considered significant (*p < 0.05; **p < 0.01), and a Bonferroni correction was used for multiple comparisons. During the experimental procedures, including surgery, histology, axon quantification, and behavioral evaluations, the evaluating researchers were blind to animal genotypes and viral vector treatments.

SUPPLEMENTAL INFORMATION Supplemental Information includes five figures and can be found with this article online at https://doi.org/10.1016/j.ymthe.2018.10.019.

AUTHOR CONTRIBUTIONS Y.O., A.S., X.J., M.H., K.S., L.M., and S.L. contributed to experimental designs, data analyses, figure making, and paper writing. S.M. and G.M.S. contributed to the viral vector generation and paper writing. M.E.S. contributed to paper writing.

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CONFLICTS OF INTEREST S.M. owns equity in a gene therapy company (Gene Therapy Research Institution) that commercializes the use of AAV vectors for gene therapy applications. S.M. has a conflict of interest to the extent that the work in this paper might increase the value of the commercial holdings. The other authors have no potential conflicts of interest to disclose.

ACKNOWLEDGMENTS

intrinsic regenerative ability revealed by quantitative proteomics. Neuron 86, 1000–1014. 16. Liu, K., Lu, Y., Lee, J.K., Samara, R., Willenberg, R., Sears-Kraxberger, I., Tedeschi, A., Park, K.K., Jin, D., Cai, B., et al. (2010). PTEN deletion enhances the regenerative ability of adult corticospinal neurons. Nat. Neurosci. 13, 1075–1081. 17. Norsworthy, M.W., Bei, F., Kawaguchi, R., Wang, Q., Tran, N.M., Li, Y., Brommer, B., Zhang, Y., Wang, C., Sanes, J.R., et al. (2017). Sox11 expression promotes regeneration of some retinal ganglion cell types but kills others. Neuron 94, 1112–1120.e4. 18. Shackelford, D.B., and Shaw, R.J. (2009). The LKB1-AMPK pathway: metabolism and growth control in tumour suppression. Nat. Rev. Cancer 9, 563–575.

We thank Drs. Umar Hayat, Nadjat Serradj, Shen Dai, and Dr. Xuebin Qin for technical support. This work was supported by research grants to S.L. from NIH (1R01NS079432 and 1R01EY024575) and from Shriners Research Foundation (SHC-85100, SHC-86300-PHI, and SHC-86200-PHI-16).

19. Hardie, D.G. (2015). AMPK: positive and negative regulation, and its role in wholebody energy homeostasis. Curr. Opin. Cell Biol. 33, 1–7.

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